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Sample records for auxin efflux transporter

  1. The Role of PIN Auxin Efflux Carriers in Polar Auxin Transport and Accumulation and Their Effect on Shaping Maize Development

    Institute of Scientific and Technical Information of China (English)

    Cristian Forestan; Serena Varotto

    2012-01-01

    In plants,proper seed development and the continuing post-embryonic organogenesis both require that different cell types are correctly differentiated in response to internal and external stimuli.Among internal stimuli,plant hormones and particularly auxin and its polar transport(PAT)have been shown to regulate a multitude of plant physiological processes during vegetative and reproductive development.Although our current auxin knowledge is almost based on the results from researches on the eudicot Arabidopsis thaliana,during the last few years,many studies tried to transfer this knowledge from model to crop species,maize in particular.Applications of auxin transport inhibitors,mutant characterization,and molecular and cell biology approaches,facilitated by the sequencing of the maize genome,allowed the identification of genes involved in auxin metabolism,signaling,and particularly in polar auxin transport.PIN auxin efflux carriers have been shown to play an essential role in regulating PAT during both seed and post-embryonic development in maize.In this review,we provide a summary of the recent findings on PIN-mediated polar auxin transport during maize development.Similarities and differences between maize and Arabidopsis are analyzed and discussed,also considering that their different plant architecture depends on the differentiation of structures whose development is controlled by auxins.

  2. The E3 Ubiquitin Ligase SCFTIR1/AFB and Membrane Sterols Play Key Roles in Auxin Regulation of Endocytosis, Recycling, and Plasma Membrane Accumulation of the Auxin Efflux Transporter PIN2 in Arabidopsis thaliana[C][W][OA

    Science.gov (United States)

    Pan, Jianwei; Fujioka, Shozo; Peng, Jianling; Chen, Jianghua; Li, Guangming; Chen, Rujin

    2009-01-01

    The PIN family of auxin efflux transporters exhibit polar plasma membrane (PM) localization and play a key role in auxin gradient-mediated developmental processes. Auxin inhibits PIN2 endocytosis and promotes its PM localization. However, the underlying mechanisms remain elusive. Here, we show that the inhibitory effect of auxin on PIN2 endocytosis was impaired in SCFTIR1/AFB auxin signaling mutants. Similarly, reducing membrane sterols impaired auxin inhibition of PIN2 endocytosis. Gas chromatography–mass spectrometry analyses indicate that membrane sterols were significantly reduced in SCFTIR1/AFB mutants, supporting a link between membrane sterols and auxin signaling in regulating PIN2 endocytosis. We show that auxin promoted PIN2 recycling from endosomes to the PM and increased PIN2 steady state levels in the PM fraction. Furthermore, we show that the positive effect of auxin on PIN2 levels in the PM was impaired by inhibiting membrane sterols or auxin signaling. Consistent with this, the sterol biosynthetic mutant fk-J79 exhibited pronounced defects in primary root elongation and gravitropic response. Our data collectively indicate that, although there are distinct processes involved in endocytic regulation of specific PM-resident proteins, the SCFTIR1/AFB-dependent processes are required for auxin regulation of endocytosis, recycling, and PM accumulation of the auxin efflux transporter PIN2 in Arabidopsis thaliana. PMID:19218398

  3. Role of the Arabidopsis PIN6 Auxin Transporter in Auxin Homeostasis and Auxin-Mediated Development

    OpenAIRE

    2013-01-01

    Plant-specific PIN-formed (PIN) efflux transporters for the plant hormone auxin are required for tissue-specific directional auxin transport and cellular auxin homeostasis. The Arabidopsis PIN protein family has been shown to play important roles in developmental processes such as embryogenesis, organogenesis, vascular tissue differentiation, root meristem patterning and tropic growth. Here we analyzed roles of the less characterised Arabidopsis PIN6 auxin transporter. PIN6 is auxin-inducible...

  4. Posttranslational modification and trafficking of PIN auxin efflux carriers.

    Science.gov (United States)

    Löfke, Christian; Luschnig, Christian; Kleine-Vehn, Jürgen

    2013-01-01

    Cell-to-cell communication is absolutely essential for multicellular organisms. Both animals and plants use chemicals called hormones for intercellular signaling. However, multicellularity of plants and animals has evolved independently, which led to establishment of distinct strategies in order to cope with variations in an ever-changing environment. The phytohormone auxin is crucial to plant development and patterning. PIN auxin efflux carrier-driven polar auxin transport regulates plant development as it controls asymmetric auxin distribution (auxin gradients), which in turn modulates a wide range of developmental processes. Internal and external cues trigger a number of posttranslational PIN auxin carrier modifications that were demonstrated to decisively influence variations in adaptive growth responses. In this review, we highlight recent advances in the analysis of posttranslational modification of PIN auxin efflux carriers, such as phosphorylation and ubiquitylation, and discuss their eminent role in directional vesicle trafficking, PIN protein de-/stabilization and auxin transport activity. We conclude with updated models, in which we attempt to integrate the mechanistic relevance of posttranslational modifications of PIN auxin carriers for the dynamic nature of plant development.

  5. Regulation of auxin transport during gravitropism

    Science.gov (United States)

    Rashotte, A.; Brady, S.; Kirpalani, N.; Buer, C.; Muday, G.

    Plants respond to changes in the gravity vector by differential growth across the gravity-stimulated organ. The plant hormone auxin, which is normally basipetally transported, changes in direction and auxin redistribution has been suggested to drive this differential growth or gravitropism. The mechanisms by which auxin transport directionality changes in response to a change in gravity vector are largely unknown. Using the model plant, Arabidopsis thaliana, we have been exploring several regulatory mechanisms that may control auxin transport. Mutations that alter protein phosphorylation suggest that auxin transport in arabidopsis roots may be controlled via phosphorylation and this signal may facilitate gravitropic bending. The protein kinase mutant pinoid (pid9) has reduced auxin transport; whereas the protein phosphatase mutant, rcn1, has elevated transport, suggesting reciprocal regulation of auxin transport by reversible protein phosphorylation. In both of these mutants, the auxin transport defects are accompanied by gravitropic defects, linking phosphorylation signaling to gravity-induced changes in auxin transport. Additionally, auxin transport may be regulated during gravity response by changes in an endogenous auxin efflux inhibitor. Flavonoids, such as quercetin and kaempferol, have been implicated in regulation of auxin transport in vivo and in vitro. Mutants that make no flavonoids have reduced root gravitropic bending. Furthermore, changes in auxin-induced gene expression and flavonoid accumulation patterns have been observed during gravity stimulation. Current studies are examining whether there are spatial and temporal changes in flavonoid accumulation that precede gravitropic bending and whether the absence of these changes are the cause of the altered gravity response in plants with mutations that block flavonoid synthesis. These results support the idea that auxin transport may be regulated during gravity response by several mechanisms including

  6. Polar auxin transport: controlling where and how much

    Science.gov (United States)

    Muday, G. K.; DeLong, A.; Brown, C. S. (Principal Investigator)

    2001-01-01

    Auxin is transported through plant tissues, moving from cell to cell in a unique polar manner. Polar auxin transport controls important growth and developmental processes in higher plants. Recent studies have identified several proteins that mediate polar auxin transport and have shown that some of these proteins are asymmetrically localized, paving the way for studies of the mechanisms that regulate auxin transport. New data indicate that reversible protein phosphorylation can control the amount of auxin transport, whereas protein secretion through Golgi-derived vesicles and interactions with the actin cytoskeleton might regulate the localization of auxin efflux complexes.

  7. Auxin efflux by PIN-FORMED proteins is activated by two different protein kinases, D6 PROTEIN KINASE and PINOID.

    Science.gov (United States)

    Zourelidou, Melina; Absmanner, Birgit; Weller, Benjamin; Barbosa, Inês C R; Willige, Björn C; Fastner, Astrid; Streit, Verena; Port, Sarah A; Colcombet, Jean; de la Fuente van Bentem, Sergio; Hirt, Heribert; Kuster, Bernhard; Schulze, Waltraud X; Hammes, Ulrich Z; Schwechheimer, Claus

    2014-06-19

    The development and morphology of vascular plants is critically determined by synthesis and proper distribution of the phytohormone auxin. The directed cell-to-cell distribution of auxin is achieved through a system of auxin influx and efflux transporters. PIN-FORMED (PIN) proteins are proposed auxin efflux transporters, and auxin fluxes can seemingly be predicted based on the--in many cells--asymmetric plasma membrane distribution of PINs. Here, we show in a heterologous Xenopus oocyte system as well as in Arabidopsis thaliana inflorescence stems that PIN-mediated auxin transport is directly activated by D6 PROTEIN KINASE (D6PK) and PINOID (PID)/WAG kinases of the Arabidopsis AGCVIII kinase family. At the same time, we reveal that D6PKs and PID have differential phosphosite preferences. Our study suggests that PIN activation by protein kinases is a crucial component of auxin transport control that must be taken into account to understand auxin distribution within the plant.

  8. Auxin efflux by PIN-FORMED proteins is activated by two different protein kinases, D6 PROTEIN KINASE and PINOID

    KAUST Repository

    Zourelidou, Melina

    2014-06-19

    The development and morphology of vascular plants is critically determined by synthesis and proper distribution of the phytohormone auxin. The directed cell-to-cell distribution of auxin is achieved through a system of auxin influx and efflux transporters. PIN-FORMED (PIN) proteins are proposed auxin efflux transporters, and auxin fluxes can seemingly be predicted based on the-in many cells-asymmetric plasma membrane distribution of PINs. Here, we show in a heterologous Xenopus oocyte system as well as in Arabidopsis thaliana inflorescence stems that PIN-mediated auxin transport is directly activated by D6 PROTEIN KINASE (D6PK) and PINOID (PID)/WAG kinases of the Arabidopsis AGCVIII kinase family. At the same time, we reveal that D6PKs and PID have differential phosphosite preferences. Our study suggests that PIN activation by protein kinases is a crucial component of auxin transport control that must be taken into account to understand auxin distribution within the plant.

  9. Novel auxin transport inhibitors phenocopy the auxin influx carrier mutation aux1.

    Science.gov (United States)

    Parry, G; Delbarre, A; Marchant, A; Swarup, R; Napier, R; Perrot-Rechenmann, C; Bennett, M J

    2001-02-01

    The hormone auxin is transported in plants through the combined actions of diffusion and specific auxin influx and efflux carriers. In contrast to auxin efflux, for which there are well documented inhibitors, understanding the developmental roles of carrier-mediated auxin influx has been hampered by the absence of specific competitive inhibitors. However, several molecules that inhibit auxin influx in cultured cells have been described recently. The physiological effects of two of these novel influx carrier inhibitors, 1-naphthoxyacetic acid (1-NOA) and 3-chloro-4-hydroxyphenylacetic acid (CHPAA), have been investigated in intact seedlings and tissue segments using classical and new auxin transport bioassays. Both molecules do disrupt root gravitropism, which is a developmental process requiring rapid auxin redistribution. Furthermore, the auxin-insensitive and agravitropic root-growth characteristics of aux1 plants were phenocopied by 1-NOA and CHPAA. Similarly, the agravitropic phenotype of inhibitor-treated seedlings was rescued by the auxin 1-naphthaleneacetic acid, but not by 2,4-dichlorophenoxyacetic acid, again resembling the relative abilities of these two auxins to rescue the phenotype of aux1. Further investigations have shown that none of these compounds block polar auxin transport, and that CHPAA exhibits some auxin-like activity at high concentrations. Whilst results indicate that 1-NOA and CHPAA represent useful tools for physiological studies addressing the role of auxin influx in planta, 1-NOA is likely to prove the more useful of the two compounds.

  10. Auxin transport is sufficient to generate a maximumand gradient guiding root growth

    NARCIS (Netherlands)

    Grieneisen, V.; Xu, J.; Marée, A.F.M.; Hogeweg, P.; Scheres, B.J.G.

    2007-01-01

    The plant growth regulator auxin controls cell identity, cell division and cell expansion. Auxin efflux facilitators (PINs) are associated with auxin maxima in distal regions of both shoots and roots. Here we model diffusion and PIN-facilitated auxin transport in and across cells within a structured

  11. Clathrin-Mediated Auxin Efflux and Maxima Regulate Hypocotyl Hook Formation and Light-Stimulated Hook Opening in Arabidopsis.

    Science.gov (United States)

    Yu, Qinqin; Zhang, Ying; Wang, Juan; Yan, Xu; Wang, Chao; Xu, Jian; Pan, Jianwei

    2016-01-04

    The establishment of auxin maxima by PIN-FORMED 3 (PIN3)- and AUXIN RESISTANT 1/LIKE AUX1 (LAX) 3 (AUX1/LAX3)-mediated auxin transport is essential for hook formation in Arabidopsis hypocotyls. Until now, however, the underlying regulatory mechanism has remained poorly understood. Here, we show that loss of function of clathrin light chain CLC2 and CLC3 genes enhanced auxin maxima and thereby hook curvature, alleviated the inhibitory effect of auxin overproduction on auxin maxima and hook curvature, and delayed blue light-stimulated auxin maxima reduction and hook opening. Moreover, pharmacological experiments revealed that auxin maxima formation and hook curvature in clc2 clc3 were sensitive to auxin efflux inhibitors 1-naphthylphthalamic acid and 2,3,5-triiodobenzoic acid but not to the auxin influx inhibitor 1-naphthoxyacetic acid. Live-cell imaging analysis further uncovered that loss of CLC2 and CLC3 function impaired PIN3 endocytosis and promoted its lateralization in the cortical cells but did not affect AUX1 localization. Taken together, these results suggest that clathrin regulates auxin maxima and thereby hook formation through modulating PIN3 localization and auxin efflux, providing a novel mechanism that integrates developmental signals and environmental cues to regulate plant skotomorphogenesis and photomorphogenesis.

  12. Plant cells use auxin efflux to explore geometry.

    Science.gov (United States)

    Zaban, Beatrix; Liu, Wenwen; Jiang, Xingyu; Nick, Peter

    2014-07-28

    Cell movement is the central mechanism for animal morphogenesis. Plant cell development rather relies on flexible alignment of cell axis adjusting cellular differentiation to directional cues. As central input, vectorial fields of mechanical stress and gradients of the phytohormone auxin have been discussed. In tissue contexts, mechanical and chemical signals will always act in concert; experimentally it is difficult to dissect their individual roles. We have designed a novel approach, based on cells, where directionality has been eliminated by removal of the cell wall. We impose a new axis using a microfluidic set-up to generate auxin gradients. Rectangular microvessels are integrated orthogonally with the gradient. Cells in these microvessels align their new axis with microvessel geometry before touching the wall. Auxin efflux is necessary for this touch-independent geometry exploration and we suggest a model, where auxin gradients can be used to align cell axis in tissues with minimized mechanical tensions.

  13. Auxin transport sites are visualized in planta using fluorescent auxin analogs

    OpenAIRE

    2014-01-01

    Fluorescent auxin analogs are designed to function as active auxins for the auxin transport system but to be inactive for auxin signaling. These fluorescent auxin analogs can mimic auxin via the transport system and be used to visualize inter- and intracellular auxin distribution in roots. These analogs allow imaging of auxin transport sites with high spatiotemporal resolution. Our fluorescent auxin system provides insight into auxin transport dynamics and subcellular auxin distribution.

  14. 生长素调控极性运输载体PIN2质膜丰度与降解%Auxin regulation of its efflux transporter PIN2 plasma membrane abundance and degradation

    Institute of Scientific and Technical Information of China (English)

    付伟; 严旭; 王超; 潘建伟

    2013-01-01

    The effects of long-term (8 h) treatments with exogenous auxins on auxin efflux transporter PIN2-GFP plasma membrane (PM) abundance,and low temperature,KCN and auxin receptors TIR1/AFBs mutation on auxin regulation of PIN2-GFP PM abundance,and ConA,a vacuolar H +-ATPase inhibitor,on vacuolar accumulation of PIN2-GFP in wild-type and auxin receptor quadruple mutants tir1afb1,2,3 cells in Arabidopsis were systematically studied.The results revealed that exogenous auxins down-regulated PIN2-GFP PM abundance through TIR1/AFB-mediated signaling and promoted PIN2-GFP internalization,intracellular membrane trafficking,and vacuolar degradation,indicating that auxin feedback regulates PIN2-GFP PM levels through TIR1/AFB-mediated signaling and inhibits excessive transport of intracellular auxin out of the cell.%主要观察了外源生长素长时间(8 h)处理对拟南芥生长素极性输出载体PIN2-GFP质膜丰度的影响,低温、KCN处理和生长素受体突变对生长素调控质膜PIN2-GFP丰度的影响,以及液泡H+-ATPase抑制剂ConA对野生型和生长素受体四突变体tir1aafb1,2,3液泡中PIN2-GFP积累的影响.结果表明:外源生长素通过其受体TIR1/AFB介导的信号途径下调质膜PIN2-GFP的丰度,促进PIN2-GFP的内吞、胞内运输和液泡降解.暗示生长素通过TIR1/AFB介导的信号途径反馈调控质膜PIN2-GFP的水平,从而阻止了胞内生长素的过多输出.

  15. Species differences in ligand specificity of auxin-controlled elongation and auxin transport: comparing Zea and Vigna

    Science.gov (United States)

    Zhao, Hu; Hertel, Rainer; Ishikawa, Hideo; Evans, Michael L.

    2002-01-01

    The plant hormone auxin affects cell elongation in both roots and shoots. In roots, the predominant action of auxin is to inhibit cell elongation while in shoots auxin, at normal physiological levels, stimulates elongation. The question of whether the primary receptor for auxin is the same in roots and shoots has not been resolved. In addition to its action on cell elongation in roots and shoots, auxin is transported in a polar fashion in both organs. Although auxin transport is well characterized in both roots and shoots, there is relatively little information on the connection, if any, between auxin transport and its action on elongation. In particular, it is not clear whether the protein mediating polar auxin movement is separate from the protein mediating auxin action on cell elongation or whether these two processes might be mediated by one and the same receptor. We examined the identity of the auxin growth receptor in roots and shoots by comparing the response of roots and shoots of the grass Zea mays L. and the legume Vigna mungo L. to indole-3-acetic acid, 2-naphthoxyacetic acid, 4,6-dichloroindoleacetic acid, and 4,7-dichloroindoleacetic acid. We also studied whether or not a single protein might mediate both auxin transport and auxin action by comparing the polar transport of indole-3-acetic acid and 2-naphthoxyacetic acid through segments from Vigna hypocotyls and maize coleoptiles. For all of the assays performed (root elongation, shoot elongation, and polar transport) the action and transport of the auxin derivatives was much greater in the dicots than in the grass species. The preservation of ligand specificity between roots and shoots and the parallels in ligand specificity between auxin transport and auxin action on growth are consistent with the hypothesis that the auxin receptor is the same in roots and shoots and that this protein may mediate auxin efflux as well as auxin action in both organ types.

  16. Maintenance of asymmetric cellular localization of an auxin transport protein through interaction with the actin cytoskeleton

    Science.gov (United States)

    Muday, G. K.

    2000-01-01

    In shoots, polar auxin transport is basipetal (that is, from the shoot apex toward the base) and is driven by the basal localization of the auxin efflux carrier complex. The focus of this article is to summarize the experiments that have examined how the asymmetric distribution of this protein complex is controlled and the significance of this polar distribution. Experimental evidence suggests that asymmetries in the auxin efflux carrier may be established through localized secretion of Golgi vesicles, whereas an attachment of a subunit of the efflux carrier to the actin cytoskeleton may maintain this localization. In addition, the idea that this localization of the efflux carrier may control both the polarity of auxin movement and more globally regulate developmental polarity is explored. Finally, evidence indicating that the gravity vector controls auxin transport polarity is summarized and possible mechanisms for the environmentally induced changes in auxin transport polarity are discussed.

  17. PIN protein phosphorylation by plant AGC3 kinases and its role in polar auxin transport

    NARCIS (Netherlands)

    Huang, Fang

    2010-01-01

    Polar cell-to-cell transport of plant hormone auxin mediated by plasma membrane (PM)-localized PIN-FORMED (PIN) auxin efflux carriers generates auxin gradients that provide positional information for various plant developmental processes. The apical-basal polar localization of the PIN proteins that

  18. Isolation and functional analysis of a Brassica juncea gene encoding a component of auxin efflux carrier

    Institute of Scientific and Technical Information of China (English)

    WEI; MIN; NI; XIAO; YA; CHEN; ZHI; HONG; XU; HONG; WEI; XUE

    2002-01-01

    Polar auxin transport plays a divergent role in plant growth and developmental processes including rootand embryo development, vascular pattern formation and cell elongation. Recently isolated Arabidopsispin gene family was believed to encode a component of auxin efflux carrier (Galweiler et al, 1998). Basedon the Arabidopsis pin1 sequence we have isolated a Brassica juncea cDNA (designated Bjpinl), whichencoded a 70-kDa putative auxin efflux carrier. Deduced BjPIN1 shared 65% identities at protein level withAtPIN1 and was highly homologous to other putative PIN proteins of Arabidopsis (with highest homologyto AtPIN3). Hydrophobic analysis showed similar structures between BjPIN1 and AtPIN proteins. Presenceof 6 exons (varying in size between 65 bp and 1229 bp) and 5 introns (sizes between 89 bp and 463 bp)in the genomic fragment was revealed by comparing the genomic and cDNA sequences. Northern blotanalysis indicated that Bjpinl was expressed in most of the tissues tested, with a relatively higher levelof transcript in flowers and a lower level in root tissues. Promoter-reporter gene fusion studies furtherrevealed the expression of Bjpinl in the mature pollen grains, young seeds, root tip, leaf vascular tissue andtrace bundle, stem epidermis, cortex and vascular cells. BjPIN1 was localized on the plasma membraneas demonstrated through fusion expression of green fluorescent protein (GFP). Auxin efflux carrier activitywas elevated in transgenic Arabidopsis expressing BjPIN1.

  19. The role of auxin transporters in monocots development

    Directory of Open Access Journals (Sweden)

    Sara eBalzan

    2014-08-01

    Full Text Available Auxin is a key regulator of plant growth and development, orchestrating cell division, elongation and differentiation, embryonic development, root and stem tropisms, apical dominance and transition to flowering. Auxin levels are higher in undifferentiated cell populations and decrease following organ initiation and tissue differentiation. This differential auxin distribution is achieved by polar auxin transport (PAT mediated by auxin transport proteins. There are 4 major families of auxin transporters in plants: PINs, ABCBs, AUX/LAXs and PILS. These families include proteins located at the plasma membrane (PM or at the Endoplasmic Reticulum (ER, which participate in auxin influx, efflux or both, from the apoplast into the cell or from the cytosol into the ER compartment. Auxin transporters have been largely studied in the dicotyledon model species Arabidopsis, but there is increasing evidence of their role in auxin regulated development in monocotyledon species. In monocots, families of auxin transporters are enlarged and often include duplicated genes and proteins with high sequence similarity. Some of these proteins underwent sub- and neo-functionalization with substantial modification to their structure and expression in organs such as adventitious roots, panicles, tassels and ears. Most of the present information on monocot auxin transporters function derives from studies conducted in rice, maize, sorghum and Brachypodium using pharmacological applications (PAT inhibitors or down-/up-regulation (over-expression and RNAi of candidate genes. Gene expression studies and comparison of predicted protein structures have also increased our knowledge of the role of PAT in monocots. However, knockout mutants and functional characterization of single genes are still scarce and the future availability of such resources will prove crucial to elucidate the role of auxin transporters in monocot development.

  20. Role of actin in auxin transport and transduction of gravity

    Science.gov (United States)

    Hu, S.; Basu, S.; Brady, S.; Muday, G.

    Transport of the plant hormone auxin is polar and the direction of the hormone movement appears to be controlled by asymmetric distribution of auxin transport protein complexes. Changes in the direction of auxin transport are believed to drive asymmetric growth in response to changes in the gravity vector. To test the possibility that asymmetric distribution of the auxin transport protein complex is mediated by attachment to the actin cytoskeleton, a variety of experimental approaches have been used. The most direct demonstration of the role of the actin cytoskeleton in localization of the protein complex is the ability of one protein in this complex to bind to affinity columns containing actin filaments. Additionally, treatments of plant tissues with drugs that fragment the actin c toskeleton reducey polar transport. In order to explore this actin interaction and the affect of gravity on auxin transport and developmental polarity, embryos of the brown alga, Fucus have been examined. Fucus zygotes are initially symmetrical, but develop asymmetry in response to environmental gradients, with light gradients being the best- characterized signal. Gravity will polarize these embryos and gravity-induced polarity is randomized by clinorotation. Auxin transport also appears necessary for environmental controls of polarity, since auxin efflux inhibitors perturb both photo- and gravity-polarization at a very discrete temporal window within six hours after fertilization. The actin cytoskeleton has previously been shown to reorganize after fertilization of Fucus embryos leading to formation of an actin patch at the site of polar outgrowth. These actin patches still form in Fucus embryos treated with auxin efflux inhibitors, yet the position of these patches is randomized. Together, these results suggest that there are connections between the actin cytoskeleton, auxin transport, and gravity oriented growth and development. (Supported by NASA Grant: NAG2-1203)

  1. The Relationship between auxin transport and maize branching.

    Science.gov (United States)

    Gallavotti, Andrea; Yang, Yan; Schmidt, Robert J; Jackson, David

    2008-08-01

    Maize (Zea mays) plants make different types of vegetative or reproductive branches during development. Branches develop from axillary meristems produced on the flanks of the vegetative or inflorescence shoot apical meristem. Among these branches are the spikelets, short grass-specific structures, produced by determinate axillary spikelet-pair and spikelet meristems. We investigated the mechanism of branching in maize by making transgenic plants expressing a native expressed endogenous auxin efflux transporter (ZmPIN1a) fused to yellow fluorescent protein and a synthetic auxin-responsive promoter (DR5rev) driving red fluorescent protein. By imaging these plants, we found that all maize branching events during vegetative and reproductive development appear to be regulated by the creation of auxin response maxima through the activity of polar auxin transporters. We also found that the auxin transporter ZmPIN1a is functional, as it can rescue the polar auxin transport defects of the Arabidopsis (Arabidopsis thaliana) pin1-3 mutant. Based on this and on the groundbreaking analysis in Arabidopsis and other species, we conclude that branching mechanisms are conserved and can, in addition, explain the formation of axillary meristems (spikelet-pair and spikelet meristems) that are unique to grasses. We also found that BARREN STALK1 is required for the creation of auxin response maxima at the flanks of the inflorescence meristem, suggesting a role in the initiation of polar auxin transport for axillary meristem formation. Based on our results, we propose a general model for branching during maize inflorescence development.

  2. Cell plate-restricted association of Arabidopsis dynamin related proteins and PIN auxin efflux carriers is required for PIN endocytic trafficking during cytokinesis.

    NARCIS (Netherlands)

    Mravec, J.; Petrasek, J.; Li, N.; Boeren, J.A.; Karlova, R.B.; Kitakura, S.; Naramoto, S.; Nodzynski, T.; Dhonukshe, P.B.; Vries, de S.C.; Zazimalova, E.; Friml, J.

    2011-01-01

    The polarized transport of the phytohormone auxin [1], which is crucial for the regulation of different stages of plant development [ [2] and [3] ], depends on the asymmetric plasma membrane distribution of the PIN-FORMED (PIN) auxin efflux carriers [4 A. Vieten, M. Sauer, P.B. Brewer and J. Friml,

  3. Cell plate-restricted association of Arabidopsis dynamin related proteins and PIN auxin efflux carriers is required for PIN endocytic trafficking during cytokinesis.

    NARCIS (Netherlands)

    Mravec, J.; Petrasek, J.; Li, N.; Boeren, J.A.; Karlova, R.B.; Kitakura, S.; Naramoto, S.; Nodzynski, T.; Dhonukshe, P.B.; Vries, de S.C.; Zazimalova, E.; Friml, J.

    2011-01-01

    The polarized transport of the phytohormone auxin [1], which is crucial for the regulation of different stages of plant development [ [2] and [3] ], depends on the asymmetric plasma membrane distribution of the PIN-FORMED (PIN) auxin efflux carriers [4 A. Vieten, M. Sauer, P.B. Brewer and J. Friml,

  4. Auxin efflux facilitator and auxin dynamism responsible for the gravity-regulated development of peg in cucumber seedlings

    Science.gov (United States)

    Takahashi, Hideyuki; Watanabe, Chiaki; Fujii, Nobuharu; Miyazawa, Yutaka

    Cucumber seedlings develop a protuberance, peg, by which seed coats are pulled out just af-ter germination. The peg is usually formed on the lower side of the transition zone between hypocotyl and root of the seedlings grown in a horizontal position. Our previous spaceflight experiment showed that unilateral positioning of a peg in cucumber seedlings occurred due to its suppression on the upper side of the transition zone because seedlings grown in microgravity developed a peg on each side of the transition zone. We also showed that auxin was a major factor responsible for peg development. There was a redistribution of auxin in the gravistimu-lated transition zone, decreasing IAA level on the upper side, and IAA application induced a peg on both lower and upper sides of the transition zone. In addition, peg was released from its suppression in the seedlings treated with inhibitors of auxin efflux. Namely, two pegs devel-oped in the TIBA-treated seedlings even when they were grown in a horizontal position. These results imply that a reduction of auxin level due to its efflux is required for the suppression of peg development on the upper side of the transition zone in a horizontal position. To under-stand molecular mechanism underlying the negative control of morphogenesis by graviresponse in cucumber seedlings, we isolated cDNAs of auxin efflux facilitators, CsPINs, from cucumber and examined the expressions of their proteins, in relation to the redistribution of endogenous auxin and peg development. We isolated six cDNAs of PIN homologues CsPIN1 to CsPIN6 from cucumber. By immunohistochemical study using some of their anti-bodies, we revealed that CsPIN1 was localized in endodermis, vascular tissue and pith around the transition zone of cucumber seedlings. In cucumber seedlings grown in a vertical position with radicles pointing down, CsPIN1 in endodermal cells was mainly localized on the plasma membrane neighboring vascular bundle but not on the plasma membrane

  5. Polar auxin transport: models and mechanisms

    NARCIS (Netherlands)

    Berkel, van K.; Boer, de R.J.; Scheres, B.; Tusscher, ten K.

    2013-01-01

    Spatial patterns of the hormone auxin are important drivers of plant development. The observed feedback between the active, directed transport that generates auxin patterns and the auxin distribution that influences transport orientation has rendered this a popular subject for modelling studies. Her

  6. Auxin transport sites are visualized in planta using fluorescent auxin analogs.

    Science.gov (United States)

    Hayashi, Ken-ichiro; Nakamura, Shouichi; Fukunaga, Shiho; Nishimura, Takeshi; Jenness, Mark K; Murphy, Angus S; Motose, Hiroyasu; Nozaki, Hiroshi; Furutani, Masahiko; Aoyama, Takashi

    2014-08-05

    The plant hormone auxin is a key morphogenetic signal that controls many aspects of plant growth and development. Cellular auxin levels are coordinately regulated by multiple processes, including auxin biosynthesis and the polar transport and metabolic pathways. The auxin concentration gradient determines plant organ positioning and growth responses to environmental cues. Auxin transport systems play crucial roles in the spatiotemporal regulation of the auxin gradient. This auxin gradient has been analyzed using SCF-type E3 ubiquitin-ligase complex-based auxin biosensors in synthetic auxin-responsive reporter lines. However, the contributions of auxin biosynthesis and metabolism to the auxin gradient have been largely elusive. Additionally, the available information on subcellular auxin localization is still limited. Here we designed fluorescently labeled auxin analogs that remain active for auxin transport but are inactive for auxin signaling and metabolism. Fluorescent auxin analogs enable the selective visualization of the distribution of auxin by the auxin transport system. Together with auxin biosynthesis inhibitors and an auxin biosensor, these analogs indicated a substantial contribution of local auxin biosynthesis to the formation of auxin maxima at the root apex. Moreover, fluorescent auxin analogs mainly localized to the endoplasmic reticulum in cultured cells and roots, implying the presence of a subcellular auxin gradient in the cells. Our work not only provides a useful tool for the plant chemical biology field but also demonstrates a new strategy for imaging the distribution of small-molecule hormones.

  7. The putative auxin efflux carrier OsPIN3t is involved in the drought stress response and drought tolerance.

    Science.gov (United States)

    Zhang, Qian; Li, Jingjing; Zhang, Wenjiao; Yan, Shuning; Wang, Rui; Zhao, Junfeng; Li, Yujing; Qi, Zhiguang; Sun, Zongxiu; Zhu, Zhengge

    2012-12-01

    The phytohormone auxin plays a critical role in plant growth and development, and its spatial distribution largely depends on the polar localization of the PIN-FORMED (PIN) auxin efflux carrier family members. In this study, we identify a putative auxin efflux carrier gene in rice, OsPIN3t, which acts in auxin polar transport but is also involved in the drought stress response in rice. We show that OsPIN3t-GFP fusion proteins are localized in plasma membranes, and this subcellular localization changes under 1-N-naphthylphthalamic acid (NPA) treatment. The tissue-specific expression patterns of OsPIN3t were also investigated using a β-glucuronidase (GUS) reporter, which showed that OsPIN3t was mainly expressed in vascular tissue. The GUS activity in OsPIN3tpro::GUS plants increased by NAA treatment and decreased by NPA treatment. Moreover, knockdown of OsPIN3t caused crown root abnormalities in the seedling stage that could be phenocopied by treatment of wild-type plants with NPA, which indicated that OsPIN3t is involved in the control of polar auxin transport. Overexpression of OsPIN3t led to improved drought tolerance, and GUS activity significantly increased when OsPIN3tpro::GUS plants were subjected to 20% polyethylene glycol stress. Taken together, these results suggest that OsPIN3t is involved in auxin transport and the drought stress response, which suggests that a polar auxin transport pathway is involved in the regulation of the response to water stress in plants. © 2012 The Authors. The Plant Journal © 2012 Blackwell Publishing Ltd.

  8. The actin cytoskeleton may control the polar distribution of an auxin transport protein

    Science.gov (United States)

    Muday, G. K.; Hu, S.; Brady, S. R.; Davies, E. (Principal Investigator)

    2000-01-01

    The gravitropic bending of plants has long been linked to the changes in the transport of the plant hormone auxin. To understand the mechanism by which gravity alters auxin movement, it is critical to know how polar auxin transport is initially established. In shoots, polar auxin transport is basipetal (i.e., from the shoot apex toward the base). It is driven by the basal localization of the auxin efflux carrier complex. One mechanism for localizing this efflux carrier complex to the basal membrane may be through attachment to the actin cytoskeleton. The efflux carrier protein complex is believed to consist of several polypeptides, including a regulatory subunit that binds auxin transport inhibitors, such as naphthylphthalamic acid (NPA). Several lines of experimentation have been used to determine if the NPA binding protein interacts with actin filaments. The NPA binding protein has been shown to partition with the actin cytoskeleton during detergent extraction. Agents that specifically alter the polymerization state of the actin cytoskeleton change the amount of NPA binding protein and actin recovered in these cytoskeletal pellets. Actin-affinity columns were prepared with polymers of actin purified from zucchini hypocotyl tissue. NPA binding activity was eluted in a single peak from the actin filament column. Cytochalasin D, which fragments the actin cytoskeleton, was shown to reduce polar auxin transport in zucchini hypocotyls. The interaction of the NPA binding protein with the actin cytoskeleton may localize it in one plane of the plasma membrane, and thereby control the polarity of auxin transport.

  9. Sequential induction of auxin efflux and influx carriers regulates lateral root emergence.

    Science.gov (United States)

    Péret, Benjamin; Middleton, Alistair M; French, Andrew P; Larrieu, Antoine; Bishopp, Anthony; Njo, Maria; Wells, Darren M; Porco, Silvana; Mellor, Nathan; Band, Leah R; Casimiro, Ilda; Kleine-Vehn, Jürgen; Vanneste, Steffen; Sairanen, Ilkka; Mallet, Romain; Sandberg, Göran; Ljung, Karin; Beeckman, Tom; Benkova, Eva; Friml, Jiří; Kramer, Eric; King, John R; De Smet, Ive; Pridmore, Tony; Owen, Markus; Bennett, Malcolm J

    2013-10-22

    In Arabidopsis, lateral roots originate from pericycle cells deep within the primary root. New lateral root primordia (LRP) have to emerge through several overlaying tissues. Here, we report that auxin produced in new LRP is transported towards the outer tissues where it triggers cell separation by inducing both the auxin influx carrier LAX3 and cell-wall enzymes. LAX3 is expressed in just two cell files overlaying new LRP. To understand how this striking pattern of LAX3 expression is regulated, we developed a mathematical model that captures the network regulating its expression and auxin transport within realistic three-dimensional cell and tissue geometries. Our model revealed that, for the LAX3 spatial expression to be robust to natural variations in root tissue geometry, an efflux carrier is required--later identified to be PIN3. To prevent LAX3 from being transiently expressed in multiple cell files, PIN3 and LAX3 must be induced consecutively, which we later demonstrated to be the case. Our study exemplifies how mathematical models can be used to direct experiments to elucidate complex developmental processes.

  10. Characterization of transmembrane auxin transport in Arabidopsis suspension-cultured cells.

    Science.gov (United States)

    Seifertová, Daniela; Skůpa, Petr; Rychtář, Jan; Laňková, Martina; Pařezová, Markéta; Dobrev, Petre I; Hoyerová, Klára; Petrášek, Jan; Zažímalová, Eva

    2014-03-15

    Polar auxin transport is a crucial process for control and coordination of plant development. Studies of auxin transport through plant tissues and organs showed that auxin is transported by a combination of phloem flow and the active, carrier-mediated cell-to-cell transport. Since plant organs and even tissues are too complex for determination of the kinetics of carrier-mediated auxin uptake and efflux on the cellular level, simplified models of cell suspension cultures are often used, and several tobacco cell lines have been established for auxin transport assays. However, there are very few data available on the specificity and kinetics of auxin transport across the plasma membrane for Arabidopsis thaliana suspension-cultured cells. In this report, the characteristics of carrier-mediated uptake (influx) and efflux for the native auxin indole-3-acetic acid and synthetic auxins, naphthalene-1-acetic and 2,4-dichlorophenoxyacetic acids (NAA and 2,4-D, respectively) in A. thaliana ecotype Landsberg erecta suspension-cultured cells (LE line) are provided. By auxin competition assays and inhibitor treatments, we show that, similarly to tobacco cells, uptake carriers have high affinity towards 2,4-D and that NAA is a good tool for studies of auxin efflux in LE cells. In contrast to tobacco cells, metabolic profiling showed that only a small proportion of NAA is metabolized in LE cells. These results show that the LE cell line is a useful experimental system for measurements of kinetics of auxin carriers on the cellular level that is complementary to tobacco cells.

  11. Connective Auxin Transport in the Shoot Facilitates Communication between Shoot Apices.

    Science.gov (United States)

    Bennett, Tom; Hines, Geneviève; van Rongen, Martin; Waldie, Tanya; Sawchuk, Megan G; Scarpella, Enrico; Ljung, Karin; Leyser, Ottoline

    2016-04-01

    The bulk polar movement of the plant signaling molecule auxin through the stem is a long-recognized but poorly understood phenomenon. Here we show that the highly polar, high conductance polar auxin transport stream (PATS) is only part of a multimodal auxin transport network in the stem. The dynamics of auxin movement through stems are inconsistent with a single polar transport regime and instead suggest widespread low conductance, less polar auxin transport in the stem, which we term connective auxin transport (CAT). The bidirectional movement of auxin between the PATS and the surrounding tissues, mediated by CAT, can explain the complex auxin transport kinetics we observe. We show that the auxin efflux carriers PIN3, PIN4, and PIN7 are major contributors to this auxin transport connectivity and that their activity is important for communication between shoot apices in the regulation of shoot branching. We propose that the PATS provides a long-range, consolidated stream of information throughout the plant, while CAT acts locally, allowing tissues to modulate and be modulated by information in the PATS.

  12. Manganese Toxicity Inhibited Root Growth by Disrupting Auxin Biosynthesis and Transport in Arabidopsis

    Science.gov (United States)

    Zhao, Jingjing; Wang, Wenying; Zhou, Huakun; Wang, Ruling; Zhang, Ping; Wang, Huichun; Pan, Xiangliang; Xu, Jin

    2017-01-01

    Mn toxicity inhibits both primary root (PR) growth and lateral root development. However, the mechanism underlying Mn-mediated root growth inhibition remains to be further elucidated. Here, we investigated the role of auxin in Mn-mediated inhibition of PR growth in Arabidopsis using physiological and genetic approaches. Mn toxicity inhibits PR elongation by reducing meristematic cell division potential. Mn toxicity also reduced auxin levels in root tips by reducing IAA biosynthesis and down-regulating the expression of auxin efflux carriers PIN4 and PIN7. Loss of function pin4 and pin7 mutants showed less inhibition of root growth than col-0 seedlings. These results indicated that this inhibitory effect of Mn toxicity on PR growth was mediated by affecting auxin biosynthesis and the expression of auxin efflux transporters PIN4 and PIN7. PMID:28316607

  13. Regulation of polar auxin transport in grapevine fruitlets (Vitis vinifera L.) and the proposed role of auxin homeostasis during fruit abscission.

    Science.gov (United States)

    Kühn, Nathalie; Serrano, Alejandra; Abello, Carlos; Arce, Aníbal; Espinoza, Carmen; Gouthu, Satyanarayana; Deluc, Laurent; Arce-Johnson, Patricio

    2016-10-28

    Indole-3-acetic acid (IAA), the most abundant auxin, is a growth promoter hormone involved in several developmental processes. Auxin homeostasis is very important to its function and this is achieved through the regulation of IAA biosynthesis, conjugation, degradation and transport. In grapevine, IAA plays an essential role during initial stages of berry development, since it delays fruitlet abscission by reducing the ethylene sensitivity in the abscission zone. For this reason, Continuous polar IAA transport to the pedicel is required. This kind of transport is controlled by IAA, which regulates its own movement by modifying the expression and localization of PIN-FORMED (PIN) auxin efflux facilitators that localize asymmetrically within the cell. On the other hand, the hormone gibberellin (GA) also activates the polar auxin transport by increasing PIN stability. In Vitis vinifera, fruitlet abscission occurs during the first two to three weeks after flowering. During this time, IAA and GA are present, however the role of these hormones in the control of polar auxin transport is unknown. In this work, the use of radiolabeled IAA showed that auxin is basipetally transported during grapevine fruitlet abscission. This observation was further supported by immunolocalization of putative VvPIN proteins that display a basipetal distribution in pericarp cells. Polar auxin transport and transcripts of four putative VvPIN genes decreased in conjunction with increased abscission, and the inhibition of polar auxin transport resulted in fruit drop. GA3 and IAA treatments reduced polar auxin transport, but only GA3 treatment decreased VvPIN transcript abundance. When GA biosynthesis was blocked, IAA was capable to increase polar auxin transport, suggesting that its effect depends on GA content. Finally, we observed significant changes in the content of several IAA-related compounds during the abscission period. These results provide evidence that auxin homeostasis plays a central

  14. The maize PIN gene family of auxin transporters

    Directory of Open Access Journals (Sweden)

    Cristian eForestan

    2012-02-01

    Full Text Available Auxin is a key regulator of plant development and its differential distribution in plant tissues, established by a polar cell-to-cell transport, can trigger a wide range of developmental processes. A few members of the two families of auxin efflux transport proteins, PIN-formed (PIN and P-glycoprotein (ABCB/PGP, have so far been characterized in maize. Nine new Zea mays auxin efflux carriers PIN family members and two maize PIN-like genes have now been identified. Four members of PIN1 (named ZmPIN1a–d cluster, one gene homologous to AtPIN2 (ZmPIN2, three orthologs of PIN5 (ZmPIN5a–c, one gene paired with AtPIN8 (ZmPIN8, and three monocot-specific PINs (ZmPIN9, ZmPIN10a and b were cloned and the phylogenetic relationships between early land plants, monocots and eudicots PIN proteins investigated, including the new maize PIN proteins. Tissue-specific expression patterns of the twelve maize PIN genes, two PIN-like genes and ZmABCB1, an ABCB auxin efflux carrier, were analyzed using semi-quantitative RT–PCR. ZmPIN gene transcripts have overlapping expression domains in the root apex, during male and female inflorescence differentiation and kernel development. However, some PIN family members have specific tissue localization: ZmPIN1d transcript marks the L1 layer of the SAM and IM during the flowering transition and the monocot-specific ZmPIN9 is expressed in the root endodermis and pericycle. The phylogenetic and gene structure analyses together with the expression pattern of the ZmPIN gene family indicate that subfunctionalization of some maize PINs can be associated to the differentiation and development of monocot-specific organs and tissues and might have occurred after the divergence between dicots and monocots.

  15. Roles of auxin transport and action in the gravity-regulated morphogenesis of cucumber seedlings

    Science.gov (United States)

    Saito, Y.; Shimizu, M.; Hotta, T.; Dai-Hee, K.; Yanai, K.; Kamada, M.; Fujii, N.; Miyazawa, Y.; Takahashi, H.

    Cucumber Cucumis sativus L seedlings develop a specialized protuberance peg on the lower side of the transition zone between the hypocotyl and the root when seeds germinate in a horizontal position The peg plays an important role in pulling seedling out from the seed coat We have reported that cucumber seedlings potentially develop a peg on each side of the transition zone but peg development on the upper side is suppressed in response to gravity Auxin is the primary factor responsible for the induction or the suppression of peg formation Here we investigated the roles of auxin transport and action in the gravity-regulated formation suppression of the peg in cucumber seedlings When cucumber seedlings were treated with inhibitors of auxin efflux carrier a peg was formed not only on the lower side but also on the upper side of the gravistimulated transition zone suggesting that activation of auxin efflux carriers is required for the suppression of peg formation To identify auxin efflux carriers involved in the suppression of peg formation by graviresponse we isolated six cucumber cDNAs of PIN auxin efflux carrier genes and investigated their mRNA accumulation and protein expression Our results show that CsPIN1 and CsPIN6 could play a role in the redistribution of auxin in the transition zone To understand auxin action on peg formation suppression we next examined the transcriptional regulators for the expressions of auxin-responsive genes The results suggest that a higher level of auxin in the lower side of the

  16. Comparative effects of auxin and abscisic acid on growth, hydrogen ion efflux and gravitropism in primary roots of maize

    Science.gov (United States)

    Evans, M. L.; Mulkey, T. J.

    1984-01-01

    In order to test the idea that auxin action on root growth may be mediated by H(+) movement, the correlation of auxin action on growth and H(+) movement in roots was examined along with changes in H(+) efflux patterns associated with the asymmetric growth which occurs during gravitropism. The effects of indoleacetic acid (IAA) and abscisic acid (AbA) on growth, H(+) secretion, and gravitropism in roots were compared. Results show a close correlation existent between H(+) efflux and growth in maize roots. In intact roots there is strong H(+) efflux from the elongation zone. Growth-promoting concentrations of IAA stimulate H(+) efflux. During gravitropism the H(+) efflux from the elongation zone becomes asymmetric; the evidence indicates that auxin redistribution contributes to the development of acid efflux asymmetry. That AbA stimulates root growth is reflected in its ability to stimulate H(+) efflux from apical root segments.

  17. Loss of GSNOR1 Function Leads to Compromised Auxin Signaling and Polar Auxin Transport.

    Science.gov (United States)

    Shi, Ya-Fei; Wang, Da-Li; Wang, Chao; Culler, Angela Hendrickson; Kreiser, Molly A; Suresh, Jayanti; Cohen, Jerry D; Pan, Jianwei; Baker, Barbara; Liu, Jian-Zhong

    2015-09-01

    Cross talk between phytohormones, nitric oxide (NO), and auxin has been implicated in the control of plant growth and development. Two recent reports indicate that NO promoted auxin signaling but inhibited auxin transport probably through S-nitrosylation. However, genetic evidence for the effect of S-nitrosylation on auxin physiology has been lacking. In this study, we used a genetic approach to understand the broader role of S-nitrosylation in auxin physiology in Arabidopsis. We compared auxin signaling and transport in Col-0 and gsnor1-3, a loss-of-function GSNOR1 mutant defective in protein de-nitrosylation. Our results showed that auxin signaling was impaired in the gsnor1-3 mutant as revealed by significantly reduced DR5-GUS/DR5-GFP accumulation and compromised degradation of AXR3NT-GUS, a useful reporter in interrogating auxin-mediated degradation of Aux/IAA by auxin receptors. In addition, polar auxin transport was compromised in gsnor1-3, which was correlated with universally reduced levels of PIN or GFP-PIN proteins in the roots of the mutant in a manner independent of transcription and 26S proteasome degradation. Our results suggest that S-nitrosylation and GSNOR1-mediated de-nitrosylation contribute to auxin physiology, and impaired auxin signaling and compromised auxin transport are responsible for the auxin-related morphological phenotypes displayed by the gsnor1-3 mutant.

  18. Auxin Biosynthesis, Accumulation, Action and Transport are Involved in Stress-Induced Microspore Embryogenesis Initiation and Progression in Brassica napus.

    Science.gov (United States)

    Rodríguez-Sanz, Héctor; Solís, María-Teresa; López, María-Fernanda; Gómez-Cadenas, Aurelio; Risueño, María C; Testillano, Pilar S

    2015-07-01

    Isolated microspores are reprogrammed in vitro by stress, becoming totipotent cells and producing embryos and plants via a process known as microspore embryogenesis. Despite the abundance of data on auxin involvement in plant development and embryogenesis, no data are available regarding the dynamics of auxin concentration, cellular localization and the expression of biosynthesis genes during microspore embryogenesis. This work involved the analysis of auxin concentration and cellular accumulation; expression of TAA1 and NIT2 encoding enzymes of two auxin biosynthetic pathways; expression of the PIN1-like efflux carrier; and the effects of inhibition of auxin transport and action by N-1-naphthylphthalamic acid (NPA) and α-(p-chlorophenoxy) isobutyric acid (PCIB) during Brassica napus microspore embryogenesis. The results indicated de novo auxin synthesis after stress-induced microspore reprogramming and embryogenesis initiation, accompanying the first cell divisions. The progressive increase of auxin concentration during progression of embryogenesis correlated with the expression patterns of TAA1 and NIT2 genes of auxin biosynthetic pathways. Auxin was evenly distributed in early embryos, whereas in heart/torpedo embryos auxin was accumulated in apical and basal embryo regions. Auxin efflux carrier PIN1-like gene expression was induced in early multicellular embryos and increased at the globular/torpedo embryo stages. Inhibition of polar auxin transport (PAT) and action, by NPA and PCIB, impaired embryo development, indicating that PAT and auxin action are required for microspore embryo progression. NPA also modified auxin embryo accumulation patterns. These findings indicate that endogenous auxin biosynthesis, action and polar transport are required in stress-induced microspore reprogramming, embryogenesis initiation and progression.

  19. Gravity-induced modification of auxin transport and distribution for peg formation in cucumber seedlings

    Science.gov (United States)

    Kamada, M.; Fujii, N.; Higashitani, A.; Takahashi, H.

    Cucumber seedlings grown in microgravity developed a peg on each side of the transition zone between hypocotyl and root, whereas seedlings grown in a horizontal position on the ground developed a peg on the concave (lower) side of the gravitropically bending transition zone. Using an auxin-inducible gene, CS-IAA1, we showed that upon gravistimulation the auxin concentration on the upper side of the horizontally placed transition zone is reduced to a level below the threshold necessary for peg formation. In this study, to elucidate the role of auxin in the lateral placement of peg formation, we measured the contents of endogenous auxin in the transition zone. The content of free IAA was lower and conjugated IAA was more abundant on the upper side of the transition zone of the gravistimulated seedlings compared with the lower side. These results support the idea that a decrease in auxin level due to a modification of auxin transport or metabolism causes the suppression of peg formation on the upper side of the transition zone in a horizontal position. Cucumber seedlings treated with auxin transport inhibitors exhibited agravitropic growth and developed a peg on each side of the transition zone. Application of auxin transport inhibitors caused an increase in CS-IAA1 mRNA (an auxin-inducible gene) at the transition zone. To analyze auxin transport system for peg formation, we isolated auxin influx carrier, CS-AUX1, and auxin efflux carrier, CS-PIN1, from cucumber plants. The accumulation of CS-AUX1 and CS-PIN1 mRNAs was observed at vascular tissue and epidermis in the transition zone. The level of CS-AUX1 mRNA was lower on the upper side of the transition zone in a horizontal position. The results suggest that the transition zone is an additional source of auxin, and that both influx and efflux of auxin in the cells of the transition zone control cytoplasmic concentration of auxin for peg formation.

  20. Master and servant: Regulation of auxin transporters by FKBPs and cyclophilins.

    Science.gov (United States)

    Geisler, Markus; Bailly, Aurélien; Ivanchenko, Maria

    2016-04-01

    Plant development and architecture are greatly influenced by the polar distribution of the essential hormone auxin. The directional influx and efflux of auxin from plant cells depends primarily on AUX1/LAX, PIN, and ABCB/PGP/MDR families of auxin transport proteins. The functional analysis of these proteins has progressed rapidly within the last decade thanks to the establishment of heterologous auxin transport systems. Heterologous co-expression allowed also for the testing of protein-protein interactions involved in the regulation of transporters and identified relationships with members of the FK506-Binding Protein (FKBP) and cyclophilin protein families, which are best known in non-plant systems as cellular receptors for the immunosuppressant drugs, FK506 and cyclosporin A, respectively. Current evidence that such interactions affect membrane trafficking, and potentially the activity of auxin transporters is reviewed. We also propose that FKBPs andcyclophilins might integrate the action of auxin transport inhibitors, such as NPA, on members of the ABCB and PIN family, respectively. Finally, we outline open questions that might be useful for further elucidation of the role of immunophilins as regulators (servants) of auxin transporters (masters).

  1. A major facilitator superfamily transporter plays a dual role in polar auxin transport and drought stress tolerance in Arabidopsis.

    Science.gov (United States)

    Remy, Estelle; Cabrito, Tânia R; Baster, Pawel; Batista, Rita A; Teixeira, Miguel C; Friml, Jiri; Sá-Correia, Isabel; Duque, Paula

    2013-03-01

    Many key aspects of plant development are regulated by the polarized transport of the phytohormone auxin. Cellular auxin efflux, the rate-limiting step in this process, has been shown to rely on the coordinated action of PIN-formed (PIN) and B-type ATP binding cassette (ABCB) carriers. Here, we report that polar auxin transport in the Arabidopsis thaliana root also requires the action of a Major Facilitator Superfamily (MFS) transporter, Zinc-Induced Facilitator-Like 1 (ZIFL1). Sequencing, promoter-reporter, and fluorescent protein fusion experiments indicate that the full-length ZIFL1.1 protein and a truncated splice isoform, ZIFL1.3, localize to the tonoplast of root cells and the plasma membrane of leaf stomatal guard cells, respectively. Using reverse genetics, we show that the ZIFL1.1 transporter regulates various root auxin-related processes, while the ZIFL1.3 isoform mediates drought tolerance by regulating stomatal closure. Auxin transport and immunolocalization assays demonstrate that ZIFL1.1 indirectly modulates cellular auxin efflux during shootward auxin transport at the root tip, likely by regulating plasma membrane PIN2 abundance. Finally, heterologous expression in yeast revealed that ZIFL1.1 and ZIFL1.3 share H(+)-coupled K(+) transport activity. Thus, by determining the subcellular and tissue distribution of two isoforms, alternative splicing dictates a dual function for the ZIFL1 transporter. We propose that this MFS carrier regulates stomatal movements and polar auxin transport by modulating potassium and proton fluxes in Arabidopsis cells.

  2. A mutation in protein phosphatase 2A regulatory subunit A affects auxin transport in Arabidopsis

    Science.gov (United States)

    Garbers, C.; DeLong, A.; Deruere, J.; Bernasconi, P.; Soll, D.; Evans, M. L. (Principal Investigator)

    1996-01-01

    The phytohormone auxin controls processes such as cell elongation, root hair development and root branching. Tropisms, growth curvatures triggered by gravity, light and touch, are also auxin-mediated responses. Auxin is synthesized in the shoot apex and transported through the stem, but the molecular mechanism of auxin transport is not well understood. Naphthylphthalamic acid (NPA) and other inhibitors of auxin transport block tropic curvature responses and inhibit root and shoot elongation. We have isolated a novel Arabidopsis thaliana mutant designated roots curl in NPA (rcn1). Mutant seedlings exhibit altered responses to NPA in root curling and hypocotyl elongation. Auxin efflux in mutant seedlings displays increased sensitivity to NPA. The rcn1 mutation was transferred-DNA (T-DNA) tagged and sequences flanking the T-DNA insert were cloned. Analysis of the RCN1 cDNA reveals that the T-DNA insertion disrupts a gene for the regulatory A subunit of protein phosphatase 2A (PP2A-A). The RCN1 gene rescues the rcn1 mutant phenotype and also complements the temperature-sensitive phenotype of the Saccharomyces cerevisiae PP2A-A mutation, tpd3-1. These data implicate protein phosphatase 2A in the regulation of auxin transport in Arabidopsis.

  3. ROP3 GTPase contributes to polar auxin transport and auxin responses and is important for embryogenesis and seedling growth in Arabidopsis.

    Science.gov (United States)

    Huang, Jia-bao; Liu, Huili; Chen, Min; Li, Xiaojuan; Wang, Mingyan; Yang, Yali; Wang, Chunling; Huang, Jiaqing; Liu, Guolan; Liu, Yuting; Xu, Jian; Cheung, Alice Y; Tao, Li-zhen

    2014-09-01

    ROP GTPases are crucial for the establishment of cell polarity and for controlling responses to hormones and environmental signals in plants. In this work, we show that ROP3 plays important roles in embryo development and auxin-dependent plant growth. Loss-of-function and dominant-negative (DN) mutations in ROP3 induced a spectrum of similar defects starting with altered cell division patterning during early embryogenesis to postembryonic auxin-regulated growth and developmental responses. These resulted in distorted embryo development, defective organ formation, retarded root gravitropism, and reduced auxin-dependent hypocotyl elongation. Our results showed that the expression of AUXIN RESPONSE FACTOR5/MONOPTEROS and root master regulators PLETHORA1 (PLT1) and PLT2 was reduced in DN-rop3 mutant embryos, accounting for some of the observed patterning defects. ROP3 mutations also altered polar localization of auxin efflux proteins (PINs) at the plasma membrane (PM), thus disrupting auxin maxima in the root. Notably, ROP3 is induced by auxin and prominently detected in root stele cells, an expression pattern similar to those of several stele-enriched PINs. Our results demonstrate that ROP3 is important for maintaining the polarity of PIN proteins at the PM, which in turn ensures polar auxin transport and distribution, thereby controlling plant patterning and auxin-regulated responses.

  4. D6PK AGCVIII kinases are required for auxin transport and phototropic hypocotyl bending in Arabidopsis.

    Science.gov (United States)

    Willige, Björn C; Ahlers, Siv; Zourelidou, Melina; Barbosa, Inês C R; Demarsy, Emilie; Trevisan, Martine; Davis, Philip A; Roelfsema, M Rob G; Hangarter, Roger; Fankhauser, Christian; Schwechheimer, Claus

    2013-05-01

    Phototropic hypocotyl bending in response to blue light excitation is an important adaptive process that helps plants to optimize their exposure to light. In Arabidopsis thaliana, phototropic hypocotyl bending is initiated by the blue light receptors and protein kinases phototropin1 (phot1) and phot2. Phototropic responses also require auxin transport and were shown to be partially compromised in mutants of the PIN-FORMED (PIN) auxin efflux facilitators. We previously described the D6 PROTEIN KINASE (D6PK) subfamily of AGCVIII kinases, which we proposed to directly regulate PIN-mediated auxin transport. Here, we show that phototropic hypocotyl bending is strongly dependent on the activity of D6PKs and the PIN proteins PIN3, PIN4, and PIN7. While early blue light and phot-dependent signaling events are not affected by the loss of D6PKs, we detect a gradual loss of PIN3 phosphorylation in d6pk mutants of increasing complexity that is most severe in the d6pk d6pkl1 d6pkl2 d6pkl3 quadruple mutant. This is accompanied by a reduction of basipetal auxin transport in the hypocotyls of d6pk as well as in pin mutants. Based on our data, we propose that D6PK-dependent PIN regulation promotes auxin transport and that auxin transport in the hypocotyl is a prerequisite for phot1-dependent hypocotyl bending.

  5. Complex regulation of Arabidopsis AGR1/PIN2-mediated root gravitropic response and basipetal auxin transport by cantharidin-sensitive protein phosphatases

    Science.gov (United States)

    Shin, Heungsop; Shin, Hwa-Soo; Guo, Zibiao; Blancaflor, Elison B.; Masson, Patrick H.; Chen, Rujin

    2005-01-01

    Polar auxin transport, mediated by two distinct plasma membrane-localized auxin influx and efflux carrier proteins/complexes, plays an important role in many plant growth and developmental processes including tropic responses to gravity and light, development of lateral roots and patterning in embryogenesis. We have previously shown that the Arabidopsis AGRAVITROPIC 1/PIN2 gene encodes an auxin efflux component regulating root gravitropism and basipetal auxin transport. However, the regulatory mechanism underlying the function of AGR1/PIN2 is largely unknown. Recently, protein phosphorylation and dephosphorylation mediated by protein kinases and phosphatases, respectively, have been implicated in regulating polar auxin transport and root gravitropism. Here, we examined the effects of chemical inhibitors of protein phosphatases on root gravitropism and basipetal auxin transport, as well as the expression pattern of AGR1/PIN2 gene and the localization of AGR1/PIN2 protein. We also examined the effects of inhibitors of vesicle trafficking and protein kinases. Our data suggest that protein phosphatases, sensitive to cantharidin and okadaic acid, are likely involved in regulating AGR1/PIN2-mediated root basipetal auxin transport and gravitropism, as well as auxin response in the root central elongation zone (CEZ). BFA-sensitive vesicle trafficking may be required for the cycling of AGR1/PIN2 between plasma membrane and the BFA compartment, but not for the AGR1/PIN2-mediated root basipetal auxin transport and auxin response in CEZ cells.

  6. Transport of the two natural auxins, indole-3-butyric acid and indole-3-acetic acid, in Arabidopsis

    Science.gov (United States)

    Rashotte, Aaron M.; Poupart, Julie; Waddell, Candace S.; Muday, Gloria K.; Brown, C. S. (Principal Investigator)

    2003-01-01

    Polar transport of the natural auxin indole-3-acetic acid (IAA) is important in a number of plant developmental processes. However, few studies have investigated the polar transport of other endogenous auxins, such as indole-3-butyric acid (IBA), in Arabidopsis. This study details the similarities and differences between IBA and IAA transport in several tissues of Arabidopsis. In the inflorescence axis, no significant IBA movement was detected, whereas IAA is transported in a basipetal direction from the meristem tip. In young seedlings, both IBA and IAA were transported only in a basipetal direction in the hypocotyl. In roots, both auxins moved in two distinct polarities and in specific tissues. The kinetics of IBA and IAA transport appear similar, with transport rates of 8 to 10 mm per hour. In addition, IBA transport, like IAA transport, is saturable at high concentrations of auxin, suggesting that IBA transport is protein mediated. Interestingly, IAA efflux inhibitors and mutations in genes encoding putative IAA transport proteins reduce IAA transport but do not alter IBA movement, suggesting that different auxin transport protein complexes are likely to mediate IBA and IAA transport. Finally, the physiological effects of IBA and IAA on hypocotyl elongation under several light conditions were examined and analyzed in the context of the differences in IBA and IAA transport. Together, these results present a detailed picture of IBA transport and provide the basis for a better understanding of the transport of these two endogenous auxins.

  7. Identification and Analysis of Medicago truncatula Auxin Transporter Gene Families Uncover their Roles in Responses to Sinorhizobium meliloti Infection.

    Science.gov (United States)

    Shen, Chenjia; Yue, Runqing; Bai, Youhuang; Feng, Rong; Sun, Tao; Wang, Xiaofei; Yang, Yanjun; Tie, Shuanggui; Wang, Huizhong

    2015-10-01

    Auxin transport plays a pivotal role in the interaction between legume species and nitrogen-fixing bacteria to form symbioses. Auxin influx carriers auxin resistant 1/like aux 1 (AUX/LAX), efflux carriers pin-formed (PIN) and efflux/conditional P-glycoprotein (PGP/ABCB) are three major protein families participating in auxin polar transport. We used the latest Medicago truncatula genome sequence to characterize and analyze the M. truncatula LAX (MtLAX), M. truncatula PIN (MtPIN) and M. truncatula ABCB (MtABCB) families. Transient expression experiments indicated that three representative auxin transporters (MtLAX3, MtPIN7 and MtABCB1) showed cell plasma membrane localizations. The expression of most MtLAX, MtPIN and MtABCB genes was up-regulated in the roots and was down-regulated in the shoots by Sinorhizobium meliloti infection in the wild type (WT). However, the expression of these genes was down-regulated in both the roots and shoots of an infection-resistant mutant, dmi3. The different expression patterns between the WT and the mutant roots indicated that auxin relocation may be involved in rhizobial infection responses. Furthermore, IAA contents were significantly up-regulated in the shoots and down-regulated in the roots after Sinorhizobium meliloti infection in the WT. Inoculation of roots with rhizobia may reduce the auxin loading from shoots to roots by inhibiting the expression of most auxin transporter genes. However, the rate of change of gene expression and IAA contents in the dmi3 mutant were obviously lower than in the WT. The identification and expression analysis of auxin transporter genes helps us to understand the roles of auxin in the regulation of nodule formation in M. truncatula.

  8. Real-time monitoring of auxin vesicular exocytotic efflux from single plant protoplasts by amperometry at microelectrodes decorated with nanowires.

    Science.gov (United States)

    Liu, Jun-Tao; Hu, Liang-Sheng; Liu, Yan-Ling; Chen, Rong-Sheng; Cheng, Zhi; Chen, Shi-Jing; Amatore, Christian; Huang, Wei-Hua; Huo, Kai-Fu

    2014-03-01

    Recent biochemical results suggest that auxin (IAA) efflux is mediated by a vesicular cycling mechanism, but no direct detection of vesicular IAA release from single plant cells in real-time has been possible up to now. A TiC@C/Pt-QANFA micro-electrochemical sensor has been developed with high sensitivity in detection of IAA, and it allows real-time monitoring and quantification of the quantal release of auxin from single plant protoplast by exocytosis.

  9. The Relationship between Auxin Transport and Maize Branching1[C][W][OA

    Science.gov (United States)

    Gallavotti, Andrea; Yang, Yan; Schmidt, Robert J.; Jackson, David

    2008-01-01

    Maize (Zea mays) plants make different types of vegetative or reproductive branches during development. Branches develop from axillary meristems produced on the flanks of the vegetative or inflorescence shoot apical meristem. Among these branches are the spikelets, short grass-specific structures, produced by determinate axillary spikelet-pair and spikelet meristems. We investigated the mechanism of branching in maize by making transgenic plants expressing a native expressed endogenous auxin efflux transporter (ZmPIN1a) fused to yellow fluorescent protein and a synthetic auxin-responsive promoter (DR5rev) driving red fluorescent protein. By imaging these plants, we found that all maize branching events during vegetative and reproductive development appear to be regulated by the creation of auxin response maxima through the activity of polar auxin transporters. We also found that the auxin transporter ZmPIN1a is functional, as it can rescue the polar auxin transport defects of the Arabidopsis (Arabidopsis thaliana) pin1-3 mutant. Based on this and on the groundbreaking analysis in Arabidopsis and other species, we conclude that branching mechanisms are conserved and can, in addition, explain the formation of axillary meristems (spikelet-pair and spikelet meristems) that are unique to grasses. We also found that BARREN STALK1 is required for the creation of auxin response maxima at the flanks of the inflorescence meristem, suggesting a role in the initiation of polar auxin transport for axillary meristem formation. Based on our results, we propose a general model for branching during maize inflorescence development. PMID:18550681

  10. Seven Things We Think We Know about Auxin Transport

    Institute of Scientific and Technical Information of China (English)

    Wendy Ann Peer; Joshua J. Blakeslee; Haibing Yang; Angus S. Murphy

    2011-01-01

    T Polar transport of the phytohormone auxin and the establishment of localized auxin maxima regulate embryonic development, stem cell maintenance, root and shoot architecture, and tropic growth responses. The past decade has been marked by dramatic progress in efforts to elucidate the complex mechanisms by which auxin transport regulates plant growth. As the understanding of auxin transport regulation has been increasingly elaborated, it has become clear that this process is involved in almost all plant growth and environmental responses in some way. However, we still lack information about some basic aspects of this fundamental regulatory mechanism. In this review, we present what we know (or what we think we know) and what we do not know about seven auxin-regulated processes. We discuss the role of auxin transport in gravitropism in primary and lateral roots, phototropism, shoot branching, leaf expansion, and venation. We also discuss the auxin reflux/fountain model at the root tip, flavonoid modulation of auxin transport processes, and outstanding aspects of post-translational regulation of auxin transporters. This discussion is not meant to be exhaustive, but highlights areas in which generally held assumptions require more substantive validation.

  11. Auxin conjugated to fluorescent dyes--a tool for the analysis of auxin transport pathways.

    Science.gov (United States)

    Sokołowska, K; Kizińska, J; Szewczuk, Z; Banasiak, A

    2014-09-01

    Auxin is a small molecule involved in most processes related to plant growth and development. Its effect usually depends on the distribution in tissues and the formation of concentration gradients. Until now there has been no tool for the direct tracking of auxin transport at the cellular and tissue level; therefore the majority of studies have been based on various indirect methods. However, due to their various restrictions, relatively little is known about the relationship between various pathways of auxin transport and specific developmental processes. We present a new research tool: fluorescently labelled auxin in the form of a conjugate with two different fluorescent tracers, FITC and RITC, which allows direct observation of auxin transport in plant tissues. Chemical analysis and biological tests have shown that our conjugates have auxin-like biological activity and transport; therefore they can be used in all experimental systems as an alternative to IAA. In addition, the conjugates are a universal tool that can be applied in studies of all plant groups and species. The conjugation procedure presented in this paper can be adapted to other fluorescent dyes, which are constantly being improved. In our opinion, the conjugates greatly expand the possibilities of research concerning the role of auxin and its transport in different developmental processes in plants.

  12. Transport and Metabolism of the Endogenous Auxin Precursor lndole-3-Butyric Acid

    Institute of Scientific and Technical Information of China (English)

    Lucia C. Strader; Bonnie Bartel

    2011-01-01

    T Plant growth and morphogenesis depend on the levels and distribution of the plant hormone auxin. Plants tightly regulate cellular levels of the active auxin indole-3-acetic acid (IAA) through synthesis, inactivation, and transport. Although the transporters that move IAA into and out of cells are well characterized and play important roles in development, little is known about the transport of IAA precursors. In this review, we discuss the accumulating evidence suggesting that the IAA precursor indole-3-butyric acid (IBA) is transported independently of the characterized IAA transport machinery along with the recent identification of specific IBA efflux carriers and enzymes suggested to metabolize IBA. These studies have revealed important roles for IBA in maintaining IAA levels and distribution within the plant to support normal development.

  13. Auxin transport inhibitors impair vesicle motility and actin cytoskeleton dynamics in diverse eukaryotes

    NARCIS (Netherlands)

    P. Dhonukshe (Pankaj); I. Grigoriev (Ilya); R. Fischer (Rainer); M. Tominaga (Motoki); D.G. Robinson (David); J. Hašek (Jiří); T. Paciorek (Tomasz); J. Petrášek (Jan); D. Seifertová (Daniela); R. Tejos (Ricardo); L.A. Meisel (Lee); E. Zažímalová (Eva); T.W.J. Gadella (Theodorus); Y.D. Stierhof; T. Ueda (Takashi); K. Oiwa (Kazuhiro); A.S. Akhmanova (Anna); R. Brock (Roland); A. Spang (Anne); J. Friml (Jiří)

    2008-01-01

    textabstractMany aspects of plant development, including patterning and tropisms, are largely dependent on the asymmetric distribution of the plant signaling molecule auxin. Auxin transport inhibitors (ATIs), which interfere with directional auxin transport, have been essential tools in formulating

  14. Tricho- and atrichoblast cell files show distinct PIN2 auxin efflux carrier exploitations and are jointly required for defined auxin-dependent root organ growth.

    Science.gov (United States)

    Löfke, Christian; Scheuring, David; Dünser, Kai; Schöller, Maria; Luschnig, Christian; Kleine-Vehn, Jürgen

    2015-08-01

    The phytohormone auxin is a vital growth regulator in plants. In the root epidermis auxin steers root organ growth. However, the mechanisms that allow adjacent tissues to integrate growth are largely unknown. Here, the focus is on neighbouring epidermal root tissues to assess the integration of auxin-related growth responses. The pharmacologic, genetic, and live-cell imaging approaches reveal that PIN2 auxin efflux carriers are differentially controlled in tricho- and atrichoblast cells. PIN2 proteins show lower abundance at the plasma membrane of trichoblast cells, despite showing higher rates of intracellular trafficking in these cells. The data suggest that PIN2 proteins display distinct cell-type-dependent trafficking rates to the lytic vacuole for degradation. Based on this insight, it is hypothesized that auxin-dependent processes are distinct in tricho- and atrichoblast cells. Moreover, genetic interference with epidermal patterning supports this assumption and suggests that tricho- and atrichoblasts have distinct importance for auxin-sensitive root growth and gravitropic responses.

  15. Heterologous expression of a membrane-spanning auxin importer: implications for functional analyses of auxin transporters.

    Science.gov (United States)

    Carrier, David John; Abu Bakar, Norliza Tendot; Lawler, Karen; Dorrian, James Matthew; Haider, Ameena; Bennett, Malcolm John; Kerr, Ian Derek

    2009-01-01

    Biochemical studies of plant auxin transporters in vivo are made difficult by the presence of multiple auxin transporters and auxin-interacting proteins. Furthermore, the expression level of most such transporters in plants is likely to be too low for purification and downstream functional analysis. Heterologous expression systems should address both of these issues. We have examined a number of such systems for their efficiency in expressing AUX1 from Arabidopsis thaliana. We find that a eukaryotic system based upon infection of insect cells with recombinant baculovirus provides a high level, easily scalable expression system capable of delivering a functional assay for AUX1. Furthermore, a transient transfection system in mammalian cells enables localization of AUX1 and AUX1-mediated transport of auxin to be investigated. In contrast, we were unable to utilise P. pastoris or L. lactis expression systems to reliably express AUX1.

  16. OsPIN5b modulates rice (Oryza sativa) plant architecture and yield by changing auxin homeostasis, transport and distribution.

    Science.gov (United States)

    Lu, Guangwen; Coneva, Viktoriya; Casaretto, José A; Ying, Shan; Mahmood, Kashif; Liu, Fang; Nambara, Eiji; Bi, Yong-Mei; Rothstein, Steven J

    2015-09-01

    Plant architecture attributes such as tillering, plant height and panicle size are important agronomic traits that determine rice (Oryza sativa) productivity. Here, we report that altered auxin content, transport and distribution affect these traits, and hence rice yield. Overexpression of the auxin efflux carrier-like gene OsPIN5b causes pleiotropic effects, mainly reducing plant height, leaf and tiller number, shoot and root biomass, seed-setting rate, panicle length and yield parameters. Conversely, reduced expression of OsPIN5b results in higher tiller number, more vigorous root system, longer panicles and increased yield. We show that OsPIN5b is an endoplasmic reticulum (ER) -localized protein that participates in auxin homeostasis, transport and distribution in vivo. This work describes an example of an auxin-related gene where modulating its expression can simultaneously improve plant architecture and yield potential in rice, and reveals an important effect of hormonal signaling on these traits.

  17. BARREN INFLORESCENCE2 interaction with ZmPIN1a suggests a role in auxin transport during maize inflorescence development.

    Science.gov (United States)

    Skirpan, Andrea; Culler, Angela Hendrickson; Gallavotti, Andrea; Jackson, David; Cohen, Jerry D; McSteen, Paula

    2009-03-01

    Polar auxin transport, mediated by the PIN-FORMED (PIN) class of auxin efflux carriers, controls organ initiation in plants. In maize, BARREN INFLORESCENCE2 (BIF2) encodes a serine/threonine protein kinase co-orthologous to PINOID (PID), which regulates the subcellular localization of AtPIN1 in Arabidopsis. We show that BIF2 phosphorylates ZmPIN1a, a maize homolog of AtPIN1, in vitro and regulates ZmPIN1a subcellular localization in vivo, similar to the role of PID in Arabidopsis. In addition, bif2 mutant inflorescences have lower auxin levels later in development. We propose that BIF2 regulates auxin transport through direct regulation of ZmPIN1a during maize inflorescence development.

  18. Melatonin regulates root meristem by repressing auxin synthesis and polar auxin transport in Arabidopsis

    Directory of Open Access Journals (Sweden)

    Qiannan Wang

    2016-12-01

    Full Text Available Melatonin (N-acetyl-5-methoxytryptamine plays important roles in regulating both biotic and abiotic stress tolerance, biological rhythms, plant growth and development. Sharing the same substrate (tryptophan for the biosynthesis, melatonin and auxin also have similar effects in plant development. However, the specific function of melatonin in modulating plant root growth and the relationship between melatonin and auxin as well as underlying mechanisms are still unclear. In this study, we found high concentration of melatonin remarkably inhibited root growth in Arabidopsis by reducing root meristem size. Further studies showed that melatonin negatively regulated auxin biosynthesis, the expression of PINFORMED (PIN proteins as well as auxin response in Arabidopsis. Moreover, the root growth of the triple mutant pin1pin3pin7 was more tolerant than that of wild type in response to melatonin treatment, suggesting the essential role of PIN1/3/7 in melatonin-mediated root growth. Combination treatment of melatonin and 5-Triiodobenzoic acid (TIBA did not enhance melatonin-mediated reduction of root meristem size, indicating that polar auxin transport (PAT may be necessary for the regulation of root meristem size by melatonin treatment. Taken together, this study indicates that melatonin regulates root growth in Arabidopsis, through auxin synthesis and polar auxin transport, at least partially.

  19. Melatonin Regulates Root Meristem by Repressing Auxin Synthesis and Polar Auxin Transport in Arabidopsis.

    Science.gov (United States)

    Wang, Qiannan; An, Bang; Wei, Yunxie; Reiter, Russel J; Shi, Haitao; Luo, Hongli; He, Chaozu

    2016-01-01

    Melatonin (N-acetyl-5-methoxytryptamine) plays important roles in regulating both biotic and abiotic stress tolerance, biological rhythms, plant growth and development. Sharing the same substrate (tryptophan) for the biosynthesis, melatonin and auxin also have similar effects in plant development. However, the specific function of melatonin in modulating plant root growth and the relationship between melatonin and auxin as well as underlying mechanisms are still unclear. In this study, we found high concentration of melatonin remarkably inhibited root growth in Arabidopsis by reducing root meristem size. Further studies showed that melatonin negatively regulated auxin biosynthesis, the expression of PINFORMED (PIN) proteins as well as auxin response in Arabidopsis. Moreover, the root growth of the triple mutant pin1pin3pin7 was more tolerant than that of wild-type in response to melatonin treatment, suggesting the essential role of PIN1/3/7 in melatonin-mediated root growth. Combination treatment of melatonin and 5-Triiodobenzoic acid (TIBA) did not enhance melatonin-mediated reduction of root meristem size, indicating that polar auxin transport (PAT) may be necessary for the regulation of root meristem size by melatonin treatment. Taken together, this study indicates that melatonin regulates root growth in Arabidopsis, through auxin synthesis and polar auxin transport, at least partially.

  20. D6 PROTEIN KINASE activates auxin transport-dependent growth and PIN-FORMED phosphorylation at the plasma membrane.

    Science.gov (United States)

    Barbosa, Inês C R; Zourelidou, Melina; Willige, Björn C; Weller, Benjamin; Schwechheimer, Claus

    2014-06-23

    The directed cell-to-cell transport of the phytohormone auxin by efflux and influx transporters is essential for proper plant growth and development. Like auxin efflux facilitators of the PIN-FORMED (PIN) family, D6 PROTEIN KINASE (D6PK) from Arabidopsis thaliana localizes to the basal plasma membrane of many cells, and evidence exists that D6PK may directly phosphorylate PINs. We find that D6PK is a membrane-bound protein that is associated with either the basal domain of the plasma membrane or endomembranes. Inhibition of the trafficking regulator GNOM leads to a rapid internalization of D6PK to endomembranes. Interestingly, the dissociation of D6PK from the plasma membrane is also promoted by auxin. Surprisingly, we find that auxin transport-dependent tropic responses are critically and reversibly controlled by D6PK and D6PK-dependent PIN phosphorylation at the plasma membrane. We conclude that D6PK abundance at the plasma membrane and likely D6PK-dependent PIN phosphorylation are prerequisites for PIN-mediated auxin transport.

  1. Auxin-driven patterning with unidirectional fluxes.

    Science.gov (United States)

    Cieslak, Mikolaj; Runions, Adam; Prusinkiewicz, Przemyslaw

    2015-08-01

    The plant hormone auxin plays an essential role in the patterning of plant structures. Biological hypotheses supported by computational models suggest that auxin may fulfil this role by regulating its own transport, but the plausibility of previously proposed models has been questioned. We applied the notion of unidirectional fluxes and the formalism of Petri nets to show that the key modes of auxin-driven patterning-the formation of convergence points and the formation of canals-can be implemented by biochemically plausible networks, with the fluxes measured by dedicated tally molecules or by efflux and influx carriers themselves. Common elements of these networks include a positive feedback of auxin efflux on the allocation of membrane-bound auxin efflux carriers (PIN proteins), and a modulation of this allocation by auxin in the extracellular space. Auxin concentration in the extracellular space is the only information exchanged by the cells. Canalization patterns are produced when auxin efflux and influx act antagonistically: an increase in auxin influx or concentration in the extracellular space decreases the abundance of efflux carriers in the adjacent segment of the membrane. In contrast, convergence points emerge in networks in which auxin efflux and influx act synergistically. A change in a single reaction rate may result in a dynamic switch between these modes, suggesting plausible molecular implementations of coordinated patterning of organ initials and vascular strands predicted by the dual polarization theory.

  2. D6PK AGCVIII Kinases Are Required for Auxin Transport and Phototropic Hypocotyl Bending in Arabidopsis[C][W

    Science.gov (United States)

    Willige, Björn C.; Ahlers, Siv; Zourelidou, Melina; Barbosa, Inês C.R.; Demarsy, Emilie; Trevisan, Martine; Davis, Philip A.; Roelfsema, M. Rob G.; Hangarter, Roger; Fankhauser, Christian; Schwechheimer, Claus

    2013-01-01

    Phototropic hypocotyl bending in response to blue light excitation is an important adaptive process that helps plants to optimize their exposure to light. In Arabidopsis thaliana, phototropic hypocotyl bending is initiated by the blue light receptors and protein kinases phototropin1 (phot1) and phot2. Phototropic responses also require auxin transport and were shown to be partially compromised in mutants of the PIN-FORMED (PIN) auxin efflux facilitators. We previously described the D6 PROTEIN KINASE (D6PK) subfamily of AGCVIII kinases, which we proposed to directly regulate PIN-mediated auxin transport. Here, we show that phototropic hypocotyl bending is strongly dependent on the activity of D6PKs and the PIN proteins PIN3, PIN4, and PIN7. While early blue light and phot-dependent signaling events are not affected by the loss of D6PKs, we detect a gradual loss of PIN3 phosphorylation in d6pk mutants of increasing complexity that is most severe in the d6pk d6pkl1 d6pkl2 d6pkl3 quadruple mutant. This is accompanied by a reduction of basipetal auxin transport in the hypocotyls of d6pk as well as in pin mutants. Based on our data, we propose that D6PK-dependent PIN regulation promotes auxin transport and that auxin transport in the hypocotyl is a prerequisite for phot1-dependent hypocotyl bending. PMID:23709629

  3. Regulation of ABCB1/PGP1-catalysed auxin transport by linker phosphorylation

    DEFF Research Database (Denmark)

    Henrichs, Sina; Wang, Bangjun; Fukao, Yoichiro

    2012-01-01

    Polar transport of the plant hormone auxin is controlled by PIN-and ABCB/PGP-efflux catalysts. PIN polarity is regulated by the AGC protein kinase, PINOID (PID), while ABCB activity was shown to be dependent on interaction with the FKBP42, TWISTED DWARF1 (TWD1). Using co-immunoprecipitation (co...... target of PID phosphorylation that determines both transporter drug binding and activity. In summary, we provide evidence that PID phosphorylation has a dual, counter-active impact on ABCB1 activity that is coordinated by TWD1-PID interaction....

  4. Auxin transport in the evolution of branching forms.

    Science.gov (United States)

    Harrison, C Jill

    2016-11-24

    I. II. III. IV. V. VI. VII. Acknowledgements References SUMMARY: Branching is one of the most striking aspects of land plant architecture, affecting resource acquisition and yield. Polar auxin transport by PIN proteins is a primary determinant of flowering plant branching patterns regulating both branch initiation and branch outgrowth. Several lines of experimental evidence suggest that PIN-mediated polar auxin transport is a conserved regulator of branching in vascular plant sporophytes. However, the mechanisms of branching and auxin transport and relationships between the two are not well known outside the flowering plants, and the paradigm for PIN-regulated branching in flowering plants does not fit bryophyte gametophytes. The evidence reviewed here suggests that divergent auxin transport routes contributed to the diversification of branching forms in distinct land plant lineages.

  5. Auxin transport through PIN-FORMED 3 (PIN3) controls shade avoidance and fitness during competition

    Science.gov (United States)

    Keuskamp, Diederik H.; Pollmann, Stephan; Voesenek, Laurentius A. C. J.; Peeters, Anton J. M.; Pierik, Ronald

    2010-01-01

    Plants grow in dense vegetations at the risk of being out-competed by neighbors. To increase their competitive power, plants display adaptive responses, such as rapid shoot elongation (shade avoidance) to consolidate light capture. These responses are induced upon detection of proximate neighbors through perception of the reduced ratio between red (R) and far-red (FR) light that is typical for dense vegetations. The plant hormone auxin is a central regulator of plant development and plasticity, but until now it has been unknown how auxin transport is controlled to regulate shade-avoidance responses. Here, we show that low R:FR detection changes the cellular location of the PIN-FORMED 3 (PIN3) protein, a regulator of auxin efflux, in Arabidopsis seedlings. As a result, auxin levels in the elongating hypocotyls are increased under low R:FR. Seedlings of the pin3-3 mutant lack this low R:FR-induced increase of endogenous auxin in the hypocotyl and, accordingly, have no elongation response to low R:FR. We hypothesize that low R:FR-induced stimulation of auxin biosynthesis drives the regulation of PIN3, thus allowing shade avoidance to occur. The adaptive significance of PIN3-mediated control of shade-avoidance is shown in plant competition studies. It was found that pin3 mutants are outcompeted by wild-type neighbors who suppress fitness of pin3-3 by 40%. We conclude that low R:FR modulates the auxin distribution by a change in the cellular location of PIN3, and that this control can be of great importance for plants growing in dense vegetations. PMID:21149713

  6. Protein ubiquitination in auxin signaling and transport

    NARCIS (Netherlands)

    Santos Maraschin, Felipe dos

    2009-01-01

    What makes plant shoots grow towards the light, and plant roots grow down into the soil? This was a question that Charles Darwin asked himself, and his experiments more than a century ago to find the answer laid the basis for the identification of the growth hormone auxin. Auxin, or indole-3-acetic

  7. The effect of polar auxin transport on adventitious branches formation in Gracilaria lichenoides in vitro.

    Science.gov (United States)

    Wang, Wenlei; Li, Huanqin; Lin, Xiangzhi; Zhang, Fang; Fang, Baishan; Wang, Zhaokai

    2016-11-01

    Seaweed tissue culture (STC) is an important micropropagation tool that has been applied for strain improvement, micropropagation and genetic engineering. Because the mechanisms associated with STC are poorly understood, its application to these organisms lags far behind that of tissue culture propagation of higher plants. Auxin, calcium (Ca(2+) ) and hydrogen peroxide (H2 O2 ) fluxes all play key roles during plant growth and development. In this study, we therefore measured indole-3-acetic acid, Ca(2+) and H2 O2 fluxes of Gracilaria lichenoides explants during adventitious branches (ABs) formation for the first time using noninvasive micro-test technology. We confirmed that polar auxin transport (PAT) also occurs in the marine red alga G. lichenoides. We additionally found that N-1-naphthylphthalamic acid may suppress auxin efflux via ABCB1 transporters and then inhibit ABs formation from the apical region of G. lichenoides segments. The involvement of Ca(2+) and H2 O2 fluxes in PAT-mediated AB formation in G. lichenoides was also investigated. We propose that complex feedback among Ca(2+) , H2 O2 and auxin signaling and response systems may occur during ABs polar formation in G. lichenoides explants, similar to that in higher plants. Our results provide innovative insights that should aid future elucidation of mechanisms operative during STC.

  8. S-nitrosylation mediates nitric oxide -auxin crosstalk in auxin signaling and polar auxin transport

    Science.gov (United States)

    Nitric oxide (NO) and auxin phytohormone cross talk has been implicated in plant development and growth. Addition and removal of NO moieties to cysteine residues of proteins, is termed S-nitrosylation and de-nitrosylation, respectively and functions as an on/off switch of protein activity. This dyna...

  9. Enquiry into the Topology of Plasma Membrane-Localized PIN Auxin Transport Components.

    Science.gov (United States)

    Nodzyński, Tomasz; Vanneste, Steffen; Zwiewka, Marta; Pernisová, Markéta; Hejátko, Jan; Friml, Jiří

    2016-11-07

    Auxin directs plant ontogenesis via differential accumulation within tissues depending largely on the activity of PIN proteins that mediate auxin efflux from cells and its directional cell-to-cell transport. Regardless of the developmental importance of PINs, the structure of these transporters is poorly characterized. Here, we present experimental data concerning protein topology of plasma membrane-localized PINs. Utilizing approaches based on pH-dependent quenching of fluorescent reporters combined with immunolocalization techniques, we mapped the membrane topology of PINs and further cross-validated our results using available topology modeling software. We delineated the topology of PIN1 with two transmembrane (TM) bundles of five α-helices linked by a large intracellular loop and a C-terminus positioned outside the cytoplasm. Using constraints derived from our experimental data, we also provide an updated position of helical regions generating a verisimilitude model of PIN1. Since the canonical long PINs show a high degree of conservation in TM domains and auxin transport capacity has been demonstrated for Arabidopsis representatives of this group, this empirically enhanced topological model of PIN1 will be an important starting point for further studies on PIN structure-function relationships. In addition, we have established protocols that can be used to probe the topology of other plasma membrane proteins in plants.

  10. Gibberellins inhibit adventitious rooting in hybrid aspen and Arabidopsis by affecting auxin transport.

    Science.gov (United States)

    Mauriat, Mélanie; Petterle, Anna; Bellini, Catherine; Moritz, Thomas

    2014-05-01

    Knowledge of processes involved in adventitious rooting is important to improve both fundamental understanding of plant physiology and the propagation of numerous plants. Hybrid aspen (Populus tremula × tremuloïdes) plants overexpressing a key gibberellin (GA) biosynthesis gene (AtGA20ox1) grow rapidly but have poor rooting efficiency, which restricts their clonal propagation. Therefore, we investigated the molecular basis of adventitious rooting in Populus and the model plant Arabidopsis. The production of adventitious roots (ARs) in tree cuttings is initiated from the basal stem region, and involves the interplay of several endogenous and exogenous factors. The roles of several hormones in this process have been characterized, but the effects of GAs have not been fully investigated. Here, we show that a GA treatment negatively affects the numbers of ARs produced by wild-type hybrid aspen cuttings. Furthermore, both hybrid aspen plants and intact Arabidopsis seedlings overexpressing AtGA20ox1, PttGID1.1 or PttGID1.3 genes (with a 35S promoter) produce few ARs, although ARs develop from the basal stem region of hybrid aspen and the hypocotyl of Arabidopsis. In Arabidopsis, auxin and strigolactones are known to affect AR formation. Our data show that the inhibitory effect of GA treatment on adventitious rooting is not mediated by perturbation of the auxin signalling pathway, or of the strigolactone biosynthetic and signalling pathways. Instead, GAs appear to act by perturbing polar auxin transport, in particular auxin efflux in hybrid aspen, and both efflux and influx in Arabidopsis.

  11. Gravistimulation changes expression of genes encoding putative carrier proteins of auxin polar transport in etiolated pea epicotyls

    Science.gov (United States)

    Hoshino, T.; Hitotsubashi, R.; Miyamoto, K.; Tanimoto, E.; Ueda, J.

    STS-95 space experiment has showed that auxin polar transport in etiolated epicotyls of pea (Pisum sativum L. cv. Alaska) seedlings is controlled by gravistimulation. In Arabidopsis thaliana auxin polar transport has considered to be regulated by efflux and influx carrier proteins in plasma membranes, AtPIN1 and AtAUX1, respectively. In order to know how gravistimuli control auxin polar transport in etiolated pea epicotyls at molecular levels, strenuous efforts have been made, resulting in successful isolation of full-length cDNAs of a putative auxin efflux and influx carriers, PsPIN2 and PsAUX1, respectively. Significantly high levels in homology were found on nucleotide and deduced amino acid sequences among PsPIN2, PsPIN1 (accession no. AY222857, Chawla and DeMason, 2003) and AtPINs, and also among PsAUX1, AtAUX1 and their related genes. Phylogenetic analyses based on the deduced amino acid sequences revealed that PsPIN2 belonged to a subclade including AtPIN3, AtPIN4 relating to lateral transport of auxin, while PsPIN1 belonged to the same clade as AtPIN1 relating to auxin polar transport. In the present study, we examined the effects of gravistimuli on the expression of PsPINs and PsAUX1 in etiolated pea seedlings by northern blot analysis. Expression of PsPIN1, PsPIN2 and PsAUX1 in hook region of 3.5-d-old etiolated pea seedlings grown under simulated microgravity conditions on a 3-D clinostat increased as compared with that of the seedlings grown under 1 g conditions. On the other hand, that of PsPIN1 and PsAUX1 in the 1st internode region under simulated microgravity conditions on a 3-D clinostat also increased, while that of PsPIN2 was affected little. These results suggest that expression of PsPIN1, PsPIN2 and PsAUX1 regulating polar/lateral transport of auxin is substantially under the control of gravity. A possible role of PsPINs and PsAUX1 of auxin polar transport in etiolated pea seedlings will also be discussed.

  12. The carrier AUXIN RESISTANT (AUX1) dominates auxin flux into Arabidopsis protoplasts.

    Science.gov (United States)

    Rutschow, Heidi L; Baskin, Tobias I; Kramer, Eric M

    2014-11-01

    The ability of the plant hormone auxin to enter a cell is critical to auxin transport and signaling. Auxin can cross the cell membrane by diffusion or via auxin-specific influx carriers. There is little knowledge of the magnitudes of these fluxes in plants. Radiolabeled auxin uptake was measured in protoplasts isolated from roots of Arabidopsis thaliana. This was done for the wild-type, under treatments with additional unlabeled auxin to saturate the influx carriers, and for the influx carrier mutant auxin resistant 1 (aux1). We also used flow cytometry to quantify the relative abundance of cells expressing AUX1-YFP in the assayed population. At pH 5.7, the majority of auxin influx into protoplasts - 75% - was mediated by the influx carrier AUX1. An additional 20% was mediated by other saturable carriers. The diffusive influx of auxin was essentially negligible at pH 5.7. The influx of auxin mediated by AUX1, expressed as a membrane permeability, was 1.5 ± 0.3 μm s(-1) . This value is comparable in magnitude to estimates of efflux permeability. Thus, auxin-transporting tissues can sustain relatively high auxin efflux and yet not become depleted of auxin.

  13. An auxin transport mechanism restricts positive orthogravitropism in lateral roots.

    Science.gov (United States)

    Rosquete, Michel Ruiz; von Wangenheim, Daniel; Marhavý, Peter; Barbez, Elke; Stelzer, Ernst H K; Benková, Eva; Maizel, Alexis; Kleine-Vehn, Jürgen

    2013-05-01

    As soon as a seed germinates, plant growth relates to gravity to ensure that the root penetrates the soil and the shoot expands aerially. Whereas mechanisms of positive and negative orthogravitropism of primary roots and shoots are relatively well understood, lateral organs often show more complex growth behavior. Lateral roots (LRs) seemingly suppress positive gravitropic growth and show a defined gravitropic set-point angle (GSA) that allows radial expansion of the root system (plagiotropism). Despite its eminent importance for root architecture, it so far remains completely unknown how lateral organs partially suppress positive orthogravitropism. Here we show that the phytohormone auxin steers GSA formation and limits positive orthogravitropism in LR. Low and high auxin levels/signaling lead to radial or axial root systems, respectively. At a cellular level, it is the auxin transport-dependent regulation of asymmetric growth in the elongation zone that determines GSA. Our data suggest that strong repression of PIN4/PIN7 and transient PIN3 expression limit auxin redistribution in young LR columella cells. We conclude that PIN activity, by temporally limiting the asymmetric auxin fluxes in the tip of LRs, induces transient, differential growth responses in the elongation zone and, consequently, controls root architecture.

  14. Inhibition of auxin transport and auxin signaling and treatment with far red light induces root coiling in the phospholipase-A mutant ppla-I-1. Significance for surface penetration?

    Science.gov (United States)

    Perrineau, F; Wimalasekera, R; Effendi, Y; Scherer, G F E

    2016-06-01

    When grown on a non-penetretable at a surface angle of 45°, Arabidopsis roots form wave-like structures and, in wild type rarely, but in certain mutants the tip root even may form circles. These circles are called coils. The formation of coils depends on the complex interaction of circumnutation, gravitropism and negative thigmotropism where - at least - gravitropism is intimately linked to auxin transport and signaling. The knockout mutant of patatin-related phospholipase-AI-1 (pplaI-1) is an auxin-signaling mutant which forms moderately increased numbers of coils on tilted agar plates. We tested the effects of the auxin efflux transport inhibitor NPA (1-naphthylphtalamic acid) and of the influx transport inhibitor 1-NOA (1-naphthoxyacetic acid) which both further increased root coil formation. The pPLAI-1 inhibitors HELSS (haloenol lactone suicide substrate=E-6-(bromomethylene)tetrahydro-3-(1-naphthalenyl)-2H-pyran-2-one) and ETYA (eicosatetraynoic acid) which are auxin signaling inhibitors also increased coil formation. In addition, far red light treatment increased coil formation. The results point out that a disturbance of auxin transport and signaling is one potential cause for root coils. As we show that the mutant pplaI-1 penetrates horizontal agar plates better than wild type plants root movements may help penetrating the soil.

  15. Ca2+-Transport through Plasma Membrane as a Test of Auxin Sensitivity

    Directory of Open Access Journals (Sweden)

    Anastasia A. Kirpichnikova

    2014-03-01

    Full Text Available Auxin is one of the crucial regulators of plant growth and development. The discovered auxin cytosolic receptor (TIR1 is not involved in the perception of the hormone signal at the plasma membrane. Instead, another receptor, related to the ABP1, auxin binding protein1, is supposed to be responsible for the perception at the plasma membrane. One of the fast and sensitive auxin-induced reactions is an increase of Ca2+ cytosolic concentration, which is suggested to be dependent on the activation of Ca2+ influx through the plasma membrane. This investigation was carried out with a plasmalemma enriched vesicle fraction, obtained from etiolated maize coleoptiles. The magnitude of Ca2+ efflux through the membrane vesicles was estimated according to the shift of potential dependent fluorescent dye diS-C3-(5. The obtained results showed that during coleoptiles ageing (3rd, 4th and 5th days of seedling etiolated growth the magnitude of Ca2+ efflux from inside-out vesicles was decreased. Addition of ABP1 led to a recovery of Ca2+ efflux to the level of the youngest and most sensitive cells. Moreover, the efflux was more sensitive, responding from 10−8 to 10−6 M 1-NAA, in vesicles containing ABP1, whereas native vesicles showed the highest efflux at 10−6 M 1-NAA. We suggest that auxin increases plasma membrane permeability to Ca2+ and that ABP1 is involved in modulation of this reaction.

  16. Plant embryogenesis requires AUX/LAX-mediated auxin influx

    NARCIS (Netherlands)

    Robert, H.S.; Grunewald, W.; Cannoot, B.; Soriano, M.; Swarup, R.; Weijers, D.; Bennett, M.; Boutilier, K.A.; Friml, J.

    2015-01-01

    The plant hormone auxin and its directional transport are known to play a crucial role in defining the embryonic axis and subsequent development of the body plan. Although the role of PIN auxin efflux transporters has been clearly assigned during embryonic shoot and root specification, the role of t

  17. Diversification and expression of the PIN, AUX/LAX and ABCB families of putative auxin transporters in Populus

    Directory of Open Access Journals (Sweden)

    Nicola eCarraro

    2012-02-01

    Full Text Available Intercellular transport of the plant hormone auxin is mediated by three families of membrane-bound protein carriers, with the PIN and ABCB families coding primarily for efflux proteins and the AUX/LAX family coding for influx proteins. In the last decade our understanding of gene and protein function for these transporters in Arabidopsis has expanded rapidly but very little is known about their role in woody plant development. Here we present a comprehensive account of all three families in the model woody species Populus, including chromosome distribution, protein structure, quantitative gene expression, and evolutionary relationships. The PIN and AUX/LAX gene families in Populus comprise 16 and 8 members respectively, and show evidence for the retention of paralogs following a relatively recent whole genome duplication. There is also evidence for differential expression across tissues within many gene pairs. The ABCB family is previously undescribed in Populus and includes 20 members, showing a much deeper evolutionary history including both tandem and whole genome duplication as well as probable loss. A striking number of these transporters are expressed in developing Populus stems and we suggest that evolutionary and structural relationships with known auxin transporters in Arabidopsis can point toward candidate genes for further study in Populus. This is especially important for the ABCBs, which is a large family and includes members in Arabidopsis that are able to transport other substrates in addition to auxin. Protein modeling, sequence alignment and expression data all point to ABCB1.1 as a likely auxin transport protein in Populus. Given that basipetal auxin flow through the cambial zone shapes the development of woody stems, it is important that we identify the full complement of proteins involved in this process. This work should lay the foundation for studies targeting specific proteins for functional characterization and in situ

  18. Auxin influx inhibitors 1-NOA, 2-NOA, and CHPAA interfere with membrane dynamics in tobacco cells.

    Science.gov (United States)

    Lanková, Martina; Smith, Richard S; Pesek, Bedrich; Kubes, Martin; Zazímalová, Eva; Petrásek, Jan; Hoyerová, Klára

    2010-08-01

    The phytohormone auxin is transported through the plant body either via vascular pathways or from cell to cell by specialized polar transport machinery. This machinery consists of a balanced system of passive diffusion combined with the activities of auxin influx and efflux carriers. Synthetic auxins that differ in the mechanisms of their transport across the plasma membrane together with polar auxin transport inhibitors have been used in many studies on particular auxin carriers and their role in plant development. However, the exact mechanism of action of auxin efflux and influx inhibitors has not been fully elucidated. In this report, the mechanism of action of the auxin influx inhibitors (1-naphthoxyacetic acid (1-NOA), 2-naphthoxyacetic acid (2-NOA), and 3-chloro-4-hydroxyphenylacetic acid (CHPAA)) is examined by direct measurements of auxin accumulation, cellular phenotypic analysis, as well as by localization studies of Arabidopsis thaliana L. auxin carriers heterologously expressed in Nicotiana tabacum L., cv. Bright Yellow cell suspensions. The mode of action of 1-NOA, 2-NOA, and CHPAA has been shown to be linked with the dynamics of the plasma membrane. The most potent inhibitor, 1-NOA, blocked the activities of both auxin influx and efflux carriers, whereas 2-NOA and CHPAA at the same concentration preferentially inhibited auxin influx. The results suggest that these, previously unknown, activities of putative auxin influx inhibitors regulate overall auxin transport across the plasma membrane depending on the dynamics of particular membrane vesicles.

  19. Expression profile of PIN, AUX/LAX and PGP auxin transporter gene families in Sorghum bicolor under phytohormone and abiotic stress.

    Science.gov (United States)

    Shen, ChenJia; Bai, YouHuang; Wang, SuiKang; Zhang, SaiNa; Wu, YunRong; Chen, Ming; Jiang, DeAn; Qi, YanHua

    2010-07-01

    Auxin is transported by the influx carriers auxin resistant 1/like aux1 (AUX/LAX), and the efflux carriers pin-formed (PIN) and P-glycoprotein (PGP), which play a major role in polar auxin transport. Several auxin transporter genes have been characterized in dicotyledonous Arabidopsis, but most are unknown in monocotyledons, especially in sorghum. Here, we analyze the chromosome distribution, gene duplication and intron/exon of SbPIN, SbLAX and SbPGP gene families, and examine their phylogenic relationships in Arabidopsis, rice and sorghum. Real-time PCR analysis demonstrated that most of these genes were differently expressed in the organs of sorghum. SbPIN3 and SbPIN9 were highly expressed in flowers, SbLAX2 and SbPGP17 were mainly expressed in stems, and SbPGP7 was strongly expressed in roots. This suggests that individual genes might participate in specific organ development. The expression profiles of these gene families were analyzed after treatment with: (a) the phytohormones indole-3-acetic acid and brassinosteroid; (b) the polar auxin transport inhibitors 1-naphthoxyacetic acids, 1-naphthylphthalamic acid and 2,3,5-triiodobenzoic acid; and (c) abscissic acid and the abiotic stresses of high salinity and drought. Most of the auxin transporter genes were strongly induced by indole-3-acetic acid and brassinosteroid, providing new evidence for the synergism of these phytohormones. Interestingly, most genes showed similar trends in expression under polar auxin transport inhibitors and each also responded to abscissic acid, salt and drought. This study provides new insights into the auxin transporters of sorghum.

  20. Dominance induction of fruitlet shedding in Malus × domestica (L. Borkh: molecular changes associated with polar auxin transport

    Directory of Open Access Journals (Sweden)

    Velasco Riccardo

    2009-11-01

    Full Text Available Abstract Background Apple fruitlet abscission is induced by dominance, a process in which hormones such as auxin, cytokinins and strigolactone play a pivotal role. The response to these hormones is controlled by transcription regulators such as Aux/IAA and ARR, whereas auxin transport is controlled by influx and efflux carriers. Results Seven partial clones encoding auxin efflux carriers (MdPIN1_A, MdPIN1_B, MdPIN10_A, MdPIN10_B, MdPIN4, MdPIN7_A and MdPIN7_B, three encoding auxin influx carriers (MdLAX1, MdLAX2 and MdLAX3 and three encoding type A ARR cytokinin response regulators (MdARR3, MdARR4 and MdARR6 were isolated by the use of degenerate primers. The organization of the PIN multigene family in apple is closer to Medicago truncatula than to Arabidopsis thaliana. The genes are differentially expressed in diverse plant organs and at different developmental stages. MdPIN1 and MdPIN7 are largely more expressed than MdPIN10 and MdPIN4. During abscission, the transcription of these genes increased in the cortex whereas in the seed a sharp fall was observed. The expression of these genes was found to be at least partially controlled by ethylene and auxin. Conclusion The ethylene burst preceding abscission of fruitlets may be responsible for the decrease in transcript level of MDPIN1, MDARR5 and MDIAA3 in seed. This situation modulates the status of the fruitlet and its fate by hampering the PAT from the seeds down through the abscission zone (AZ and this brings about the shedding of the fruitlet.

  1. Auxin distribution and transport during embryogenesis and seed germi-nation of Arabidopsis

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Auxin distribution during embryogenesis and seed germination were studied with transgenic Arabidopsis plants expressing GUS gene driven by a synthetic DR5 promoter, an auxin responsive promoter. The results showed that GUS activity is higher in ends of hypophysis and cotyledon primordia of heart-, torpedo- and cotyledon-stage embryos, leaf tip area, lateral root primordia, root apex and cotyledon of young seedlings.And GUS accumulated in root apex of the seedlings grown on auxin transport inhibitor containing media.All these suggested that above-mentioned part of the organs and tissues have a higher level of auxin, and auxin polar transport inhibitor could cause the accumulation of auxin in root apex. And auxin transport inhibitor also resulted in aberration of Arabidopsis leaf pattern formation, root gravitropism and elongation.

  2. Polar auxin transport is essential for gall formation by Pantoea agglomerans on Gypsophila.

    Science.gov (United States)

    Chalupowicz, Laura; Weinthal, Dan; Gaba, Victor; Sessa, Guido; Barash, Isaac; Manulis-Sasson, Shulamit

    2013-02-01

    The virulence of the bacterium Pantoea agglomerans pv. gypsophilae (Pag) on Gypsophila paniculata depends on a type III secretion system (T3SS) and its effectors. The hypothesis that plant-derived indole-3-acetic acid (IAA) plays a major role in gall formation was examined by disrupting basipetal polar auxin transport with the specific inhibitors 2,3,5-triiodobenzoic acid (TIBA) and N-1-naphthylphthalamic acid (NPA). On inoculation with Pag, galls developed in gypsophila stems above but not below lanolin rings containing TIBA or NPA, whereas, in controls, galls developed above and below the rings. In contrast, TIBA and NPA could not inhibit tumour formation in tomato caused by Agrobacterium tumefaciens. The colonization of gypsophila stems by Pag was reduced below, but not above, the lanolin-TIBA ring. Following Pag inoculation and TIBA treatment, the expression of hrpL (a T3SS regulator) and pagR (a quorum-sensing transcriptional regulator) decreased four-fold and that of pthG (a T3SS effector) two-fold after 24 h. Expression of PIN2 (a putative auxin efflux carrier) increased 35-fold, 24 h after Pag inoculation. However, inoculation with a mutant in the T3SS effector pthG reduced the expression of PIN2 by two-fold compared with wild-type infection. The results suggest that pthG might govern the elevation of PIN2 expression during infection, and that polar auxin transport-derived IAA is essential for gall initiation. © 2012 THE AUTHORS. MOLECULAR PLANT PATHOLOGY © 2012 BSPP AND BLACKWELL PUBLISHING LTD.

  3. Strigolactone can promote or inhibit shoot branching by triggering rapid depletion of the auxin efflux protein PIN1 from the plasma membrane.

    Directory of Open Access Journals (Sweden)

    Naoki Shinohara

    Full Text Available Plants continuously extend their root and shoot systems through the action of meristems at their growing tips. By regulating which meristems are active, plants adjust their body plans to suit local environmental conditions. The transport network of the phytohormone auxin has been proposed to mediate this systemic growth coordination, due to its self-organising, environmentally sensitive properties. In particular, a positive feedback mechanism termed auxin transport canalization, which establishes auxin flow from active shoot meristems (auxin sources to the roots (auxin sinks, has been proposed to mediate competition between shoot meristems and to balance shoot and root growth. Here we provide strong support for this hypothesis by demonstrating that a second hormone, strigolactone, regulates growth redistribution in the shoot by rapidly modulating auxin transport. A computational model in which strigolactone action is represented as an increase in the rate of removal of the auxin export protein, PIN1, from the plasma membrane can reproduce both the auxin transport and shoot branching phenotypes observed in various mutant combinations and strigolactone treatments, including the counterintuitive ability of strigolactones either to promote or inhibit shoot branching, depending on the auxin transport status of the plant. Consistent with this predicted mode of action, strigolactone signalling was found to trigger PIN1 depletion from the plasma membrane of xylem parenchyma cells in the stem. This effect could be detected within 10 minutes of strigolactone treatment and was independent of protein synthesis but dependent on clathrin-mediated membrane trafficking. Together these results support the hypothesis that growth across the plant shoot system is balanced by competition between shoot apices for a common auxin transport path to the root and that strigolactones regulate shoot branching by modulating this competition.

  4. Flux-based transport enhancement as a plausible unifying mechanism for auxin transport in meristem development.

    Directory of Open Access Journals (Sweden)

    Szymon Stoma

    2008-10-01

    Full Text Available Plants continuously generate new organs through the activity of populations of stem cells called meristems. The shoot apical meristem initiates leaves, flowers, and lateral meristems in highly ordered, spiralled, or whorled patterns via a process called phyllotaxis. It is commonly accepted that the active transport of the plant hormone auxin plays a major role in this process. Current hypotheses propose that cellular hormone transporters of the PIN family would create local auxin maxima at precise positions, which in turn would lead to organ initiation. To explain how auxin transporters could create hormone fluxes to distinct regions within the plant, different concepts have been proposed. A major hypothesis, canalization, proposes that the auxin transporters act by amplifying and stabilizing existing fluxes, which could be initiated, for example, by local diffusion. This convincingly explains the organised auxin fluxes during vein formation, but for the shoot apical meristem a second hypothesis was proposed, where the hormone would be systematically transported towards the areas with the highest concentrations. This implies the coexistence of two radically different mechanisms for PIN allocation in the membrane, one based on flux sensing and the other on local concentration sensing. Because these patterning processes require the interaction of hundreds of cells, it is impossible to estimate on a purely intuitive basis if a particular scenario is plausible or not. Therefore, computational modelling provides a powerful means to test this type of complex hypothesis. Here, using a dedicated computer simulation tool, we show that a flux-based polarization hypothesis is able to explain auxin transport at the shoot meristem as well, thus providing a unifying concept for the control of auxin distribution in the plant. Further experiments are now required to distinguish between flux-based polarization and other hypotheses.

  5. Molecular cloning and characterization of the genes encoding an auxin efflux carrier and the auxin influx carriers associated with the adventitious root formation in mango (Mangifera indica L.) cotyledon segments.

    Science.gov (United States)

    Li, Yun-He; Zou, Ming-Hong; Feng, Bi-Hong; Huang, Xia; Zhang, Zhi; Sun, Guang-Ming

    2012-06-01

    Polar auxin transport (PAT) plays an important role in the adventitious root formation of mango cotyledon segments, but the molecular mechanism remains unclear. In this study, we cloned a gene encoding an auxin efflux carrier (designated as MiPIN1), and we cloned four genes encoding auxin influx carriers (designated as MiAUX1, MiAUX2, MiAUX3 and MiAUX4). The results of a phylogenetic tree analysis indicated that MiPIN1 and the MiAUXs belong to plant PIN and AUXs/LAXs groups. Quantitative real-time PCR indicated that the expression of MiPIN1 and the MiAUXs was lowest at 0 days but sharply increased on and after day 4. During the root formation in the mango cotyledon segments, the MiPIN1 expression in the distal cut surface (DCS) was always higher than the expression in the proximal cut surface (PCS) whereas the expression of the MiAUXs in the PCS was usually higher than in the DCS. This expression pattern might be result in the PAT from the DCS to the PCS, which is essential for the adventitious root formation in the PCS. Our previous study indicated that a pre-treatment of embryos with indole-3-butyric acid (IBA) significantly promoted adventitious rooting in PCS whereas a pre-treatment with 2,3,5-triiodobenzoic acid (TIBA) completely inhibited this rooting. In this study, however, IBA and TIBA pre-treatments slightly changed the expression of MiPIN1. In contrast, while the MiAUX3 and MiAUX4 expression levels were significantly up-regulated by the IBA pre-treatment, the expression levels were down-regulated by the TIBA pre-treatment. These findings imply that MiAUX3 and MiAUX4 are more sensitive to the IBA and TIBA treatments and that they might play important roles during adventitious root formation in mango cotyledon segments. Copyright © 2012 Elsevier Masson SAS. All rights reserved.

  6. Reduced phototropism in pks mutants may be due to altered auxin-regulated gene expression or reduced lateral auxin transport.

    Science.gov (United States)

    Kami, Chitose; Allenbach, Laure; Zourelidou, Melina; Ljung, Karin; Schütz, Frédéric; Isono, Erika; Watahiki, Masaaki K; Yamamoto, Kotaro T; Schwechheimer, Claus; Fankhauser, Christian

    2014-02-01

    Phototropism allows plants to orient their photosynthetic organs towards the light. In Arabidopsis, phototropins 1 and 2 sense directional blue light such that phot1 triggers phototropism in response to low fluence rates, while both phot1 and phot2 mediate this response under higher light conditions. Phototropism results from asymmetric growth in the hypocotyl elongation zone that depends on an auxin gradient across the embryonic stem. How phototropin activation leads to this growth response is still poorly understood. Members of the phytochrome kinase substrate (PKS) family may act early in this pathway, because PKS1, PKS2 and PKS4 are needed for a normal phototropic response and they associate with phot1 in vivo. Here we show that PKS proteins are needed both for phot1- and phot2-mediated phototropism. The phototropic response is conditioned by the developmental asymmetry of dicotyledonous seedlings, such that there is a faster growth reorientation when cotyledons face away from the light compared with seedlings whose cotyledons face the light. The molecular basis for this developmental effect on phototropism is unknown; here we show that PKS proteins play a role at the interface between development and phototropism. Moreover, we present evidence for a role of PKS genes in hypocotyl gravi-reorientation that is independent of photoreceptors. pks mutants have normal levels of auxin and normal polar auxin transport, however they show altered expression patterns of auxin marker genes. This situation suggests that PKS proteins are involved in auxin signaling and/or lateral auxin redistribution.

  7. The Control of Auxin Transport in Parasitic and Symbiotic Root–Microbe Interactions

    Directory of Open Access Journals (Sweden)

    Jason Liang Pin Ng

    2015-08-01

    Full Text Available Most field-grown plants are surrounded by microbes, especially from the soil. Some of these, including bacteria, fungi and nematodes, specifically manipulate the growth and development of their plant hosts, primarily for the formation of structures housing the microbes in roots. These developmental processes require the correct localization of the phytohormone auxin, which is involved in the control of cell division, cell enlargement, organ development and defense, and is thus a likely target for microbes that infect and invade plants. Some microbes have the ability to directly synthesize auxin. Others produce specific signals that indirectly alter the accumulation of auxin in the plant by altering auxin transport. This review highlights root–microbe interactions in which auxin transport is known to be targeted by symbionts and parasites to manipulate the development of their host root system. We include case studies for parasitic root–nematode interactions, mycorrhizal symbioses as well as nitrogen fixing symbioses in actinorhizal and legume hosts. The mechanisms to achieve auxin transport control that have been studied in model organisms include the induction of plant flavonoids that indirectly alter auxin transport and the direct targeting of auxin transporters by nematode effectors. In most cases, detailed mechanisms of auxin transport control remain unknown.

  8. Alignment between PIN1 polarity and microtubule orientation in the shoot apical meristem reveals a tight coupling between morphogenesis and auxin transport.

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    Marcus G Heisler

    Full Text Available Morphogenesis during multicellular development is regulated by intercellular signaling molecules as well as by the mechanical properties of individual cells. In particular, normal patterns of organogenesis in plants require coordination between growth direction and growth magnitude. How this is achieved remains unclear. Here we show that in Arabidopsis thaliana, auxin patterning and cellular growth are linked through a correlated pattern of auxin efflux carrier localization and cortical microtubule orientation. Our experiments reveal that both PIN1 localization and microtubule array orientation are likely to respond to a shared upstream regulator that appears to be biomechanical in nature. Lastly, through mathematical modeling we show that such a biophysical coupling could mediate the feedback loop between auxin and its transport that underlies plant phyllotaxis.

  9. Role of auxin and protons in plant shoot gravitropism

    Science.gov (United States)

    Rayle, D. L.; Migliaccio, F.; Watson, E.

    1982-01-01

    Experiments designed to probe the relationship between asymmetric acid efflux and auxin redistribution during gravitropism are reported. Gravistimulation of sunflower hypocotyls results in the retardation of growth on the upper surface and the acceleration of growth on the lower surface relative to a vertically oriented control. Auxin and H(+) both elicit growth over a similarly broad region of the hypocotyl. The correspondence between auxin, H(+), and gravisensitive tissues is consistent with the notion that auxin redistribution may initiate asymmetric acid efflux during gravistimulation. Data are presented showing a redistribution of C-14-IAA and H-3-IAA occurs within 20-30 minutes of gravistimulation. Data on the effects of selected inhibitors of shoot gravitropism are also presented. Taken together, the data suggest that lateral transport of auxin initiates asymmetric acid efflux in gravitropically stimulated shoots.

  10. Revisiting Apoplastic Auxin Signaling Mediated by AUXIN BINDING PROTEIN 1.

    Science.gov (United States)

    Feng, Mingxiao; Kim, Jae-Yean

    2015-10-01

    It has been suggested that AUXIN BINDING PROTEIN 1 (ABP1) functions as an apoplastic auxin receptor, and is known to be involved in the post-transcriptional process, and largely independent of the already well-known SKP-cullin-F-box-transport inhibitor response (TIR1) /auxin signaling F-box (AFB) (SCF(TIR1/AFB)) pathway. In the past 10 years, several key components downstream of ABP1 have been reported. After perceiving the auxin signal, ABP1 interacts, directly or indirectly, with plasma membrane (PM)-localized transmembrane proteins, transmembrane kinase (TMK) or SPIKE1 (SPK1), or other unidentified proteins, which transfer the signal into the cell to the Rho of plants (ROP). ROPs interact with their effectors, such as the ROP interactive CRIB motif-containing protein (RIC), to regulate the endocytosis/exocytosis of the auxin efflux carrier PIN-FORMED (PIN) proteins to mediate polar auxin transport across the PM. Additionally, ABP1 is a negative regulator of the traditional SCF(TIR1/AFB) auxin signaling pathway. However, Gao et al. (2015) very recently reported that ABP1 is not a key component in auxin signaling, and the famous abp1-1 and abp1-5 mutant Arabidopsis lines are being called into question because of possible additional mutantion sites, making it necessary to reevaluate ABP1. In this review, we will provide a brief overview of the history of ABP1 research.

  11. Foliar modifications induced by inhibition of polar transport of auxin

    Institute of Scientific and Technical Information of China (English)

    NIDIAN; LINGJIANWANG; 等

    1999-01-01

    The effects of auxin polar transport inhibitors,9-hydroxy-fluorene-9-carboxylic acid (HFCA);2,3,5-triiodobenzoic acid(TIBA) and trans-cinnamic acid (CA) on leaf pattern formation were investigated with shoots formed from cultured leaf explants of tobacco and cultured pedicel explants of Orychophragmus violaceus,and the seedlings of tobacco and Brassica chinensis,Although the effective concentration varies with the inhibitors used,all of the inhibitors induced the formation of trumpet-shaped and/or fused leaves.The frequency of trumpet-shaped leaf formation was related to the concentration of inhibitors in the medium.Histological observation of tobacco seedlings showed that there was only one main vascular bundle and several minor vascular bundles in normal leaves of the control,but there were several vascular bundles of more or less the same size in the trumpet-shaped leaves of treated ones.These results indicated that auxin polar transport played an important role on bilateral symmetry of leaf growth.

  12. Activation of Big Grain1 significantly improves grain size by regulating auxin transport in rice.

    Science.gov (United States)

    Liu, Linchuan; Tong, Hongning; Xiao, Yunhua; Che, Ronghui; Xu, Fan; Hu, Bin; Liang, Chengzhen; Chu, Jinfang; Li, Jiayang; Chu, Chengcai

    2015-09-01

    Grain size is one of the key factors determining grain yield. However, it remains largely unknown how grain size is regulated by developmental signals. Here, we report the identification and characterization of a dominant mutant big grain1 (Bg1-D) that shows an extra-large grain phenotype from our rice T-DNA insertion population. Overexpression of BG1 leads to significantly increased grain size, and the severe lines exhibit obviously perturbed gravitropism. In addition, the mutant has increased sensitivities to both auxin and N-1-naphthylphthalamic acid, an auxin transport inhibitor, whereas knockdown of BG1 results in decreased sensitivities and smaller grains. Moreover, BG1 is specifically induced by auxin treatment, preferentially expresses in the vascular tissue of culms and young panicles, and encodes a novel membrane-localized protein, strongly suggesting its role in regulating auxin transport. Consistent with this finding, the mutant has increased auxin basipetal transport and altered auxin distribution, whereas the knockdown plants have decreased auxin transport. Manipulation of BG1 in both rice and Arabidopsis can enhance plant biomass, seed weight, and yield. Taking these data together, we identify a novel positive regulator of auxin response and transport in a crop plant and demonstrate its role in regulating grain size, thus illuminating a new strategy to improve plant productivity.

  13. Suitable experimental design for determination of auxin polar transport in space using a spacecraft.

    Science.gov (United States)

    Shimazu, T; Miyamoto, K; Hoson, T; Kamisaka, S; Ueda, J

    2000-03-01

    It is necessary to establish a suitable experimental design for the determination of auxin (indole-3-acetic acid: IAA) polar transport in space using a spacecraft in concerning with the role of gravity. Problems in space experiments are as follows: I) Selection of suitable plant species; II) Preservation of integrity of plant segments for activities of auxin polar transport; III) Stop of auxin polar transport of the segments after the transport experiment in space. Segments of etiolated pea epicotyls and etiolated maize coleoptiles showed relatively high activities of auxin polar transport among dicotyledonous and monocotyledonous plants tested, respectively. The activities decreased dramatically when the segments were pre-stored at 25 degrees C only for 1 day. On the other hand, the storage at low temperature (5 degrees C) in the presence of antioxidants or chelating agents, especially EGTA, maintained relatively high activities of auxin polar transport in pea epicotyl segments. Low temperature (5 degrees C) substantially inhibited the activity of auxin polar transport. Based on the results in this study, a suitable experimental design for the space experiment of auxin polar transport using a spacecraft is also proposed.

  14. Genetic and chemical reductions in protein phosphatase activity alter auxin transport, gravity response, and lateral root growth

    Science.gov (United States)

    Rashotte, A. M.; DeLong, A.; Muday, G. K.; Brown, C. S. (Principal Investigator)

    2001-01-01

    Auxin transport is required for important growth and developmental processes in plants, including gravity response and lateral root growth. Several lines of evidence suggest that reversible protein phosphorylation regulates auxin transport. Arabidopsis rcn1 mutant seedlings exhibit reduced protein phosphatase 2A activity and defects in differential cell elongation. Here we report that reduced phosphatase activity alters auxin transport and dependent physiological processes in the seedling root. Root basipetal transport was increased in rcn1 or phosphatase inhibitor-treated seedlings but showed normal sensitivity to the auxin transport inhibitor naphthylphthalamic acid (NPA). Phosphatase inhibition reduced root gravity response and delayed the establishment of differential auxin-induced gene expression across a gravity-stimulated root tip. An NPA treatment that reduced basipetal transport in rcn1 and cantharidin-treated wild-type plants also restored a normal gravity response and asymmetric auxin-induced gene expression, indicating that increased basipetal auxin transport impedes gravitropism. Increased auxin transport in rcn1 or phosphatase inhibitor-treated seedlings did not require the AGR1/EIR1/PIN2/WAV6 or AUX1 gene products. In contrast to basipetal transport, root acropetal transport was normal in phosphatase-inhibited seedlings in the absence of NPA, although it showed reduced NPA sensitivity. Lateral root growth also exhibited reduced NPA sensitivity in rcn1 seedlings, consistent with acropetal transport controlling lateral root growth. These results support the role of protein phosphorylation in regulating auxin transport and suggest that the acropetal and basipetal auxin transport streams are differentially regulated.

  15. Aluminium toxicity targets PIN2 in Arabidopsis root apices: Effects on PIN2 endocytosis, vesicular recycling,and polar auxin transport

    Institute of Scientific and Technical Information of China (English)

    SHEN Hong; HOU NingYan; Markus SCHLICHT; WAN YingLang; Stefano MANCUSO; Frantisek BALUSKA

    2008-01-01

    The most obvious symptom of AI toxicity is the inhibition of root growth.However,the mechanism of AI-inhibiting root growth remains to be elucidated.In this study,auxin transport and vesicle movement of an auxin-efflux carrier (PIN2) were investigated in Arabidopsis roots in response to AI stress.Results indicated that AI inhibited the apical transport of auxin in root tips of Arabidopsis significantly.The severe inhibition was localized in the cells of transition zone,where the concentration of auxin was only 34% that of the control.Brefeldin A (BFA),an inhibitor of vesicle transport,induced the dot-like structure of PIN2 vesicle significantly.Al decreased the size of dot-like structure of PIN2 vesicles.Re-sults of real-time RT-PCR and Western-blotting analysis showed that Al increased the transcript level of PIN2 and the accumulation of PIN2 protein in horizontal direction of plasma membrane,but decreased its distribution in endosomes,suggesting that AI inhibited the transport of PIN2 vesicles from plasma membrane to endosomes.Results of cytoskeleton-depolymering drugs indicated that it was via the pathway of disruption of actin microfilaments that AI inhibited the transport of PIN2 vesicles.Exposed to AI stress,the cells of elongation zone had less AI uptake and less transport frequency of vesicles than cells of transition zone.Taken together,our results suggested that AI inhibited root growth mainly by modulating the transport of PIN2 vesicles between plasma membrane and endosomes,thus block-ing auxin transport and root growth.

  16. SAUR39, a small auxin-up RNA gene, acts as a negative regulator of auxin synthesis and transport in rice.

    Science.gov (United States)

    Kant, Surya; Bi, Yong-Mei; Zhu, Tong; Rothstein, Steven J

    2009-10-01

    The phytohormone auxin plays a critical role for plant growth by regulating the expression of a set of genes. One large auxin-responsive gene family of this type is the small auxin-up RNA (SAUR) genes, although their function is largely unknown. The expression of the rice (Oryza sativa) SAUR39 gene showed rapid induction by transient change in different environmental factors, including auxin, nitrogen, salinity, cytokinin, and anoxia. Transgenic rice plants overexpressing the SAUR39 gene resulted in lower shoot and root growth, altered shoot morphology, smaller vascular tissue, and lower yield compared with wild-type plants. The SAUR39 gene was expressed at higher levels in older leaves, unlike auxin biosynthesis, which occurs largely in the meristematic region. The transgenic plants had a lower auxin level and a reduced polar auxin transport as well as the down-regulation of some putative auxin biosynthesis and transporter genes. Biochemical analysis also revealed that transgenic plants had lower chlorophyll content, higher levels of anthocyanin, abscisic acid, sugar, and starch, and faster leaf senescence compared with wild-type plants at the vegetative stage. Most of these phenomena have been shown to be negatively correlated with auxin level and transport. Transcript profiling revealed that metabolic perturbations in overexpresser plants were largely due to transcriptional changes of genes involved in photosynthesis, senescence, chlorophyll production, anthocyanin accumulation, sugar synthesis, and transport. The lower growth and yield of overexpresser plants was largely recovered by exogenous auxin application. Taken together, the results suggest that SAUR39 acts as a negative regulator for auxin synthesis and transport.

  17. Localized auxin peaks in concentration-based transport models of the shoot apical meristem.

    Science.gov (United States)

    Draelants, Delphine; Avitabile, Daniele; Vanroose, Wim

    2015-05-06

    We study the formation of auxin peaks in a generic class of concentration-based auxin transport models, posed on static plant tissues. Using standard asymptotic analysis, we prove that, on bounded domains, auxin peaks are not formed via a Turing instability in the active transport parameter, but via simple corrections to the homogeneous steady state. When the active transport is small, the geometry of the tissue encodes the peaks' amplitude and location: peaks arise where cells have fewer neighbours, that is, at the boundary of the domain. We test our theory and perform numerical bifurcation analysis on two models that are known to generate auxin patterns for biologically plausible parameter values. In the same parameter regimes, we find that realistic tissues are capable of generating a multitude of stationary patterns, with a variable number of auxin peaks, that can be selected by different initial conditions or by quasi-static changes in the active transport parameter. The competition between active transport and production rate determines whether peaks remain localized or cover the entire domain. In particular, changes in the auxin production that are fast with respect to the cellular life cycle affect the auxin peak distribution, switching from localized spots to fully patterned states. We relate the occurrence of localized patterns to a snaking bifurcation structure, which is known to arise in a wide variety of nonlinear media, but has not yet been reported in plant models.

  18. Non-invasive quantification of endogenous root auxin transport using an integrated flux microsensor technique.

    Science.gov (United States)

    McLamore, Eric S; Diggs, Alfred; Calvo Marzal, Percy; Shi, Jin; Blakeslee, Joshua J; Peer, Wendy A; Murphy, Angus S; Porterfield, D Marshall

    2010-09-01

    Indole-3-acetic acid (IAA) is a primary phytohormone that regulates multiple aspects of plant development. Because polar transport of IAA is an essential determinant of organogenesis and dynamic tropic growth, methods to monitor IAA movement in vivo are in demand. A self-referencing electrochemical microsensor was optimized to non-invasively measure endogenous IAA flux near the surface of Zea mays roots without the addition of exogenous IAA. Enhanced sensor surface modification, decoupling of acquired signals, and integrated flux analyses were combined to provide direct, real time quantification of endogenous IAA movement in B73 maize inbred and brachytic2 (br2) auxin transport mutant roots. BR2 is localized in epidermal and hypodermal tissues at the root apex. br2 roots exhibit reduced shootward IAA transport at the root apex in radiotracer experiments and reduced gravitropic growth. IAA flux data indicates that maximal transport occurs in the distal elongation zone of maize roots, and net transport in/out of br2 roots was decreased compared to B73. Integration of short term real time flux data in this zone revealed oscillatory patterns, with B73 exhibiting shorter oscillatory periods and greater amplitude than br2. IAA efflux and influx were inhibited using 1-N-naphthylphthalamic acid (NPA), and 2-naphthoxyacetic acid (NOA), respectively. A simple harmonic oscillation model of these data produced a correlation between modeled and measured values of 0.70 for B73 and 0.69 for br2. These results indicate that this technique is useful for real-time IAA transport monitoring in surface tissues and that this approach can be performed simultaneously with current live imaging techniques.

  19. Simulation of organ patterning on the floral meristem using a polar auxin transport model.

    Directory of Open Access Journals (Sweden)

    Simon van Mourik

    Full Text Available An intriguing phenomenon in plant development is the timing and positioning of lateral organ initiation, which is a fundamental aspect of plant architecture. Although important progress has been made in elucidating the role of auxin transport in the vegetative shoot to explain the phyllotaxis of leaf formation in a spiral fashion, a model study of the role of auxin transport in whorled organ patterning in the expanding floral meristem is not available yet. We present an initial simulation approach to study the mechanisms that are expected to play an important role. Starting point is a confocal imaging study of Arabidopsis floral meristems at consecutive time points during flower development. These images reveal auxin accumulation patterns at the positions of the organs, which strongly suggests that the role of auxin in the floral meristem is similar to the role it plays in the shoot apical meristem. This is the basis for a simulation study of auxin transport through a growing floral meristem, which may answer the question whether auxin transport can in itself be responsible for the typical whorled floral pattern. We combined a cellular growth model for the meristem with a polar auxin transport model. The model predicts that sepals are initiated by auxin maxima arising early during meristem outgrowth. These form a pre-pattern relative to which a series of smaller auxin maxima are positioned, which partially overlap with the anlagen of petals, stamens, and carpels. We adjusted the model parameters corresponding to properties of floral mutants and found that the model predictions agree with the observed mutant patterns. The predicted timing of the primordia outgrowth and the timing and positioning of the sepal primordia show remarkable similarities with a developing flower in nature.

  20. Auxin polar transport in arabidopsis under simulated microgravity conditions - relevance to growth and development

    Science.gov (United States)

    Miyamoto, K.; Oka, M.; Yamamoto, R.; Masuda, Y.; Hoson, T.; Kamisaka, S.; Ueda, J.

    1999-01-01

    Activity of auxin polar transport in inflorescence axes of Arabidopsis thaliana grown under simulated microgravity conditions was studied in relation to the growth and development. Seeds were germinated and allowed to grow on an agar medium in test tubes on a horizontal clinostat. Horizontal clinostat rotation substantially reduced the growth of inflorescence axes and the productivity of seeds of Arabidopsis thaliana (ecotypes Landsberg erecta and Columbia), although it little affected seed germination, development of rosette leaves and flowering. The activity of auxin polar transport in inflorescence axes decreased when Arabidopsis plants were grown on a horizontal clinostat from germination stage, being ca. 60% of 1 g control. On the other hand, the auxin polar transport in inflorescence axes of Arabidopsis grown in 1 g conditions was not affected when the segments were exposed to various gravistimuli, including 3-dimensional clinorotation, during transport experiments. Pin-formed mutant of Arabidopsis, having a unique structure of the inflorescence axis with no flower and extremely low levels of the activity of auxin polar transport in inflorescence axes and endogenous auxin, did not continue its vegetative growth under clinostat rotation. These facts suggest that the development of the system of auxin polar transport in Arabidopsis is affected by microgravity, resulting in the inhibition of growth and development, especially during reproductive growth.

  1. AUX/LAX family of auxin influx carriers-an overview

    Directory of Open Access Journals (Sweden)

    Ranjan eSwarup

    2012-10-01

    Full Text Available Auxin regulates several aspects of plant growth and development. Auxin is unique among plant hormones for exhibiting polar transport. Indole-3-acetic acid, the major form of auxin in higher plants, is a weak acid and its intercellular movement is facilitated by auxin influx and efflux carriers.. Polarity of auxin movement is provided by asymmetric localisation of auxin carriers (mainly PIN efflux carriers. PIN-FORMED (PIN and P-GLYCOPROTEIN (PGP family of proteins are major auxin efflux carriers whereas AUXIN1/LIKE-AUX1 (AUX/LAX are major auxin influx carriers.. Genetic and biochemical evidence show that each member of the AUX/LAX family is a functional auxin influx carrier and mediate auxin related developmental programmes in different organs and tissues. Of the four AUX/LAX genes, AUX1 regulates root gravitropism, root hair development and leaf phyllotaxy whereas LAX2 regulates vascular development in cotyledons. Both AUX1 and LAX3 have been implicated in lateral root development as well as apical hook formation whereas both AUX1 and LAX1 and possibly LAX2 are required for leaf phyllotactic patterning.

  2. The auxin-resistant diageotropica mutant of tomato responds to gravity via an auxin-mediated pathway

    Science.gov (United States)

    Rice, M. S.; Lomax, T. L.

    2000-01-01

    Hypocotyls of the diageotropica (dgt) mutant of tomato (Lycopersicon esculentum Mill.) do not elongate in response to exogenous auxin, but can respond to gravity. This appears paradoxical in light of the Cholodny-Went hypothesis, which states that shoot gravicurvature results from asymmetric stimulation of elongation by auxin. While light-grown dgt seedlings can achieve correct gravitropic reorientation, the response is slow compared to wild-type seedlings. The sensitivity of dgt seedlings to inhibition of gravicurvature by immersion in auxin or auxin-transport inhibitors is similar to that of wild-type plants, indicating that both an auxin gradient and auxin transport are required for the gravitropic response and that auxin uptake, efflux, and at least one auxin receptor are functional in dgt. Furthermore, dgt gravicurvature is the result of asymmetrically increased elongation as would be expected for an auxin-mediated response. Our results suggest differences between elongation in response to exogenous auxin (absent in dgt) and elongation in response to gravistimulation (present but attenuated in dgt) and confirm the presence of two phases during the gravitropic response, both of which are dependent on functional auxin transport.

  3. Redirection of auxin flow in Arabidopsis thaliana roots after infection by root-knot nematodes

    NARCIS (Netherlands)

    Kyndt, Tina; Goverse, Aska; Haegeman, Annelies; Warmerdam, Sonja; Wanjau, Cecilia; Jahani, Mona; Engler, Gilbert; Almeida Engler, De Janice; Gheysen, Godelieve

    2016-01-01

    Plant-parasitic root-knot nematodes induce the formation of giant cells within the plant root, and it has been recognized that auxin accumulates in these feeding sites. Here, we studied the role of the auxin transport system governed by AUX1/LAX3 influx proteins and different PIN efflux proteins dur

  4. ERECTA family genes regulate auxin transport in the shoot apical meristem and forming leaf primordia.

    Science.gov (United States)

    Chen, Ming-Kun; Wilson, Rebecca L; Palme, Klaus; Ditengou, Franck Anicet; Shpak, Elena D

    2013-08-01

    Leaves are produced postembryonically at the flanks of the shoot apical meristem. Their initiation is induced by a positive feedback loop between auxin and its transporter PIN-FORMED1 (PIN1). The expression and polarity of PIN1 in the shoot apical meristem is thought to be regulated primarily by auxin concentration and flow. The formation of an auxin maximum in the L1 layer of the meristem is the first sign of leaf initiation and is promptly followed by auxin flow into the inner tissues, formation of the midvein, and appearance of the primordium bulge. The ERECTA family genes (ERfs) encode leucine-rich repeat receptor-like kinases, and in Arabidopsis (Arabidopsis thaliana), this gene family consists of ERECTA (ER), ERECTA-LIKE1 (ERL1), and ERL2. Here, we show that ERfs regulate auxin transport during leaf initiation. The shoot apical meristem of the er erl1 erl2 triple mutant produces leaf primordia at a significantly reduced rate and with altered phyllotaxy. This phenotype is likely due to deficiencies in auxin transport in the shoot apex, as judged by altered expression of PIN1, the auxin reporter DR5rev::GFP, and the auxin-inducible genes MONOPTEROS, INDOLE-3-ACETIC ACID INDUCIBLE1 (IAA1), and IAA19. In er erl1 erl2, auxin presumably accumulates in the L1 layer of the meristem, unable to flow into the vasculature of a hypocotyl. Our data demonstrate that ERfs are essential for PIN1 expression in the forming midvein of future leaf primordia and in the vasculature of emerging leaves.

  5. Transcript profiles of auxin efflux carrier and IAA-amido synthetase genes suggest the role of auxin on apple (Malus x domestica) fruit maturation patterns

    Science.gov (United States)

    Plant growth regulators are known to regulate fruit development at various stages including flowering, fruit growth, maturation and ripening. Recent transcriptome studies indicated that auxin might play an essential role in regulating apple fruit maturation and ripening beside the well-defined role...

  6. Genome-wide identification and expression profiling analysis of ZmPIN, ZmPILS, ZmLAX and ZmABCB auxin transporter gene families in maize (Zea mays L. under various abiotic stresses.

    Directory of Open Access Journals (Sweden)

    Runqing Yue

    Full Text Available The auxin influx carriers auxin resistant 1/like aux 1 (AUX/LAX, efflux carriers pin-formed (PIN (together with PIN-like proteins and efflux/conditional P-glycoprotein (ABCB are major protein families involved in auxin polar transport. However, how they function in responses to exogenous auxin and abiotic stresses in maize is largely unknown. In this work, the latest updated maize (Zea mays L. reference genome sequence was used to characterize and analyze the ZmLAX, ZmPIN, ZmPILS and ZmABCB family genes from maize. The results showed that five ZmLAXs, fifteen ZmPINs, nine ZmPILSs and thirty-five ZmABCBs were mapped on all ten maize chromosomes. Highly diversified gene structures, nonconservative transmembrane helices and tissue-specific expression patterns suggested the possibility of function diversification for these genes. Quantitative real-time polymerase chain reaction (qRT-PCR was used to analyze the expression patterns of ZmLAX, ZmPIN, ZmPILS and ZmABCB genes under exogenous auxin and different environmental stresses. The expression levels of most ZmPIN, ZmPILS, ZmLAX and ZmABCB genes were induced in shoots and were reduced in roots by various abiotic stresses (drought, salt and cold stresses. The opposite expression response patterns indicated the dynamic auxin transport between shoots and roots under abiotic stresses. Analysis of the expression patterns of ZmPIN, ZmPILS, ZmLAX and ZmABCB genes under drought, salt and cold treatment may help us to understand the possible roles of maize auxin transporter genes in responses and tolerance to environmental stresses.

  7. MAB4-induced auxin sink generates local auxin gradients in Arabidopsis organ formation.

    Science.gov (United States)

    Furutani, Masahiko; Nakano, Yasukazu; Tasaka, Masao

    2014-01-21

    In Arabidopsis, leaves and flowers form cyclically in the shoot meristem periphery and are triggered by local accumulations of the plant hormone auxin. Auxin maxima are established by the auxin efflux carrier PIN-formed1 (PIN1). During organ formation, two distinct types of PIN1 polarization occur. First, convergence of PIN1 polarity in the surface of the meristem creates local auxin peaks. Second, basipetal PIN1 polarization causes auxin to move away from the surface in the middle of an incipient organ primordium, thought to contribute to vascular formation. Several mathematical models have been developed in attempts to explain the PIN1 localization pattern. However, the molecular mechanisms that control these dynamic changes are unknown. Here, we show that loss-of-function in the MACCHI-BOU 4 (MAB4) family genes, which encode nonphototropic hypocotyl 3-like proteins and regulate PIN endocytosis, cause deletion of basipetal PIN1 polarization, resulting in extensive auxin accumulation all over the meristem surface from lack of a sink for auxin. These results indicate that the MAB4 family genes establish inward auxin transport from the L1 surface of incipient organ primordia by basipetal PIN1 polarization, and that this behavior is essential for the progression of organ development. Furthermore, the expression of the MAB4 family genes depends on auxin response. Our results define two distinct molecular mechanisms for PIN1 polarization during organ development and indicate that an auxin response triggers the switching between these two mechanisms.

  8. Auxin-responsive DR5 promoter coupled with transport assays suggest separate but linked routes of auxin transport during woody stem development in Populus.

    Science.gov (United States)

    Spicer, Rachel; Tisdale-Orr, Tracy; Talavera, Christian

    2013-01-01

    Polar auxin transport (PAT) is a major determinant of plant morphology and internal anatomy with important roles in vascular patterning, tropic growth responses, apical dominance and phyllotactic arrangement. Woody plants present a highly complex system of vascular development in which isolated bundles of xylem and phloem gradually unite to form concentric rings of conductive tissue. We generated several transgenic lines of hybrid poplar (Populus tremula x alba) with the auxin-responsive DR5 promoter driving GUS expression in order to visualize an auxin response during the establishment of secondary growth. Distinct GUS expression in the cambial zone and developing xylem-side derivatives supports the current view of this tissue as a major stream of basipetal PAT. However, we also found novel sites of GUS expression in the primary xylem parenchyma lining the outer perimeter of the pith. Strands of primary xylem parenchyma depart the stem as a leaf trace, and showed GUS expression as long as the leaves to which they were connected remained attached (i.e., until just prior to leaf abscission). Tissue composed of primary xylem parenchyma strands contained measurable levels of free indole-3-acetic acid (IAA) and showed basipetal transport of radiolabeled auxin ((3)H-IAA) that was both significantly faster than diffusion and highly sensitive to the PAT inhibitor NPA. Radiolabeled auxin was also able to move between the primary xylem parenchyma in the interior of the stem and the basipetal stream in the cambial zone, an exchange that was likely mediated by ray parenchyma cells. Our results suggest that (a) channeling of leaf-derived IAA first delineates isolated strands of pre-procambial tissue but then later shifts to include basipetal transport through the rapidly expanding xylem elements, and (b) the transition from primary to secondary vascular development is gradual, with an auxin response preceding the appearance of a unified and radially-organized vascular cambium.

  9. Flavonoids and Auxin Transport Inhibitors Rescue Symbiotic Nodulation in the Medicago truncatula Cytokinin Perception Mutant cre1.

    Science.gov (United States)

    Ng, Jason Liang Pin; Hassan, Samira; Truong, Thy T; Hocart, Charles H; Laffont, Carole; Frugier, Florian; Mathesius, Ulrike

    2015-08-01

    Initiation of symbiotic nodules in legumes requires cytokinin signaling, but its mechanism of action is largely unknown. Here, we tested whether the failure to initiate nodules in the Medicago truncatula cytokinin perception mutant cre1 (cytokinin response1) is due to its altered ability to regulate auxin transport, auxin accumulation, and induction of flavonoids. We found that in the cre1 mutant, symbiotic rhizobia cannot locally alter acro- and basipetal auxin transport during nodule initiation and that these mutants show reduced auxin (indole-3-acetic acid) accumulation and auxin responses compared with the wild type. Quantification of flavonoids, which can act as endogenous auxin transport inhibitors, showed a deficiency in the induction of free naringenin, isoliquiritigenin, quercetin, and hesperetin in cre1 roots compared with wild-type roots 24 h after inoculation with rhizobia. Coinoculation of roots with rhizobia and the flavonoids naringenin, isoliquiritigenin, and kaempferol, or with the synthetic auxin transport inhibitor 2,3,5,-triiodobenzoic acid, rescued nodulation efficiency in cre1 mutants and allowed auxin transport control in response to rhizobia. Our results suggest that CRE1-dependent cytokinin signaling leads to nodule initiation through the regulation of flavonoid accumulation required for local alteration of polar auxin transport and subsequent auxin accumulation in cortical cells during the early stages of nodulation.

  10. Ethylene regulates root growth through effects on auxin biosynthesis and transport-dependent auxin distribution

    OpenAIRE

    2007-01-01

    In plants, each developmental process integrates a network of signaling events that are regulated by different phytohormones, and interactions among hormonal pathways are essential to modulate their effect. Continuous growth of roots results from the postembryonic activity of cells within the root meristem that is controlled by the coordinated action of several phytohormones, including auxin and ethylene. Although their interaction has been studied intensively, the molecular and cellular mech...

  11. Regulation of Auxin Transport by Phosphorylation and Flavonoids during Gravitropism in Arabidopsis

    Science.gov (United States)

    Muday, Gloria K.

    2005-01-01

    The focus of this research includes: 1) Regulation of Axin transport by flavonoids during gravitropism; 2) Phosphorylation control of auxin transport during gravity response; 3) Ethylene regulation of gravitropic curvature; 4) IBA transport and gravitropic response; and 5) Other collaborative projects.

  12. Extracellular ATP inhibits root gravitropism at concentrations that inhibit polar auxin transport

    Science.gov (United States)

    Tang, Wenqiang; Brady, Shari R.; Sun, Yu; Muday, Gloria K.; Roux, Stanley J.

    2003-01-01

    Raising the level of extracellular ATP to mM concentrations similar to those found inside cells can block gravitropism of Arabidopsis roots. When plants are grown in Murashige and Skoog medium supplied with 1 mM ATP, their roots grow horizontally instead of growing straight down. Medium with 2 mM ATP induces root curling, and 3 mM ATP stimulates lateral root growth. When plants are transferred to medium containing exogenous ATP, the gravity response is reduced or in some cases completely blocked by ATP. Equivalent concentrations of ADP or inorganic phosphate have slight but usually statistically insignificant effects, suggesting the specificity of ATP in these responses. The ATP effects may be attributable to the disturbance of auxin distribution in roots by exogenously applied ATP, because extracellular ATP can alter the pattern of auxin-induced gene expression in DR5-beta-glucuronidase transgenic plants and increase the response sensitivity of plant roots to exogenously added auxin. The presence of extracellular ATP also decreases basipetal auxin transport in a dose-dependent fashion in both maize (Zea mays) and Arabidopsis roots and increases the retention of [(3)H]indole-3-acetic acid in root tips of maize. Taken together, these results suggest that the inhibitory effects of extracellular ATP on auxin distribution may happen at the level of auxin export. The potential role of the trans-plasma membrane ATP gradient in auxin export and plant root gravitropism is discussed.

  13. Brassinosteroids stimulate plant tropisms through modulation of polar auxin transport in Brassica and Arabidopsis.

    Science.gov (United States)

    Li, Li; Xu, Jian; Xu, Zhi-Hong; Xue, Hong-Wei

    2005-10-01

    Brassinosteroids (BRs) are important plant growth regulators in multiple developmental processes. Previous studies have indicated that BR treatment enhanced auxin-related responses, but the underlying mechanisms remain unknown. Using (14)C-labeled indole-3-acetic acid and Arabidopsis thaliana plants harboring an auxin-responsive reporter construct, we show that the BR brassinolide (BL) stimulates polar auxin transport capacities and modifies the distribution of endogenous auxin. In plants treated with BL or defective in BR biosynthesis or signaling, the transcription of PIN genes, which facilitate functional auxin transport in plants, was differentially regulated. In addition, BL enhanced plant tropistic responses by promoting the accumulation of the PIN2 protein from the root tip to the elongation zone and stimulating the expression and dispersed localization of ROP2 during tropistic responses. Constitutive overexpression of ROP2 results in enhanced polar accumulation of PIN2 protein in the root elongation region and increased gravitropism, which is significantly affected by latrunculin B, an inhibitor of F-actin assembly. The ROP2 dominant negative mutants (35S-ROP2-DA/DN) show delayed tropistic responses, and this delay cannot be reversed by BL addition, strongly supporting the idea that ROP2 modulates the functional localization of PIN2 through regulation of the assembly/reassembly of F-actins, thereby mediating the BR effects on polar auxin transport and tropistic responses.

  14. Bioassay for investigation of auxin transport in single cell layers

    Directory of Open Access Journals (Sweden)

    Alina B. Wodzicki

    2014-02-01

    Full Text Available Auxin was collected from the cambial region of Pinus sylvestris by applying agar strips to the cut surfaces of stem sections which comprised a single layer of 2 to 4-mm long, mainly intact fusiform cells. Sections of the agar strips were either bioassayed immediately to determine their auxin content or stored for several months at -80oC, extracted with 80% MeOH and redissolved in hot agar prior to bioassay. Auxin concentrations were determined by Went's oat coleoptile test, as described by Funke, which was modified considerably to give highly reproducible results. The modifications proved essential for good replication of results and are described in detail together with the use of the bioassay to determine changes in cambial cell polarity during ageing and senescence in P. sylvestris.

  15. Auxin physiology of the tomato mutant diageotropica

    Science.gov (United States)

    Daniel, S. G.; Rayle, D. L.; Cleland, R. E.

    1989-01-01

    The tomato (Lycopersicon esculentum, Mill.) mutant diageotropica (dgt) exhibits biochemical, physiological, and morphological abnormalities that suggest the mutation may have affected a primary site of auxin perception or action. We have compared two aspects of the auxin physiology of dgt and wild-type (VFN8) seedlings: auxin transport and cellular growth parameters. The rates of basipetal indole-3-acetic acid (IAA) polar transport are identical in hypocotyl sections of the two genotypes, but dgt sections have a slightly greater capacity for IAA transport. 2,3,5-Triiodobenzoic acid and ethylene reduce transport in both mutant and wild-type sections. The kinetics of auxin uptake into VFN8 and dgt sections are nearly identical. These results make it unlikely that an altered IAA efflux carrier or IAA uptake symport are responsible for the pleiotropic effects resulting from the dgt mutation. The lack of auxin-induced cell elongation in dgt plants is not due to insufficient turgor, as the osmotic potential of dgt cell sap is less (more negative) than that of VFN8. An auxin-induced increase in wall extensibility, as measured by the Instron technique, only occurs in the VFN8 plants. These data suggest dgt hypocotyls suffer a defect in the sequence of events culminating in auxin-induced cell wall loosening.

  16. Action of Abscisic Acid on Auxin Transport and its Relation to Phototropism

    DEFF Research Database (Denmark)

    Naqvi, S. M.; Engvild, Kjeld Christensen

    1974-01-01

    The action of abscisic acid on the kinetics of auxin transport through Zea mays L. (cv. Goudster) coleoptiles has been investigated. Abscisic acid applied simultaneously with indoleacetic acid-2-14C in the donor block reduced the transport intensity without materially affecting the basipetal...

  17. Auxin influx carriers control vascular patterning and xylem differentiation in Arabidopsis thaliana.

    Directory of Open Access Journals (Sweden)

    Norma Fàbregas

    2015-04-01

    Full Text Available Auxin is an essential hormone for plant growth and development. Auxin influx carriers AUX1/LAX transport auxin into the cell, while auxin efflux carriers PIN pump it out of the cell. It is well established that efflux carriers play an important role in the shoot vascular patterning, yet the contribution of influx carriers to the shoot vasculature remains unknown. Here, we combined theoretical and experimental approaches to decipher the role of auxin influx carriers in the patterning and differentiation of vascular tissues in the Arabidopsis inflorescence stem. Our theoretical analysis predicts that influx carriers facilitate periodic patterning and modulate the periodicity of auxin maxima. In agreement, we observed fewer and more spaced vascular bundles in quadruple mutants plants of the auxin influx carriers aux1lax1lax2lax3. Furthermore, we show AUX1/LAX carriers promote xylem differentiation in both the shoot and the root tissues. Influx carriers increase cytoplasmic auxin signaling, and thereby differentiation. In addition to this cytoplasmic role of auxin, our computational simulations propose a role for extracellular auxin as an inhibitor of xylem differentiation. Altogether, our study shows that auxin influx carriers AUX1/LAX regulate vascular patterning and differentiation in plants.

  18. Auxin influx carriers control vascular patterning and xylem differentiation in Arabidopsis thaliana.

    Directory of Open Access Journals (Sweden)

    Norma Fàbregas

    2015-04-01

    Full Text Available Auxin is an essential hormone for plant growth and development. Auxin influx carriers AUX1/LAX transport auxin into the cell, while auxin efflux carriers PIN pump it out of the cell. It is well established that efflux carriers play an important role in the shoot vascular patterning, yet the contribution of influx carriers to the shoot vasculature remains unknown. Here, we combined theoretical and experimental approaches to decipher the role of auxin influx carriers in the patterning and differentiation of vascular tissues in the Arabidopsis inflorescence stem. Our theoretical analysis predicts that influx carriers facilitate periodic patterning and modulate the periodicity of auxin maxima. In agreement, we observed fewer and more spaced vascular bundles in quadruple mutants plants of the auxin influx carriers aux1lax1lax2lax3. Furthermore, we show AUX1/LAX carriers promote xylem differentiation in both the shoot and the root tissues. Influx carriers increase cytoplasmic auxin signaling, and thereby differentiation. In addition to this cytoplasmic role of auxin, our computational simulations propose a role for extracellular auxin as an inhibitor of xylem differentiation. Altogether, our study shows that auxin influx carriers AUX1/LAX regulate vascular patterning and differentiation in plants.

  19. Separating the roles of acropetal and basipetal auxin transport on gravitropism with mutations in two Arabidopsis multidrug resistance-like ABC transporter genes.

    Science.gov (United States)

    Lewis, Daniel R; Miller, Nathan D; Splitt, Bessie L; Wu, Guosheng; Spalding, Edgar P

    2007-06-01

    Two Arabidopsis thaliana ABC transporter genes linked to auxin transport by various previous results were studied in a reverse-genetic fashion. Mutations in Multidrug Resistance-Like1 (MDR1) reduced acropetal auxin transport in roots by 80% without affecting basipetal transport. Conversely, mutations in MDR4 blocked 50% of basipetal transport without affecting acropetal transport. Developmental and auxin distribution phenotypes associated with these altered auxin flows were studied with a high-resolution morphometric system and confocal microscopy, respectively. Vertically grown mdr1 roots produced positive and negative curvatures threefold greater than the wild type, possibly due to abnormal auxin distribution observed in the elongation zone. However, upon 90 degrees reorientation, mdr1 gravitropism was inseparable from the wild type. Thus, acropetal auxin transport maintains straight growth but contributes surprisingly little to gravitropism. Conversely, vertically maintained mdr4 roots grew as straight as the wild type, but their gravitropism was enhanced. Upon reorientation, curvature in this mutant developed faster, was distributed more basally, and produced a greater total angle than the wild type. An amplified auxin asymmetry may explain the mdr4 hypertropism. Double mutant analysis indicated that the two auxin transport streams are more independent than interdependent. The hypothesis that flavanols regulate MDR-dependent auxin transport was supported by the epistatic relationship of mdr4 to the tt4 phenylpropanoid pathway mutation.

  20. Model of polar auxin transport coupled to mechanical forces retrieves robust morphogenesis along the Arabidopsis root

    Science.gov (United States)

    Romero-Arias, J. Roberto; Hernández-Hernández, Valeria; Benítez, Mariana; Alvarez-Buylla, Elena R.; Barrio, Rafael A.

    2017-03-01

    Stem cells are identical in many scales, they share the same molecular composition, DNA, genes, and genetic networks, yet they should acquire different properties to form a functional tissue. Therefore, they must interact and get some external information from their environment, either spatial (dynamical fields) or temporal (lineage). In this paper we test to what extent coupled chemical and physical fields can underlie the cell's positional information during development. We choose the root apical meristem of Arabidopsis thaliana to model the emergence of cellular patterns. We built a model to study the dynamics and interactions between the cell divisions, the local auxin concentration, and physical elastic fields. Our model recovers important aspects of the self-organized and resilient behavior of the observed cellular patterns in the Arabidopsis root, in particular, the reverse fountain pattern observed in the auxin transport, the PIN-FORMED (protein family of auxin transporters) polarization pattern and the accumulation of auxin near the region of maximum curvature in a bent root. Our model may be extended to predict altered cellular patterns that are expected under various applied auxin treatments or modified physical growth conditions.

  1. Quercetin Promotes Auxin Transport in Arabidopsis thaliana%槲皮素促进拟南芥生长素极性运输的研究

    Institute of Scientific and Technical Information of China (English)

    高静; 黄华孙; 程汉

    2008-01-01

    Study on the role of quercentin in polar auxin transportation. Arabidopsis was cultured on medium supplemented with quercetin to observe the growth of hypocotyls, 14C-IAA transport assays were conducted to measure the auxin transport activity. The results showed that Arabidopsis mutant auxl which had been deficient in auxin influx transportion obviously recovered the ability after cultured on the medium with quercetin. The polar auxin transport was promoted by the addition of quercetin. These results indicated that quereetin could promote polar auxin transport in vivo.

  2. Overexpression of 3β-hydroxysteroid dehydrogenases/C-4 decarboxylases causes growth defects possibly due to abnormal auxin transport in Arabidopsis.

    Science.gov (United States)

    Kim, Bokyung; Kim, Gyusik; Fujioka, Shozo; Takatsuto, Suguru; Choe, Sunghwa

    2012-07-01

    Sterols play crucial roles as membrane components and precursors of steroid hormones (e.g., brassinosteroids, BR). Within membranes, sterols regulate membrane permeability and fluidity by interacting with other lipids and proteins. Sterols are frequently enriched in detergent-insoluble membranes (DIMs), which organize molecules involved in specialized signaling processes, including auxin transporters. To be fully functional, the two methyl groups at the C-4 position of cycloartenol, a precursor of plant sterols, must be removed by bifunctional 3β-hydroxysteroid dehydrogenases/C-4 decarboxylases (3βHSD/D). To understand the role of 3βHSD/D in Arabidopsis development, we analyzed the phenotypes of knock-out mutants and overexpression lines of two 3βHSD/D genes (At1g47290 and At2g26260). Neither single nor double knock-out mutants displayed a noticeable phenotype; however, overexpression consistently resulted in plants with wrinkled leaves and short inflorescence internodes. Interestingly, the internode growth defects were opportunistic; even within a plant, some stems were more severely affected than others. Endogenous levels of BRs were not altered in the overexpression lines, suggesting that the growth defect is not primarily due to a flaw in BR biosynthesis. To determine if overexpression of the sterol biosynthetic genes affects the functions of membrane-localized auxin transporters, we subjected plants to the auxin efflux carrier inhibitor, 1-N-naphthylphthalamic acid (NPA). Where-as the gravity vectors of wild-type roots became randomly scattered in response to NPA treatment, those of the overexpression lines continued to grow in the direction of gravity. Overexpression of the two Arabidopsis 3βHSD/D genes thus appears to affect auxin transporter activity, possibly by altering sterol composition in the membranes.

  3. Fusarium Oxysporum Volatiles Enhance Plant Growth Via Affecting Auxin Transport and Signaling.

    Science.gov (United States)

    Bitas, Vasileios; McCartney, Nathaniel; Li, Ningxiao; Demers, Jill; Kim, Jung-Eun; Kim, Hye-Seon; Brown, Kathleen M; Kang, Seogchan

    2015-01-01

    Volatile organic compounds (VOCs) have well-documented roles in plant-plant communication and directing animal behavior. In this study, we examine the less understood roles of VOCs in plant-fungal relationships. Phylogenetically and ecologically diverse strains of Fusarium oxysporum, a fungal species complex that often resides in the rhizosphere of assorted plants, produce volatile compounds that augment shoot and root growth of Arabidopsis thaliana and tobacco. Growth responses of A. thaliana hormone signaling mutants and expression patterns of a GUS reporter gene under the auxin-responsive DR5 promoter supported the involvement of auxin signaling in F. oxysporum volatile-mediated growth enhancement. In addition, 1-naphthylthalamic acid, an inhibitor of auxin efflux, negated F. oxysporum volatile-mediated growth enhancement in both plants. Comparison of the profiles of volatile compounds produced by F. oxysporum strains that differentially affected plant growth suggests that the relative compositions of both growth inhibitory and stimulatory compounds may determine the degree of plant growth enhancement. Volatile-mediated signaling between fungi and plants may represent a potentially conserved, yet mostly overlooked, mechanism underpinning plant-fungus interactions and fungal niche adaption.

  4. Fusarium oxysporum volatiles enhance plant growth via affecting auxin transport and signaling

    Directory of Open Access Journals (Sweden)

    Vasileios eBitas

    2015-11-01

    Full Text Available Volatile organic compounds (VOCs have well-documented roles in plant-plant communication and directing animal behavior. In this study, we examine the less understood roles of VOCs in plant-fungal relationships. Phylogenetically and ecologically diverse strains of Fusarium oxysporum, a fungal species complex that often resides in the rhizosphere of assorted plants, produce volatile compounds that augment shoot and root growth of Arabidopsis thaliana and tobacco. Growth responses of A. thaliana hormone signaling mutants and expression patterns of a GUS reporter gene under the auxin-responsive DR5 promoter supported the involvement of auxin signaling in F. oxysporum volatile-mediated growth enhancement. In addition, 1-naphthylthalamic acid, an inhibitor of auxin efflux, negated F. oxysporum volatile-mediated growth enhancement in both plants. Comparison of the profiles of volatile compounds produced by F. oxysporum strains that differentially affected plant growth suggests that the relative compositions of both growth inhibitory and stimulatory compounds may determine the degree of plant growth enhancement. Volatile-mediated signaling between fungi and plants may represent a potentially conserved, yet mostly overlooked, mechanism underpinning plant-fungus interactions and fungal niche adaption.

  5. Simulation of organ patterning on the floral meristem using a polar auxin transport model

    NARCIS (Netherlands)

    S. van Mourik (Simon); K. Kauffman; A.D.J. van Dijk (Aalt); G.C. Angenent; R.M.H. Merks (Roeland); J. Molenaar (Gijs)

    2012-01-01

    htmlabstractAn intriguing phenomenon in plant development is the timing and positioning of lateral organ initiation, which is a fundamental aspect of plant architecture. Although important progress has been made in elucidating the role of auxin transport in the vegetative shoot to explain the

  6. Basipetal auxin transport is required for gravitropism in roots of Arabidopsis

    Science.gov (United States)

    Rashotte, A. M.; Brady, S. R.; Reed, R. C.; Ante, S. J.; Muday, G. K.; Davies, E. (Principal Investigator)

    2000-01-01

    Auxin transport has been reported to occur in two distinct polarities, acropetally and basipetally, in two different root tissues. The goals of this study were to determine whether both polarities of indole-3-acetic acid (IAA) transport occur in roots of Arabidopsis and to determine which polarity controls the gravity response. Global application of the auxin transport inhibitor naphthylphthalamic acid (NPA) to roots blocked the gravity response, root waving, and root elongation. Immediately after the application of NPA, the root gravity response was completely blocked, as measured by an automated video digitizer. Basipetal [(3)H]IAA transport in Arabidopsis roots was inhibited by NPA, whereas the movement of [(14)C]benzoic acid was not affected. Inhibition of basipetal IAA transport by local application of NPA blocked the gravity response. Inhibition of acropetal IAA transport by application of NPA at the root-shoot junction only partially reduced the gravity response at high NPA concentrations. Excised root tips, which do not receive auxin from the shoot, exhibited a normal response to gravity. The Arabidopsis mutant eir1, which has agravitropic roots, exhibited reduced basipetal IAA transport but wild-type levels of acropetal IAA transport. These results support the hypothesis that basipetally transported IAA controls root gravitropism in Arabidopsis.

  7. N-MYC down-regulated-like proteins regulate meristem initiation by modulating auxin transport and MAX2 expression.

    Directory of Open Access Journals (Sweden)

    Yashwanti Mudgil

    Full Text Available BACKGROUND: N-MYC down-regulated-like (NDL proteins interact with the Gβ subunit (AGB1 of the heterotrimeric G protein complex and play an important role in AGB1-dependent regulation of lateral root formation by affecting root auxin transport, auxin gradients and the steady-state levels of mRNA encoding the PIN-FORMED 2 and AUXIN 1 auxin transport facilitators. Auxin transport in aerial tissue follows different paths and utilizes different transporters than in roots; therefore, in the present study, we analyzed whether NDL proteins play an important role in AGB1-dependent, auxin-mediated meristem development. METHODOLOGY/PRINCIPAL FINDINGS: Expression levels of NDL gene family members need to be tightly regulated, and altered expression (both over-expression and down-regulation confers ectopic growth. Over-expression of NDL1 disrupts vegetative and reproductive organ development. Reduced expression of the NDL gene family members results in asymmetric leaf emergence, twinning of rosette leaves, defects in leaf formation, and abnormal silique distribution. Reduced expression of the NDL genes in the agb1-2 (null allele mutant rescues some of the abnormal phenotypes, such as silique morphology, silique distribution, and peduncle angle, suggesting that proper levels of NDL proteins are maintained by AGB1. We found that all of these abnormal aerial phenotypes due to altered NDL expression were associated with increases in basipetal auxin transport, altered auxin maxima and altered MAX2 expression within the inflorescence stem. CONCLUSION/SIGNIFICANCE: NDL proteins, together with AGB1, act as positive regulators of meristem initiation and branching. AGB1 and NDL1 positively regulate basipetal inflorescence auxin transport and modulate MAX2 expression in shoots, which in turn regulates organ and lateral meristem formation by the establishment and maintenance of auxin gradients.

  8. In Vivo Exposure of Kaempferol Is Driven by Phase II Metabolic Enzymes and Efflux Transporters.

    Science.gov (United States)

    Zheng, Liang; Zhu, Lijun; Zhao, Min; Shi, Jian; Li, Yuhuan; Yu, Jia; Jiang, Huangyu; Wu, Jinjun; Tong, Yunli; Liu, Yuting; Hu, Ming; Lu, Linlin; Liu, Zhongqiu

    2016-09-01

    Kaempferol is a well-known flavonoid; however, it lacks extensive pharmacokinetic studies. Phase II metabolic enzymes and efflux transporters play an important role in the disposition of flavonoids. This study aimed to investigate the mechanism by which phase II metabolic enzymes and efflux transporters determine the in vivo exposure of kaempferol. Pharmacokinetic analysis in Sprague-Dawley rats revealed that kaempferol was mostly biotransformed to conjugates, namely, kaempferol-3-glucuronide (K-3-G), kaempferol-7-glucuronide (K-7-G), and kaempferol-7-sulfate, in plasma. K-3-G represented the major metabolite. Compared with that in wild-type mice, pharmacokinetics in knockout FVB mice demonstrated that the absence of multidrug resistance protein 2 (MRP2) and breast cancer resistance protein (BCRP) significantly increased the area under the curve (AUC) of the conjugates. The lack of MRP1 resulted in a much lower AUC of the conjugates. Intestinal perfusion in rats revealed that the glucuronide conjugates were mainly excreted in the small intestine, but 7-sulfate was mainly excreted in the colon. In Caco-2 monolayers, K-7-G efflux toward the apical (AP) side was significantly higher than K-3-G efflux. In contrast, K-3-G efflux toward the basolateral (BL) side was significantly higher than K-7-G efflux. The BL-to-AP efflux was significantly reduced in the presence of the MRP2 inhibitor LTC4. The AP-to-BL efflux was significantly decreased in the presence of the BL-side MRPs inhibitor MK571. The BCRP inhibitor Ko143 decreased the glucuronide conjugate efflux. Therefore, kaempferol is mainly exposed as K-3-G in vivo, which is driven by phase II metabolic enzymes and efflux transporters (i.e., BCRP and MRPs).

  9. Early embryo development in Fucus distichus is auxin sensitive

    Science.gov (United States)

    Basu, Swati; Sun, Haiguo; Brian, Leigh; Quatrano, Ralph L.; Muday, Gloria K.

    2002-01-01

    Auxin and polar auxin transport have been implicated in controlling embryo development in land plants. The goal of these studies was to determine if auxin and auxin transport are also important during the earliest stages of development in embryos of the brown alga Fucus distichus. Indole-3-acetic acid (IAA) was identified in F. distichus embryos and mature tissues by gas chromatography-mass spectroscopy. F. distichus embryos accumulate [(3)H]IAA and an inhibitor of IAA efflux, naphthylphthalamic acid (NPA), elevates IAA accumulation, suggesting the presence of an auxin efflux protein complex similar to that found in land plants. F. distichus embryos normally develop with a single unbranched rhizoid, but growth on IAA leads to formation of multiple rhizoids and growth on NPA leads to formation of embryos with branched rhizoids, at concentrations that are active in auxin accumulation assays. The effects of IAA and NPA are complete before 6 h after fertilization (AF), which is before rhizoid germination and cell division. The maximal effects of IAA and NPA are between 3.5 and 5 h AF and 4 and 5.5 h AF, respectively. Although, the location of the planes of cell division was significantly altered in NPA- and IAA-treated embryos, these abnormal divisions occurred after abnormal rhizoid initiation and branching was observed. The results of this study suggest that auxin acts in the formation of apical basal patterns in F. distichus embryo development.

  10. BEX1/ARF1A1C is Required for BFA-Sensitive Recycling of PIN Auxin Transporters and Auxin-Mediated Development in Arabidopsis

    Science.gov (United States)

    Tanaka, Hirokazu; Nodzyński, Tomasz; Kitakura, Saeko; Feraru, Mugurel I.; Sasabe, Michiko; Ishikawa, Tomomi; Kleine-Vehn, Jürgen; Kakimoto, Tatsuo; Friml, Jiří

    2014-01-01

    Correct positioning of membrane proteins is an essential process in eukaryotic organisms. The plant hormone auxin is distributed through intercellular transport and triggers various cellular responses. Auxin transporters of the PIN-FORMED (PIN) family localize asymmetrically at the plasma membrane (PM) and mediate the directional transport of auxin between cells. A fungal toxin, brefeldin A (BFA), inhibits a subset of guanine nucleotide exchange factors for ADP-ribosylation factor small GTPases (ARF GEFs) including GNOM, which plays a major role in localization of PIN1 predominantly to the basal side of the PM. The Arabidopsis genome encodes 19 ARF-related putative GTPases. However, ARF components involved in PIN1 localization have been genetically poorly defined. Using a fluorescence imaging-based forward genetic approach, we identified an Arabidopsis mutant, bfa-visualized exocytic trafficking defective1 (bex1), in which PM localization of PIN1–green fluorescent protein (GFP) as well as development is hypersensitive to BFA. We found that in bex1 a member of the ARF1 gene family, ARF1A1C, was mutated. ARF1A1C localizes to the trans-Golgi network/early endosome and Golgi apparatus, acts synergistically to BEN1/MIN7 ARF GEF and is important for PIN recycling to the PM. Consistent with the developmental importance of PIN proteins, functional interference with ARF1 resulted in an impaired auxin response gradient and various developmental defects including embryonic patterning defects and growth arrest. Our results show that ARF1A1C is essential for recycling of PIN auxin transporters and for various auxin-dependent developmental processes. PMID:24369434

  11. Auxin and its transport play a role in plant tolerance to arsenite-induced oxidative stress in Arabidopsis thaliana.

    Science.gov (United States)

    Krishnamurthy, Aparna; Rathinasabapathi, Bala

    2013-10-01

    The role of auxin in plant development is well known; however, its possible function in root response to abiotic stress is poorly understood. In this study, we demonstrate a novel role of auxin transport in plant tolerance to oxidative stress caused by arsenite. Plant response to arsenite [As(III)] was evaluated by measuring root growth and markers for stress on seedlings treated with control or As(III)-containing medium. Auxin transporter mutants aux1, pin1 and pin2 were significantly more sensitive to As(III) than the wild type (WT). Auxin transport inhibitors significantly reduced plant tolerance to As(III) in the WT, while exogenous supply of indole-3-acetic acid improved As(III) tolerance of aux1 and not that of WT. Uptake assays using H(3) -IAA showed As(III) affected auxin transport in WT roots. As(III) increased the levels of H2 O2 in WT but not in aux1, suggesting a positive role for auxin transport through AUX1 on plant tolerance to As(III) stress via reactive oxygen species (ROS)-mediated signalling. Compared to the WT, the mutant aux1 was significantly more sensitive to high-temperature stress and salinity, also suggesting auxin transport influences a common element shared by plant tolerance to arsenite, salinity and high-temperature stress.

  12. Effect of proinflammatory cytokine IL-6 on efflux transport of rebamipide in Caco-2 cells.

    Science.gov (United States)

    Miyake, Masateru; Nakai, Daisuke

    2017-09-01

    1. Effect of IL-6, a pro-inflammatory cytokine, on efflux transport of rebamipide, an antiulcer drug, was investigated in Caco-2 cells. 2. Rebamipide had a greater basal-to-apical than apical-to-basal transport rate. Efflux transport of rebamipide was inhibited by cyclosporine A, a P-gp inhibitor, and probenecid, which is a general MRP inhibitor, but not by Ko143, a BCRP inhibitor. 3. By the addition of IL-6, mannitol transport was slightly increased in a concentration-dependent manner in both directions of absorption and efflux. The addition of IL-6 did not change efflux transport of rebamipide even though efflux transport of digoxin, a typical substrate of P-gp, was significantly decreased by the addition of IL-6, indicating decrease of the function of P-gp. 4. Therefore, it was suggested that increase of MRP(s)-mediated transport compensates for the decrease of P-gp mediated transport of rebamipide. These findings suggested that rebamipide absorption is unlikely to be changed in IBD patients.

  13. Auxin biosynthesis and storage forms.

    Science.gov (United States)

    Korasick, David A; Enders, Tara A; Strader, Lucia C

    2013-06-01

    The plant hormone auxin drives plant growth and morphogenesis. The levels and distribution of the active auxin indole-3-acetic acid (IAA) are tightly controlled through synthesis, inactivation, and transport. Many auxin precursors and modified auxin forms, used to regulate auxin homeostasis, have been identified; however, very little is known about the integration of multiple auxin biosynthesis and inactivation pathways. This review discusses the many ways auxin levels are regulated through biosynthesis, storage forms, and inactivation, and the potential roles modified auxins play in regulating the bioactive pool of auxin to affect plant growth and development.

  14. Deciphering the role of RND efflux transporters in Burkholderia cenocepacia.

    Directory of Open Access Journals (Sweden)

    Silvia Bazzini

    Full Text Available Burkholderia cenocepacia J2315 is representative of a highly problematic group of cystic fibrosis (CF pathogens. Eradication of B. cenocepacia is very difficult with the antimicrobial therapy being ineffective due to its high resistance to clinically relevant antimicrobial agents and disinfectants. RND (Resistance-Nodulation-Cell Division efflux pumps are known to be among the mediators of multidrug resistance in gram-negative bacteria. Since the significance of the 16 RND efflux systems present in B. cenocepacia (named RND-1 to -16 has been only partially determined, the aim of this work was to analyze mutants of B. cenocepacia strain J2315 impaired in RND-4 and RND-9 efflux systems, and assess their role in the efflux of toxic compounds. The transcriptomes of mutants deleted individually in RND-4 and RND-9 (named D4 and D9, and a double-mutant in both efflux pumps (named D4-D9, were compared to that of the wild-type B. cenocepacia using microarray analysis. Microarray data were confirmed by qRT-PCR, phenotypic experiments, and by Phenotype MicroArray analysis. The data revealed that RND-4 made a significant contribution to the antibiotic resistance of B. cenocepacia, whereas RND-9 was only marginally involved in this process. Moreover, the double mutant D4-D9 showed a phenotype and an expression profile similar to D4. The microarray data showed that motility and chemotaxis-related genes appeared to be up-regulated in both D4 and D4-D9 strains. In contrast, these gene sets were down-regulated or expressed at levels similar to J2315 in the D9 mutant. Biofilm production was enhanced in all mutants. Overall, these results indicate that in B. cenocepacia RND pumps play a wider role than just in drug resistance, influencing additional phenotypic traits important for pathogenesis.

  15. Effect of drug efflux transporters on placental transport of antiretroviral agent abacavir.

    Science.gov (United States)

    Neumanova, Zuzana; Cerveny, Lukas; Greenwood, Susan L; Ceckova, Martina; Staud, Frantisek

    2015-11-01

    Abacavir is as a frequent part of combination antiretroviral therapy used in pregnant women. The aim of this study was to investigate, using in vitro, in situ and ex vivo experimental approaches, whether the transplacental pharmacokinetics of abacavir is affected by ATP-binding cassette (ABC) efflux transporters functionally expressed in the placenta: P-glycoprotein (ABCB1), breast cancer resistance protein (ABCG2), multidrug resistance-associated protein 2 (ABCC2) and multidrug resistance-associated protein 5 (ABCC5). In vitro transport assays revealed that abacavir is a substrate of human ABCB1 and ABCG2 transporters but not of ABCC2 or ABCC5. In addition, in situ experiments using dually perfused rat term placenta confirmed interactions of abacavir with placental Abcb1/Abcg2. In contrast, uptake studies in human placental villous fragments did not reveal any interaction of abacavir with efflux transporters suggesting a large contribution of passive diffusion and/or influx mechanisms to net transplacental abacavir transfer.

  16. Differential effects of auxin polar transport inhibitors on rooting in some Crassulaceae species

    OpenAIRE

    Marian Saniewski; Justyna Góraj; Elżbieta Węgrzynowicz-Lesiak; Kensuke Miyamoto; Junichi Ueda

    2014-01-01

    Effects of auxin polar transport inhibitors, 2,3,5-triio-dobenzoic acid (TIBA), 1-N-naphthylphthalamic acid (NPA) and methyl 2-chloro-9-hydroxyfluorene-9-carboxylate (morphactin IT 3456), as a lanolin paste, on root formation in cuttings of some species of Crassulaceae, such as Bryophyllum daigremontianum, B. calycinum, Kalanchoe blossfeldiana and K. tubiflora, were studied. Cuttings of these plants were easily rooted in water without any treatment. TIBA and morphactin IT 3456 completely inhi...

  17. Assembly & Transport Mechanism of Tripartite Drug Efflux Systems

    OpenAIRE

    Misra, Rajeev; Bavro, Vassiliy N.

    2009-01-01

    Multidrug efflux (MDR) pumps remove a variety of compounds from the cell into the external environment. There are five different classes of MDR pumps in bacteria, and quite often a single bacterial species expresses multiple classes of pumps. Although under normal circumstances MDR pumps confer low-level intrinsic resistance to drugs, the presence of drugs and mutations in regulatory genes lead to high level expression of MDR pumps that can pose problems with therapeutic treatments. This revi...

  18. Redirection of auxin flow in Arabidopsis thaliana roots after infection by root-knot nematodes

    Science.gov (United States)

    Kyndt, Tina; Goverse, Aska; Haegeman, Annelies; Warmerdam, Sonja; Wanjau, Cecilia; Jahani, Mona; Engler, Gilbert; de Almeida Engler, Janice; Gheysen, Godelieve

    2016-01-01

    Plant-parasitic root-knot nematodes induce the formation of giant cells within the plant root, and it has been recognized that auxin accumulates in these feeding sites. Here, we studied the role of the auxin transport system governed by AUX1/LAX3 influx proteins and different PIN efflux proteins during feeding site development in Arabidopsis thaliana roots. Data generated via promoter–reporter line and protein localization analyses evoke a model in which auxin is being imported at the basipetal side of the feeding site by the concerted action of the influx proteins AUX1 and LAX3, and the efflux protein PIN3. Mutants in auxin influx proteins AUX1 and LAX3 bear significantly fewer and smaller galls, revealing that auxin import into the feeding sites is needed for their development and expansion. The feeding site development in auxin export (PIN) mutants was only slightly hampered. Expression of some PINs appears to be suppressed in galls, probably to prevent auxin drainage. Nevertheless, a functional PIN4 gene seems to be a prerequisite for proper nematode development and gall expansion, most likely by removing excessive auxin to stabilize the hormone level in the feeding site. Our data also indicate a role of local auxin peaks in nematode attraction towards the root. PMID:27312670

  19. Enhanced cholesterol efflux to HDL through the ABCA1 transporter in hypertriglyceridemia of type 2 diabetes.

    Science.gov (United States)

    Yassine, Hussein N; Belopolskaya, Alexandra; Schall, Christina; Stump, Craig S; Lau, Serrine S; Reaven, Peter D

    2014-05-01

    Our objective was to examine the role of hypertriglyceridemia on the capacity of HDL to facilitate ABCA-1 mediated cholesterol efflux in type 2 diabetes (T2DM). HDL mediated cholesterol efflux through the ABCA-1 transporter was measured using BHK cell lines in samples of 71 participants with T2DM in the presence or absence of high triglyceride levels (TG). Additionally, HDL mediated efflux was measured in 13 diabetic and non-diabetic participants fasting and four hours after a high-fat test challenge. HDL mediated cholesterol efflux function was increased in participants with T2DM with hypertriglyceridemia when compared to participants with T2DM without hypertriglyceridemia (efflux ratio mean±standard deviation (SD), T2DM+TG: 1.17±0.25 vs. T2DM - TG: 1.03±0.19, p=0.0098). In the fat challenge study, we observed a significant increase in ABCA-1 mediated cholesterol efflux capacity following an ingestion of high-fat test meal by participants in both groups of T2DM (n=6, efflux ratio, mean±SD, pre: 0.86±0.4 vs. post: 1.34±0.6, p=0.01) and non-diabetic participants (n=7, efflux ratio mean±SD pre: 1.24±0.31 vs. post: 1.39±0.42, p=0.04) that was partly explained by the difference in CETP activity (r=0.6, p=0.03). Our study suggests that high triglyceride levels facilitate ABCA-1 mediated efflux function of HDL in part by activating CETP. Copyright © 2014 Elsevier Inc. All rights reserved.

  20. Transport of cisplatin by the copper efflux transporter ATP7B.

    Science.gov (United States)

    Safaei, Roohangiz; Otani, Shinji; Larson, Barrett J; Rasmussen, Michael L; Howell, Stephen B

    2008-02-01

    ATP7B is a P-type ATPase that mediates the efflux of copper. Recent studies have demonstrated that ATP7B regulates the cellular efflux of cisplatin (DDP) and controls sensitivity to the cytotoxic effects of this drug. To determine whether DDP is a substrate for ATP7B, DDP transport was assayed in vesicles isolated from Sf9 cells infected with a baculovirus that expressed either the wild-type ATP7B or a mutant ATP7B that was unable to transport copper as a result of conversion of the transmembrane metal binding CPC motif to CPA. Only the wild-type ATP7B-expressing vesicles exhibited copper-dependent ATPase activity, copper-induced acyl-phosphate formation, and ATP-dependent transport of copper. The amount of DDP that became bound was higher for vesicles expressing either type of ATP7B than for those not expressing either form of ATP7B, but only the vesicles expressing wild-type ATP7B mediated ATP-dependent accumulation of the drug. At pH 4.6, the vesicles expressing the wild-type ATP7B exhibited ATP-dependent accumulation of DDP with an apparent K(m) of 1.2 +/- 0.5 (S.E.M.) muM and V(max) of 0.03 +/- 0.002 (S.E.M.) nmol/mg of protein/min. DDP also induced the acyl-phosphorylation of ATP7B but at a much slower rate than copper. Copper and DDP each inhibited the ATP-dependent transport of the other. These results establish that DDP is a substrate for ATP7B but is transported at a much slower rate than copper.

  1. Export of a single drug molecule in two transport cycles by a multidrug efflux pump.

    Science.gov (United States)

    Fluman, Nir; Adler, Julia; Rotenberg, Susan A; Brown, Melissa H; Bibi, Eitan

    2014-08-08

    Secondary multidrug transporters use ion concentration gradients to energize the removal from cells of various antibiotics. The Escherichia coli multidrug transporter MdfA exchanges a single proton with a single monovalent cationic drug molecule. This stoichiometry renders the efflux of divalent cationic drugs energetically unfavourable, as it requires exchange with at least two protons. Here we show that surprisingly, MdfA catalyses efflux of divalent cations, provided that they have a unique architecture: the two charged moieties must be separated by a long linker. These drugs are exchanged for two protons despite the apparent inability of MdfA to exchange two protons for a single drug molecule. Our results suggest that these drugs are transported in two consecutive transport cycles, where each cationic moiety is transported as if it were a separate substrate. We propose that secondary transport can adopt a processive-like mode of action, thus expanding the substrate spectrum of multidrug transporters.

  2. Differential effects of auxin polar transport inhibitors on rooting in some Crassulaceae species

    Directory of Open Access Journals (Sweden)

    Marian Saniewski

    2014-07-01

    Full Text Available Effects of auxin polar transport inhibitors, 2,3,5-triio-dobenzoic acid (TIBA, 1-N-naphthylphthalamic acid (NPA and methyl 2-chloro-9-hydroxyfluorene-9-carboxylate (morphactin IT 3456, as a lanolin paste, on root formation in cuttings of some species of Crassulaceae, such as Bryophyllum daigremontianum, B. calycinum, Kalanchoe blossfeldiana and K. tubiflora, were studied. Cuttings of these plants were easily rooted in water without any treatment. TIBA and morphactin IT 3456 completely inhibited root formation in the cuttings of these plants but NPA did not when these inhibitors were applied around the stem below the leaves. When TIBA and morphactin were applied around the stem near the top, but leaves were present below the treatment, the root formation was observed in B. calycinum and K. blossfeldiana but in a smaller degree than in control cuttings. These results strongly suggest that endogenous auxin is required for root formation in cuttings of Crassulaceae plants. The differential mode of action of NPA is discussed together with its effect on auxin polar transport.

  3. Structures and transport dynamics of a Campylobacter jejuni multidrug efflux pump

    Energy Technology Data Exchange (ETDEWEB)

    Su, Chih-Chia; Yin, Linxiang; Kumar, Nitin; Dai, Lei; Radhakrishnan, Abhijith; Bolla, Jani Reddy; Lei, Hsiang-Ting; Chou, Tsung-Han; Delmar, Jared A.; Rajashankar, Kanagalaghatta R.; Zhang, Qijing; Shin, Yeon-Kyun; Yu, Edward W. (Cornell); (Iowa State)

    2017-08-01

    Resistance-nodulation-cell division efflux pumps are integral membrane proteins that catalyze the export of substrates across cell membranes. Within the hydrophobe-amphiphile efflux subfamily, these resistance-nodulation-cell division proteins largely form trimeric efflux pumps. The drug efflux process has been proposed to entail a synchronized motion between subunits of the trimer to advance the transport cycle, leading to the extrusion of drug molecules. Here we use X-ray crystallography and single-molecule fluorescence resonance energy transfer imaging to elucidate the structures and functional dynamics of the Campylobacter jejuni CmeB multidrug efflux pump. We find that the CmeB trimer displays a very unique conformation. A direct observation of transport dynamics in individual CmeB trimers embedded in membrane vesicles indicates that each CmeB subunit undergoes conformational transitions uncoordinated and independent of each other. On the basis of our findings and analyses, we propose a model for transport mechanism where CmeB protomers function independently within the trimer.

  4. Auxin and nitric oxide control indeterminate nodule formation

    Directory of Open Access Journals (Sweden)

    Spena Angelo

    2007-05-01

    Full Text Available Abstract Background Rhizobia symbionts elicit root nodule formation in leguminous plants. Nodule development requires local accumulation of auxin. Both plants and rhizobia synthesise auxin. We have addressed the effects of bacterial auxin (IAA on nodulation by using Sinorhizobium meliloti and Rhizobium leguminosarum bacteria genetically engineered for increased auxin synthesis. Results IAA-overproducing S. meliloti increased nodulation in Medicago species, whilst the increased auxin synthesis of R. leguminosarum had no effect on nodulation in Phaseolus vulgaris, a legume bearing determinate nodules. Indeterminate legumes (Medicago species bearing IAA-overproducing nodules showed an enhanced lateral root development, a process known to be regulated by both IAA and nitric oxide (NO. Higher NO levels were detected in indeterminate nodules of Medicago plants formed by the IAA-overproducing rhizobia. The specific NO scavenger cPTIO markedly reduced nodulation induced by wild type and IAA-overproducing strains. Conclusion The data hereby presented demonstrate that auxin synthesised by rhizobia and nitric oxide positively affect indeterminate nodule formation and, together with the observation of increased expression of an auxin efflux carrier in roots bearing nodules with higher IAA and NO content, support a model of nodule formation that involves auxin transport regulation and NO synthesis.

  5. Role of copper transporters in the uptake and efflux of platinum containing drugs.

    Science.gov (United States)

    Safaei, Roohangiz

    2006-03-08

    Cellular mechanisms for the uptake, intracellular distribution and efflux of the platinum (Pt) containing compounds cisplatin (DDP), carboplatin (CBDCA) and oxaliplatin (LOHP) are unknown. Current data suggest that specialized transporters/carriers mediate the transport of Pt drugs across the cellular membranes. Specific roles for the copper (Cu) transporters CTR1, ATP7A and ATP7B have been demonstrated during recent years. The finding that in cultured cells and tumor samples a correlation can be found between the expression of Cu transporters and the degree of the acquired resistance to Pt drug suggests that the Cu transporters are important constituents of the program that regulates sensitivity to Pt drugs. A model is presented that describes the function of Cu transporters in the regulation of Pt drug uptake and efflux.

  6. Importance of Non-Diffusive Transport for Soil CO2 Efflux in a Temperate Mountain Grassland

    Science.gov (United States)

    Roland, Marilyn; Vicca, Sara; Bahn, Michael; Ladreiter-Knauss, Thomas; Schmitt, Michael; Janssens, Ivan A.

    2015-04-01

    A key focus in climate change is on the dynamics and predictions of the soil CO2 efflux (SCE) from terrestrial ecosystems. Limited knowledge of CO2 transport through the soil restricts our understanding of the various biotic and abiotic processes underlying these emissions. Diffusion is often thought to be the main transport mechanism for trace gases in soils, an assumption that is reflected in the increasing popularity of the flux-gradient approach (FGA). Based on Fick's law, the FGA calculates soil CO2 efflux from CO2 concentration profiles, given good estimates of the diffusion coefficient. The latter can be calculated via different commonly used models, and solid-state sensors allow continuous high-frequency measurements of soil CO2 concentrations with minimal disturbance to the soil conditions in a cost-effective way. Fast growing evidence of pressure pumping and advection, makes it impossible to disregard non-diffusive gas transport when evaluating diel and day-to-day dynamics of soil CO2 emissions. We have analyzed combined measurements from solid-state sensors and soil chambers to gain insight in the CO2 transport mechanisms in a grassland site in the Austrian Alps. The FGA-derived efflux underestimated the chamber efflux by 10 to 87% at our site, depending on which model was used for calculation of the diffusion coefficient. We found that the actual transport rates correlated well with irradiation and wind speed, even more when the soil moisture content was below 33%. These findings suggest that bulk soil air transport was enhanced by pressure changes induced by wind shear at the surface and by local heating of the soil surface. Considering the importance of non-diffusive transport processes is a prerequisite when using solid-state CO2 concentration measurements to estimate soil CO2 efflux at any given site.

  7. Helix stabilization of amphipathic peptides by hydrocarbon stapling increases cholesterol efflux by the ABCA1 transporter.

    Science.gov (United States)

    Sviridov, D O; Ikpot, I Z; Stonik, J; Drake, S K; Amar, M; Osei-Hwedieh, D O; Piszczek, G; Turner, S; Remaley, A T

    2011-07-08

    Apolipoprotein mimetic peptides are short amphipathic peptides that efflux cholesterol from cells by the ABCA1 transporter and are being investigated as therapeutic agents for cardiovascular disease. We examined the role of helix stabilization of these peptides in cholesterol efflux. A 23-amino acid long peptide (Ac-VLEDSFKVSFLSALEEYTKKLNTQ-NH2) based on the last helix of apoA-I (A10) was synthesized, as well as two variants, S1A10 and S2A10, in which the third and fourth and third and fifth turn of each peptide, respectively, were covalently joined by hydrocarbon staples. By CD spectroscopy, the stapled variants at 24 °C were more helical in aqueous buffer than A10 (A10 17%, S1A10 62%, S2A10 97%). S1A10 and S2A10 unlike A10 were resistant to proteolysis by pepsin and chymotrypsin. S1A10 and S2A10 showed more than a 10-fold increase in cholesterol efflux by the ABCA1 transporter compared to A10. In summary, hydrocarbon stapling of amphipathic peptides increases their helicity, makes them resistant to proteolysis and enhances their ability to promote cholesterol efflux by the ABCA1 transporter, indicating that this peptide modification may be useful in the development of apolipoprotein mimetic peptides.

  8. The ABCG2 efflux transporter from rabbit placenta: Cloning and functional characterization.

    Science.gov (United States)

    Halwachs, Sandra; Kneuer, Carsten; Gohlsch, Katrin; Müller, Marian; Ritz, Vera; Honscha, Walther

    2016-02-01

    In human placenta, the ATP-binding cassette efflux transporter ABCG2 is highly expressed in syncytiotrophoblast cells and mediates cellular excretion of various drugs and toxins. Hence, physiological ABCG2 activity substantially contributes to the fetoprotective placenta barrier function during gestation. Developmental toxicity studies are often performed in rabbit. However, despite its toxicological relevance, there is no data so far on functional ABCG2 expression in this species. Therefore, we cloned ABCG2 from placenta tissues of chinchilla rabbit. Sequencing showed 84-86% amino acid sequence identity to the orthologues from man, rat and mouse. We transduced the rabbit ABCG2 clone (rbABCG2) in MDCKII cells and stable rbABCG2 gene and protein expression was shown by RT-PCR and Western blot analysis. The rbABCG2 efflux activity was demonstrated with the Hoechst H33342 assay using the specific ABCG2 inhibitor Ko143. We further tested the effect of established human ABCG2 (hABCG2) drug substrates including the antibiotic danofloxacin or the histamine H2-receptor antagonist cimetidine on H33342 accumulation in MDCKII-rbABCG2 or -hABCG2 cells. Human therapeutic plasma concentrations of all tested drugs caused a comparable competitive inhibition of H33342 excretion in both ABCG2 clones. Altogether, we first showed functional expression of the ABCG2 efflux transporter in rabbit placenta. Moreover, our data suggest a similar drug substrate spectrum of the rabbit and the human ABCG2 efflux transporter.

  9. Syntaxin 1A interaction with the dopamine transporter promotes amphetamine-induced dopamine efflux.

    Science.gov (United States)

    Binda, Francesca; Dipace, Concetta; Bowton, Erica; Robertson, Sabrina D; Lute, Brandon J; Fog, Jacob U; Zhang, Minjia; Sen, Namita; Colbran, Roger J; Gnegy, Margaret E; Gether, Ulrik; Javitch, Jonathan A; Erreger, Kevin; Galli, Aurelio

    2008-10-01

    The soluble N-ethylmaleimide-sensitive factor attachment protein receptor protein syntaxin 1A (SYN1A) interacts with and regulates the function of transmembrane proteins, including ion channels and neurotransmitter transporters. Here, we define the first 33 amino acids of the N terminus of the dopamine (DA) transporter (DAT) as the site of direct interaction with SYN1A. Amphetamine (AMPH) increases the association of SYN1A with human DAT (hDAT) in a heterologous expression system (hDAT cells) and with native DAT in murine striatal synaptosomes. Immunoprecipitation of DAT from the biotinylated fraction shows that the AMPH-induced increase in DAT/SYN1A association occurs at the plasma membrane. In a superfusion assay of DA efflux, cells overexpressing SYN1A exhibited significantly greater AMPH-induced DA release with respect to control cells. By combining the patch-clamp technique with amperometry, we measured DA release under voltage clamp. At -60 mV, a physiological resting potential, AMPH did not induce DA efflux in hDAT cells and DA neurons. In contrast, perfusion of exogenous SYN1A (3 microM) into the cell with the whole-cell pipette enabled AMPH-induced DA efflux at -60 mV in both hDAT cells and DA neurons. It has been shown recently that Ca2+/calmodulin-dependent protein kinase II (CaMKII) is activated by AMPH and regulates AMPH-induced DA efflux. Here, we show that AMPH-induced association between DAT and SYN1A requires CaMKII activity and that inhibition of CaMKII blocks the ability of exogenous SYN1A to promote DA efflux. These data suggest that AMPH activation of CaMKII supports DAT/SYN1A association, resulting in a mode of DAT capable of DA efflux.

  10. Auxin Polar Transport In Stamen Formation And Development: How Many Actors?

    Directory of Open Access Journals (Sweden)

    Maura eCardarelli

    2014-07-01

    Full Text Available In flowering plants, proper development of stamens, the male reproductive organs, is required for successful sexual reproduction. In Arabidopsis thaliana normally six stamen primordia arise in the third whorl of floral organs and subsequently differentiate into stamen filaments and anthers, where male meiosis occurs, thus ending the early developmental phase. This early phase is followed by a late developmental phase, which consists of a rapid elongation of stamen filaments coordinated with anther dehiscence and pollen maturation, and terminates with mature pollen grain release at anthesis. Increasing evidence suggests that auxin transport is necessary for both early and late phases of stamen development. It has been shown that different members of PIN (PIN-FORMED family are involved in the early phase, whereas members of both PIN and P-glycoproteins of the ABCB (PGP transporter families are required during the late developmental phase. In this review we provide an overview of the increasing knowledge on auxin transporters involved in Arabidopsis stamen formation and development and we discuss their role and functional conservation across plant species.

  11. Stochastic and deterministic multiscale models for systems biology: an auxin-transport case study

    Directory of Open Access Journals (Sweden)

    King John R

    2010-03-01

    Full Text Available Abstract Background Stochastic and asymptotic methods are powerful tools in developing multiscale systems biology models; however, little has been done in this context to compare the efficacy of these methods. The majority of current systems biology modelling research, including that of auxin transport, uses numerical simulations to study the behaviour of large systems of deterministic ordinary differential equations, with little consideration of alternative modelling frameworks. Results In this case study, we solve an auxin-transport model using analytical methods, deterministic numerical simulations and stochastic numerical simulations. Although the three approaches in general predict the same behaviour, the approaches provide different information that we use to gain distinct insights into the modelled biological system. We show in particular that the analytical approach readily provides straightforward mathematical expressions for the concentrations and transport speeds, while the stochastic simulations naturally provide information on the variability of the system. Conclusions Our study provides a constructive comparison which highlights the advantages and disadvantages of each of the considered modelling approaches. This will prove helpful to researchers when weighing up which modelling approach to select. In addition, the paper goes some way to bridging the gap between these approaches, which in the future we hope will lead to integrative hybrid models.

  12. Pinoid kinase regulates root gravitropism through modulation of PIN2-dependent basipetal auxin transport in Arabidopsis thaliana

    Science.gov (United States)

    Muday, Gloria; Sukumar, Poornima; Edwards, Karin; Delong, Alison; Rahman, Abidur

    Reversible protein phosphorylation is a key regulatory mechanism governing polar auxin transport. We tested the hypothesis that PINOID (PID)-mediated phosphorylation and RCN1- regulated dephosphorylation might antagonistically regulate auxin transport and gravity response in seedling roots. Here we show that basipetal IAA transport and gravitropism are reduced in pid mutant seedlings, while acropetal transport and lateral root development are unchanged. Treatment of wild-type seedlings with the protein kinase inhibitor, staurosporine, phenocopied the reduced auxin transport and gravity response of pid-9 and reduced formation of asymmetric DR5-revGFP expression at the root tip after reorientation relative to gravity. Gravitropism and auxin transport in pid are resistant to further inhibition by staurosporine. Gravity response defects of rcn1 and pid-9 are partially rescued by treatment with staurosporine or the phosphatase inhibitor, cantharidin, respectively, and in the pid-9 rcn1 double mutant. Furthermore, the effect of staurosporine is lost in pin2, and a PIN2::GFP fusion protein accumulates in endomembrane compartments after staurosporine treatment. In the pid-9 mutant, immunological techniques find a similar PIN2 localization. These data suggest that staurosporine inhibits gravitropism and basipetal IAA transport by blocking PID action and altering PIN2 localization and support the model that PID and RCN1 reciprocally regulate root gravitropic curvature.

  13. Flavonoids Redirect PIN-mediated Polar Auxin Fluxes during Root Gravitropic Responses*S⃞

    Science.gov (United States)

    Santelia, Diana; Henrichs, Sina; Vincenzetti, Vincent; Sauer, Michael; Bigler, Laurent; Klein, Markus; Bailly, Aurélien; Lee, Youngsook; Friml, Jir̆í; Geisler, Markus; Martinoia, Enrico

    2008-01-01

    The rate, polarity, and symmetry of the flow of the plant hormone auxin are determined by the polar cellular localization of PIN-FORMED (PIN) auxin efflux carriers. Flavonoids, a class of secondary plant metabolites, have been suspected to modulate auxin transport and tropic responses. Nevertheless, the identity of specific flavonoid compounds involved and their molecular function and targets in vivo are essentially unknown. Here we show that the root elongation zone of agravitropic pin2/eir1/wav6/agr1 has an altered pattern and amount of flavonol glycosides. Application of nanomolar concentrations of flavonols to pin2 roots is sufficient to partially restore root gravitropism. By employing a quantitative cell biological approach, we demonstrate that flavonoids partially restore the formation of lateral auxin gradients in the absence of PIN2. Chemical complementation by flavonoids correlates with an asymmetric distribution of the PIN1 protein. pin2 complementation probably does not result from inhibition of auxin efflux, as supply of the auxin transport inhibitor N-1-naphthylphthalamic acid failed to restore pin2 gravitropism. We propose that flavonoids promote asymmetric PIN shifts during gravity stimulation, thus redirecting basipetal auxin streams necessary for root bending. PMID:18718912

  14. Auxin and chloroplast movements.

    Science.gov (United States)

    Eckstein, Aleksandra; Krzeszowiec, Weronika; Waligórski, Piotr; Gabryś, Halina

    2016-03-01

    Auxin is involved in a wide spectrum of physiological processes in plants, including responses controlled by the blue light photoreceptors phototropins: phototropic bending and stomatal movement. However, the role of auxin in phototropin-mediated chloroplast movements has never been studied. To address this question we searched for potential interactions between auxin and the chloroplast movement signaling pathway using different experimental approaches and two model plants, Arabidopsis thaliana and Nicotiana tabacum. We observed that the disturbance of auxin homeostasis by shoot decapitation caused a decrease in chloroplast movement parameters, which could be rescued by exogenous auxin application. In several cases, the impairment of polar auxin transport, by chemical inhibitors or in auxin carrier mutants, had a similar negative effect on chloroplast movements. This inhibition was not correlated with changes in auxin levels. Chloroplast relocations were also affected by the antiauxin p-chlorophenoxyisobutyric acid and mutations in genes encoding some of the elements of the SCF(TIR1)-Aux/IAA auxin receptor complex. The observed changes in chloroplast movement parameters are not prominent, which points to a modulatory role of auxin in this process. Taken together, the obtained results suggest that auxin acts indirectly to regulate chloroplast movements, presumably by regulating gene expression via the SCF(TIR1)-Aux/IAA-ARF pathway. Auxin does not seem to be involved in controlling the expression of phototropins.

  15. Characterization of zebrafish Abcc4 as an efflux transporter of organochlorine pesticides.

    Science.gov (United States)

    Lu, Xing; Long, Yong; Lin, Li; Sun, Rongze; Zhong, Shan; Cui, Zongbin

    2014-01-01

    DDT and lindane are highly toxic organochlorine pesticides and posing adverse effects on the environment and public health due to their frequent usage in developing countries. ABCC4/MRP4 is an organic anion transporter that mediates cellular efflux of a wide range of exogenous and endogenous compounds such as cyclic nucleotides and anti-cancer drugs; however, it remains unclear whether ABCC4 and its orthologs function in the detoxification of organochlorine pesticides. Here, we demonstrated the roles of zebrafish Abcc4 in cellular efflux of DDT and lindane. Zebrafish abcc4 was maternally expressed in the oocytes and its transcripts were detected in the lens, pancreas, gills, liver, intestine and bladder of developing embryos and in adult tissues examined. DDT and lindane were able to induce the expression of abcc4 gene and overexpression of Abcc4 significantly decreased the cytotoxicity and accumulation of DDT and lindane in LLC-PK1 cells and developing embryos. In contrast, overexpression of an Abcc4-G1188D mutant abolished its transporter function without effects on its substrate binding activity, and sensitized LLC-PK1 cells and developing embryos to toxic pesticides. Moreover, glutathione (GSH) was involved in the efflux of cellular pesticides and ATPase activity in developing embryos can be induced by DDT or lindane. Thus, zebrafish Abcc4 plays crucial roles in cellular efflux of organochlorine pesticides and can be used a potential molecular marker for the monitor of DDT and lindane contamination in the aquatic environment.

  16. Crystal structures of the CusA efflux pump suggest methionine-mediated metal transport

    Energy Technology Data Exchange (ETDEWEB)

    Long, Feng; Su, Chih-Chia; Zimmermann, Michael T.; Boyken, Scott E.; Rajashankar, Kanagalaghatta R.; Jernigan, Robert L.; Yu, Edward W. (Cornell); (Iowa State)

    2010-09-23

    Gram-negative bacteria, such as Escherichia coli, frequently use tripartite efflux complexes in the resistance-nodulation-cell division (RND) family to expel various toxic compounds from the cell. The efflux system CusCBA is responsible for extruding biocidal Cu(I) and Ag(I) ions. No previous structural information was available for the heavy-metal efflux (HME) subfamily of the RND efflux pumps. Here we describe the crystal structures of the inner-membrane transporter CusA in the absence and presence of bound Cu(I) or Ag(I). These CusA structures provide new structural information about the HME subfamily of RND efflux pumps. The structures suggest that the metal-binding sites, formed by a three-methionine cluster, are located within the cleft region of the periplasmic domain. This cleft is closed in the apo-CusA form but open in the CusA-Cu(I) and CusA-Ag(I) structures, which directly suggests a plausible pathway for ion export. Binding of Cu(I) and Ag(I) triggers significant conformational changes in both the periplasmic and transmembrane domains. The crystal structure indicates that CusA has, in addition to the three-methionine metal-binding site, four methionine pairs - three located in the transmembrane region and one in the periplasmic domain. Genetic analysis and transport assays suggest that CusA is capable of actively picking up metal ions from the cytosol, using these methionine pairs or clusters to bind and export metal ions. These structures suggest a stepwise shuttle mechanism for transport between these sites.

  17. Shoot-supplied ammonium targets the root auxin influx carrier AUX1 and inhibits lateral root emergence in Arabidopsis

    KAUST Repository

    Li, Baohai

    2011-03-24

    Deposition of ammonium (NH4 +) from the atmosphere is a substantial environmental problem. While toxicity resulting from root exposure to NH4 + is well studied, little is known about how shoot-supplied ammonium (SSA) affects root growth. In this study, we show that SSA significantly affects lateral root (LR) development. We show that SSA inhibits lateral root primordium (LRP) emergence, but not LRP initiation, resulting in significantly impaired LR number. We show that the inhibition is independent of abscisic acid (ABA) signalling and sucrose uptake in shoots but relates to the auxin response in roots. Expression analyses of an auxin-responsive reporter, DR5:GUS, and direct assays of auxin transport demonstrated that SSA inhibits root acropetal (rootward) auxin transport while not affecting basipetal (shootward) transport or auxin sensitivity of root cells. Mutant analyses indicated that the auxin influx carrier AUX1, but not the auxin efflux carriers PIN-FORMED (PIN)1 or PIN2, is required for this inhibition of LRP emergence and the observed auxin response. We found that AUX1 expression was modulated by SSA in vascular tissues rather than LR cap cells in roots. Taken together, our results suggest that SSA inhibits LRP emergence in Arabidopsis by interfering with AUX1-dependent auxin transport from shoot to root. © 2011 Blackwell Publishing Ltd.

  18. Silicon efflux transporters isolated from two pumpkin cultivars contrasting in Si uptake.

    Science.gov (United States)

    Mitani-Ueno, Namiki; Yamaji, Naoki; Ma, Jian Feng

    2011-07-01

    The accumulation of silicon (Si) differs greatly with plant species and cultivars due to different ability of the roots to take up Si. In Si accumulating plants such as rice, barley and maize, Si uptake is mediated by the influx (Lsi1) and efflux (Lsi2) transporters. Here we report isolation and functional analysis of two Si efflux transporters (CmLsi2-1 and CmLsi2-2) from two pumpkin (Cucurbita moschata Duch.) cultivars contrasting in Si uptake. These cultivars are used for rootstocks of bloom and bloomless cucumber, respectively. Different from mutations in the Si influx transporter CmLsi1, there was no difference in the sequence of either CmLsi2 between two cultivars. Both CmLsi2-1 and CmLsi2-2 showed an efflux transport activity for Si and they were expressed in both the roots and shoots. These results confirm our previous finding that mutation in CmLsi1, but not in CmLsi2-1 and CmLsi2-2 are responsible for bloomless phenotype resulting from low Si uptake.

  19. A Role for Auxin in Flower Development

    Institute of Scientific and Technical Information of China (English)

    Youfa Cheng; Yunde Zhao

    2007-01-01

    Auxin has long been implicated in many aspects of plant growth and development including flower development. However, the exact roles of auxin in flower development have not been well defined until the recent identification of auxin biosynthesis mutants. Auxin is necessary for the initiation of floral primordia,and the disruption of auxin biosynthesis, polar auxin transport or auxin signaling leads to the failure of flower formation. Auxin also plays an essential role in specifying the number and identity of floral organs.Further analysis of the relationship between the auxin pathways and the known flower development genes will provide critical information regarding mechanisms of organogenesis and pattern formation in plants.

  20. The auxin response factor MONOPTEROS controls meristem function and organogenesis in both the shoot and root through the direct regulation of PIN genes.

    Science.gov (United States)

    Krogan, Naden T; Marcos, Danielle; Weiner, Aaron I; Berleth, Thomas

    2016-10-01

    The regulatory effect auxin has on its own transport is critical in numerous self-organizing plant patterning processes. However, our understanding of the molecular mechanisms linking auxin signal transduction and auxin transport is still fragmentary, and important regulatory genes remain to be identified. To track a key link between auxin signaling and auxin transport in development, we established an Arabidopsis thaliana genetic background in which fundamental patterning processes in both shoot and root were essentially abolished and the expression of PIN FORMED (PIN) auxin efflux facilitators was dramatically reduced. In this background, we demonstrate that activating a steroid-inducible variant of the auxin response factor (ARF) MONOPTEROS (MP) is sufficient to restore patterning and PIN gene expression. Further, we show that MP binds to distinct promoter elements of multiple genetically defined PIN genes. Our work identifies a direct regulatory link between central, well-characterized genes involved in auxin signal transduction and auxin transport. The steroid-inducible MP system directly demonstrates the importance of this molecular link in multiple patterning events in embryos, shoots and roots, and provides novel options for interrogating the properties of self-regulated auxin-based patterning in planta. © 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.

  1. Low-fluence red light increases the transport and biosynthesis of auxin.

    Science.gov (United States)

    Liu, Xing; Cohen, Jerry D; Gardner, Gary

    2011-10-01

    In plants, light is an important environmental signal that induces photomorphogenesis and interacts with endogenous signals, including hormones. We found that light increased polar auxin transport in dark-grown Arabidopsis (Arabidopsis thaliana) and tomato (Solanum lycopersicum) hypocotyls. In tomato, this increase was induced by low-fluence red or blue light followed by 1 d of darkness. It was reduced in phyA, phyB1, and phyB2 tomato mutants and was reversed by far-red light applied immediately after the red or blue light exposure, suggesting that phytochrome is involved in this response. We further found that the free indole-3-acetic acid (IAA) level in hypocotyl regions below the hook was increased by red light, while the level of conjugated IAA was unchanged. Analysis of IAA synthesized from [¹³C]indole or [¹³C]tryptophan (Trp) revealed that both Trp-dependent and Trp-independent IAA biosynthesis were increased by low-fluence red light in the top section (meristem, cotyledons, and hook), and the Trp-independent pathway appears to become the primary route for IAA biosynthesis after red light exposure. IAA biosynthesis in tissues below the top section was not affected by red light, suggesting that the increase of free IAA in this region was due to increased transport of IAA from above. Our study provides a comprehensive view of light effects on the transport and biosynthesis of IAA, showing that red light increases both IAA biosynthesis in the top section and polar auxin transport in hypocotyls, leading to unchanged free IAA levels in the top section and increased free IAA levels in the lower hypocotyl regions.

  2. Importance of nondiffusive transport for soil CO2 efflux in a temperate mountain grassland.

    Science.gov (United States)

    Roland, Marilyn; Vicca, Sara; Bahn, Michael; Ladreiter-Knauss, Thomas; Schmitt, Michael; Janssens, Ivan A

    2015-03-01

    Soil respiration and its biotic and abiotic drivers have been an important research topic in recent years. While the bulk of these efforts has focused on the emission of CO2 from soils, the production and subsequent transport of CO2 from soil to atmosphere received far less attention. However, to understand processes underlying emissions of CO2 from terrestrial ecosystems, both processes need to be fully evaluated. In this study, we tested to what extent the transport of CO2 in a grassland site in the Austrian Alps could be modeled based on the common assumption that diffusion is the main transport mechanism for trace gases in soils. Therefore, we compared the CO2 efflux calculated from the soil CO2 concentration gradient with the CO2 efflux from chamber measurements. We used four commonly used diffusion-driven models for the flux-gradient approach. Models generally underestimated the soil chamber effluxes and their amplitudes, indicating that processes other than diffusion were responsible for the transport of CO2. We further observed that transport rates correlated well with irradiation and, below a soil moisture content of 33%, with wind speed. This suggests that mechanisms such as bulk soil air transport, due to pressure pumping or thermal expansion of soil air due to local surface heating, considerably influence soil CO2 transport at this site. Our results suggest that nondiffusive transport may be an important mechanism influencing diel and day-to-day dynamics of soil CO2 emissions, leading to a significant mismatch (10-87% depending on the model used) between the two approaches at short time scales.

  3. A rho scaffold integrates the secretory system with feedback mechanisms in regulation of auxin distribution.

    Directory of Open Access Journals (Sweden)

    Ora Hazak

    2010-01-01

    Full Text Available Development in multicellular organisms depends on the ability of individual cells to coordinate their behavior by means of small signaling molecules to form correctly patterned tissues. In plants, a unique mechanism of directional transport of the signaling molecule auxin between cells connects cell polarity and tissue patterning and thus is required for many aspects of plant development. Direction of auxin flow is determined by polar subcellular localization of PIN auxin efflux transporters. Dynamic PIN polar localization results from the constitutive endocytic cycling to and from the plasma membrane, but it is not well understood how this mechanism connects to regulators of cell polarity. The Rho family small GTPases ROPs/RACs are master regulators of cell polarity, however their role in regulating polar protein trafficking and polar auxin transport has not been established. Here, by analysis of mutants and transgenic plants, we show that the ROP interactor and polarity regulator scaffold protein ICR1 is required for recruitment of PIN proteins to the polar domains at the plasma membrane. icr1 mutant embryos and plants display an a array of severe developmental aberrations that are caused by compromised differential auxin distribution. ICR1 functions at the plasma membrane where it is required for exocytosis but does not recycle together with PINs. ICR1 expression is quickly induced by auxin but is suppressed at the positions of stable auxin maxima in the hypophysis and later in the embryonic and mature root meristems. Our results imply that ICR1 is part of an auxin regulated positive feedback loop realized by a unique integration of auxin-dependent transcriptional regulation into ROP-mediated modulation of cell polarity. Thus, ICR1 forms an auxin-modulated link between cell polarity, exocytosis, and auxin transport-dependent tissue patterning.

  4. myo-Inositol-1-phosphate synthase is required for polar auxin transport and organ development

    KAUST Repository

    Chen, Hao

    2010-06-01

    myo-Inositol-1-phosphate synthase is a conserved enzyme that catalyzes the first committed and rate-limiting step in inositol biosynthesis. Despite its wide occurrence in all eukaryotes, the role of myo-inositol-1-phosphate synthase and de novo inositol biosynthesis in cell signaling and organism development has been unclear. In this study, we isolated loss-of-function mutants in the Arabidopsis MIPS1 gene from different ecotypes. It was found that all mips1 mutants are defective in embryogenesis, cotyledon venation patterning, root growth, and root cap development. The mutant roots are also agravitropic and have reduced basipetal auxin transport. mips1 mutants have significantly reduced levels of major phosphatidylinositols and exhibit much slower rates of endocytosis. Treatment with brefeldin A induces slower PIN2 protein aggregation in mips1, indicating altered PIN2 trafficking. Our results demonstrate that MIPS1 is critical for maintaining phosphatidylinositol levels and affects pattern formation in plants likely through regulation of auxin distribution. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc.

  5. Expression of ABC Efflux transporters in placenta from women with insulin-managed diabetes.

    Science.gov (United States)

    Anger, Gregory J; Cressman, Alex M; Piquette-Miller, Micheline

    2012-01-01

    Drug efflux transporters in the placenta can significantly influence the materno-fetal transfer of a diverse array of drugs and other xenobiotics. To determine if clinically important drug efflux transporter expression is altered in pregnancies complicated by gestational diabetes mellitus (GDM-I) or type 1 diabetes mellitus (T1DM-I), we compared the expression of multidrug resistance protein 1 (MDR1), multidrug resistance-associated protein 2 (MRP2) and the breast cancer resistance protein (BCRP) via western blotting and quantitative real-time polymerase chain reaction in samples obtained from insulin-managed diabetic pregnancies to healthy term-matched controls. At the level of mRNA, we found significantly increased expression of MDR1 in the GDM-I group compared to both the T1DM-I (pchanges in the placental protein expression of MDR1, MRP2, and BCRP were not detected (p>0.05). Interestingly, there was a significant, positive correlation observed between plasma hemoglobin A1c levels (a retrospective marker of glycemic control) and both BCRP protein expression (r = 0.45, pmanaged DM groups. Collectively, the data suggest that the expression of placental efflux transporters is not altered in pregnancies complicated by diabetes when hyperglycemia is managed; however, given the relationship between BCRP expression and plasma hemoglobin A1c levels it is plausible that their expression could change in poorly managed diabetes.

  6. Influence of the gastrointestinal microflora and efflux transporters on the absorption of scutellarin and scutellarein.

    Science.gov (United States)

    You, Hai-Sheng; Xing, Jian-Feng; Lu, Jun; Dong, Wei-Hua; Liu, Jun-Tian; Dong, Ya-Lin

    2014-09-01

    Scutellarin (SG) and its aglycone, Scutellarein (S), are flavonoids of therapeutic cardiocerebrovascular disease. SG was hydrolyzed by bacterial enzyme into S which was absorbed in the intestine. The aim of this study was to determine the effects of the microflora in the intestinal lumen and the efflux transporter of intestinal epithelial cells on the absorption process of SG and S. After oral administration of antibiotics in Sprague-Dawley rats, the reduced bacterial enzyme formation significantly hinders the absorption of SG, whereas scarcely that of S. The absorption study in situ single-pass intestinal perfusion revealed that S could be absorbed throughout the intestine of rats. The effective intestinal permeability of S in the jejunum was much lower than in the other sections of the GI tract. The efflux transporter promoted SG secretion into lumen from enterocytes, which hindered the absorption of both SG and S into the bloodstream. The efflux transporter protein inhibitor (verapamil, probenecid and reserpine) remarkably enhanced the absorption of S and the bioconversion of S into SG in both the rat intestine and Caco-2-monolayer models.

  7. Low levels of graphene and graphene oxide inhibit cellular xenobiotic defense system mediated by efflux transporters.

    Science.gov (United States)

    Liu, Su; Jiang, Wei; Wu, Bing; Yu, Jing; Yu, Haiyan; Zhang, Xu-Xiang; Torres-Duarte, Cristina; Cherr, Gary N

    2016-01-01

    Low levels of graphene and graphene oxide (GO) are considered to be environmentally safe. In this study, we analyzed the potential effects of graphene and GO at relatively low concentrations on cellular xenobiotic defense system mediated by efflux transporters. The results showed that graphene (graphene and GO at the nontoxic concentrations could increase calcein-AM (CAM, an indicator of membrane ATP-binding cassette (ABC) transporter) activity) accumulation, indicating inhibition of ABC transporters' efflux capabilities. This inhibition was observed even at 0.005 μg/mL graphene and 0.05 μg/mL GO, which are 100 times and 400 times lower than their lowest toxic concentration from cytotoxicity experiments, respectively. The inhibition of ABC transporters significantly increased the toxicity of paraquat and arsenic, known substrates of ABC transporters. The inhibition of ABC transporters was found to be based on graphene and GO damaging the plasma membrane structure and fluidity, thus altering functions of transmembrane ABC transporters. This study demonstrates that low levels of graphene and GO are not environmentally safe since they can significantly make cell more susceptible to other xenobiotics, and this chemosensitizing activity should be considered in the risk assessment of graphene and GO.

  8. Polar transport of auxin across gravistimulated roots of maize and its enhancement by calcium

    Science.gov (United States)

    Lee, J. S.; Evans, M. L.

    1985-01-01

    The effect of Ca on the polar movement of [3H]indoleacetic acid ([3H]IAA) in gravistimulated roots was examined using 3-day-old seedlings of maize (Zea mays L.). Transport of label was measured by placing an agar donor block containing [3H]IAA on one side of the elongation zone and measuring movement of label across the root into an agar receiver block on the opposite side. In vertically oriented roots, movement of label across the elongation zone into the receiver was slight and was not enhanced by incorporating 10 millimolar CaCl2 into the receiver block. In horizontally oriented roots, movement of label across the root was readily detectable and movement to a receiver on the bottom was about 3-fold greater than movement in the opposite direction. This polarity was abolished in roots from which the caps were removed prior to gravistimulation. When CaCl2 was incorporated into the receivers, movement of label across horizontally oriented intact roots was increased about 3-fold in both the downward and upward direction. The ability of Ca to enhance the movement of label from [3H]IAA increased with increasing Ca concentration in the receiver up to 5 to 10 millimolar CaCl2. With the inclusion of CaCl2 in the receiver blocks, gravity-induced polar movement of label into receiver blocks from applied [3H]IAA was detectable within 30 minutes, and asymmetric distribution of label within the tissue was detectable within 20 minutes. The results indicate that gravistimulation induces a physiological asymmetry in the auxin transport system of maize roots and that Ca increases the total transport of auxin across the root.

  9. ROTUNDA3 function in plant development by phosphatase 2A-mediated regulation of auxin transporter recycling.

    Science.gov (United States)

    Karampelias, Michael; Neyt, Pia; De Groeve, Steven; Aesaert, Stijn; Coussens, Griet; Rolčík, Jakub; Bruno, Leonardo; De Winne, Nancy; Van Minnebruggen, Annemie; Van Montagu, Marc; Ponce, María Rosa; Micol, José Luis; Friml, Jiří; De Jaeger, Geert; Van Lijsebettens, Mieke

    2016-03-08

    The shaping of organs in plants depends on the intercellular flow of the phytohormone auxin, of which the directional signaling is determined by the polar subcellular localization of PIN-FORMED (PIN) auxin transport proteins. Phosphorylation dynamics of PIN proteins are affected by the protein phosphatase 2A (PP2A) and the PINOID kinase, which act antagonistically to mediate their apical-basal polar delivery. Here, we identified the ROTUNDA3 (RON3) protein as a regulator of the PP2A phosphatase activity in Arabidopsis thaliana. The RON3 gene was map-based cloned starting from the ron3-1 leaf mutant and found to be a unique, plant-specific gene coding for a protein with high and dispersed proline content. The ron3-1 and ron3-2 mutant phenotypes [i.e., reduced apical dominance, primary root length, lateral root emergence, and growth; increased ectopic stages II, IV, and V lateral root primordia; decreased auxin maxima in indole-3-acetic acid (IAA)-treated root apical meristems; hypergravitropic root growth and response; increased IAA levels in shoot apices; and reduced auxin accumulation in root meristems] support a role for RON3 in auxin biology. The affinity-purified PP2A complex with RON3 as bait suggested that RON3 might act in PIN transporter trafficking. Indeed, pharmacological interference with vesicle trafficking processes revealed that single ron3-2 and double ron3-2 rcn1 mutants have altered PIN polarity and endocytosis in specific cells. Our data indicate that RON3 contributes to auxin-mediated development by playing a role in PIN recycling and polarity establishment through regulation of the PP2A complex activity.

  10. Caveolin-1 and ATP binding cassette transporter A1 and G1-mediated cholesterol efflux.

    Science.gov (United States)

    Wang, Faqi; Gu, Hong-mei; Zhang, Da-wei

    2014-01-01

    Atherosclerosis is one major cause of cardiovascular diseases, the leading cause of death in industrialized countries. Reverse cholesterol transport (RCT) is thought to be one primary pathway to protect against atherosclerosis. The first and rate-limiting step of RCT is ATP-binding cassette transport A1 (ABCA1) and ABCG1-mediated cholesterol efflux from the cells. Recently, caveolin-1 (CAV1), a scaffolding protein that organizes and concentrates certain caveolin-interacting signaling molecules and receptors within caveolae membranes, has been shown to regulate ABCA1 and ABCG1-mediated cholesterol efflux probably via interacting with them. In the present review, we summarize the current knowledge and views on the regulatory role of CAV1 on the cholesterol homeostasis with emphasis on the association of CAV1 with ABCA1 and ABCG1. We conclude that the dominance of the positive regulation by CAV1 on the ABCA1 and ABCG1-mediated cholesterol efflux is depending on the species, cell types, as well as the levels of CAV1 expression.

  11. Decarboxylation and transport of auxin in segments of sunflower and cabbage roots.

    Science.gov (United States)

    Iversen, T H; Aasheim, T

    1970-12-01

    The movement of (14)C from indole-3-acetic acid (IAA) (14)C has been examined in 5 mm root segments of dark-grown seedlings of Helianthus annuus and Brassica oleracea. Contaminants from distilled water, phosphate buffer and the razor-blade cutter increase the decarboxylation of IAA-(14)C, and cutting of root segments results in an activation of IAA-destroying enzymes at the cut surfaces. When these sources of errors were eliminated the following was shown: a) Both in sunflower and cabbage there is a slight acropetal flux of (14)C through the root segments into the agar receiver blocks. The amount of (14)C found in the receiver blocks increases with the lenght of the transport period. b) When the root segments, after the transport period, are cut in two equal parts and these assayed separately, the amounts of (14)C in the two parts indicate a greater acropetal than basipetal transport. c) The total radioactivity of the receiver blocks is in part due to IAA-(14)C and in part to (14)CO2, the latter being a result of enzymatic destruction of auxin. d) Addition of ferulic acid, an inhibitor of IAA oxidases, to the receiver blocks markedly inhibits the decarboxylation of IAA-(14)C and thus increases the amount transported. This effect is more pronounced after a 20 hr than after a 6 hr transport period.

  12. Flavonol-induced changes in PIN2 polarity and auxin transport in the Arabidopsis thaliana rol1-2 mutant require phosphatase activity

    Science.gov (United States)

    Kuhn, Benjamin M.; Nodzyński, Tomasz; Errafi, Sanae; Bucher, Rahel; Gupta, Shibu; Aryal, Bibek; Dobrev, Petre; Bigler, Laurent; Geisler, Markus; Zažímalová, Eva; Friml, Jiří; Ringli, Christoph

    2017-01-01

    The phytohormone auxin is a major determinant and regulatory component important for plant development. Auxin transport between cells is mediated by a complex system of transporters such as AUX1/LAX, PIN, and ABCB proteins, and their localization and activity is thought to be influenced by phosphatases and kinases. Flavonols have been shown to alter auxin transport activity and changes in flavonol accumulation in the Arabidopsis thaliana rol1-2 mutant cause defects in auxin transport and seedling development. A new mutation in ROOTS CURL IN NPA 1 (RCN1), encoding a regulatory subunit of the phosphatase PP2A, was found to suppress the growth defects of rol1-2 without changing the flavonol content. rol1-2 rcn1-3 double mutants show wild type-like auxin transport activity while levels of free auxin are not affected by rcn1-3. In the rol1-2 mutant, PIN2 shows a flavonol-induced basal-to-apical shift in polar localization which is reversed in the rol1-2 rcn1-3 to basal localization. In vivo analysis of PINOID action, a kinase known to influence PIN protein localization in a PP2A-antagonistic manner, revealed a negative impact of flavonols on PINOID activity. Together, these data suggest that flavonols affect auxin transport by modifying the antagonistic kinase/phosphatase equilibrium. PMID:28165500

  13. A novel putative auxin carrier family regulates intracellular auxin homeostasis in plants

    OpenAIRE

    2012-01-01

    The phytohormone auxin acts as a prominent signal, providing, by its local accumulation or depletion in selected cells, a spatial and temporal reference for changes in the developmental program. The distribution of auxin depends on both auxin metabolism (biosynthesis, conjugation and degradation) and cellular auxin transport. We identified in silico a novel putative auxin transport facilitator family, called PIN-LIKES (PILS). Here we illustrate that PILS proteins are required for auxin-depend...

  14. Auxin as an inducer of asymmetrical division generating the subsidiary cells in stomatal complexes of Zea mays.

    Science.gov (United States)

    Livanos, Pantelis; Giannoutsou, Eleni; Apostolakos, Panagiotis; Galatis, Basil

    2015-01-01

    The data presented in this work revealed that in Zea mays the exogenously added auxins indole-3-acetic acid (IAA) and 1-napthaleneacetic acid (NAA), promoted the establishment of subsidiary cell mother cell (SMC) polarity and the subsequent subsidiary cell formation, while treatment with auxin transport inhibitors 2,3,5-triiodobenzoic acid (TIBA) and 1-napthoxyacetic acid (NOA) specifically blocked SMC polarization and asymmetrical division. Furthermore, in young guard cell mother cells (GMCs) the PIN1 auxin efflux carriers were mainly localized in the transverse GMC faces, while in the advanced GMCs they appeared both in the transverse and the lateral ones adjacent to SMCs. Considering that phosphatidyl-inositol-3-kinase (PI3K) is an active component of auxin signal transduction and that phospholipid signaling contributes in the establishment of polarity, treatments with the specific inhibitor of the PI3K LY294002 were carried out. The presence of LY294002 suppressed polarization of SMCs and prevented their asymmetrical division, whereas combined treatment with exogenously added NAA and LY294002 restricted the promotional auxin influence on subsidiary cell formation. These findings support the view that auxin is involved in Z. mays subsidiary cell formation, probably functioning as inducer of the asymmetrical SMC division. Collectively, the results obtained from treatments with auxin transport inhibitors and the appearance of PIN1 proteins in the lateral GMC faces indicate a local transfer of auxin from GMCs to SMCs. Moreover, auxin signal transduction seems to be mediated by the catalytic function of PI3K.

  15. The cotton transcription factor TCP14 functions in auxin-mediated epidermal cell differentiation and elongation.

    Science.gov (United States)

    Wang, Miao-Ying; Zhao, Pi-Ming; Cheng, Huan-Qing; Han, Li-Bo; Wu, Xiao-Min; Gao, Peng; Wang, Hai-Yun; Yang, Chun-Lin; Zhong, Nai-Qin; Zuo, Jian-Ru; Xia, Gui-Xian

    2013-07-01

    Plant-specific TEOSINTE-BRANCHED1/CYCLOIDEA/PCF (TCP) transcription factors play crucial roles in development, but their functional mechanisms remain largely unknown. Here, we characterized the cellular functions of the class I TCP transcription factor GhTCP14 from upland cotton (Gossypium hirsutum). GhTCP14 is expressed predominantly in fiber cells, especially at the initiation and elongation stages of development, and its expression increased in response to exogenous auxin. Induced heterologous overexpression of GhTCP14 in Arabidopsis (Arabidopsis thaliana) enhanced initiation and elongation of trichomes and root hairs. In addition, root gravitropism was severely affected, similar to mutant of the auxin efflux carrier PIN-FORMED2 (PIN2) gene. Examination of auxin distribution in GhTCP14-expressing Arabidopsis by observation of auxin-responsive reporters revealed substantial alterations in auxin distribution in sepal trichomes and root cortical regions. Consistent with these changes, expression of the auxin uptake carrier AUXIN1 (AUX1) was up-regulated and PIN2 expression was down-regulated in the GhTCP14-expressing plants. The association of GhTCP14 with auxin responses was also evidenced by the enhanced expression of auxin response gene IAA3, a gene in the AUXIN/INDOLE-3-ACETIC ACID (Aux/IAA) family. Electrophoretic mobility shift assays showed that GhTCP14 bound the promoters of PIN2, IAA3, and AUX1, and transactivation assays indicated that GhTCP14 had transcription activation activity. Taken together, these results demonstrate that GhTCP14 is a dual-function transcription factor able to positively or negatively regulate expression of auxin response and transporter genes, thus potentially acting as a crucial regulator in auxin-mediated differentiation and elongation of cotton fiber cells.

  16. Anti-cancer drug disposition: In vitro and in vivo functions of ABC efflux and OATP uptake transporters

    NARCIS (Netherlands)

    Durmus, S.

    2014-01-01

    ATP-binding cassette (ABC) efflux and Organic Anion-Transporting Polypeptide (OATP) uptake transporters are two major membrane transporter superfamilies that have major roles in the absorption, disposition and toxicity of drugs. They are widely expressed in several pharmacokinetically relevant organ

  17. Voluntary exercise increases cholesterol efflux but not macrophage reverse cholesterol transport in vivo in mice

    Directory of Open Access Journals (Sweden)

    Kuipers Folkert

    2010-07-01

    Full Text Available Abstract Physical exercise beneficially impacts on the plasma lipoprotein profile as well as on the incidence of cardiovascular events and is therefore recommended in primary and secondary prevention strategies against atherosclerotic cardiovascular disease. However, the underlying mechanisms of the protective effect of exercise remain largely unknown. Therefore, the present study tested the hypothesis that voluntary exercise in mice impacts on cholesterol efflux and in vivo reverse cholesterol transport (RCT. After two weeks of voluntary wheel running (average 10.1 ± 1.4 km/day plasma triglycerides were lower (p

  18. Sucrose efflux mediated by SWEET proteins as a key step for phloem transport.

    Science.gov (United States)

    Chen, Li-Qing; Qu, Xiao-Qing; Hou, Bi-Huei; Sosso, Davide; Osorio, Sonia; Fernie, Alisdair R; Frommer, Wolf B

    2012-01-13

    Plants transport fixed carbon predominantly as sucrose, which is produced in mesophyll cells and imported into phloem cells for translocation throughout the plant. It is not known how sucrose migrates from sites of synthesis in the mesophyll to the phloem, or which cells mediate efflux into the apoplasm as a prerequisite for phloem loading by the SUT sucrose-H(+) (proton) cotransporters. Using optical sucrose sensors, we identified a subfamily of SWEET sucrose efflux transporters. AtSWEET11 and 12 localize to the plasma membrane of the phloem. Mutant plants carrying insertions in AtSWEET11 and 12 are defective in phloem loading, thus revealing a two-step mechanism of SWEET-mediated export from parenchyma cells feeding H(+)-coupled import into the sieve element-companion cell complex. We discuss how restriction of intercellular transport to the interface of adjacent phloem cells may be an effective mechanism to limit the availability of photosynthetic carbon in the leaf apoplasm in order to prevent pathogen infections.

  19. Substrate-dependent dynamics of the multidrug efflux transporter AcrB of Escherichia coli.

    Science.gov (United States)

    Yamamoto, Kentaro; Tamai, Rei; Yamazaki, Megumi; Inaba, Takehiko; Sowa, Yoshiyuki; Kawagishi, Ikuro

    2016-02-26

    The resistance-nodulation-cell division (RND)-type xenobiotic efflux system plays a major role in the multidrug resistance of gram-negative bacteria. The only constitutively expressed RND system of Escherichia coli consists of the inner membrane transporter AcrB, the membrane fusion protein AcrA, and the outer membrane channel TolC. The latter two components are shared with another RND-type transporter AcrD, whose expression is induced by environmental stimuli. Here, we demonstrate how RND-type ternary complexes, which span two membranes and the cell wall, form in vivo. Total internal reflection fluorescence (TIRF) microscopy revealed that most fluorescent foci formed by AcrB fused to green fluorescent protein (GFP) were stationary in the presence of TolC but showed lateral displacements when tolC was deleted. The fraction of stationary AcrB-GFP foci decreased with increasing levels of AcrD. We propose that the AcrB-containing complex becomes unstable upon the induction of AcrD, which presumably replaces AcrB, a process we call "transporter exchange." This instability is suppressed by AcrB-specific substrates, suggesting that the ternary complex is stabilised when it is in action. These results suggest that the assembly of the RND-type efflux system is dynamically regulated in response to external stimuli, shedding new light on the adaptive antibiotic resistance of bacteria.

  20. Involvement of organic anion transporters in the efflux of uremic toxins across the blood-brain barrier.

    Science.gov (United States)

    Deguchi, Tsuneo; Isozaki, Kouya; Yousuke, Kouno; Terasaki, Tetsuya; Otagiri, Masaki

    2006-02-01

    Renal failure causes multiple physiological changes involving CNS dysfunction. In cases of uremia, there is close correlation between plasma levels of uremic toxins [e.g. 3-carboxy-4-methyl-5-propyl-2-furanpropionate (CMPF), hippurate (HA) and indoleacetate (IA)] and the degree of uremic encephalopathy, suggesting that uremic toxins are involved in uremic encephalopathy. In order to evaluate the relevance of uremic toxins to CNS dysfunction, we investigated directional transport of uremic toxins across the blood-brain barrier (BBB) using in vivo integration plot analysis and the brain efflux index method. We observed saturable efflux transport of [(3)H]CMPF, [(14)C]HA and [(3)H]IA, which was inhibited by probenecid. For all uremic toxins evaluated, apparent efflux clearance across the BBB was greater than apparent influx clearance, suggesting that these toxins are predominantly transported from the brain to blood across the BBB. Saturable efflux transport of [(3)H]CMPF, [(14)C]HA and [(3)H]IA was completely inhibited by benzylpenicillin, which is a substrate of rat organic anion transporter 3 (rOat3). Taurocholate and digoxin, which are common substrates of rat organic anion transporting polypeptide (rOatp), partially inhibited the efflux of [(3)H]CMPF. Transport experiments using a Xenopus laevis oocyte expression system revealed that CMPF, HA and IA are substrates of rOat3, and that CMPF (but not HA or IA) is a substrate of rOap2. These results suggest that rOat3 mediates brain-to-blood transport of uremic toxins, and that rOatp2 is involved in efflux of CMPF. Thus, conditions typical of uremia can cause inhibition of brain-to-blood transport involving rOat3 and/or rOatp2, leading to accumulation of endogenous metabolites and drugs in the brain.

  1. PKC phosphorylates residues in the N-terminal of the DA transporter to regulate amphetamine-induced DA efflux.

    Science.gov (United States)

    Wang, Qiang; Bubula, Nancy; Brown, Jason; Wang, Yunliang; Kondev, Veronika; Vezina, Paul

    2016-05-27

    The DA transporter (DAT), a phosphoprotein, controls extracellular dopamine (DA) levels in the central nervous system through transport or reverse transport (efflux). Multiple lines of evidence support the claim that PKC significantly contributes to amphetamine-induced DA efflux. Other signaling pathways, involving CaMKII and ERK, have also been shown to regulate DAT mediated efflux. Here we assessed the contribution of putative PKC residues (S4, S7, S13) in the N-terminal of the DAT to amphetamine-induced DA efflux by transfecting DATs containing different serine to alanine (S-A) point mutations into DA pre-loaded HEK-293 cells and incubating these cells in amphetamine (2μM). The effects of a S-A mutation at the non-PKC residue S12 and a threonine to alanine (T-A) mutation at the ERK T53 residue were also assessed for comparison. WT-DATs were used as controls. In an initial experiment, we confirmed that inhibiting PKC with Go6976 (130nM) significantly reduced amphetamine-induced DA efflux. In subsequent experiments, cells transfected with the S4A, S12A, S13A, T53A and S4,7,13A mutants showed a reduction in amphetamine-induced DA efflux similar to that observed with Go6976. Interestingly, cells transfected with the S7A mutant, identified by some as a PKC-PKA residue, showed unperturbed WT-DAT levels of amphetamine-induced DA efflux. These results indicate that phosphorylation by PKC of select residues in the DAT N-terminal can regulate amphetamine-induced efflux. PKC can act either independently or in concert with other kinases such as ERK to produce this effect.

  2. The arabidopsis IDD14, IDD15, and IDD16 cooperatively regulate lateral organ morphogenesis and gravitropism by promoting auxin biosynthesis and transport.

    Directory of Open Access Journals (Sweden)

    Dayong Cui

    Full Text Available The plant hormone auxin plays a critical role in regulating various aspects of plant growth and development, and the spatial accumulation of auxin within organs, which is primarily attributable to local auxin biosynthesis and polar transport, is largely responsible for lateral organ morphogenesis and the establishment of plant architecture. Here, we show that three Arabidopsis INDETERMINATE DOMAIN (IDD transcription factors, IDD14, IDD15, and IDD16, cooperatively regulate auxin biosynthesis and transport and thus aerial organ morphogenesis and gravitropic responses. Gain-of-function of each IDD gene in Arabidopsis results in small and transversally down-curled leaves, whereas loss-of-function of these IDD genes causes pleiotropic phenotypes in aerial organs and defects in gravitropic responses, including altered leaf shape, flower development, fertility, and plant architecture. Further analyses indicate that these IDD genes regulate spatial auxin accumulation by directly targeting YUCCA5 (YUC5, TRYPTOPHAN AMINOTRANSFERASE of ARABIDOPSIS1 (TAA1, and PIN-FORMED1 (PIN1 to promote auxin biosynthesis and transport. Moreover, mutation or ectopic expression of YUC suppresses the organ morphogenic phenotype and partially restores the gravitropic responses in gain- or loss-of-function idd mutants, respectively. Taken together, our results reveal that a subfamily of IDD transcription factors plays a critical role in the regulation of spatial auxin accumulation, thereby controlling organ morphogenesis and gravitropic responses in plants.

  3. P-glycoprotein mediates brain-to-blood efflux transport of buprenorphine across the blood-brain barrier.

    Science.gov (United States)

    Suzuki, Toyofumi; Zaima, Chika; Moriki, Yoshiaki; Fukami, Toshiro; Tomono, Kazuo

    2007-01-01

    The involvement of P-glycoprotein (P-gp) in buprenorphine (BNP) transport at the blood-brain barrier (BBB) in rats was investigated in vivo by means of both the brain uptake index technique and the brain efflux index technique. P-gp inhibitors, such as cyclosporin A, quinidine and verapamil, enhanced the apparent brain uptake of [3H]BNP by 1.5-fold. The increment of the BNP uptake by the brain suggests the involvement of a P-gp efflux mechanism of BNP transport at the BBB. [3H]BNP was eliminated with an apparent elimination half-life of 27.5 min after microinjection into the parietal cortex area 2 regions of the rat brain. The apparent efflux clearance of [3H]BNP across the BBB was 0.154 ml/min/g brain, which was calculated from the elimination rate constant (2.52 x 10- 2 min- 1) and the distribution volume in the brain (6.11 ml/g brain). The efflux transport of [3H]BNP was inhibited by range from 32 to 64% in the presence of P-gp inhibitors. The present results suggest that BNP is transported from the brain across the BBB via a P-gp-mediated efflux transport system, at least in part.

  4. Arabidopsis TWISTED DWARF1 functionally interacts with Auxin Exporter ABCB1 on the root plasma membrane

    DEFF Research Database (Denmark)

    Wang, Bangjun; Bailly, Aurélien; Zwiewka, Marta

    2013-01-01

    Plant architecture is influenced by the polar, cell-to-cell transport of auxin that is primarily provided and regulated by plasma membrane efflux catalysts of the PIN-FORMED and B family of ABC transporter (ABCB) classes. The latter were shown to require the functionality of the FK506 binding...... assays, we demonstrate a predominant lateral, mainly outward-facing, plasma membrane location for TWD1 in the root epidermis characterized by the lateral marker ABC transporter G36/PLEIOTROPIC DRUG-RESISTANCE8/PENETRATION3. At these epidermal plasma membrane domains, TWD1 colocalizes with nonpolar ABCB1....... In planta bioluminescence resonance energy transfer analysis was used to verify specific ABC transporter B1 (ABCB1)-TWD1 interaction. Our data support a model in which TWD1 promotes lateral ABCB-mediated auxin efflux via protein-protein interaction at the plasma membrane, minimizing reflux from the root...

  5. Role of auxin during intercellular infection of Discaria trinervis by Frankia

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    Leandro eImanishi

    2014-08-01

    Full Text Available Nitrogen-fixing nodules induced by Frankia in the actinorhizal plant Discaria trinervis result from a primitive intercellular root invasion pathway that does not involve root hair deformation and infection threads. Here, we analyzed the role of auxin in this intercellular infection pathway at the molecular level and compared it with our previous work in the intracellular infected actinorhizal plant Casuarina glauca. Immunolocalisation experiments showed that auxin accumulated in Frankia-infected cells in both systems. We then characterized the expression of auxin transporters in D. trinervis nodules. No activation of the heterologous CgAUX1 promoter was detected in infected cells in D. trinervis. These results were confirmed with the endogenous D. trinervis gene, DtAUX1. However, DtAUX1 was expressed in the nodule meristem. Consistently, transgenic D. trinervis plants containing the auxin response marker DR5:VENUS showed expression of the reporter gene in the meristem. Immunolocalisation experiments using an antibody against the auxin efflux carrier PIN1, revealed the presence of this transporter in the plasma membrane of infected cells. Finally, we used in silico cellular models to analyse auxin fluxes in D. trinervis nodules. Our results point to the existence of divergent roles of auxin in intercellularly- and intracellularly-infected actinorhizal plants, an ancestral infection pathways leading to root nodule symbioses.

  6. Role of auxin during intercellular infection of Discaria trinervis by Frankia

    Science.gov (United States)

    Imanishi, Leandro; Perrine-Walker, Francine M.; Ndour, Adama; Vayssières, Alice; Conejero, Genevieve; Lucas, Mikaël; Champion, Antony; Laplaze, Laurent; Wall, Luis; Svistoonoff, Sergio

    2014-01-01

    Nitrogen-fixing nodules induced by Frankia in the actinorhizal plant Discaria trinervis result from a primitive intercellular root invasion pathway that does not involve root hair deformation and infection threads. Here, we analyzed the role of auxin in this intercellular infection pathway at the molecular level and compared it with our previous work in the intracellular infected actinorhizal plant Casuarina glauca. Immunolocalisation experiments showed that auxin accumulated in Frankia-infected cells in both systems. We then characterized the expression of auxin transporters in D. trinervis nodules. No activation of the heterologous CgAUX1 promoter was detected in infected cells in D. trinervis. These results were confirmed with the endogenous D. trinervis gene, DtAUX1. However, DtAUX1 was expressed in the nodule meristem. Consistently, transgenic D. trinervis plants containing the auxin response marker DR5:VENUS showed expression of the reporter gene in the meristem. Immunolocalisation experiments using an antibody against the auxin efflux carrier PIN1, revealed the presence of this transporter in the plasma membrane of infected cells. Finally, we used in silico cellular models to analyse auxin fluxes in D. trinervis nodules. Our results point to the existence of divergent roles of auxin in intercellularly- and intracellularly-infected actinorhizal plants, an ancestral infection pathways leading to root nodule symbioses. PMID:25191330

  7. The acropetal effects of indole-3-acetic acid in isolated shoot segments of Acer pseudoplatanus L. II. Possible regulation by a vectorial fieid of auxin waves

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    Jacek A. Adamczyk

    2014-02-01

    Full Text Available The acropetal effects of auxin on elongation of axillary buds and on modulation of the wave-like pattern of basipetal efflux of natural auxin to agar from Acer pseudoplatanus L. shoots were studied. When synthetic IAA was applied to cut surfaces of one of two branches the elongation growth of buds situated on the opposite branch was retarded, suggesting regulation independent of the direct action of the molecules of the applied IAA. Oscillations in basipetal transport of natural auxin along the stem segments were observed corroborating the results of other authors using different tree species. Apical application of synthetic IAA for 1 hour to the lateral branch caused a phase shift of the wave-like pattern of basipetal efflux of natural auxin, when the stem segment above the treated branch was sectioned. The same effect was observed evoked by the laterally growing branch which is interpreted as an effect of natural auxin produced by the actively growing shoot. These modulations could be propagated acropetally at a rate excluding direct action of auxin molecules at the sites of measurement. The results seem to corroborate the hypothesis suggesting that auxin is involved in acropetal regulation of shoot apex growth through its effect upon modulation of the vectorial field which arises when the auxin-waves translocate in cambium.

  8. The effect of NGATHA altered activity in auxin signaling pathways within the Arabidopsis gynoecium

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    Irene eMartinez-Fernandez

    2014-05-01

    Full Text Available The four NGATHA genes (NGA form a small subfamily within the large family of B3-domain transcription factors of Arabidopsis thaliana. NGA genes act redundantly to direct the development of the apical tissues of the gynoecium, the style and the stigma. Previous studies indicate that NGA genes could exert this function at least partially by directing the synthesis of auxin at the distal end of the developing gynoecium through the upregulation of two different YUCCA genes, which encode flavin monooxygenases involved in auxin biosynthesis. We have compared three developing pistil transcriptome data sets from wildtype, nga quadruple mutants and a 35S::NGA3 line. The differentially expressed genes showed a significant enrichment for auxin-related genes, supporting the idea of NGA genes as major regulators of auxin accumulation and distribution within the developing gynoecium.We have introduced reporter lines for several of these differentially expressed genes involved in synthesis, transport and response to auxin in NGA gain- and loss-of-function backgrounds. We present here a detailed map of the response of these reporters to NGA misregulation that could help to clarify the role of NGA in auxin-mediated gynoecium morphogenesis. Our data point to a very reduced auxin synthesis in the developing apical gynoecium of nga mutants, likely responsible for the lack of DR5rev::GFP reporter activity observed in these mutants. In addition, NGA altered activity affects the expression of protein kinases that regulate the cellular localization of auxin efflux regulators, and thus likely impact auxin transport. Finally, protein accumulation in pistils of several ARFs was differentially affected by nga mutations or NGA overexpression, suggesting that these accumulation patterns depend not only on auxin distribution but could be also regulated by transcriptional networks involving NGA factors.

  9. Cellular Auxin Homeostasis:Gatekeeping Is Housekeeping

    Institute of Scientific and Technical Information of China (English)

    Michel Ruiz Rosquete; Elke Barbez; Jürgen Kleine-Vehn

    2012-01-01

    The phytohormone auxin is essential for plant development and contributes to nearly every aspect of the plant life cycle.The spatio-temporal distribution of auxin depends on a complex interplay between auxin metabolism and cell-to-cell auxin transport.Auxin metabolism and transport are both crucial for plant development;however,it largely remains to be seen how these processes are integrated to ensure defined cellular auxin levels or even gradients within tissues or organs.In this review,we provide a glance at very diverse topics of auxin biology,such as biosynthesis,conjugation,oxidation,and transport of auxin.This broad,but certainly superficial,overview highlights the mutual importance of auxin metabolism and transport.Moreover,it allows pinpointing how auxin metabolism and transport get integrated to jointly regulate cellular auxin homeostasis.Even though these processes have been so far only separately studied,we assume that the phytohormonal crosstalk integrates and coordinates auxin metabolism and transport.Besides the integrative power of the global hormone signaling,we additionally introduce the hypothetical concept considering auxin transport components as gatekeepers for auxin responses.

  10. Effective Cellular Uptake and Efflux of Thyroid Hormone by Human Monocarboxylate Transporter 10

    Science.gov (United States)

    Friesema, Edith C. H.; Jansen, Jurgen; Jachtenberg, Jan-willem; Visser, W. Edward; Kester, Monique H. A.; Visser, Theo J.

    2008-01-01

    Cellular entry of thyroid hormone is mediated by plasma membrane transporters, among others a T-type (aromatic) amino acid transporter. Monocarboxylate transporter 10 (MCT10) has been reported to transport aromatic amino acids but not iodothyronines. Within the MCT family, MCT10 is most homologous to MCT8, which is a very important iodothyronine transporter but does not transport amino acids. In view of this paradox, we decided to reinvestigate the possible transport of thyroid hormone by human (h) MCT10 in comparison with hMCT8. Transfection of COS1 cells with hMCT10 cDNA resulted in 1) the production of an approximately 55 kDa protein located to the plasma membrane as shown by immunoblotting and confocal microscopy, 2) a strong increase in the affinity labeling of intracellular type I deiodinase by N-bromoacetyl-[125I]T3, 3) a marked stimulation of cellular T4 and, particularly, T3 uptake, 4) a significant inhibition of T3 uptake by phenylalanine, tyrosine, and tryptophan of 12.5%, 22.2%, and 51.4%, respectively, and 5) a marked increase in the intracellular deiodination of T4 and T3 by different deiodinases. Cotransfection studies using the cytosolic thyroid hormone-binding protein μ-crystallin (CRYM) indicated that hMCT10 facilitates both cellular uptake and efflux of T4 and T3. In the absence of CRYM, hMCT10 and hMCT8 increased T3 uptake after 5 min incubation up to 4.0- and 1.9-fold, and in the presence of CRYM up to 6.9- and 5.8-fold, respectively. hMCT10 was less active toward T4 than hMCT8. These findings establish that hMCT10 is at least as active a thyroid hormone transporter as hMCT8, and that both transporters facilitate iodothyronine uptake as well as efflux. PMID:18337592

  11. Advances on Carriers of Plant Auxin Transport%植物生长素运输载体研究进展

    Institute of Scientific and Technical Information of China (English)

    陈晓阳; 谢文军; 罗海山; 罗红兵

    2011-01-01

    生长素运输载体通过影响极性运输而作用于植物组织器官的形成、生长等过程.植物中存在三种类型生长素运输载体:AUX1(Auxin Resistant)蛋白、PIN (PIN-FORMED)蛋白家族和MDR/PGP(Multidrug-Resistant/P-glycoprotein)蛋白家族.对拟南芥、玉米、水稻中生长素运输载体的最新研究进行了综述,并对未来可能研究领域提出了展望.%Auxin transport carriers modulate organogenesis and growth by affecting polar auxin transport. Three types of auxin transport carriers have been identified: AUX1 (Auxin Resistant) protein PIN (PIN-FORMED) protein families and MDR/PGP(Multidrug-Resistant/P-glycoprotein) protein families. Recent advances on auxin transport carriers in Arabidopsis, Maize.Rice are summarized,and research perspectives in this field are discussed in this review.

  12. Comprehensive Analysis and Expression Profiling of the OsLAX and OsABCB Auxin Transporter Gene Families in Rice (Oryza sativa under Phytohormone Stimuli and Abiotic Stresses

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    Chenglin eChai

    2016-05-01

    Full Text Available The plant hormone auxin regulates many aspects of plant growth and developmental processes. Auxin gradient is formed in plant as a result of polar auxin transportation by three types of auxin transporters such as OsLAX, OsPIN, and OsABCB. We report here the analysis of two rice auxin transporter gene families, OsLAX and OsABCB, using bioinformatics tools, publicly accessible microarray data, and quantitative RT-PCR. There are 5 putative OsLAXs and 22 putative OsABCBs in rice genome, which were mapped on 8 chromosomes. The exon-intron structure of OsLAX genes and properties of deduced proteins were relatively conserved within grass family, while that of OsABCB genes varied greatly. Both constitutive and organ/tissue specific expression patterns were observed in OsLAXs and OsABCBs. Analysis of evolutionarily closely related gene pairs together with organ/tissue specific expression revealed possible function gaining and function losing events during rice evolution. Most OsLAX and OsABCB genes were regulated by drought and salt stress, as well as hormonal stimuli [auxin and Abscisic Acid (ABA], which suggests extensive crosstalk between abiotic stresses and hormone signaling pathways. The existence of large number of auxin and stress related cis-regulatory elements in promoter regions might account for their massive responsiveness of these genes to these environmental stimuli, indicating complexity of regulatory networks involved in various developmental and physiological processes. The comprehensive analysis of OsLAX and OsABCB auxin transporter genes in this study would be helpful for understanding the biological significance of these gene families in hormone signaling and adaptation of rice plants to unfavorable environments.

  13. Auxin polar transport of etiolated Ageotropum pea epicotyls is not affected by gravistimulation: Relevance to automorphosis-like growth and development

    Science.gov (United States)

    Miyamoto, Kensuke; Hoshino, Tomoki; Takahashi, Yoshinori; Ueda, Junichi

    There appears to be a close relationship between automorphosis and changes in auxin polar transport due to the fact that microgravity conditions cause both changes in the activity of auxin polar transport and in automorphosis of etiolated Alaska pea epicotyls. In addition, the application of inhibitors of auxin polar transport results in automorphosis-like growth and development. To elucidate the role of auxin polar transport in gravimorphogenesis in etiolated pea seedlings, we have studied the effects of gravistimulation on growth and development, and auxin polar transport in epicotyls of an agravitropic pea mutant " Ageotropum" seedlings and the normal "Alaska" seedlings. When the embryo axes in seeds of Alaska pea were set in a vertical (parallel to the direction of gravity) or a horizontal (vertical to the direction of gravity) position, and allowed to germinate and grow under 1 g conditions in the dark for 3 or 6.5 days, the epicotyls grew upward due to negative gravitropic responses regardless of gravistimulation during seed germination. On the other hand, epicotyls of etiolated Ageotropum pea seedlings showed automorphosis-like bending away from the cotyledons regardless of gravistimulation during seed germination. Automorphosis-like epicotyl bending of etiolated Ageotropum pea seedlings was also unaffected by clinorotation on a three-dimensional (3-D) clinostat. The activity of auxin polar transport in the 2nd internodes of 6.5-d-old etiolated Ageotropum pea seedlings was lower than those of Alaska pea seedlings, and was not affected by clinorotation on a 3-D clinostat or by changes in gravity conditions during seed germination. These findings strongly support our previous studies that showed that normal auxin polar transport is required for the normal graviresponse of epicotyls in etiolated pea seedlings.

  14. Raltegravir permeability across blood-tissue barriers and the potential role of drug efflux transporters.

    Science.gov (United States)

    Hoque, M Tozammel; Kis, Olena; De Rosa, María F; Bendayan, Reina

    2015-05-01

    The objectives of this study were to investigate raltegravir transport across several blood-tissue barrier models and the potential interactions with drug efflux transporters. Raltegravir uptake, accumulation, and permeability were evaluated in vitro in (i) P-glycoprotein (P-gp), breast cancer resistance protein (BCRP), multidrug resistance-associated protein 1 (MRP1), or MRP4-overexpressing MDA-MDR1 (P-gp), HEK-ABCG2, HeLa-MRP1, or HEK-MRP4 cells, respectively; (ii) cell culture systems of the human blood-brain (hCMEC/D3), mouse blood-testicular (TM4), and human blood-intestinal (Caco-2) barriers; and (iii) rat jejunum and ileum segments using an in situ single-pass intestinal perfusion model. [(3)H]Raltegravir accumulation by MDA-MDR1 (P-gp) and HEK-ABCG2-overexpressing cells was significantly enhanced in the presence of PSC833 {6-[(2S,4R,6E)-4-methyl-2-(methylamino)-3-oxo-6-octenoic acid]-7-L-valine-cyclosporine}, a P-gp inhibitor, or Ko143 [(3S,6S,12aS)-1,2,3,4,6,7,12,12a-octahydro-9-methoxy-6-(2-methylpropyl)-1,4-dioxopyrazino[1',2':1,6]pyrido[3,4-b]indole-3-propanoic acid 1,1-dimethylethyl ester], a BCRP inhibitor, suggesting the inhibition of a P-gp- or BCRP-mediated efflux process, respectively. Furthermore, [(3)H]raltegravir accumulation by human cerebral microvessel endothelial hCMEC/D3 and mouse Sertoli TM4 cells was significantly increased by PSC833 and Ko143. In human intestinal Caco-2 cells grown on Transwell filters, PSC833, but not Ko143, significantly decreased the [(3)H]raltegravir efflux ratios. In rat intestinal segments, [(3)H]raltegravir in situ permeability was significantly enhanced by the concurrent administration of PSC833 and Ko143. In contrast, in the transporter inhibition assays, raltegravir (10 to 500 μM) did not increase the accumulation of substrate for P-gp (rhodamine-6G), BCRP ([(3)H]mitoxantrone), or MRP1 [2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF)] by MDA-MDR1 (P-gp)-, HEK-ABCG2-, or HeLa-MRP1-overexpressing

  15. Bacterial glyphosate resistance conferred by overexpression of an E. coli membrane efflux transporter.

    Science.gov (United States)

    Staub, Jeffrey M; Brand, Leslie; Tran, Minhtien; Kong, Yifei; Rogers, Stephen G

    2012-04-01

    Glyphosate herbicide-resistant crop plants, introduced commercially in 1994, now represent approximately 85% of the land area devoted to transgenic crops. Herbicide resistance in commercial glyphosate-resistant crops is due to expression of a variant form of a bacterial 5-enolpyruvylshikimate-3-phosphate synthase with a significantly decreased binding affinity for glyphosate at the target site of the enzyme. As a result of widespread and recurrent glyphosate use, often as the only herbicide used for weed management, increasing numbers of weedy species have evolved resistance to glyphosate. Weed resistance is most often due to changes in herbicide translocation patterns, presumed to be through the activity of an as yet unidentified membrane transporter in plants. To provide insight into glyphosate resistance mechanisms and identify a potential glyphosate transporter, we screened Escherichia coli genomic DNA for alternate sources of glyphosate resistance genes. Our search identified a single non-target gene that, when overexpressed in E. coli and Pseudomonas, confers high-level glyphosate resistance. The gene, yhhS, encodes a predicted membrane transporter of the major facilitator superfamily involved in drug efflux. We report here that an alternative mode of glyphosate resistance in E. coli is due to reduced accumulation of glyphosate in cells that overexpress this membrane transporter and discuss the implications for potential alternative resistance mechanisms in other organisms such as plants.

  16. P-glycoprotein in sheep liver and small intestine: gene expression and transport efflux activity.

    Science.gov (United States)

    Ballent, M; Wilkens, M R; Maté, L; Muscher, A S; Virkel, G; Sallovitz, J; Schröder, B; Lanusse, C; Lifschitz, A

    2013-12-01

    The role of the transporter P-glycoprotein (P-gp) in the disposition kinetics of different drugs therapeutically used in veterinary medicine has been demonstrated. Considering the anatomo-physiological features of the ruminant species, the constitutive expression of P-gp (ABCB1) along the sheep gastrointestinal tract was studied. Additionally, the effect of repeated dexamethasone (DEX) administrations on the ABCB1 gene expression in the liver and small intestine was also assessed. The ABCB1 mRNA expression was determined by real-time quantitative PCR. P-gp activity was evaluated in diffusion chambers to determine the efflux of rhodamine 123 (Rho 123) in the ileum from experimental sheep. The constitutive ABCB1 expression was 65-fold higher in the liver than in the intestine (ileum). The highest ABCB1 mRNA expression along the small intestine was observed in the ileum (between 6- and 120-fold higher). The treatment with DEX did not elicit a significant effect on the P-gp gene expression levels in any of the investigated gastrointestinal tissues. Consistently, no significant differences were observed in the intestinal secretion of Rho 123, between untreated control (Peff S-M = 3.99 × 10(-6)  ± 2.07 × 10(-6) ) and DEX-treated animals (Peff S-M = 6.00 × 10(-6)  ± 2.5 × 10(-6) ). The understanding of the efflux transporters expression and activity along the digestive tract may help to elucidate clinical implications emerging from drug interactions in livestock.

  17. Identification of a novel topoisomerase inhibitor effective in cells overexpressing drug efflux transporters.

    Directory of Open Access Journals (Sweden)

    Walid Fayad

    Full Text Available BACKGROUND: Natural product structures have high chemical diversity and are attractive as lead structures for discovery of new drugs. One of the disease areas where natural products are most frequently used as therapeutics is oncology. METHOD AND FINDINGS: A library of natural products (NCI Natural Product set was screened for compounds that induce apoptosis of HCT116 colon carcinoma cells using an assay that measures an endogenous caspase-cleavage product. One of the apoptosis-inducing compounds identified in the screen was thaspine (taspine, an alkaloid from the South American tree Croton lechleri. The cortex of this tree is used for medicinal purposes by tribes in the Amazonas basin. Thaspine was found to induce conformational activation of the pro-apoptotic proteins Bak and Bax, mitochondrial cytochrome c release and mitochondrial membrane permeabilization in HCT116 cells. Analysis of the gene expression signature of thaspine-treated cells suggested that thaspine is a topoisomerase inhibitor. Inhibition of both topoisomerase I and II was observed using in vitro assays, and thaspine was found to have a reduced cytotoxic effect on a cell line with a mutated topoisomerase II enzyme. Interestingly, in contrast to the topoisomerase II inhibitors doxorubicin, etoposide and mitoxantrone, thaspine was cytotoxic to cell lines overexpressing the PgP or MRP drug efflux transporters. We finally show that thaspine induces wide-spread apoptosis in colon carcinoma multicellular spheroids and that apoptosis is induced in two xenograft mouse models in vivo. CONCLUSIONS: The alkaloid thaspine from the cortex of Croton lechleri is a dual topoisomerase inhibitor effective in cells overexpressing drug efflux transporters and induces wide-spread apoptosis in multicellular spheroids.

  18. The Allelochemical MDCA Inhibits Lignification and Affects Auxin Homeostasis1[OPEN

    Science.gov (United States)

    Steenackers, Ward; Corneillie, Sander; Van de Wouwer, Dorien; Zažímalová, Eva

    2016-01-01

    The phenylpropanoid 3,4-(methylenedioxy)cinnamic acid (MDCA) is a plant-derived compound first extracted from roots of Asparagus officinalis and further characterized as an allelochemical. Later on, MDCA was identified as an efficient inhibitor of 4-COUMARATE-CoA LIGASE (4CL), a key enzyme of the general phenylpropanoid pathway. By blocking 4CL, MDCA affects the biosynthesis of many important metabolites, which might explain its phytotoxicity. To decipher the molecular basis of the allelochemical activity of MDCA, we evaluated the effect of this compound on Arabidopsis thaliana seedlings. Metabolic profiling revealed that MDCA is converted in planta into piperonylic acid (PA), an inhibitor of CINNAMATE-4-HYDROXYLASE (C4H), the enzyme directly upstream of 4CL. The inhibition of C4H was also reflected in the phenolic profile of MDCA-treated plants. Treatment of in vitro grown plants resulted in an inhibition of primary root growth and a proliferation of lateral and adventitious roots. These observed growth defects were not the consequence of lignin perturbation, but rather the result of disturbing auxin homeostasis. Based on DII-VENUS quantification and direct measurement of cellular auxin transport, we concluded that MDCA disturbs auxin gradients by interfering with auxin efflux. In addition, mass spectrometry was used to show that MDCA triggers auxin biosynthesis, conjugation, and catabolism. A similar shift in auxin homeostasis was found in the c4h mutant ref3-2, indicating that MDCA triggers a cross talk between the phenylpropanoid and auxin biosynthetic pathways independent from the observed auxin efflux inhibition. Altogether, our data provide, to our knowledge, a novel molecular explanation for the phytotoxic properties of MDCA. PMID:27506238

  19. Auxin polar transport of etiolated epicotyls of ageotropum pea seedlings is not affected by gravistimulation: Relevance to automorphosis-like growth and development

    Science.gov (United States)

    Miyamoto, K.; Hoshino, T.; Takahashi, Y.; Ueda, J.

    Both true microgravity conditions in space STS-95 space experiment and simulated ones on a three-dimensional 3-D clinostat have been demonstrated to induce automorphosis in etiolated pea Pisum sativum L cv Alaska seedlings represented as epicotyl bending as well as changes in root growth direction and inhibition of hook formation and to alter the activities of auxin polar transport of epicotyls The fact that the application of inhibitors of auxin polar transport phenocopied automorphosis together with the result of detail kinetic analyses of epicotyl bending on the 3-D clinostat suggests that automorphosis of etiolated pea epicotyls is due to suppression of a negative gravitropic response on 1 g conditions and graviresponse of etiolated pea seedlings under 1 g conditions requires normal activities of auxin polar transport To study the role of auxin polar transport in graviresponse in early growth stage of etiolated pea seedlings effect of gravistimulation on auxin polar transport in epicotyls of Alaska pea seedlings was studied in comparison with that of the agravitropic pea mutant ageotropum seedlings Dry pea seeds whose embryo axes were set in a horizontal position referred to as horizontal position or an inclinational one to the gravity vector referred to as inclinational position allowed to germinate and grow in the dark for 2 5 days Epicotyls of etiolated Alaska pea seedlings grown under horizontal position showed negative gravitropisum due to relatively larger elongation in the proximal side to the cotyledons

  20. Identification of an Efflux Transporter LmrB Regulating Stress Response and Extracellular Polysaccharide Synthesis in Streptococcus mutans

    Directory of Open Access Journals (Sweden)

    Jia Liu

    2017-06-01

    Full Text Available Efflux transporters have been implicated in regulating bacterial virulence properties such as resistance to antibiotics, biofilm formation and colonization. The pathogenicity of Streptococcus mutans, the primary etiologic agent of human dental caries, relies on the bacterium’s ability to form biofilms on tooth surface. However, the studies on efflux transporters in S. mutans are scare and the function of these transporters remained to be clarified. In this study, we identified an efflux transporter (LmrB in S. mutans through cloning the lmrB gene into Escherichia coli. Introducing lmrB into E. coli conferred a multidrug-resistant phenotype and resulted in higher EtBr efflux activity which could be suppressed by efflux inhibitor. To explore whether LmrB was involved in S. mutans virulence properties regulation, we constructed the lmrB inactivation mutant and examined the phenotypes of the mutant. It was found that LmrB deficiency resulted in increased IPS storage and prolonged acid production. Enhanced biofilm formation characterized by increased extracellular polysaccharides (EPS production and elevated resistance to hydrogen peroxide and antimicrobials were also observed in lmrB mutant. To gain a better understanding of the global role of LmrB, a transcriptome analysis was performed using lmrB mutant strain. The expression of 107 genes was up- or down-regulated in the lmrB mutant compared with the wild type. Notably, expression of genes in several genomic islands was differentially modulated, such as stress-related GroELS and scnRK, sugar metabolism associated glg operons and msmREFGK transporter. The results presented here indicate that LmrB plays a vital global role in the regulation of several important virulence properties in S. mutans.

  1. Arabidopsis thaliana AUCSIA-1 regulates auxin biology and physically interacts with a kinesin-related protein.

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    Barbara Molesini

    Full Text Available Aucsia is a green plant gene family encoding 44-54 amino acids long miniproteins. The sequenced genomes of most land plants contain two Aucsia genes. RNA interference of both tomato (Solanum lycopersicum Aucsia genes (SlAucsia-1 and SlAucsia-2 altered auxin sensitivity, auxin transport and distribution; it caused parthenocarpic development of the fruit and other auxin-related morphological changes. Here we present data showing that the Aucsia-1 gene of Arabidopsis thaliana alters, by itself, root auxin biology and that the AtAUCSIA-1 miniprotein physically interacts with a kinesin-related protein. The AtAucsia-1 gene is ubiquitously expressed, although its expression is higher in roots and inflorescences in comparison to stems and leaves. Two allelic mutants for AtAucsia-1 gene did not display visible root morphological alterations; however both basipetal and acropetal indole-3-acetic acid (IAA root transport was reduced as compared with wild-type plants. The transcript steady state levels of the auxin efflux transporters ATP BINDING CASSETTE subfamily B (ABCB ABCB1, ABCB4 and ABCB19 were reduced in ataucsia-1 plants. In ataucsia-1 mutant, lateral root growth showed an altered response to i exogenous auxin, ii an inhibitor of polar auxin transport and iii ethylene. Overexpression of AtAucsia-1 inhibited primary root growth. In vitro and in vivo protein-protein interaction experiments showed that AtAUCSIA-1 interacts with a 185 amino acids long fragment belonging to a 2712 amino acids long protein of unknown function (At4g31570. Bioinformatics analysis indicates that the AtAUCSIA-1 interacting protein (AtAUCSIA-1IP clusters with a group of CENP-E kinesin-related proteins. Gene ontology predictions for the two proteins are consistent with the hypothesis that the AtAUCSIA-1/AtAUCSIA-1IP complex is involved in the regulation of the cytoskeleton dynamics underlying auxin biology.

  2. The boron efflux transporter ROTTEN EAR is required for maize inflorescence development and fertility.

    Science.gov (United States)

    Chatterjee, Mithu; Tabi, Zara; Galli, Mary; Malcomber, Simon; Buck, Amy; Muszynski, Michael; Gallavotti, Andrea

    2014-07-01

    Although boron has a relatively low natural abundance, it is an essential plant micronutrient. Boron deficiencies cause major crop losses in several areas of the world, affecting reproduction and yield in diverse plant species. Despite the importance of boron in crop productivity, surprisingly little is known about its effects on developing reproductive organs. We isolated a maize (Zea mays) mutant, called rotten ear (rte), that shows distinct defects in vegetative and reproductive development, eventually causing widespread sterility in its inflorescences, the tassel and the ear. Positional cloning revealed that rte encodes a membrane-localized boron efflux transporter, co-orthologous to the Arabidopsis thaliana BOR1 protein. Depending on the availability of boron in the soil, rte plants show a wide range of phenotypic defects that can be fully rescued by supplementing the soil with exogenous boric acid, indicating that rte is crucial for boron transport into aerial tissues. rte is expressed in cells surrounding the xylem in both vegetative and reproductive tissues and is required for meristem activity and organ development. We show that low boron supply to the inflorescences results in widespread defects in cell and cell wall integrity, highlighting the structural importance of boron in the formation of fully fertile reproductive organs.

  3. Atypical dopamine efflux caused by 3,4-methylenedioxypyrovalerone (MDPV) via the human dopamine transporter.

    Science.gov (United States)

    Shekar, Aparna; Aguilar, Jenny I; Galli, Greta; Cozzi, Nicholas V; Brandt, Simon D; Ruoho, Arnold E; Baumann, Michael H; Matthies, Heinrich J G; Galli, Aurelio

    2017-10-01

    Synthetic cathinones are similar in chemical structure to amphetamines, and their behavioral effects are associated with enhanced dopaminergic signaling. The past ten years of research on the common constituent of bath salts, MDPV (the synthetic cathinone 3,4-methylenedioxypyrovalerone), has aided the understanding of how synthetic cathinones act at the dopamine (DA) transporter (DAT). Several groups have described the ability of MDPV to block the DAT with high-affinity. In this study, we demonstrate for the first time a new mode of action of MDPV, namely its ability to promote DAT-mediated DA efflux. Using single cell amperometric assays, we determined that low concentrations of MDPV (1nM) can cause reverse transport of DA via DAT. Notably, administration of MDPV leads to hyperlocomotion in Drosophila melanogaster. These data describe further how MDPV acts at the DAT, possibly paving the way for novel treatment strategies for individuals who abuse bath salts. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. Multidrug efflux transporters limit accumulation of inorganic, but not organic, mercury in sea urchin embryos.

    Science.gov (United States)

    Bosnjak, Ivana; Uhlinger, Kevin R; Heim, Wesley; Smital, Tvrtko; Franekić-Colić, Jasna; Coale, Kenneth; Epel, David; Hamdoun, Amro

    2009-11-01

    Mercuric compounds are persistent global pollutants that accumulate in marine organisms and in humans who consume them. While the chemical cycles and speciation of mercury in the oceans are relatively well described, the cellular mechanisms that govern which forms of mercury accumulate in cells and why they persist are less understood. In this study we examined the role of multidrug efflux transport in the differential accumulation of inorganic (HgCl(2)) and organic (CH(3)HgCl) mercury in sea urchin (Strongylocentrotus purpuratus) embryos. We found that inhibition of MRP/ABCC-type transporters increases intracellular accumulation of inorganic mercury but had no effect on accumulation of organic mercury. Similarly, pharmacological inhibition of metal conjugating enzymes by ligands GST/GSH significantly increases this antimitotic potency of inorganic mercury, but had no effect on the potency of organic mercury. Our results point to MRP-mediated elimination of inorganic mercury conjugates as a cellular basis for differences in the accumulation and potency of the two major forms of mercury found in marine environments.

  5. Rooting of carnation cuttings: The auxin signal

    OpenAIRE

    Acosta, Manuel; Oliveros-Valenzuela, M Rocío; Nicolás, Carlos; Sánchez-Bravo, José

    2009-01-01

    The rooting of stem cuttings is a common vegetative propagation practice in many ornamental species. Among other signals, auxin polarly transported through the stem plays a key role in the formation and growth of adventitious roots. Unlike in other plant species, auxin from mature leaves plays a decisive role in the rooting of carnation (Dianthus caryophyllus. L) cuttings. The gene DcAUX1, which codifies an auxin influx carrier involved in polar auxin transport, has now been cloned and charac...

  6. A radiotracer method to study efflux transport of iodide liberated from thyroid hormones via deiodination metabolism in the brain.

    Science.gov (United States)

    Okamura, Toshimitsu; Igarashi, Jun; Kikuchi, Tatsuya; Fukushi, Kiyoshi; Arano, Yasushi; Irie, Toshiaki

    2009-06-05

    Thyroid hormones (TH) play an important role in the development and functional maintenance of the central nervous system. The purpose of this study was to develop a radiotracer method for studying the in vivo efflux transport of iodide liberated by the TH metabolism in the brain. The rationale of our method is as follows: a radioiodinated compound can enter the brain and rapidly release iodide in situ; the iodide efflux rate can be estimated from the clearance of brain radioactivity after disappearance of the iodinated compound. 6-[(125)I]Iodo-9-pentylpurine ([(125)I]9Pe6IP) was designed to enter the brain and release (125)I(-) by the reaction with glutathione and synthesized from the corresponding bromo derivative in a Br/(125)I exchange reaction. The brain kinetics of radioactivity and radioactive metabolites were investigated after intravenous injection of [(125)I]9Pe6IP into mice. The iodide efflux rate was estimated in mice pretreated with perchlorate, an inhibitor of iodide transport from the brain. High brain uptake (5.3% injected dose/g) was observed at 1 min, and almost complete conversion of [(125)I]9Pe6IP to (125)I(-) occurred 10 min after injection. The (125)I(-) uptake from the blood was negligible. (125)I(-) was eliminated from the brain along a single-exponential curve with a half-life of 6.0 min. Furthermore, dose-dependent inhibition of (125)I(-) efflux was observed in mice pretreated with perchlorate. We conclude that 9Pe6IP labeled with (124)I (positron emitter) or (123)I (single-photon emitter) may be useful for studying the in vivo efflux transport of iodide in the brain using nuclear medicine imaging devices.

  7. Differential behavior within a grapevine cluster: decreased ethylene-related gene expression dependent on auxin transport is correlated with low abscission of first developed berries.

    Directory of Open Access Journals (Sweden)

    Nathalie Kühn

    Full Text Available In grapevine, fruit abscission is known to occur within the first two to three weeks after flowering, but the reason why some berries in a cluster persist and others abscise is not yet understood. Ethylene sensitivity modulates abscission in several fruit species, based on a mechanism where continuous polar auxin transport across the pedicel results in a decrease in ethylene perception, which prevents abscission. In grapevine, flowering takes about four to seven days in a single cluster, thus while some flowers are developing into berries, others are just starting to open. So, in this work it was assessed whether uneven flowering accounted for differences in berry abscission dependent on polar auxin transport and ethylene-related gene expression. For this, flowers that opened in a cluster were tagged daily, which allowed to separately analyze berries, regarding their ability to persist. It was found that berries derived from flowers that opened the day that flowering started--named as "first berries"--had lower abscission rate than berries derived from flowers that opened during the following days--named as "late berries". Use of radiolabeled auxin showed that "first berries" had higher polar auxin transport, correlated with lower ethylene content and lower ethylene-related transcript abundance than "late berries". When "first berries" were treated with a polar auxin transport inhibitor they showed higher ethylene-related transcript abundance and were more prone to abscise than control berries. This study provides new insights on fruit abscission control. Our results indicate that polar auxin transport sustains the ability of "first berries" to persist in the cluster during grapevine abscission and also suggest that this could be associated with changes in ethylene-related gene expression.

  8. Altered regulation of hepatic efflux transporters disrupts acetaminophen disposition in pediatric nonalcoholic steatohepatitis.

    Science.gov (United States)

    Canet, Mark J; Merrell, Matthew D; Hardwick, Rhiannon N; Bataille, Amy M; Campion, Sarah N; Ferreira, Daniel W; Xanthakos, Stavra A; Manautou, Jose E; A-Kader, H Hesham; Erickson, Robert P; Cherrington, Nathan J

    2015-06-01

    Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease, representing a spectrum of liver pathologies that include simple hepatic steatosis and the more advanced nonalcoholic steatohepatitis (NASH). The current study was conducted to determine whether pediatric NASH also results in altered disposition of acetaminophen (APAP) and its two primary metabolites, APAP-sulfate and APAP-glucuronide. Pediatric patients with hepatic steatosis (n = 9) or NASH (n = 3) and healthy patients (n = 12) were recruited in a small pilot study design. All patients received a single 1000-mg dose of APAP. Blood and urine samples were collected at 1, 2, and 4 hours postdose, and APAP and APAP metabolites were determined by high-performance liquid chromatography. Moreover, human liver tissues from patients diagnosed with various stages of NAFLD were acquired from the Liver Tissue Cell Distribution System to investigate the regulation of the membrane transporters, multidrug resistance-associated protein 2 and 3 (MRP2 and MRP3, respectively). Patients with the more severe disease (i.e., NASH) had increased serum and urinary levels of APAP-glucuronide along with decreased serum levels of APAP-sulfate. Moreover, an induction of hepatic MRP3 and altered canalicular localization of the biliary efflux transporter, MRP2, describes the likely mechanism for the observed increase in plasma retention of APAP-glucuronide, whereas altered regulation of sulfur activation genes may explain decreased sulfonation activity in NASH. APAP-glucuronide and APAP-sulfate disposition is altered in NASH and is likely due to hepatic membrane transporter dysregulation as well as altered intracellular sulfur activation.

  9. The transparent testa4 mutation prevents flavonoid synthesis and alters auxin transport and the response of Arabidopsis roots to gravity and light.

    Science.gov (United States)

    Buer, Charles S; Muday, Gloria K

    2004-05-01

    We examined whether flavonoids act as endogenous auxin transport regulators during gravity vector and light intensity changes in Arabidopsis thaliana roots. Flavonoid deficient transparent testa4 [tt4(2YY6)] seedlings had elevated root basipetal auxin transport compared with the wild type, consistent with the absence of a negative auxin transport regulator. The tt4(2YY6) roots had delayed gravitropism that was chemically complemented with a flavonoid intermediate. Flavonoid accumulation was found in wild-type columella cells, the site of gravity perception, and in epidermal and cortical cells, the site of differential growth, but flavonoid accumulation was absent in tt4(2YY6) roots. Flavonoid accumulation was higher in gravity-stimulated root tips as compared with vertical controls, with maximum differences coinciding with the timing of gravitropic bending, and was located in epidermal cells. Exogenous indole-3-acetic acid (IAA) also elevated flavonoid accumulation, suggesting that flavonoid changes in response to gravity might be partly as a result of changing IAA distribution. Acropetal IAA transport was also elevated in roots of tt4(2YY6). Flavonoid synthesis was repressed in the dark, as were differences in root acropetal transport in tt4(2YY6). These results are consistent with light- and gravity-induced flavonoid stimulation that alters auxin transport in roots and dependent physiological processes, including gravitropic bending and root development.

  10. The role of multidrug resistance protein (MRP-1) as an active efflux transporter on blood-brain barrier (BBB) permeability.

    Science.gov (United States)

    Lingineni, Karthik; Belekar, Vilas; Tangadpalliwar, Sujit R; Garg, Prabha

    2017-01-03

    Drugs acting on central nervous system (CNS) may take longer duration to reach the market as these compounds have a higher attrition rate in clinical trials due to the complexity of the brain, side effects, and poor blood-brain barrier (BBB) permeability compared to non-CNS-acting compounds. The roles of active efflux transporters with BBB are still unclear. The aim of the present work was to develop a predictive model for BBB permeability that includes the MRP-1 transporter, which is considered as an active efflux transporter. A support vector machine model was developed for the classification of MRP-1 substrates and non-substrates, which was validated with an external data set and Y-randomization method. An artificial neural network model has been developed to evaluate the role of MRP-1 on BBB permeation. A total of nine descriptors were selected, which included molecular weight, topological polar surface area, ClogP, number of hydrogen bond donors, number of hydrogen bond acceptors, number of rotatable bonds, P-gp, BCRP, and MRP-1 substrate probabilities for model development. We identified 5 molecules that fulfilled all criteria required for passive permeation of BBB, but they all have a low logBB value, which suggested that the molecules were effluxed by the MRP-1 transporter.

  11. Assessment of three Resistance-Nodulation-Cell Division drug efflux transporters of Burkholderia cenocepacia in intrinsic antibiotic resistance

    Directory of Open Access Journals (Sweden)

    Venturi Vittorio

    2009-09-01

    Full Text Available Abstract Background Burkholderia cenocepacia are opportunistic Gram-negative bacteria that can cause chronic pulmonary infections in patients with cystic fibrosis. These bacteria demonstrate a high-level of intrinsic antibiotic resistance to most clinically useful antibiotics complicating treatment. We previously identified 14 genes encoding putative Resistance-Nodulation-Cell Division (RND efflux pumps in the genome of B. cenocepacia J2315, but the contribution of these pumps to the intrinsic drug resistance of this bacterium remains unclear. Results To investigate the contribution of efflux pumps to intrinsic drug resistance of B. cenocepacia J2315, we deleted 3 operons encoding the putative RND transporters RND-1, RND-3, and RND-4 containing the genes BCAS0591-BCAS0593, BCAL1674-BCAL1676, and BCAL2822-BCAL2820. Each deletion included the genes encoding the RND transporter itself and those encoding predicted periplasmic proteins and outer membrane pores. In addition, the deletion of rnd-3 also included BCAL1672, encoding a putative TetR regulator. The B. cenocepacia rnd-3 and rnd-4 mutants demonstrated increased sensitivity to inhibitory compounds, suggesting an involvement of these proteins in drug resistance. Moreover, the rnd-3 and rnd-4 mutants demonstrated reduced accumulation of N-acyl homoserine lactones in the growth medium. In contrast, deletion of the rnd-1 operon had no detectable phenotypes under the conditions assayed. Conclusion Two of the three inactivated RND efflux pumps in B. cenocepacia J2315 contribute to the high level of intrinsic resistance of this strain to some antibiotics and other inhibitory compounds. Furthermore, these efflux systems also mediate accumulation in the growth medium of quorum sensing molecules that have been shown to contribute to infection. A systematic study of RND efflux systems in B. cenocepacia is required to provide a full picture of intrinsic antibiotic resistance in this opportunistic

  12. Overexpression of OsWRKY72 gene interferes in the abscisic acid signal and auxin transport pathway of Arabidopsis

    Indian Academy of Sciences (India)

    Song Yu; Chen Ligang; Zhang Liping; Yu Diqiu

    2010-09-01

    Through activating specific transcriptional programmes, plants can launch resistance mechanisms to stressful environments and acquire a new equilibrium between development and defence. To screen the rice WRKY transcription factor which functions in abiotic stress tolerance and modulates the abscisic acid (ABA) response, we generated a whole array of 35S-OsWRKY transgenic Arabidopsis. In this study, we report that 35S-OsWRKY72 transgenic Arabidopsis, whose seed germination was retarded under normal conditions, emerged more sensitive to mannitol, NaCl, ABA stresses and sugar starvation than vector plants. Meanwhile, 35S-OsWRKY72 transgenic Arabidopsis displayed early flowering, reduced apical dominance, lost high temperature-induced hypocotyl elongation response, and enhanced gravitropism response, which were similar to the auxin-related gene mutants aux1, axr1 and bud1. Further, semi-quantitative RT-PCR showed that the expression patterns of three auxin-related genes AUX1, AXR1 and BUD1 were significantly altered in rosette leaves and inflorescences of 35S-OsWRKY72 plants compared with control Arabidopsis, and two ABA-related genes ABA2 and ABI4 were induced in 35S-OsWRKY72 seedlings. In addition, northern blot analysis indicated that, in rice, OsWRKY72 was inducible by polyethylene glycol (PEG), NaCl, naphthalene acetic acid (NAA), ABA and 42°C, similar to its orthologue AtWRKY75 in Arabidopsis, implying that these two WRKY genes might be required for multiple physiological processes in their plants. Together, these results suggest that OsWRKY72 interferes in the signal cross-talk between the ABA signal and auxin transport pathway in transgenic Arabidopsis.

  13. Overexpression of OsWRKY72 gene interferes in the abscisic acid signal and auxin transport pathway of Arabidopsis.

    Science.gov (United States)

    Yu, Song; Ligang, Chen; Liping, Zhang; Diqiu, Yu

    2010-09-01

    Through activating specific transcriptional programmes, plants can launch resistance mechanisms to stressful environments and acquire a new equilibrium between development and defence. To screen the rice WRKY transcription factor which functions in abiotic stress tolerance and modulates the abscisic acid (ABA) response, we generated a whole array of 35S-OsWRKY transgenic Arabidopsis. In this study, we report that 35S-OsWRKY72 transgenic Arabidopsis, whose seed germination was retarded under normal conditions, emerged more sensitive to mannitol, NaCl, ABA stresses and sugar starvation than vector plants. Meanwhile, 35S-OsWRKY72 transgenic Arabidopsis displayed early flowering, reduced apical dominance, lost high temperature-induced hypocotyl elongation response, and enhanced gravitropism response, which were similar to the auxin-related gene mutants aux1, axr1 and bud1. Further, semi-quantitative RT-PCR showed that the expression patterns of three auxin-related genes AUX1, AXR1 and BUD1 were significantly altered in rosette leaves and infl orescences of 35S-OsWRKY72 plants compared with control Arabidopsis, and two ABA-related genes ABA2 and ABI4 were induced in 35S-OsWRKY72 seedlings. In addition, northern blot analysis indicated that, in rice, OsWRKY72 was inducible by polyethylene glycol (PEG), NaCl, naphthalene acetic acid (NAA), ABA and 42 degrees C, similar to its orthologue AtWRKY75 in Arabidopsis, implying that these two WRKY genes might be required for multiple physiological processes in their plants. Together, these results suggest that OsWRKY72 interferes in the signal cross-talk between the ABA signal and auxin transport pathway in transgenic Arabidopsis.

  14. 3D structure of AcrB: the archetypal multidrug efflux transporter of Escherichia coli likely captures substrates from periplasm.

    Science.gov (United States)

    Elkins, Christopher A; Nikaido, Hiroshi

    2003-02-01

    Recent advances in structural biology have extended our understanding of the multiple drug efflux complex, AcrAB-TolC, of Escherichia coli. This tripartite complex and its homologs are the major mechanisms that give most Gram-negative bacteria their characteristic intrinsic resistance to a variety of lipophilic drugs, dyes, and detergents. Most recently, the structure of the transporter AcrB was elucidated at high resolution [Nature 419(2002)587]. It is a particularly significant achievement since integral membrane proteins are notoriously elusive structures for crystallography. The striking features of this trimeric pump, such as the presence of potential substrate-binding sites in the periplasmic domain and the possibility of direct interaction with the end of TolC tunnel, refine our understanding of the mode of action of this tripartite efflux transport complex.

  15. Extra-Renal Elimination of Uric Acid via Intestinal Efflux Transporter BCRP/ABCG2

    Science.gov (United States)

    Hosomi, Atsushi; Nakanishi, Takeo; Fujita, Takuya; Tamai, Ikumi

    2012-01-01

    Urinary excretion accounts for two-thirds of total elimination of uric acid and the remainder is excreted in feces. However, the mechanism of extra-renal elimination is poorly understood. In the present study, we aimed to clarify the mechanism and the extent of elimination of uric acid through liver and intestine using oxonate-treated rats and Caco-2 cells as a model of human intestinal epithelium. In oxonate-treated rats, significant amounts of externally administered and endogenous uric acid were recovered in the intestinal lumen, while biliary excretion was minimal. Accordingly, direct intestinal secretion was thought to be a substantial contributor to extra-renal elimination of uric acid. Since human efflux transporter BCRP/ABCG2 accepts uric acid as a substrate and genetic polymorphism causing a decrease of BCRP activity is known to be associated with hyperuricemia and gout, the contribution of rBcrp to intestinal secretion was examined. rBcrp was confirmed to transport uric acid in a membrane vesicle study, and intestinal regional differences of expression of rBcrp mRNA were well correlated with uric acid secretory activity into the intestinal lumen. Bcrp1 knockout mice exhibited significantly decreased intestinal secretion and an increased plasma concentration of uric acid. Furthermore, a Bcrp inhibitor, elacridar, caused a decrease of intestinal secretion of uric acid. In Caco-2 cells, uric acid showed a polarized flux from the basolateral to apical side, and this flux was almost abolished in the presence of elacridar. These results demonstrate that BCRP contributes at least in part to the intestinal excretion of uric acid as extra-renal elimination pathway in humans and rats. PMID:22348008

  16. Arabidopsis phosphatidylinositol monophosphate 5-kinase 2 is involved in root gravitropism through regulation of polar auxin transport by affecting the cycling of PIN proteins

    Institute of Scientific and Technical Information of China (English)

    Yu Mei; Wen-Jing Jia; Yu-Jia Chu; Hong-Wei Xue

    2012-01-01

    Phosphatidylinositol monophosphate 5-kinase(PIP5K)catalyzes the synthesis of PI-4,5-bisphosphate(PtdIns(4,5)P2)by phosphorylation of PI-4-phosphate at the 5 position of the inositol ring,and is involved in regulating multiple developmental processes and stress responses.We here report on the functional characterization of Arabidopsis PIP5K2,which is expressed during lateral root initiation and elongation,and whose expression is enhanced by exogenous auxin.The knockout mutant pip5k2 shows reduced lateral root formation,which could be recovered with exogenous auxin,and interestingly,delayed root gravity response that could not be recovered with exogenous auxin.Crossing with the DR5-GUS marker line and measurement of free IAA content confirmed the reduced auxin accumulation in pip5k2.In addition,analysis using the membrane-selective dye FM4-64 revealed the decelerated vesicle trafficking caused by PtdIns(4,5)P2 reduction,which hence results in suppressed cycling of PIN proteins(PIN2 and 3),and delayed redistribution of PIN2 and auxin under gravistimulation in pipSk2 roots.On the contrary,PtdIns(4,5)P2 significantly enhanced the vesicle trafficking and cycling of PIN proteins.These results demonstrate that PIP5K2 is involved in regulating lateral root formation and root gravity response,and reveal a critical role of PIP5K2/Ptdlns(4,5)P2 in root development through regulation of PIN proteins,providing direct evidence of crosstalk between the phosphatidylinositol signaling pathway and auxin response,and new insights into the control of polar auxin transport.

  17. The auxin transporter, OsAUX1, is involved in primary root and root hair elongation and in Cd stress responses in rice (Oryza sativa L.).

    Science.gov (United States)

    Yu, ChenLiang; Sun, ChenDong; Shen, Chenjia; Wang, Suikang; Liu, Fang; Liu, Yan; Chen, YunLong; Li, Chuanyou; Qian, Qian; Aryal, Bibek; Geisler, Markus; Jiang, De An; Qi, YanHua

    2015-09-01

    Auxin and cadmium (Cd) stress play critical roles during root development. There are only a few reports on the mechanisms by which Cd stress influences auxin homeostasis and affects primary root (PR) and lateral root (LR) development, and almost nothing is known about how auxin and Cd interfere with root hair (RH) development. Here, we characterize rice osaux1 mutants that have a longer PR and shorter RHs in hydroponic culture, and that are more sensitive to Cd stress compared to wild-type (Dongjin). OsAUX1 expression in root hair cells is different from that of its paralogous gene, AtAUX1, which is expressed in non-hair cells. However, OsAUX1, like AtAUX1, localizes at the plasma membrane and appears to function as an auxin tranporter. Decreased auxin distribution and contents in the osaux1 mutant result in reduction of OsCyCB1;1 expression and shortened PRs, LRs and RHs under Cd stress, but may be rescued by treatment with the membrane-permeable auxin 1-naphthalene acetic acid. Treatment with the auxin transport inhibitors 1-naphthoxyacetic acid and N-1-naphthylphthalamic acid increased the Cd sensitivity of WT rice. Cd contents in the osaux1 mutant were not altered, but reactive oxygen species-mediated damage was enhanced, further increasing the sensitivity of the osaux1 mutant to Cd stress. Taken together, our results indicate that OsAUX1 plays an important role in root development and in responses to Cd stress.

  18. Solanum tuberosum StCDPK1 is regulated by miR390 at the posttranscriptional level and phosphorylates the auxin efflux carrier StPIN4 in vitro, a potential downstream target in potato development.

    Science.gov (United States)

    Santin, Franco; Bhogale, Sneha; Fantino, Elisa; Grandellis, Carolina; Banerjee, Anjan K; Ulloa, Rita M

    2017-02-01

    Among many factors that regulate potato tuberization, calcium and calcium-dependent protein kinases (CDPKs) play an important role. CDPK activity increases at the onset of tuber formation with StCDPK1 expression being strongly induced in swollen stolons. However, not much is known about the transcriptional and posttranscriptional regulation of StCDPK1 or its downstream targets in potato development. To elucidate further, we analyzed its expression in different tissues and stages of the life cycle. Histochemical analysis of StCDPK1::GUS (β-glucuronidase) plants demonstrated that StCDPK1 is strongly associated with the vascular system in stems, roots, during stolon to tuber transition, and in tuber sprouts. In agreement with the observed GUS profile, we found specific cis-acting elements in StCDPK1 promoter. In silico analysis predicted miR390 to be a putative posttranscriptional regulator of StCDPK1. Quantitative real time-polymerase chain reaction (qRT-PCR) analysis showed ubiquitous expression of StCDPK1 in different tissues which correlated well with Western blot data except in leaves. On the contrary, miR390 expression exhibited an inverse pattern in leaves and tuber eyes suggesting a possible regulation of StCDPK1 by miR390. This was further confirmed by Agrobacterium co-infiltration assays. In addition, in vitro assays showed that recombinant StCDPK1-6xHis was able to phosphorylate the hydrophilic loop of the auxin efflux carrier StPIN4. Altogether, these results indicate that StCDPK1 expression is varied in a tissue-specific manner having significant expression in vasculature and in tuber eyes; is regulated by miR390 at posttranscriptional level and suggest that StPIN4 could be one of its downstream targets revealing the overall role of this kinase in potato development. © 2016 Scandinavian Plant Physiology Society.

  19. Deficiency in the manganese efflux transporter SLC30A10 induces severe hypothyroidism in mice.

    Science.gov (United States)

    Hutchens, Steven; Liu, Chunyi; Jursa, Thomas; Shawlot, William; Chaffee, Beth K; Yin, Weiling; Gore, Andrea C; Aschner, Michael; Smith, Donald R; Mukhopadhyay, Somshuvra

    2017-06-09

    Manganese is an essential metal that becomes toxic at elevated levels. Loss-of-function mutations in SLC30A10, a cell-surface-localized manganese efflux transporter, cause a heritable manganese metabolism disorder resulting in elevated manganese levels and parkinsonian-like movement deficits. The underlying disease mechanisms are unclear; therefore, treatment is challenging. To understand the consequences of loss of SLC30A10 function at the organism level, we generated Slc30a10 knock-out mice. During early development, knock-outs were indistinguishable from controls. Surprisingly, however, after weaning and compared with controls, knock-out mice failed to gain weight, were smaller, and died prematurely (by ∼6-8 weeks of age). At 6 weeks, manganese levels in the brain, blood, and liver of the knock-outs were ∼20-60-fold higher than controls. Unexpectedly, histological analyses revealed that the brain and liver of the knock-outs were largely unaffected, but their thyroid exhibited extensive alterations. Because hypothyroidism leads to growth defects and premature death in mice, we assayed for changes in thyroid and pituitary hormones. At 6 weeks and compared with controls, the knock-outs had markedly reduced thyroxine levels (∼50-80%) and profoundly increased thyroid-stimulating hormone levels (∼800-1000-fold), indicating that Slc30a10 knock-out mice develop hypothyroidism. Importantly, a low-manganese diet produced lower tissue manganese levels in the knock-outs and rescued the phenotype, suggesting that manganese toxicity was the underlying cause. Our unanticipated discovery highlights the importance of determining the role of thyroid dysfunction in the onset and progression of manganese-induced disease and identifies Slc30a10 knock-out mice as a new model for studying thyroid biology. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  20. phot1 inhibition of ABCB19 primes lateral auxin fluxes in the shoot apex required for phototropism.

    Directory of Open Access Journals (Sweden)

    John M Christie

    2011-06-01

    Full Text Available It is well accepted that lateral redistribution of the phytohormone auxin underlies the bending of plant organs towards light. In monocots, photoreception occurs at the shoot tip above the region of differential growth. Despite more than a century of research, it is still unresolved how light regulates auxin distribution and where this occurs in dicots. Here, we establish a system in Arabidopsis thaliana to study hypocotyl phototropism in the absence of developmental events associated with seedling photomorphogenesis. We show that auxin redistribution to the epidermal sites of action occurs at and above the hypocotyl apex, not at the elongation zone. Within this region, we identify the auxin efflux transporter ATP-BINDING CASSETTE B19 (ABCB19 as a substrate target for the photoreceptor kinase PHOTOTROPIN 1 (phot1. Heterologous expression and physiological analyses indicate that phosphorylation of ABCB19 by phot1 inhibits its efflux activity, thereby increasing auxin levels in and above the hypocotyl apex to halt vertical growth and prime lateral fluxes that are subsequently channeled to the elongation zone by PIN-FORMED 3 (PIN3. Together, these results provide new insights into the roles of ABCB19 and PIN3 in establishing phototropic curvatures and demonstrate that the proximity of light perception and differential phototropic growth is conserved in angiosperms.

  1. Auxin Acts through MONOPTEROS to Regulate Plant Cell Polarity and Pattern Phyllotaxis.

    Science.gov (United States)

    Bhatia, Neha; Bozorg, Behruz; Larsson, André; Ohno, Carolyn; Jönsson, Henrik; Heisler, Marcus G

    2016-12-05

    The periodic formation of plant organs such as leaves and flowers gives rise to intricate patterns that have fascinated biologists and mathematicians alike for hundreds of years [1]. The plant hormone auxin plays a central role in establishing these patterns by promoting organ formation at sites where it accumulates due to its polar, cell-to-cell transport [2-6]. Although experimental evidence as well as modeling suggest that feedback from auxin to its transport direction may help specify phyllotactic patterns [7-12], the nature of this feedback remains unclear [13]. Here we reveal that polarization of the auxin efflux carrier PIN-FORMED 1 (PIN1) is regulated by the auxin response transcription factor MONOPTEROS (MP) [14]. We find that in the shoot, cell polarity patterns follow MP expression, which in turn follows auxin distribution patterns. By perturbing MP activity both globally and locally, we show that localized MP activity is necessary for the generation of polarity convergence patterns and that localized MP expression is sufficient to instruct PIN1 polarity directions non-cell autonomously, toward MP-expressing cells. By expressing MP in the epidermis of mp mutants, we further show that although MP activity in a single-cell layer is sufficient to promote polarity convergence patterns, MP in sub-epidermal tissues helps anchor these polarity patterns to the underlying cells. Overall, our findings reveal a patterning module in plants that determines organ position by orienting transport of the hormone auxin toward cells with high levels of MP-mediated auxin signaling. We propose that this feedback process acts broadly to generate periodic plant architectures.

  2. Auxin Transport and Ribosome Biogenesis Mutant/Reporter Lines to Study Plant Cell Growth and Proliferation under Altered Gravity

    Science.gov (United States)

    Valbuena, Miguel A.; Manzano, Ana I.; van Loon, Jack JWA.; Saez-Vasquez, Julio; Carnero-Diaz, Eugenie; Herranz, Raul; Medina, F. J.

    2013-02-01

    We tested different Arabidopsis thaliana strains to check their availability for space use in the International Space Station (ISS). We used mutants and reporter gene strains affecting factors of cell proliferation and cell growth, to check variations induced by an altered gravity vector. Seedlings were grown either in a Random Positioning Machine (RPM), under simulated microgravity (μg), or in a Large Diameter Centrifuge (LDC), under hypergravity (2g). A combination of the two devices (μgRPM+LDC) was also used. Under all gravity alterations, seedling roots were longer than in control 1g conditions, while the levels of the nucleolar protein nucleolin were depleted. Alterations in the pattern of expression of PIN2, an auxin transporter, and of cyclin B1, a cell cycle regulator, were shown. All these alterations are compatible with previous space data, so the use of these strains will be useful in the next experiments in ISS, under real microgravity.

  3. The PIN1 family gene PvPIN1 is involved in auxin-dependent root emergence and tillering in switchgrass

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    Kaijie Xu

    2016-03-01

    Full Text Available Abstract Switchgrass (Panicum virgatum L.; family Poaceae is a warm-season C4 perennial grass. Tillering plays an important role in determining the morphology of aboveground parts and the final biomass yield of switchgrass. Auxin distribution in plants can affect a variety of important growth and developmental processes, including the regulation of shoot and root branching, plant resistance and biological yield. Auxin transport and gradients in plants are mediated by influx and efflux carriers. PvPIN1, a switchgrass PIN1-like gene that is involved in regulating polar transport, is a putative auxin efflux carrier. Neighbor-joining analysis using sequences deposited in NCBI databases showed that the PvPIN1gene belongs to the PIN1 family and is evolutionarily closer to the Oryza sativa japonica group. Tiller emergence and development was significantly promoted in plants subjected toPvPIN1 RNA interference (RNAi, which yielded a phenotype similar to that of wild-type plants treated with the auxin transport inhibitor TIBA (2,3,5-triiodobenzoic acid. A transgenic approach that inducedPvPIN1 gene overexpression or suppression altered tiller number and the shoot/root ratio. These data suggest that PvPIN1plays an important role in auxin-dependent adventitious root emergence and tillering.

  4. A Division in PIN-Mediated Auxin Patterning during Organ Initiation in Grasses

    Science.gov (United States)

    O'Connor, Devin L.; Runions, Adam; Sluis, Aaron; Bragg, Jennifer; Vogel, John P.

    2014-01-01

    The hormone auxin plays a crucial role in plant morphogenesis. In the shoot apical meristem, the PIN-FORMED1 (PIN1) efflux carrier concentrates auxin into local maxima in the epidermis, which position incipient leaf or floral primordia. From these maxima, PIN1 transports auxin into internal tissues along emergent paths that pattern leaf and stem vasculature. In Arabidopsis thaliana, these functions are attributed to a single PIN1 protein. Using phylogenetic and gene synteny analysis we identified an angiosperm PIN clade sister to PIN1, here termed Sister-of-PIN1 (SoPIN1), which is present in all sampled angiosperms except for Brassicaceae, including Arabidopsis. Additionally, we identified a conserved duplication of PIN1 in the grasses: PIN1a and PIN1b. In Brachypodium distachyon, SoPIN1 is highly expressed in the epidermis and is consistently polarized toward regions of high expression of the DR5 auxin-signaling reporter, which suggests that SoPIN1 functions in the localization of new primordia. In contrast, PIN1a and PIN1b are highly expressed in internal tissues, suggesting a role in vascular patterning. PIN1b is expressed in broad regions spanning the space between new primordia and previously formed vasculature, suggesting a role in connecting new organs to auxin sinks in the older tissues. Within these regions, PIN1a forms narrow canals that likely pattern future veins. Using a computer model, we reproduced the observed spatio-temporal expression and localization patterns of these proteins by assuming that SoPIN1 is polarized up the auxin gradient, and PIN1a and PIN1b are polarized to different degrees with the auxin flux. Our results suggest that examination and modeling of PIN dynamics in plants outside of Brassicaceae will offer insights into auxin-driven patterning obscured by the loss of the SoPIN1 clade in Brassicaceae. PMID:24499933

  5. Specificity patterns indicate that auxin exporters and receptors are the same proteins.

    Science.gov (United States)

    Hössel, D; Schmeiser, C; Hertel, R

    2005-01-01

    A study of transport and action of synthetic auxin analogues can help to identify transporters and receptors of this plant hormone. Both aspects--transportability and action on growth--were tested with 2-naphthoxyacetic acid (2-NOA) and compared across several plant species. 2-NOA stimulates elongation effectively at low concentrations in petioles of the gymnosperm Ginkgo biloba L., in hypocotyls or internodes of the dicot legumes, mung bean (Vigna mungo L.) and pea (Pisum sativum L.), in cotyledons of onion (Allium cepa L.) and in leaf bases of chive (Allium schoenoprasum L.), the latter two of the monocot order Asparagales. In contrast, elongation of coleoptile segments of maize (Zea mays L.) is poorly responsive to 2-NOA. Significant auxin-like transport of 2-NOA was observed in segments of mung bean hypocotyls, pea internodes, and chive leaf bases, but not in segments of the grass coleoptiles. Thus, for the two assays, elongation and polar transportability, the same difference in ligand specificity was observed between the grass and all other species assayed. This finding supports the hypothesis that a common protein mediates auxin efflux as well as auxin action on elongation.

  6. Auxin response under osmotic stress.

    Science.gov (United States)

    Naser, Victoria; Shani, Eilon

    2016-08-01

    The phytohormone auxin (indole-3-acetic acid, IAA) is a small organic molecule that coordinates many of the key processes in plant development and adaptive growth. Plants regulate the auxin response pathways at multiple levels including biosynthesis, metabolism, transport and perception. One of the most striking aspects of plant plasticity is the modulation of development in response to changing growth environments. In this review, we explore recent findings correlating auxin response-dependent growth and development with osmotic stresses. Studies of water deficit, dehydration, salt, and other osmotic stresses point towards direct and indirect molecular perturbations in the auxin pathway. Osmotic stress stimuli modulate auxin responses by affecting auxin biosynthesis (YUC, TAA1), transport (PIN), perception (TIR/AFB, Aux/IAA), and inactivation/conjugation (GH3, miR167, IAR3) to coordinate growth and patterning. In turn, stress-modulated auxin gradients drive physiological and developmental mechanisms such as stomata aperture, aquaporin and lateral root positioning. We conclude by arguing that auxin-mediated growth inhibition under abiotic stress conditions is one of the developmental and physiological strategies to acclimate to the changing environment.

  7. Quantitative evaluation of the combination between cytotoxic drug and efflux transporter inhibitors based on a tumour growth inhibition model.

    Science.gov (United States)

    Sostelly, Alexandre; Payen, Léa; Guitton, Jérôme; Di Pietro, Attilio; Falson, Pierre; Honorat, Mylène; Boumendjel, Ahcène; Gèze, Annabelle; Freyer, Gilles; Tod, Michel

    2014-04-01

    ATP-Binding Cassette transporters such as ABCG2 confer resistance to various anticancer drugs including irinotecan and its active metabolite, SN38. Early quantitative evaluation of efflux transporter inhibitors-cytotoxic combination requires quantitative drug-disease models. A proof-of-concept study has been carried out for studying the effect of a new ABCG2 transporter inhibitor, MBLI87 combined to irinotecan in mice xenografted with cells overexpressing ABCG2. Mice were treated with irinotecan alone or combined to MBLI87, and tumour size was periodically measured. To model those data, a tumour growth inhibition model was developed. Unperturbed tumour growth was modelled using Simeoni's model. Drug effect kinetics was accounted for by a Kinetic-Pharmacodynamic approach. Effect of inhibitor was described with a pharmacodynamic interaction model where inhibitor enhances activity of cytotoxic. This model correctly predicted tumour growth dynamics from our study. MBLI87 increased irinotecan potency by 20% per μmol of MBLI87. This model retains enough complexity to simultaneously describe tumour growth and effect of this type of drug combination. It can thus be used as a template to early evaluate efflux transporter inhibitors in-vivo.

  8. Basipetal auxin versus acropetal cytokinin transport, and their interaction with NO3 fertilisation in cotyledon senescence and sink:source relationships in cucumber (Cucumis sativus L.).

    Science.gov (United States)

    Bangerth, K-F

    2015-11-01

    The paramount role of cytokinins (CKs) in initiation, as well as prevention, of senescence is well established. In recent years, experimental methods have become available to raise and lower the CK concentration and experimentally manipulate senescence. Decapitating the apical shoot and adding the synthetic auxin naphthylacetic acid to the cut stem reduced endogenous CKs to low levels. Conversely, if no auxin was applied, xylem and leaf CK levels increased dramatically, indicating that basipolar auxin transport is a key determinant in the synthesis of CKs and is potentially more important than NO(3). Manipulating the concentration of applied NO(3) caused considerable variation in leaf CK levels and concomitant changes in senescence. These and other results suggest that the frequently discussed decrease in nitrogen use efficiency (NUE) may be more highly regulated by CKs than by NO(3). Analysis of the re-metabolisation and re-allocation of chlorophyll, proteins, amino acids and starch in three different cucumber cultivars indirectly showed that these metabolites were significantly affected by the concentration of CKs in the leaves. Further research in this area may allow leaf senescence and plant yield to be more efficiently regulated by manipulating CKs and/or basipolar auxin transport instead of nitrate.

  9. Impaired N-linked glycosylation of uptake and efflux transporters in human non-alcoholic fatty liver disease.

    Science.gov (United States)

    Clarke, John D; Novak, Petr; Lake, April D; Hardwick, Rhiannon N; Cherrington, Nathan J

    2017-07-01

    N-linked glycosylation of proteins is critical for proper protein folding and trafficking to the plasma membrane. Drug transporters are one class of proteins that have reduced function when glycosylation is impaired. N-linked glycosylation of plasma proteins has also been investigated as a biomarker for several liver diseases, including non-alcoholic fatty liver disease (NAFLD). The purpose of this study was to assess the transcriptomic expression of genes involved in protein processing and glycosylation, and to determine the glycosylation status of key drug transporters during human NAFLD progression. Human liver samples diagnosed as healthy, steatosis, and non-alcoholic steatohepatitis (NASH) were analysed for gene expression of glycosylation-related genes and for protein glycosylation using immunoblot. Genes involved in protein processing in the ER and biosynthesis of N-glycans were significantly enriched for down-regulation in NAFLD progression. Included in the down regulated N-glycan biosynthesis category were genes involved in the oligosaccharyltransferase complex, N-glycan quality control, N-glycan precursor biosynthesis, N-glycan trimming to the core, and N-glycan extension from the core. N-glycan degradation genes were unaltered in the progression to NASH. Immunoblot analysis of the uptake transporters organic anion transporting polypeptide-1B1 (OATP1B1), OATP1B3, OATP2B1, and Sodium/Taurocholate Co-transporting Polypeptide (NTCP) and the efflux transporter multidrug resistance-associated protein 2 (MRP2) demonstrated a significant loss of glycosylation following the progression to NASH. These data suggest that the loss of glycosylation of key uptake and efflux transporters in humans NASH may influence transporter function and contribute to altered drug disposition observed in NASH. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  10. Maize ZmALMT2 is a root anion transporter that mediates constitutive root malate efflux

    Science.gov (United States)

    Aluminum (Al) toxicity is a primary limitation to crop productivity on acid soils throughout the plant. Root efflux of organic acid anions constitutes a mechanism by which plants cope with toxic aluminum (Al) ions on acid soils. In this study, we have characterized ZmALMT2 (a member of aluminum-acti...

  11. GH3-Mediated Auxin Conjugation Can Result in Either Transient or Oscillatory Transcriptional Auxin Responses.

    Science.gov (United States)

    Mellor, Nathan; Bennett, Malcolm J; King, John R

    2016-02-01

    The conjugation of the phytohormone auxin to amino acids via members of the gene family GH3 is an important component in the auxin-degradation pathway in the model plant species Arabidopsis thaliana, as well as many other plant species. Since the GH3 genes are themselves up-regulated in response to auxin, providing a negative feedback on intracellular auxin levels, it is hypothesised that the GH3s have a role in auxin homoeostasis. To investigate this, we develop a mathematical model of auxin signalling and response that includes the auxin-inducible negative feedback from GH3 on the rate of auxin degradation. In addition, we include a positive feedback on the rate of auxin input via the auxin influx transporter LAX3, shown previously to be expressed in response to auxin and to have an important role during lateral root emergence. In the absence of the LAX3 positive feedback, we show that the GH3 negative feedback suffices to generate a transient transcriptional response to auxin in the shape of damped oscillations of the model system. When LAX3 positive feedback is present, sustained oscillations of the system are possible. Using steady-state analyses, we identify and discuss key parameters affecting the oscillatory behaviour of the model. The transient peak of auxin and subsequent transcriptional response caused by the up-regulation of GH3 represents a possible protective homoeostasis mechanism that may be used by plant cells in response to excess auxin.

  12. Protection against oxidative stress in beta thalassemia/hemoglobin E erythrocytes by inhibitors of glutathione efflux transporters.

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    Chatchai Muanprasat

    Full Text Available In beta thalassemia/hemoglobin E (Hb E, abnormally high levels of oxidative stress account for accelerated senescence and increased destruction of erythrocytes. The present study aimed to investigate the role of glutathione efflux transporters, namely cystic fibrosis transmembrane conductance regulator (CFTR and multidrug resistance-associated protein 1 (MRP1, in the control of glutathione levels and protection against oxidative challenges in beta thalassemia/Hb E erythrocytes. We found that CFTR protein was expressed in the erythrocytes of beta thalassemia/Hb E patients. Treatments with GlyH-101 (50 µM, a small molecule CFTR inhibitor, and MK571 (50 µM, an MRP1 inhibitor, reduced H(2O(2-induced free radical generation in the erythrocytes by ∼80% and 50%, respectively. Furthermore, combined treatment with GlyH-101 and MK571 completely abolished the induction of reactive oxygen radicals. Increased oxidative stress in the erythrocytes following H(2O(2 challenges was accompanied by a decrease in intracellular level of reduced glutathione (GSH, which was prevented by treatments with GlyH-101 and MK571. CMFDA-based assays revealed that GlyH-101 and MK571 reduced H(2O(2-induced glutathione efflux from the erythrocytes by 87% and 66%, respectively. Interestingly, H(2O(2-induced osmotic tolerance of erythrocytes, a sign of erythrocyte aging, was ameliorated by treatment with GlyH-101. Our study indicates that oxidative stress induces glutathione efflux via CFTR and MRP1 in beta thalassemia/Hb E erythrocytes. Pharmacological inhibition of glutathione efflux represents a potential therapy to delay aging and premature destruction of erythrocytes in beta thalassemia/Hb E.

  13. Roles of pollen-specific boron efflux transporter, OsBOR4, in the rice fertilization process.

    Science.gov (United States)

    Tanaka, Nobuhiro; Uraguchi, Shimpei; Saito, Akihiro; Kajikawa, Masataka; Kasai, Koji; Sato, Yutaka; Nagamura, Yoshiaki; Fujiwara, Toru

    2013-12-01

    Arabidopsis thaliana BOR1 was the first boron (B) transporter identified in living systems. There are four AtBOR1-like genes, OsBOR1, 2, 3 and 4, present in the rice genome. We characterized the activity, expression and physiological function of OsBOR4. OsBOR4 is an active efflux transporter of B. Quantitative PCR analysis and OsBOR4 promoter-green fluorescent protein (GFP) fusion revealed that OsBOR4 was both highly and specifically expressed in pollen. We obtained five Tos17 insertion mutants of osbor4. The pollen grains were viable and development of floral organs was normal in the homozygous osbor4 mutants. We observed that in all Tos17 insertion lines tested, the frequency of osbor4 homozygous plants was lower than expected in the progeny of self-fertilized heterozygous plants. These results establish that OsBOR4 is essential for normal reproductive processes. Pollen from osbor4 homozygous plants elongated fewer tubes on wild-type stigmas, and tube elongation of mutant pollen was less efficient compared with the wild-type pollen, suggesting reduced competence of osbor4 mutant pollen. The reduced competence of mutant pollen was further supported by the crosses of independent Tos17-inserted alleles of OsBOR4. Our results suggest that OsBOR4, a boron efflux transporter, is required for normal pollen germination and/or tube elongation.

  14. Computational Study of Correlated Domain Motions in the AcrB Efflux Transporter

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    Robert Schulz

    2015-01-01

    Full Text Available As active part of the major efflux system in E. coli bacteria, AcrB is responsible for the uptake and pumping of toxic substrates from the periplasm toward the extracellular space. In combination with the channel protein TolC and membrane fusion protein AcrA, this efflux pump is able to help the bacterium to survive different kinds of noxious compounds. With the present study we intend to enhance the understanding of the interactions between the domains and monomers, for example, the transduction of mechanical energy from the transmembrane domain into the porter domain, correlated motions of different subdomains within monomers, and cooperative effects between monomers. To this end, targeted molecular dynamics simulations have been employed either steering the whole protein complex or specific parts thereof. By forcing only parts of the complex towards specific conformational states, the risk for transient artificial conformations during the simulations is reduced. Distinct cooperative effects between the monomers in AcrB have been observed. Possible allosteric couplings have been identified providing microscopic insights that might be exploited to design more efficient inhibitors of efflux systems.

  15. Effects of natural nuclear factor-kappa B inhibitors on anticancer drug efflux transporter human P-glycoprotein.

    Science.gov (United States)

    Nabekura, Tomohiro; Hiroi, Takashi; Kawasaki, Tatsuya; Uwai, Yuichi

    2015-03-01

    Drug efflux transporter P-glycoprotein plays an important role in cancer chemotherapy. The nuclear factor-κB (NF-κB) transcription factors play critical roles in development and progression of cancer. In this study, the effects of natural compounds that can inhibit NF-κB activation on the function of P-glycoprotein were investigated using human MDR1 gene-transfected KB/MDR1 cells. The accumulation of daunorubicin or rhodamine 123, fluorescent substrates of P-glycoprotein, in KB/MDR1 cells increased in the presence of caffeic acid phenetyl ester (CAPE), licochalcone A, anacardic acid, celastrol, xanthohumol, magnolol, and honokiol in a concentration-dependent manner. In contrast, lupeol, zerumbone, thymoquinone, emodin, and anethol had no effects. The ATPase activities of P-glycoprotein were stimulated by CAPE, licochalcone A, anacardic acid, celastrol, xanthohumol, magnolol, and honokiol. Tumor necrosis factor (TNF)-α stimulated NF-κB activation was inhibited by CAPE, licochalcone A, anacardic acid, and xanthohumol. KB/MDR1 cells were sensitized to vinblastine cytotoxicity by CAPE, licochalcone A, anacardic acid, xanthohumol, magnolol, and honokiol, showing that these natural NF-κB inhibitors reverse multidrug resistance. These results suggest that natural compounds, such as CAPE, licochalcone A, and anacardic acid, have dual inhibitory effects on the anticancer drug efflux transporter P-glycoprotein and NF-κB activation, and may become useful to enhance the efficacy of cancer chemotherapy.

  16. Fungicide efflux and the MgMFS1 transporter contribute to the multidrug resistance phenotype in Zymoseptoria tritici field isolates.

    Science.gov (United States)

    Omrane, Selim; Sghyer, Hind; Audéon, Colette; Lanen, Catherine; Duplaix, Clémentine; Walker, Anne-Sophie; Fillinger, Sabine

    2015-08-01

    Septoria leaf blotch is mainly controlled by fungicides. Zymoseptoria tritici, which is responsible for this disease, displays strong adaptive capacity to fungicide challenge. It developed resistance to most fungicides due to target site modifications. Recently, isolated strains showed cross-resistance to fungicides with unrelated modes of action, suggesting a resistance mechanism known as multidrug resistance (MDR). We show enhanced prochloraz efflux, sensitive to the modulators amitryptiline and chlorpromazine, for two Z. tritici strains, displaying an MDR phenotype in addition to the genotypes CYP51(I381V Y461H) or CYP51(I381V ΔY459/) (G460) , respectively, hereafter named MDR6 and MDR7. Efflux was also inhibited by verapamil in the MDR7 strain. RNA sequencing lead to the identification of several transporter genes overexpressed in both MDR strains. The expression of the MgMFS1 gene was the strongest and constitutively high in MDR field strains. Its inactivation in the MDR6 strain abolished resistance to fungicides with different modes of action supporting its involvement in MDR in Z. tritici. A 519 bp insert in the MgMFS1 promoter was detected in half of the tested MDR field strains, but absent from sensitive field strains, suggesting that the insert is correlated with the observed MDR phenotype. Besides MgMfs1, other transporters and mutations may be involved in MDR in Z. tritici.

  17. Bacillus cereus efflux protein BC3310 - a multidrug transporter of the unknown major facilitator family, UMF-2

    Directory of Open Access Journals (Sweden)

    Jasmin K Kroeger

    2015-10-01

    Full Text Available Phylogenetic classification divides the major facilitator superfamily (MFS into 82 families, including 25 families that are comprised of transporters with no characterized functions. This study describes functional data for BC3310 from Bacillus cereus ATCC 14579, a member of the unknown major facilitator family 2 (UMF 2. BC3310 was shown to be a multidrug efflux pump conferring resistance to ethidium bromide, SDS and silver nitrate when heterologously expressed in E. coli DH5α ΔacrAB. A conserved aspartate residue (D105 in putative transmembrane helix 4 was identified, which was essential for the energy dependent ethidium bromide efflux by BC3310. Transport proteins of the MFS comprise specific sequence motifs. Sequence analysis of UMF 2 proteins revealed that they carry a variant of the MFS motif A, which may be used as a marker to distinguish easily between this family and other MFS proteins. Genes orthologous to bc3310 are highly conserved within the B. cereus group of organisms and thus belong to the core genome, suggesting an important conserved functional role in the normal physiology of these bacteria.

  18. Inhibition of auxin transport from the ovary or from the apical shoot induces parthenocarpic fruit-set in tomato mediated by gibberellins.

    Science.gov (United States)

    Serrani, Juan Carlos; Carrera, Esther; Ruiz-Rivero, Omar; Gallego-Giraldo, Lina; Peres, Lázaro Eustáquio Pereira; García-Martínez, José Luis

    2010-06-01

    Fruit-set in tomato (Solanum lycopersicum) depends on gibberellins and auxins (GAs). Here, we show, using the cv MicroTom, that application of N-1-naphthylphthalamic acid (NPA; an inhibitor of auxin transport) to unpollinated ovaries induced parthenocarpic fruit-set, associated with an increase of indole-3-acetic acid (IAA) content, and that this effect was negated by paclobutrazol (an inhibitor of GA biosynthesis). NPA-induced ovaries contained higher content of GA(1) (an active GA) and transcripts of GA biosynthetic genes (SlCPS, SlGA20ox1, and -2). Interestingly, application of NPA to pollinated ovaries prevented their growth, potentially due to supraoptimal IAA accumulation. Plant decapitation and inhibition of auxin transport by NPA from the apical shoot also induced parthenocarpic fruit growth of unpollinated ovaries. Application of IAA to the severed stump negated the plant decapitation effect, indicating that the apical shoot prevents unpollinated ovary growth through IAA transport. Parthenocarpic fruit growth induced by plant decapitation was associated with high levels of GA(1) and was counteracted by paclobutrazol treatment. Plant decapitation also produced changes in transcript levels of genes encoding enzymes of GA biosynthesis (SlCPS and SlGA20ox1) in the ovary, quite similar to those found in NPA-induced fruits. All these results suggest that auxin can have opposing effects on fruit-set, either inducing (when accumulated in the ovary) or repressing (when transported from the apical shoot) that process, and that GAs act as mediators in both cases. The effect of NPA application and decapitation on fruit-set induction was also observed in MicroTom lines bearing introgressed DWARF and SELF-PRUNING wild-type alleles.

  19. Effects of pharmaceuticals and personal care products (PPCPs) on multixenobiotic resistance (MXR) related efflux transporter activity in zebrafish (Danio rerio) embryos.

    Science.gov (United States)

    Cunha, V; Burkhardt-Medicke, K; Wellner, P; Santos, M M; Moradas-Ferreira, P; Luckenbach, T; Ferreira, M

    2017-02-01

    Certain ATP binding cassette (ABC) transporter proteins, such as zebrafish Abcb4, are efflux pumps acting as a cellular defence against a wide range of different, potentially toxic chemical compounds thus mediating so called multixenobiotic resistance (MXR). Certain chemicals target MXR proteins and, as so called chemosensitisers, inhibit the activity of these proteins thus increasing the toxicity of other chemicals that would normally be effluxed. In this study 14 pharmaceuticals and personal care products (PPCPs) that are being increasingly detected in aquatic systems, were assessed for interference with the MXR system of zebrafish (Danio rerio). Concentration dependent effects of test compounds were recorded with the dye accumulation assay using zebrafish embryos and in ATPase assays with recombinant zebrafish Abcb4. In the dye accumulation assay embryos at 24h post fertilisation (hpf) were exposed to 8µm rhodamine 123 along with test compounds for 2h. The rhodamine 123 tissue levels upon the exposure served as a measure for MXR transporter efflux activity of the embryo (low rhodamine levels - high activity; high levels - low activity). The known ABC protein inhibitors MK571, vinblastine and verapamil served as positive controls. All tested PPCPs affected rhodamine 123 accumulation in embryos. For seven compounds rhodamine tissue levels were either both decreased and increased depending on the compound concentration indicating both stimulation and inhibition of rhodamine 123 efflux by those compounds, only increased (inhibition, six compounds) or only decreased (stimulation, one compound). Recombinant zebrafish Abcb4 was obtained with the baculovirus expression system and PPCPs were tested for stimulation/inhibition of basal transporter ATPase activity and for inhibition of the transporter ATPase activity stimulated with verapamil. Eight of the tested PPCPs showed effects on Abcb4 ATPase activity indicating that their effects in the dye accumulation assay may

  20. The role of polar auxin transport through pedicels of Prunus avium L. in relation to fruit development and retention.

    Science.gov (United States)

    Else, Mark A; Stankiewicz-Davies, Anna P; Crisp, Carol M; Atkinson, Christopher J

    2004-09-01

    It was investigated whether premature fruit abscission in Prunus avium L. was triggered by a reduction in polar auxin transport (PAT). The capacity of pedicels to transport tritiated IAA ([3H]-IAA) via the PAT pathway was measured at intervals throughout flower and fruit development. The extent of passive diffusion, assessed by concurrent applications of [14C]-benzoic acid ([14C]-BA), was negligible. Transported radioactivity recovered from agar blocks eluted at the same retention time as authentic [3H]-IAA during HPLC fractionation. The capacity for PAT was already high 7 d before anthesis and increased further following the fertilization of flowers at anthesis. PAT intensity was greatest immediately following fertilization and at the beginning of the cell expansion phase of fruit growth; the transport intensity in fruitlets destined to abscind was negligible. The amount of endogenous IAA moving through the PAT pathway was greatest during the first 3 weeks after fertilization and was again high at the beginning of the fruit expansion stage. IAA export in the phloem increased following fertilization then declined below detectable levels. ABA export in the phloem increased markedly during stone formation and at the onset of fruit expansion. TIBA applied to pedicels of fruit in situ promoted fruitlet abscission in 2000 but not in 2001, despite PAT capacity being reduced by over 98% in the treated pedicels. The application of TIBA to pedicels did not affect fruit expansion. The role of PAT and IAA in relation to the development and retention of Prunus avium fruit is discussed.

  1. In vitro transport activity of the fully assembled MexAB-OprM efflux pump from Pseudomonas aeruginosa

    Science.gov (United States)

    Verchère, Alice; Dezi, Manuela; Adrien, Vladimir; Broutin, Isabelle; Picard, Martin

    2015-04-01

    Antibiotic resistance is a major public health issue and many bacteria responsible for human infections have now developed a variety of antibiotic resistance mechanisms. For instance, Pseudomonas aeruginosa, a disease-causing Gram-negative bacteria, is now resistant to almost every class of antibiotics. Much of this resistance is attributable to multidrug efflux pumps, which are tripartite membrane protein complexes that span both membranes and actively expel antibiotics. Here we report an in vitro procedure to monitor transport by the tripartite MexAB-OprM pump. By combining proteoliposomes containing the MexAB and OprM portions of the complex, we are able to assay energy-dependent substrate translocation in a system that mimics the dual-membrane architecture of Gram-negative bacteria. This assay facilitates the study of pump transport dynamics and could be used to screen pump inhibitors with potential clinical use in restoring therapeutic activity of old antibiotics.

  2. Methane efflux from marine sediments in passive and active margins: Estimations from bioenergetic reaction-transport simulations

    Science.gov (United States)

    Dale, A. W.; Van Cappellen, P.; Aguilera, D. R.; Regnier, P.

    2008-01-01

    A simplified version of a kinetic-bioenergetic reaction model for anaerobic oxidation of methane (AOM) in marine sediments [Dale, A.W., Regnier, P., Van Cappellen, P., 2006. Bioenergetic controls on anaerobic oxidation of methane (AOM) in coastal marine sediments: a theoretical analysis. Am. J. Sci. 306, 246-294.] is used to assess the impact of transport processes on biomass distributions, AOM rates and methane release fluxes from the sea floor. The model explicitly represents the functional microbial groups and the kinetic and bioenergetic limitations of the microbial metabolic pathways involved in AOM. Model simulations illustrate the dominant control exerted by the transport regime on the activity and abundance of AOM communities. Upward fluid flow at active seep systems restricts AOM to a narrow subsurface reaction zone and sustains high rates of methane oxidation. In contrast, pore-water transport dominated by molecular diffusion leads to deeper and broader zones of AOM, characterized by much lower rates and biomasses. Under steady-state conditions, less than 1% of the upward dissolved methane flux reaches the water column, irrespective of the transport regime. However, a sudden increase in the advective flux of dissolved methane, for example as a result of the destabilization of methane hydrates, causes a transient efflux of methane from the sediment. The benthic efflux of dissolved methane is due to the slow growth kinetics of the AOM community and lasts on the order of 60 years. This time window is likely too short to allow for a significant escape of pore-water methane following a large scale gas hydrate dissolution event such as the one that may have accompanied the Paleocene/Eocene Thermal Maximum (PETM).

  3. CgOpt1, a putative oligopeptide transporter from Colletotrichum gloeosporioides that is involved in responses to auxin and pathogenicity

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    Maor Rudy

    2009-08-01

    Full Text Available Abstract Background The fungus Colletotrichum gloeosporioides f. sp. aeschynomene produces high levels of indole-3-acetic acid (IAA in axenic cultures and during plant infection. We generated a suppression subtractive hybridization library enriched for IAA-induced genes and identified a clone, which was highly expressed in IAA-containing medium. Results The corresponding gene showed similarity to oligopeptide transporters of the OPT family and was therefore named CgOPT1. Expression of CgOPT1 in mycelia was low, and was enhanced by external application of IAA. cgopt1-silenced mutants produced less spores, had reduced pigmentation, and were less pathogenic to plants than the wild-type strain. IAA enhanced spore formation and caused changes in colony morphology in the wild-type strain, but had no effect on spore formation or colony morphology of the cgopt1-silenced mutants. Conclusion Our results show that IAA induces developmental changes in C. gloeosporioides. These changes are blocked in cgopt1-silenced mutants, suggesting that this protein is involved in regulation of fungal response to IAA. CgOPT1 is also necessary for full virulence, but it is unclear whether this phenotype is related to auxin.

  4. Auxin Biogenesis

    Science.gov (United States)

    Bower, Peter J.; Brown, Hugh M.; Purves, William K.

    1976-01-01

    Subcellular fractionation of cucumber (Cucumis sativus L.) seedlings was achieved, and two of the enzymes in the auxin biosynthetic pathway were localized. NADH-specific indoleacetaldehyde reductase activity was observed only in the cytosol fractions obtained from separated hypocotyl and cotyledon tissue. In contrast, a portion of the NADPH-specific indoleacetaldehyde reductase activity was associated with a microsomal fraction derived from these tissues. The NADPH-specific indoleacetaldehyde reductase was consistently found to be more firmly associated with the microsomal fraction derived from hypocotyls than with that from the cotyledons. These results indicate a division of the terminal steps of auxin biogenesis into at least two subcellular compartments. PMID:16659584

  5. A tetratricopeptide repeat domain-containing protein SSR1 located in mitochondria is involved in root development and auxin polar transport in Arabidopsis.

    Science.gov (United States)

    Zhang, Min; Wang, Cuiping; Lin, Qingfang; Liu, Aihua; Wang, Ting; Feng, Xuanjun; Liu, Jie; Han, Huiling; Ma, Yan; Bonea, Diana; Zhao, Rongmin; Hua, Xuejun

    2015-08-01

    Auxin polar transport mediated by a group of Pin-formed (PIN) transporters plays important roles in plant root development. However, the mechanism underlying the PIN expression and targeting in response to different developmental and environmental stimuli is still not fully understood. Here, we report a previously uncharacterized gene SSR1, which encodes a mitochondrial protein with tetratricopeptide repeat (TPR) domains, and show its function in root development in Arabidopsis thaliana. In ssr1-2, a SSR1 knock-out mutant, the primary root growth was dramatically inhibited due to severely impaired cell proliferation and cell elongation. Significantly lowered level of auxin was found in ssr1-2 roots by auxin measurement and was further supported by reduced expression of DR5-driven reporter gene. As a result, the maintenance of the root stem cell niche is compromised in ssr1-2. It is further revealed that the expression level of several PIN proteins, namely, PIN1, PIN2, PIN3, PIN4 and PIN7, were markedly reduced in ssr1-2 roots. In particular, we showed that the reduced protein level of PIN2 on cell membrane in ssr1-2 is due to impaired retrograde trafficking, possibly resulting from a defect in retromer sorting system, which destines PIN2 for degradation in vacuoles. In conclusion, our results indicated that SSR1 is functioning in root development in Arabidopsis, possibly by affecting PIN protein expression and subcellular targeting.

  6. The expression of efflux and uptake transporters are regulated by statins in Caco-2 and hepG2 cells

    Institute of Scientific and Technical Information of China (English)

    Alice Cristina RODRIGUES; Rui CURI; Fabiana Dalla Vecchia GENVIGIR; Mario Hiroyuki HIRATA; Rosario Dominguez Crespo HIRATA

    2009-01-01

    Aim:Statin disposition and response are greatly determined by the activities of drug metabolizing enzymes and effiux/uptake transporters.There is little information on the regulation of these proteins in human ceils after statin therapy.In this study,the effects of atorvastatin and simvastatin on mRNA expression of efflux (ABCB1,ABCG2 and ABCC2) and uptake (SLCO1B1,SLCO2B1 and SLC22A1) drug transporters in Caco-2 and HepG2 cells were investigated.Methods:Quantitative real-time PCR was used to measure mRNA levels after exposure of HepG2 and Caco-2 cells to statins Results:Differences in mRNA basal levels of the transporters were as follows:ABCC2>ABCG2>ABCB1>SLCOIB1>>>SLC22A1>SLC 02B1 for HepG2 cells,and SLCO2B1>>ABCC2>ABCB1>ABCG2>>>SLC22A1 for Caco-2 cells.While for HepG2 cells,ABCC2,ABCG2 and SLCO2B1 mRNA levels were significantly up-regulated at 1,10 and 20 μmol/L after 12 or 24 h treatment,in Caco-2 cells,only the efflux transporter ABCB1 was significantly down-regulated by two-fold following a 12 h treatment with atorvastatin.Interestingly,whereas treatment with simvastatin had no effect on mRNA levels of the transporters in HepG2 cells,in Caco-2 cells the statin signifi cantly down-regulated ABCB1,ABCC2,SLC22A1,and SLCO2B1 mRNA levels after 12 or 24 h treatment.Conclusion:These findings reveal that statins exhibits differential effects on mRNA expression of drug transporters,and this effect depends on the cell type.Furthermore,alterations in the expression levels of drug transporters in the liver and/or intestine may contribute to the variability in oral disposition of statins.

  7. Direct Comparison of Manganese Detoxification/Efflux Proteins and Molecular Characterization of ZnT10 Protein as a Manganese Transporter.

    Science.gov (United States)

    Nishito, Yukina; Tsuji, Natsuko; Fujishiro, Hitomi; Takeda, Taka-Aki; Yamazaki, Tomohiro; Teranishi, Fumie; Okazaki, Fumiko; Matsunaga, Ayu; Tuschl, Karin; Rao, Rajini; Kono, Satoshi; Miyajima, Hiroaki; Narita, Hiroshi; Himeno, Seiichiro; Kambe, Taiho

    2016-07-08

    Manganese homeostasis involves coordinated regulation of specific proteins involved in manganese influx and efflux. However, the proteins that are involved in detoxification/efflux have not been completely resolved nor has the basis by which they select their metal substrate. Here, we compared six proteins, which were reported to be involved in manganese detoxification/efflux, by evaluating their ability to reduce manganese toxicity in chicken DT40 cells, finding that human ZnT10 (hZnT10) was the most significant contributor. A domain swapping and substitution analysis between hZnT10 and the zinc-specific transporter hZnT1 showed that residue Asn(43), which corresponds to the His residue constituting the potential intramembranous zinc coordination site in other ZnT transporters, is necessary to impart hZnT10's unique manganese mobilization activity; residues Cys(52) and Leu(242) in transmembrane domains II and V play a subtler role in controlling the metal specificity of hZnT10. Interestingly, the His → Asn reversion mutant in hZnT1 conferred manganese transport activity and loss of zinc transport activity. These results provide important information about manganese detoxification/efflux mechanisms in vertebrate cells as well as the molecular characterization of hZnT10 as a manganese transporter.

  8. Keeping it all together: auxin-actin crosstalk in plant development.

    Science.gov (United States)

    Zhu, Jinsheng; Geisler, Markus

    2015-08-01

    Polar auxin transport and the action of the actin cytoskeleton are tightly interconnected, which is documented by the finding that auxin transporters reach their final destination by active movement of secretory vesicles along F-actin tracks. Moreover, auxin transporter polarity and flexibility is thought to depend on transporter cycling that requires endocytosis and exocytosis of vesicles. In this context, we have reviewed the current literature on an involvement of the actin cytoskeleton in polar auxin transport and identify known similarities and differences in its structure, function and dynamics in comparison to non-plant organisms. By describing how auxin modulates actin expression and actin organization and how actin and its stability affects auxin-transporter endocytosis and recycling, we discuss the current knowledge on regulatory auxin-actin feedback loops. We focus on known effects of auxin and of auxin transport inhibitors on the stability and organization of actin and examine the functionality of auxin and/or auxin transport inhibitor-binding proteins with respect to their suitability to integrate auxin/auxin transport inhibitor action. Finally, we indicate current difficulties in the interpretation of organ, time and concentration-dependent auxin/auxin transport inhibitor treatments and formulate simple future experimental guidelines.

  9. Regulation of ATP-binding cassette transporters and cholesterol efflux by glucose in primary human monocytes and murine bone marrow-derived macrophages

    Science.gov (United States)

    Individuals with type 2 diabetes mellitus are at increased risk of developing atherosclerosis. This may be partially attributable to suppression of macrophage ATP-binding cassette (ABC) transporter mediated cholesterol efflux by sustained elevated blood glucose concentrations. Two models were used...

  10. Pattern formation in a cell based auxin transport model with numerical bifurcation analysis

    CERN Document Server

    Draelants, Delphine; Beemster, Gerrit T S; Vanroose, Wim

    2012-01-01

    Transport models of growth hormones can be used to reproduce the hormone accumulations that occur in plant organs. Mostly, these accumulation patterns are calculated using time step methods, even though only the resulting steady state patterns of the model are of interest. We examine the steady state solutions of the hormone transport model of Smith et al (2006) for a one-dimensional row of plant cells. We search for the steady state solutions as a function of three of the model parameters by using numerical continuation methods and bifurcation analysis. These methods are more adequate for solving steady state problems than time step methods. We discuss a trivial solution where the concentrations of hormones are equal in all cells and examine its stability region. We identify two generic bifurcation scenarios through which the trivial solution loses its stability. The trivial solution becomes either a steady state pattern with regular spaced peaks or a pattern where the concentration is periodic in time.

  11. Cellular and regional specific changes in multidrug efflux transporter expression during recovery of vasogenic edema in the rat hippocampus and piriform cortex.

    Science.gov (United States)

    Kim, Yeon-Joo; Kim, Ji-Eun; Choi, Hui-Chul; Song, Hong-Ki; Kang, Tae-Cheon

    2015-06-01

    In the present study, we investigated the characteristics of drug efflux transporter expressions following status epilepticus (SE). In the hippocampus and piriform cortex (PC), vasogenic edema peaked 3-4 days after SE. The expression of breast cancer resistance protein (BCRP), multidrug resistance protein-4 (MRP4), and p-glycoprotein (p-GP) were decreased 4 days after SE when vasogenic edema was peaked, but subsequently increased 4 weeks after SE. Multidrug resistance protein-1 (MRP1) expression gradually decreased in endothelial cells until 4 weeks after SE. These findings indicate that SE-induced vasogenic edema formation transiently reduced drug efflux pump expressions in endothelial cells. Subsequently, during recovery of vasogenic edema drug efflux pump expressions were differentially upregulated in astrocytes, neuropils, and endothelial cells. Therefore, we suggest that vasogenic edema formation may be a risk factor in pharmacoresistent epilepsy.

  12. Auxin at the Shoot Apical Meristem

    Science.gov (United States)

    Vernoux, Teva; Besnard, Fabrice; Traas, Jan

    2010-01-01

    Plants continuously generate new tissues and organs through the activity of populations of undifferentiated stem cells, called meristems. Here, we discuss the so-called shoot apical meristem (SAM), which generates all the aerial parts of the plant. It has been known for many years that auxin plays a central role in the functioning of this meristem. Auxin is not homogeneously distributed at the SAM and it is thought that this distribution is interpreted in terms of differential gene expression and patterned growth. In this context, auxin transporters of the PIN and AUX families, creating auxin maxima and minima, are crucial regulators. However, auxin transport is not the only factor involved. Auxin biosynthesis genes also show specific, patterned activities, and local auxin synthesis appears to be essential for meristem function as well. In addition, auxin perception and signal transduction defining the competence of cells to react to auxin, add further complexity to the issue. To unravel this intricate signaling network at the SAM, systems biology approaches, involving not only molecular genetics but also live imaging and computational modeling, have become increasingly important. PMID:20452945

  13. The role of auxin signaling in early embryo pattern formation

    NARCIS (Netherlands)

    Smit, Margot E.; Weijers, Dolf

    2015-01-01

    Pattern formation of the early Arabidopsis embryo generates precursors to all major cell types, and is profoundly controlled by the signaling molecule auxin. Here we discuss recent milestones in our understanding of auxin-dependent embryo patterning. Auxin biosynthesis, transport and response mec

  14. Drug trafficking in mice: In vivo functions of OATP uptake and ABC efflux transporters

    NARCIS (Netherlands)

    Iusuf, D.

    2013-01-01

    In recent years, there has been increasing attention for drug uptake transporters of the Organic Anion-Transporting Polypeptide (human OATP, mouse Oatp, gene names SLCO, Slco) superfamily. Especially the OATP1A and OATP1B subfamilies turn out to have important physiological and pharmacological

  15. Transcriptional Responses to the Auxin Hormone

    NARCIS (Netherlands)

    Weijers, Dolf; Wagner, Doris

    2016-01-01

    Auxin is arguably the most important signaling molecule in plants, and the last few decades have seen remarkable breakthroughs in understanding its production, transport, and perception. Recent investigations have focused on transcriptional responses to auxin, providing novel insight into the fun

  16. Moving out: from sterol transport to drug resistance - the ABCG subfamily of efflux pumps.

    Science.gov (United States)

    Moitra, Karobi; Silverton, Latoya; Limpert, Katy; Im, Kate; Dean, Michael

    2011-01-01

    The ATP binding cassette (ABC) proteins are typically ATP-driven transmembrane pumps that have been evolutionarily conserved from bacteria to humans. In humans these transporters are subdivided into seven subfamilies, ranging from A to G. The ABCG subfamily of transporters is the primary focus of this review. This subfamily of proteins has been conserved throughout evolution and plays a central role in several cellular processes, such as sterol homeostasis and multidrug resistance. Functional polymorphisms/mutations in some of these G-subfamily transporters have clinical consequences in humans.

  17. The ABC transporter ATR1 is necessary for efflux of the toxin cercosporin in the fungus Cercospora nicotianae.

    Science.gov (United States)

    Amnuaykanjanasin, Alongkorn; Daub, Margaret E

    2009-02-01

    The Cercospora nicotianae mutant deficient for the CRG1 transcription factor has marked reductions in both resistance and biosynthesis of the toxin cercosporin. We cloned and sequenced full-length copies of two genes, ATR1 and CnCFP, previously identified from a subtractive library between the wild type (WT) and a crg1 mutant. ATR1 is an ABC transporter gene and has an open reading frame (ORF) of 4368bp with one intron. CnCFP encodes a MFS transporter with homology to Cercospora kikuchii CFP, previously implicated in cercosporin export, and has an ORF of 1975bp with three introns. Disruption of ATR1 indicated atr1-null mutants had dramatic reductions in cercosporin production (25% and 20% of WT levels) in solid and liquid cultures, respectively. The ATR1 disruptants also showed moderately higher sensitivity to cercosporin. Constitutive expression of ATR1 in the crg1 mutant restored cercosporin biosynthesis and moderately increased resistance. In contrast, CnCFP overexpression in the mutant did not restore toxin production, however, it moderately enhanced toxin resistance. The results together indicate ATR1 acts as a cercosporin efflux pump in this fungus and plays a partial role in resistance.

  18. The Boron Efflux Transporter ROTTEN EAR Is Required for Maize Inflorescence Development and Fertility[C][W][OPEN

    Science.gov (United States)

    Chatterjee, Mithu; Tabi, Zara; Galli, Mary; Malcomber, Simon; Buck, Amy; Muszynski, Michael; Gallavotti, Andrea

    2014-01-01

    Although boron has a relatively low natural abundance, it is an essential plant micronutrient. Boron deficiencies cause major crop losses in several areas of the world, affecting reproduction and yield in diverse plant species. Despite the importance of boron in crop productivity, surprisingly little is known about its effects on developing reproductive organs. We isolated a maize (Zea mays) mutant, called rotten ear (rte), that shows distinct defects in vegetative and reproductive development, eventually causing widespread sterility in its inflorescences, the tassel and the ear. Positional cloning revealed that rte encodes a membrane-localized boron efflux transporter, co-orthologous to the Arabidopsis thaliana BOR1 protein. Depending on the availability of boron in the soil, rte plants show a wide range of phenotypic defects that can be fully rescued by supplementing the soil with exogenous boric acid, indicating that rte is crucial for boron transport into aerial tissues. rte is expressed in cells surrounding the xylem in both vegetative and reproductive tissues and is required for meristem activity and organ development. We show that low boron supply to the inflorescences results in widespread defects in cell and cell wall integrity, highlighting the structural importance of boron in the formation of fully fertile reproductive organs. PMID:25035400

  19. High Density Lipoproteins and Arteriosclerosis: Role of Cholesterol Efflux and Reverse Cholesterol Transport

    National Research Council Canada - National Science Library

    von Eckardstein, Arnold; Nofer, Jerzy Roch; Assmann, Gerd

    2001-01-01

    Abstract—High density lipoprotein (HDL) cholesterol is an important risk factor for coronary heart disease, and HDL exerts various potentially antiatherogenic properties, including the mediation of reverse transport of cholesterol...

  20. Efflux Transporter of Siderophore Staphyloferrin A in Staphylococcus aureus Contributes to Bacterial Fitness in Abscesses and Epithelial Cells.

    Science.gov (United States)

    Nakaminami, Hidemasa; Chen, Chunhui; Truong-Bolduc, Que Chi; Kim, Eu Suk; Wang, Yin; Hooper, David C

    2017-08-01

    The siderophores staphyloferrin A (SA) and staphyloferrin B (SB) of Staphylococcus aureus are essential for iron acquisition in the iron-restricted environment of the host, such as in subcutaneous abscesses. SA and SB are secreted by SfaA and SbnD transporters, respectively. To assess the further function of SfaA and SbnD in S. aureus fitness, we tested its effect on murine abscess models and intracellular replication in epithelial cells. Bacterial fitness in abscesses and in epithelial cells was studied, by comparing the parental strains RN6390 and MW2 and their ΔsfaA and ΔsbnD mutants using competition assays in a murine abscess model and invasion and replication assays with human lung adenocarcinoma cell line A549. In the murine abscess model using equal inocula of a ΔsfaA or ΔsbnD mutant and the wild-type RN6390 strain, the ΔsfaA mutant exhibited growth defects of 2.2-fold. Additionally, replication of the ΔsfaA mutant within A549 cells was decreased 3.0-fold. In complementation experiments, the ΔsfaA mutant carrying plasmid-borne sfaA restored the growth fitness in abscesses and epithelial cells. The ΔsbnD mutant, in contrast, showed no growth defect in either abscesses or epithelial cells. Our findings demonstrate that the efflux transporter of the siderophore SA contributes to the ability of S. aureus to replicate in abscesses and epithelial cells. Furthermore, fitness of S. aureus in these sites of replication is not compromised by the absence of transporter SbnD. Copyright © 2017 American Society for Microbiology.

  1. Syntaxin 1A interaction with the dopamine transporter promotes amphetamine-induced dopamine efflux

    DEFF Research Database (Denmark)

    Binda, Francesca; Dipace, Concetta; Bowton, Erica

    2008-01-01

    of the dopamine (DA) transporter (DAT) as the site of direct interaction with SYN1A. Amphetamine (AMPH) increases the association of SYN1A with human DAT (hDAT) in a heterologous expression system (hDAT cells) and with native DAT in murine striatal synaptosomes. Immunoprecipitation of DAT from the biotinylated...

  2. Comprehensive Transcriptome Analysis of Auxin Responses in Arabidopsis

    Institute of Scientific and Technical Information of China (English)

    Ivan A.Paponov; Martina Paponov; William Teale; Margit Menges; Sohini Chakrabortee; James A.H.Murray; Klaus Palme

    2008-01-01

    In plants,the hormone auxin shapes gene expression to regulate growth and development.Despite the detailed characterization of auxin-inducible genes,a comprehensive overview of the temporal and spatial dynamics of auxinregulated gene expression is lacking.Here,we analyze transcriptome data from many publicly available Arabidopsis profiling experiments and assess tissue-specific gene expression both in response to auxin concentration and exposure time and in relation to other plant growth regulators.Our analysis shows that the primary response to auxin over a wide range of auxin application conditions and in specific tissues comprises almost exclusively the up-regulation of genes and identifies the most robust auxin marker genes.Tissue-specific auxin responses correlate with differential expression of Aux/IAA genes and the subsequent regulation of context- and sequence-specific patterns of gene expression.Changes in transcript levels were consistent with a distinct sequence of conjugation,increased transport capacity and down-regulation of biosynthesis in the temperance of high cellular auxin concentrations.Our data show that auxin regulates genes associated with the biosynthesis,catabolism and signaling pathways of other phytohormones.We present a transcriptional overview of the auxin response.Specific interactions between auxin and other phytohormones are highlighted,particularly the regulation of their metabolism.Our analysis provides a roadmap for auxin-dependent processes that underpins the concept of an 'auxin code'-a tissue-specific fingerprint of gene expression that initiates specific developmental processes.

  3. The role of auxin signaling in early embryo pattern formation.

    Science.gov (United States)

    Smit, Margot E; Weijers, Dolf

    2015-12-01

    Pattern formation of the early Arabidopsis embryo generates precursors to all major cell types, and is profoundly controlled by the signaling molecule auxin. Here we discuss recent milestones in our understanding of auxin-dependent embryo patterning. Auxin biosynthesis, transport and response mechanisms interact to generate local auxin accumulation in the early embryo. New auxin-dependent reporters help identifying these sites, while atomic structures of transcriptional response mediators help explain the diverse outputs of auxin signaling. Key auxin outputs are control of cell identity and cell division orientation, and progress has been made towards understanding the cellular basis of each. Importantly, a number of studies have combined computational modeling and experiments to analyze the developmental role, genetic circuitry and molecular mechanisms of auxin-dependent cell division control.

  4. Opioid analgesics and P-glycoprotein efflux transporters: a potential systems-level contribution to analgesic tolerance.

    Science.gov (United States)

    Mercer, Susan L; Coop, Andrew

    2011-01-01

    Chronic clinical pain remains poorly treated. Despite attempts to develop novel analgesic agents, opioids remain the standard analgesics of choice in the clinical management of chronic and severe pain. However, mu opioid analgesics have undesired side effects including, but not limited to, respiratory depression, physical dependence and tolerance. A growing body of evidence suggests that P-glycoprotein (P-gp), an efflux transporter, may contribute a systems-level approach to the development of opioid tolerance. Herein, we describe current in vitro and in vivo methodology available to analyze interactions between opioids and P-gp and critically analyze P-gp data associated with six commonly used mu opioids to include morphine, methadone, loperamide, meperidine, oxycodone, and fentanyl. Recent studies focused on the development of opioids lacking P-gp substrate activity are explored, concentrating on structure-activity relationships to develop an optimal opioid analgesic lacking this systems-level contribution to tolerance development. Continued work in this area will potentially allow for delineation of the mechanism responsible for opioid-related P-gp up-regulation and provide further support for evidence based medicine supporting clinical opioid rotation.

  5. Effect of Efflux Transporter Inhibition on the Distribution of Fluconazole in the Rat Brain.

    Science.gov (United States)

    Wang, Wei; Zheng, Na; Zhang, Jiatang; Huang, Xusheng; Yu, Shengyuan

    2017-03-24

    Multidrug resistance-associated proteins (MRPs) and organic anion transporters (OATs) are expressed on the blood-brain barrier (BBB) and blood-cerebrospinal fluid barrier (BCSFB), preventing the entry of or the pumping out of numerous molecules. Fluconazole is widely used to treat fungal meningoencephalitis. The effect of these transporters on the distribution of fluconazole in the brain is unclear. We used microdialysis to compare the distribution of fluconazole in the rat brain with and without co-administration of probenecid, a MRP and OAT inhibitor. Additionally, we also observed the difference in fluconazole distribution between the two barriers. The results showed that probenecid increased the penetration of fluconazole into the BBB but did not alter the penetration of fluconazole into the BCSFB of rats. The penetration of the BBB and BCSFB by fluconazole did not statistically differ according to physiological condition. These results demonstrate that transporters that can be inhibited by probenecid may be involved in fluconazole resistance at the BBB and provide a laboratory basis for predicting brain extracellular fluid (ECF) concentration using the cerebrospinal fluid (CSF) concentration of fluconazole.

  6. The auxin transporter, OsAUX1, is involved in primary root and root hair elongation and in Cd stress responses in rice (Oryza sativa L.)

    National Research Council Canada - National Science Library

    Yu, ChenLiang; Sun, ChenDong; Shen, Chenjia; Wang, Suikang; Liu, Fang; Liu, Yan; Chen, YunLong; Li, Chuanyou; Qian, Qian; Aryal, Bibek; Geisler, Markus; Jiang, De An; Qi, YanHua

    2015-01-01

    Auxin and cadmium (Cd) stress play critical roles during root development. There are only a few reports on the mechanisms by which Cd stress influences auxin homeostasis and affects primary root ( PR ) and lateral root ( LR...

  7. A ROP GTPase-dependent auxin signaling pahtway regulates the subcellular distribution of PIN2 in Arabidopsis roots

    NARCIS (Netherlands)

    Lin, D.; Nagawa, S.; Chen, J.; Cao, L.; Scheres, B.

    2012-01-01

    PIN-FORMED (PIN) protein-mediated auxin polar transport is critically important for development, pattern formation, and morphogenesis in plants. Auxin has been implicated in the regulation of polar auxin transport by inhibiting PIN endocytosis [1 and 2], but how auxin regulates this process is

  8. A ROP GTPase-dependent auxin signaling pahtway regulates the subcellular distribution of PIN2 in Arabidopsis roots

    NARCIS (Netherlands)

    Lin, D.; Nagawa, S.; Chen, J.; Cao, L.; Scheres, B.

    2012-01-01

    PIN-FORMED (PIN) protein-mediated auxin polar transport is critically important for development, pattern formation, and morphogenesis in plants. Auxin has been implicated in the regulation of polar auxin transport by inhibiting PIN endocytosis [1 and 2], but how auxin regulates this process is poorl

  9. Aryl hydrocarbon receptor-mediated up-regulation of ATP-driven xenobiotic efflux transporters at the blood-brain barrier.

    Science.gov (United States)

    Wang, Xueqian; Hawkins, Brian T; Miller, David S

    2011-02-01

    Many widespread and persistent organic pollutants, e.g., 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), activate the aryl hydrocarbon receptor (AhR), causing it to translocate to the cell nucleus, where it transactivates target genes. AhR's ability to target the blood-brain barrier is essentially unexplored. We show here that exposing isolated rat brain capillaries to 0.05-0.5 nM TCDD roughly doubled transport activity and protein expression of P-glycoprotein, an ATP-driven drug efflux pump and a critical determinant of drug entry into the CNS. These effects were abolished by actinomycin D or cycloheximide or by the AhR antagonists resveratrol and α-naphthoflavone. Brain capillaries from TCDD-dosed rats (1-5 μg/kg, i.p.) exhibited increased transport activity and protein expression of 3 xenobiotic efflux pumps, P-glycoprotein, multidrug resistance-associated protein 2, and breast cancer resistance polypeptide, as well as expression of Cyp1a1 and Cyp1b1, both AhR target genes. Consistent with increased P-glycoprotein expression in capillaries from TCDD-dosed rats, in situ brain perfusion indicated significantly reduced brain accumulation of verapamil, a P-glycoprotein substrate. These findings suggest a new paradigm for the field of environmental toxicology: toxicants acting through AhR to target xenobiotic efflux transporters at the blood-brain barrier and thus reduce brain accumulation of CNS-acting therapeutic drugs.

  10. Efflux Transporters Regulate Arsenite Induced Genotoxicity in Double Negative and Double Positive T Cells.

    Science.gov (United States)

    Xu, Huan; Medina, Sebastian; Lauer, Fredine T; Douillet, Christelle; Jian Liu, Ke; Hudson, Laurie G; Stýblo, Miroslav; Aleksunes, Lauren M; Burchiel, Scott W

    2017-04-29

    Arsenite (As+3) exposure is known to cause immunotoxicity in human and animal models. Our previous studies demonstrated that As+3 at 50 to 500 nM concentrations induced both genotoxicity and non-genotoxicity in mouse thymus cells. Developing T cells at CD4-CD8- double negative (DN) stage, the first stage after early T cells are transported from bone marrow to thymus, were found to be more sensitive to As+3 toxicity than the T cells at CD4+CD8+ double positive (DP) stage in vitro. Induction of Mdr1 (Abcb1) and Mrp1 (Abcc1), two multidrug resistance transporters and exporters of As+3, was associated with the reversal of As+3-induced double strand breaks and DNA damage. In order to confirm that the thymus cell populations have different sensitivity to As+3in vivo, male C57BL/6J mice were exposed to 0, 100 and 500 ppb As+3 in drinking water for 30 d. A significant decrease in DN cell percentage was observed with exposure to 500 ppb As+3. Low to moderate concentrations of As+3 were shown to induce higher genotoxicity in sorted DN cells than DP cells in vitro. Calcein AM uptake and Mdr1/Mrp1 mRNA quantification results revealed that DN cells not only had limited As+3 exporter activity, but also lacked the ability to activate these exporters with As+3 treatments, resulting in a higher accumulation of intracellular As+3. Knockdown study of As+3 exporters in the DN thymic cell line, D1 using siRNA, demonstrated that Mdr1 and Mrp1 regulate intracellular As+3 accumulation and genotoxicity. Taken together, the results indicate that transporter regulation is an important mechanism for differential genotoxicity induced by As+3 in thymocytes at different developmental stages. © The Author 2017. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  11. Do trees grow on money? Auxin as the currency of the cellular economy.

    Science.gov (United States)

    Stewart, Jodi L; Nemhauser, Jennifer L

    2010-02-01

    Auxin plays a role in nearly every aspect of a plant's life. Signals from the developmental program, physiological status, and encounters with other organisms all converge on the auxin pathway. The molecular mechanisms facilitating these interactions are diverse; yet, common themes emerge. Auxin can be regulated by modulating rates of biosynthesis, conjugation, and transport, as well as sensitivity of a cell to the auxin signal. In this article, we describe some well-studied examples of auxin's interactions with other pathways.

  12. Auxin-Cytokinin Interaction Regulates Meristem Development

    Institute of Scientific and Technical Information of China (English)

    Ying-Hua Su; Yu-Bo Liu; Xian-Sheng Zhang

    2011-01-01

    Plant hormones regulate many aspects of plant growth and development. Both auxin and cytokinin have been known for a long time to act either synergistically or antagonistically to control several significant developmental processes, such as the formation and maintenance of meristem. Over the past few years, exciting progress has been made to reveal the molecular mechanisms underlying the auxin-cytokinin action and interaction. In this review, we shall briefly discuss the major progress made in auxin and cytokinin biosynthesis, auxin transport, and auxin and cytokinin signaling.The frameworks for the complicated interaction of these two hormones in the control of shoot apical meristem and root apical meristem formation as well as their roles in in vitro organ regeneration are the major focus of this review.

  13. Endosidin1 defines a compartment involved in endocytosis of the brassinosteroid receptor BRI1 and the auxin transporters PIN2 and AUX1.

    Science.gov (United States)

    Robert, Stéphanie; Chary, S Narasimha; Drakakaki, Georgia; Li, Shundai; Yang, Zhenbiao; Raikhel, Natasha V; Hicks, Glenn R

    2008-06-17

    Although it is known that proteins are delivered to and recycled from the plasma membrane (PM) via endosomes, the nature of the compartments and pathways responsible for cargo and vesicle sorting and cellular signaling is poorly understood. To define and dissect specific recycling pathways, chemical effectors of proteins involved in vesicle trafficking, especially through endosomes, would be invaluable. Thus, we identified chemicals affecting essential steps in PM/endosome trafficking, using the intensely localized PM transport at the tips of germinating pollen tubes. The basic mechanisms of this localized growth are likely similar to those of non-tip growing cells in seedlings. The compound endosidin 1 (ES1) interfered selectively with endocytosis in seedlings, providing a unique tool to dissect recycling pathways. ES1 treatment induced the rapid agglomeration of the auxin translocators PIN2 and AUX1 and the brassinosteroid receptor BRI1 into distinct endomembrane compartments termed "endosidin bodies"; however, the markers PIN1, PIN7, and other PM proteins were unaffected. Endosidin bodies were defined by the syntaxin SYP61 and the V-ATPase subunit VHA-a1, two trans-Golgi network (TGN)/endosomal proteins. Interestingly, brassinosteroid (BR)-induced gene expression was inhibited by ES1 and treated seedlings displayed a brassinolide (BL)-insensitive phenotype similar to a bri1 loss-of-function mutant. No effect was detected in auxin signaling. Thus, PIN2, AUX1, and BRI1 use interactive pathways involving an early SYP61/VHA-a1 endosomal compartment.

  14. N-glycan containing a core α1,3-fucose residue is required for basipetal auxin transport and gravitropic response in rice (Oryza sativa).

    Science.gov (United States)

    Harmoko, Rikno; Yoo, Jae Yong; Ko, Ki Seong; Ramasamy, Nirmal Kumar; Hwang, Bo Young; Lee, Eun Ji; Kim, Ho Soo; Lee, Kyung Jin; Oh, Doo-Byoung; Kim, Dool-Yi; Lee, Sanghun; Li, Yang; Lee, Sang Yeol; Lee, Kyun Oh

    2016-10-01

    In plants, α1,3-fucosyltransferase (FucT) catalyzes the transfer of fucose from GDP-fucose to asparagine-linked GlcNAc of the N-glycan core in the medial Golgi. To explore the physiological significance of this processing, we isolated two Oryza sativa (rice) mutants (fuct-1 and fuct-2) with loss of FucT function. Biochemical analyses of the N-glycan structure confirmed that α1,3-fucose is missing from the N-glycans of allelic fuct-1 and fuct-2. Compared with the wild-type cv Kitaake, fuct-1 displayed a larger tiller angle, shorter internode and panicle lengths, and decreased grain filling as well as an increase in chalky grains with abnormal shape. The mutant allele fuct-2 gave rise to similar developmental abnormalities, although they were milder than those of fuct-1. Restoration of a normal tiller angle in fuct-1 by complementation demonstrated that the phenotype is caused by the loss of FucT function. Both fuct-1 and fuct-2 plants exhibited reduced gravitropic responses. Expression of the genes involved in tiller and leaf angle control was also affected in the mutants. We demonstrate that reduced basipetal auxin transport and low auxin accumulation at the base of the shoot in fuct-1 account for both the reduced gravitropic response and the increased tiller angle.

  15. Cooperation between prokaryotic (Lde) and eukaryotic (MRP) efflux transporters in J774 macrophages infected with Listeria monocytogenes: studies with ciprofloxacin and moxifloxacin.

    Science.gov (United States)

    Lismond, Ann; Tulkens, Paul M; Mingeot-Leclercq, Marie-Paule; Courvalin, Patrice; Van Bambeke, Françoise

    2008-09-01

    Antibiotic efflux is observed in both eukaryotic and prokaryotic cells, modulating accumulation and resistance. The present study examines whether eukaryotic and prokaryotic fluoroquinolone transporters can cooperate in the context of an intracellular infection. We have used (i) J774 macrophages (comparing a ciprofloxacin-resistant cell line overexpressing an MRP-like transporter with wild-type cells with basal expression), (ii) Listeria monocytogenes (comparing a clinical isolate [CLIP21369] displaying ciprofloxacin resistance associated with overexpression of the Lde efflux system with a wild-type strain [EGD]), (iii) ciprofloxacin (substrate of both Lde and MRP) and moxifloxacin (nonsubstrate), and (iv) probenecid and reserpine (preferential inhibitors of MRP and Lde, respectively). The ciprofloxacin MICs for EGD were unaffected by reserpine, while those for CLIP21369 were decreased approximately fourfold (and made similar to those of EGD). Neither probenecid nor reserpine affected the moxifloxacin MICs against EGD or CLIP21369. In dose-response studies (0.01x to 100x MIC) in broth, reserpine fully restored the susceptibility of CLIP21369 to ciprofloxacin (no effect on EGD) but did not influence the activity of moxifloxacin. In studies with intracellular bacteria, reserpine, probenecid, and their combination increased the activity of ciprofloxacin in wild-type and ciprofloxacin-resistant macrophages in parallel with an increase in ciprofloxacin accumulation in macrophages for EGD and an increase in accumulation and decrease in MIC (in broth) for CLIP21369. Moxifloxacin accumulation and intracellular activity were consistently not affected by the inhibitors. A bacterial efflux pump may thus actively cooperate with a eukaryotic efflux transporter to reduce the activity of a common substrate (ciprofloxacin) toward an intracellular bacterial target.

  16. Effect of Apolipoprotein A-I on ATP Binding Cassette Transporter A1 Degradation and Cholesterol Efflux in THP-1 Macrophage-derived Foam Cells

    Institute of Scientific and Technical Information of China (English)

    Chao-Ke TANG; Chang-Geng RUAN; Yong-Zong YANG; Guo-Hua TANG; Guang-Hui IY; Zuo WANG; Lu-Shan LIU; Shuang WAN; Zhong-Hua YUAN; Xiu-Sheng HE; Jun-Hao YANG

    2004-01-01

    Cholesterol-loaded macrophage foam cells are a central componentof atherosclerotic lesions ATP binding cassette transporter A1(ABCA1),the defective molecule in Tangier disease,mediates the efflux ofphospholipid and cholesterol from cells to apolipoprotein A-I(apoA-I),reversing foam cell formation.This study investigated the effect of apoA-I on ABCA1 degradation and cholesterol efflux in THP-1 macrophagederived foam cells.After exposure of the cultured THP-1 macrophage-derived foam cells to apoA-I for different time,cholesterol efflux,ABCA1 mRNA and protein levels were determined by FJ-2107P type liquid scintillator,RT-PCR and Western blot,respectively.The mean ABCA1 fluorescence intensity on THP-1macrophage-derived foam cells was detected by flow cytometry.Results showed that apoA-I markedly increased ABCAl-mediated cholesterol efflux from THP-1 macrophage-derived foam cells.This was accompanied by an increase in the content of ABCA1.ApoA-I did not alter ABCA 1 mRNA abundance.Significantly,thiol protease inhibitors increased the level ofABCA1 protein and slowed its decay in THP-1macrophage-derived foam cells,whereas none of the proteosome-specific inhibitor lactacystin,other protease inhibitors,or the lysosomal inhibitor NH4Cl showed such effects.The apoA-I-mediated cellular cholesterol efflux was enhanced by thiol protease inhibitors.Our results suggested that thiol protease inhibitors mightprovide an alternative way to upregulate ABCA1 protein.This strategy is especially appealing since it may mimic the stabilizing effect of the natural ligands apoA-I.

  17. Multidrug efflux pumps: the structures of prokaryotic ATP-binding cassette transporter efflux pumps and implications for our understanding of eukaryotic P-glycoproteins and homologues.

    Science.gov (United States)

    Kerr, Ian D; Jones, Peter M; George, Anthony M

    2010-02-01

    One of the Holy Grails of ATP-binding cassette transporter research is a structural understanding of drug binding and transport in a eukaryotic multidrug resistance pump. These transporters are front-line mediators of drug resistance in cancers and represent an important therapeutic target in future chemotherapy. Although there has been intensive biochemical research into the human multidrug pumps, their 3D structure at atomic resolution remains unknown. The recent determination of the structure of a mouse P-glycoprotein at subatomic resolution is complemented by structures for a number of prokaryotic homologues. These structures have provided advances into our knowledge of the ATP-binding cassette exporter structure and mechanism, and have provided the template data for a number of homology modelling studies designed to reconcile biochemical data on these clinically important proteins.

  18. Effects of stachyose on absorption and transportation of tea catechins in mice: possible role of Phase II metabolic enzymes and efflux transporters inhibition by stachyose

    Science.gov (United States)

    Li, Wenfeng; Lu, Yalong; Huang, Di; Han, Xiao; Yang, Xingbin

    2016-01-01

    Background Nutritional and absorption-promoting properties of stachyose combined with tea catechins (TC) have been revealed. However, the mechanism involved in non-digestible oligosaccharides-mediated enhancement of flavonoid absorption has largely remained elusive. Methods This study was designed to investigate the molecular mechanism of stachyose in enhancing absorption and transportation of TC in mice. Mice were orally pre-treated with stachyose (50, 250, and 500 mg/kg·bw) for 0–8 weeks, and 1 h before sacrifice, mice were treated with TC (250 mg/kg·bw). Results Gas chromatography-mass spectrometry analysis showed that serum concentrations of epicatechin, epigallocatechin, epicatechin gallate, and epigallocatechin gallate were dose- and time-dependently elevated with stachyose pre-treatment in mice. Furthermore, pre-treatment with stachyose in mice reduced intestinal sulfotransferase and uridine diphosphate-glucuronosyltransferase levels by 3.3–43.2% and 23.9–30.4%, relative to control mice, respectively. Moreover, intestinal P-glycoprotein and multidrug resistance-associated protein-1 contents were decreased in mice by pre-administration of stachyose in dose- and time-dependent manner. Conclusions This is the first time to demonstrate that suppression of Phase II metabolic enzymes and efflux transporters of TC in the intestine can play a major role in increasing absorption of TC by stachyose feeding. PMID:27782875

  19. Effects of stachyose on absorption and transportation of tea catechins in mice: possible role of Phase II metabolic enzymes and efflux transporters inhibition by stachyose

    Directory of Open Access Journals (Sweden)

    Wenfeng Li

    2016-10-01

    Full Text Available Background: Nutritional and absorption-promoting properties of stachyose combined with tea catechins (TC have been revealed. However, the mechanism involved in non-digestible oligosaccharides-mediated enhancement of flavonoid absorption has largely remained elusive. Methods: This study was designed to investigate the molecular mechanism of stachyose in enhancing absorption and transportation of TC in mice. Mice were orally pre-treated with stachyose (50, 250, and 500 mg/kg·bw for 0–8 weeks, and 1 h before sacrifice, mice were treated with TC (250 mg/kg·bw. Results: Gas chromatography-mass spectrometry analysis showed that serum concentrations of epicatechin, epigallocatechin, epicatechin gallate, and epigallocatechin gallate were dose- and time-dependently elevated with stachyose pre-treatment in mice. Furthermore, pre-treatment with stachyose in mice reduced intestinal sulfotransferase and uridine diphosphate-glucuronosyltransferase levels by 3.3–43.2% and 23.9–30.4%, relative to control mice, respectively. Moreover, intestinal P-glycoprotein and multidrug resistance-associated protein-1 contents were decreased in mice by pre-administration of stachyose in dose- and time-dependent manner. Conclusions: This is the first time to demonstrate that suppression of Phase II metabolic enzymes and efflux transporters of TC in the intestine can play a major role in increasing absorption of TC by stachyose feeding.

  20. Selecting surfactants for the maximum inhibition of the activity of the multidrug resistance efflux pump transporter, P-glycoprotein: conceptual development

    Directory of Open Access Journals (Sweden)

    Shireesh Apte

    2010-09-01

    Full Text Available Amphiphilic excipients, such as surfactants, have been shown to be inhibitors of the multidrug resistance (MDR efflux pump transporter protein, P glycoprotein (Pgp. In vitro studies using many surfactants have demonstrated that those with an optimum hydrophilic-lipophilic balance (HLB exhibit greater efflux pump inhibition than those that are either very hydrophobic, or very hydrophilic, although the correlation of HLB to Pgp inhibition activity remains weak. Using the data from multiple in vitro studies, a model has been conceptualized that underscores the attributes of both the HLB and the critical micellar concentration (CMC, occurring in tandem, and unable of being varied independently, as key determinants toward prediction of surfactant Pgp inhibition activity. The algorithm that formalizes this concept provides a ‘semi-rational’ method of choosing surfactants for a specific type of cancer for maximum inhibition of MDR.

  1. Unraveling carbohydrate transport mechanisms in young beech trees (Fagus sylvatica f. purpurea) by 13CO2 efflux measurements from stem and soil

    Science.gov (United States)

    Thoms, Ronny; Muhr, Jan; Keitel, Claudia; Kayler, Zachary; Gavrichkova, Olga; Köhler, Michael; Gessler, Arthur; Gleixner, Gerd

    2016-04-01

    Transport mechanisms of soluble carbohydrates and diurnal CO2 efflux from tree stems and surrounding soil are well studied. However, the effect of transport carbohydrates on respiration and their interaction with storage processes is largely unknown. Therefore, we performed a set of 13CO2 pulse labeling experiments on young trees of European beech (Fagus sylvatica f. purpurea). We labeled the whole tree crowns in a closed transparent plastic chamber with 99% 13CO2 for 30 min. In one experiment, only a single branch was labeled and removed 36 hours after labeling. In all experiments, we continuously measured the 13CO2 efflux from stem, branch and soil and sampled leaf and stem material every 3 h for 2 days, followed by a daily sampling of leaves in the successive 5 days. The compound specific δ 13C value of extracted soluble carbohydrates from leaf and stem material was measured by high-performance liquid chromatography linked with an isotope ratio mass spectrometer (HPLC-IRMS). The 13CO2 signal from soil respiration occurred only few hours after labeling indicating a very high transport rate of carbohydrates from leaf to roots and to the rhizosphere. The label was continuously depleted within the next 5 days. In contrast, we observed a remarkable oscillating pattern of 13CO2 efflux from the stem with maximum 13CO2 enrichment at noon and minima at night time. This oscillation suggests that enriched carbohydrates are respired during the day, whereas in the night the enriched sugars are not respired. The observed oscillation in stem 13CO2 enrichment remained unchanged even when only single branches were labelled and cut right afterwards. Thus, storage and conversion of carbohydrates only occurred within the stem. The δ13C patterns of extracted soluble carbohydrates showed, that a transformation of transitory starch to carbohydrates and vice versa was no driver of the oscillating 13CO2 efflux from the stem. Carbohydrates might have been transported in the phloem to

  2. Hydrophobic amino acids in the hinge region of the 5A apolipoprotein mimetic peptide are essential for promoting cholesterol efflux by the ABCA1 transporter.

    Science.gov (United States)

    Sviridov, Denis O; Andrianov, Alexander M; Anishchenko, Ivan V; Stonik, John A; Amar, Marcelo J A; Turner, Scott; Remaley, Alan T

    2013-01-01

    The bihelical apolipoprotein mimetic peptide 5A effluxes cholesterol from cells and reduces inflammation and atherosclerosis in animal models. We investigated how hydrophobic residues in the hinge region between the two helices are important in the structure and function of this peptide. By simulated annealing analysis and molecular dynamics modeling, two hydrophobic amino acids, F-18 and W-21, in the hinge region were predicted to be relatively surface-exposed and to interact with the aqueous solvent. Using a series of 5A peptide analogs in which F-18 or W-21 was changed to either F, W, A, or E, only peptides with hydrophobic amino acids in these two positions were able to readily bind and solubilize phospholipid vesicles. Compared with active peptides containing F or W, peptides containing E in either of these two positions were more than 10-fold less effective in effluxing cholesterol by the ABCA1 transporter. Intravenous injection of 5A in C57BL/6 mice increased plasma-free cholesterol (5A: 89.9 ± 13.6 mg/dl; control: 38.7 ± 4.3 mg/dl (mean ± S.D.); P < 0.05) and triglycerides (5A: 887.0 ± 172.0 mg/dl; control: 108.9 ± 9.9 mg/dl; P < 0.05), whereas the EE peptide containing E in both positions had no effect. Finally, 5A increased cholesterol efflux approximately 2.5-fold in vivo from radiolabeled macrophages, whereas the EE peptide was inactive. These results provide a rationale for future design of therapeutic apolipoprotein mimetic peptides and provide new insights into the interaction of hydrophobic residues on apolipoproteins with phospholipids in the lipid microdomain created by the ABCA1 transporter during the cholesterol efflux process.

  3. 滇杨倒置插穗的生长素极性运输%The Polarity of Auxin Transport in Inverted Cuttings of Populus yunnanensis

    Institute of Scientific and Technical Information of China (English)

    李佳蔓; 员涛; 王军民; 郑元; 周安佩; 纵丹; 何承忠

    2015-01-01

    生长素是唯一具有极性运输的内源激素,在植株生长发育过程中发挥着重要的调控作用。为探讨倒置插穗在生根发芽过程中生长素的极性运输规律,以滇杨1年生根萌苗主干制作插穗,分别正向放置和倒向放置后进行水培,采用高效液相色谱法(HPLC)测定正置、倒置插穗不同部位树皮与侧芽内生长素(IAA)和脱落酸(ABA)含量的动态变化。结果表明,不同时期滇杨正置、倒置插穗树皮和侧芽内IAA和ABA总含量的动态变化规律存在差异,极性对倒置插穗的生理生化活性有一定的影响。滇杨正置插穗树皮和侧芽内IAA的运输符合极性运输规律。倒置插穗分别在21 d和28 d之前,其树皮和侧芽内IAA的运输符合极性运输规律,即从形态学上端(插穗的下端)向形态学下端(插穗的上端)运输;之后,倒置插穗内重新建立了IAA的运输方向,即从形态学下端(插穗的上端)向形态学上端(插穗的下端)运输。正置与倒置插穗不同部位树皮和侧芽内ABA的含量从上到下依次递增,运输规律遵循向重力性。研究结果初步表明:滇杨倒置插穗内IAA的极性运输可能会发生逆转。%Polar transport within plant tissues is unique to auxin, and it plays an important role to regulate plant growth and development. In order to investigate the polar auxin transport regularity in inverted cutting of Popu-lus yunnanensis for the duration of rooting and shooting, cuttings made of one-year-old main stem of P. yunna-nensis rootlings, upright and inverted cuttings cultured in water, high performance liquid chromatography (HPLC) was used to measure the dynamic changes of IAA and ABA contents in different parts of bark and lat-eral buds of upright and inverted cuttings. The results showed that differences existed in amount contents of IAA and ABA in bark and lateral buds between upright cuttings and inverted cuttings, and the polarity made

  4. Changes in starch and inositol 1,4,5-trisphosphate levels and auxin transport are interrelated in graviresponding oat (Avena sativa) shoots.

    Science.gov (United States)

    Yun, Hye Sup; Joo, Se-Hwan; Kaufman, Peter B; Kim, Tae-Wuk; Kirakosyan, Ara; Philosoph-Hadas, Sonia; Kim, Seong-Ki; Chang, Soo Chul

    2006-11-01

    This study was conducted to unravel a mechanism for the gravitropic curvature response in oat (Avena sativa) shoot pulvini. For this purpose, we examined the downward movement of starch-filled chloroplast gravisensors, differential changes in inositol 1,4,5-trisphosphate (IP(3)) levels, transport of indole-3-acetic acid (IAA) and gravitropic curvature. Upon gravistimulation, the ratio for IAA levels in lower halves versus those in upper halves (L/U) increased from 1.0 at 0 h and reached a maximum value of 1.45 at 8 h. When shoots were grown in the dark for 10 d, to deplete starch in the chloroplast, the gravity-induced L/U of IAA was reduced to 1.0. N-naphthylphthalamic acid (NPA) and 2,3,5-triiodobenzoic acid (TIBA), both auxin transport inhibitors, significantly reduced the amount of gravitropic curvature and gravity-induced lateral IAA transport, but did not reduce the gravity-induced late change in the L/U ratio of IP(3) levels. U73122, a specific phospholipase C (PLC) inhibitor, decreased gravity-induced curvature. Because U73122 reduced the ratio of L/U of IAA imposed by gravistimulation, it is clear that IAA transport is correlated with changes in IP(3) levels upon gravistimulation. These results indicate that gravistimulation-induced differential lateral IAA transport may result from the onset of graviperception in the chloroplast gravisensors coupled with gravity-induced asymmetric changes in IP(3) levels in oat shoot pulvini.

  5. Auxin metabolism rates and implications for plant development

    Directory of Open Access Journals (Sweden)

    Eric M Kramer

    2015-03-01

    Full Text Available Studies of auxin metabolism rarely express their results as a metabolic rate, although the data obtained would often permit such a calculation to be made. We analyze data from 31 previously published papers to quantify the rates of auxin biosynthesis, conjugation, conjugate hydrolysis, and catabolism in seed plants. Most metabolic pathways have rates in the range 10 nM/h to 1 μM/h, with the exception of auxin conjugation, which has rates as high as ~100 μM/h. The highest rates of auxin conjugation suggests that auxin metabolic sinks may be very small, perhaps as small as a single cell. By contrast, the relatively low rate of auxin biosynthesis requires plants to conserve and recycle auxin during long-distance transport. The consequences for plant development are discussed.

  6. Auxin activity: Past, present, and future.

    Science.gov (United States)

    Enders, Tara A; Strader, Lucia C

    2015-02-01

    Long before its chemical identity was known, the phytohormone auxin was postulated to regulate plant growth. In the late 1800s, Sachs hypothesized that plant growth regulators, present in small amounts, move differentially throughout the plant to regulate growth. Concurrently, Charles Darwin and Francis Darwin were discovering that light and gravity were perceived by the tips of shoots and roots and that the stimulus was transmitted to other tissues, which underwent a growth response. These ideas were improved upon by Boysen-Jensen and Paál and were later developed into the Cholodny-Went hypothesis that tropisms were caused by the asymmetric distribution of a growth-promoting substance. These observations led to many efforts to identify this elusive growth-promoting substance, which we now know as auxin. In this review of auxin field advances over the past century, we start with a seminal paper by Kenneth Thimann and Charles Schneider titled "The relative activities of different auxins" from the American Journal of Botany, in which they compare the growth altering properties of several auxinic compounds. From this point, we explore the modern molecular understanding of auxin-including its biosynthesis, transport, and perception. Finally, we end this review with a discussion of outstanding questions and future directions in the auxin field. Over the past 100 yr, much of our progress in understanding auxin biology has relied on the steady and collective advance of the field of auxin researchers; we expect that the next 100 yr of auxin research will likewise make many exciting advances.

  7. Phyllotaxis involves auxin drainage through leaf primordia

    DEFF Research Database (Denmark)

    Deb, Yamini; Marti, Dominik; Frenz, Martin;

    2015-01-01

    of phyllotaxis invoke the accumulation of auxin at leaf initials and removal of auxin through their developing vascular strand, the midvein. We have developed a precise microsurgical tool to ablate the midvein at high spatial and temporal resolution in order to test its function in leaf formation and phyllotaxis...... and to an increase in their width. Phyllotaxis was transiently affected after midvein ablations, but readjusted after two plastochrons. These results indicate that the developing midvein is involved in the basipetal transport of auxin through young primordia, which contributes to phyllotactic spacing and stability...

  8. Morphometric Analysis of Auxin-Mediated Development

    Science.gov (United States)

    Lewis, Daniel

    Auxin controls many aspects of plant development through its effects on growth. Its distribution is controlled by specific tissue and organ level polar transport streams. The responses to environmental cues such as gravity light, nutrient availability are largely controlled by coordinated regulation of distinct auxin transport streams. Many plant responses to the environment involve changes in shape. Much can be learned about the underlying processes controlling plant form if the response is measured with sufficient resolution. Computer-aided analysis of digital images or 'machine vision' can be used to greatly increase the speed and consistency of data from a morphometric study of plant form. Advances in image acquisition and analysis pioneered at UW-Madison have allowed unprecedented resolution of the growth and gravitropism of Arabidopsis. A reverse genetic analysis was used to determine if the MDR-like ABC transporters influence auxin distribution important for plant development and the response to environmental cues in Arabidopsis. Mutations in MDR1 (At3g28860) reduce acropetal auxin transport in the root. This is correlated with deviation from the vertical axis. Mutations in MDR4 (At2g47000) reduce basipetal auxin transport in the root. This is correlated with hypergravitropism. It was theorized that reduced transport whithin the elongation zone is responsible for the increased curvature. Flavanols were found to regulate gravitropism upstream of MDR4. The mdr1 mdr4 double mutant showed additive but not synergistic phenotypes, suggesting that the two auxin transport streams are more independent than interdependent. MDR proteins seem to enhance auxin transport in situations where PIN-type effux alone is insufficient.

  9. Dynamic regulation of auxin oxidase and conjugating enzymes AtDAO1 and GH3 modulates auxin homeostasis

    Science.gov (United States)

    Mellor, Nathan; Band, Leah R.; Pěnčík, Aleš; Rashed, Afaf; Holman, Tara; Wilson, Michael H.; Voß, Ute; Bishopp, Anthony; King, John R.

    2016-01-01

    The hormone auxin is a key regulator of plant growth and development, and great progress has been made understanding auxin transport and signaling. Here, we show that auxin metabolism and homeostasis are also regulated in a complex manner. The principal auxin degradation pathways in Arabidopsis include oxidation by Arabidopsis thaliana gene DIOXYGENASE FOR AUXIN OXIDATION 1/2 (AtDAO1/2) and conjugation by Gretchen Hagen3s (GH3s). Metabolic profiling of dao1-1 root tissues revealed a 50% decrease in the oxidation product 2-oxoindole-3-acetic acid (oxIAA) and increases in the conjugated forms indole-3-acetic acid aspartic acid (IAA-Asp) and indole-3-acetic acid glutamic acid (IAA-Glu) of 438- and 240-fold, respectively, whereas auxin remains close to the WT. By fitting parameter values to a mathematical model of these metabolic pathways, we show that, in addition to reduced oxidation, both auxin biosynthesis and conjugation are increased in dao1-1. Transcripts of AtDAO1 and GH3 genes increase in response to auxin over different timescales and concentration ranges. Including this regulation of AtDAO1 and GH3 in an extended model reveals that auxin oxidation is more important for auxin homoeostasis at lower hormone concentrations, whereas auxin conjugation is most significant at high auxin levels. Finally, embedding our homeostasis model in a multicellular simulation to assess the spatial effect of the dao1-1 mutant shows that auxin increases in outer root tissues in agreement with the dao1-1 mutant root hair phenotype. We conclude that auxin homeostasis is dependent on AtDAO1, acting in concert with GH3, to maintain auxin at optimal levels for plant growth and development. PMID:27651495

  10. Gibberellins regulate lateral root formation in Populus through interactions with auxin and other hormones.

    Science.gov (United States)

    Gou, Jiqing; Strauss, Steven H; Tsai, Chung Jui; Fang, Kai; Chen, Yiru; Jiang, Xiangning; Busov, Victor B

    2010-03-01

    The role of gibberellins (GAs) in regulation of lateral root development is poorly understood. We show that GA-deficient (35S:PcGA2ox1) and GA-insensitive (35S:rgl1) transgenic Populus exhibited increased lateral root proliferation and elongation under in vitro and greenhouse conditions, and these effects were reversed by exogenous GA treatment. In addition, RNA interference suppression of two poplar GA 2-oxidases predominantly expressed in roots also decreased lateral root formation. GAs negatively affected lateral root formation by inhibiting lateral root primordium initiation. A whole-genome microarray analysis of root development in GA-modified transgenic plants revealed 2069 genes with significantly altered expression. The expression of 1178 genes, including genes that promote cell proliferation, growth, and cell wall loosening, corresponded to the phenotypic severity of the root traits when transgenic events with differential phenotypic expression were compared. The array data and direct hormone measurements suggested crosstalk of GA signaling with other hormone pathways, including auxin and abscisic acid. Transgenic modification of a differentially expressed gene encoding an auxin efflux carrier suggests that GA modulation of lateral root development is at least partly imparted by polar auxin transport modification. These results suggest a mechanism for GA-regulated modulation of lateral root proliferation associated with regulation of plant allometry during the stress response.

  11. Evidence of oxidative attenuation of auxin signalling.

    Science.gov (United States)

    Peer, Wendy Ann; Cheng, Yan; Murphy, Angus S

    2013-06-01

    Indole-3-acetic acid (IAA) is the principle auxin in Arabidopsis and is synthesized primarily in meristems and nodes. Auxin is transported to distal parts of the plant in response to developmental programming or environmental stimuli to activate cell-specific responses. As with any signalling event, the signal must be attenuated to allow the system to reset. Local auxin accumulations are thus reduced by conjugation or catabolism when downstream responses have reached their optima. In most cell types, localized auxin accumulation increases both reactive oxygen species (ROS) and an irreversible catabolic product 2-oxindole-3-acid acid (oxIAA). oxIAA is inactive and does not induce expression of the auxin-responsive reporters DR5 or 2XD0. Here it is shown that oxIAA is not transported from cell to cell, although it appears to be a substrate for the ATP-binding cassette subfamily G (ABCG) transporters that are positioned primarily on the outer lateral surface of the root epidermis. However, oxIAA and oxIAA-Glc levels are higher in ABCB mutants that accumulate auxin due to defective cellular export. Auxin-induced ROS production appears to be at least partially mediated by the NAD(P)H oxidase RbohD. oxIAA levels are higher in mutants that lack ROS-scavenging flavonoids (tt4) and are lower in mutants that accumulate excess flavonols (tt3). These data suggest a model where IAA signalling is attenuated by IAA catabolism to oxIAA. Flavonoids appear to buffer ROS accumulations that occur with localized increases in IAA. This buffering of IAA oxidation would explain some growth responses observed in flavonoid-deficient mutants that cannot be explained by their established role in partially inhibiting auxin transport.

  12. The role of auxin and cytokinin signalling in specifying the root architecture of Arabidopsis thaliana

    KAUST Repository

    Muraro, Daniele

    2013-01-01

    Auxin and cytokinin are key hormonal signals that control the cellular architecture of the primary root and the initiation of new lateral root organs in the plant Arabidopsis thaliana. Both developmental processes are regulated by cross-talk between these hormones and their signalling pathways. In this paper, sub-cellular and multi-cellular mathematical models are developed to investigate how interactions between auxin and cytokinin influence the size and location of regions of division and differentiation within the primary root, and describe how their cross-regulation may cause periodic branching of lateral roots. We show how their joint activity may influence tissue-specific oscillations in gene expression, as shown in Moreno-Risueno et al. (2010) and commented upon in Traas and Vernoux (2010), and we propose mechanisms that may generate synchronisation of such periodic behaviours inside a cell and with its neighbours. Using a multi-cellular model, we also analyse the roles of cytokinin and auxin in specifying the three main regions of the primary root (elongation, transition and division zones), our simulation results being in good agreement with independent experimental observations. We then use our model to generate testable predictions concerning the effect of varying the concentrations of the auxin efflux transporters on the sizes of the different root regions. In particular, we predict that over-expression of the transporters will generate a longer root with a longer elongation zone and a smaller division zone than that of a wild type root. This root will contain fewer cells than its wild type counterpart. We conclude that our model can provide a useful tool for investigating the response of cell division and elongation to perturbations in hormonal signalling. © 2012 Elsevier Ltd.

  13. Auxin: simply complicated.

    Science.gov (United States)

    Sauer, Michael; Robert, Stéphanie; Kleine-Vehn, Jürgen

    2013-06-01

    Auxin is a plant hormone involved in an extraordinarily broad variety of biological mechanisms. These range from basic cellular processes, such as endocytosis, cell polarity, and cell cycle control over localized responses such as cell elongation and differential growth, to macroscopic phenomena such as embryogenesis, tissue patterning, and de novo formation of organs. Even though the history of auxin research reaches back more than a hundred years, we are still far from a comprehensive understanding of how auxin governs such a wide range of responses. Some answers to this question may lie in the auxin molecule itself. Naturally occurring auxin-like substances have been found and they may play roles in specific developmental and cellular processes. The molecular mode of auxin action can be further explored by the utilization of synthetic auxin-like molecules. A second area is the perception of auxin, where we know of three seemingly independent receptors and signalling systems, some better understood than others, but each of them probably involved in distinct physiological processes. Lastly, auxin is actively modified, metabolized, and intracellularly compartmentalized, which can have a great impact on its availability and activity. In this review, we will give an overview of these rather recent and emerging areas of auxin research and try to formulate some of the open questions. Without doubt, the manifold facets of auxin biology will not cease to amaze us for a long time to come.

  14. Evolution and structural diversification of PILS putative auxin carriers in plants

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    Elena eFeraru

    2012-10-01

    Full Text Available The phytohormone auxin contributes to virtually every aspect of the plant development. The spatiotemporal distribution of auxin depends on a complex interplay between auxin metabolism and intercellular auxin transport. Intracellular auxin compartmentalization provides another link between auxin transport processes and auxin metabolism. The PIN-LIKES (PILS putative auxin carriers localize to the endoplasmic reticulum (ER and contribute to cellular auxin homeostasis. PILS proteins regulate intracellular auxin accumulation, the rate of auxin conjugation and subsequently, affect nuclear auxin signalling. Here, we investigate sequence diversification of the PILS family in Arabidopsis thaliana and provide insights into the evolution of these novel putative auxin carriers in plants. Our data suggest that PILS proteins are conserved throughout the plant lineage and expanded during higher plant evolution. PILS proteins diversified early during plant evolution into three clades. Besides the ancient Clade I encompassing non-land plant species, PILS proteins evolved into two clades. The diversification of Clade II and Clade III occurred already at the level of non-vascular plant evolution and, hence, both clades contain vascular and non-vascular plant species. Nevertheless, Clade III contains fewer non- and increased numbers of vascular plants, indicating higher importance of Clade III for vascular plant evolution. Notably, PILS proteins are distinct and appear evolutionarily older than the prominent PIN-FORMED auxin carriers. Moreover, we revealed particular PILS sequence divergence in Arabidopsis and assume that these alterations could contribute to distinct gene regulations and protein functions.

  15. Inhibition of the Human ABC Efflux Transporters P-gp and BCRP by the BDE-47 Hydroxylated Metabolite 6-OH-BDE-47: Considerations for Human Exposure.

    Science.gov (United States)

    Marchitti, Satori A; Mazur, Christopher S; Dillingham, Caleb M; Rawat, Swati; Sharma, Anshika; Zastre, Jason; Kenneke, John F

    2017-01-01

    High body burdens of polybrominated diphenyl ethers (PBDEs) in infants and young children have led to increased concern over their potential impact on human development. PBDE exposure can alter the expression of genes involved in thyroid homeostasis, including those of ATP-binding cassette (ABC) transporters, which mediate cellular xenobiotic efflux. However, little information exists on how PBDEs interact with ABC transporters such as P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP). The purpose of this study was to evaluate the interactions of 2,2',4,4'-tetrabromodiphenyl ether (BDE-47) and its hydroxylated metabolite 6-OH-BDE-47 with P-gp and BCRP, using human MDR1- and BCRP-expressing membrane vesicles and stably transfected NIH-3T3-MDR1 and MDCK-BCRP cells. In P-gp membranes, BDE-47 did not affect P-gp activity; however, 6-OH-BDE-47 inhibited P-gp activity at low µM concentrations (IC50 = 11.7 µM). In BCRP membranes, BDE-47 inhibited BCRP activity; however, 6-OH-BDE-47 was a stronger inhibitor [IC50 = 45.9 µM (BDE-47) vs. IC50 = 9.4 µM (6-OH-BDE-47)]. Intracellular concentrations of known P-gp and BCRP substrates [((3)H)-paclitaxel and ((3)H)-prazosin, respectively] were significantly higher (indicating less efflux) in NIH-3T3-MDR1 and MDCK-BCRP cells in the presence of 6-OH-BDE-47, but not BDE-47. Collectively, our results indicate that the BDE-47 metabolite 6-OH-BDE-47 is an inhibitor of both P-gp and BCRP efflux activity. These findings suggest that some effects previously attributed to BDE-47 in biological systems may actually be due to 6-OH-BDE-47. Considerations for human exposure are discussed.

  16. Increased Systemic Exposure of Methotrexate by a Polyphenol-Rich Herb via Modulation on Efflux Transporters Multidrug Resistance-Associated Protein 2 and Breast Cancer Resistance Protein.

    Science.gov (United States)

    Yu, Chung-Ping; Hsieh, Yun-Chung; Shia, Chi-Sheng; Hsu, Pei-Wen; Chen, Jen-Yuan; Hou, Yu-Chi; Hsieh, Yo-Wen

    2016-01-01

    Scutellariae radix (SR, roots of Scutellaria baicalensis Georgi), a popular Chinese medicine, contains plenty of flavonoids such as baicalin, wogonoside, baicalein, and wogonin. Methotrexate (MTX), an important immunosuppressant with a narrow therapeutic index, is a substrate of multidrug resistance-associated proteins (MRPs) and breast cancer resistance protein (BCRP). This study investigated the effect of SR on MTX pharmacokinetics and the underlying mechanisms. Rats were orally administered MTX alone and with 1.0 or 2.0 g/kg of SR. The serum concentrations of MTX were determined by a fluorescence polarization immunoassay. Cell models were used to explore the involvement of MRP2 and BCRP in the interaction. The results showed that 1.0 g/kg of SR significantly increased Cmax, AUC(0-30), AUC(0-2880), and mean residence time (MRT) of MTX by 50%, 45%, 501%, and 347%, respectively, and 2.0 g/kg of SR significantly enhanced the AUC(0-2880) and MRT by 242% and 293%, respectively, but decreased AUC(0-30) by 41%. Cell line studies indicated that SR activated the BCRP-mediated efflux transport, whereas the serum metabolites of SR inhibited both the BCRP- and MRP2-mediated efflux transports. In conclusion, SR ingestion increased the systemic exposure and MRT of MTX via modulation on MRP2 and BCRP.

  17. In vitro drug response and efflux transporters associated with drug resistance in pediatric high grade glioma and diffuse intrinsic pontine glioma.

    Directory of Open Access Journals (Sweden)

    Susanna J E Veringa

    Full Text Available Pediatric high-grade gliomas (pHGG, including diffuse intrinsic pontine gliomas (DIPG, are the leading cause of cancer-related death in children. While it is clear that surgery (if possible, and radiotherapy are beneficial for treatment, the role of chemotherapy for these tumors is still unclear. Therefore, we performed an in vitro drug screen on primary glioma cells, including three DIPG cultures, to determine drug sensitivity of these tumours, without the possible confounding effect of insufficient drug delivery. This screen revealed a high in vitro cytotoxicity for melphalan, doxorubicine, mitoxantrone, and BCNU, and for the novel, targeted agents vandetanib and bortezomib in pHGG and DIPG cells. We subsequently determined the expression of the drug efflux transporters P-gp, BCRP1, and MRP1 in glioma cultures and their corresponding tumor tissues. Results indicate the presence of P-gp, MRP1 and BCRP1 in the tumor vasculature, and expression of MRP1 in the glioma cells themselves. Our results show that pediatric glioma and DIPG tumors per se are not resistant to chemotherapy. Treatment failure observed in clinical trials, may rather be contributed to the presence of drug efflux transporters that constitute a first line of drug resistance located at the blood-brain barrier or other resistance mechanism. As such, we suggest that alternative ways of drug delivery may offer new possibilities for the treatment of pediatric high-grade glioma patients, and DIPG in particular.

  18. Dopamine transporters are involved in the onset of hypoxia-induced dopamine efflux in striatum as revealed by in vivo microdialysis.

    Science.gov (United States)

    Orset, Cyrille; Parrot, Sandrine; Sauvinet, Valérie; Cottet-Emard, Jean-Marie; Bérod, Anne; Pequignot, Jean-Marc; Denoroy, Luc

    2005-06-01

    Although many studies have revealed alterations in neurotransmission during ischaemia, few works have been devoted to the neurochemical effects of mild hypoxia, a situation encountered during life in altitude or in several pathologies. In that context, the present work was undertaken to determine the in vivo mechanisms underlying the striatal dopamine efflux induced by mild hypoxaemic hypoxia. For that purpose, the extracellular concentrations of dopamine and its metabolite 3,4-dihydroxyphenyl acetic acid were simultaneously measured using brain microdialysis during acute hypoxic exposure (10% O(2), 1h) in awake rats. Hypoxia induced a +80% increase in dopamine. Application of the dopamine transporters inhibitor, nomifensine (10 microM), just before the hypoxia prevented the rise in dopamine during the early part of hypoxia; in contrast the application of nomifensine after the beginning of hypoxia, failed to alter the increase in dopamine. Application of the voltage-dependent Na(+) channel blocker tetrodotoxin abolished the increase in dopamine, whether administered just before or after the beginning of hypoxia. These data show that the neurochemical mechanisms of the dopamine efflux may change over the course of the hypoxic exposure, dopamine transporters being involved only at the beginning of hypoxia.

  19. Centrifuge-induced hypergravity and glutamate efflux by reversal of high-affinity, sodium-dependent transporters from rat brain synaptosomes.

    Science.gov (United States)

    Borisova, T.; Himmelreich, N.

    Glutamate uptake by high affinity sodium-dependent glutamate transporters is essential for termination of the synaptic transmission. Glutamate transporters may also contribute to an increase in extracellular glutamate. Glutamate efflux can occur by reversal of the sodium-dependent glutamate transporters during ATP depletion and dissipation of the sodium gradient across the cell membrane. Depolarization-induced calcium independent release of neurotransmitter from synaptosomal cytosolic pool is Na+-dependent and due to reverse of the neurotransmitter transporters also. We used monovalent organic cations N-methyl-D-glucamine (NMDG) to replace extracellular sodium, suggesting that the reducing of Na+ elucidate further the mechanism underlying Ca2+-independent glutamate release. A reduction in extracellular sodium would facilitate reversal of sodium-dependent transporters with extrusion of glutamate. We have compared the basal release of glutamate in Ca2+-free Na+-supplemented and NMDG-supplemented medium in control and after exposure of animals to long-arm centrifuge-induced hypergravity (ten G, during one hour). Replacement of sodium by NMDG enhanced basal level of neurotransmitter. The value of basal level increased to 110± 4% and 140± 2% in the medium with NMDG in comparison with Na+ under the control and hypergravity conditions, respectively. It is likely to reflect the enhancement of the neurotransmitter level in cytosolic pool. Thermodynamic considerations show that the extracellular level of a amino acid neurotransmitter, such as glutamate, that can be generated by transporter reversal are directly proportional to the intracellular concentration of the intracellular concentration of amino acid. KCl-stimulated glutamate release from cytosolic pool increased not statistically after hypergravity loading. We examined the effects of transporter inhibitors DL-threo-beta-benzyloxyaspartate ( DL-TBOA) on the release to elucidate whether reverse transport via the

  20. Lysine63-linked ubiquitylation of PIN2 auxin carrier protein governs hormonally controlled adaptation of Arabidopsis root growth.

    Science.gov (United States)

    Leitner, Johannes; Petrášek, Jan; Tomanov, Konstantin; Retzer, Katarzyna; Pařezová, Markéta; Korbei, Barbara; Bachmair, Andreas; Zažímalová, Eva; Luschnig, Christian

    2012-05-22

    Cross-talk between plant cells and their surroundings requires tight regulation of information exchange at the plasma membrane (PM), which involves dynamic adjustments of PM protein localization and turnover to modulate signal perception and solute transport at the interface between cells and their surroundings. In animals and fungi, turnover of PM proteins is controlled by reversible ubiquitylation, which signals endocytosis and delivery to the cell's lytic compartment, and there is emerging evidence for related mechanisms in plants. Here, we describe the fate of Arabidopsis PIN2 protein, required for directional cellular efflux of the phytohormone auxin, and identify cis- and trans-acting mediators of PIN2 ubiquitylation. We demonstrate that ubiquitin acts as a principal signal for PM protein endocytosis in plants and reveal dynamic adjustments in PIN2 ubiquitylation coinciding with variations in vacuolar targeting and proteolytic turnover. We show that control of PIN2 proteolytic turnover via its ubiquitylation status is of significant importance for auxin distribution in root meristems and for environmentally controlled adaptations of root growth. Moreover, we provide experimental evidence indicating that PIN2 vacuolar sorting depends on modification specifically by lysine(63)-linked ubiquitin chains. Collectively, our results establish lysine(63)-linked PM cargo ubiquitylation as a regulator of polar auxin transport and adaptive growth responses in higher plants.

  1. PatA and PatB form a functional heterodimeric ABC multidrug efflux transporter responsible for the resistance of Streptococcus pneumoniae to fluoroquinolones.

    Science.gov (United States)

    Boncoeur, Emilie; Durmort, Claire; Bernay, Benoît; Ebel, Christine; Di Guilmi, Anne Marie; Croizé, Jacques; Vernet, Thierry; Jault, Jean-Michel

    2012-10-02

    All bacterial multidrug ABC transporters have been shown to work as either homodimers or heterodimers. Two possibly linked genes, patA and patB from Streptococcus pneumococcus, that encode half-ABC transporters have been shown previously to be involved in fluoroquinolone resistance. We showed that the ΔpatA, ΔpatB, or ΔpatA/ΔpatB mutant strains were more sensitive to unstructurally related compounds, i.e., ethidium bromide or fluoroquinolones, than the wild-type R6 strain. Inside-out vesicles prepared from Escherichia coli expressing PatA and/or PatB transported Hoechst 33342, a classical substrate of multidrug transporters, only when both PatA and PatB were coexpressed. This transport was inhibited either by orthovanadate or by reserpine, and mutation of the conserved Walker A lysine residue of either PatA or PatB fully abrogated Hoechst 33342 transport. PatA, PatB, and the PatA/PatB heterodimer were purified from detergent-solubilized E. coli membrane preparations. Protein dimers were identified in all cases, albeit in different proportions. In contrast to the PatA/PatB heterodimers, homodimers of PatA or PatB failed to show a vanadate-sensitive ATPase activity. Thus, PatA and PatB need to interact together to make a functional drug efflux transporter, and they work only as heterodimers.

  2. Phytochrome B promotes branching in Arabidopsis by suppressing auxin signaling.

    Science.gov (United States)

    Krishna Reddy, Srirama; Finlayson, Scott A

    2014-03-01

    Many plants respond to competition signals generated by neighbors by evoking the shade avoidance syndrome, including increased main stem elongation and reduced branching. Vegetation-induced reduction in the red light:far-red light ratio provides a competition signal sensed by phytochromes. Plants deficient in phytochrome B (phyB) exhibit a constitutive shade avoidance syndrome including reduced branching. Because auxin in the polar auxin transport stream (PATS) inhibits axillary bud outgrowth, its role in regulating the phyB branching phenotype was tested. Removing the main shoot PATS auxin source by decapitation or chemically inhibiting the PATS strongly stimulated branching in Arabidopsis (Arabidopsis thaliana) deficient in phyB, but had a modest effect in the wild type. Whereas indole-3-acetic acid (IAA) levels were elevated in young phyB seedlings, there was less IAA in mature stems compared with the wild type. A split plate assay of bud outgrowth kinetics indicated that low auxin levels inhibited phyB buds more than the wild type. Because the auxin response could be a result of either the auxin signaling status or the bud's ability to export auxin into the main shoot PATS, both parameters were assessed. Main shoots of phyB had less absolute auxin transport capacity compared with the wild type, but equal or greater capacity when based on the relative amounts of native IAA in the stems. Thus, auxin transport capacity was unlikely to restrict branching. Both shoots of young phyB seedlings and mature stem segments showed elevated expression of auxin-responsive genes and expression was further increased by auxin treatment, suggesting that phyB suppresses auxin signaling to promote branching.

  3. Possible involvement of cationic-drug sensitive transport systems in the blood-to-brain influx and brain-to-blood efflux of amantadine across the blood-brain barrier.

    Science.gov (United States)

    Suzuki, Toyofumi; Fukami, Toshiro; Tomono, Kazuo

    2015-03-01

    The purpose of this study was to characterize the brain-to-blood efflux transport of amantadine across the blood-brain barrier (BBB). The apparent in vivo efflux rate constant for [(3) H]amantadine from the rat brain (keff ) was found to be 1.53 × 10(-2) min(-1) after intracerebral microinjection using the brain efflux index method. The efflux of [(3) H]amantadine was inhibited by 1-methyl-4-phenylpyridinium (MPP(+) ), a cationic neurotoxin, suggesting that amantadine transport from the brain to the blood across the BBB potentially involves the rat plasma membrane monoamine transporter (rPMAT). On the other hand, other selected substrates for organic cation transporters (OCTs) and organic anion transporters (OATs), as well as inhibitors of P-glycoprotein (P-gp), did not affect the efflux transport of [(3) H]amantadine. In addition, in vitro studies using an immortalized rat brain endothelial cell line (GPNT) showed that the uptake and retention of [(3) H]amantadine by the cells was not changed by the addition of cyclosporin, which is an inhibitor of P-gp. However, cyclosporin affected the uptake and retention of rhodamine123. Finally, the initial brain uptake of [(3) H]amantadine was determined using an in situ mouse brain perfusion technique. Notably, the brain uptake clearance for [(3) H]amantadine was significantly decreased with the co-perfusion of quinidine or verapamil, which are cationic P-gp inhibitors, while MPP(+) did not have a significant effect. It is thus concluded that while P-gp is not involved, it is possible that rPMAT and the cationic drug-sensitive transport system participate in the brain-to-blood efflux and the blood-to-brain influx of amantadine across the BBB, respectively.

  4. Exendin-4 Ameliorates Lipotoxicity-induced Glomerular Endothelial Cell Injury by Improving ABC Transporter A1-mediated Cholesterol Efflux in Diabetic apoE Knockout Mice.

    Science.gov (United States)

    Yin, Qing-Hua; Zhang, Rui; Li, Li; Wang, Yi-Ting; Liu, Jing-Ping; Zhang, Jie; Bai, Lin; Cheng, Jing-Qiu; Fu, Ping; Liu, Fang

    2016-12-16

    ATP-binding cassette transporter A1 (ABCA1), which promotes cholesterol efflux from cells and inhibits inflammatory responses, is highly expressed in the kidney. Research has shown that exendin-4, a glucagon-like peptide-1 receptor (GLP-1R) agonist, promotes ABCA1 expression in multiple tissues and organs; however, the mechanisms underlying exendin-4 induction of ABCA1 expression in glomerular endothelial cells are not fully understood. In this study we investigated the effect of exendin-4 on ABCA1 in glomerular endothelial cells of diabetic kidney disease (DKD) and the possible mechanism. We observed a marked increase in glomerular lipid deposits in tissues of patients with DKD and diabetic apolipoprotein E knock-out (apoE(-/-)) mice by Oil Red O staining and biochemical analysis of cholesterol. We found significantly decreased ABCA1 expression in glomerular endothelial cells of diabetic apoE(-/-) mice and increased renal lipid, cholesterol, and inflammatory cytokine levels. Exendin-4 decreased renal cholesterol accumulation and inflammation and increased cholesterol efflux by up-regulating ABCA1. In human glomerular endothelial cells, GLP-1R-mediated signaling pathways (e.g. Ca(2+)/calmodulin-dependent protein kinase, cAMP/PKA, PI3K/AKT, and ERK1/2) were involved in cholesterol efflux and inflammatory responses by regulating ABCA1 expression. We propose that exendin-4 increases ABCA1 expression in glomerular endothelial cells, which plays an important role in alleviating renal lipid accumulation, inflammation, and proteinuria in mice with type 2 diabetes. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  5. Forward genetic screen for auxin-deficient mutants by cytokinin.

    Science.gov (United States)

    Wu, Lei; Luo, Pan; Di, Dong-Wei; Wang, Li; Wang, Ming; Lu, Cheng-Kai; Wei, Shao-Dong; Zhang, Li; Zhang, Tian-Zi; Amakorová, Petra; Strnad, Miroslav; Novák, Ondřej; Guo, Guang-Qin

    2015-07-06

    Identification of mutants with impairments in auxin biosynthesis and dynamics by forward genetic screening is hindered by the complexity, redundancy and necessity of the pathways involved. Furthermore, although a few auxin-deficient mutants have been recently identified by screening for altered responses to shade, ethylene, N-1-naphthylphthalamic acid (NPA) or cytokinin (CK), there is still a lack of robust markers for systematically isolating such mutants. We hypothesized that a potentially suitable phenotypic marker is root curling induced by CK, as observed in the auxin biosynthesis mutant CK-induced root curling 1 / tryptophan aminotransferase of Arabidopsis 1 (ckrc1/taa1). Phenotypic observations, genetic analyses and biochemical complementation tests of Arabidopsis seedlings displaying the trait in large-scale genetic screens showed that it can facilitate isolation of mutants with perturbations in auxin biosynthesis, transport and signaling. However, unlike transport/signaling mutants, the curled (or wavy) root phenotypes of auxin-deficient mutants were significantly induced by CKs and could be rescued by exogenous auxins. Mutants allelic to several known auxin biosynthesis mutants were re-isolated, but several new classes of auxin-deficient mutants were also isolated. The findings show that CK-induced root curling provides an effective marker for discovering genes involved in auxin biosynthesis or homeostasis.

  6. RAC/ROP GTPases and Auxin Signaling[W

    Science.gov (United States)

    Wu, Hen-ming; Hazak, Ora; Cheung, Alice Y.; Yalovsky, Shaul

    2011-01-01

    Auxin functions as a key morphogen in regulating plant growth and development. Studies on auxin-regulated gene expression and on the mechanism of polar auxin transport and its asymmetric distribution within tissues have provided the basis for realizing the molecular mechanisms underlying auxin function. In eukaryotes, members of the Ras and Rho subfamilies of the Ras superfamily of small GTPases function as molecular switches in many signaling cascades that regulate growth and development. Plants do not have Ras proteins, but they contain Rho-like small G proteins called RACs or ROPs that, like fungal and metazoan Rhos, are regulators of cell polarity and may also undertake some Ras functions. Here, we discuss the advances made over the last decade that implicate RAC/ROPs as mediators for auxin-regulated gene expression, rapid cell surface-located auxin signaling, and directional auxin transport. We also describe experimental data indicating that auxin–RAC/ROP crosstalk may form regulatory feedback loops and theoretical modeling that attempts to connect local auxin gradients with RAC/ROP regulation of cell polarity. We hope that by discussing these experimental and modeling studies, this perspective will stimulate efforts to further refine our understanding of auxin signaling via the RAC/ROP molecular switch. PMID:21478442

  7. Auxin response factors.

    Science.gov (United States)

    Chandler, John William

    2016-05-01

    Auxin signalling involves the activation or repression of gene expression by a class of auxin response factor (ARF) proteins that bind to auxin response elements in auxin-responsive gene promoters. The release of ARF repression in the presence of auxin by the degradation of their cognate auxin/indole-3-acetic acid repressors forms a paradigm of transcriptional response to auxin. However, this mechanism only applies to activating ARFs, and further layers of complexity of ARF function and regulation are being revealed, which partly reflect their highly modular domain structure. This review summarizes our knowledge concerning ARF binding site specificity, homodimer and heterodimer multimeric ARF association and cooperative function and how activator ARFs activate target genes via chromatin remodelling and evolutionary information derived from phylogenetic comparisons from ARFs from diverse species. ARFs are regulated in diverse ways, and their importance in non-auxin-regulated pathways is becoming evident. They are also embedded within higher-order transcription factor complexes that integrate signalling pathways from other hormones and in response to the environment. The ways in which new information concerning ARFs on many levels is causing a revision of existing paradigms of auxin response are discussed.

  8. Characterization of a pituitary-tumor-derived cell line, TtT/GF, that expresses Hoechst efflux ABC transporter subfamily G2 and stem cell antigen 1.

    Science.gov (United States)

    Mitsuishi, Hideo; Kato, Takako; Chen, Mo; Cai, Li-Yi; Yako, Hideji; Higuchi, Masashi; Yoshida, Saishu; Kanno, Naoko; Ueharu, Hiroki; Kato, Yukio

    2013-11-01

    The anterior lobe of the pituitary gland is composed of five types of endocrine cells and of non-endocrine folliculo-stellate cells that produce various local signaling molecules. The TtT/GF cell line is derived from pituitary tumors, produces no hormones and has folliculo-stellate cell-like characteristics. The biological function of TtT/GF cells remains elusive but several properties have been postulated (support of endocrine cells, control of cell proliferation, scavenger function). Recently, we observed that TtT/GF cells have high resistance to the antibiotic G418 and low influx for Hoechst 33342, indicating the presence of ATP-binding cassette (ABC) transporters that efflux multiple drugs, i.e., a property similar to that of stem/progenitor cells. Therefore, we examine TtT/GF cells for the presence of ABC transporters, for the efflux ability of Hoechst 33342 and for those genes characteristic of TtT/GF cells. Real-time polymerase chain reaction (PCR) for ABC transporters demonstrated that Abcb1a, Abcb1b and Abcg2, regarded as stem cell markers, were characteristically expressed in TtT/GF cells but not in Tpit/F1 and LβT2 cells. Furthermore, the remarkable low-efflux ability of Hoechst 33342 from TtT/GF cells was confirmed by using inhibitors and contrasted with the abilities of Tpit/F1 and LβT2 cells. The high and specific expression of stem cell antigen 1 (Sca1) in TtT/GF cells was confirmed by real-time PCR. We also demonstrated those genes that are expressed abundantly and characteristically in TtT/GF, suggesting that TtT/GF cells have unique characteristics similar to those of stem/progenitor cells of endothelial or mesenchymal origin. Thus, the present study has revealed an intriguing property of TtT/GF cells, providing a new clue for an understanding of the function of this cell line.

  9. Vacuolar CAX1 and CAX3 influence auxin transport in guard cells via regulation of apoplastic pH

    Science.gov (United States)

    Cation exchangers CAX1 and CAX3 are vacuolar ion transporters involved in ion homeostasis in plants. Widely expressed in the plant, they mediate calcium transport from the cytosol to the vacuole lumen using the proton gradient across the tonoplast. Here, we report an unexpected role of CAX1 and CAX3...

  10. Charged Amino Acids (R83, E567, D617, E625, R669, and K678) of CusA Are Required for Metal Ion Transport in the Cus Efflux System

    Energy Technology Data Exchange (ETDEWEB)

    Su, Chih-Chia; Long, Feng; Lei, Hsiang-Ting; Reddy Bolla, Jani; Do, Sylvia V.; Rajashankar, Kanagalaghatta R.; Yu, Edward W. (Cornell); (Iowa State)

    2012-10-23

    Gram-negative bacteria expel various toxic chemicals via tripartite efflux pumps belonging to the resistance-nodulation-cell division superfamily. These pumps span both the inner and outer membranes of the cell. The three components of these tripartite systems are an inner-membrane, substrate-binding transporter (or pump); a periplasmic membrane fusion protein (or adaptor); and an outer-membrane-anchored channel. These three efflux proteins interact in the periplasmic space to form the three-part complexes. We previously presented the crystal structures of both the inner-membrane transporter CusA and membrane fusion protein CusB of the CusCBA tripartite efflux system from Escherichia coli. We also described the co-crystal structure of the CusBA adaptor-transporter, revealing that the trimeric CusA efflux pump assembles with six CusB protein molecules to form the complex CusB{sub 6}-CusA{sub 3}. We here report three different conformers of the crystal structures of CusBA-Cu(I), suggesting a mechanism on how Cu(I) binding initiates a sequence of conformational transitions in the transport cycle. Genetic analysis and transport assays indicate that charged residues, in addition to the methionine pairs and clusters, are essential for extruding metal ions out of the cell.

  11. Cadmium interferes with maintenance of auxin homeostasis in Arabidopsis seedlings.

    Science.gov (United States)

    Hu, Yan Feng; Zhou, Guoying; Na, Xiao Fan; Yang, Lijing; Nan, Wen Bin; Liu, Xu; Zhang, Yong Qiang; Li, Jiao Long; Bi, Yu Rong

    2013-07-15

    Auxin and its homeostasis play key roles in many aspects of plant growth and development. Cadmium (Cd) is a phytotoxic heavy metal and its inhibitory effects on plant growth and development have been extensively studied. However, the underlying molecular mechanism of the effects of Cd stress on auxin homeostasis is still unclear. In the present study, we found that the root elongation, shoot weight, hypocotyl length and chlorophyll content in wild-type (WT) Arabidopsis seedlings were significantly reduced after exposure to Cd stress. However, the lateral root (LR) formation was markedly promoted by Cd stress. The level and distribution of auxin were both greatly altered in primary root tips and cotyledons of Cd-treated plants. The results also showed that after Cd treatment, the IAA content was significantly decreased, which was accompanied by increases in the activity of the IAA oxidase and alteration in the expression of several putative auxin biosynthetic and catabolic genes. Application of the auxin transport inhibitor, 1-naphthylphthalamic acid (NPA) and 1-naphthoxyacetic acid (1-NOA), reversed the effects of Cd on LR formation. Additionally, there was less promotion of LR formation by Cd treatment in aux1-7 and pin2 mutants than that in the WT. Meanwhile, Cd stress also altered the expression of PINs and AUX1 in Arabidopsis roots, implying that the auxin transport pathway is required for Cd-modulated LR development. Taken together, these findings suggest that Cd stress disturbs auxin homeostasis through affecting auxin level, distribution, metabolism, and transport in Arabidopsis seedling.

  12. Modelling of Arabidopsis LAX3 expression suggests auxin homeostasis.

    Science.gov (United States)

    Mellor, Nathan; Péret, Benjamin; Porco, Silvana; Sairanen, Ilkka; Ljung, Karin; Bennett, Malcolm; King, John

    2015-02-07

    Emergence of new lateral roots from within the primary root in Arabidopsis has been shown to be regulated by the phytohormone auxin, via the expression of the auxin influx carrier LAX3, mediated by the ARF7/19 IAA14 signalling module (Swarup et al., 2008). A single cell model of the LAX3 and IAA14 auxin response was formulated and used to demonstrate that hysteresis and bistability may explain the experimentally observed 'all-or-nothing' LAX3 spatial expression pattern in cortical cells containing a gradient of auxin concentrations. The model was tested further by using a parameter fitting algorithm to match model output with qRT-PCR mRNA expression data following exogenous auxin treatment. It was found that the model is able to show good agreement with the data, but only when the exogenous auxin signal is degraded over time, at a rate higher than that measured in the experimental medium, suggesting the triggering of an endogenous auxin homeostasis mechanism. Testing the model over a more physiologically relevant range of extracellular auxin shows bistability and hysteresis still occur when using the optimised parameters, providing the rate of LAX3 active auxin transport is sufficiently high relative to passive diffusion.

  13. Interaction of BDE-47 and its Hydroxylated Metabolite 6-OH-BDE-47 with the Human ABC Efflux Transporters P-gp and BCRP: Considerations for Human Exposure and Risk Assessment

    Science.gov (United States)

    ATP binding cassette (ABC) transporters, including P-glycoprotein (P-gp; also known as MDR1, ABCB1) and breast cancer resistance protein (BCRP; also known as ABCG2), are membrane-bound proteins that mediate the cellular efflux of xenobiotics as an important defense against chemic...

  14. Binding of PDZ-RhoGEF to ATP-binding cassette transporter A1 (ABCA1) induces cholesterol efflux through RhoA activation and prevention of transporter degradation.

    Science.gov (United States)

    Okuhira, Keiichiro; Fitzgerald, Michael L; Tamehiro, Norimasa; Ohoka, Nobumichi; Suzuki, Kazuhiro; Sawada, Jun-ichi; Naito, Mikihiko; Nishimaki-Mogami, Tomoko

    2010-05-21

    ATP-binding cassette transporter A1 (ABCA1)-mediated lipid efflux to apolipoprotein A1 (apoA-I) initiates the biogenesis of high density lipoprotein. Here we show that the Rho guanine nucleotide exchange factors PDZ-RhoGEF and LARG bind to the C terminus of ABCA1 by a PDZ-PDZ interaction and prevent ABCA1 protein degradation by activating RhoA. ABCA1 is a protein with a short half-life, and apoA-I stabilizes ABCA1 protein; however, depletion of PDZ-RhoGEF/LARG by RNA interference suppressed the apoA-I stabilization of ABCA1 protein in human primary fibroblasts. Exogenous PDZ-RhoGEF expression activated RhoA and increased ABCA1 protein levels and cholesterol efflux activity. Likewise, forced expression of a constitutively active RhoA mutant significantly increased ABCA1 protein levels, whereas a dominant negative RhoA mutant decreased them. The constitutively active RhoA retarded ABCA1 degradation, thus accounting for its ability to increase ABCA1 protein. Moreover, stimulation with apoA-I transiently activated RhoA, and the pharmacological inhibition of RhoA or the dominant negative RhoA blocked the ability of apoA-I to stabilize ABCA1. Finally, depletion of RhoA or RhoGEFs/RhoA reduces the cholesterol efflux when transcriptional regulation via PPARgamma is eliminated. Taken together, our results have identified a novel physical and functional interaction between ABCA1 and PDZ-RhoGEF/LARG, which activates RhoA, resulting in ABCA1 stabilization and cholesterol efflux activity.

  15. Endoplasmic reticulum localization and activity of maize auxin biosynthetic enzymes.

    Science.gov (United States)

    Kriechbaumer, Verena; Seo, Hyesu; Park, Woong June; Hawes, Chris

    2015-09-01

    Auxin is a major growth hormone in plants and the first plant hormone to be discovered and studied. Active research over >60 years has shed light on many of the molecular mechanisms of its action including transport, perception, signal transduction, and a variety of biosynthetic pathways in various species, tissues, and developmental stages. The complexity and redundancy of the auxin biosynthetic network and enzymes involved raises the question of how such a system, producing such a potent agent as auxin, can be appropriately controlled at all. Here it is shown that maize auxin biosynthesis takes place in microsomal as well as cytosolic cellular fractions from maize seedlings. Most interestingly, a set of enzymes shown to be involved in auxin biosynthesis via their activity and/or mutant phenotypes and catalysing adjacent steps in YUCCA-dependent biosynthesis are localized to the endoplasmic reticulum (ER). Positioning of auxin biosynthetic enzymes at the ER could be necessary to bring auxin biosynthesis in closer proximity to ER-localized factors for transport, conjugation, and signalling, and allow for an additional level of regulation by subcellular compartmentation of auxin action. Furthermore, it might provide a link to ethylene action and be a factor in hormonal cross-talk as all five ethylene receptors are ER localized.

  16. The gut microbiota ellagic acid-derived metabolite urolithin A and its sulfate conjugate are substrates for the drug efflux transporter breast cancer resistance protein (ABCG2/BCRP).

    Science.gov (United States)

    González-Sarrías, Antonio; Miguel, Verónica; Merino, Gracia; Lucas, Ricardo; Morales, Juan C; Tomás-Barberán, Francisco; Alvarez, Ana I; Espín, Juan C

    2013-05-08

    The breast cancer resistance protein (BCRP/ABCG2) is a drug efflux transporter that can affect the pharmacological and toxicological properties of many molecules. Urolithins, metabolites produced by the gut microbiota from ellagic acid (EA) and ellagitannins, have been acknowledged with in vivo anti-inflammatory and cancer chemopreventive properties. This study evaluated whether urolithins (Uro-A, -B, -C, and -D) and their main phase II metabolites Uro-A sulfate, Uro-A glucuronide, and Uro-B glucuronide as well as their precursor EA were substrates for ABCG2/BCRP. Parental and Bcrp1-transduced MDCKII cells were used for active transport assays. Uro-A and, to a lesser extent, Uro-A sulfate showed a significant increase in apically directed translocation in Bcrp1-transduced cells. Bcrp1 did not show affinity for the rest of the tested compounds. Data were confirmed for murine, human, bovine, and ovine BCRP-transduced subclones as well as with the use of the selective BCRP inhibitor Ko143. The transport inhibition by Uro-A was analyzed by flow cytometry compared to Ko143 using the antineoplastic agent mitoxantrone as a model substrate. Results showed that Uro-A was able to inhibit mitoxantrone transport in a dose-dependent manner. This study reports for the first time that Uro-A and its sulfate conjugate are ABCG2/BCRP substrates. The results suggest that physiologically relevant concentrations of these gut microbiota-derived metabolites could modulate ABCG2/BCRP-mediated transport processes and mechanisms of cancer drug resistance. Further in vivo investigations are warranted.

  17. CHX14 is a plasma membrane K-efflux transporter that regulates K+ redistribution in "Arabidopsis thaliana"

    Science.gov (United States)

    Potassium (K(+)) is essential for plant growth and development, yet the molecular identity of many K(+) transporters remains elusive. Here we characterized cation/H(+) exchanger (CHX) 14 as a plasma membrane K(+) transporter. "CHX14" expression was induced by elevated K(+) and histochemical analysis...

  18. Local auxin sources orient the apical-basal axis in Arabidopsis embryos.

    Science.gov (United States)

    Robert, Hélène S; Grones, Peter; Stepanova, Anna N; Robles, Linda M; Lokerse, Annemarie S; Alonso, Jose M; Weijers, Dolf; Friml, Jiří

    2013-12-16

    Establishment of the embryonic axis foreshadows the main body axis of adults both in plants and in animals, but underlying mechanisms are considered distinct. Plants utilize directional, cell-to-cell transport of the growth hormone auxin to generate an asymmetric auxin response that specifies the embryonic apical-basal axis. The auxin flow directionality depends on the polarized subcellular localization of PIN-FORMED (PIN) auxin transporters. It remains unknown which mechanisms and spatial cues guide cell polarization and axis orientation in early embryos. Herein, we provide conceptually novel insights into the formation of embryonic axis in Arabidopsis by identifying a crucial role of localized tryptophan-dependent auxin biosynthesis. Local auxin production at the base of young embryos and the accompanying PIN7-mediated auxin flow toward the proembryo are required for the apical auxin response maximum and the specification of apical embryonic structures. Later in embryogenesis, the precisely timed onset of localized apical auxin biosynthesis mediates PIN1 polarization, basal auxin response maximum, and specification of the root pole. Thus, the tight spatiotemporal control of distinct local auxin sources provides a necessary, non-cell-autonomous trigger for the coordinated cell polarization and subsequent apical-basal axis orientation during embryogenesis and, presumably, also for other polarization events during postembryonic plant life.

  19. Identification of the hepatic efflux transporters of organic anions using double-transfected Madin-Darby canine kidney II cells expressing human organic anion-transporting polypeptide 1B1 (OATP1B1)/multidrug resistance-associated protein 2, OATP1B1/multidrug resistance 1, and OATP1B1/breast cancer resistance protein.

    Science.gov (United States)

    Matsushima, Soichiro; Maeda, Kazuya; Kondo, Chihiro; Hirano, Masaru; Sasaki, Makoto; Suzuki, Hiroshi; Sugiyama, Yuichi

    2005-09-01

    Until recently, it was generally believed that the transport of various organic anions across the bile canalicular membrane was mainly mediated by multidrug resistance-associated protein 2 (MRP2/ABCC2). However, a number of new reports have shown that some organic anions are also substrates of multidrug resistance 1 (MDR1/ABCB1) and/or breast cancer resistance protein (BCRP/ABCG2), implying MDR1 and BCRP could also be involved in the biliary excretion of organic anions in humans. In the present study, we constructed new double-transfected Madin-Darby canine kidney II (MDCKII) cells expressing organic anion-transporting polypeptide 1B1 (OATP1B1)/MDR1 and OATP1B1/BCRP, and we investigated the transcellular transport of four kinds of organic anions, estradiol-17beta-d-glucuronide (EG), estrone-3-sulfate (ES), pravastatin (PRA), and cerivastatin (CER), to identify which efflux transporters mediate the biliary excretion of compounds using double-transfected cells. We observed the vectorial transport of EG and ES in all the double transfectants. MRP2 showed the highest efflux clearance of EG among these efflux transporters, whereas BCRP-mediated clearance of ES was the highest in these double transfectants. In addition, two kinds of 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors, CER and PRA, were also substrates of all these efflux transporters. The rank order of the efflux clearance of PRA mediated by each transporter was the same as that of EG, whereas the contribution of MDR1 to the efflux of CER was relatively greater than for PRA. This experimental system is very useful for identifying which transporters are involved in the biliary excretion of organic anions that cannot easily penetrate the plasma membrane.

  20. Partial loss-of-function alleles reveal a role for GNOM in auxin transport-related, post-embryonic development of Arabidopsis

    DEFF Research Database (Denmark)

    Geldner, Niko; Richter, Sandra; Vieten, Anne

    2004-01-01

    characterised newly isolated weak gnom alleles as well as trans-heterozygotes of complementing strong alleles. These genotypes form a phenotypic series of GNOM activity in post-embryonic development, with auxin-related defects, especially in the maintenance of primary root meristem activity...

  1. Integrating two patterning processes in the flower. (refereed by Editor) Addendum to: Simulation of organ patterning on the floral meristem using a polar auxin transport model.

    NARCIS (Netherlands)

    Mourik, van S.; Kaufmann, K.; Dijk, van A.D.J.; Angenent, G.C.; Merks, R.M.H.; Molenaar, J.

    2012-01-01

    Spatial organ arrangement plays an important role in flower development. The position and the identity of floral organs is influenced by various processes, in particular the expression of MADS-box transcription factors for identity and dynamics of the plant hormone auxin for positioning. We are curr

  2. P-glycoprotein mediated efflux limits the transport of the novel anti-Parkinson's disease candidate drug FLZ across the physiological and PD pathological in vitro BBB models.

    Science.gov (United States)

    Liu, Qian; Hou, Jinfeng; Chen, Xiaoguang; Liu, Gengtao; Zhang, Dan; Sun, Hua; Zhang, Jinlan

    2014-01-01

    FLZ, a novel anti-Parkinson's disease (PD) candidate drug, has shown poor blood-brain barrier (BBB) penetration based on the pharmacokinetic study using rat brain. P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) are two important transporters obstructing substrates entry into the CNS as well as in relation to PD neuropathology. However, it is unclear whether P-gp and BCRP are involved in low BBB permeability of FLZ and what the differences of FLZ brain penetration are between normal and Parkinson's conditions. For this purpose, in vitro BBB models mimicking physiological and PD pathological-related BBB properties were constructed by C6 astroglial cells co-cultured with primary normal or PD rat cerebral microvessel endothelial cells (rCMECs) and in vitro permeability experiments of FLZ were carried out. High transepithelial electrical resistance (TEER) and low permeability for sodium fluorescein (NaF) confirmed the BBB functionality of the two models. Significantly greater expressions of P-gp and BCRP were detected in PD rCMECs associated with the lower in vitro BBB permeability of FLZ in pathological BBB model compared with physiological model. In transport studies only P-gp blocker effectively inhibited the efflux of FLZ, which was consistent with the in vivo permeability data. This result was also confirmed by ATPase assays, suggesting FLZ is a substrate for P-gp but not BCRP. The present study first established in vitro BBB models reproducing PD-related changes of BBB functions in vivo and demonstrated that poor brain penetration of FLZ and low BBB permeability were due to the P-gp transport.

  3. P-glycoprotein mediated efflux limits the transport of the novel anti-Parkinson's disease candidate drug FLZ across the physiological and PD pathological in vitro BBB models.

    Directory of Open Access Journals (Sweden)

    Qian Liu

    Full Text Available FLZ, a novel anti-Parkinson's disease (PD candidate drug, has shown poor blood-brain barrier (BBB penetration based on the pharmacokinetic study using rat brain. P-glycoprotein (P-gp and breast cancer resistance protein (BCRP are two important transporters obstructing substrates entry into the CNS as well as in relation to PD neuropathology. However, it is unclear whether P-gp and BCRP are involved in low BBB permeability of FLZ and what the differences of FLZ brain penetration are between normal and Parkinson's conditions. For this purpose, in vitro BBB models mimicking physiological and PD pathological-related BBB properties were constructed by C6 astroglial cells co-cultured with primary normal or PD rat cerebral microvessel endothelial cells (rCMECs and in vitro permeability experiments of FLZ were carried out. High transepithelial electrical resistance (TEER and low permeability for sodium fluorescein (NaF confirmed the BBB functionality of the two models. Significantly greater expressions of P-gp and BCRP were detected in PD rCMECs associated with the lower in vitro BBB permeability of FLZ in pathological BBB model compared with physiological model. In transport studies only P-gp blocker effectively inhibited the efflux of FLZ, which was consistent with the in vivo permeability data. This result was also confirmed by ATPase assays, suggesting FLZ is a substrate for P-gp but not BCRP. The present study first established in vitro BBB models reproducing PD-related changes of BBB functions in vivo and demonstrated that poor brain penetration of FLZ and low BBB permeability were due to the P-gp transport.

  4. Multidrug Resistance-Associated Protein 4 (MRP4/ABCC4) Controls Efflux Transport of Hesperetin Sulfates in Sulfotransferase 1A3-Overexpressing Human Embryonic Kidney 293 Cells.

    Science.gov (United States)

    Sun, Hua; Wang, Xiao; Zhou, Xiaotong; Lu, Danyi; Ma, Zhiguo; Wu, Baojian

    2015-10-01

    Sulfonation is an important metabolic pathway for hesperetin. However, the mechanisms for the cellular disposition of hesperetin and its sulfate metabolites are not fully established. In this study, disposition of hesperetin via the sulfonation pathway was investigated using human embryonic kidney (HEK) 293 cells overexpressing sulfotransferase 1A3. Two monosulfates, hesperetin-3'-O-sulfate (H-3'-S) and hesperetin-7-O-sulfate (H-7-S), were rapidly generated and excreted into the extracellular compartment upon incubation of the cells with hesperetin. Regiospecific sulfonation of hesperetin by the cell lysate followed the substrate inhibition kinetics (Vmax = 0.66 nmol/min per mg, Km = 12.9 μM, and Ksi= 58.1 μM for H-3'-S; Vmax = 0.29 nmol/min per mg, Km = 14.8 μM, and Ksi= 49.1 μM for H-7-S). The pan-multidrug resistance-associated protein (MRP) inhibitor MK-571 at 20 μM essentially abolished cellular excretion of both H-3'-S and H-7-S (the excretion activities were only 6% of the control), whereas the breast cancer resistance protein-selective inhibitor Ko143 had no effects on sulfate excretion. In addition, knockdown of MRP4 led to a substantial reduction (>47.1%; P transport by MRP4 according to the vesicular transport assay. Moreover, sulfonation of hesperetin and excretion of its metabolites were well characterized by a two-compartment pharmacokinetic model that integrated drug uptake and sulfonation with MRP4-mediated sulfate excretion. In conclusion, the exporter MRP4 controlled efflux transport of hesperetin sulfates in HEK293 cells. Due to significant expression in various organs/tissues (including the liver and kidney), MRP4 should be a determining factor for the elimination and body distribution of hesperetin sulfates.

  5. Aintegumenta and Aintegumenta-Like6 regulate auxin-mediated flower development in Arabidopsis

    Directory of Open Access Journals (Sweden)

    Krizek Beth A

    2011-06-01

    Full Text Available Abstract Background Two related genes encoding AP2/ERF-type transcription factors, AINTEGUMENTA (ANT and AINTEGUMENTA-LIKE6 (AIL6, are important regulators of floral growth and patterning in Arabidopsis. Evidence suggests that these genes promote several aspects of flower development in response to auxin. To investigate the interplay of ANT, AIL6 and auxin during floral development, I have examined the phenotypic consequences of disrupting polar auxin transport in ant, ail6 and ant ail6 mutants by either genetic or chemical means. Results Plants containing mutations in ANT or AIL6 alone or in both genes together exhibit increased sensitivity to disruptions in polar auxin transport. Both genes promote shoot growth, floral meristem initiation and floral meristem patterning in combination with auxin transport. However, differences in the responses of ant and ail6 single mutants to perturbations in auxin transport suggest that these two genes also have non-overlapping activities in each of these developmental processes. Conclusions The enhanced sensitivity of ant and ail6 mutants to alterations in polar auxin transport suggests that these mutants have defects in some aspect of auxin physiology. The inability of ant ail6 double mutants to initiate flowers in backgrounds disrupted for auxin transport confirm the proposed roles for these two genes in floral meristem initiation.

  6. Arabidopsis ABCB21 is a facultative auxin importer/exporter regulated by cytoplasmic auxin concentration.

    Science.gov (United States)

    Kamimoto, Yoshihisa; Terasaka, Kazuyoshi; Hamamoto, Masafumi; Takanashi, Kojiro; Fukuda, Shoju; Shitan, Nobukazu; Sugiyama, Akifumi; Suzuki, Hideyuki; Shibata, Daisuke; Wang, Bangjun; Pollmann, Stephan; Geisler, Markus; Yazaki, Kazufumi

    2012-12-01

    The phytohormone auxin is critical for plant growth and many developmental processes. Members of the P-glycoprotein (PGP/ABCB) subfamily of ATP-binding cassette (ABC) transporters have been shown to function in the polar movement of auxin by transporting auxin over the plasma membrane in both monocots and dicots. Here, we characterize a new Arabidopsis member of the ABCB subfamily, ABCB21/PGP21, a close homolog of ABCB4, for which conflicting transport directionalities have been reported. ABCB21 is strongly expressed in the abaxial side of cotyledons and in junctions of lateral organs in the aerial part, whereas in roots it is specifically expressed in pericycle cells. Membrane fractionation by sucrose density gradient centrifugation followed by Western blot showed that ABCB21 is a plasma membrane-localized ABC transporter. A transport assay with Arabidopsis protoplasts suggested that ABCB21 was involved in IAA transport in an outward direction, while naphthalene acetic acid (NAA) was a less preferable substrate for ABCB21. Further functional analysis of ABCB21 using yeast import and export assays showed that ABCB21 mediates the 1-N-naphthylphthalamic acid (NPA)-sensitive translocation of auxin in an inward direction when the cytoplasmic IAA concentration is low, whereas this transporter mediates outward transport under high internal IAA. An increase in the cytoplasmic IAA concentration by pre-loading of IAA into yeast cells abolished the IAA uptake activity by ABCB21 as well as ABCB4. These findings suggest that ABCB21 functions as a facultative importer/exporter controlling auxin concentrations in plant cells.

  7. Killing them with kindness? In-hive medications may inhibit xenobiotic efflux transporters and endanger honey bees.

    Directory of Open Access Journals (Sweden)

    David J Hawthorne

    Full Text Available BACKGROUND: Honey bees (Apis mellifera have recently experienced higher than normal overwintering colony losses. Many factors have been evoked to explain the losses, among which are the presence of residues of pesticides and veterinary products in hives. Multiple residues are present at the same time, though most often in low concentrations so that no single product has yet been associated with losses. Involvement of a combination of residues to losses may however not be excluded. To understand the impact of an exposure to combined residues on honey bees, we propose a mechanism-based strategy, focusing here on Multi-Drug Resistance (MDR transporters as mediators of those interactions. METHODOLOGY/PRINCIPAL FINDINGS: Using whole-animal bioassays, we demonstrate through inhibition by verapamil that the widely used organophosphate and pyrethroid acaricides coumaphos and τ-fluvalinate, and three neonicotinoid insecticides: imidacloprid, acetamiprid and thiacloprid are substrates of one or more MDR transporters. Among the candidate inhibitors of honey bee MDR transporters is the in-hive antibiotic oxytetracycline. Bees prefed oxytetracycline were significantly sensitized to the acaricides coumaphos and τ-fluvalinate, suggesting that the antibiotic may interfere with the normal excretion or metabolism of these pesticides. CONCLUSIONS/SIGNIFICANCE: Many bee hives receive regular treatments of oxytetracycline and acaricides for prevention and treatment of disease and parasites. Our results suggest that seasonal co-application of these medicines to bee hives could increase the adverse effects of these and perhaps other pesticides. Our results also demonstrate the utility of a mechanism-based strategy. By identifying pesticides and apicultural medicines that are substrates and inhibitors of xenobiotic transporters we prioritize the testing of those chemical combinations most likely to result in adverse interactions.

  8. Auxin activates the plasma membrane H+-ATPase by phosphorylation during hypocotyl elongation in Arabidopsis.

    Science.gov (United States)

    Takahashi, Koji; Hayashi, Ken-ichiro; Kinoshita, Toshinori

    2012-06-01

    The phytohormone auxin is a major regulator of diverse aspects of plant growth and development. The ubiquitin-ligase complex SCF(TIR1/AFB) (for Skp1-Cul1-F-box protein), which includes the TRANSPORT INHIBITOR RESPONSE1/AUXIN SIGNALING F-BOX (TIR1/AFB) auxin receptor family, has recently been demonstrated to be critical for auxin-mediated transcriptional regulation. Early-phase auxin-induced hypocotyl elongation, on the other hand, has long been explained by the acid-growth theory, for which proton extrusion by the plasma membrane H(+)-ATPase is a functional prerequisite. However, the mechanism by which auxin mediates H(+)-ATPase activation has yet to be elucidated. Here, we present direct evidence for H(+)-ATPase activation in etiolated hypocotyls of Arabidopsis (Arabidopsis thaliana) by auxin through phosphorylation of the penultimate threonine during early-phase hypocotyl elongation. Application of the natural auxin indole-3-acetic acid (IAA) to endogenous auxin-depleted hypocotyl sections induced phosphorylation of the penultimate threonine of the H(+)-ATPase and increased H(+)-ATPase activity without altering the amount of the enzyme. Changes in both the phosphorylation level of H(+)-ATPase and IAA-induced elongation were similarly concentration dependent. Furthermore, IAA-induced H(+)-ATPase phosphorylation occurred in a tir1-1 afb2-3 double mutant, which is severely defective in auxin-mediated transcriptional regulation. In addition, α-(phenylethyl-2-one)-IAA, the auxin antagonist specific for the nuclear auxin receptor TIR1/AFBs, had no effect on IAA-induced H(+)-ATPase phosphorylation. These results suggest that the TIR1/AFB auxin receptor family is not involved in auxin-induced H(+)-ATPase phosphorylation. Our results define the activation mechanism of H(+)-ATPase by auxin during early-phase hypocotyl elongation; this is the long-sought-after mechanism that is central to the acid-growth theory.

  9. Pavement cells: a model system for non-transcriptional auxin signalling and crosstalks.

    Science.gov (United States)

    Chen, Jisheng; Wang, Fei; Zheng, Shiqin; Xu, Tongda; Yang, Zhenbiao

    2015-08-01

    Auxin (indole acetic acid) is a multifunctional phytohormone controlling various developmental patterns, morphogenetic processes, and growth behaviours in plants. The transcription-based pathway activated by the nuclear TRANSPORT INHIBITOR RESISTANT 1/auxin-related F-box auxin receptors is well established, but the long-sought molecular mechanisms of non-transcriptional auxin signalling remained enigmatic until very recently. Along with the establishment of the Arabidopsis leaf epidermal pavement cell (PC) as an exciting and amenable model system in the past decade, we began to gain insight into non-transcriptional auxin signalling. The puzzle-piece shape of PCs forms from intercalated or interdigitated cell growth, requiring local intra- and inter-cellular coordination of lobe and indent formation. Precise coordination of this interdigitated pattern requires auxin and an extracellular auxin sensing system that activates plasma membrane-associated Rho GTPases from plants and subsequent downstream events regulating cytoskeletal reorganization and PIN polarization. Apart from auxin, mechanical stress and cytokinin have been shown to affect PC interdigitation, possibly by interacting with auxin signals. This review focuses upon signalling mechanisms for cell polarity formation in PCs, with an emphasis on non-transcriptional auxin signalling in polarized cell expansion and pattern formation and how different auxin pathways interplay with each other and with other signals.

  10. Exposure Characteristics of the Analogous β-Carboline Alkaloids Harmaline and Harmine Based on the Efflux Transporter of Multidrug Resistance Protein 2

    Directory of Open Access Journals (Sweden)

    Shuping Li

    2017-08-01

    Full Text Available Harmaline and harmine occur naturally in plants and are distributed endogenously in human and animal tissues. The two β-carboline alkaloids possess potential for treating Alzheimer's disease, Parkinson's disease, depression and other central nervous system diseases. However, studies have showed that the two compounds have similar structures but with quite different bioavailability. The aim of this study was to elucidate the exposure difference and characterize the in vitro transport, metabolism, and pharmacokinetic properties of harmaline and harmine. The results showed that the harmaline and harmine transport across the Caco-2 and MDCK cell monolayers was varied as the time, concentration, pH and temperature changed. The absorption of harmaline and harmine was significantly decreased when ES (OATPs inhibitor, TEA (OCTs/OCTNs substrate, NaN3 (adenosine triphosphate inhibitor, or sodium vanadate (ATPase Na+/K+-dependent inhibitor was added. However, when given MK571 and probenecid (the typical MRP2 inhibitor, the PappAB of harmine was increased (1.62- and 1.27-folds, and the efflux ratio was decreased from 1.59 to 0.98 and from 1.59 to 1.19, respectively. In addition, the uptake ratio of harmine at 1 μM was >2.65 in the membrane vesicles expressing human MRP2. Furthermore, harmine could slightly up-regulate the expression of MRP2, which implying harmine might be the substrate of MRP2. Particularly, the CLint-value for harmine was ~1.49-folds greater than that of harmaline in human liver microsomes. It was worth noting that the F-value of harmine was increased 1.96-folds after harmine co-administration with probenecid. To summarize, comprehensive analysis indicated that harmaline and harmine were absorbed by transcellular passive diffusion and a pH- and Na+-dependent mechanism might be mediated by OATPs and OCTs/OCTNs. MRP2 but MDR1 or BCRP might be involved in the transport of harmine. Furthermore, harmine was more unstable and easily

  11. The inter-kingdom volatile signal indole promotes root development by interfering with auxin signalling.

    Science.gov (United States)

    Bailly, Aurélien; Groenhagen, Ulrike; Schulz, Stefan; Geisler, Markus; Eberl, Leo; Weisskopf, Laure

    2014-12-01

    Recently, emission of volatile organic compounds (VOCs) has emerged as a mode of communication between bacteria and plants. Although some bacterial VOCs that promote plant growth have been identified, their underlying mechanism of action is unknown. Here we demonstrate that indole, which was identified using a screen for Arabidopsis growth promotion by VOCs from soil-borne bacteria, is a potent plant-growth modulator. Its prominent role in increasing the plant secondary root network is mediated by interfering with the auxin-signalling machinery. Using auxin reporter lines and classic auxin physiological and transport assays we show that the indole signal invades the plant body, reaches zones of auxin activity and acts in a polar auxin transport-dependent bimodal mechanism to trigger differential cellular auxin responses. Our results suggest that indole, beyond its importance as a bacterial signal molecule, can serve as a remote messenger to manipulate plant growth and development.

  12. Small molecules unravel complex interplay between auxin biology and endomembrane trafficking.

    Science.gov (United States)

    Doyle, Siamsa M; Vain, Thomas; Robert, Stéphanie

    2015-08-01

    The establishment and maintenance of controlled auxin gradients within plant tissues are essential for a multitude of developmental processes. Auxin gradient formation is co-ordinated via local biosynthesis and transport. Cell to cell auxin transport is facilitated and precisely regulated by complex endomembrane trafficking mechanisms that target auxin carrier proteins to their final destinations. In turn, auxin and cross-talk with other phytohormones regulate the endomembrane trafficking of auxin carriers. Dissecting such rapid and complicated processes is challenging for classical genetic experiments due to trafficking pathway diversity, gene functional redundancy, and lethality in loss-of-function mutants. Many of these difficulties can be bypassed via the use of small molecules to modify or disrupt the function or localization of proteins. Here, we will review examples of the knowledge acquired by the use of such chemical tools in this field, outlining the advantages afforded by chemical biology approaches.

  13. Multidrug efflux pumps in Gram-negative bacteria and their role in antibiotic resistance.

    Science.gov (United States)

    Blair, Jessica M A; Richmond, Grace E; Piddock, Laura J V

    2014-01-01

    Gram-negative bacteria express a plethora of efflux pumps that are capable of transporting structurally varied molecules, including antibiotics, out of the bacterial cell. This efflux lowers the intracellular antibiotic concentration, allowing bacteria to survive at higher antibiotic concentrations. Overexpression of some efflux pumps can cause clinically relevant levels of antibiotic resistance in Gram-negative pathogens. This review discusses the role of efflux in resistance of clinical isolates of Gram-negative bacteria, the regulatory mechanisms that control efflux pump expression, the recent advances in our understanding of efflux pump structure and how inhibition of efflux is a promising future strategy for tackling multidrug resistance in Gram-negative pathogens.

  14. Glutathione Efflux and Cell Death

    Science.gov (United States)

    2012-01-01

    Abstract Significance: Glutathione (GSH) depletion is a central signaling event that regulates the activation of cell death pathways. GSH depletion is often taken as a marker of oxidative stress and thus, as a consequence of its antioxidant properties scavenging reactive species of both oxygen and nitrogen (ROS/RNS). Recent Advances: There is increasing evidence demonstrating that GSH loss is an active phenomenon regulating the redox signaling events modulating cell death activation and progression. Critical Issues: In this work, we review the role of GSH depletion by its efflux, as an important event regulating alterations in the cellular redox balance during cell death independent from oxidative stress and ROS/RNS formation. We discuss the mechanisms involved in GSH efflux during cell death progression and the redox signaling events by which GSH depletion regulates the activation of the cell death machinery. Future Directions: The evidence summarized here clearly places GSH transport as a central mechanism mediating redox signaling during cell death progression. Future studies should be directed toward identifying the molecular identity of GSH transporters mediating GSH extrusion during cell death, and addressing the lack of sensitive approaches to quantify GSH efflux. Antioxid. Redox Signal. 17, 1694–1713. PMID:22656858

  15. An electrically tight in vitro blood-brain barrier model displays net brain-to-blood efflux of substrates for the ABC transporters, P-gp, Bcrp and Mrp-1.

    Science.gov (United States)

    Helms, Hans Christian; Hersom, Maria; Kuhlmann, Louise Borella; Badolo, Lasina; Nielsen, Carsten Uhd; Brodin, Birger

    2014-09-01

    Efflux transporters of the ATP-binding cassette superfamily including breast cancer resistance protein (Bcrp/Abcg2), P-glycoprotein (P-gp/Abcb1) and multidrug resistance-associated proteins (Mrp's/Abcc's) are expressed in the blood-brain barrier (BBB). The aim of this study was to investigate if a bovine endothelial/rat astrocyte in vitro BBB co-culture model displayed polarized transport of known efflux transporter substrates. The co-culture model displayed low mannitol permeabilities of 0.95 ± 0.1 · 10(-6) cm·s(-1) and high transendothelial electrical resistances of 1,177 ± 101 Ω·cm(2). Bidirectional transport studies with (3)H-digoxin, (3)H-estrone-3-sulphate and (3)H-etoposide revealed polarized transport favouring the brain-to-blood direction for all substrates. Steady state efflux ratios of 2.5 ± 0.2 for digoxin, 4.4 ± 0.5 for estrone-3-sulphate and 2.4 ± 0.1 for etoposide were observed. These were reduced to 1.1 ± 0.08, 1.4 ± 0.2 and 1.5 ± 0.1, by addition of verapamil (digoxin), Ko143 (estrone-3-sulphate) or zosuquidar + reversan (etoposide), respectively. Brain-to-blood permeability of all substrates was investigated in the presence of the efflux transporter inhibitors verapamil, Ko143, zosuquidar, reversan and MK 571 alone or in combinations. Digoxin was mainly transported via P-gp, estrone-3-sulphate via Bcrp and Mrp's and etoposide via P-gp and Mrp's. The expression of P-gp, Bcrp and Mrp-1 was confirmed using immunocytochemistry. The findings indicate that P-gp, Bcrp and at least one isoform of Mrp are functionally expressed in our bovine/rat co-culture model and that the model is suitable for investigations of small molecule transport.

  16. Perturbation of auxin homeostasis by overexpression of wild-type IAA15 results in impaired stem cell differentiation and gravitropism in roots.

    Directory of Open Access Journals (Sweden)

    Da-Wei Yan

    Full Text Available Aux/IAAs interact with auxin response factors (ARFs to repress their transcriptional activity in the auxin signaling pathway. Previous studies have focused on gain-of-function mutations of domain II and little is known about whether the expression level of wild-type Aux/IAAs can modulate auxin homeostasis. Here we examined the perturbation of auxin homeostasis by ectopic expression of wild-type IAA15. Root gravitropism and stem cell differentiation were also analyzed. The transgenic lines were less sensitive to exogenous auxin and exhibited low-auxin phenotypes including failures in gravity response and defects in stem cell differentiation. Overexpression lines also showed an increase in auxin concentration and reduced polar auxin transport. These results demonstrate that an alteration in the expression of wild-type IAA15 can disrupt auxin homeostasis.

  17. Efflux in fungi: la piece de resistance.

    Directory of Open Access Journals (Sweden)

    Jeffrey J Coleman

    2009-06-01

    Full Text Available Pathogens must be able to overcome both host defenses and antimicrobial treatment in order to successfully infect and maintain colonization of the host. One way fungi accomplish this feat and overcome intercellular toxin accumulation is efflux pumps, in particular ATP-binding cassette transporters and transporters of the major facilitator superfamily. Members of these two superfamilies remove many toxic compounds by coupling transport with ATP hydrolysis or a proton gradient, respectively. Fungal genomes encode a plethora of members of these families of transporters compared to other organisms. In this review we discuss the role these two fungal superfamilies of transporters play in virulence and resistance to antifungal agents. These efflux transporters are responsible not only for export of compounds involved in pathogenesis such as secondary metabolites, but also export of host-derived antimicrobial compounds. In addition, we examine the current knowledge of these transporters in resistance of pathogens to clinically relevant antifungal agents.

  18. Efflux Transport Characterization of Resveratrol Glucuronides in UDP-Glucuronosyltransferase 1A1 Transfected HeLa Cells: Application of a Cellular Pharmacokinetic Model to Decipher the Contribution of Multidrug Resistance-Associated Protein 4.

    Science.gov (United States)

    Wang, Shuai; Li, Feng; Quan, Enxi; Dong, Dong; Wu, Baojian

    2016-04-01

    Resveratrol undergoes extensive metabolism to form biologically active glucuronides in humans. However, the transport mechanisms for resveratrol glucuronides are not fully established. Here, we aimed to characterize the efflux transport of resveratrol glucuronides using UGT1A1-overexpressing HeLa cells (HeLa1A1 cells), and to determine the contribution of multidrug resistance-associated protein (MRP) 4 to cellular excretion of the glucuronides. Two glucuronide isomers [i.e., resveratrol 3-O-glucuronide (R3G) and resveratrol 4'-O-glucuronide (R4'G)] were excreted into the extracellular compartment after incubation of resveratrol (1-100 μM) with HeLa1A1 cells. The excretion rate was linearly related to the level of intracellular glucuronide, indicating that glucuronide efflux was a nonsaturable process. MK-571 (a dual inhibitor of UGT1A1 and MRPs) significantly decreased the excretion rates of R3G and R4'G while increasing their intracellular levels. Likewise, short-hairpin RNA (shRNA)-mediated silencing of MRP4 caused a significant reduction in glucuronide excretion but an elevation in glucuronide accumulation. Furthermore, β-glucuronidase expressed in the cells catalyzed the hydrolysis of the glucuronides back to the parent compound. A cellular pharmacokinetic model integrating resveratrol transport/metabolism with glucuronide hydrolysis/excretion was well fitted to the experimental data, allowing derivation of the efflux rate constant values in the absence or presence of shRNA targeting MRP4. It was found that a large percentage of glucuronide excretion (43%-46%) was attributed to MRP4. In conclusion, MRP4 participated in cellular excretion of R3G and R4'G. Integration of mechanistic pharmacokinetic modeling with transporter knockdown was a useful method to derive the contribution percentage of an exporter to overall glucuronide excretion.

  19. Identification of auxin responsive genes in Arabidopsis by cDNA array

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    The plant hormone auxin influences a variety of developmental and physiological processes. But the mechanism of its action is quite unclear. In order to identify and analyze the expression of auxin responsive genes, a cDNA array approach was used to screen for genes with altered expression from Arabidopsis suspension culture after IAA treatment and was identified 50 differentially expressed genes from 13824 cDNA clones. These genes were related to signal transduction, stress responses, senescence, photosynthesis, protein biosynthesis and transportation. The results provide the molecular evidence that auxin influences a variety of physiological processes and pave a way for further investigation of the mechanism of auxin action. Furthermore,we found that the expression of a ClpC (regulation subunit of Clp protease) was repressed by exogenous auxin, but increased in dark-induced senescing leaves. This suggests that ClpC may be a senescence-associated gene and can be regulated by auxin.

  20. Dynamic Regulation of Auxin Response during Rice Development Revealed by Newly Established Hormone Biosensor Markers

    Science.gov (United States)

    Yang, Jing; Yuan, Zheng; Meng, Qingcai; Huang, Guoqiang; Périn, Christophe; Bureau, Charlotte; Meunier, Anne-Cécile; Ingouff, Mathieu; Bennett, Malcolm J.; Liang, Wanqi; Zhang, Dabing

    2017-01-01

    The hormone auxin is critical for many plant developmental processes. Unlike the model eudicot plant Arabidopsis (Arabidopsis thaliana), auxin distribution and signaling in rice tissues has not been systematically investigated due to the absence of suitable auxin response reporters. In this study we observed the conservation of auxin signaling components between Arabidopsis and model monocot crop rice (Oryza sativa), and generated complementary types of auxin biosensor constructs, one derived from the Aux/IAA-based biosensor DII-VENUS but constitutively driven by maize ubiquitin-1 promoter, and the other termed DR5-VENUS in which a synthetic auxin-responsive promoter (DR5rev) was used to drive expression of the yellow fluorescent protein (YFP). Using the obtained transgenic lines, we observed that during the vegetative development, accumulation of DR5-VENUS signal was at young and mature leaves, tiller buds and stem base. Notably, abundant DR5-VENUS signals were observed in the cytoplasm of cortex cells surrounding lateral root primordia (LRP) in rice. In addition, auxin maxima and dynamic re-localization were seen at the initiation sites of inflorescence and spikelet primordia including branch meristems (BMs), female and male organs. The comparison of these observations among Arabidopsis, rice and maize suggests the unique role of auxin in regulating rice lateral root emergence and reproduction. Moreover, protein localization of auxin transporters PIN1 homologs and GFP tagged OsAUX1 overlapped with DR5-VENUS during spikelet development, helping validate these auxin response reporters are reliable markers in rice. This work firstly reveals the direct correspondence between auxin distribution and rice reproductive and root development at tissue and cellular level, and provides high-resolution auxin tools to probe fundamental developmental processes in rice and to establish links between auxin, development and agronomical traits like yield or root architecture. PMID

  1. Effects of Auxins on PIN-FORMED2 (PIN2) Dynamics Are Not Mediated by Inhibiting PIN2 Endocytosis.

    Science.gov (United States)

    Jásik, Ján; Bokor, Boris; Stuchlík, Stanislav; Mičieta, Karol; Turňa, Ján; Schmelzer, Elmon

    2016-10-01

    By using the photoconvertible fluorescence protein Dendra2 as a tag we demonstrated that neither the naturally occurring auxins indole-3-acetic acid and indole-3-butyric acid, nor the synthetic auxin analogs 1-naphthaleneacetic acid and 2,4-dichlorophenoxyacetic acid nor compounds inhibiting polar auxin transport such as 2,3,5-triiodobenzoic acid and 1-N-naphthylphthalamic acid, were able to inhibit endocytosis of the putative auxin transporter PIN-FORMED2 (PIN2) in Arabidopsis (Arabidopsis thaliana) root epidermis cells. All compounds, except Indole-3-butyric acid, repressed the recovery of the PIN2-Dendra2 plasma membrane pool after photoconversion when they were used in high concentrations. The synthetic auxin analogs 1-naphthaleneacetic acid and 2,4-dichlorophenoxyacetic acid showed the strongest inhibition. Auxins and auxin transport inhibitors suppressed also the accumulation of both newly synthesized and endocytotic PIN2 pools in Brefeldin A compartments (BFACs). Furthermore, we demonstrated that all compounds are also interfering with BFAC formation. The synthetic auxin analogs caused the highest reduction in the number and size of BFACs. We concluded that auxins and inhibitors of auxin transport do affect PIN2 turnover in the cells, but it is through the synthetic rather than the endocytotic pathway. The study also confirmed inappropriateness of the BFA-based approach to study PIN2 endocytosis because the majority of PIN2 accumulating in BFACs is newly synthesized and not derived from the plasma membrane. © 2016 American Society of Plant Biologists. All Rights Reserved.

  2. Rhizobacterial volatile emissions regulate auxin homeostasis and cell expansion in Arabidopsis.

    Science.gov (United States)

    Zhang, Huiming; Kim, Mi-Seong; Krishnamachari, Venkat; Payton, Paxton; Sun, Yan; Grimson, Mark; Farag, Mohamed A; Ryu, Choong-Min; Allen, Randy; Melo, Itamar S; Paré, Paul W

    2007-09-01

    Certain plant growth-promoting rhizobacteria (PGPR), in the absence of physical contact with a plant stimulate growth via volatile organic compound (VOC) emissions, through largely unknown mechanisms. To probe how PGPR VOCs trigger growth in plants, RNA transcript levels of Arabidopsis seedlings exposed to Bacillus subtilus (strain GB03) were examined using oligonucleotide microarrays. In screening over 26,000 protein-coded transcripts, a group of approximately 600 differentially expressed genes related to cell wall modifications, primary and secondary metabolism, stress responses, hormone regulation and other expressed proteins were identified. Transcriptional and histochemical data indicate that VOCs from the PGPR strain GB03 trigger growth promotion in Arabidopsis by regulating auxin homeostasis. Specifically, gene expression for auxin synthesis was up regulated in aerial regions of GB03-exposed plants; auxin accumulation decreased in leaves and increased in roots with GB03 exposure as revealed in a transgenic DR5::GUS Arabidopsis line, suggesting activation of basipetal auxin transport. Application of the auxin transport inhibitor 1-naphthylphthalamic acid (NPA) restricted auxin accumulation to sites of synthesis thereby preventing GB03-mediated decreases in shoot auxin levels as well as thwarting GB03-mediated growth promotion. In addition, microarray data revealed coordinated regulation of cell wall loosening enzymes that implicated cell expansion with GB03 exposure, which was confirmed by comparative cytological measurements. The discovery that bacterial VOCs, devoid of auxin or other known plant hormones regulate auxin homeostasis and cell expansion provides a new paradigm as to how rhizobacteria promote plant growth.

  3. DIFFERENCES IN ERYTHROCYTE SODIUM-TRANSPORT BETWEEN HUMAN PLASMA AND ARTIFICIAL MEDIUM - THE ROLE AND CHARACTER OF SODIUM-EFFLUX AND INFLUX STIMULATING PLASMA FACTORS

    NARCIS (Netherlands)

    TEPPER, T; JILDERDA, JF; HUISMAN, RM; VANDERHEM, GK; DEZEEUW, D

    1992-01-01

    The main objective of this study was to further characterize the plasma factor(s) which stimulate sodium efflux from erythrocytes, which we reported previously. Dialysis of plasma against an artificial medium using membranes with varying molecular mass cut-off points revealed relative molecular

  4. Antagonistic regulation of PIN phosphorylation by PP2A and PINOID directs auxin flux

    NARCIS (Netherlands)

    Michniewicz, M.; Zago, M.K.; Abas, L.; Weijers, D.; Schweighofer, A.; Meskiene, I.; Heisler, M.G.; Ohno, C.; Zhang, J.; Huang, F.; Schwab, R.; Weigel, D.; Meyerowitz, E.M.; Luschnig, C.; Offringa, R.; Friml, J.

    2007-01-01

    In plants, cell polarity and tissue patterning are connected by intercellular flow of the phytohormone auxin, whose directional signaling depends on polar subcellular localization of PIN auxin transport proteins. The mechanism of polar targeting of PINs or other cargos in plants is largely

  5. Local Auxin Sources Orient the Apical-Basal Axis in Arabidopsis Embryos

    NARCIS (Netherlands)

    Robert, H.S.; Grones, P.; Stepanova, A.N.; Robles, L.M.; Lokerse, A.S.; Alonso, J.M.; Weijers, D.; Friml, J.

    2013-01-01

    Establishment of the embryonic axis foreshadows the main body axis of adults both in plants and in animals, but underlying mechanisms are considered distinct. Plants utilize directional, cell-to-cell transport of the growth hormone auxin [1 and 2] to generate an asymmetric auxin response that specif

  6. Antagonistic regulation of PIN phosphorylation by PP2A and PINOID directs auxin flux

    NARCIS (Netherlands)

    Michniewicz, M.; Zago, M.K.; Abas, L.; Weijers, D.; Schweighofer, A.; Meskiene, I.; Heisler, M.G.; Ohno, C.; Zhang, J.; Huang, F.; Schwab, R.; Weigel, D.; Meyerowitz, E.M.; Luschnig, C.; Offringa, R.; Friml, J.

    2007-01-01

    In plants, cell polarity and tissue patterning are connected by intercellular flow of the phytohormone auxin, whose directional signaling depends on polar subcellular localization of PIN auxin transport proteins. The mechanism of polar targeting of PINs or other cargos in plants is largely unidentif

  7. Lipopolysaccharide impairs amyloid beta efflux from brain: altered vascular sequestration, cerebrospinal fluid reabsorption, peripheral clearance and transporter function at the blood–brain barrier

    Directory of Open Access Journals (Sweden)

    Erickson Michelle A

    2012-06-01

    Full Text Available Abstract Background Defects in the low density lipoprotein receptor-related protein-1 (LRP-1 and p-glycoprotein (Pgp clearance of amyloid beta (Aβ from brain are thought to contribute to Alzheimer’s disease (AD. We have recently shown that induction of systemic inflammation by lipopolysaccharide (LPS results in impaired efflux of Aβ from the brain. The same treatment also impairs Pgp function. Here, our aim is to determine which physiological routes of Aβ clearance are affected following systemic inflammation, including those relying on LRP-1 and Pgp function at the blood–brain barrier. Methods CD-1 mice aged between 6 and 8 weeks were treated with 3 intraperitoneal injections of 3 mg/kg LPS at 0, 6, and 24 hours and studied at 28 hours. 125I-Aβ1-42 or 125I-alpha-2-macroglobulin injected into the lateral ventricle of the brain (intracerebroventricular (ICV or into the jugular vein (intravenous (IV was used to quantify LRP-1-dependent partitioning between the brain vasculature and parenchyma and peripheral clearance, respectively. Disappearance of ICV-injected 14 C-inulin from brain was measured to quantify bulk flow of cerebrospinal fluid (CSF. Brain microvascular protein expression of LRP-1 and Pgp was measured by immunoblotting. Endothelial cell localization of LRP-1 was measured by immunofluorescence microscopy. Oxidative modifications to LRP-1 at the brain microvasculature were measured by immunoprecipitation of LRP-1 followed by immunoblotting for 4-hydroxynonenal and 3-nitrotyrosine. Results We found that LPS: caused an LRP-1-dependent redistribution of ICV-injected Aβ from brain parenchyma to brain vasculature and decreased entry into blood; impaired peripheral clearance of IV-injected Aβ; inhibited reabsorption of CSF; did not significantly alter brain microvascular protein levels of LRP-1 or Pgp, or oxidative modifications to LRP-1; and downregulated LRP-1 protein levels and caused LRP-1 mislocalization in cultured brain

  8. Auxin and ethylene induce flavonol accumulation through distinct transcriptional networks.

    Science.gov (United States)

    Lewis, Daniel R; Ramirez, Melissa V; Miller, Nathan D; Vallabhaneni, Prashanthi; Ray, W Keith; Helm, Richard F; Winkel, Brenda S J; Muday, Gloria K

    2011-05-01

    Auxin and ethylene are key regulators of plant growth and development, and thus the transcriptional networks that mediate responses to these hormones have been the subject of intense research. This study dissected the hormonal cross talk regulating the synthesis of flavonols and examined their impact on root growth and development. We analyzed the effects of auxin and an ethylene precursor on roots of wild-type and hormone-insensitive Arabidopsis (Arabidopsis thaliana) mutants at the transcript, protein, and metabolite levels at high spatial and temporal resolution. Indole-3-acetic acid (IAA) and 1-aminocyclopropane-1-carboxylic acid (ACC) differentially increased flavonol pathway transcripts and flavonol accumulation, altering the relative abundance of quercetin and kaempferol. The IAA, but not ACC, response is lost in the transport inhibitor response1 (tir1) auxin receptor mutant, while ACC responses, but not IAA responses, are lost in ethylene insensitive2 (ein2) and ethylene resistant1 (etr1) ethylene signaling mutants. A kinetic analysis identified increases in transcripts encoding the transcriptional regulators MYB12, Transparent Testa Glabra1, and Production of Anthocyanin Pigment after hormone treatments, which preceded increases in transcripts encoding flavonoid biosynthetic enzymes. In addition, myb12 mutants were insensitive to the effects of auxin and ethylene on flavonol metabolism. The equivalent phenotypes for transparent testa4 (tt4), which makes no flavonols, and tt7, which makes kaempferol but not quercetin, showed that quercetin derivatives are the inhibitors of basipetal root auxin transport, gravitropism, and elongation growth. Collectively, these experiments demonstrate that auxin and ethylene regulate flavonol biosynthesis through distinct signaling networks involving TIR1 and EIN2/ETR1, respectively, both of which converge on MYB12. This study also provides new evidence that quercetin is the flavonol that modulates basipetal auxin transport.

  9. Auxin regulates distal stem cell differentiation in Arabidopsis roots.

    Science.gov (United States)

    Ding, Zhaojun; Friml, Jirí

    2010-06-29

    The stem cell niche in the root meristem is critical for the development of the plant root system. The plant hormone auxin acts as a versatile trigger in many developmental processes, including the regulation of root growth, but its role in the control of the stem cell activity remains largely unclear. Here we show that local auxin levels, determined by biosynthesis and intercellular transport, mediate maintenance or differentiation of distal stem cells in the Arabidopsis thaliana roots. Genetic analysis shows that auxin acts upstream of the major regulators of the stem cell activity, the homeodomain transcription factor WOX5, and the AP-2 transcription factor PLETHORA. Auxin signaling for differentiation of distal stem cells requires the transcriptional repressor IAA17/AXR3 as well as the ARF10 and ARF16 auxin response factors. ARF10 and ARF16 activities repress the WOX5 transcription and restrict it to the quiescent center, where WOX5, in turn, is needed for the activity of PLETHORA. Our investigations reveal that long-distance auxin signals act upstream of the short-range network of transcriptional factors to mediate the differentiation of distal stem cells in roots.

  10. Theoretical and experimental evidence indicates that there is no detectable auxin gradient in the angiosperm female gametophyte.

    Science.gov (United States)

    Lituiev, Dmytro S; Krohn, Nádia G; Müller, Bruno; Jackson, David; Hellriegel, Barbara; Dresselhaus, Thomas; Grossniklaus, Ueli

    2013-11-01

    The plant life cycle alternates between a diploid sporophytic and a haploid gametophytic generation. The female gametophyte (FG) of flowering plants is typically formed through three syncytial mitoses, followed by cellularisation that forms seven cells belonging to four cell types. The specification of cell fates in the FG has been suggested to depend on positional information provided by an intrinsic auxin concentration gradient. The goal of this study was to develop mathematical models that explain the formation of this gradient in a syncytium. Two factors were proposed to contribute to the maintenance of the auxin gradient in Arabidopsis FGs: polar influx at early stages and localised auxin synthesis at later stages. However, no gradient could be generated using classical, one-dimensional theoretical models under these assumptions. Thus, we tested other hypotheses, including spatial confinement by the large central vacuole, background efflux and localised degradation, and investigated the robustness of cell specification under different parameters and assumptions. None of the models led to the generation of an auxin gradient that was steep enough to allow sufficiently robust patterning. This led us to re-examine the response to an auxin gradient in developing FGs using various auxin reporters, including a novel degron-based reporter system. In agreement with the predictions of our models, auxin responses were not detectable within the FG of Arabidopsis or maize, suggesting that the effects of manipulating auxin production and response on cell fate determination might be indirect.

  11. Targeting efflux pumps to overcome antifungal drug resistance.

    Science.gov (United States)

    Holmes, Ann R; Cardno, Tony S; Strouse, J Jacob; Ivnitski-Steele, Irena; Keniya, Mikhail V; Lackovic, Kurt; Monk, Brian C; Sklar, Larry A; Cannon, Richard D

    2016-08-01

    Resistance to antifungal drugs is an increasingly significant clinical problem. The most common antifungal resistance encountered is efflux pump-mediated resistance of Candida species to azole drugs. One approach to overcome this resistance is to inhibit the pumps and chemosensitize resistant strains to azole drugs. Drug discovery targeting fungal efflux pumps could thus result in the development of azole-enhancing combination therapy. Heterologous expression of fungal efflux pumps in Saccharomyces cerevisiae provides a versatile system for screening for pump inhibitors. Fungal efflux pumps transport a range of xenobiotics including fluorescent compounds. This enables the use of fluorescence-based detection, as well as growth inhibition assays, in screens to discover compounds targeting efflux-mediated antifungal drug resistance. A variety of medium- and high-throughput screens have been used to identify a number of chemical entities that inhibit fungal efflux pumps.

  12. SLOW MOTION Is Required for Within-Plant Auxin Homeostasis and Normal Timing of Lateral Organ Initiation at the Shoot Meristem in Arabidopsis[C][W

    Science.gov (United States)

    Lohmann, Daniel; Stacey, Nicola; Breuninger, Holger; Jikumaru, Yusuke; Müller, Dörte; Sicard, Adrien; Leyser, Ottoline; Yamaguchi, Shinjiro; Lenhard, Michael

    2010-01-01

    The regular arrangement of leaves and flowers around a plant's stem is a fascinating expression of biological pattern formation. Based on current models, the spacing of lateral shoot organs is determined by transient local auxin maxima generated by polar auxin transport, with existing primordia draining auxin from their vicinity to restrict organ formation close by. It is unclear whether this mechanism encodes not only spatial information but also temporal information about the plastochron (i.e., the interval between the formation of successive primordia). Here, we identify the Arabidopsis thaliana F-box protein SLOW MOTION (SLOMO) as being required for a normal plastochron. SLOMO interacts genetically with components of polar auxin transport, and mutant shoot apices contain less free auxin. However, this reduced auxin level at the shoot apex is not due to increased polar auxin transport down the stem, suggesting that it results from reduced synthesis. Independently reducing the free auxin level in plants causes a similar lengthening of the plastochron as seen in slomo mutants, suggesting that the reduced auxin level in slomo mutant shoot apices delays the establishment of the next auxin maximum. SLOMO acts independently of other plastochron regulators, such as ALTERED MERISTEM PROGRAM1 or KLUH/CYP78A5. We propose that SLOMO contributes to auxin homeostasis in the shoot meristem, thus ensuring a normal rate of the formation of auxin maxima and organ initiation. PMID:20139162

  13. Pathways of arsenic uptake and efflux.

    Science.gov (United States)

    Yang, Hung-Chi; Fu, Hsueh-Liang; Lin, Yung-Feng; Rosen, Barry P

    2012-01-01

    Arsenic is the most prevalent environmental toxic substance and ranks first on the U.S. Environmental Protection Agency's Superfund List. Arsenic is a carcinogen and a causative agent of numerous human diseases. Paradoxically arsenic is used as a chemotherapeutic agent for treatment of acute promyelocytic leukemia. Inorganic arsenic has two biological important oxidation states: As(V) (arsenate) and As(III) (arsenite). Arsenic uptake is adventitious because the arsenate and arsenite are chemically similar to required nutrients. Arsenate resembles phosphate and is a competitive inhibitor of many phosphate-utilizing enzymes. Arsenate is taken up by phosphate transport systems. In contrast, at physiological pH, the form of arsenite is As(OH)(3), which resembles organic molecules such as glycerol. Consequently, arsenite is taken into cells by aquaglyceroporin channels. Arsenic efflux systems are found in nearly every organism and evolved to rid cells of this toxic metalloid. These efflux systems include members of the multidrug resistance protein family and the bacterial exchangers Acr3 and ArsB. ArsB can also be a subunit of the ArsAB As(III)-translocating ATPase, an ATP-driven efflux pump. The ArsD metallochaperone binds cytosolic As(III) and transfers it to the ArsA subunit of the efflux pump. Knowledge of the pathways and transporters for arsenic uptake and efflux is essential for understanding its toxicity and carcinogenicity and for rational design of cancer chemotherapeutic drugs. Copyright © 2012 Elsevier Inc. All rights reserved.

  14. An Electrically Tight In Vitro Blood-Brain Barrier Model Displays Net Brain-to-Blood Efflux of Substrates for the ABC Transporters, P-gp, Bcrp and Mrp-1

    DEFF Research Database (Denmark)

    Helms, Hans Christian; Hersom, Maria; Kuhlmann, Louise Borella;

    2014-01-01

    Efflux transporters of the ATP-binding cassette superfamily including breast cancer resistance protein (Bcrp/Abcg2), P-glycoprotein (P-gp/Abcb1) and multidrug resistance-associated proteins (Mrp's/Abcc's) are expressed in the blood-brain barrier (BBB). The aim of this study was to investigate......, zosuquidar, reversan and MK 571 alone or in combinations. Digoxin was mainly transported via P-gp, estrone-3-sulphate via Bcrp and Mrp's and etoposide via P-gp and Mrp's. The expression of P-gp, Bcrp and Mrp-1 was confirmed using immunocytochemistry. The findings indicate that P-gp, Bcrp and at least one...... isoform of Mrp are functionally expressed in our bovine/rat co-culture model and that the model is suitable for investigations of small molecule transport....

  15. AUXIN STIMULATION OF ETHYLENE EVOLUTION

    Science.gov (United States)

    mechanism of auxin action on the enhancement of ethylene production is the formation of enzymes involved in ethylene biogenesis....The stimulation of ethylene production by auxin was inhibited by actinomycin D and other inhibitors of protein synthesis. It is concluded that the

  16. The importance of localized auxin production for morphogenesis of reproductive organs and embryos in Arabidopsis.

    Science.gov (United States)

    Robert, Hélène S; Crhak Khaitova, Lucie; Mroue, Souad; Benková, Eva

    2015-08-01

    Plant sexual reproduction involves highly structured and specialized organs: stamens (male) and gynoecia (female, containing ovules). These organs synchronously develop within protective flower buds, until anthesis, via tightly coordinated mechanisms that are essential for effective fertilization and production of viable seeds. The phytohormone auxin is one of the key endogenous signalling molecules controlling initiation and development of these, and other, plant organs. In particular, its uneven distribution, resulting from tightly controlled production, metabolism and directional transport, is an important morphogenic factor. In this review we discuss how developmentally controlled and localized auxin biosynthesis and transport contribute to the coordinated development of plants' reproductive organs, and their fertilized derivatives (embryos) via the regulation of auxin levels and distribution within and around them. Current understanding of the links between de novo local auxin biosynthesis, auxin transport and/or signalling is presented to highlight the importance of the non-cell autonomous action of auxin production on development and morphogenesis of reproductive organs and embryos. An overview of transcription factor families, which spatiotemporally define local auxin production by controlling key auxin biosynthetic enzymes, is also presented.

  17. Auxin control of root development.

    Science.gov (United States)

    Overvoorde, Paul; Fukaki, Hidehiro; Beeckman, Tom

    2010-06-01

    A plant's roots system determines both the capacity of a sessile organism to acquire nutrients and water, as well as providing a means to monitor the soil for a range of environmental conditions. Since auxins were first described, there has been a tight connection between this class of hormones and root development. Here we review some of the latest genetic, molecular, and cellular experiments that demonstrate the importance of generating and maintaining auxin gradients during root development. Refinements in the ability to monitor and measure auxin levels in root cells coupled with advances in our understanding of the sources of auxin that contribute to these pools represent important contributions to our understanding of how this class of hormones participates in the control of root development. In addition, we review the role of identified molecular components that convert auxin gradients into local differentiation events, which ultimately defines the root architecture.

  18. Modified host cells with efflux pumps

    Science.gov (United States)

    Dunlop, Mary J.; Keasling, Jay D.; Mukhopadhyay, Aindrila

    2016-08-30

    The present invention provides for a modified host cell comprising a heterologous expression of an efflux pump capable of transporting an organic molecule out of the host cell wherein the organic molecule at a sufficiently high concentration reduces the growth rate of or is lethal to the host cell.

  19. Drug efflux proteins in multidrug resistant bacteria

    NARCIS (Netherlands)

    vanVeen, HW; Konings, WN

    1997-01-01

    Bacteria contain an array of transport proteins in their cytoplasmic membrane. Many of these proteins play an important role in conferring resistance to toxic compounds. The multidrug efflux systems encountered in prokaryotic cells are very similar to those observed in eukaryotic cells. Therefore, a

  20. Drug efflux proteins in multidrug resistant bacteria

    NARCIS (Netherlands)

    vanVeen, HW; Konings, WN

    Bacteria contain an array of transport proteins in their cytoplasmic membrane. Many of these proteins play an important role in conferring resistance to toxic compounds. The multidrug efflux systems encountered in prokaryotic cells are very similar to those observed in eukaryotic cells. Therefore, a

  1. In Vitro Methods to Study the Interplay of Drug Metabolism and Efflux in the Intestine

    NARCIS (Netherlands)

    Siissalo, Sanna; Heikkinen, Aki T.

    2013-01-01

    This review provides an overview of the in vitro methods currently used in studies of intestinal drug metabolism and active efflux with a special emphasis on the efflux-metabolism interplay. These methods include e. g. expressed enzymes or efflux transporters, fractionated intestinal cells, cell lin

  2. 13-hydroxy linoleic acid increases expression of the cholesterol transporters ABCA1, ABCG1 and SR-BI and stimulates apoA-I-dependent cholesterol efflux in RAW264.7 macrophages

    Directory of Open Access Journals (Sweden)

    Kämmerer Ines

    2011-11-01

    Full Text Available Abstract Background Synthetic activators of peroxisome proliferator-activated receptors (PPARs stimulate cholesterol removal from macrophages through PPAR-dependent up-regulation of liver × receptor α (LXRα and subsequent induction of cholesterol exporters such as ATP-binding cassette transporter A1 (ABCA1 and scavenger receptor class B type 1 (SR-BI. The present study aimed to test the hypothesis that the hydroxylated derivative of linoleic acid (LA, 13-HODE, which is a natural PPAR agonist, has similar effects in RAW264.7 macrophages. Methods RAW264.7 macrophages were treated without (control or with LA or 13-HODE in the presence and absence of PPARα or PPARγ antagonists and determined protein levels of LXRα, ABCA1, ABCG1, SR-BI, PPARα and PPARγ and apolipoprotein A-I mediated lipid efflux. Results Treatment of RAW264.7 cells with 13-HODE increased PPAR-transactivation activity and protein concentrations of LXRα, ABCA1, ABCG1 and SR-BI when compared to control treatment (P Conclusion 13-HODE induces cholesterol efflux from macrophages via the PPAR-LXRα-ABCA1/SR-BI-pathway.

  3. WOX5-1AA17 Feedback Circuit-Mediated CellularAuxin Response Is Crucial for the Patterning ofRoot Stem Cell Niches in Arabidopsis

    Institute of Scientific and Technical Information of China (English)

    2014-01-01

    In plants, the patterning of stem cell-enriched meristems requires a graded auxin response maximum thatemerges from the concerted action of polar auxin transport, auxin biosynthesis, auxin metabolism, and cellular auxinresponse machinery. However, mechanisms underlying this auxin response maximum-mediated root stem cell mainte-nance are not fully understood. Here, we present unexpected evidence that WUSCHEL-RELATED HOMEOBOX 5 (WOX5)transcription factor modulates expression of auxin biosynthetic genes in the quiescent center (QC) of the root and thusprovides a robust mechanism for the maintenance of auxin response maximum in the root tip. This WOX5 action is bal-anced through the activity of indole-3-acetic acid 17 (IAA17) auxin response repressor. Our combined genetic, cell biol-ogy, and computational modeling studies revealed a previously uncharacterized feedback loop linking WOX5-mediatedauxin production to IAA17-dependent repression of auxin responses. This WOX5-1AA17 feedback circuit further assuresthe maintenance of auxin response maximum in the root tip and thereby contributes to the maintenance of distal stemcell (DSC) populations. Our experimental studies and in silico computer simulations both demonstrate that the WOX5-iAA17 feedback circuit is essential for the maintenance of auxin gradient in the root tip and the auxin-mediated root DSCdifferentiation.

  4. 7-Rhamnosylated Flavonols Modulate Homeostasis of the Plant Hormone Auxin and Affect Plant Development.

    Science.gov (United States)

    Kuhn, Benjamin M; Errafi, Sanae; Bucher, Rahel; Dobrev, Petre; Geisler, Markus; Bigler, Laurent; Zažímalová, Eva; Ringli, Christoph

    2016-03-04

    Flavonols are a group of secondary metabolites that affect diverse cellular processes. They are considered putative negative regulators of the transport of the phytohormone auxin, by which they influence auxin distribution and concomitantly take part in the control of plant organ development. Flavonols are accumulating in a large number of glycosidic forms. Whether these have distinct functions and diverse cellular targets is not well understood. The rol1-2 mutant of Arabidopsis thaliana is characterized by a modified flavonol glycosylation profile that is inducing changes in auxin transport and growth defects in shoot tissues. To determine whether specific flavonol glycosides are responsible for these phenotypes, a suppressor screen was performed on the rol1-2 mutant, resulting in the identification of an allelic series of UGT89C1, a gene encoding a flavonol 7-O-rhamnosyltransferase. A detailed analysis revealed that interfering with flavonol rhamnosylation increases the concentration of auxin precursors and auxin metabolites, whereas auxin transport is not affected. This finding provides an additional level of complexity to the possible ways by which flavonols influence auxin distribution and suggests that flavonol glycosides play an important role in regulating plant development.

  5. Sodium efflux in plant roots: what do we really know?

    Science.gov (United States)

    Britto, D T; Kronzucker, H J

    2015-08-15

    The efflux of sodium (Na(+)) ions across the plasma membrane of plant root cells into the external medium is surprisingly poorly understood. Nevertheless, Na(+) efflux is widely regarded as a major mechanism by which plants restrain the rise of Na(+) concentrations in the cytosolic compartments of root cells and, thus, achieve a degree of tolerance to saline environments. In this review, several key ideas and bodies of evidence concerning root Na(+) efflux are summarized with a critical eye. Findings from decades past are brought to bear on current thinking, and pivotal studies are discussed, both "purely physiological", and also with regard to the SOS1 protein, the only major Na(+) efflux transporter that has, to date, been genetically characterized. We find that the current model of rapid transmembrane sodium cycling (RTSC), across the plasma membrane of root cells, is not adequately supported by evidence from the majority of efflux studies. An alternative hypothesis cannot be ruled out, that most Na(+) tracer efflux from the root in the salinity range does not proceed across the plasma membrane, but through the apoplast. Support for this idea comes from studies showing that Na(+) efflux, when measured with tracers, is rarely affected by the presence of inhibitors or the ionic composition in saline rooting media. We conclude that the actual efflux of Na(+) across the plasma membrane of root cells may be much more modest than what is often reported in studies using tracers, and may predominantly occur in the root tips, where SOS1 expression has been localized.

  6. Efflux transport of chrysin and apigenin sulfates in HEK293 cells overexpressing SULT1A3: The role of multidrug resistance-associated protein 4 (MRP4/ABCC4).

    Science.gov (United States)

    Li, Wan; Sun, Hua; Zhang, Xingwang; Wang, Huan; Wu, Baojian

    2015-11-01

    Efflux transport is a critical determinant to the pharmacokinetics of sulfate conjugates. Here we aimed to establish SULT1A3 stably transfected HEK293 cells, and to determine the contributions of BCRP and MRP transporters to excretion of chrysin and apigenin sulfates. The cDNA of SULT1A3 was stably introduced into HEK293 cells using a lentiviral vector, generating a sulfonation active cell line (i.e., SULT293 cells). Identification of sulfate transporters was achieved through chemical inhibition (using chemical inhibitors) and biological inhibition (using short-hairpin RNAs (shRNAs)) methods. Sulfated metabolites were rapidly generated and excreted upon incubation of SULT293 cells with chrysin and apigenin. Ko143 (a selective BCRP inhibitor) did not show inhibitory effects on sulfate disposition, whereas the pan-MRP inhibitor MK-571 caused significant reductions (38.5-64.3%, pHEK293 cells were an appropriate tool to study SULT1A3-mediated sulfonation and to characterize BCRP/MRP4-mediated sulfate transport.

  7. 生长素对水稻根系生长发育调控的研究进展%Advances on Molecular Mechanism of Auxin in Rice Root

    Institute of Scientific and Technical Information of China (English)

    康书静; 钱前; 朱丽

    2014-01-01

    水稻根系是非常重要的吸收营养和水分的器官,其生长与发育受多个植物激素协同调控。本文综述了生长素对水稻根形态建成调控的分子遗传学研究进展,影响生长素合成的YUCCA基因是通过控制生长素在植株体内的浓度来改变根的形态。而生长素运输主要通过调控生长素输出载体PIN和生长素输入载体AUXI的极性分布,影响侧根和不定根的发育,以及根的向重性。此外,TIR1、Aux/IAA与ARF互作在生长素信号转导中起重要的调控作用。其他激素可以通过信号转导途径影响生长素的分布,调控根系统的建成。但目前水稻相关基因的克隆进展缓慢,对生长素调控网络认识还不够清晰。因此,更为深入的研究水稻生长素相关基因将对理解水稻根系发育的分子机制具有重要意义。%Rice root is the main organ for nutrient and water absorption, its growth and development are regulated by several plant hormones collaboratively. Of which,auxin is the extensively studied. In this paper, the author elucidate the development process and molecular mechanism of rice root architecture by analyzing the regulating genes that have been cloned. Comprehensive analysis re-veals that the root shape is altered by auxin biosynthesis genes that regulate auxin concentration. Auxin transport regulate the devel-opment of lateral root and crown root through controlling auxin influx and efflux. Auxin signal transduction, which affects the root sys-tem, is regulated by TIR1、Aux/IAA and ARF. Besides the distrubition of auxin is influenced by acting with other plant hormones. However,these cloned root shape genes in rice is still insufficient, and the outline of molecular regulation network of auxin is not clear. Therefore,it is important to further understand molecular mechanism of auxin in rice root by screening and collecting rice auxin mutants with root mutations.

  8. Glucose and auxin signaling interaction in controlling Arabidopsis thaliana seedlings root growth and development.

    Directory of Open Access Journals (Sweden)

    Bhuwaneshwar S Mishra

    Full Text Available BACKGROUND: Plant root growth and development is highly plastic and can adapt to many environmental conditions. Sugar signaling has been shown to affect root growth and development by interacting with phytohormones such as gibberellins, cytokinin and abscisic acid. Auxin signaling and transport has been earlier shown to be controlling plant root length, number of lateral roots, root hair and root growth direction. PRINCIPAL FINDINGS: Increasing concentration of glucose not only controls root length, root hair and number of lateral roots but can also modulate root growth direction. Since root growth and development is also controlled by auxin, whole genome transcript profiling was done to find out the extent of interaction between glucose and auxin response pathways. Glucose alone could transcriptionally regulate 376 (62% genes out of 604 genes affected by IAA. Presence of glucose could also modulate the extent of regulation 2 fold or more of almost 63% genes induced or repressed by IAA. Interestingly, glucose could affect induction or repression of IAA affected genes (35% even if glucose alone had no significant effect on the transcription of these genes itself. Glucose could affect auxin biosynthetic YUCCA genes family members, auxin transporter PIN proteins, receptor TIR1 and members of a number of gene families including AUX/IAA, GH3 and SAUR involved in auxin signaling. Arabidopsis auxin receptor tir1 and response mutants, axr2, axr3 and slr1 not only display a defect in glucose induced change in root length, root hair elongation and lateral root production but also accentuate glucose induced increase in root growth randomization from vertical suggesting glucose effects on plant root growth and development are mediated by auxin signaling components. CONCLUSION: Our findings implicate an important role of the glucose interacting with auxin signaling and transport machinery to control seedling root growth and development in changing nutrient

  9. Modeling halotropism: a key role for root tip architecture and reflux loop remodeling in redistributing auxin.

    Science.gov (United States)

    van den Berg, Thea; Korver, Ruud A; Testerink, Christa; Ten Tusscher, Kirsten H W J

    2016-09-15

    A key characteristic of plant development is its plasticity in response to various and dynamically changing environmental conditions. Tropisms contribute to this flexibility by allowing plant organs to grow from or towards environmental cues. Halotropism is a recently described tropism in which plant roots bend away from salt. During halotropism, as in most other tropisms, directional growth is generated through an asymmetric auxin distribution that generates differences in growth rate and hence induces bending. Here, we develop a detailed model of auxin transport in the Arabidopsis root tip and combine this with experiments to investigate the processes generating auxin asymmetry during halotropism. Our model points to the key role of root tip architecture in allowing the decrease in PIN2 at the salt-exposed side of the root to result in a re-routing of auxin to the opposite side. In addition, our model demonstrates how feedback of auxin on the auxin transporter AUX1 amplifies this auxin asymmetry, while a salt-induced transient increase in PIN1 levels increases the speed at which this occurs. Using AUX1-GFP imaging and pin1 mutants, we experimentally confirmed these model predictions, thus expanding our knowledge of the cellular basis of halotropism.

  10. Inter-regulation of the unfolded protein response and auxin signaling.

    Science.gov (United States)

    Chen, Yani; Aung, Kyaw; Rolčík, Jakub; Walicki, Kathryn; Friml, Jiří; Brandizzi, Federica

    2014-01-01

    The unfolded protein response (UPR) is a signaling network triggered by overload of protein-folding demand in the endoplasmic reticulum (ER), a condition termed ER stress. The UPR is critical for growth and development; nonetheless, connections between the UPR and other cellular regulatory processes remain largely unknown. Here, we identify a link between the UPR and the phytohormone auxin, a master regulator of plant physiology. We show that ER stress triggers down-regulation of auxin receptors and transporters in Arabidopsis thaliana. We also demonstrate that an Arabidopsis mutant of a conserved ER stress sensor IRE1 exhibits defects in the auxin response and levels. These data not only support that the plant IRE1 is required for auxin homeostasis, they also reveal a species-specific feature of IRE1 in multicellular eukaryotes. Furthermore, by establishing that UPR activation is reduced in mutants of ER-localized auxin transporters, including PIN5, we define a long-neglected biological significance of ER-based auxin regulation. We further examine the functional relationship of IRE1 and PIN5 by showing that an ire1 pin5 triple mutant enhances defects of UPR activation and auxin homeostasis in ire1 or pin5. Our results imply that the plant UPR has evolved a hormone-dependent strategy for coordinating ER function with physiological processes.

  11. Structural Biology of Nuclear Auxin Action.

    Science.gov (United States)

    Dinesh, Dhurvas Chandrasekaran; Villalobos, Luz Irina A Calderón; Abel, Steffen

    2016-04-01

    Auxin coordinates plant development largely via hierarchical control of gene expression. During the past decades, the study of early auxin genes paired with the power of Arabidopsis genetics have unraveled key nuclear components and molecular interactions that perceive the hormone and activate primary response genes. Recent research in the realm of structural biology allowed unprecedented insight into: (i) the recognition of auxin-responsive DNA elements by auxin transcription factors; (ii) the inactivation of those auxin response factors by early auxin-inducible repressors; and (iii) the activation of target genes by auxin-triggered repressor degradation. The biophysical studies reviewed here provide an impetus for elucidating the molecular determinants of the intricate interactions between core components of the nuclear auxin response module.

  12. PI3K signaling supports amphetamine-induced dopamine efflux.

    Science.gov (United States)

    Lute, Brandon J; Khoshbouei, Habibeh; Saunders, Christine; Sen, Namita; Lin, Richard Z; Javitch, Jonathan A; Galli, Aurelio

    2008-08-01

    The dopamine (DA) transporter (DAT) is a major molecular target of the psychostimulant amphetamine (AMPH). AMPH, as a result of its ability to reverse DAT-mediated inward transport of DA, induces DA efflux thereby increasing extracellular DA levels. This increase is thought to underlie the behavioral effects of AMPH. We have demonstrated previously that insulin, through phosphatidylinositol 3-kinase (PI3K) signaling, regulates DA clearance by fine-tuning DAT plasma membrane expression. PI3K signaling may represent a novel mechanism for regulating DA efflux evoked by AMPH, since only active DAT at the plasma membrane can efflux DA. Here, we show in both a heterologous expression system and DA neurons that inhibition of PI3K decreases DAT cell surface expression and, as a consequence, AMPH-induced DA efflux.

  13. Adenosine Diphosphate Ribosylation Factor-GTPaseActivating Protein Stimulates the Transport of AUX1Endosome, Which Relies on Actin Cytoskeletal Organization in Rice Root DevelopmentF

    Institute of Scientific and Technical Information of China (English)

    Cheng Du; Yunyuan XU; Yingdian Wang; Kang Chong

    2011-01-01

    Polar auxin transport,which depends on polarized subcellular distribution of AUXIN RESISTANT 1/LIKE AUX1 (AUX1/LAX) influx carriers and PIN-FORMED (PIN) efflux carriers,mediates various processes of plant growth and development.Endosomal recycling of PIN1 is mediated by an adenosine diphosphate (ADP)ribosylation factor (ARF)-GTPase exchange factor protein,GNOM.However,the mediation of auxin influx carrier recycling is poorly understood.Here,we report that overexpression of OsAGAP,an ARF-GTPase-activating protein in rice,stimulates vesicle transport from the plasma membrane to the Golgi apparatus in protoplasts and transgenic plants and induces the accumulation of early endosomes and AUX1.AUX1 endosomes could partially colocalize with FM4-64 labeled early endosome after actin disruption.Furthermore,OsAGAP is involved in actin cytoskeletal organization,and its overexpression tends to reduce the thickness and bundling of actin filaments.Fluorescence recovery after photobleaching analysis revealed exocytosis of the AUX1 recycling endosome was not affected in the OsAGAP overexpression cells,and was only slightly promoted when the actin filaments were completely disrupted by Lat B.Thus,we propose that AUX1 accumulation in the OsAGAP overexpression and actin disrupted cells may be due to the fact that endocytosis of the auxin influx carrier AUX1 early endosome was greatly promoted by actin cytoskeleton disruption.

  14. Solitary Inhibition of the Breast Cancer Resistance Protein Efflux Transporter Results in a Clinically Significant Drug-Drug Interaction with Rosuvastatin by Causing up to a 2-Fold Increase in Statin Exposure.

    Science.gov (United States)

    Elsby, Robert; Martin, Paul; Surry, Dominic; Sharma, Pradeep; Fenner, Katherine

    2016-03-01

    The intestinal efflux transporter breast cancer resistance protein (BCRP) restricts the absorption of rosuvastatin. Of the transporters important to rosuvastatin disposition, fostamatinib inhibited BCRP (IC50 = 50 nM) and organic anion-transporting polypeptide 1B1 (OATP1B1; IC50 > 10 μM), but not organic anion transporter 3, in vitro, predicting a drug-drug interaction (DDI) in vivo through inhibition of BCRP only. Consequently, a clinical interaction study between fostamatinib and rosuvastatin was performed (and reported elsewhere). This confirmed the critical role BCRP plays in statin absorption, as inhibition by fostamatinib resulted in a significant 1.96-fold and 1.88-fold increase in rosuvastatin area under the plasma concentration-time curve (AUC) and Cmax, respectively. An in vitro BCRP inhibition assay, using polarized Caco-2 cells and rosuvastatin as probe substrate, was subsequently validated with literature inhibitors and used to determine BCRP inhibitory potencies (IC50) of the perpetrator drugs eltrombopag, darunavir, lopinavir, clopidogrel, ezetimibe, fenofibrate, and fluconazole. OATP1B1 inhibition was also determined using human embryonic kidney 293-OATP1B1 cells versus estradiol 17β-glucuronide. Calculated parameters of maximum enterocyte concentration [Igut max], maximum unbound hepatic inlet concentration, transporter fraction excreted value, and determined IC50 value were incorporated into mechanistic static equations to compute theoretical increases in rosuvastatin AUC due to inhibition of BCRP and/or OATP1B1. Calculated theoretical increases in exposure correctly predicted the clinically observed changes in rosuvastatin exposure and suggested intestinal BCRP inhibition (not OATP1B1) to be the mechanism underlying the DDIs with these drugs. In conclusion, solitary inhibition of the intestinal BCRP transporter can result in clinically significant DDIs with rosuvastatin, causing up to a maximum 2-fold increase in exposure, which may warrant

  15. ACTION OF AUXIN ON LEAF ABSCISSION

    Science.gov (United States)

    Experiments have been conducted to investigate a two-stage effect of auxin on abscission. The two stages were demonstrated on greenhouse-grown Black...the second stage - the stage which is stimulated by auxin . Similar experiments were performed with petioles of various lengths and ages. The...implications of these results indicate possible sites of auxin action on leaf abscission. (Author)

  16. Exploring the role of auxin in the androgynophore movement in Passiflora

    Directory of Open Access Journals (Sweden)

    Livia C.T. Scorza

    2015-09-01

    Full Text Available The flowers of the species belonging to the genus Passiflorashow a range of features that are thought to have arisen as adaptations to different pollinators. Some Passiflora species belonging to the subgenus Decaloba sect. Xerogona, show touch-sensitive motile androgynophores. We tested the role of auxin polar transport in the modulation of the androgynophore movement by applying auxin (IAA or an inhibitor of auxin polar transport (NPA in the flowers. We recorded the movement of the androgynophore during mechano-stimulation and analyzed the duration, speed, and the angle formed by the androgynophore before and after the movement, and found that both IAA and NPA increase the amplitude of the movement in P. sanguinolenta. We hypothesize that auxin might have a role in modulating the fitness of these Decaloba species to different pollination syndromes and demonstrate that an interspecific hybrid between insect- and hummingbird-pollinated Xerogona species present a heterosis effect on the speed of the androgynophore movement.

  17. The Ferroportin Metal Efflux Proteins Function in Iron and Cobalt Homeostasis in Arabidopsis

    Science.gov (United States)

    Relatively little is known about how metals such as iron are effluxed from cells, a necessary step for transport from the root to the shoot. Ferroportin is the sole iron efflux transporter in animals, and there are two closely related orthologs in Arabidopsis, FPN1 and FPN2. FPN1 localizes to the pl...

  18. Dynamics of auxin movement in the gravistimulated leaf-sheath pulvinus of oat (Avena sativa)

    Science.gov (United States)

    Brock, Thomas C.; Kapen, E. H.; Ghosheh, Najati S.; Kaufman, Peter B.

    1991-01-01

    The role of auxin redistribution in the graviresponse of the leaf-sheath pulvinus of oat was evaluated using H-3-indole-3-acetic acid (H-3-IAA) preloaded into isolated pulvini. Results obtained reveal that, while lateral transport of auxin occurs following gravistimulation, it is not necessary for a graviresponse. Localized changes in tissue responsiveness or the conversion of conjugated hormone to free hormone may suffice to drive the graviresponse.

  19. Aluminium-induced reduction of plant growth in alfalfa (Medicago sativa) is mediated by interrupting auxin transport and accumulation in roots.

    Science.gov (United States)

    Wang, Shengyin; Ren, Xiaoyan; Huang, Bingru; Wang, Ge; Zhou, Peng; An, Yuan

    2016-07-20

    The objective of this study was to investigate Al(3+)-induced IAA transport, distribution, and the relation of these two processes to Al(3+)-inhibition of root growth in alfalfa. Alfalfa seedlings with or without apical buds were exposed to 0 or 100 μM AlCl3 and were foliar sprayed with water or 6 mg L(-1) IAA. Aluminium stress resulted in disordered arrangement of cells, deformed cell shapes, altered cell structure, and a shorter length of the meristematic zone in root tips. Aluminium stress significantly decreased the IAA concentration in apical buds and root tips. The distribution of IAA fluorescence signals in root tips was disturbed, and the IAA transportation from shoot base to root tip was inhibited. The highest intensity of fluorescence signals was detected in the apical meristematic zone. Exogenous application of IAA markedly alleviated the Al(3+)-induced inhibition of root growth by increasing IAA accumulation and recovering the damaged cell structure in root tips. In addition, Al(3+) stress up-regulated expression of AUX1 and PIN2 genes. These results indicate that Al(3+)-induced reduction of root growth could be associated with the inhibitions of IAA synthesis in apical buds and IAA transportation in roots, as well as the imbalance of IAA distribution in root tips.

  20. miRNA-mediated auxin signalling repression during Vat-mediated aphid resistance in Cucumis melo.

    Science.gov (United States)

    Sattar, Sampurna; Addo-Quaye, Charles; Thompson, Gary A

    2016-06-01

    Resistance to Aphis gossypii in melon is attributed to the presence of the single dominant R gene virus aphid transmission (Vat), which is biologically expressed as antibiosis, antixenosis and tolerance. However, the mechanism of resistance is poorly understood at the molecular level. Aphid-induced transcriptional changes, including differentially expressed miRNA profiles that correspond to resistance interaction have been reported in melon. The potential regulatory roles of miRNAs in Vat-mediated aphid resistance were further revealed by identifying the specific miRNA degradation targets. A total of 70 miRNA:target pairs, including 28 novel miRNA:target pairs, for the differentially expressed miRNAs were identified: 11 were associated with phytohormone regulation, including six miRNAs that potentially regulate auxin interactions. A model for a redundant regulatory system of miRNA-mediated auxin insensitivity is proposed that incorporates auxin perception, auxin modification and auxin-regulated transcription. Chemically inhibiting the transport inhibitor response-1 (TIR-1) auxin receptor in susceptible melon tissues provides in vivo support for the model of auxin-mediated impacts on A. gossypii resistance.

  1. The effects of auxin and strigolactones on tuber initiation and stolon architecture in potato.

    Science.gov (United States)

    Roumeliotis, Efstathios; Kloosterman, Bjorn; Oortwijn, Marian; Kohlen, Wouter; Bouwmeester, Harro J; Visser, Richard G F; Bachem, Christian W B

    2012-07-01

    Various transcriptional networks and plant hormones have been implicated in controlling different aspects of potato tuber formation. Due to its broad impact on many plant developmental processes, a role for auxin in tuber initiation has been suggested but never fully resolved. Here, auxin concentrations were measured throughout the plant prior to and during the process of tuber formation. Auxin levels increase dramatically in the stolon prior to tuberization and remain relatively high during subsequent tuber growth, suggesting a promoting role for auxin in tuber formation. Furthermore, in vitro tuberization experiments showed higher levels of tuber formation from axillary buds of explants where the auxin source (stolon tip) had been removed. This phenotype could be rescued by application of auxin on the ablated stolon tips. In addition, a synthetic strigolactone analogue applied on the basal part of the stolon resulted in fewer tubers. The experiments indicate that a system for the production and directional transport of auxin exists in stolons and acts synergistically with strigolactones to control the outgrowth of the axillary stolon buds, similar to the control of above-ground shoot branching.

  2. Distinct Characteristics of Indole-3-Acetic Acid and Phenylacetic Acid, Two Common Auxins in Plants

    Science.gov (United States)

    Sugawara, Satoko; Mashiguchi, Kiyoshi; Tanaka, Keita; Hishiyama, Shojiro; Sakai, Tatsuya; Hanada, Kousuke; Kinoshita-Tsujimura, Kaori; Yu, Hong; Dai, Xinhua; Takebayashi, Yumiko; Takeda-Kamiya, Noriko; Kakimoto, Tatsuo; Kawaide, Hiroshi; Natsume, Masahiro; Estelle, Mark; Zhao, Yunde; Hayashi, Ken-ichiro; Kamiya, Yuji; Kasahara, Hiroyuki

    2015-01-01

    The phytohormone auxin plays a central role in many aspects of plant growth and development. IAA is the most studied natural auxin that possesses the property of polar transport in plants. Phenylacetic acid (PAA) has also been recognized as a natural auxin for >40 years, but its role in plant growth and development remains unclear. In this study, we show that IAA and PAA have overlapping regulatory roles but distinct transport characteristics as auxins in plants. PAA is widely distributed in vascular and non-vascular plants. Although the biological activities of PAA are lower than those of IAA, the endogenous levels of PAA are much higher than those of IAA in various plant tissues in Arabidopsis. PAA and IAA can regulate the same set of auxin-responsive genes through the TIR1/AFB pathway in Arabidopsis. IAA actively forms concentration gradients in maize coleoptiles in response to gravitropic stimulation, whereas PAA does not, indicating that PAA is not actively transported in a polar manner. The induction of the YUCCA (YUC) genes increases PAA metabolite levels in Arabidopsis, indicating that YUC flavin-containing monooxygenases may play a role in PAA biosynthesis. Our results provide new insights into the regulation of plant growth and development by different types of auxins. PMID:26076971

  3. Distinct Characteristics of Indole-3-Acetic Acid and Phenylacetic Acid, Two Common Auxins in Plants.

    Science.gov (United States)

    Sugawara, Satoko; Mashiguchi, Kiyoshi; Tanaka, Keita; Hishiyama, Shojiro; Sakai, Tatsuya; Hanada, Kousuke; Kinoshita-Tsujimura, Kaori; Yu, Hong; Dai, Xinhua; Takebayashi, Yumiko; Takeda-Kamiya, Noriko; Kakimoto, Tatsuo; Kawaide, Hiroshi; Natsume, Masahiro; Estelle, Mark; Zhao, Yunde; Hayashi, Ken-Ichiro; Kamiya, Yuji; Kasahara, Hiroyuki

    2015-08-01

    The phytohormone auxin plays a central role in many aspects of plant growth and development. IAA is the most studied natural auxin that possesses the property of polar transport in plants. Phenylacetic acid (PAA) has also been recognized as a natural auxin for >40 years, but its role in plant growth and development remains unclear. In this study, we show that IAA and PAA have overlapping regulatory roles but distinct transport characteristics as auxins in plants. PAA is widely distributed in vascular and non-vascular plants. Although the biological activities of PAA are lower than those of IAA, the endogenous levels of PAA are much higher than those of IAA in various plant tissues in Arabidopsis. PAA and IAA can regulate the same set of auxin-responsive genes through the TIR1/AFB pathway in Arabidopsis. IAA actively forms concentration gradients in maize coleoptiles in response to gravitropic stimulation, whereas PAA does not, indicating that PAA is not actively transported in a polar manner. The induction of the YUCCA (YUC) genes increases PAA metabolite levels in Arabidopsis, indicating that YUC flavin-containing monooxygenases may play a role in PAA biosynthesis. Our results provide new insights into the regulation of plant growth and development by different types of auxins. © The Author 2015. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.

  4. Auxins as Signals in Arbuscular Mycorrhiza Formation

    Science.gov (United States)

    Güther, Mike

    2007-01-01

    Plant hormones such as auxin derivatives are likely signals during the establishment of an arbuscular mycorrhizal (AM) symbiosis. Although reports on auxin levels during AM in different plant species are contradictory, the contribution of auxins to the establishment of an AM symbiosis might be an important factor especially for the development of lateral roots which are the preferred infection sites for the fungi. In addition to evidence that different auxins could be elevated after colonization with AM fungi, there are also overlapping gene expression patterns between auxin-treated and AM-inoculated roots that provide further clues on auxin-triggered colonization events. Using an auxin-inducible promoter-reporter system it was shown that the reporter was strongly induced in AM colonized roots, although co-localization with AM fungi was not observed. Our data are discussed in frame of a model together with other plant hormones which might be involved in the AM colonization processes. PMID:19704695

  5. An attenuated mutant of the Rv1747 ATP-binding cassette transporter of Mycobacterium tuberculosis and a mutant of its cognate kinase, PknF, show increased expression of the efflux pump-related iniBAC operon

    Science.gov (United States)

    Spivey, Vicky L; Whalan, Rachael H; Hirst, Elizabeth M A; Smerdon, Stephen J; Buxton, Roger S

    2013-01-01

    The ATP-binding cassette transporter Rv1747 is required for the growth of Mycobacterium tuberculosis in mice and in macrophages. Its structure suggests it is an exporter. Rv1747 forms a two-gene operon with pknF coding for the serine/threonine protein kinase PknF, which positively modulates the function of the transporter. We show that deletion of Rv1747 or pknF results in a number of transcriptional changes which could be complemented by the wild type allele, most significantly up-regulation of the iniBAC genes. This operon is inducible by isoniazid and ethambutol and by a broad range of inhibitors of cell wall biosynthesis and is required for efflux pump functioning. However, neither the Rv1747 or pknF mutant showed increased susceptibility to a range of drugs and cell wall stress reagents including isoniazid and ethambutol, cell wall structure and cell division appear normal by electron microscopy, and no differences in lipoarabinomannan were found. Transcription from the pknF promoter was not induced by a range of stress reagents. We conclude that the loss of Rv1747 affects cell wall biosynthesis leading to the production of intermediates that cause induction of iniBAC transcription and implicates it in exporting a component of the cell wall, which is necessary for virulence. PMID:23915284

  6. Genetic evidence for auxin involvement in arbuscular mycorrhiza initiation.

    Science.gov (United States)

    Hanlon, Meredith T; Coenen, Catharina

    2011-02-01

    • Formation of arbuscular mycorrhiza (AM) is controlled by a host of small, diffusible signaling molecules, including phytohormones. To test the hypothesis that the plant hormone auxin controls mycorrhiza development, we assessed mycorrhiza formation in two mutants of tomato (Solanum lycopersicum): diageotropica (dgt), an auxin-resistant mutant, and polycotyledon (pct), a mutant with hyperactive polar auxin transport. • Mutant and wild-type (WT) roots were inoculated with spores of the AM fungus Glomus intraradices. Presymbiotic root-fungus interactions were observed in root organ culture (ROC) and internal fungal colonization was quantified both in ROC and in intact seedlings. • In ROC, G. intraradices stimulated presymbiotic root branching in pct but not in dgt roots. pct roots stimulated production of hyphal fans indicative of appressorium formation and were colonized more rapidly than WT roots. By contrast, approaching hyphae reversed direction to grow away from cultured dgt roots and failed to colonize them. In intact seedlings, pct and dgt roots were colonized poorly, but development of hyphae, arbuscules, and vesicles was morphologically normal within roots of both mutants. • We conclude that auxin signaling within host roots is required for the early stages of AM formation, including during presymbiotic signal exchange. © 2010 The Authors. New Phytologist © 2010 New Phytologist Trust.

  7. Multi-Scale Characean Experimental System: From Electrophysiology of Membrane Transporters to Cell-to-Cell Connectivity, Cytoplasmic Streaming and Auxin Metabolism

    Science.gov (United States)

    Beilby, Mary J.

    2016-01-01

    The morphology of characean algae could be mistaken for a higher plant: stem-like axes with leaf-like branchlets anchored in the soil by root-like rhizoids. However, all of these structures are made up of giant multinucleate cells separated by multicellular nodal complexes. The excised internodal cells survive long enough for the nodes to give rise to new thallus. The size of the internodes and their thick cytoplasmic layer minimize impalement injury and allow specific micro-electrode placement. The cell structure can be manipulated by centrifugation, perfusion of cell contents or creation of cytoplasmic droplets, allowing access to both vacuolar and cytoplasmic compartments and both sides of the cell membranes. Thousands of electrical measurements on intact or altered cells and cytoplasmic droplets laid down basis to modern plant electrophysiology. Furthermore, the giant internodal cells and whole thalli facilitate research into many other plant properties. As nutrients have to be transported from rhizoids to growing parts of the thallus and hormonal signals need to pass from cell to cell, Characeae possess very fast cytoplasmic streaming. The mechanism was resolved in the characean model. Plasmodesmata between the internodal cells and nodal complexes facilitate transport of ions, nutrients and photosynthates across the nodes. The internal structure was found to be similar to those of higher plants. Recent experiments suggest a strong circadian influence on metabolic pathways producing indole-3-acetic acid (IAA) and serotonin/melatonin. The review will discuss the impact of the characean models arising from fragments of cells, single cells, cell-to-cell transport or whole thalli on understanding of plant evolution and physiology. PMID:27504112

  8. Multi-scale characean experimental system: from electrophysiology of membrane transporters to cell-to-cell connectivity, cytoplasmic streaming and auxin metabolism.

    Directory of Open Access Journals (Sweden)

    Mary Jane Beilby

    2016-07-01

    Full Text Available The morphology of characean algae could be mistaken for a higher plant: stem-like axes with leaf-like branchlets anchored in the soil by root-like rhizoids. However, all of these structures are made up of giant multinucleate cells separated by multicellular nodal complexes. The excised internodal cells survive long enough for the nodes to give rise to new thallus. The size of the internodes and their thick cytoplasmic layer minimize impalement injury and allow specific micro-electrode placement. The cell structure can be manipulated by centrifugation, perfusion of cell contents or creation of cytoplasmic droplets, allowing access to both vacuolar and cytoplasmic compartments and both sides of the cell membranes. Thousands of electrical measurements on intact or altered cells and cytoplasmic droplets laid down basis to modern plant electrophysiology. Furthermore, the giant internodal cells and whole thalli facilitate research into many other plant properties. As nutrients have to be transported from rhizoids to growing parts of the thallus and hormonal signals need to pass from cell to cell, Characeae possess very fast cytoplasmic streaming. The mechanism was resolved in the characean model. Plasmodesmata between the internodal cells and nodal complexes facilitate transport of ions, nutrients and photosynthates across the nodes. The internal structure was found to be similar to those of higher plants. Recent experiments suggest a strong circadian influence on metabolic pathways producing indole-3-acetic acid (IAA and serotonin/melatonin. The review will discuss the impact of the characean models arising from fragments of cells, single cells, cell-to-cell transport or whole thalli on understanding of plant evolution and physiology.

  9. Multi-Scale Characean Experimental System: From Electrophysiology of Membrane Transporters to Cell-to-Cell Connectivity, Cytoplasmic Streaming and Auxin Metabolism.

    Science.gov (United States)

    Beilby, Mary J

    2016-01-01

    The morphology of characean algae could be mistaken for a higher plant: stem-like axes with leaf-like branchlets anchored in the soil by root-like rhizoids. However, all of these structures are made up of giant multinucleate cells separated by multicellular nodal complexes. The excised internodal cells survive long enough for the nodes to give rise to new thallus. The size of the internodes and their thick cytoplasmic layer minimize impalement injury and allow specific micro-electrode placement. The cell structure can be manipulated by centrifugation, perfusion of cell contents or creation of cytoplasmic droplets, allowing access to both vacuolar and cytoplasmic compartments and both sides of the cell membranes. Thousands of electrical measurements on intact or altered cells and cytoplasmic droplets laid down basis to modern plant electrophysiology. Furthermore, the giant internodal cells and whole thalli facilitate research into many other plant properties. As nutrients have to be transported from rhizoids to growing parts of the thallus and hormonal signals need to pass from cell to cell, Characeae possess very fast cytoplasmic streaming. The mechanism was resolved in the characean model. Plasmodesmata between the internodal cells and nodal complexes facilitate transport of ions, nutrients and photosynthates across the nodes. The internal structure was found to be similar to those of higher plants. Recent experiments suggest a strong circadian influence on metabolic pathways producing indole-3-acetic acid (IAA) and serotonin/melatonin. The review will discuss the impact of the characean models arising from fragments of cells, single cells, cell-to-cell transport or whole thalli on understanding of plant evolution and physiology.

  10. Aluminium-induced reduction of plant growth in alfalfa (Medicago sativa) is mediated by interrupting auxin transport and accumulation in roots

    OpenAIRE

    Shengyin Wang; Xiaoyan Ren; Bingru Huang; Ge Wang; Peng Zhou; Yuan An

    2016-01-01

    The objective of this study was to investigate Al3+-induced IAA transport, distribution, and the relation of these two processes to Al3+-inhibition of root growth in alfalfa. Alfalfa seedlings with or without apical buds were exposed to 0 or 100 μM AlCl3 and were foliar sprayed with water or 6 mg L−1 IAA. Aluminium stress resulted in disordered arrangement of cells, deformed cell shapes, altered cell structure, and a shorter length of the meristematic zone in root tips. Aluminium stress signi...

  11. The effect of carbon monoxide integrating nitric oxide through auxin signal in Arabidopsis to modulate iron deficiency

    Directory of Open Access Journals (Sweden)

    Liming eYang

    2016-03-01

    Full Text Available Carbon monoxide (CO and nitric oxide (NO are essential modulators that regulate the plant response to iron deficiency (-Fe. Auxin is a phytohormone that plays important roles in plant growth and development. We report here that in Arabidopsis –Fe enhanced heme oxygenase-dependent CO generation and auxin transport through redistribution of PIN1 protein, which subsequently increased NO accumulation; NO signaling regulated the activity of ferric chelate reductase (FCR and the expression of Fe-uptake genes including basic helix-loop-helix transcription factor (FIT and the ferric reduction oxidase 2 (FRO2. Over-expression of HY1 encoding heme oxygenase, or treatment with CO donor enhanced basipetal auxin transport, FCR activity, and the expressions of FIT and FRO2 under –Fe. Such effects were compromised in the mutant aux1-7 impaired in auxin transport or in the mutant noa1 or nia1/nia2 defective in NO biosynthesis. -Fe failed to promote auxin transport and FCR activity in hy1 mutant; such inability was reversed in the double mutant of hy1/yucca1 with elevated auxin production, or in hy1/cue1 mutant with NO over-accumulation. Taken together, our results suggest that CO modulates NO signaling through auxin to cope with Fe deficiency in Arabidopsis.

  12. A Simple Auxin Transcriptional Response System Regulates Multiple Morphogenetic Processes in the Liverwort Marchantia polymorpha.

    Science.gov (United States)

    Flores-Sandoval, Eduardo; Eklund, D Magnus; Bowman, John L

    2015-05-01

    In land plants comparative genomics has revealed that members of basal lineages share a common set of transcription factors with the derived flowering plants, despite sharing few homologous structures. The plant hormone auxin has been implicated in many facets of development in both basal and derived lineages of land plants. We functionally characterized the auxin transcriptional response machinery in the liverwort Marchantia polymorpha, a member of the basal lineage of extant land plants. All components known from flowering plant systems are present in M. polymorpha, but they exist as single orthologs: a single MpTOPLESS (TPL) corepressor, a single MpTRANSPORT inhibitor response 1 auxin receptor, single orthologs of each class of auxin response factor (ARF; MpARF1, MpARF2, MpARF3), and a single negative regulator auxin/indole-3-acetic acid (MpIAA). Phylogenetic analyses suggest this simple system is the ancestral condition for land plants. We experimentally demonstrate that these genes act in an auxin response pathway--chimeric fusions of the MpTPL corepressor with heterodimerization domains of MpARF1, MpARF2, or their negative regulator, MpIAA, generate auxin insensitive plants that lack the capacity to pattern and transition into mature stages of development. Our results indicate auxin mediated transcriptional regulation acts as a facilitator of branching, differentiation and growth, rather than acting to determine or specify tissues during the haploid stage of the M. polymorpha life cycle. We hypothesize that the ancestral role of auxin is to modulate a balance of differentiated and pluri- or totipotent cell states, whose fates are determined by interactions with combinations of unrelated transcription factors.

  13. Transcriptomic Analysis in Strawberry Fruits Reveals Active Auxin Biosynthesis and Signaling in the Ripe Receptacle

    Directory of Open Access Journals (Sweden)

    Elizabeth Estrada-Johnson

    2017-05-01

    Full Text Available The role of auxin in ripening strawberry (Fragaria ×ananassa fruits has been restricted to the early stages of development where the growth of the receptacle is dependent on the delivery of auxin from the achenes. At later stages, during enlargement of the receptacle, other hormones have been demonstrated to participate to different degrees, from the general involvement of gibberellins and abscisic acid to the more specific of ethylene. Here we report the involvement of auxin at the late stages of receptacle ripening. The auxin content of the receptacle remains constant during ripening. Analysis of the transcriptome of ripening strawberry fruit revealed the changing expression pattern of the genes of auxin synthesis, perception, signaling and transport along with achene and receptacle development from the green to red stage. Specific members of the corresponding gene families show active transcription in the ripe receptacle. For the synthesis of auxin, two genes encoding tryptophan aminotransferases, FaTAA1 and FaTAR2, were expressed in the red receptacle, with FaTAR2 expression peaking at this stage. Transient silencing of this gene in ripening receptacle was accompanied by a diminished responsiveness to auxin. The auxin activity in the ripening receptacle is supported by the DR5-directed expression of a GUS reporter gene in the ripening receptacle of DR5-GUS transgenic strawberry plants. Clustering by co-expression of members of the FaAux/IAA and FaARF families identified five members whose transcriptional activity was increased with the onset of receptacle ripening. Among these, FaAux/IAA11 and FaARF6a appeared, by their expression level and fold-change, as the most likely candidates for their involvement in the auxin activity in the ripening receptacle. The association of the corresponding ARF6 gene in Arabidopsis to cell elongation constitutes a suggestive hypothesis for FaARF6a involvement in the same cellular process in the growing and

  14. Repression of the Auxin Response Pathway Increases Arabidopsis Susceptibility to Necrotrophic Fungi

    Institute of Scientific and Technical Information of China (English)

    Francisco Llorente; Paul Muskett; Andrea Sánchez-Vallet; Gemma López; Brisa Ramos; Clara Sánchez-Rodríguez; Lucia Jordá; Jane Parker; Antonio Molina

    2008-01-01

    In plants, resistance to necrotrophic pathogens depends on the interplay between different hormone systems, such as those regulated by salicylic acid (SA), jasmonic acid (JA), ethylene, and abscisic acid. Repression of auxin signaling by the SA pathway was recently shown to contribute to antibacterial resistance. Here, we demonstrate that Arabidopsis auxin signaling mutants axrl, axr2, and axr6 that have defects in the auxin-stimulated SCF (Skpl-Cullin-F-box) ubiquitination pathway exhibit increased susceptibility to the necrotrophic fungi Plectosphaerella cucumerina and Botrytis cinerea. Also, stabilization of the auxin transcriptional repressor AXR3 that is normally targeted for removal by the SCF-ubiquitin/proteasome machinery occurs upon P. cucumerina infection. Pharmacological inhibition of auxin transport or proteasome function each compromise necrotroph resistance of wild-type plants to a similar extent as in non-treated auxin response mutants. These results suggest that auxin signaling is important for resistance to the necrotrophic fungi P. cucumerina and B. cinerea. SGTlb (one of two Arabidopsis SGT1 genes encoding HSP90/HSC70 co-chaperones) promotes the functions of SCF E3-ubiquitin ligase complexes in auxin and JA responses and resistance conditioned by certain Resistance (R) genes to biotrophic pathogens. We find that sgtlb mutants are as resistant to P. cucumerina as wild-type plants. Conversely, auxin/SCF signaling mutants are uncompromised in RPP4-triggered resistance to the obligate biotrophic oomycete, Hyaloperonospora parasitica. Thus, the predominant action of SGTlb in R gene-conditioned resistance to oomycetes appears to be at a site other than assisting SCF E3-ubiquitin ligases. However, genetic additivity of sgtlb axr1 double mutants in susceptibility to H. parasitica suggests that SCF-mediated ubiquitination contributes to limiting biotrophic pathogen colonization once plant-pathogen compatibility is established.

  15. Role of the Mmr efflux pump in drug resistance in Mycobacterium tuberculosis.

    Science.gov (United States)

    Rodrigues, Liliana; Villellas, Cristina; Bailo, Rebeca; Viveiros, Miguel; Aínsa, José A

    2013-02-01

    Efflux pumps are membrane proteins capable of actively transporting a broad range of substrates from the cytoplasm to the exterior of the cell. Increased efflux activity in response to drug treatment may be the first step in the development of bacterial drug resistance. Previous studies showed that the efflux pump Mmr was significantly overexpressed in strains exposed to isoniazid. In the work to be described, we constructed mutants lacking or overexpressing Mmr in order to clarify the role of this efflux pump in the development of resistance to isoniazid and other drugs in M. tuberculosis. The mmr knockout mutant showed an increased susceptibility to ethidium bromide, tetraphenylphosphonium, and cetyltrimethylammonium bromide (CTAB). Overexpression of mmr caused a decreased susceptibility to ethidium bromide, acriflavine, and safranin O that was obliterated in the presence of the efflux inhibitors verapamil and carbonyl cyanide m-chlorophenylhydrazone. Isoniazid susceptibility was not affected by the absence or overexpression of mmr. The fluorometric method allowed the detection of a decreased efflux of ethidium bromide in the knockout mutant, whereas the overexpressed strain showed increased efflux of this dye. This increased efflux activity was inhibited in the presence of efflux inhibitors. Under our experimental conditions, we have found that efflux pump Mmr is mainly involved in the susceptibility to quaternary compounds such as ethidium bromide and disinfectants such as CTAB. The contribution of this efflux pump to isoniazid resistance in Mycobacterium tuberculosis still needs to be further elucidated.

  16. 胆固醇逆转运的新途径:胆固醇经肠道直接排出体外?%Novel Pathway for Reverse Cholesterol Transport: Directly Transintestinal Cholesterol Efflux?

    Institute of Scientific and Technical Information of China (English)

    司艳红; 商战平; 秦树存

    2011-01-01

    过多的胆固醇沉积在动脉壁对机体极为有害,可以引起动脉粥样硬化甚至心血管疾病.而胆固醇逆转运(reverse cholesterol transport,RCT)可以逆转此过程.传统的RCT是指胆固醇由外周组织转运回肝脏进行再循环或以胆汁酸的形式随粪便排出体外的过程,此过程受多种因子调控.近几年研究发现,胆固醇还可由血经过肠道直接分泌(transintestinal cholesterol efflux,TICE)通路随粪便排出体外,此过程对外界刺激更敏感.RCT已经成为防治动脉粥样硬化研究的新靶点,TICE有可能成为更有效的RCT调控通路.%The presence of excess cholesterol in cells, and in particular in macrophages in the arterial vessel wall, might be harmful. The accumulation of cholesterol in arteries can lead to atherosclerosis, and in turn, to other cardiovascular diseases. Reverse cholesterol transport ( RCT) can reverse this processing. By the pathway of classic RCT, excess cholesterol is collected from peripheral tissues and delivered back to the liver and hepatobiliary tract for excretion via the feces. This processing could be affected by many factors. For a long time this removal via the hepatobiliary secretion was considered to be the sole route involved in the RCT. In the last few years it has become evident that a nonbiliary cholesterol secretion pathway exists. That is the transintestinal cholesterol efflux ( TICE) pathway. Moreover, recent studies have shown that TICE is also sensitive to stimulation. Therefore, RCT, especially TICE, was seen as a suitable and approachable target for the development of drugs aimed at the prevention of atherosclerosis.

  17. Role of Human Breast Cancer Related Protein versus P-Glycoprotein as an Efflux Transporter for Benzylpenicillin: Potential Importance at the Blood-Brain Barrier.

    Directory of Open Access Journals (Sweden)

    Yangfang Li

    Full Text Available While the blood-brain barrier (BBB protects the brain by controlling the access of solutes and toxic substances to brain, it also limits drug entry to treat central nervous system disorders. Many drugs are substrates for ATP-binding cassette (ABC transporters at the BBB that limit their entry into the brain. The role of those transporters in limiting the entry of the widely prescribed therapeutic, benzylpenicillin, has produced conflicting results. This study investigated the possible potential involvement of P-glycoprotein (P-gp and breast cancer resistance protein (BCRP, two ABC transporters, in benzylpenicillin transport at BBB in human using MDCKII cells overexpressing those transporters as well as pharmacological inhibition. MDCKII cells overexpressing human BCRP (MDCKII-BCRP but not those overexpressing human P-gp (MDCKII-MDR cells had reduced [3H]benzylpenicillin uptake. Similarly, inhibiting BCRP increased [3H]benzylpenicillin uptake in MDCKII-BCRP cells, while inhibiting P-gp in MDCKII-MDR cells had no effect on uptake although there was evidence that benzylpenicillin is a substrate for canine P-gp. While inhibiting BCRP affected [3H]benzylpenicillin cell concentrations it did not affect transepithelial flux in MDCKII-BCRP cells. In summary, the results indicate that human BCRP and not human P-gp is involved in benzylpenicillin transport. However, targeting BCRP alone was not sufficient to alter transepithelial flux in MDCKII cells. Whether it would be sufficient to alter blood-to-brain flux at the human BBB remains to be investigated.

  18. Inhibition of auxin movement from the shoot into the root inhibits lateral root development in Arabidopsis

    Science.gov (United States)

    Reed, R. C.; Brady, S. R.; Muday, G. K.

    1998-01-01

    In roots two distinct polar movements of auxin have been reported that may control different developmental and growth events. To test the hypothesis that auxin derived from the shoot and transported toward the root controls lateral root development, the two polarities of auxin transport were uncoupled in Arabidopsis. Local application of the auxin-transport inhibitor naphthylphthalamic acid (NPA) at the root-shoot junction decreased the number and density of lateral roots and reduced the free indoleacetic acid (IAA) levels in the root and [3H]IAA transport into the root. Application of NPA to the basal half of or at several positions along the root only reduced lateral root density in regions that were in contact with NPA or in regions apical to the site of application. Lateral root development was restored by application of IAA apical to NPA application. Lateral root development in Arabidopsis roots was also inhibited by excision of the shoot or dark growth and this inhibition was reversible by IAA. Together, these results are consistent with auxin transport from the shoot into the root controlling lateral root development.

  19. The rice FISH BONE gene encodes a tryptophan aminotransferase, which affects pleiotropic auxin-related processes.

    Science.gov (United States)

    Yoshikawa, Takanori; Ito, Momoyo; Sumikura, Tsuyoshi; Nakayama, Akira; Nishimura, Takeshi; Kitano, Hidemi; Yamaguchi, Isomaro; Koshiba, Tomokazu; Hibara, Ken-Ichiro; Nagato, Yasuo; Itoh, Jun-Ichi

    2014-06-01

    Auxin is a fundamental plant hormone and its localization within organs plays pivotal roles in plant growth and development. Analysis of many Arabidopsis mutants that were defective in auxin biosynthesis revealed that the indole-3-pyruvic acid (IPA) pathway, catalyzed by the TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS (TAA) and YUCCA (YUC) families, is the major biosynthetic pathway of indole-3-acetic acid (IAA). In contrast, little information is known about the molecular mechanisms of auxin biosynthesis in rice. In this study, we identified a auxin-related rice mutant, fish bone (fib). FIB encodes an orthologue of TAA genes and loss of FIB function resulted in pleiotropic abnormal phenotypes, such as small leaves with large lamina joint angles, abnormal vascular development, small panicles, abnormal organ identity and defects in root development, together with a reduction in internal IAA levels. Moreover, we found that auxin sensitivity and polar transport activity were altered in the fib mutant. From these results, we suggest that FIB plays a pivotal role in IAA biosynthesis in rice and that auxin biosynthesis, transport and sensitivity are closely interrelated.

  20. Arsenic efflux governed by the arsenic resistance determinant of Staphylococcus aureus plasmid pI258.

    OpenAIRE

    Bröer, S; Ji, G.; Bröer, A; Silver, S

    1993-01-01

    The arsenic resistance operon of Staphylococcus aureus plasmid pI258 determined lowered net cellular uptake of 73As by an active efflux mechanism. Arsenite was exported from the cells; intracellular arsenate was first reduced to arsenite and then transported out of the cells. Resistant cells showed lower accumulation of 73As originating from both arsenate and arsenite. Active efflux from cells loaded with arsenite required the presence of the plasmid-determined arsB gene. Efflux of arsenic or...

  1. Molecular response to imatinib & its correlation with mRNA expression levels of imatinib influx & efflux transporters in patients with chronic myeloid leukaemia in chronic phase

    Directory of Open Access Journals (Sweden)

    Hemant Malhotra

    2015-01-01

    Full Text Available Background & objectives: Imatinib is the standard first-line treatment for chronic myeloid leukaemia (CML patients. About 20 to 30 per cent patients develop resistance to imatinib and fail imatinib treatment. One of the mechanisms proposed is varying expression levels of the drug transporters. This study was aimed to determine the expression levels of imatinib transporter genes (OCT1, ABCB1, ABCG2 in CML patients and to correlate these levels with molecular response. Methods: Sixty three CML chronic phase patients who were on 400 mg/day imatinib for more than two years were considered for gene expression analysis study for OCT1, ABCB1 and ABCG2 genes. These were divided into responders and non-responders. The relative transcript expression levels of the three genes were compared between these two categories. The association between the expression values of these three genes was also determined. Results: No significant difference in the expression levels of OCT1, ABCB1 and ABCG2 was found between the two categories. The median transcript expression levels of OCT1, ABCB1 and ABCG2 genes in responders were 26.54, 10.78 and 0.64 versus 33.48, 7.09 and 0.53 in non-responders, respectively. A positive association was observed between the expression of the ABCB1 and ABCG2 transporter genes (r=0.407, P<0.05 while no association was observed between the expression of either of the ABC transporter genes with the OCT1 gene. Interpretation & conclusions: Our findings demonstrated that the mRNA expression levels of imatinib transporter genes were not correlated with molecular response in CML patients. Further studies need to be done on a large sample of CML patients to confirm these findings.

  2. Transcriptomic analysis reveals ethylene as stimulator and auxin as regulator of adventitious root formation in petunia cuttings

    Directory of Open Access Journals (Sweden)

    Uwe eDruege

    2014-09-01

    Full Text Available Adventitious root (AR formation in the stem base of cuttings is the basis for propagation of many plant species and petunia is used as model to study this developmental process. Following AR formation from 2 to 192 hours after excision (hpe of cuttings, transcriptome analysis by microarray revealed a change of the character of the rooting zone from stem base to root identity. The greatest shift in the number of differentially expressed genes was observed between 24 and 72 hpe, when the categories storage, mineral nutrient acquisition, anti-oxidative and secondary metabolism, and biotic stimuli showed a notable high number of induced genes. Analyses of phytohormone-related genes disclosed multifaceted changes of the auxin transport system, auxin conjugation and the auxin signal perception machinery indicating a reduction in auxin sensitivity and phase-specific responses of particular auxin-regulated genes. Genes involved in ethylene biosynthesis and action showed a more uniform pattern as a high number of respective genes were generally induced during the whole process of AR formation. The important role of ethylene for stimulating AR formation was demonstrated by the application of inhibitors of ethylene biosynthesis and perception as well as of the precursor aminocyclopropane-1-carboxylic acid, all changing the number and length of AR. A model is proposed showing the putative role of polar auxin transport and resulting auxin accumulation in initiation of subsequent changes in auxin homeostasis and signal perception with a particular role of Aux/IAA expression. These changes might in turn guide the entrance into the different phases of AR formation. Ethylene biosynthesis, which is stimulated by wounding and does probably also respond to other stresses and auxin, acts as important stimulator of AR formation probably via the expression of ethylene responsive transcription factor genes, whereas the timing of different phases seems to be controlled

  3. Prolonged Oral Administration of a Pan-Retinoic Acid Receptor Antagonist Inhibits Spermatogenesis in Mice With a Rapid Recovery and Changes in the Expression of Influx and Efflux Transporters.

    Science.gov (United States)

    Chung, Sanny S W; Wang, Xiangyuan; Wolgemuth, Debra J

    2016-04-01

    We have previously shown that oral administration of a pan-retinoic acid receptor antagonist in mice daily at 2.5 mg/kg for 4 weeks reversibly inhibited spermatogenesis, with no detectable side effects. To elucidate the lowest dose and the longest dosing regimen that inhibits spermatogenesis but results in complete restoration of fertility upon cessation of administration of the drug, we examined the effects of daily doses as low as 1.0 mg/kg with dosing periods of 4, 8, and 16 weeks. We observed 100% sterility in all regimens, with restoration of fertility upon cessation of the drug treatment even for as long as 16 weeks. There was no change in testosterone levels in these males and the progeny examined from 2 of the recovered males were healthy and fertile, with normal testicular weight and testicular histology. Strikingly, a more rapid recovery, as assessed by mating studies, was observed at the lower dose and longer dosing periods. Insight into possible mechanisms underlying this rapid recovery was obtained at 2 levels. First, histological examination revealed that spermatogenesis was not as severely disrupted at the lower dose and with the longer treatment regimens. Second, gene expression analysis revealed that the more rapid recovery may involve the interplay of ATP-binding cassette efflux and solute carrier influx transporters in the testes.

  4. The glucosinolate breakdown product indole-3-carbinol acts as an auxin antagonist in roots of Arabidopsis thaliana.

    Science.gov (United States)

    Katz, Ella; Nisani, Sophia; Yadav, Brijesh S; Woldemariam, Melkamu G; Shai, Ben; Obolski, Uri; Ehrlich, Marcelo; Shani, Eilon; Jander, Georg; Chamovitz, Daniel A

    2015-05-01

    The glucosinolate breakdown product indole-3-carbinol functions in cruciferous vegetables as a protective agent against foraging insects. While the toxic and deterrent effects of glucosinolate breakdown on herbivores and pathogens have been studied extensively, the secondary responses that are induced in the plant by indole-3-carbinol remain relatively uninvestigated. Here we examined the hypothesis that indole-3-carbinol plays a role in influencing plant growth and development by manipulating auxin signaling. We show that indole-3-carbinol rapidly and reversibly inhibits root elongation in a dose-dependent manner, and that this inhibition is accompanied by a loss of auxin activity in the root meristem. A direct interaction between indole-3-carbinol and the auxin perception machinery was suggested, as application of indole-3-carbinol rescues auxin-induced root phenotypes. In vitro and yeast-based protein interaction studies showed that indole-3-carbinol perturbs the auxin-dependent interaction of Transport Inhibitor Response (TIR1) with auxin/3-indoleacetic acid (Aux/IAAs) proteins, further supporting the possibility that indole-3-carbinol acts as an auxin antagonist. The results indicate that chemicals whose production is induced by herbivory, such as indole-3-carbinol, function not only to repel herbivores, but also as signaling molecules that directly compete with auxin to fine tune plant growth and development.

  5. Role of Auxin in orchid development.

    Science.gov (United States)

    Darling-Novak, Stacey; Luna, Lila J; Gamage, Roshan N

    2014-08-25

    Auxin's capacity to regulate aspects of plant development has been well characterized in model plant systems. In contrast, orchids have received considerably less attention, but the realization that many orchid species are endangered has led to culture-based propagation studies which have unveiled some functions for auxin in this system. This mini-review summarizes the many auxin-mediated developmental responses in orchids that are consistent with model systems; however, it also brings to the forefront auxin responses that are unique to orchid development, namely protocorm formation and ovary/ovule maturation. With regard to shoot establishment, we also assess auxin's involvement in orchid germination, PLB formation, and somatic embryogenesis. Further, it makes evident that auxin flow during germination of the undifferentiated, but mature, orchid embryo mirrors late embryogenesis of typical angiosperms. Also discussed is the use of orchid protocorms in future phytohormone studies to better understand the mechanisms behind meristem formation and organogenesis.

  6. Overcoming the heterologous bias: An in vivo functional analysis of multidrug efflux transporter, CgCdr1p in matched pair clinical isolates of Candida glabrata

    Energy Technology Data Exchange (ETDEWEB)

    Puri, Nidhi; Manoharlal, Raman; Sharma, Monika [Membrane Biology Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi 110 067 (India); Sanglard, Dominique [Institut de Microbiologie, Centre Hospitalier Universitaire Vaudois, 1011 Lausanne (Switzerland); Prasad, Rajendra, E-mail: rp47jnu@gmail.com [Membrane Biology Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi 110 067 (India)

    2011-01-07

    Research highlights: {yields} First report to demonstrate an in vivo expression system of an ABC multidrug transporter CgCdr1p of C. glabrata. {yields} First report on the structure and functional characterization of CgCdr1p. {yields} Functional conservation of divergent but typical residues of CgCdr1p. {yields} CgCdr1p elicits promiscuity towards substrates and has a large drug binding pocket with overlapping specificities. -- Abstract: We have taken advantage of the natural milieu of matched pair of azole sensitive (AS) and azole resistant (AR) clinical isolates of Candida glabrata for expressing its major ABC multidrug transporter, CgCdr1p for structure and functional analysis. This was accomplished by tagging a green fluorescent protein (GFP) downstream of ORF of CgCDR1 and integrating the resultant fusion protein at its native chromosomal locus in AS and AR backgrounds. The characterization confirmed that in comparison to AS isolate, CgCdr1p-GFP was over-expressed in AR isolates due to its hyperactive native promoter and the GFP tag did not affect its functionality in either construct. We observed that in addition to Rhodamine 6 G (R6G) and Fluconazole (FLC), a recently identified fluorescent substrate of multidrug transporters Nile Red (NR) could also be expelled by CgCdr1p. Competition assays with these substrates revealed the presence of overlapping multiple drug binding sites in CgCdr1p. Point mutations employing site directed mutagenesis confirmed that the role played by unique amino acid residues critical to ATP catalysis and localization of ABC drug transporter proteins are well conserved in C. glabrata as in other yeasts. This study demonstrates a first in vivo novel system where over-expression of GFP tagged MDR transporter protein can be driven by its own hyperactive promoter of AR isolates. Taken together, this in vivo system can be exploited for the structure and functional analysis of CgCdr1p and similar proteins wherein the arte-factual concerns

  7. Control of Angiogenesis by AIBP-mediated Cholesterol Efflux

    Science.gov (United States)

    Fang, Longhou; Choi, Soo-Ho; Baek, Ji Sun; Liu, Chao; Almazan, Felicidad; Ulrich, Florian; Wiesner, Philipp; Taleb, Adam; Deer, Elena; Pattison, Jennifer; Torres-Vázquez, Jesús; Li, Andrew C.; Miller, Yury I.

    2013-01-01

    Cholesterol is a structural component of the cell, indispensable for normal cellular function, but its excess often leads to abnormal proliferation, migration, inflammatory responses and/or cell death. To prevent cholesterol overload, ATP-binding cassette (ABC) transporters mediate cholesterol efflux from the cells to apolipoprotein A-I (ApoA-I) and to the ApoA-I-containing high-density lipoprotein (HDL)1-3. Maintaining efficient cholesterol efflux is essential for normal cellular function4-6. However, the role of cholesterol efflux in angiogenesis and the identity of its local regulators are poorly understood. Here we show that ApoA-I binding protein (AIBP) accelerates cholesterol efflux from endothelial cells (EC) to HDL and thereby regulates angiogenesis. AIBP/HDL-mediated cholesterol depletion reduces lipid rafts, interferes with VEGFR2 dimerization and signaling, and inhibits VEGF-induced angiogenesis in vitro and mouse aortic neovascularization ex vivo. Remarkably, Aibp regulates the membrane lipid order in embryonic zebrafish vasculature and functions as a non-cell autonomous regulator of zebrafish angiogenesis. Aibp knockdown results in dysregulated sprouting/branching angiogenesis, while forced Aibp expression inhibits angiogenesis. Dysregulated angiogenesis is phenocopied in Abca1/Abcg1-deficient embryos, and cholesterol levels are increased in Aibp-deficient and Abca1/Abcg1-deficient embryos. Our findings demonstrate that secreted AIBP positively regulates cholesterol efflux from EC and that effective cholesterol efflux is critical for proper angiogenesis. PMID:23719382

  8. Altered localisation of the copper efflux transporters ATP7A and ATP7B associated with cisplatin resistance in human ovarian carcinoma cells

    Directory of Open Access Journals (Sweden)

    Reedijk Jan

    2008-06-01

    Full Text Available Abstract Background Copper homeostasis proteins ATP7A and ATP7B are assumed to be involved in the intracellular transport of cisplatin. The aim of the present study was to assess the relevance of sub cellular localisation of these transporters for acquired cisplatin resistance in vitro. For this purpose, localisation of ATP7A and ATP7B in A2780 human ovarian carcinoma cells and their cisplatin-resistant variant, A2780cis, was investigated. Methods Sub cellular localisation of ATP7A and ATP7B in sensitive and resistant cells was investigated using confocal fluorescence microscopy after immunohistochemical staining. Co-localisation experiments with a cisplatin analogue modified with a carboxyfluorescein-diacetate residue were performed. Cytotoxicity of the fluorescent cisplatin analogue in A2780 and A2780cis cells was determined using an MTT-based assay. The significance of differences was analysed using Student's t test or Mann-Whitney test as appropriate, p values of Results In the sensitive cells, both transporters are mainly localised in the trans-Golgi network, whereas they are sequestrated in more peripherally located vesicles in the resistant cells. Altered localisation of ATP7A and ATP7B in A2780cis cells is likely to be a consequence of major abnormalities in intracellular protein trafficking related to a reduced lysosomal compartment in this cell line. Changes in sub cellular localisation of ATP7A and ATP7B may facilitate sequestration of cisplatin in the vesicular structures of A2780cis cells, which may prevent drug binding to genomic DNA and thereby contribute to cisplatin resistance. Conclusion Our results indicate that alterations in sub cellular localisation of transport proteins may contribute to cisplatin resistance in vitro. Investigation of intracellular protein localisation in primary tumour cell cultures and tumour tissues may help to develop markers of clinically relevant cisplatin resistance. Detection of resistant tumours

  9. A mass spectrometry-based assay for improved quantitative measurements of efflux pump inhibition.

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    Adam R Brown

    Full Text Available Bacterial efflux pumps are active transport proteins responsible for resistance to selected biocides and antibiotics. It has been shown that production of efflux pumps is up-regulated in a number of highly pathogenic bacteria, including methicillin resistant Staphylococcus aureus. Thus, the identification of new bacterial efflux pump inhibitors is a topic of great interest. Existing assays to evaluate efflux pump inhibitory activity rely on fluorescence by an efflux pump substrate. When employing these assays to evaluate efflux pump inhibitory activity of plant extracts and some purified compounds, we observed severe optical interference that gave rise to false negative results. To circumvent this problem, a new mass spectrometry-based method was developed for the quantitative measurement of bacterial efflux pump inhibition. The assay was employed to evaluate efflux pump inhibitory activity of a crude extract of the botanical Hydrastis Canadensis, and to compare the efflux pump inhibitory activity of several pure flavonoids. The flavonoid quercetin, which appeared to be completely inactive with a fluorescence-based method, showed an IC50 value of 75 μg/mL with the new method. The other flavonoids evaluated (apigenin, kaempferol, rhamnetin, luteolin, myricetin, were also active, with IC50 values ranging from 19 μg/mL to 75 μg/mL. The assay described herein could be useful in future screening efforts to identify efflux pump inhibitors, particularly in situations where optical interference precludes the application of methods that rely on fluorescence.

  10. Efflux as a mechanism of antimicrobial drug resistance in clinical relevant microorganisms: the role of efflux inhibitors.

    Science.gov (United States)

    Willers, Clarissa; Wentzel, Johannes Frederik; du Plessis, Lissinda Hester; Gouws, Chrisna; Hamman, Josias Hendrik

    2017-01-01

    Microbial resistance against antibiotics is a serious threat to the effective treatment of infectious diseases. Several mechanisms exist through which microorganisms can develop resistance against antimicrobial drugs, of which the overexpression of genes to produce efflux pumps is a major concern. Several efflux transporters have been identified in microorganisms, which infer resistance against specific antibiotics and even multidrug resistance. Areas covered: This paper focuses on microbial resistance against antibiotics by means of the mechanism of efflux and gives a critical overview of studies conducted to overcome this problem by combining efflux pump inhibitors with antibiotics. Information was obtained from a literature search done with MEDLINE, Pubmed, Scopus, ScienceDirect, OneSearch and EBSCO host. Expert opinion: Efflux as a mechanism of multidrug resistance has presented a platform for improved efficacy against resistant microorganisms by co-administration of efflux pump inhibitors with antimicrobial agents. Although proof of concept has been shown for this approach with in vitro experiments, further research is needed to develop more potent inhibitors with low toxicity which is clinically effective.

  11. Ezetimibe Promotes Brush Border Membrane-to-Lumen Cholesterol Efflux in the Small Intestine.

    Directory of Open Access Journals (Sweden)

    Takanari Nakano

    Full Text Available Ezetimibe inhibits Niemann-Pick C1-like 1 (NPC1L1, an apical membrane cholesterol transporter of enterocytes, thereby reduces intestinal cholesterol absorption. This treatment also increases extrahepatic reverse cholesterol transport via an undefined mechanism. To explore this, we employed a trans-intestinal cholesterol efflux (TICE assay, which directly detects circulation-to-intestinal lumen 3H-cholesterol transit in a cannulated jejunal segment, and found an increase of TICE by 45%. To examine whether such increase in efflux occurs at the intestinal brush border membrane(BBM-level, we performed luminal perfusion assays, similar to TICE but the jejunal wall was labelled with orally-given 3H-cholesterol, and determined elevated BBM-to-lumen cholesterol efflux by 3.5-fold with ezetimibe. Such increased efflux probably promotes circulation-to-lumen cholesterol transit eventually; thus increases TICE. Next, we wondered how inhibition of NPC1L1, an influx transporter, resulted in increased efflux. When we traced orally-given 3H-cholesterol in mice, we found that lumen-to-BBM 3H-cholesterol transit was rapid and less sensitive to ezetimibe treatment. Comparison of the efflux and fractional cholesterol absorption revealed an inverse correlation, indicating the efflux as an opposite-regulatory factor for cholesterol absorption efficiency and counteracting to the naturally-occurring rapid cholesterol influx to the BBM. These suggest that the ezetimibe-stimulated increased efflux is crucial in reducing cholesterol absorption. Ezetimibe-induced increase in cholesterol efflux was approximately 2.5-fold greater in mice having endogenous ATP-binding cassette G5/G8 heterodimer, the major sterol efflux transporter of enterocytes, than the knockout counterparts, suggesting that the heterodimer confers additional rapid BBM-to-lumen cholesterol efflux in response to NPC1L1 inhibition. The observed framework for intestinal cholesterol fluxes may provide ways to

  12. Ezetimibe Promotes Brush Border Membrane-to-Lumen Cholesterol Efflux in the Small Intestine.

    Science.gov (United States)

    Nakano, Takanari; Inoue, Ikuo; Takenaka, Yasuhiro; Ono, Hiraku; Katayama, Shigehiro; Awata, Takuya; Murakoshi, Takayuki

    2016-01-01

    Ezetimibe inhibits Niemann-Pick C1-like 1 (NPC1L1), an apical membrane cholesterol transporter of enterocytes, thereby reduces intestinal cholesterol absorption. This treatment also increases extrahepatic reverse cholesterol transport via an undefined mechanism. To explore this, we employed a trans-intestinal cholesterol efflux (TICE) assay, which directly detects circulation-to-intestinal lumen 3H-cholesterol transit in a cannulated jejunal segment, and found an increase of TICE by 45%. To examine whether such increase in efflux occurs at the intestinal brush border membrane(BBM)-level, we performed luminal perfusion assays, similar to TICE but the jejunal wall was labelled with orally-given 3H-cholesterol, and determined elevated BBM-to-lumen cholesterol efflux by 3.5-fold with ezetimibe. Such increased efflux probably promotes circulation-to-lumen cholesterol transit eventually; thus increases TICE. Next, we wondered how inhibition of NPC1L1, an influx transporter, resulted in increased efflux. When we traced orally-given 3H-cholesterol in mice, we found that lumen-to-BBM 3H-cholesterol transit was rapid and less sensitive to ezetimibe treatment. Comparison of the efflux and fractional cholesterol absorption revealed an inverse correlation, indicating the efflux as an opposite-regulatory factor for cholesterol absorption efficiency and counteracting to the naturally-occurring rapid cholesterol influx to the BBM. These suggest that the ezetimibe-stimulated increased efflux is crucial in reducing cholesterol absorption. Ezetimibe-induced increase in cholesterol efflux was approximately 2.5-fold greater in mice having endogenous ATP-binding cassette G5/G8 heterodimer, the major sterol efflux transporter of enterocytes, than the knockout counterparts, suggesting that the heterodimer confers additional rapid BBM-to-lumen cholesterol efflux in response to NPC1L1 inhibition. The observed framework for intestinal cholesterol fluxes may provide ways to modulate the flux

  13. Dynamic control of auxin distribution imposes a bilateral-to-radial symmetry switch during gynoecium development.

    Science.gov (United States)

    Moubayidin, Laila; Ostergaard, Lars

    2014-11-17

    Symmetry formation is a remarkable feature of biological life forms associated with evolutionary advantages and often with great beauty. Several examples exist in which organisms undergo a transition in symmetry during development. Such transitions are almost exclusively in the direction from radial to bilateral symmetry. Here, we describe the dynamics of symmetry establishment during development of the Arabidopsis gynoecium. We show that the apical style region undergoes an unusual transition from a bilaterally symmetric stage ingrained in the gynoecium due to its evolutionary origin to a radially symmetric structure. We also identify two transcription factors, INDEHISCENT and SPATULA, that are both necessary and sufficient for the radialization process. Our work furthermore shows that these two transcription factors control style symmetry by directly regulating auxin distribution. Establishment of specific auxin-signaling foci and the subsequent development of a radially symmetric auxin ring at the style are required for the transition to radial symmetry, because genetic manipulations of auxin transport can either cause loss of radialization in a wild-type background or rescue mutants with radialization defects. Whereas many examples have described how auxin provides polarity and specific identity to cells in a range of developmental contexts, our data presented here demonstrate that auxin can also be recruited to impose uniform identity to a group of cells that are otherwise differentially programmed. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

  14. SIZ1 regulation of phosphate starvation-induced root architecture remodeling involves the control of auxin accumulation.

    Science.gov (United States)

    Miura, Kenji; Lee, Jiyoung; Gong, Qingqiu; Ma, Shisong; Jin, Jing Bo; Yoo, Chan Yul; Miura, Tomoko; Sato, Aiko; Bohnert, Hans J; Hasegawa, Paul M

    2011-02-01

    Phosphate (Pi) limitation causes plants to modulate the architecture of their root systems to facilitate the acquisition of Pi. Previously, we reported that the Arabidopsis (Arabidopsis thaliana) SUMO E3 ligase SIZ1 regulates root architecture remodeling in response to Pi limitation; namely, the siz1 mutations cause the inhibition of primary root (PR) elongation and the promotion of lateral root (LR) formation. Here, we present evidence that SIZ1 is involved in the negative regulation of auxin patterning to modulate root system architecture in response to Pi starvation. The siz1 mutations caused greater PR growth inhibition and LR development of seedlings in response to Pi limitation. Similar root phenotypes occurred if Pi-deficient wild-type seedlings were supplemented with auxin. N-1-Naphthylphthalamic acid, an inhibitor of auxin efflux activity, reduced the Pi starvation-induced LR root formation of siz1 seedlings to a level equivalent to that seen in the wild type. Monitoring of the auxin-responsive reporter DR5::uidA indicated that auxin accumulates in PR tips at early stages of the Pi starvation response. Subsequently, DR5::uidA expression was observed in the LR primordia, which was associated with LR elongation. The time-sequential patterning of DR5::uidA expression occurred earlier in the roots of siz1 as compared with the wild type. In addition, microarray analysis revealed that several other auxin-responsive genes, including genes involved in cell wall loosening and biosynthesis, were up-regulated in siz1 relative to wild-type seedlings in response to Pi starvation. Together, these results suggest that SIZ1 negatively regulates Pi starvation-induced root architecture remodeling through the control of auxin patterning.

  15. PHABULOSA Mediates an Auxin Signaling Loop to Regulate Vascular Patterning in Arabidopsis.

    Science.gov (United States)

    Müller, Christina Joy; Valdés, Ana Elisa; Wang, Guodong; Ramachandran, Prashanth; Beste, Lisa; Uddenberg, Daniel; Carlsbecker, Annelie

    2016-02-01

    Plant vascular tissues, xylem and phloem, differentiate in distinct patterns from procambial cells as an integral transport system for water, sugars, and signaling molecules. Procambium formation is promoted by high auxin levels activating class III homeodomain leucine zipper (HD-ZIP III) transcription factors (TFs). In the root of Arabidopsis (Arabidopsis thaliana), HD-ZIP III TFs dose-dependently govern the patterning of the xylem axis, with higher levels promoting metaxylem cell identity in the central axis and lower levels promoting protoxylem at its flanks. It is unclear, however, by what mechanisms the HD-ZIP III TFs control xylem axis patterning. Here, we present data suggesting that an important mechanism is their ability to moderate the auxin response. We found that changes in HD-ZIP III TF levels affect the expression of genes encoding core auxin response molecules. We show that one of the HD-ZIP III TFs, PHABULOSA, directly binds the promoter of both MONOPTEROS (MP)/AUXIN RESPONSE FACTOR5, a key factor in vascular formation, and IAA20, encoding an auxin/indole acetic acid protein that is stable in the presence of auxin and able to interact with and repress MP activity. The double mutant of IAA20 and its closest homolog IAA30 forms ectopic protoxylem, while overexpression of IAA30 causes discontinuous protoxylem and occasional ectopic metaxylem, similar to a weak loss-of-function mp mutant. Our results provide evidence that HD-ZIP III TFs directly affect the auxin response and mediate a feed-forward loop formed by MP and IAA20 that may focus and stabilize the auxin response during vascular patterning and the differentiation of xylem cell types.

  16. [Efflux systems in Serratia marcescens].

    Science.gov (United States)

    Mardanova, A M; Bogomol'naia, L M; Romanova, Iu D; Sharipova, M R

    2014-01-01

    A widespread bacterium Serratia marcescens (family Enterobacteriaceae) is an opportunistic and exhibits multiple drug resistance. Active removal of antibiotics and other antimicrobials from pathogen and exhibits multiple drug resistance. Active removal of antibiotics and other antimicrobials from the cells by efflux systems is one of the mechanisms responsible for microbial resistance to these compounds. Among enterobacteria, efflux systems of Escherichia coli and Salmonella enterica var. Typhimurium have been studied most extensively. Few efflux systems that belong to different families have been reported for S. marcescens. In this review, we analyzed available literature about S. marcescens efflux systems and carried out the comparative analysis of the genes encoding the RND type systems in different Serratia species and in other enterobacteria. Bioinformatical analysis of the S. marcescens genome allowed us to identify the previously unknown efflux systems based on their homology with the relevant E. coli genes. Identification of additional efflux systems in S. marcescens genome will promote our understanding of physiology of these bacteria, will detect new molecular mechanisms of resistance and will reveal their resistance potential.

  17. Nongenomic mechanisms of physiological estrogen-mediated dopamine efflux

    Directory of Open Access Journals (Sweden)

    Watson Cheryl S

    2009-06-01

    Full Text Available Abstract Background Neurological diseases and neuropsychiatric disorders that vary depending on female life stages suggest that sex hormones may influence the function of neurotransmitter regulatory machinery such as the dopamine transporter (DAT. Results In this study we tested the rapid nongenomic effects of several physiological estrogens [estradiol (E2, estrone (E1, and estriol (E3] on dopamine efflux via the DAT in a non-transfected, NGF-differentiated, rat pheochromocytoma (PC12 cell model that expresses membrane estrogen receptors (ERs α, β, and GPR30. We examined kinase, ionic, and physical interaction mechanisms involved in estrogenic regulation of the DAT function. E2-mediated dopamine efflux is DAT-specific and not dependent on extracellular Ca2+-mediated exocytotic release from vesicular monoamine transporter vesicles (VMATs. Using kinase inhibitors we also showed that E2-mediated dopamine efflux is dependent on protein kinase C and MEK activation, but not on PI3K or protein kinase A. In plasma membrane there are ligand-independent associations of ERα and ERβ (but not GPR30 with DAT. Conditions which cause efflux (a 9 min 10-9 M E2 treatment cause trafficking of ERα (stimulatory to the plasma membrane and trafficking of ERβ (inhibitory away from the plasma membrane. In contrast, E1 and E3 can inhibit efflux with a nonmonotonic dose pattern, and cause DAT to leave the plasma membrane. Conclusion Such mechanisms explain how gender biases in some DAT-dependent diseases can occur.

  18. Nongenomic mechanisms of physiological estrogen-mediated dopamine efflux.

    Science.gov (United States)

    Alyea, Rebecca A; Watson, Cheryl S

    2009-06-16

    Neurological diseases and neuropsychiatric disorders that vary depending on female life stages suggest that sex hormones may influence the function of neurotransmitter regulatory machinery such as the dopamine transporter (DAT). In this study we tested the rapid nongenomic effects of several physiological estrogens [estradiol (E2), estrone (E1), and estriol (E3)] on dopamine efflux via the DAT in a non-transfected, NGF-differentiated, rat pheochromocytoma (PC12) cell model that expresses membrane estrogen receptors (ERs) alpha, beta, and GPR30. We examined kinase, ionic, and physical interaction mechanisms involved in estrogenic regulation of the DAT function. E2-mediated dopamine efflux is DAT-specific and not dependent on extracellular Ca2+-mediated exocytotic release from vesicular monoamine transporter vesicles (VMATs). Using kinase inhibitors we also showed that E2-mediated dopamine efflux is dependent on protein kinase C and MEK activation, but not on PI3K or protein kinase A. In plasma membrane there are ligand-independent associations of ERalpha and ERbeta (but not GPR30) with DAT. Conditions which cause efflux (a 9 min 10(-9) M E2 treatment) cause trafficking of ERalpha (stimulatory) to the plasma membrane and trafficking of ERbeta (inhibitory) away from the plasma membrane. In contrast, E1 and E3 can inhibit efflux with a nonmonotonic dose pattern, and cause DAT to leave the plasma membrane. Such mechanisms explain how gender biases in some DAT-dependent diseases can occur.

  19. Gravitropism: lateral thinking in auxin transport.

    Science.gov (United States)

    Moore, Ian

    2002-07-09

    Plant stems and roots orient themselves with respect to directional illumination and gravity by differential growth on either side of the organ. A model formulated in the 1930s proposed that tropic growth curvature arose from growth hormone redistribution. Studies with Arabidopsis are beginning to reveal the mechanism.

  20. The role of the polymorphic efflux transporter P-glycoprotein on the brain accumulation of d-methylphenidate and d-amphetamine.

    Science.gov (United States)

    Zhu, Hao-Jie; Wang, Jun-Sheng; DeVane, C Lindsay; Williard, Robin L; Donovan, Jennifer L; Middaugh, Lawrence D; Gibson, Brian B; Patrick, Kennerly S; Markowitz, John S

    2006-07-01

    The psychostimulant medications methylphenidate (MPH) and amphetamine (AMP), available in various ratios or enantiopure formulations of their respective active dextrorotary isomers, constitute the majority of agents used in the treatment of attention-deficit/hyperactivity disorder (ADHD). Substantial interindividual variability occurs in their pharmacokinetics and tolerability. Little is known regarding the potential role of drug transporters such as P-glycoprotein (P-gp) in psychostimulant pharmacokinetics and response. Therefore, experiments were carried out in P-gp knockout (KO) mice versus wild-type (WT) mice after intraperitoneal dosing (2.5 mg/kg) of d-MPH or (3.0 mg/kg) of d-AMP. After the administration of each psychostimulant, locomotor activity was assessed at 30-min intervals for 2 h. Total brain-to-plasma drug concentration ratios were determined at 10-, 30-, and 80-min postdosing time-points. The results showed no statistically supported genotypic difference in d-AMP-induced locomotor activity stimulation or in brain-to-plasma ratio of d-AMP. As for d-MPH, the P-gp KO mice had 33% higher brain concentrations (p gp compared with that for WT mice. These data indicate that P-gp has no apparent effect on the pharmacokinetics and pharmacodynamics of d-AMP. In addition, d-MPH is a relatively weak P-gp substrate, and its entry into the brain may be limited by P-gp. Furthermore, the mechanism by which d-MPH-induced locomotor activity was attenuated in P-gp KO mice remains to be elucidated.

  1. Centrifuge-induced hypergravity: [ 3H]GABA and L-[ 14C]glutamate uptake, exocytosis and efflux mediated by high-affinity, sodium-dependent transporters

    Science.gov (United States)

    Borisova, T. A.; Himmelreich, N. H.

    The effects of centrifuge-induced hypergravity on the presynaptic events have been investigated in order to provide further insight into regulation of glutamate and GABA neurotransmission and correlation between excitatory and inhibitory responses under artificial gravity conditions. Exposure of animals to hypergravity (centrifugation of rats at 10 G for 1 h) has been found to cause changes in the synaptic processes of brain, in particular neurotransmitter release and uptake in rat brain synaptosomes. Hypergravity loading resulted in more than two-fold enhancement of GABA transporter activity ( Vmax increased from 1.4 ± 0.3 nmol/min/mg of protein in the control group to 3.3 ± 0.59 nmol/min/mg of protein for the animals exposed to hypergravity ( P ⩽ 0.05)). The maximal velocity of L-[ 14C]glutamate uptake decreased from 12.5 ± 3.2 to 5.6 ± 0.9 nmol/min/mg of protein under artificial gravity conditions. Depolarization-evoked exocytotic release of the neurotransmitters has also changed in response to hypergravity. It increased for GABA (7.2 ± 0.54% and 11.74 ± 1.2% of total accumulated label for control and hypergravity, respectively ( P ⩽ 0.05)), but reduced for glutamate (14.4 ± 0.7% and 6.2 ± 1.9%, for control and hypergravity, respectively). Thus, comparative analysis of the neurotransmitter uptake and release has demonstrated that short-term centrifuge-induced 10 G hypergravity loading intensified inhibitory and attenuated excitatory processes in nerve terminals. The activation or reduction of neurotransmitter uptake appeared to be coupled with similarly directed alterations of the neurotransmitter release.

  2. Auxin regulation of cell polarity in plants.

    Science.gov (United States)

    Pan, Xue; Chen, Jisheng; Yang, Zhenbiao

    2015-12-01

    Auxin is well known to control pattern formation and directional growth at the organ/tissue levels via the nuclear TIR1/AFB receptor-mediated transcriptional responses. Recent studies have expanded the arena of auxin actions as a trigger or key regulator of cell polarization and morphogenesis. These actions require non-transcriptional responses such as changes in the cytoskeleton and vesicular trafficking, which are commonly regulated by ROP/Rac GTPase-dependent pathways. These findings beg for the question about the nature of auxin receptors that regulate these responses and renew the interest in ABP1 as a cell surface auxin receptor, including the work showing auxin-binding protein 1 (ABP1) interacts with the extracellular domain of the transmembrane kinase (TMK) receptor-like kinases in an auxin-dependent manner, as well as the debate on this auxin binding protein discovered about 40 years ago. This review highlights recent work on the non-transcriptional auxin signaling mechanisms underscoring cell polarity and shape formation in plants.

  3. Identification of efflux proteins using efficient radial basis function networks with position-specific scoring matrices and biochemical properties.

    Science.gov (United States)

    Ou, Yu-Yen; Chen, Shu-An; Chang, Yun-Min; Velmurugan, Devadasan; Fukui, Kazuhiko; Michael Gromiha, M

    2013-09-01

    Efflux proteins are membrane proteins, which are involved in the transportation of multidrugs. The annotation of efflux proteins in genomic sequences would aid to understand the function. Although the percentage of membrane proteins in genomes is estimated to be 25-30%, there is no information about the content of efflux proteins. For annotating such class of proteins it is necessary to develop a reliable method to identify efflux proteins from amino acid sequence information. In this work, we have developed a method based on radial basis function networks using position specific scoring matrices (PSSM) and amino acid properties. We noticed that the C-terminal domain of efflux proteins contain vital information for discrimination. Our method showed an accuracy of 78 and 92% in discriminating efflux proteins from transporters and membrane proteins, respectively using fivefold cross-validation. We utilized our method for annotating the genomes E. coli and P. aeruginosa and it predicted 8.7 and 9.2% of proteins as efflux proteins in these genomes, respectively. The predicted efflux proteins have been compared with available experimental data and we observed a very good agreement between them. Further, we developed a web server for classifying efflux proteins and it is freely available at http://rbf.bioinfo.tw/∼sachen/EFFLUXpredict/Efflux-RBF.php. We suggest that our method could be an effective tool for annotating efflux proteins in genomic sequences.

  4. Characterization of auxin-binding proteins from zucchini plasma membrane

    Science.gov (United States)

    Hicks, G. R.; Rice, M. S.; Lomax, T. L.

    1993-01-01

    We have previously identified two auxin-binding polypeptides in plasma membrane (PM) preparations from zucchini (Cucurbita pepo L.) (Hicks et al. 1989, Proc. Natl. Acad. Sci. USA 86, 4948-4952). These polypeptides have molecular weights of 40 kDa and 42 kDa and label specifically with the photoaffinity auxin analog 5-N3-7-3H-IAA (azido-IAA). Azido-IAA permits both the covalent and radioactive tagging of auxin-binding proteins and has allowed us to characterize further the 40-kDa and 42-kDa polypeptides, including the nature of their attachment to the PM, their relationship to each other, and their potential function. The azido-IAA-labeled polypeptides remain in the pelleted membrane fraction following high-salt and detergent washes, which indicates a tight and possibly integral association with the PM. Two-dimensional electrophoresis of partially purified azido-IAA-labeled protein demonstrates that, in addition to the major isoforms of the 40-kDa and 42-kDa polypeptides, which possess isoelectric points (pIs) of 8.2 and 7.2, respectively, several less abundant isoforms that display unique pIs are apparent at both molecular masses. Tryptic and chymotryptic digestion of the auxin-binding proteins indicates that the 40-kDa and 42-kDa polypeptides are closely related or are modifications of the same polypeptide. Phase extraction with the nonionic detergent Triton X-114 results in partitioning of the azido-IAA-labeled polypeptides into the aqueous (hydrophilic) phase. This apparently paradoxical behavior is also exhibited by certain integral membrane proteins that aggregate to form channels. The results of gel filtration indicate that the auxin-binding proteins do indeed aggregate strongly and that the polypeptides associate to form a dimer or multimeric complex in vivo. These characteristics are consistent with the hypothesis that the 40-kDa and 42-kDa polypeptides are subunits of a multimeric integral membrane protein which has an auxin-binding site, and which may

  5. Moderate alcohol consumption increases cholesterol efflux mediated by ABCA1

    NARCIS (Netherlands)

    Beulens, J.W.J.; Sierksma, A.; Tol, van A.; Fournier, C.

    2004-01-01

    Moderate alcohol consumption increases HDL cholesterol, which is involved in reverse cholesterol transport (RCT). The aim of this study was to investigate the effect of moderate alcohol consumption on cholesterol efflux, using J774 mouse macrophages and Fu5AH cells, and on other parameters in the RC

  6. Research Progresses on Auxin Response Factors

    Institute of Scientific and Technical Information of China (English)

    Hai-Bin Wei; Bai-Ming Cui; Yan-Li Ren; Juan-Hua Li; Wei-Bin Liao; Nan-Fei Xu; Ming Peng

    2006-01-01

    Auxin response factors (ARFs), a family of transcription factors, have been discovered recently. The ARFs bind specifically to the auxin response elements (AuxREs) within promoters of primary auxin responsive genes and function as activators or repressors. The ARFs contain three domains, namely a conserved Nterminal DNA-binding domain, a non-conserved middle region, and a conserved C-terminal dirnerization domain. The ARFs can form a protein complex with auxin/indoleacetic acid through homodimerization or heterodimerization. The particular protein-protein interaction may play a key role in modulating the expression of early auxin responsive genes. The identification of ARF mutations in Arabidopsis helps to demonstrate/dissect the function of ARFs in the normal growth and development of plants. Phylogenetic analysis also reveals some interesting protein evolution points in the ARF family.

  7. NCP1/AtMOB1A Plays Key Roles in Auxin-Mediated Arabidopsis Development.

    Science.gov (United States)

    Cui, Xiaona; Guo, Zhiai; Song, Lizhen; Wang, Yanli; Cheng, Youfa

    2016-03-01

    MOB1 protein is a core component of the Hippo signaling pathway in animals where it is involved in controlling tissue growth and tumor suppression. Plant MOB1 proteins display high sequence homology to animal MOB1 proteins, but little is known regarding their role in plant growth and development. Herein we report the critical roles of Arabidopsis MOB1 (AtMOB1A) in auxin-mediated development in Arabidopsis. We found that loss-of-function mutations in AtMOB1A completely eliminated the formation of cotyledons when combined with mutations in PINOID (PID), which encodes a Ser/Thr protein kinase that participates in auxin signaling and transport. We showed that atmob1a was fully rescued by its Drosophila counterpart, suggesting functional conservation. The atmob1a pid double mutants phenocopied several well-characterized mutant combinations that are defective in auxin biosynthesis or transport. Moreover, we demonstrated that atmob1a greatly enhanced several other known auxin mutants, suggesting that AtMOB1A plays a key role in auxin-mediated plant development. The atmob1a single mutant displayed defects in early embryogenesis and had shorter root and smaller flowers than wild type plants. AtMOB1A is uniformly expressed in embryos and suspensor cells during embryogenesis, consistent with its role in embryo development. AtMOB1A protein is localized to nucleus, cytoplasm, and associated to plasma membrane, suggesting that it plays roles in these subcellular localizations. Furthermore, we showed that disruption of AtMOB1A led to a reduced sensitivity to exogenous auxin. Our results demonstrated that AtMOB1A plays an important role in Arabidopsis development by promoting auxin signaling.

  8. Composite structure of auxin response elements.

    Science.gov (United States)

    Ulmasov, T; Liu, Z B; Hagen, G; Guilfoyle, T J

    1995-10-01

    The auxin-responsive soybean GH3 gene promoter is composed of multiple auxin response elements (AuxREs), and each AuxRE contributes incrementally to the strong auxin inducibility to the promoter. Two independent AuxREs of 25 bp (D1) and 32 bp (D4) contain the sequence TGTCTC. Results presented here show that the TGTCTC element in D1 and D4 is required but not sufficient for auxin inducibility in carrot protoplast transient expression assays. Additional nucleotides upstream of TGTCTC are also required for auxin inducibility. These upstream sequences showed constitutive activity and no auxin inducibility when part or all of the TGTCTC element was mutated or deleted. In D1, the constitutive element overlaps the 5' portion of TGTCTC; in D4, the constitutive element is separated from TGTCTC. An 11-bp element in D1, CCTCGTGTCTC, conferred auxin inducibility to a minimal cauliflower mosaic virus 35S promoter in transgenic tobacco seedlings as well as in carrot protoplasts (i.e., transient expression assays). Both constitutive elements bound specifically to plant nuclear proteins, and the constitutive element in D1 bound to a recombinant soybean basic leucine zipper transcription factor with G-box specificity. To demonstrate further the composite nature of AuxREs and the ability of the TGTCTC element to confer auxin inducibility, we created a novel AuxRE by placing a yeast GAL4 DNA binding site adjacent to the TGTCTC element. Expression of a GAL4-c-Rel transactivator in the presence of this novel AuxRE resulted in auxin-inducible expression. Our results indicate that at least some AuxREs have a composite structure consisting of a constitutive element adjacent to a conserved TGTCTC element that confers auxin inducibility.

  9. ArsP: a methylarsenite efflux permease.

    Science.gov (United States)

    Chen, Jian; Madegowda, Mahendra; Bhattacharjee, Hiranmoy; Rosen, Barry P

    2015-11-01

    Trivalent organoarsenic compounds are far more toxic than either pentavalent organoarsenicals or inorganic arsenite. Many microbes methylate inorganic arsenite (As(III)) to more toxic and carcinogenic methylarsenite (MAs(III)). Additionally, monosodium methylarsenate (MSMA or MAs(V)) has been used widely as an herbicide and is reduced by microbial communities to MAs(III). Roxarsone (3-nitro-4-hydroxybenzenearsonic acid) is a pentavalent aromatic arsenical that is used as antimicrobial growth promoter for poultry and swine, and its active form is the trivalent species Rox(III). A bacterial permease, ArsP, from Campylobacter jejuni, was recently shown to confer resistance to roxarsone. In this study, C. jejuni arsP was expressed in Escherichia coli and shown to confer resistance to MAs(III) and Rox(III) but not to inorganic As(III) or pentavalent organoarsenicals. Cells of E. coli expressing arsP did not accumulate trivalent organoarsenicals. Everted membrane vesicles from those cells accumulated MAs(III) > Rox(III) with energy supplied by NADH oxidation, reflecting efflux from cells. The vesicles did not transport As(III), MAs(V) or pentavalent roxarsone. Mutation or modification of the two conserved cysteine residues resulted in loss of transport activity, suggesting that they play a role in ArsP function. Thus, ArsP is the first identified efflux system specific for trivalent organoarsenicals.

  10. Halogenated auxins affect microtubules and root elongation in Lactuca sativa

    Science.gov (United States)

    Zhang, N.; Hasenstein, K. H.

    2000-01-01

    We studied the effect of 4,4,4-trifluoro-3-(indole-3-)butyric acid (TFIBA), a recently described root growth stimulator, and 5,6-dichloro-indole-3-acetic acid (DCIAA) on growth and microtubule (MT) organization in roots of Lactuca sativa L. DCIAA and indole-3-butyric acid (IBA) inhibited root elongation and depolymerized MTs in the cortex of the elongation zone, inhibited the elongation of stele cells, and promoted xylem maturation. Both auxins caused the plane of cell division to shift from anticlinal to periclinal. In contrast, TFIBA (100 micromolar) promoted elongation of primary roots by 40% and stimulated the elongation of lateral roots, even in the presence of IBA, the microtubular inhibitors oryzalin and taxol, or the auxin transport inhibitor naphthylphthalamic acid. However, TFIBA inhibited the formation of lateral root primordia. Immunostaining showed that TFIBA stabilized MTs orientation perpendicular to the root axis, doubled the cortical cell length, but delayed xylem maturation. The data indicate that the auxin-induced inhibition of elongation and swelling of roots results from reoriented phragmoplasts, the destabilization of MTs in elongating cells, and promotion of vessel formation. In contrast, TFIBA induced promotion of root elongation by enhancing cell length, prolonging transverse MT orientation, delaying cell and xylem maturation.

  11. Halogenated auxins affect microtubules and root elongation in Lactuca sativa

    Science.gov (United States)

    Zhang, N.; Hasenstein, K. H.

    2000-01-01

    We studied the effect of 4,4,4-trifluoro-3-(indole-3-)butyric acid (TFIBA), a recently described root growth stimulator, and 5,6-dichloro-indole-3-acetic acid (DCIAA) on growth and microtubule (MT) organization in roots of Lactuca sativa L. DCIAA and indole-3-butyric acid (IBA) inhibited root elongation and depolymerized MTs in the cortex of the elongation zone, inhibited the elongation of stele cells, and promoted xylem maturation. Both auxins caused the plane of cell division to shift from anticlinal to periclinal. In contrast, TFIBA (100 micromolar) promoted elongation of primary roots by 40% and stimulated the elongation of lateral roots, even in the presence of IBA, the microtubular inhibitors oryzalin and taxol, or the auxin transport inhibitor naphthylphthalamic acid. However, TFIBA inhibited the formation of lateral root primordia. Immunostaining showed that TFIBA stabilized MTs orientation perpendicular to the root axis, doubled the cortical cell length, but delayed xylem maturation. The data indicate that the auxin-induced inhibition of elongation and swelling of roots results from reoriented phragmoplasts, the destabilization of MTs in elongating cells, and promotion of vessel formation. In contrast, TFIBA induced promotion of root elongation by enhancing cell length, prolonging transverse MT orientation, delaying cell and xylem maturation.

  12. Small-molecule auxin inhibitors that target YUCCA are powerful tools for studying auxin function.

    Science.gov (United States)

    Kakei, Yusuke; Yamazaki, Chiaki; Suzuki, Masashi; Nakamura, Ayako; Sato, Akiko; Ishida, Yosuke; Kikuchi, Rie; Higashi, Shouichi; Kokudo, Yumiko; Ishii, Takahiro; Soeno, Kazuo; Shimada, Yukihisa

    2015-11-01

    Auxin is essential for plant growth and development, this makes it difficult to study the biological function of auxin using auxin-deficient mutants. Chemical genetics have the potential to overcome this difficulty by temporally reducing the auxin function using inhibitors. Recently, the indole-3-pyruvate (IPyA) pathway was suggested to be a major biosynthesis pathway in Arabidopsis thaliana L. for indole-3-acetic acid (IAA), the most common member of the auxin family. In this pathway, YUCCA, a flavin-containing monooxygenase (YUC), catalyzes the last step of conversion from IPyA to IAA. In this study, we screened effective inhibitors, 4-biphenylboronic acid (BBo) and 4-phenoxyphenylboronic acid (PPBo), which target YUC. These compounds inhibited the activity of recombinant YUC in vitro, reduced endogenous IAA content, and inhibited primary root elongation and lateral root formation in wild-type Arabidopsis seedlings. Co-treatment with IAA reduced the inhibitory effects. Kinetic studies of BBo and PPBo showed that they are competitive inhibitors of the substrate IPyA. Inhibition constants (Ki ) of BBo and PPBo were 67 and 56 nm, respectively. In addition, PPBo did not interfere with the auxin response of auxin-marker genes when it was co-treated with IAA, suggesting that PPBo is not an inhibitor of auxin sensing or signaling. We propose that these compounds are a class of auxin biosynthesis inhibitors that target YUC. These small molecules are powerful tools for the chemical genetic analysis of auxin function.

  13. Functionally different PIN proteins control auxin flux during bulbil development in Agave tequilana.

    Science.gov (United States)

    Abraham Juárez, María Jazmín; Hernández Cárdenas, Rocío; Santoyo Villa, José Natzul; O'Connor, Devin; Sluis, Aaron; Hake, Sarah; Ordaz-Ortiz, José; Terry, Leon; Simpson, June

    2015-07-01

    In Agave tequilana, reproductive failure or inadequate flower development stimulates the formation of vegetative bulbils at the bracteoles, ensuring survival in a hostile environment. Little is known about the signals that trigger this probably unique phenomenon in agave species. Here we report that auxin plays a central role in bulbil development and show that the localization of PIN1-related proteins is consistent with altered auxin transport during this process. Analysis of agave transcriptome data led to the identification of the A. tequilana orthologue of PIN1 (denoted AtqPIN1) and a second closely related gene from a distinct clade reported as 'Sister of PIN1' (denoted AtqSoPIN1). Quantitative real-time reverse transcription-PCR (RT-qPCR) analysis showed different patterns of expression for each gene during bulbil formation, and heterologous expression of the A. tequilana PIN1 and SoPIN1 genes in Arabidopsis thaliana confirmed functional differences between these genes. Although no free auxin was detected in induced pedicel samples, changes in the levels of auxin precursors were observed. Taken as a whole, the data support the model that AtqPIN1 and AtqSoPIN1 have co-ordinated but distinct functions in relation to auxin transport during the initial stages of bulbil formation.

  14. Competitive inhibition of the luminal efflux by multidrug and toxin extrusions, but not basolateral uptake by organic cation transporter 2, is the likely mechanism underlying the pharmacokinetic drug-drug interactions caused by cimetidine in the kidney.

    Science.gov (United States)

    Ito, Sumito; Kusuhara, Hiroyuki; Yokochi, Miyu; Toyoshima, Junko; Inoue, Katsuhisa; Yuasa, Hiroaki; Sugiyama, Yuichi

    2012-02-01

    Cimetidine, an H₂ receptor antagonist, has been used to investigate the tubular secretion of organic cations in human kidney. We report a systematic comprehensive analysis of the inhibition potency of cimetidine for the influx and efflux transporters of organic cations [human organic cation transporter 1 (hOCT1) and hOCT2 and human multidrug and toxin extrusion 1 (hMATE1) and hMATE2-K, respectively]. Inhibition constants (K(i)) of cimetidine were determined by using five substrates [tetraethylammonium (TEA), metformin, 1-methyl-4-phenylpyridinium, 4-(4-(dimethylamino)styryl)-N-methylpyridinium, and m-iodobenzylguanidine]. They were 95 to 146 μM for hOCT2, providing at most 10% inhibition based on its clinically reported plasma unbound concentrations (3.6-7.8 μM). In contrast, cimetidine is a potent inhibitor of MATE1 and MATE2-K with K(i) values (μM) of 1.1 to 3.8 and 2.1 to 6.9, respectively. The same tendency was observed for mouse Oct1 (mOct1), mOct2, and mouse Mate1. Cimetidine showed a negligible effect on the uptake of metformin by mouse kidney slices at 20 μM. Cimetidine was administered to mice by a constant infusion to achieve a plasma unbound concentration of 21.6 μM to examine its effect on the renal disposition of Mate1 probes (metformin, TEA, and cephalexin) in vivo. The kidney- and liver-to-plasma ratios of metformin both were increased 2.4-fold by cimetidine, whereas the renal clearance was not changed. Cimetidine also increased the kidney-to-plasma ratio of TEA and cephalexin 8.0- and 3.3-fold compared with a control and decreased the renal clearance from 49 to 23 and 11 to 6.6 ml/min/kg, respectively. These results suggest that the inhibition of MATEs, but not OCT2, is a likely mechanism underlying the drug-drug interactions with cimetidine in renal elimination.

  15. ABCG1 is involved in vitamin E efflux.

    Science.gov (United States)

    Olivier, Maryline; BottG, Remain; Frisdal, Eric; Nowick, Marion; Plengpanich, Wanee; Desmarchelier, Charles; Roi, Stéphanie; Quinn, Carmel M; Gelissen, Ingrid; Jessup, Wendy; Van Eck, Miranda; Guérin, Maryse; Le Goff, Wilfried; Reboul, Emmanuelle

    2014-12-01

    Vitamin E membrane transport has been shown to involve the cholesterol transporters SR-BI, ABCA1 and NPC1L1. Our aim was to investigate the possible participation of another cholesterol transporter in cellular vitamin E efflux: ABCG1. In Abcgl-deficient mice, vitamin E concentration was reduced in plasma lipoproteins whereas most tissues displayed a higher vitamin E content compared to wild-type mice. α- and γ-tocopherol efflux was increased in CHO cells overexpressing human ABCG1 compared to control cells. Conversely, α- and γ- tocopherol efflux was decreased in ABCG1-knockdown human cells (Hep3B hepatocytes and THP-1 macro- phages). Interestingly, α- and γ-tocopherol significantly downregulated ABCG1 and ABCA1 expression levels in Hep3B and THP-1, an effect confirmed in vivo in rats given vitamin E for 5 days. This was likely due to reduced LXR activation by oxysterols, as Hep3B cells and rat liver treated with vitamin E displayed a significantly reduced content in oxysterols compared to their respective controls. Overall, the present study reveals for the first time that ABCG1 is involved in cellular vitamin E efflux.

  16. Modulation of Bacterial Multidrug Resistance Efflux Pumps of the Major Facilitator Superfamily

    Directory of Open Access Journals (Sweden)

    Sanath Kumar

    2013-01-01

    Full Text Available Bacterial infections pose a serious public health concern, especially when an infectious disease has a multidrug resistant causative agent. Such multidrug resistant bacteria can compromise the clinical utility of major chemotherapeutic antimicrobial agents. Drug and multidrug resistant bacteria harbor several distinct molecular mechanisms for resistance. Bacterial antimicrobial agent efflux pumps represent a major mechanism of clinical resistance. The major facilitator superfamily (MFS is one of the largest groups of solute transporters to date and includes a significant number of bacterial drug and multidrug efflux pumps. We review recent work on the modulation of multidrug efflux pumps, paying special attention to those transporters belonging primarily to the MFS.

  17. A sister of PIN1 gene in tomato (Solanum lycopersicum) defines leaf and flower organ initiation patterns by maintaining epidermal auxin flux.

    Science.gov (United States)

    Martinez, Ciera C; Koenig, Daniel; Chitwood, Daniel H; Sinha, Neelima R

    2016-11-01

    The spatiotemporal localization of the plant hormone auxin acts as a positional cue during early leaf and flower organogenesis. One of the main contributors to auxin localization is the auxin efflux carrier PIN-FORMED1 (PIN1). Phylogenetic analysis has revealed that PIN1 genes are split into two sister clades; PIN1 and the relatively uncharacterized Sister-Of-PIN1 (SoPIN1). In this paper we identify entire-2 as a loss-of-function SlSoPIN1a (Solyc10g078370) mutant in Solanum lycopersicum. The entire-2 plants are unable to specify proper leaf initiation leading to a frequent switch from the wild type spiral phyllotactic pattern to distichous and decussate patterns. Leaves in entire-2 are large and less complex and the leaflets display spatial deformities in lamina expansion, vascular development, and margin specification. During sympodial growth in entire-2 the specification of organ position and identity is greatly affected resulting in variable branching patterns on the main sympodial and inflorescence axes. To understand how SlSoPIN1a functions in establishing proper auxin maxima we used the auxin signaling reporter DR5: Venus to visualize differences in auxin localization between entire-2 and wild type. DR5: Venus visualization shows a widening of auxin localization which spreads to subepidermal tissue layers during early leaf and flower organogenesis, showing that SoPIN1 functions to focus auxin signaling to the epidermal layer. The striking spatial deformities observed in entire-2 help provide a mechanistic framework for explaining the function of the SoPIN1 clade in S.lycopersicum. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  18. Plant AGC protein kinases orient auxin-mediated differential growth and organogenesis

    NARCIS (Netherlands)

    Galván Ampudia, Carlos Samuel

    2009-01-01

    In view of their predominant sessile lifestyle, plants need to be able to adapt to changes in their environment. Environmental signals such as light and gravity modulate plant growth and architecture by redirecting polar cell-to-cell transport of auxin, thus causing changes in the distribution of th

  19. Tripartite assembly of RND multidrug efflux pumps

    Science.gov (United States)

    Daury, Laetitia; Orange, François; Taveau, Jean-Christophe; Verchère, Alice; Monlezun, Laura; Gounou, Céline; Marreddy, Ravi K. R.; Picard, Martin; Broutin, Isabelle; Pos, Klaas M.; Lambert, Olivier

    2016-02-01

    Tripartite multidrug efflux systems of Gram-negative bacteria are composed of an inner membrane transporter, an outer membrane channel and a periplasmic adaptor protein. They are assumed to form ducts inside the periplasm facilitating drug exit across the outer membrane. Here we present the reconstitution of native Pseudomonas aeruginosa MexAB-OprM and Escherichia coli AcrAB-TolC tripartite Resistance Nodulation and cell Division (RND) efflux systems in a lipid nanodisc system. Single-particle analysis by electron microscopy reveals the inner and outer membrane protein components linked together via the periplasmic adaptor protein. This intrinsic ability of the native components to self-assemble also leads to the formation of a stable interspecies AcrA-MexB-TolC complex suggesting a common mechanism of tripartite assembly. Projection structures of all three complexes emphasize the role of the periplasmic adaptor protein as part of the exit duct with no physical interaction between the inner and outer membrane components.

  20. Neutron Radiation Affects the Expression of Genes Involved in the Response to Auxin, Senescence and Oxidative Stress in Arabidopsis

    Science.gov (United States)

    Fortunati, A.; Tassone, P.; Migliaccio, F.

    2008-06-01

    Researches were conducted on the effect of neutron radiation on the expression of genes auxin activated or connected with the process of senescence in Arabidopsis plants. The research was done by applying the real-time polymerase chain reaction (PCR) technique. The results indicated that the auxin response factors (ARFs) genes are clearly downregulated, whereas the indolacetic acid-induced (Aux/IAAs) genes in some cases were upregulated. By contrast in the mutants for auxin transport aux1 and eir1 the ARFs genes were upregulated. In addition, both in the wildtype and mutants, some already known genes activated by stress and senescence were significantly upregulated. On the basis of these researches we conclude that the process of senescence induced by irradiation is, at least in part, controlled by the physiology of the hormone auxin.

  1. The role of Phe82 and Phe351 in auxin-induced substrate perception by TIR1 ubiquitin ligase: a novel insight from molecular dynamics simulations.

    Directory of Open Access Journals (Sweden)

    Ge-Fei Hao

    Full Text Available It is well known that Auxin plays a key role in controlling many aspects of plant growth and development. Crystal structures of Transport inhibitor response 1 (TIR1, a true receptor of auxin, were very recently determined for TIR1 alone and in complexes with auxin and different synthetic analogues and an Auxin/Indole-3-Acetic Acid (Aux/IAA substrate peptide. However, the dynamic conformational changes of the key residues of TIR1 that take place during the auxin and substrate perception by TIR1 and the detailed mechanism of these changes are still unclear. In the present study, various computational techniques were integrated to uncover the detailed molecular mechanism of the auxin and Aux/IAA perception process; these simulations included molecular dynamics (MD simulations on complexes and the free enzyme, the molecular mechanics Poisson Boltzmann surface area (MM-PBSA calculations, normal mode analysis, and hydrogen bond energy (HBE calculations. The computational simulation results provided a reasonable explanation for the structure-activity relationships of auxin and its synthetic analogues in view of energy. In addition, a more detailed model for auxin and Aux/IAA perception was also proposed, indicating that Phe82 and Phe351 played a pivotal role in Aux/IAA perception. Upon auxin binding, Phe82 underwent conformational changes to accommodate the subsequent binding of Aux/IAA. As a result, auxin enhances the TIR1-Aux/IAA interactions by acting as a "molecular glue". Besides, Phe351 acts as a "fastener" to further improve the substrate binding. The structural and mechanistic insights obtained from the present study will provide valuable clues for the future design of promising auxin analogues.

  2. Arabidopsis monothiol glutaredoxin, AtGRXS17, is critical for temperature-dependent postembryonic growth and development via modulating auxin response.

    Science.gov (United States)

    Cheng, Ning-Hui; Liu, Jian-Zhong; Liu, Xing; Wu, Qingyu; Thompson, Sean M; Lin, Julie; Chang, Joyce; Whitham, Steven A; Park, Sunghun; Cohen, Jerry D; Hirschi, Kendal D

    2011-06-10

    Global environmental temperature changes threaten innumerable plant species. Although various signaling networks regulate plant responses to temperature fluctuations, the mechanisms unifying these diverse processes are largely unknown. Here, we demonstrate that an Arabidopsis monothiol glutaredoxin, AtGRXS17 (At4g04950), plays a critical role in redox homeostasis and hormone perception to mediate temperature-dependent postembryonic growth. AtGRXS17 expression was induced by elevated temperatures. Lines altered in AtGRXS17 expression were hypersensitive to elevated temperatures and phenocopied mutants altered in the perception of the phytohormone auxin. We show that auxin sensitivity and polar auxin transport were perturbed in these mutants, whereas auxin biosynthesis was not altered. In addition, atgrxs17 plants displayed phenotypes consistent with defects in proliferation and/or cell cycle control while accumulating higher levels of reactive oxygen species and cellular membrane damage under high temperature. Together, our findings provide a nexus between reactive oxygen species homeostasis, auxin signaling, and temperature responses.

  3. DAR2 acts as an important node connecting cytokinin, auxin, SHY2 and PLT1/2 in root meristem size control.

    Science.gov (United States)

    Peng, Yuancheng; Chen, Liangliang; Lu, Yaru; Ma, Wenying; Tong, Yiping; Li, Yunhai

    2013-06-01

    Cytokinin and auxin antagonistically affect cell proliferation and differentiation and thus regulate root meristem size by influencing the abundance of SHORT HYPOCOTYL2 (SHY2/IAA3). SHY2 affects auxin distribution in the root meristem by repressing the auxin-inducible expression of PIN-FORMED (PIN) auxin transport genes. The PLETHORA (PLT1/2) genes influence root meristem growth by promoting stem cells and transit-amplifying cells. However, the factors connecting cytokinin, auxin, SHY2 and PLT1/2 are largely unknown. In a recent study, we have shown that the DA1-related protein 2 (DAR2) acts downstream of cytokinin and SHY2 but upstream of PLT1/2 to affect root meristem size. Here, we discuss the possible molecular mechanisms by which Arabidopsis DAR2 controls root meristem size.

  4. Phloem-associated auxin response maxima determine radial positioning of lateral roots in maize.