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Sample records for tonoplast

  1. Regulation of transport processes across the tonoplast

    Science.gov (United States)

    Neuhaus, H. Ekkehard; Trentmann, Oliver

    2014-01-01

    In plants, the vacuole builds up the cellular turgor and represents an important component in cellular responses to diverse stress stimuli. Rapid volume changes of cells, particularly of motor cells, like guard cells, are caused by variation of osmolytes and consequently of the water contents in the vacuole. Moreover, directed solute uptake into or release out of the large central vacuole allows adaptation of cytosolic metabolite levels according to the current physiological requirements and specific cellular demands. Therefore, solute passage across the vacuolar membrane, the tonoplast, has to be tightly regulated. Important principles in vacuolar transport regulation are changes of tonoplast transport protein abundances by differential expression of genes or changes of their activities, e.g., due to post-translational modification or by interacting proteins. Because vacuolar transport is in most cases driven by an electro-chemical gradient altered activities of tonoplast proton pumps significantly influence vacuolar transport capacities. Intense studies on individual tonoplast proteins but also unbiased system biological approaches have provided important insights into the regulation of vacuolar transport. This short review refers to selected examples of tonoplast proteins and their regulation, with special focus on protein phosphorylation. PMID:25309559

  2. Tonoplast aquaporins facilitate lateral root emergence

    DEFF Research Database (Denmark)

    Reinhardt, Hagen; Hachez, Charles; Bienert, Manuela Désirée

    2016-01-01

    Aquaporins (AQPs) are water channels allowing fast and passive diffusion of water across cell membranes. It was hypothesized that AQPs contribute to cell elongation processes by allowing water influx across the plasma membrane and the tonoplast to maintain adequate turgor pressure. Here, we report...... mutants showed no or minor reduction in growth of the main root. This phenotype was due to the retardation of LRP emergence. Live cell imaging revealed that tight spatiotemporal control of TIP abundance in the tonoplast of the different LRP cells is pivotal to mediating this developmental process. While...... lateral root emergence is correlated to a reduction of AtTIP1;1 and AtTIP1;2 protein levels in LRPs, expression of AtTIP2;1 is specifically needed in a restricted cell population at the base, then later at the flanks, of developing LRPs. Interestingly, the LRP emergence phenotype of the triple tip mutants...

  3. Modulation in the activity of purified tonoplast H+-ATPase by tonoplast glycolipids prepared from cultured rice (Oryza sativa L. var. Boro) cells.

    Science.gov (United States)

    Yamaguchi, M; Kasamo, K

    2001-05-01

    Glycolipids, phospholipids, and neutral lipids were extracted from the tonoplast fraction of cultured rice cells (Oryza sativa L. var. Boro). Acyl steryl glucoside (ASG) and glucocerebroside (GlcCer) were also prepared from this fraction. We determined the effects of these tonoplast lipids on the activity of H+-ATPase which was delipidated and purified from the tonoplast fraction. Exogenously added tonoplast phospholipids stimulated the activity of purified tonoplast H+-ATPase, but tonoplast glycolipids did not. When tonoplast glycolipids or tonoplast ASG was added in the presence of tonoplast phospholipids, they decreased the phospholipid-induced activation of the tonoplast H+-ATPase; tonoplast GlcCer only caused a small decrease. Steryl glucoside (SG) did not cause any decrease in this activation. Phospholipids, ASG, and GlcCer made up 35 mol%, 20 mol% and 7 mol% of the total lipids of the tonoplast fraction of cultured rice cells, respectively, and these glycolipid levels were enough to depress the phospholipid-induced activation of the tonoplast H+-ATPASE: These results revealed that H+-ATPase activity in the tonoplast may be modulated toward activation and depression by tonoplast phospholipids and glycolipids, respectively. The acylation of SG would be responsible for the depression in the phospholipid-induced H+-ATPase activity.

  4. A tonoplast intrinsic protein in Gardenia jasminoides

    Science.gov (United States)

    Gao, Lan; Li, Hao-Ming

    2017-08-01

    Physiological and molecular studies proved that plasma membrane intrinsic proteins (PIPs) and tonoplast intrinsic proteins (TIPs) subfamily of aquaporins play key functions in plant water homeostasis. Five specialized subgroups (TIP1-5) of TIPs have been found in higher plants, in which the TIP1 and TIP2 isoforms are the largest arbitrary groups. TIPs have high water-transport activity than PIPs, some TIPs can transport other small molecule such as urea, ammonia, hydrogen peroxide, and carbon dioxide. In this work, the structure of the putative tonoplast aquaporin from Gardenia jasminoides (GjTIP) was analyzed. Its transcript level has increased during fruit maturation. A phylogenetic analysis indicates that the protein belongs to TIP1 subfamily. A three-dimensional model structure of GjTIP was built based on crystal structure of an ammonia-permeable AtTIP2-1 from Arabidopsis thaliana. The model structure displayed as a homo-tetramer, each monomer has six trans-membrane and two half-membrane-spanning α helices. The data suggests that the GjTIP has tendency to be a mixed function aquaporin, might involve in water, urea and hydrogen peroxide transport, and the gating machanism founded in some AQPs involving pH and phosphorylation response have not been proved in GjTIP.

  5. Identification and characterization of polyphosphoinositides in the tonoplast membrane

    International Nuclear Information System (INIS)

    Cho, Myeon; Memon, A.R.; Boss, W.F.

    1989-01-01

    In vitro data indicate that the tonoplast membrane is the major target site for IP 3 action and Ca +2 is released from this compartment. If this is true, then several questions arise: (1) Is the plasma membrane or tonoplast the source of IP 3 ? (2) If the plasma membrane is the source of IP 3 , how does IP 3 get to the vacuole? In this connection, the results from in vivo labeling of carrot cells with [ 3 H] inositol show that the major phospholipid components in the tonoplast are PI and lysoPIP. In vitro phosphorylation studies using [γ- 32 P]ATP indicate that PI kinase is present in the tonoplast membrane even though PIP was not detected with inositol labeling. If PIP and PIP 2 are not present in the tonoplast, then IP 3 must diffuse or be transported from the plasma membrane. Since IP 3 is rapidly metabolized by cytosolic phosphatases there may be an IP 3 -sensitive calcium store other than the vacuole closely associated with the plasma membrane

  6. Regulation of transport processes across the tonoplast membrane

    Directory of Open Access Journals (Sweden)

    Oliver eTrentmann

    2014-09-01

    Full Text Available In plants, the vacuole builds up the cellular turgor and represents an important component in cellular responses to diverse stress stimuli. Rapid volume changes of cells, particularly of motor cells, like guard cells, are caused by variation of osmolytes and consequently of the water contents in the vacuole. Moreover, directed solute uptake into or release out of the large central vacuole allows adaptation of cytosolic metabolite levels according to the current physiological requirements and specific cellular demands. Therefore, solute passage across the vacuolar membrane, the tonoplast, has to be tightly regulated. Important principles in vacuolar transport regulation are changes of tonoplast transport protein abundances by differential expression of genes or changes of their activities, e.g. due to post-translational modification or by interacting proteins. Because vacuolar transport is in most cases driven by an electro-chemical gradient altered activities of tonoplast proton pumps significantly influence vacuolar transport capacities. Intense studies on individual tonoplast proteins but also unbiased system biological approaches have provided important insights into the regulation of vacuolar transport. This short review refers to selected examples of tonoplast proteins and their regulation, with special focus on protein phosphorylation.

  7. Probing the functional subunits of the tonoplast H+-ATPase

    International Nuclear Information System (INIS)

    Randall, S.K.; Lai, S.; Sze, H.

    1986-01-01

    The tonoplast ATPase of oat roots is composed of at least three polypeptides of 72, 60, and 16 kDa. The 16 kDA polypeptide covalently binds N,N'-dicyclohexylcarbodiimide and is postulated to be a component of the proton channel. Initial studies to identify other subunits indicate that both the 72 and 60 kDa subunits covalently bind 14 C]-7-chloro-4-nitrobenzo-2-oxa-1,3-diazole and [ 14 C]N-ethylamleimide, inhibitors of the tonoplast ATPase. ATP prevents binding of these inhibitors suggesting that both the 72 and 60 kDa subunits are involved in substrate binding. Polyclonal antibody has been made to the 72 kDa subunit. Western blot analysis of tonoplast vesicles reveals single reactive polypeptide (72 kDa). The antibody shows no cross-reactivity towards either the mitochondrial F 1 -ATPase or the plasma membrane ATPase. This antibody specifically inhibits ATP hydrolysis and ATP-dependent H + pumping in native tonoplast vesicles. The authors conclude that the 72 kDa subunit is intimately associated with the catalytic (or ATP-binding) site

  8. PHYSIOLOGY OF ION TRANSPORT ACROSS THE TONOPLAST OF HIGHER PLANTS.

    Science.gov (United States)

    Barkla, Bronwyn J.; Pantoja, Omar

    1996-06-01

    The vacuole of plant cells plays an important role in the homeostasis of the cell. It is involved in the regulation of cytoplasmic pH, sequestration of toxic ions and xenobiotics, regulation of cell turgor, storage of amino acids, sugars and CO2 in the form of malate, and possibly as a source for elevating cytoplasmic calcium. All these activities are driven by two primary active transport mechanisms present in the vacuolar membrane (tonoplast). These two mechanisms employ high-energy metabolites to pump protons into the vacuole, establishing a proton electrochemical potential that mediates the transport of a diverse range of solutes. Within the past few years, great advances at the molecular and functional levels have been made on the characterization and identification of these mechanisms. The aim of this review is to summarize these studies in the context of the physiology of the plant cell.

  9. Quantitative proteomics of the tonoplast reveals a role for glycolytic enzymes in salt tolerance.

    Science.gov (United States)

    Barkla, Bronwyn J; Vera-Estrella, Rosario; Hernández-Coronado, Marcela; Pantoja, Omar

    2009-12-01

    To examine the role of the tonoplast in plant salt tolerance and identify proteins involved in the regulation of transporters for vacuolar Na(+) sequestration, we exploited a targeted quantitative proteomics approach. Two-dimensional differential in-gel electrophoresis analysis of free flow zonal electrophoresis separated tonoplast fractions from control, and salt-treated Mesembryanthemum crystallinum plants revealed the membrane association of glycolytic enzymes aldolase and enolase, along with subunits of the vacuolar H(+)-ATPase V-ATPase. Protein blot analysis confirmed coordinated salt regulation of these proteins, and chaotrope treatment indicated a strong tonoplast association. Reciprocal coimmunoprecipitation studies revealed that the glycolytic enzymes interacted with the V-ATPase subunit B VHA-B, and aldolase was shown to stimulate V-ATPase activity in vitro by increasing the affinity for ATP. To investigate a physiological role for this association, the Arabidopsis thaliana cytoplasmic enolase mutant, los2, was characterized. These plants were salt sensitive, and there was a specific reduction in enolase abundance in the tonoplast from salt-treated plants. Moreover, tonoplast isolated from mutant plants showed an impaired ability for aldolase stimulation of V-ATPase hydrolytic activity. The association of glycolytic proteins with the tonoplast may not only channel ATP to the V-ATPase, but also directly upregulate H(+)-pump activity.

  10. Quantitative Proteomics of the Tonoplast Reveals a Role for Glycolytic Enzymes in Salt Tolerance[C][W

    Science.gov (United States)

    Barkla, Bronwyn J.; Vera-Estrella, Rosario; Hernández-Coronado, Marcela; Pantoja, Omar

    2009-01-01

    To examine the role of the tonoplast in plant salt tolerance and identify proteins involved in the regulation of transporters for vacuolar Na+ sequestration, we exploited a targeted quantitative proteomics approach. Two-dimensional differential in-gel electrophoresis analysis of free flow zonal electrophoresis separated tonoplast fractions from control, and salt-treated Mesembryanthemum crystallinum plants revealed the membrane association of glycolytic enzymes aldolase and enolase, along with subunits of the vacuolar H+-ATPase V-ATPase. Protein blot analysis confirmed coordinated salt regulation of these proteins, and chaotrope treatment indicated a strong tonoplast association. Reciprocal coimmunoprecipitation studies revealed that the glycolytic enzymes interacted with the V-ATPase subunit B VHA-B, and aldolase was shown to stimulate V-ATPase activity in vitro by increasing the affinity for ATP. To investigate a physiological role for this association, the Arabidopsis thaliana cytoplasmic enolase mutant, los2, was characterized. These plants were salt sensitive, and there was a specific reduction in enolase abundance in the tonoplast from salt-treated plants. Moreover, tonoplast isolated from mutant plants showed an impaired ability for aldolase stimulation of V-ATPase hydrolytic activity. The association of glycolytic proteins with the tonoplast may not only channel ATP to the V-ATPase, but also directly upregulate H+-pump activity. PMID:20028841

  11. Identification and Functional Characterization of a Tonoplast Dicarboxylate Transporter in Tomato (Solanum lycopersicum)

    OpenAIRE

    Liu, Ruiling; Li, Boqiang; Qin, Guozheng; Zhang, Zhanquan; Tian, Shiping

    2017-01-01

    Acidity plays an important role in flavor and overall organoleptic quality of fruit and is mainly due to the presence of organic acids. Understanding the molecular basis of organic acid metabolism is thus of primary importance for fruit quality improvement. Here, we cloned a putative tonoplast dicarboxylate transporter gene (SlTDT) from tomato, and submitted it to the NCBI database (GenBank accession number: KC733165). SlTDT protein contained 13 putative transmembrane domains in silico analys...

  12. Identification and Functional Characterization of a Tonoplast Dicarboxylate Transporter in Tomato (Solanum lycopersicum).

    Science.gov (United States)

    Liu, Ruiling; Li, Boqiang; Qin, Guozheng; Zhang, Zhanquan; Tian, Shiping

    2017-01-01

    Acidity plays an important role in flavor and overall organoleptic quality of fruit and is mainly due to the presence of organic acids. Understanding the molecular basis of organic acid metabolism is thus of primary importance for fruit quality improvement. Here, we cloned a putative tonoplast dicarboxylate transporter gene ( SlTDT ) from tomato, and submitted it to the NCBI database (GenBank accession number: KC733165). SlTDT protein contained 13 putative transmembrane domains in silico analysis. Confocal microscopic study using green fluorescent fusion proteins revealed that SlTDT was localized on tonoplast. The expression patterns of SlTDT in tomato were analyzed by RT-qPCR. The results indicated that SlTDT expressed in leaves, roots, flowers and fruits at different ripening stages, suggesting SlTDT may be associated with the development of different tissues. To further explore the function of SlTDT , we constructed both overexpression and RNAi vectors and obtained transgenic tomato plants by agrobacterium-mediated method. Gas chromatography-mass spectrometer (GC-MS) analysis showed that overexpression of SlTDT significantly increased malate content, and reduced citrate content in tomato fruit. By contrast, repression of SlTDT in tomato reduced malate content of and increased citrate content. These results indicated that SlTDT played an important role in remobilization of malate and citrate in fruit vacuoles.

  13. Purification of the N,N'-dicyclohexylcarbodiimide-binding proteolipid of a higher plant tonoplast H+-ATPase

    International Nuclear Information System (INIS)

    Rea, P.A.; Griffith, C.J.; Sanders, D.

    1987-01-01

    The H+-ATPase of Beta vacuolar membrane (tonoplast) comprises at least three functionally distinct subunits of Mr = 67,000, 57,000, and 16,000, respectively. The hydrophobic carboxyl reagent N,N'-dicyclohexylcarbodiimide (DCCD) inactivates the enzyme with pseudo-first order kinetics, and the concentration dependence of the reaction indicates that DCCD interacts with a single site on the enzyme to exert its inhibitory effect. The apparent pseudo-first order rate constant (k0) is reciprocally dependent on membrane protein concentration, which is expected if a large fraction of the DCCD partitions into the lipid phase. k0 has a nominal value of 1000 M-1 min-1 at a protein concentration of 250 micrograms/ml, although when phase partitioning is taken into account, the true, protein concentration-independent value of k0 is calculated to be about an order of magnitude lower. [ 14 C]DCCD primarily labels the Mr = 16,000 polypeptide of native tonoplast vesicles. Binding is venturicidin-insensitive and occurs at a rate similar to the rate of enzyme inactivation, implying that inhibition is a direct result of covalent modification of the Mr = 16,000 polypeptide. Labeling of the containing Mr = 8,000 subunit of mitochondrial F0F1-ATPase is, on the other hand, faster by a factor of 5 and totally abolished by venturicidin. These results confirm that the Mr = 16,000 polypeptide which copurifies with tonoplast H+-ATPase activity is a subunit of the enzyme. Most of the DCCD-reactive Mr = 16,000 subunit is extracted from acetone:ethanol-washed tonoplast vesicles by chloroform:methanol. [ 14 C]DCCD bound to the Mr = 16,000 polypeptide is enriched in the chloroform:methanol extract by 5-fold compared with native tonoplast and the specific activity (nmol of [ 14 C]DCCD/mg of protein) can be increased a further 37-fold by chromatography on DEAE-Sephadex

  14. Comparative study of the active cadmium efflux systems operating at the plasma membrane and tonoplast of cucumber root cells.

    Science.gov (United States)

    Migocka, Magdalena; Papierniak, Anna; Kosatka, Ewelina; Klobus, Grazyna

    2011-10-01

    The strategies developed by plants to avoid the toxicity of cadmium (Cd) and other heavy metals involve active sequestration of metals into the apoplast and vacuoles. The protein systems excluding heavy metals from the cell cytosol localize to the plasma membrane and tonoplast and are energized either by ATP or by the electrochemical gradient generated by H(+)-ATPase or by V-ATPase and pyrophosphatase (PPase), respectively. In this work, a comparative study on the contribution of both the plasma membrane and tonoplast in the active detoxification of plant cells after treatment with Cd was performed. The studies using plants treated and untreated with Cd reveal that both, H(+)-coupled and MgATP-driven efflux of Cd across plasma membranes and tonoplast is markedly stimulated in the presence of Cd in the environment. Previous studies on plasma-membrane localized H(+)-coupled Cd efflux together with the present data demonstrating tonoplast H(+)/Cd(2+) antiport activity suggest that H(+)-coupled secondary transport of Cd displays a lower affinity for Cd when compared with Cd primary pumps driven by MgATP. In addition, it is shown that MgATP-energized Cd efflux across both membranes is significantly enhanced by cysteine, dithiothreitol, and glutathione. These results suggest that Cd is excluded from the cytosol through an energy-dependent system as a free ion as well as a complexed form. Although both membranes contribute in the active exclusion of ionized and complexed Cd from the cytosol, the overall calculation of Cd accumulation in the everted plasma membranes and vacuolar vesicles suggests that the tonoplast and vacuole have a major function in Cd efflux from the cytosol in the roots of cucumber subjected to Cd stress.

  15. THE TONOPLAST TRANSPORT SYSTEMS OF PLANT VACUOLES AND THEIR POTENTIAL APPLICATION IN BIOTECHNOLOGY

    Directory of Open Access Journals (Sweden)

    S. V. Isayenkov

    2013-06-01

    Full Text Available The pivotal role of plant vacuoles in plant survival was discussed in the review. Particularly, the providing of cellular turgor, accumulation of inorganic osmolytes and nutrients are the primary tasks of these cellular organelles. The main mechanisms of tonoplast transport systems were described. The known transport pathways of minerals, heavy metals, vitamins and other organic compounds were classified and outlined. The main systems of membrane vacuolar transport were reviewed. The outline of the physiological functions and features of vacuolar membrane transport proteins were performed. The physiological role of transport of minerals, nutrients and other compounds into vacuoles were discussed. This article reviews the main types of plant vacuoles and their functional role in plant cell. Current state and progress in vacuolar transport research was outlined. The examples of application for rinciples and mechanisms of vacuolar membrane transport in plant biotechnology were iven. The perspectives and approaches in plant and food biotechnology concerning transport and physiology of vacuoles are discussed.

  16. Upregulation of a tonoplast-localized cytochrome P450 during petal senescence in Petunia inflata

    Directory of Open Access Journals (Sweden)

    Ishida Hiroyuki

    2006-04-01

    Full Text Available Abstract Background Gene expression in Petunia inflata petals undergoes major changes following compatible pollination. Severe flower wilting occurs reproducibly within 36 hours, providing an excellent model for investigation of petal senescence and programmed cell death. Expression of a number of genes and various enzyme activities involved in the degradation and remobilization of macromolecules have been found to be upregulated during the early stages of petal senescence. Results By performing differential display of cDNAs during Petunia inflata petal senescence, a highly upregulated gene encoding a cytochrome P450 was identified. Analysis of the complete cDNA sequence revealed that the predicted protein is a member of the CYP74C family (CYP74C9 and is highly similar to a tomato CYP74C allene oxide synthase (AOS that is known to be active on 9-hydroperoxides. Cloning of the petunia genomic DNA revealed an intronless gene with a promoter region that carries signals found in stress-responsive genes and potential binding sites for Myb transcription factors. Transcripts were present at detectable levels in root and stem, but were 40 times more abundant in flowers 36 hours after pollination. Ethylene and jasmonate treatment resulted in transitory increases in expression in detached flowers. A protein fusion of the CYP74C coding region to a C-terminal GFP was found to be located in the tonoplast. Conclusion Though oxylipins, particularly jasmonates, are known to be involved in stress responses, the role of other products of CYP74 enzymes is less well understood. The identification of a CYP74C family member as a highly upregulated gene during petal senescence suggests that additional products of fatty acid metabolism may play important roles during programmed cell death. In contrast to the chloroplast localization of AOS proteins in the CYP74A subfamily, GFP fusion data indicates that the petunia CYP74C9 enzyme is in the tonoplast. This result

  17. A rice tonoplastic calcium exchanger, OsCCX2 mediates Ca2+/cation transport in yeast

    Science.gov (United States)

    Yadav, Akhilesh K.; Shankar, Alka; Jha, Saroj K.; Kanwar, Poonam; Pandey, Amita; Pandey, Girdhar K.

    2015-01-01

    In plant cell, cations gradient in cellular compartments is maintained by synergistic action of various exchangers, pumps and channels. The Arabidopsis exchanger family members (AtCCX3 and AtCCX5) were previously studied and belong to CaCA (calcium cation exchangers) superfamily while none of the rice CCXs has been functionally characterized for their cation transport activities till date. Rice genome encode four CCXs and only OsCCX2 transcript showed differential expression under abiotic stresses and Ca2+ starvation conditions. The OsCCX2 localized to tonoplast and suppresses the Ca2+ sensitivity of K667 (low affinity Ca2+ uptake deficient) yeast mutant under excess CaCl2 conditions. In contrast to AtCCXs, OsCCX2 expressing K667 yeast cells show tolerance towards excess Na+, Li+, Fe2+, Zn2+ and Co2+ and suggest its ability to transport both mono as well as divalent cations in yeast. Additionally, in contrast to previously characterized AtCCXs, OsCCX2 is unable to complement yeast trk1trk2 double mutant suggesting inability to transport K+ in yeast system. These finding suggest that OsCCX2 having distinct metal transport properties than previously characterized plant CCXs. OsCCX2 can be used as potential candidate for enhancing the abiotic stress tolerance in plants as well as for phytoremediation of heavy metal polluted soil. PMID:26607171

  18. Determination of the individual electrical and transport properties of the plasmalemma and the tonoplast of the giant marine alga Ventricaria ventricosa by means of the integrated perfusion/charge-pulse technique: evidence for a multifolded tonoplast.

    Science.gov (United States)

    Ryser, C; Wang, J; Mimietz, S; Zimmermann, U

    1999-03-15

    The charge-pulse relaxation spectrum of nonperfused and perfused (turgescent) cells of the giant marine alga Ventricaria ventricosa showed two main exponential decays with time constants of approximately 0.1 msec and 10 msec, respectively, when the cells were bathed in artificial sea water (pH 8). Variation of the external pH did not change the relaxation pattern (in contrast to other giant marine algae). Addition of nystatin (a membrane-impermeable and pore-forming antibiotic) to the vacuolar perfusion solution resulted in the disappearance of the slow exponential, whereas external nystatin decreased dramatically the time constant of the fast one. This indicated (by analogy to corresponding experiments with Valonia utricularis, J. Wang, I. Spiess, C. Ryser, U. Zimmermann, J. Membrane Biol. 157: 311-321, 1997) that the fast relaxation must be assigned to the RC-properties of the plasmalemma and the slow one to those of the tonoplast. Consistent with this, external variation of [K+]o or of [Cl-]o as well as external addition of K+- or Cl--channel/carrier inhibitors (TEA, Ba2+, DIDS) affected only the fast relaxation, but not the slow one. In contrast, addition of these inhibitors to the vacuolar perfusion solution had no measurable effect on the charge-pulse relaxation spectrum. The analysis of the data in terms of the "two membrane model" showed that K+- and (to a smaller extent) Cl--conducting elements dominated the plasmalemma conductance. The analysis of the charge-pulse relaxation spectra also yielded the following area-specific data for the capacitance and the conductance for the plasmalemma and tonoplast (by assuming that both membranes have a planar surface): (plasmalemma) Cp = 0.82 * 10(-2) F m-2, Rp = 1.69 * 10(-2) Omega m2, Gp = 5.9 * 10(4) mS m-2, (tonoplast) Ct = 7. 1 * 10(-2) F m-2, Rt = 14.9 * 10(-2) Omega m2 and Gt = 0.67 * 10(4) mS m-2. The electrical data for the tonoplast show that (in contrast to the literature) the area-specific membrane

  19. Photolabeling of tonoplast from sugar beet cell suspensions by [3H]5-(N-methyl-N-isobutyl)-amiloride, an inhibitor of the vacuolar Na+/H+ antiport

    International Nuclear Information System (INIS)

    Barkla, B.J.; Charuk, J.H.M.; Blumwald, E.; Cragoe, E.J. Jr.

    1990-01-01

    The effects of 5-(N-methyl-N-isobutyl)-amiloride (MIA), an amiloride analog, was tested on the Na + /H + antiport activity of intact vacuoles and tonoplast vesicles isolated from sugar beet (Beta vulgaris L.) cell suspension cultures. MIA inhibited Na + /H + exchange in a competitive manner with a K i of 2.5 and 5.9 micromolar for ΔpH-dependent 22 Na + influx in tonoplast vesicles and Na + -dependent H + efflux in intact vacuoles, respectively. Scatchard analysis of the binding of [ 3 H]MIA to tonoplast membranes revealed a high affinity binding component with a K d of 1.3 micromolar. The close relationship between the dissociation constant value obtained and the constants of inhibition for MIA obtained by fluorescence quenching and isotope exchange suggests that the high affinity component represents a class of sites associated with the tonoplast Na + /H + antiport. Photolabeling of the tonoplast with [ 3 H]MIA revealed two sets of polypeptides with a different affinity to amiloride and its analog

  20. Photolabeling of tonoplast from sugar beet cell suspensions by [h]5-(N-methyl-N-isobutyl)-amiloride, an inhibitor of the vacuolar na/h antiport.

    Science.gov (United States)

    Barkla, B J; Charuk, J H; Cragoe, E J; Blumwald, E

    1990-07-01

    The effects of 5-(N-methyl-N-isobutyl)-amiloride (MIA), an amiloride analog, was tested on the Na(+)/H(+) antiport activity of intact vacuoles and tonoplast vesicles isolated from sugar beet (Beta vulgaris L.) cell suspension cultures. MIA inhibited Na(+)/H(+) exchange in a competitive manner with a K(i) of 2.5 and 5.9 micromolar for DeltapH-dependent (22)Na(+) influx in tonoplast vesicles and Na(+)-dependent H(+) efflux in intact vacuoles, respectively. Scatchard analysis of the binding of [(3)H]MIA to tonoplast membranes revealed a high affinity binding component with a K(d) of 1.3 micromolar. The close relationship between the dissociation constant value obtained and the constants of inhibition for MIA obtained by fluorescence quenching and isotope exchange suggests that the high affinity component represents a class of sites associated with the tonoplast Na(+)/H(+) antiport. Photolabeling of the tonoplast with [(3)H]MIA revealed two sets of polypeptides with a different affinity to amiloride and its analog.

  1. Photolabeling of Tonoplast from Sugar Beet Cell Suspensions by [3H]5-(N-Methyl-N-Isobutyl)-Amiloride, an Inhibitor of the Vacuolar Na+/H+ Antiport 1

    Science.gov (United States)

    Barkla, Bronwyn J.; Charuk, Jeffrey H. M.; Cragoe, Edward J.; Blumwald, Eduardo

    1990-01-01

    The effects of 5-(N-methyl-N-isobutyl)-amiloride (MIA), an amiloride analog, was tested on the Na+/H+ antiport activity of intact vacuoles and tonoplast vesicles isolated from sugar beet (Beta vulgaris L.) cell suspension cultures. MIA inhibited Na+/H+ exchange in a competitive manner with a Ki of 2.5 and 5.9 micromolar for ΔpH-dependent 22Na+ influx in tonoplast vesicles and Na+-dependent H+ efflux in intact vacuoles, respectively. Scatchard analysis of the binding of [3H]MIA to tonoplast membranes revealed a high affinity binding component with a Kd of 1.3 micromolar. The close relationship between the dissociation constant value obtained and the constants of inhibition for MIA obtained by fluorescence quenching and isotope exchange suggests that the high affinity component represents a class of sites associated with the tonoplast Na+/H+ antiport. Photolabeling of the tonoplast with [3H]MIA revealed two sets of polypeptides with a different affinity to amiloride and its analog. Images Figure 7 PMID:16667602

  2. In vitro and in vivo phosphorylation of polypeptides in plasma membrane and tonoplast-enriched fractions from barley roots

    International Nuclear Information System (INIS)

    Garbarino, J.E.; Hurkman, W.J.; Tanaka, C.K.; DuPont, F.M.

    1991-01-01

    Phosphorylation of polypeptides in membrane fractions from barley (Hordeum vulgare L. cv CM 72) roots was compared in in vitro and in vivo assays to assess the potential role of protein kinases in modification of membrane transport. Membrane fractions enriched in endoplasmic reticulum, tonoplast, and plasma membrane were isolated using sucrose gradients and the membrane polypeptides separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis. When the membrane fractions were incubated with γ[p 32 P]ATP, phosphorylation occurred almost exclusively in the plasma membrane fraction. Phosphorylation of a band at 38 kilodaltons increased as the concentration of Mg 2+ was decreased from millimolar to micromolar levels. Phosphorylation of bands at 125, 86, 58, 46 and 28 kilodaltons required millimolar Mg 2+ concentrations and was greatly enhanced by Ca 2+ . When roots of intact plants were labeled with [ 32 P]orthophosphate, polypeptides at approximately 135, 166, 90, 46 to 53, 32, 28, and 19 kilodaltons were labeled in the plasma membrane fraction and polypeptides at approximately 73, 66, and 48 kilodaltons were labeled in the tonoplast fraction. Treatment of the roots of intact plants with 150 millimolar NaCl resulted in increased phosphorylation of some polypeptides while treatment with 100 mM NaCl had no effect

  3. Alfalfa mosaic virus replicase proteins, P1 and P2, localize to the tonoplast in the presence of virus RNA

    International Nuclear Information System (INIS)

    Ibrahim, Amr; Hutchens, Heather M.; Howard Berg, R.; Sue Loesch-Fries, L.

    2012-01-01

    To identify the virus components important for assembly of the Alfalfa mosaic virus replicase complex, we used live cell imaging of Arabidopsis thaliana protoplasts that expressed various virus cDNAs encoding native and GFP-fusion proteins of P1 and P2 replicase proteins and full-length virus RNAs. Expression of P1-GFP alone resulted in fluorescent vesicle-like bodies in the cytoplasm that colocalized with FM4-64, an endocytic marker, and RFP-AtVSR2, RabF2a/Rha1-mCherry, and RabF2b/Ara7-mCherry, all of which localize to multivesicular bodies (MVBs), which are also called prevacuolar compartments, that mediate traffic to the lytic vacuole. GFP-P2 was driven from the cytosol to MVBs when expressed with P1 indicating that P1 recruited GFP-P2. P1-GFP localized on the tonoplast, which surrounds the vacuole, in the presence of infectious virus RNA, replication competent RNA2, or P2 and replication competent RNA1 or RNA3. This suggests that a functional replication complex containing P1, P2, and a full-length AMV RNA assembles on MVBs to traffic to the tonoplast.

  4. Alfalfa mosaic virus replicase proteins, P1 and P2, localize to the tonoplast in the presence of virus RNA

    Energy Technology Data Exchange (ETDEWEB)

    Ibrahim, Amr [Department of Botany and Plant Pathology, Purdue University, West Lafayette, IN 47907 (United States); Present address: Genomics Facility, Agricultural Genetic Engineering Research Institute, Agricultural Research Center, Giza 12619 (Egypt); Hutchens, Heather M. [Department of Botany and Plant Pathology, Purdue University, West Lafayette, IN 47907 (United States); Howard Berg, R. [Integrated Microscopy Facility, Donald Danforth Plant Science Center, Saint Louis, MO 63132 (United States); Sue Loesch-Fries, L., E-mail: loeschfr@purdue.edu [Department of Botany and Plant Pathology, Purdue University, West Lafayette, IN 47907 (United States)

    2012-11-25

    To identify the virus components important for assembly of the Alfalfa mosaic virus replicase complex, we used live cell imaging of Arabidopsis thaliana protoplasts that expressed various virus cDNAs encoding native and GFP-fusion proteins of P1 and P2 replicase proteins and full-length virus RNAs. Expression of P1-GFP alone resulted in fluorescent vesicle-like bodies in the cytoplasm that colocalized with FM4-64, an endocytic marker, and RFP-AtVSR2, RabF2a/Rha1-mCherry, and RabF2b/Ara7-mCherry, all of which localize to multivesicular bodies (MVBs), which are also called prevacuolar compartments, that mediate traffic to the lytic vacuole. GFP-P2 was driven from the cytosol to MVBs when expressed with P1 indicating that P1 recruited GFP-P2. P1-GFP localized on the tonoplast, which surrounds the vacuole, in the presence of infectious virus RNA, replication competent RNA2, or P2 and replication competent RNA1 or RNA3. This suggests that a functional replication complex containing P1, P2, and a full-length AMV RNA assembles on MVBs to traffic to the tonoplast.

  5. A Tonoplast P3B-ATPase Mediates Fusion of Two Types of Vacuoles in Petal Cells

    Directory of Open Access Journals (Sweden)

    Marianna Faraco

    2017-06-01

    Full Text Available It is known that plant cells can contain multiple distinct vacuoles; however, the abundance of multivacuolar cells and the mechanisms underlying vacuolar differentiation and communication among different types of vacuoles remain unknown. PH1 and PH5 are tonoplast P-ATPases that form a heteromeric pump that hyper-acidifies the central vacuole (CV of epidermal cells in petunia petals. Here, we show that the sorting of this pump and other vacuolar proteins to the CV involves transit through small vacuoles: vacuolinos. Vacuolino formation is controlled by transcription factors regulating pigment synthesis and transcription of PH1 and PH5. Trafficking of proteins from vacuolinos to the central vacuole is impaired by misexpression of vacuolar SNAREs as well as mutants for the PH1 component of the PH1-PH5 pump. The finding that PH1-PH5 and these SNAREs interact strongly suggests that structural tonoplast proteins can act as tethering factors in the recognition of different vacuolar types.

  6. Impaired Malate and Fumarate Accumulation Due to the Mutation of the Tonoplast Dicarboxylate Transporter Has Little Effects on Stomatal Behavior.

    Science.gov (United States)

    Medeiros, David B; Barros, Kallyne A; Barros, Jessica Aline S; Omena-Garcia, Rebeca P; Arrivault, Stéphanie; Sanglard, Lílian M V P; Detmann, Kelly C; Silva, Willian Batista; Daloso, Danilo M; DaMatta, Fábio M; Nunes-Nesi, Adriano; Fernie, Alisdair R; Araújo, Wagner L

    2017-11-01

    Malate is a central metabolite involved in a multiplicity of plant metabolic pathways, being associated with mitochondrial metabolism and playing significant roles in stomatal movements. Vacuolar malate transport has been characterized at the molecular level and is performed by at least one carrier protein and two channels in Arabidopsis ( Arabidopsis thaliana ) vacuoles. The absence of the Arabidopsis tonoplast Dicarboxylate Transporter (tDT) in the tdt knockout mutant was associated previously with an impaired accumulation of malate and fumarate in leaves. Here, we investigated the consequences of this lower accumulation on stomatal behavior and photosynthetic capacity as well as its putative metabolic impacts. Neither the stomatal conductance nor the kinetic responses to dark, light, or high CO 2 were highly affected in tdt plants. In addition, we did not observe any impact on stomatal aperture following incubation with abscisic acid, malate, or citrate. Furthermore, an effect on photosynthetic capacity was not observed in the mutant lines. However, leaf mitochondrial metabolism was affected in the tdt plants. Levels of the intermediates of the tricarboxylic acid cycle were altered, and increases in both light and dark respiration were observed. We conclude that manipulation of the tonoplastic organic acid transporter impacted mitochondrial metabolism, while the overall stomatal and photosynthetic capacity were unaffected. © 2017 American Society of Plant Biologists. All Rights Reserved.

  7. Tonoplast Na+/H+ Antiport Activity and Its Energization by the Vacuolar H+-ATPase in the Halophytic Plant Mesembryanthemum crystallinum L.

    Science.gov (United States)

    Barkla, B. J.; Zingarelli, L.; Blumwald, E.; Smith, JAC.

    1995-10-01

    Tonoplast vesicles were isolated from leaf mesophyll tissue of the inducible Crassulacean acid metabolism plant Mesembryanthemum crystallinum to investigate the mechanism of vacuolar Na+ accumulation in this halophilic species. In 8-week-old plants exposed to 200 mM NaCl for 2 weeks, tonoplast H+-ATPase activity was approximately doubled compared with control plants of the same age, as determined by rates of both ATP hydrolysis and ATP-dependent H+ transport. Evidence was also obtained for the presence of an electroneutral Na+/H+ antiporter at the tonoplast that is constitutively expressed, since extravesicular Na+ was able to dissipate a pre-existing transmembrane pH gradient. Initial rates of H+ efflux showed saturation kinetics with respect to extravesicular Na+ concentration and were 2.1-fold higher from vesicles of salt-treated plants compared with the controls. Na+-dependent H+ efflux also showed a high selectivity for Na+ over K+, was insensitive to the transmembrane electrical potential difference, and was more than 50% inhibited by 200 [mu]M N-amidino-3,5-diamino-6-chloropyrazinecarboxamide hydrochloride. The close correlation between increased Na+/H+ antiport and H+-ATPase activities in response to salt treatment suggests that accumulation of the very high concentrations of vacuolar Na+ found in M. crystallinum is energized by the H+ electrochemical gradient across the tonoplast.

  8. Overexpression of a Panax ginseng tonoplast aquaporin alters salt tolerance, drought tolerance and cold acclimation ability in transgenic Arabidopsis plants.

    Science.gov (United States)

    Peng, Yanhui; Lin, Wuling; Cai, Weiming; Arora, Rajeev

    2007-08-01

    Water movement across cellular membranes is regulated largely by a family of water channel proteins called aquaporins (AQPs). Since several abiotic stresses such as, drought, salinity and freezing, manifest themselves via altering water status of plant cells and are linked by the fact that they all result in cellular dehydration, we overexpressed an AQP (tonoplast intrinsic protein) from Panax ginseng, PgTIP1, in transgenic Arabidopsis thaliana plants to test its role in plant's response to drought, salinity and cold acclimation (induced freezing tolerance). Under favorable conditions, PgTIP1 overexpression significantly increased plant growth as determined by the biomass production, and leaf and root morphology. PgTIP1 overexpression had beneficial effect on salt-stress tolerance as indicated by superior growth status and seed germination of transgenic plants under salt stress; shoots of salt-stressed transgenic plants also accumulated greater amounts of Na(+) compared to wild-type plants. Whereas PgTIP1 overexpression diminished the water-deficit tolerance of plants grown in shallow (10 cm deep) pots, the transgenic plants were significantly more tolerant to water stress when grown in 45 cm deep pots. The rationale for this contrasting response, apparently, comes from the differences in the root morphology and leaf water channel activity (speed of dehydration/rehydration) between the transgenic and wild-type plants. Plants overexpressed with PgTIP1 exhibited lower (relative to wild-type control) cold acclimation ability; however, this response was independent of cold-regulated gene expression. Our results demonstrate a significant function of PgTIP1 in growth and development of plant cells, and suggest that the water movement across tonoplast (via AQP) represents a rate-limiting factor for plant vigor under favorable growth conditions and also significantly affect responses of plant to drought, salt and cold stresses.

  9. Active glucose transport and proton pumping in tonoplast membrane of Zea mays L. coleoptiles are inhibited by anti-H+-ATPase antibodies

    International Nuclear Information System (INIS)

    Rausch, T.; Butcher, D.N.; Taiz, L.

    1987-01-01

    A tonoplast enriched fraction was obtained from Zea mays L. coleoptiles by isopycnic centrifugation of microsomal membranes in a sucrose step gradient. At the 18/26% interface chloride-stimulated and nitrate-inhibited proton pumping activity coincided with a Mg 2+ -ATP dependent accumulation of 3-O-methyl-D-glucose (OMG) as determined by a membrane filtration technique using 14 C-labeled substrate. OMG transport showed an apparently saturable component with a K/sub m/ of 110 micromolar, and was completely inhibited by 10 micromolar carbonyl cyanide m-chlorophenylhydrazone. Polyclonal antibodies against solubilized native tonoplast H + -ATPase and its 62 and 72 kilodalton subunits were assayed for their ability to inhibit proton pumping and OMG accumulation. Antibodies against both the native enzyme and the putative catalytic subunit strongly inhibited proton pumping and OMG transport whereas antibodies against the 62 kilodalton subunit had only a slight effect on both processes

  10. Biochar and lignite affect H+-ATPase and H+-PPase activities in root tonoplast and nutrient contents of mung bean under salt stress.

    Science.gov (United States)

    Torabian, Shahram; Farhangi-Abriz, Salar; Rathjen, Judith

    2018-05-31

    This research was conducted to evaluate effects of biochar (50 and 100 g kg -1 soil) and lignite (50 and 100 g kg -1 soil) treatments on H + -ATPase and H + -PPase activity of root tonoplast, nutrient content, and performance of mung bean under salt stress. High saline conditions increased H + -ATPase and H + -PPase activities in root tonoplast, sodium (Na) content, reactive oxygen species (H 2 O 2 and O 2 - ) generation, relative electrolyte leakage (REL) and 2,2-Diphenyl-1-picrylhydrazyl (DPPH) activity in root and leaf, but decreased relative water content (RWC), chlorophyll content index, leaf area, potassium (K), calcium (Ca), magnesium (Mg), zinc (Zn) and iron (Fe) content of plant tissues, root and shoot dry weight of mung bean. Lignite and biochar treatments decreased the H + -ATPase and H + -PPase activities of root tonoplast under salt stress. Moreover, these treatments increased the cation exchange capacity of soil and nutrient values in plant tissues. Biochar and lignite diminished the generation of reactive oxygen species and DPPH activity in root and leaf cells, and these superior effects improved chlorophyll content index, leaf area and growth of mung bean under both conditions. In general, the results of this study demonstrated that biochar and lignite decreased the entry of Na ion into the cells, enriched plant cells with nutrients, and consequently improved mung bean performance under salt toxicity. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

  11. Photolabeling of tonoplast from sugar beet cell suspensions by [3H]-MIA, an inhibitor of the vacuolar Na+/H+ antiport

    International Nuclear Information System (INIS)

    Barkla, B.J.; Blumwald, E.

    1990-01-01

    A radiolabeled amiloride analog, [ 3 H]-MIA, was used for equilibrium binding studies and photolabeling of purified tonoplast vesicles. Scatchard analysis revealed a high affinity binding component with a K 4 of 1.4 μM which is closely related to constants of inhibition obtained for Na + -dependent H + efflux (5.9 μM) and pH-dependent 22 Na + influx (2.5 μM). This suggests that the high affinity component represents a class of sites associated with the Na + /H + antiport. Photolabeling of tonoplast with [ 3 H]-MIA in the presence of amiloride revealed the presence of two classes of receptors with distinct affinities for MIA, possibly representing the Na + /H + antiport and the Na + channel. In order to identify these receptors, amiloride analogues specific for the Na + /H + antiport or the Na + channel are being used to protect differentially against labeling of tonoplast proteins by photo-irradiation of [ 3 H]-MIA

  12. Structural studies of the vacuolar membrane ATPase from Neurospora crassa and comparison with the tonoplast membrane ATPase and Zea mays

    International Nuclear Information System (INIS)

    Bowman, E.J.; Mandala, S.; Taiz, L.; Bowman, B.J.

    1986-01-01

    The H + translocating ATPase located on vacuolar membranes of Neurospora crassa was partially purified by solubilization in two detergents, Triton X-100 and N-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, followed by centrifugation on sucrose density gradients. Two polypeptides of M/sub r/ ≅ 70,000 and ≅ 62,000 consistently migrated with activity, along with several minor bands of lower molecular weight. Radioactively labeled inhibitors of ATPase activity, N-[ 14 C]ethylmaleimide and 7-chloro-4-nitro[ 14 C]benzo-2-oxa-1,3-diazole, labeled the M/sub r/ ≅ 70,000 polypeptide; this labeling was reduced in the presence of ATP. N,N'-[ 14 C]dicyclohexylcarbodiimide labeled a polypeptide of M/sub r/ ≅ 15,000. Estimation of the functional size of the vacuolar membrane ATPase by radiation inactivation gave a value of M/sub r/ 5.2 x 10 5 , 10-15% larger than the mitochondrial ATPase. The Neurospora vacuolar ATPase showed no crossreactivity with antiserum to plasma membrane or mitochrondrial ATPase but stongly crossreacted with antiserum against a polypeptide of M/sub r/ ≅ 70,000 associated with the tonoplast ATPase of corn coleoptiles. These results suggest that fungal and plant vacuolar ATPases may be large multisubunit complexes, somewhat similar to, but immunologically distinct from, known F 0 F 1 ATPases

  13. Overexpression of the tonoplast aquaporin AtTIP5;1 conferred tolerance to boron toxicity in Arabidopsis.

    Science.gov (United States)

    Pang, Yongqi; Li, Lijuan; Ren, Fei; Lu, Pingli; Wei, Pengcheng; Cai, Jinghui; Xin, Lingguo; Zhang, Juan; Chen, Jia; Wang, Xuechen

    2010-06-01

    Boron (B) toxicity to plants is responsible for low crop productivity in many regions of the world. Here we report a novel and effective means to alleviate the B toxicity to plants under high B circumstance. Functional characterization of AtTIP5;1, an aquaporin gene, revealed that overexpression of AtTIP5;1 (OxAtTIP5;1) in Arabidopsis significantly increased its tolerance to high B toxicity. Compared to wild-type plants, OxAtTIP5;1 plants exhibited longer hypocotyls, accelerated development, increased silique production under high B treatments. GUS staining and quantitative RT-PCR (qRT-PCR) results demonstrated that the expression of AtTIP5;1 was induced by high B concentration treatment. Subcellular localization analysis revealed that the AtTIP5;1-GFP fusion protein was localized on the tonoplast membrane, which was consistent with the prediction based on bioinformatics. Taken together, our results suggest that AtTIP5;1 is involved in B transport pathway possibly via vacuolar compartmentation for B, and that overexpression of AtTIP5;1 in plants may provide an effective way to overcome the problem resulting from high B concentration toxicity. Copyright 2010 Institute of Genetics and Developmental Biology and the Genetics Society of China. Published by Elsevier Ltd. All rights reserved.

  14. Isolation of Highly Purified Fractions of Plasma Membrane and Tonoplast from the Same Homogenate of Soybean Hypocotyls by Free-Flow Electrophoresis 1

    Science.gov (United States)

    Sandelius, Anna Stina; Penel, Claude; Auderset, Guy; Brightman, Andrew; Millard, Merle; Morré, D. James

    1986-01-01

    A procedure is described whereby highly purified fractions of plasma membrane and tonoplast were isolated from hypocotyls of dark-grown soybean (Glycine max L. var Wayne) by the technique of preparative free-flow electrophoresis. Fractions migrating the slowest toward the anode were enriched in thick (10 nanometers) membranes identified as plasma membranes based on ability to bind N-1-naphthylphthalamic acid (NPA), glucan synthetase-II, and K+-stimulated, vanadate-inhibited Mg2+ ATPase, reaction with phosphotungstic acid at low pH on electron microscope sections, and morphological evaluations. Fractions migrating farthest toward the anode (farthest from the point of sample injection) were enriched in membrane vesicles with thick (7-9 nanometers) membranes that did not stain with phosphotungstic acid at low pH, contained a nitrate-inhibited, Cl-stimulated ATPase and had the in situ morphological characteristics of tonoplast including the presence of flocculent contents. These vesicles neither bound NPA nor contained levels of glucan synthetase II above background. Other membranous cell components such as dictyosomes (fucosyltransferase, latent nucleosidediphosphate phosphatase), endoplasmic reticulum vesicles (NADH- and NADPH- cytochrome c reductase), mitochondria (succinate-2(p-indophenyl)-3-p-nitrophenyl)-5-phenyl tetrazolium-reductase and cytochrome oxidase) and plastids (carotenoids and monogalactosyl diglyceride synthetase) were identified on the basis of appropriate marker constituents and, except for plastid thylakoids, had thin (marker activities. From electron microscope morphometry (using both membrane measurements and staining with phosphotungstic acid at low pH) and analysis of marker enzymes, both plasma membrane and tonoplast fractions were estimated to be about 90% pure. Neither fraction appeared to be contaminated by the other by more than 3%. Images Fig. 1 Fig. 3 Fig. 4 Fig. 5 Fig. 9 PMID:16664771

  15. DsSWEET17, a Tonoplast-Localized Sugar Transporter from Dianthus spiculifolius, Affects Sugar Metabolism and Confers Multiple Stress Tolerance in Arabidopsis

    Directory of Open Access Journals (Sweden)

    Aimin Zhou

    2018-05-01

    Full Text Available Plant SWEETs (Sugars Will Eventually be Exported Transporters affect the growth of plants by regulating the transport of sugar from source to sink and its intracellular transport between different organelles. In this study, DsSWEET17 from Dianthus spiculifolius was identified and characterized. Real-time quantitative PCR analysis revealed that the expression of DsSWEET17 was affected by exogenous application of fructose and glucose as well as under salt, osmotic, and oxidation stress. Colocalization experiments showed that the DsSWEET17-GFP (green fluorescent protein fusion protein was localized to the FM4-64-labeled tonoplasts in Arabidopsis. Compared to the wild type, the transgenic Arabidopsis seedlings overexpressing DsSWEET17 had longer roots, greater fresh weight, and a faster root growth upon exogenous application of fructose. Furthermore, transgenic Arabidopsis seedlings had significantly higher fructose accumulation than was observed for the wild-type seedlings. The analysis of root length revealed that transgenic Arabidopsis had higher tolerance to salt, osmotic, and oxidative stresses. Taken together, our results suggest that DsSWEET17 may be a tonoplast sugar transporter, and its overexpression affects sugar metabolism and confers multiple stress tolerance in Arabidopsis.

  16. DsSWEET17, a Tonoplast-Localized Sugar Transporter from Dianthus spiculifolius, Affects Sugar Metabolism and Confers Multiple Stress Tolerance in Arabidopsis.

    Science.gov (United States)

    Zhou, Aimin; Ma, Hongping; Feng, Shuang; Gong, Shufang; Wang, Jingang

    2018-05-24

    Plant SWEETs (Sugars Will Eventually be Exported Transporters) affect the growth of plants by regulating the transport of sugar from source to sink and its intracellular transport between different organelles. In this study, DsSWEET17 from Dianthus spiculifolius was identified and characterized. Real-time quantitative PCR analysis revealed that the expression of DsSWEET17 was affected by exogenous application of fructose and glucose as well as under salt, osmotic, and oxidation stress. Colocalization experiments showed that the DsSWEET17-GFP (green fluorescent protein) fusion protein was localized to the FM4-64-labeled tonoplasts in Arabidopsis . Compared to the wild type, the transgenic Arabidopsis seedlings overexpressing DsSWEET17 had longer roots, greater fresh weight, and a faster root growth upon exogenous application of fructose. Furthermore, transgenic Arabidopsis seedlings had significantly higher fructose accumulation than was observed for the wild-type seedlings. The analysis of root length revealed that transgenic Arabidopsis had higher tolerance to salt, osmotic, and oxidative stresses. Taken together, our results suggest that DsSWEET17 may be a tonoplast sugar transporter, and its overexpression affects sugar metabolism and confers multiple stress tolerance in Arabidopsis .

  17. Knock-down of a tonoplast localized low-affinity nitrate transporter OsNPF7.2 affects rice growth under high nitrate ssupply

    Directory of Open Access Journals (Sweden)

    Rui Hu

    2016-10-01

    Full Text Available The large nitrate transporter 1/peptide transporter family (NPF has been shown to transport diverse substrates, including nitrate, amino acids, peptides, phytohormones, and glucosinolates. However, the rice (Oryza sativa root-specific expressed member OsNPF7.2 has not been characterized. Here, our data show that OsNPF7.2 is a tonoplast localized low-affinity nitrate transporter, and affects rice growth under high nitrate supply. The expression analysis showed that OsNPF7.2 was mainly expressed in the elongation and maturation zones of roots, especially in the root sclerenchyma, cortex and stele. It was also induced by high concentrations of nitrate. Subcellular localization analysis showed that OsNPF7.2 was localized on the tonoplast of large and small vacuoles. Heterogenous expression in Xenopus laevis oocytes suggested that OsNPF7.2 was a low-affinity nitrate transporter. Knock-down of OsNPF7.2 retarded rice growth under high concentrations of nitrate. Therefore, we deduce that OsNPF7.2 plays a role in intracellular allocation of nitrate in roots, and thus influences rice growth under high nitrate supply.

  18. Arabidopsis senescence-associated protein DMP1 is involved in membrane remodeling of the ER and tonoplast

    Directory of Open Access Journals (Sweden)

    Kasaras Alexis

    2012-04-01

    Full Text Available Abstract Background Arabidopsis DMP1 was discovered in a genome-wide screen for senescence-associated membrane proteins. DMP1 is a member of a novel plant-specific membrane protein family of unknown function. In rosette leaves DMP1 expression increases from very low background level several 100fold during senescence progression. Results Expression of AtDMP1 fused to eGFP in Nicotiana benthamiana triggers a complex process of succeeding membrane remodeling events affecting the structure of the endoplasmic reticulum (ER and the vacuole. Induction of spherical structures (“bulbs”, changes in the architecture of the ER from tubular to cisternal elements, expansion of smooth ER, formation of crystalloid ER, and emergence of vacuolar membrane sheets and foamy membrane structures inside the vacuole are proceeding in this order. In some cells it can be observed that the process culminates in cell death after breakdown of the entire ER network and the vacuole. The integrity of the plasma membrane, nucleus and Golgi vesicles are retained until this stage. In Arabidopsis thaliana plants expressing AtDMP1-eGFP by the 35S promoter massive ER and vacuole vesiculation is observed during the latest steps of leaf senescence, whereas earlier in development ER and vacuole morphology are not perturbed. Expression by the native DMP1 promoter visualizes formation of aggregates termed “boluses” in the ER membranes and vesiculation of the entire ER network, which precedes disintegration of the central vacuole during the latest stage of senescence in siliques, rosette and cauline leaves and in darkened rosette leaves. In roots tips, DMP1 is strongly expressed in the cortex undergoing vacuole biogenesis. Conclusions Our data suggest that DMP1 is directly or indirectly involved in membrane fission during breakdown of the ER and the tonoplast during leaf senescence and in membrane fusion during vacuole biogenesis in roots. We propose that these properties of DMP1

  19. Modulation of proton pumping across proteoliposome membranes reconstituted with tonoplast H(+)-ATPase from cultured rice (Oryza sativa L. var. Boro) cells by acyl steryl glucoside and steryl glucoside.

    Science.gov (United States)

    Yamaguchi, Mineo; Kasamo, Kunihiro

    2002-07-01

    Tonoplast H(+)-ATPase purified from cultured rice cells (Oryza sativa L. var. Boro) was reconstituted into asolectin liposomes containing steryl glucoside (SG) or acyl steryl glucoside (ASG), and the effects of SG and ASG on proton pumping, ATP-hydrolysis activity and proton permeability of the proteoliposome membranes were investigated. In the proteoliposomes containing 10 mol% SG, proton pumping and ATP-hydrolysis activity were increased to around 140% of those in SG-free proteoliposomes. In the proteoliposomes containing ASG, proton pumping and ATP-hydrolysis activity were decreased to one-tenth of those in ASG-free proteoliposomes at 15 mol% ASG; however, activity increased again slightly in the range between 20 and 40 mol% ASG. The change in proton pumping across the proteoliposome membrane is not due to a change of proteoliposome size nor to the location of the catalytic site of the tonoplast H(+)-ATPase in the proteoliposomes. SG and ASG also reduced the passive proton permeability of the proteoliposomes. These results show that SG and ASG modulate proton pumping across the tonoplast toward stimulation and depression, respectively, and they reduce the passive proton permeability of the tonoplast.

  20. Alamethicin permeabilizes the plasma membrane and mitochondria but not the tonoplast in tobacco (Nicotiana tabacum L. cv Bright Yellow) suspension cells

    DEFF Research Database (Denmark)

    Matic, S.; Geisler, D.A.; Møller, I.M.

    2005-01-01

    remained intact, as indicated by an unaffected tonoplast proton gradient. Low-flux permeabilization of plasma membranes and mitochondria at moderate AlaM concentrations was reversible and did not affect cell vigour. Higher AlaM concentrations induced cell death. After the addition of catalase that removes...... concentrations. Possible uses and limitations of this method for plant cell research are discussed.......The ion channel-forming peptide AlaM (alamethicin) is known to permeabilize isolated mitochondria as well as animal cells. When intact tobacco (Nicotiana tabacum L.) Bright Yellow-2 cells were treated with AlaM, the cells became permeable for low-molecular-mass molecules as shown by induced leakage...

  1. Reduced Tonoplast Fast-Activating and Slow-Activating Channel Activity Is Essential for Conferring Salinity Tolerance in a Facultative Halophyte, Quinoa1[C][W][OA

    Science.gov (United States)

    Bonales-Alatorre, Edgar; Shabala, Sergey; Chen, Zhong-Hua; Pottosin, Igor

    2013-01-01

    Halophyte species implement a “salt-including” strategy, sequestering significant amounts of Na+ to cell vacuoles. This requires a reduction of passive Na+ leak from the vacuole. In this work, we used quinoa (Chenopodium quinoa) to investigate the ability of halophytes to regulate Na+-permeable slow-activating (SV) and fast-activating (FV) tonoplast channels, linking it with Na+ accumulation in mesophyll cells and salt bladders as well as leaf photosynthetic efficiency under salt stress. Our data indicate that young leaves rely on Na+ exclusion to salt bladders, whereas old ones, possessing far fewer salt bladders, depend almost exclusively on Na+ sequestration to mesophyll vacuoles. Moreover, although old leaves accumulate more Na+, this does not compromise their leaf photochemistry. FV and SV channels are slightly more permeable for K+ than for Na+, and vacuoles in young leaves express less FV current and with a density unchanged in plants subjected to high (400 mm NaCl) salinity. In old leaves, with an intrinsically lower density of the FV current, FV channel density decreases about 2-fold in plants grown under high salinity. In contrast, intrinsic activity of SV channels in vacuoles from young leaves is unchanged under salt stress. In vacuoles of old leaves, however, it is 2- and 7-fold lower in older compared with young leaves in control- and salt-grown plants, respectively. We conclude that the negative control of SV and FV tonoplast channel activity in old leaves reduces Na+ leak, thus enabling efficient sequestration of Na+ to their vacuoles. This enables optimal photosynthetic performance, conferring salinity tolerance in quinoa species. PMID:23624857

  2. Impaired pH homeostasis in Arabidopsis lacking the vacuolar dicarboxylate transporter and analysis of carboxylic acid transport across the tonoplast.

    Science.gov (United States)

    Hurth, Marco Alois; Suh, Su Jeoung; Kretzschmar, Tobias; Geis, Tina; Bregante, Monica; Gambale, Franco; Martinoia, Enrico; Neuhaus, H Ekkehard

    2005-03-01

    Arabidopsis (Arabidopsis thaliana) mutants lacking the tonoplastic malate transporter AttDT (A. thaliana tonoplast dicarboxylate transporter) and wild-type plants showed no phenotypic differences when grown under standard conditions. To identify putative metabolic changes in AttDT knock-out plants, we provoked a metabolic scenario connected to an increased consumption of dicarboxylates. Acidification of leaf discs stimulated dicarboxylate consumption and led to extremely low levels of dicarboxylates in mutants. To investigate whether reduced dicarboxylate concentrations in mutant leaf cells and, hence, reduced capacity to produce OH(-) to overcome acidification might affect metabolism, we measured photosynthetic oxygen evolution under conditions where the cytosol is acidified. AttDT::tDNA protoplasts showed a much stronger inhibition of oxygen evolution at low pH values when compared to wild-type protoplasts. Apparently citrate, which is present in higher amounts in knock-out plants, is not able to replace dicarboxylates to overcome acidification. To raise more information on the cellular level, we performed localization studies of carboxylates. Although the total pool of carboxylates in mutant vacuoles was nearly unaltered, these organelles contained a lower proportion of malate and fumarate and a higher proportion of citrate when compared to wild-type vacuoles. These alterations concur with the observation that radioactively labeled malate and citrate are transported into Arabidopsis vacuoles by different carriers. In addition, wild-type vacuoles and corresponding organelles from AttDT::tDNA mutants exhibited similar malate channel activities. In conclusion, these results show that Arabidopsis vacuoles contain at least two transporters and a channel for dicarboxylates and citrate and that the activity of AttDT is critical for regulation of pH homeostasis.

  3. Impaired pH Homeostasis in Arabidopsis Lacking the Vacuolar Dicarboxylate Transporter and Analysis of Carboxylic Acid Transport across the Tonoplast1

    Science.gov (United States)

    Hurth, Marco Alois; Suh, Su Jeoung; Kretzschmar, Tobias; Geis, Tina; Bregante, Monica; Gambale, Franco; Martinoia, Enrico; Neuhaus, H. Ekkehard

    2005-01-01

    Arabidopsis (Arabidopsis thaliana) mutants lacking the tonoplastic malate transporter AttDT (A. thaliana tonoplast dicarboxylate transporter) and wild-type plants showed no phenotypic differences when grown under standard conditions. To identify putative metabolic changes in AttDT knock-out plants, we provoked a metabolic scenario connected to an increased consumption of dicarboxylates. Acidification of leaf discs stimulated dicarboxylate consumption and led to extremely low levels of dicarboxylates in mutants. To investigate whether reduced dicarboxylate concentrations in mutant leaf cells and, hence, reduced capacity to produce OH− to overcome acidification might affect metabolism, we measured photosynthetic oxygen evolution under conditions where the cytosol is acidified. AttDT::tDNA protoplasts showed a much stronger inhibition of oxygen evolution at low pH values when compared to wild-type protoplasts. Apparently citrate, which is present in higher amounts in knock-out plants, is not able to replace dicarboxylates to overcome acidification. To raise more information on the cellular level, we performed localization studies of carboxylates. Although the total pool of carboxylates in mutant vacuoles was nearly unaltered, these organelles contained a lower proportion of malate and fumarate and a higher proportion of citrate when compared to wild-type vacuoles. These alterations concur with the observation that radioactively labeled malate and citrate are transported into Arabidopsis vacuoles by different carriers. In addition, wild-type vacuoles and corresponding organelles from AttDT::tDNA mutants exhibited similar malate channel activities. In conclusion, these results show that Arabidopsis vacuoles contain at least two transporters and a channel for dicarboxylates and citrate and that the activity of AttDT is critical for regulation of pH homeostasis. PMID:15728336

  4. The tonoplast intrinsic aquaporin (TIP) subfamily of Eucalyptus grandis: Characterization of EgTIP2, a root-specific and osmotic stress-responsive gene.

    Science.gov (United States)

    Rodrigues, Marcela I; Bravo, Juliana P; Sassaki, Flávio T; Severino, Fábio E; Maia, Ivan G

    2013-12-01

    Aquaporins have important roles in various physiological processes in plants, including growth, development and adaptation to stress. In this study, a gene encoding a root-specific tonoplast intrinsic aquaporin (TIP) from Eucalyptus grandis (named EgTIP2) was investigated. The root-specific expression of EgTIP2 was validated over a panel of five eucalyptus organ/tissues. In eucalyptus roots, EgTIP2 expression was significantly induced by osmotic stress imposed by PEG treatment. Histochemical analysis of transgenic tobacco lines (Nicotiana tabacum SR1) harboring an EgTIP2 promoter:GUS reporter cassette revealed major GUS staining in the vasculature and in root tips. Consistent with its osmotic-stress inducible expression in eucalyptus, EgTIP2 promoter activity was up-regulated by mannitol treatment, but was down-regulated by abscisic acid. Taken together, these results suggest that EgTIP2 might be involved in eucalyptus response to drought. Additional searches in the eucalyptus genome revealed the presence of four additional putative TIP coding genes, which could be individually assigned to the classical TIP1-5 groups. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  5. A tonoplast sugar transporter underlies a sugar accumulation QTL in watermelon

    Science.gov (United States)

    The molecular mechanism controlling accumulation of soluble sugars in watermelon (Citrullus lanatus) fruit, a trait associated with sweet-dessert watermelon domestication, is still unknown. We re-sequenced 96 recombinant inbred lines, derived from a cross between sweet and unsweet watermelon accessi...

  6. Tonoplast- and plasma membrane-localized aquaporin-family transporters in blue hydrangea sepals of aluminum hyperaccumulating plant.

    Directory of Open Access Journals (Sweden)

    Takashi Negishi

    Full Text Available Hydrangea (Hydrangea macrophylla is tolerant of acidic soils in which toxicity generally arises from the presence of the soluble aluminum (Al ion. When hydrangea is cultivated in acidic soil, its resulting blue sepal color is caused by the Al complex formation of anthocyanin. The concentration of vacuolar Al in blue sepal cells can reach levels in excess of approximately 15 mM, suggesting the existence of an Al-transport and/or storage system. However, until now, no Al transporter has been identified in Al hyperaccumulating plants, animals or microorganisms. To identify the transporter being responsible for Al hyperaccumulation, we prepared a cDNA library from blue sepals according to the sepal maturation stage, and then selected candidate genes using a microarray analysis and an in silico study. Here, we identified the vacuolar and plasma membrane-localized Al transporters genes vacuolar Al transporter (VALT and plasma membrane Al transporter 1 (PALT1, respectively, which are both members of the aquaporin family. The localization of each protein was confirmed by the transient co-expression of the genes. Reverse transcription-PCR and immunoblotting results indicated that VALT and PALT1 are highly expressed in sepal tissue. The overexpression of VALT and PALT1 in Arabidopsis thaliana conferred Al-tolerance and Al-sensitivity, respectively.

  7. Amino acid transport across the tonoplast of vacuoles isolated from barley mesophyll protoplasts: Uptake of alanine, leucine, and glutamine

    International Nuclear Information System (INIS)

    Dietz, K.J.; Jaeger, R.; Kaiser, G.; Martinoia, E.

    1990-01-01

    Mesophyll protoplasts from leaves of well-fertilized barley (Hordeum vulgare L.) plants contained amino acids at concentrations as high as 120 millimoles per liter. With the exception of glutamic acid, which is predominantly localized in the cytoplasm, a major part of all other amino acids was contained inside the large central vacuole. Alanine, leucine, and glutamine are the dominant vacuolar amino acids in barley. Their transport into isolated vacuoles was studied using 14 C-labeled amino acids. Uptake was slow in the absence of ATP. A three- to sixfold stimulation of uptake was observed after addition of ATP or adenylyl imidodiphosphate an ATP analogue not being hydrolyzed by ATPases. Other nucleotides were ineffective in increasing the rate of uptake. ATP-Stimulated amino acid transport was not dependent on the transtonoplast pH or membrane potential. p-Chloromercuriphenylsulfonic acid and n-ethyl maleimide increased transport independently of ATP. Neutral amino acids such as valine or leucine effectively decreased the rate of alanine transport. Glutamine and glycine were less effective or not effective as competitive inhibitors of alanine transport. The results indicate the existence of a uniport translocator specific for neutral or basic amino acids that is under control of metabolic effectors

  8. TRANSPARENT TESTA 13 is a tonoplast P3A -ATPase required for vacuolar deposition of proanthocyanidins in Arabidopsis thaliana seeds

    NARCIS (Netherlands)

    Appelhagen, I.; Nordholt, N.; Seidel, T.; Spelt, K.; Koes, R.; Quattrochio, F.; Sagasser, M.; Weisshaar, B.

    2015-01-01

    Intracellular pH homeostasis is essential for all living cells. In plants, pH is usually maintained by three structurally distinct and differentially localized types of proton pump: P-type H(+) -ATPases in the plasma membrane, and multimeric vacuolar-type H(+) -ATPases (V-ATPases) and vacuolar H(+)

  9. Molecular cloning and characterization of the plasma membrane ...

    African Journals Online (AJOL)

    User

    2011-05-09

    May 9, 2011 ... Southern blot analysis indicated that OvPIP gene was present in O. ... Key words: Orychophragmus violaceus, plasma membrane, tonoplast aquaporins .... fractionated by 0.8% agarose gel electrophoresis and transferred to.

  10. The Arabidopsis cax1 mutant exhibits impaired ion homeostasis, development, and hormonal responses and reveals interplay among vacuolar transporters.

    Science.gov (United States)

    Cheng, Ning-Hui; Pittman, Jon K; Barkla, Bronwyn J; Shigaki, Toshiro; Hirschi, Kendal D

    2003-02-01

    The Arabidopsis Ca(2+)/H(+) transporter CAX1 (Cation Exchanger1) may be an important regulator of intracellular Ca(2+) levels. Here, we describe the preliminary localization of CAX1 to the tonoplast and the molecular and biochemical characterization of cax1 mutants. We show that these mutants exhibit a 50% reduction in tonoplast Ca(2+)/H(+) antiport activity, a 40% reduction in tonoplast V-type H(+)-translocating ATPase activity, a 36% increase in tonoplast Ca(2+)-ATPase activity, and increased expression of the putative vacuolar Ca(2+)/H(+) antiporters CAX3 and CAX4. Enhanced growth was displayed by the cax1 lines under Mn(2+) and Mg(2+) stress conditions. The mutants exhibited altered plant development, perturbed hormone sensitivities, and altered expression of an auxin-regulated promoter-reporter gene fusion. We propose that CAX1 regulates myriad plant processes and discuss the observed phenotypes with regard to the compensatory alterations in other transporters.

  11. The Arabidopsis cax1 Mutant Exhibits Impaired Ion Homeostasis, Development, and Hormonal Responses and Reveals Interplay among Vacuolar Transporters

    Science.gov (United States)

    Cheng, Ning-Hui; Pittman, Jon K.; Barkla, Bronwyn J.; Shigaki, Toshiro; Hirschi, Kendal D.

    2003-01-01

    The Arabidopsis Ca2+/H+ transporter CAX1 (Cation Exchanger1) may be an important regulator of intracellular Ca2+ levels. Here, we describe the preliminary localization of CAX1 to the tonoplast and the molecular and biochemical characterization of cax1 mutants. We show that these mutants exhibit a 50% reduction in tonoplast Ca2+/H+ antiport activity, a 40% reduction in tonoplast V-type H+-translocating ATPase activity, a 36% increase in tonoplast Ca2+-ATPase activity, and increased expression of the putative vacuolar Ca2+/H+ antiporters CAX3 and CAX4. Enhanced growth was displayed by the cax1 lines under Mn2+ and Mg2+ stress conditions. The mutants exhibited altered plant development, perturbed hormone sensitivities, and altered expression of an auxin-regulated promoter-reporter gene fusion. We propose that CAX1 regulates myriad plant processes and discuss the observed phenotypes with regard to the compensatory alterations in other transporters. PMID:12566577

  12. Reference: 531 [Arabidopsis Phenome Database[Archive

    Lifescience Database Archive (English)

    Full Text Available 1 double mutant was more sensitive to Zn than either of the single mutants, suggesting ZIF1 influences a dis...a Zn ligand complex into vacuoles. A novel major facilitator superfamily protein at the tonoplast influences

  13. Identification of RAPD markers linked to salinity tolerance in wheat

    African Journals Online (AJOL)

    armghan_shehzad

    The individual plants from F2 population segregation for salinity tolerance and the parents ..... plasma membrane Na+/H+ antiporters that are energized by proton gradients ... tonoplast by vascular Na+/ H+ antiporters into the cell vacuoles ...

  14. Expression of Water Channel Proteins in Mesembryanthemum crystallinum1

    Science.gov (United States)

    Kirch, Hans-Hubert; Vera-Estrella, Rosario; Golldack, Dortje; Quigley, Francoise; Michalowski, Christine B.; Barkla, Bronwyn J.; Bohnert, Hans J.

    2000-01-01

    We have characterized transcripts for nine major intrinsic proteins (MIPs), some of which function as water channels (aquaporins), from the ice plant Mesembryanthemum crystallinum. To determine the cellular distribution and expression of these MIPs, oligopeptide-based antibodies were generated against MIP-A, MIP-B, MIP-C, or MIP-F, which, according to sequence and functional characteristics, are located in the plasma membrane (PM) and tonoplast, respectively. MIPs were most abundant in cells involved in bulk water flow and solute flux. The tonoplast MIP-F was found in all cells, while signature cell types identified different PM-MIPs: MIP-A predominantly in phloem-associated cells, MIP-B in xylem parenchyma, and MIP-C in the epidermis and endodermis of immature roots. Membrane protein analysis confirmed MIP-F as tonoplast located. MIP-A and MIP-B were found in tonoplast fractions and also in fractions distinct from either the tonoplast or PM. MIP-C was most abundant but not exclusive to PM fractions, where it is expected based on its sequence signature. We suggest that within the cell, MIPs are mobile, which is similar to aquaporins cycling through animal endosomes. MIP cycling and the differential regulation of these proteins observed under conditions of salt stress may be fundamental for the control of tissue water flux. PMID:10806230

  15. Cell-free transfer of sterols by plant fractions

    International Nuclear Information System (INIS)

    Morre, D.J.; Wilkinson, F.E.; Morre, D.M.; Moreau, P.; Sandelius, A.S.; Penel, C.; Greppin, H.

    1990-01-01

    Microsomes from etiolated hypocotyls of soybean or leaves of light-grown spinach radiolabeled in vivo with [ 3 H]acetate or in vitro with [ 3 H]squalene or [ 3 H]cholesterol as donor transferred radioactivity to unlabeled acceptor membranes immobilized on nitrocellulose. Most efficient transfer was with plasma membrane or tonoplast as the acceptor. The latter were highly purified by aqueous two-phase partition (plasma membrane) and preparative free-flow electrophoresis (tonoplast and plasma membrane). Plasma membrane- and tonoplast-free microsomes and purified mitochondria were less efficient acceptors. Sterol transfer was verified by thin-layer chromatography of extracted lipids. Transfer was time- and temperature-dependent, required ATP but was not promoted by cytosol. The nature of the donor (endoplasmic reticulum, Golgi apparatus or both) and of the transfer mechanism is under investigation

  16. Effects of ammonium nitrate, cesium chloride and ...

    African Journals Online (AJOL)

    Jane

    2011-10-12

    Oct 12, 2011 ... Full Length Research Paper. Effects of ... constitutes 2 to 10% of plant dry weight (Leigh et al.,. 1984 ... membrane proteins has been reported, including K+ channels ... have functions in the plasma membrane and tonoplast. (Senn et al. .... analysis of K+ uptake in a solution containing 50 µM K+ showed a ...

  17. Vacuole Integrity Maintained by DUF300 Proteins Is Required for Brassinosteroid Signaling Regulation

    Czech Academy of Sciences Publication Activity Database

    Liu, Q.; Vain, T.; Viotti, C.; Doyle, S. M.; Tarkowská, Danuše; Novák, Ondřej; Zipfel, C.; Sitbon, F.; Robert, S.; Hofius, D.

    2018-01-01

    Roč. 11, č. 4 (2018), s. 553-567 ISSN 1674-2052 R&D Projects: GA MŠk(CZ) LO1204 Institutional support: RVO:61389030 Keywords : Arabidopsis * brassinosteroid signaling * DUF300 proteins * tonoplast * vacuole integrity Subject RIV: EB - Genetics ; Molecular Biology OBOR OECD: Plant sciences, botany Impact factor: 8.827, year: 2016

  18. Intracellular localization of Na + /H + antiporter from Malus zumi ...

    African Journals Online (AJOL)

    In this study, we examined the intracellular localization of the product of Na+/H+ antiporter gene (MzNHX1) cloned from Malus zumi. Analysis using yeast cells expressing a fusion protein of MzNHX1 and green fluorescent protein confirmed the localization of MzNHX1 on the tonoplast.

  19. Salt stress in Plantago - The role of membranes, channels and pumps

    NARCIS (Netherlands)

    Prins, HBA

    1995-01-01

    In the present article the cellular mechanism of Na+ transport across the plasma membrane and tonoplast of root cells of Plantago media (salt sensitive) and Plantago maritima (salt tolerant) is discussed based on findings obtained mainly by patch clamp technique. It is conluded that the combination

  20. Ion Exchangers NHX1 and NHX2 Mediate Active Potassium Uptake into Vacuoles to Regulate Cell Turgor and Stomatal Function in Arabidopsis[W][OA

    Science.gov (United States)

    Barragán, Verónica; Leidi, Eduardo O.; Andrés, Zaida; Rubio, Lourdes; De Luca, Anna; Fernández, José A.; Cubero, Beatriz; Pardo, José M.

    2012-01-01

    Intracellular NHX proteins are Na+,K+/H+ antiporters involved in K+ homeostasis, endosomal pH regulation, and salt tolerance. Proteins NHX1 and NHX2 are the two major tonoplast-localized NHX isoforms. Here, we show that NHX1 and NHX2 have similar expression patterns and identical biochemical activity, and together they account for a significant amount of the Na+,K+/H+ antiport activity in tonoplast vesicles. Reverse genetics showed functional redundancy of NHX1 and NHX2 genes. Growth of the double mutant nhx1 nhx2 was severely impaired, and plants were extremely sensitive to external K+. By contrast, nhx1 nhx2 mutants showed similar sensitivity to salinity stress and even greater rates of Na+ sequestration than the wild type. Double mutants had reduced ability to create the vacuolar K+ pool, which in turn provoked greater K+ retention in the cytosol, impaired osmoregulation, and compromised turgor generation for cell expansion. Genes NHX1 and NHX2 were highly expressed in guard cells, and stomatal function was defective in mutant plants, further compromising their ability to regulate water relations. Together, these results show that tonoplast-localized NHX proteins are essential for active K+ uptake at the tonoplast, for turgor regulation, and for stomatal function. PMID:22438021

  1. Intracellular localization of Na /H antiporter from Malus zumi (MzNHX1)

    African Journals Online (AJOL)

    PRECIOUS

    2009-12-15

    Dec 15, 2009 ... protein since 1980s. The exogenous protein can be located on different membrane in yeast cells, which is impossible in bacteria. There are ... porter protein which locates on the plasma membrane or the tonoplast, it is wise to use eGFP as fluorescence mark in yeast cells, because that the technology of this.

  2. Exchangers man the pumps: Functional interplay between proton pumps and proton-coupled Ca(2+) exchangers

    Science.gov (United States)

    Tonoplast-localised proton-coupled Ca(2+) transporters encoded by cation/H(+) exchanger (CAX) genes play a critical role in sequestering Ca(2+) into the vacuole. These transporters may function in coordination with Ca(2+) release channels, to shape stimulus-induced cytosolic Ca(2+) elevations. Recen...

  3. The plant vacuolar Na+/H+ antiport.

    Science.gov (United States)

    Barkla, B J; Apse, M P; Manolson, M F; Blumwald, E

    1994-01-01

    Salt stress imposes severe limitations on plant growth, however, the extent of growth reduction depends upon the soil salinity level and the plant species. One of the mechanisms employed by salt tolerant plants is the effective vacuolar compartmentalization of sodium. The sequestration of sodium into the vacuole occurs by the operation of a Na+/H+ antiport located at the tonoplast. Evidence for a plant vacuolar Na+/H+ antiport has been demonstrated in tissues, intact vacuoles and isolated tonoplast vesicles. In sugar beet cell suspensions, the activity of the vacuolar Na+/H+ antiport increased with increasing NaCl concentrations in the growth medium. This increased activity was correlated with the increased synthesis of a 170 kDa tonoplast polypeptide. In vivo labelling of tonoplast proteins showed the enhanced synthesis of the 170 kDa polypeptide not only upon exposure of the cells to salt, but also when the cells were grown in the presence of amiloride. Exposure of the cells to amiloride also resulted in increased vacuolar Na+/H+ antiport activity. Polyclonal antibodies raised against the 170 kDa polypeptide almost completely inhibited the antiport activity, suggesting the association of this protein with the plant vacuolar Na+/H+ antiport. Antibodies against the Na+/H+ antiport-associated polypeptide were used to screen a Beta lambda ZAP expression library. A partial clone of 1.65 kb was sequenced and found to encode a polypeptide with a putative transmembrane domain and a large hydrophilic C terminus. This clone showed no homology to any previously cloned gene at either the nucleic acid or the amino acid level.

  4. Exchangers man the pumps: Functional interplay between proton pumps and proton-coupled Ca2+ exchangers

    OpenAIRE

    Barkla, Bronwyn J; Hirschi, Kendal D; Pittman, Jon K

    2008-01-01

    Tonoplast-localised proton-coupled Ca2+ transporters encoded by cation/H+ exchanger (CAX) genes play a critical role in sequestering Ca2+ into the vacuole. These transporters may function in coordination with Ca2+ release channels, to shape stimulus-induced cytosolic Ca2+ elevations. Recent analysis of Arabidopsis CAX knockout mutants, particularly cax1 and cax3, identified a variety of phenotypes including sensitivity to abiotic stresses, which indicated that these transporters might play a ...

  5. Hydrolytic enzymes in the central vacuole of plant cells

    International Nuclear Information System (INIS)

    Boller, T.; Kende, H.

    1979-01-01

    The hydrolase content of vacuoles isolated from protoplasts of suspension-cultured tobacco cells, of tulip petals, and pineapple leaves, and the sedimentation behavior of tobacco tonoplasts were studied. Three precautions were found to be important for the analysis of vacuolar hydrolases and of the tonoplast: (a) purification of protoplasts in a Ficoll gradient was necessary to remove cell debris which contained contaminating hydrolases adsorbed from the fungal cell-wall-degrading enzyme preparation; (b) hydrolase activities in the homogenates of the intact cells or the tissue used and of the purified protoplasts had to be compared to verify the absence of contaminating hydrolases in the protoplast preparation; and (c) vacuoles obtained from the protoplasts by an osmotic shock had to be purified from the lysate in a Ficoll gradient. Since the density of the central vacuole approximates that of the protoplasts, about a 10% contamination of the vacuolar preparation by surviving protoplasts could not be eliminated. The intracellular activities of the following acid hydrolases were primarily localized in the vacuole of tobacco cells: α-mannosidase, β-N-acetylglucosaminidase, β-fructosidase, nuclease, phosphatase, phosphodiesterase. A similar composition of acid hydrolases was found in vacuoles obtained from protoplasts of tulip petals. Proteinase, a hydrolase with low activity in tobacco cells and tulip petals was found to be vacuolar in pineapple leaves, a tissue containing high levels of this enzyme. None of the vacuolar enzymes investigated ws found to be bound to the tonoplast. When vacuoles were isolated from cells labeled with radioactive choline, the vacuolar membrane was found to contain radioactivity. On sucrose gradients, the label incorporated into tonoplasts banded around a density of 1.10 grams per cubic centimeter

  6. Elektrophysiologische Untersuchungen an einzelligen marinen Riesenalgen

    OpenAIRE

    Ryser, Christoph

    2002-01-01

    Die kombinierte Mikroperfusions-/Ladungspulstechnik an einzelligen marinen Riesenalgen ermöglicht die getrennte Darstellung der elektrischen Eigenschaften von Plasmalemma und Tonoplast durch gezielte Manipulation des vakuolären und externen Mediums. Dabei kann der für die Physiologie der Zellen wichtige hydrostatische Innendruck (Turgor) ständig kontrolliert werden. Die Applikation eines Ladungspulses resultierte bei Valonia utricularis und Ventricaria ventricosa in einer biphasischen Relaxat...

  7. Akkumulation von L-Malat und D-Lactat in Arabidopsis thaliana und Laccase/HBT-vermittelte Delignifizierung von Spartina alterniflora und Phragmites australis

    OpenAIRE

    Heil, Alexander

    2016-01-01

    The current work contains two projects "Accumulation of L-malate and D-lactate in Arabidopsis thaliana" (A) "Laccase/HBT mediated delignification of Spartina alterniflora and Phragmites australis" (B). In project A, L-malate and D-lactate accumulated in A. thaliana plants. The accumulation of L-malate is carried out by modification of the plant metabolism with the enzymes PEPC, MDH and the tonoplast dicarboxylate transporter (TDT). Gene pepci2 (Hydrilla verticillata), mdh5 (Zea mays) and tdt ...

  8. Identification of a 170-kDa protein associated with the vacuolar Na+/H+ antiport of Beta vulgaris.

    Science.gov (United States)

    Barkla, B J; Blumwald, E

    1991-12-15

    The effect of the addition of amiloride to the growth medium was tested on the Na+/H+ antiport activity of tonoplast vesicles isolated from sugar beet (beta vulgaris L.) cell suspensions. Cells grown in the presence of NaCl and amiloride displayed an increased antiport activity. Analysis of the kinetic data showed that while the affinity of the antiport for Na+ ions did not change, the maximal velocity of the Na+/H+ exchange increased markedly. These results suggest the addition of more antiport molecules to the tonoplast and/or an increase in the turnover rate of the Na+/H+ exchange. The increase in activity of the antiport by the presence of amiloride was correlated with the enhanced synthesis of a tonoplast 170-kDa polypeptide. The increased synthesis of this polypeptide was detected not only upon exposure of the cells to amiloride but also when the cells were exposed to high NaCl concentrations. Polyclonal antibodies against the 170-kDa polypeptide almost completely inhibited the antiport activity. These results suggest the association of the 170-kDa polypeptide with the vacuolar Na+/H+ antiport.

  9. Vacuolar biogenesis and aquaporin expression at early germination of broad bean seeds.

    Science.gov (United States)

    Novikova, Galina V; Tournaire-Roux, Colette; Sinkevich, Irina A; Lityagina, Snejana V; Maurel, Christophe; Obroucheva, Natalie

    2014-09-01

    A key event in seed germination is water uptake-mediated growth initiation in embryonic axes. Vicia faba var. minor (broad bean) seeds were used for studying cell growth, vacuolar biogenesis, expression and function of tonoplast water channel proteins (aquaporins) in embryonic axes during seed imbibition, radicle emergence and growth. Hypocotyl and radicle basal cells showed vacuole restoration from protein storage vacuoles, whereas de novo vacuole formation from provacuoles was observed in cells newly produced by root meristem. cDNA fragments of seven novel aquaporin isoforms including five Tonoplast Intrinsic Proteins (TIP) from three sub-types were amplified by PCR. The expression was probed using q-RT-PCR and when possible with isoform-specific antibodies. Decreased expression of TIP3s was associated to the transformation of protein storage vacuoles to vacuoles, whereas enhanced expression of a TIP2 homologue was closely linked to the fast cell elongation. Water channel functioning checked by inhibitory test with mercuric chloride showed closed water channels prior to growth initiation and active water transport into elongating cells. The data point to a crucial role of tonoplast aquaporins during germination, especially during growth of embryonic axes, due to accelerated water uptake and vacuole enlargement resulting in rapid cell elongation. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

  10. Enhanced separation of membranes during free flow zonal electrophoresis in plants.

    Science.gov (United States)

    Barkla, Bronwyn J; Vera-Estrella, Rosario; Pantoja, Omar

    2007-07-15

    Free flow zonal electrophoresis (FFZE) is a versatile technique that allows for the separation of cells, organelles, membranes, and proteins based on net surface charge during laminar flow through a thin aqueous layer. We have been optimizing the FFZE technique to enhance separation of plant vacuolar membranes (tonoplast) from other endomembranes to pursue a directed proteomics approach to identify novel tonoplast transporters. Addition of ATP to a mixture of endomembranes selectively enhanced electrophoretic mobility of acidic vesicular compartments during FFZE toward the positive electrode. This has been attributed to activation of the V-ATPase generating a more negative membrane potential outside the vesicles, resulting in enhanced migration of acidic vesicles, including tonoplast, to the anode (Morré, D. J.; Lawrence, J.; Safranski, K.; Hammond, T.; Morré, D. M. J. Chromatogr., A 1994, 668, 201-213). We confirm that ATP does induce a redistribution of membranes during FFZE of microsomal membranes isolated from several plant species, including Arabidopsis thaliana, Thellungiella halophila, Mesembryanthemum crystallinum, and Ananas comosus. However, we demonstrate, using V-ATPase-specific inhibitors, nonhydrolyzable ATP analogs, and ionophores to dissipate membrane potential, that the ATP-dependent migrational shift of membranes under FFZE is not due to activation of the V-ATPase. Addition of EDTA to chelate Mg2+, leading to the production of the tetravalent anionic form of ATP, resulted in a further enhancement of membrane migration toward the anode, and manipulation of cell surface charge by addition of polycations also influenced the ATP-dependent migration of membranes. We propose that ATP enhances the mobility of endomembranes by screening positive surface charges on the membrane surface.

  11. Functional characterization of a wheat NHX antiporter gene TaNHX2 that encodes a K(+/H(+ exchanger.

    Directory of Open Access Journals (Sweden)

    Yuanyuan Xu

    Full Text Available The subcellular localization of a wheat NHX antiporter, TaNHX2, was studied in Arabidopsis protoplasts, and its function was evaluated using Saccharomyces cerevisiae as a heterologous expression system. Fluorescence patterns of TaNHX2-GFP fusion protein in Arabidopsis cells indicated that TaNHX2 localized at endomembranes. TaNHX2 has significant sequence homology to NHX sodium exchangers from Arabidopsis, is abundant in roots and leaves and is induced by salt or dehydration treatments. Western blot analysis showed that TaNHX2 could be expressed in transgenic yeast cells. Expressed TaNHX2 protein suppressed the salt sensitivity of a yeast mutant strain by increasing its K(+ content when exposed to salt stress. TaNHX2 also increased the tolerance of the strain to potassium stress. However, the expression of TaNHX2 did not affect the sodium concentration in transgenic cells. Western blot analysis for tonoplast proteins indicated that the TaNHX2 protein localized at the tonoplast of transgenic yeast cells. The tonoplast vesicles from transgenic yeast cells displayed enhanced K(+/H(+ exchange activity but very little Na(+/H(+ exchange compared with controls transformed with the empty vector; Na(+/H(+ exchange was not detected with concentrations of less than 37.5 mM Na(+ in the reaction medium. Our data suggest that TaNHX2 is a endomembrane-bound protein and may primarily function as a K(+/H(+ antiporter, which is involved in cellular pH regulation and potassium nutrition under normal conditions. Under saline conditions, the protein mediates resistance to salt stress through the intracellular compartmentalization of potassium to regulate cellular pH and K(+ homeostasis.

  12. Transport systems of Ventricaria ventricosa: I/V analysis of both membranes in series as a function of [K(+)](o).

    Science.gov (United States)

    Beilby, M J; Bisson, M A

    1999-09-01

    The current-voltage (I/V) profiles of Ventricaria (formerly Valonia) membranes were measured at a range of external potassium concentrations, [K(+)](o), from 0.1 to 100 mm. The conductance-voltage (G/V) characteristics were computed to facilitate better resolution of the profile change with time after exposure to different [K(+)](o). The resistance-voltage (R/V) characteristics were computed to attempt resolution of plasmalemma and tonoplast. Four basic electrophysiological stages emerged: (1) Uniform low resistance between -60 and +60 mV after the cell impalement. (2) High resistance between +50 and +150 for [K(+)](o) from 0.1 to 1.0 mm and hypotonic media. (3) High resistance between -150 and -20 mV for [K(+)](o) of 10 mm (close to natural seawater) and hypertonic media. (4) High resistance between -150 and +170 mV at [K(+)](o) of 100 mm. The changes between these states were slow, requiring minutes to hours and sometimes exhibiting spontaneous oscillations of the membrane p.d. (potential difference). Our analysis of the I/V data supports a previous hypothesis, that Ventricaria tonoplast is the more resistive membrane containing a pump, which transports K(+) into the vacuole to regulate turgor. We associate state (1) with the plasmalemma conductance being dominant and the K(+) pump at the tonoplast short-circuited probably by a K(+) channel, state (2) with the K(+) pump "off" or short-circuited at p.d.s more negative than +50 mV, state (3) with the K(+) pump "on, " and state (4) with the pump dominant, but affected by high K(+). A model for the Ventricaria membrane system is proposed.

  13. The study of the morphological features of autophagy as a type of programmed death of plant cells under the condition of bacterial infection

    Directory of Open Access Journals (Sweden)

    Сергій Іванович Шевченко

    2016-09-01

    Full Text Available The ultrastructure of the destruction of the plant cells protoplast is studied under the condition of bacterial infection. According to the autophagy processes in animal cells, the morphological ways of plant cells autophagy – vacuolization of cytoplasm, condensation and decondensation of the nuclear mass, multivesicular nucleation, phagophore expansion and macroautophagosome ripening, autophagolysosome formation by the way of tonoplast invagination, mitophagy phenomenon are determined. The places of the final degradation of the ruined cytoplasm in the vacuoles of destroyed cells are shown

  14. The Tr-cp 14 cysteine protease in white clover (Trifolium repens) is localized to the endoplasmic reticulum and is associated with programmed cell death during development of tracheary elements

    DEFF Research Database (Denmark)

    Mulisch, Maria; Asp, Torben; Krupinska, Karin

    2013-01-01

    family of cysteine proteases with homology to XCP1 and XCP2 from Arabidopsis thaliana and p48h-17 from Zinnia elegans, which previously have been reported to be associated with tracheary element differentiation. The proform as well as the processed form of the protein was detected in petioles, flowers....... Immunogold studies suggest that the protease prior to the burst of the vacuole was associated to the ER cisternae. After disruption of the tonoplast, it was found in the cytoplasm, and, in later stages, associated with disintegrating material dispersed throughout the cell....

  15. Identification of a 170-kDa protein associated with the vacuolar Na+/H+ antiport of Beta vulgaris.

    OpenAIRE

    Barkla, B J; Blumwald, E

    1991-01-01

    The effect of the addition of amiloride to the growth medium was tested on the Na+/H+ antiport activity of tonoplast vesicles isolated from sugar beet (beta vulgaris L.) cell suspensions. Cells grown in the presence of NaCl and amiloride displayed an increased antiport activity. Analysis of the kinetic data showed that while the affinity of the antiport for Na+ ions did not change, the maximal velocity of the Na+/H+ exchange increased markedly. These results suggest the addition of more antip...

  16. Towards the production of salt-tolerant crops.

    Science.gov (United States)

    Barkla, B J; Vera-Estrella, R; Pantoja, O

    1999-01-01

    Crop production is affected by numerous environmental factors, with soil salinity and drought having the most detrimental effects. Attempts to improve yield under stress conditions by plant breeding have been unsuccessful, primarily due to the multigenic origin of the adaptive responses. The transfer of genes through genetic engineering of crop plants appears more feasible. Important adaptive mechanisms targeted for potential gene transfer would be the tonoplast Na+/H+ antiport, compatible solute synthesis and, regulation of water channel activity and expression, mechanisms involved in cellular osmoregulation. In this review we discuss recent advances in our understanding of these adaptive mechanisms.

  17. Model for Stress-induced Protein Degradation in Lemna minor1

    Science.gov (United States)

    Cooke, Robert J.; Roberts, Keith; Davies, David D.

    1980-01-01

    Transfer of Lemna minor fronds to adverse or stress conditions produces a large increase in the rate of protein degradation. Cycloheximide partially inhibits stress-induced protein degradation and also partially inhibits the protein degradation which occurs in the absence of stress. The increased protein degradation does not appear to be due to an increase in activity of soluble proteolytic enzymes. Biochemical evidence indicates that stress, perhaps acting via hormones, affects the permeability of certain membranes, particularly the tonoplast. A general model for stress-induced protein degradation is presented in which changes in membrane properties allow vacuolar proteolytic enzymes increased access to cytoplasmic proteins. PMID:16661588

  18. Hydrolytic enzymes in the central vacuole of plant cells.

    Science.gov (United States)

    Boller, T; Kende, H

    1979-06-01

    The hydrolase content of vacuoles isolated from protoplasts of suspension-cultured tobacco cells, of tulip petals, and of pineapple leaves, and the sedimentation behavior of tobacco tonoplasts were studied. Three precautions were found to be important for the analysis of vacuolar hydrolases and of the tonoplast. (a) Purification of protoplasts in a Ficoll gradient was necessary to remove cell debris which contained contaminating hydrolases adsorbed from the fungal cell-wall-degrading enzyme preparation. (b) Hydrolase activities in the homogenates of the intact cells or the tissue used and of the purified protoplasts had to be compared to verify the absence of contaminating hydrolases in the protoplast preparation. (c) Vacuoles obtained from the protoplasts by an osmotic shock had to be purified from the lysate in a Ficoll gradient. Since the density of the central vacuole approximates that of the protoplasts, about a 10% contamination of the vacuolar preparation by surviving protoplasts could not be eliminated and had to be taken into account when the distribution of enzymes and of radioactivity was calculated.THE INTRACELLULAR ACTIVITIES OF THE FOLLOWING ACID HYDROLASES WERE PRIMARILY LOCALIZED IN THE VACUOLE OF TOBACCO CELLS: alpha-mannosidase, beta-N-acetylglucosaminidase, beta-fructosidase, nuclease, phosphatase, phosphodiesterase. A similar composition of acid hydrolases was found in vacuoles obtained from protoplasts of tulip petals. Proteinase, a hydrolase with low activity in tobacco cells and tulip petals and therefore difficult to localize unequivocally, was found to be vacuolar in pineapple leaves, a tissue containing high levels of this enzyme. Our data support the hypothesis that the central vacuole of higher plant cells has an enzyme composition analogous to that of the animal lysosome.None of the vacuolar enzymes investigated was found to be bound to the tonoplast. When vacuoles were isolated from cells labeled with radioactive choline, the vacuolar

  19. Nitrate-induced changes in protein synthesis and translation of RNA in maize roots

    International Nuclear Information System (INIS)

    McClure, P.R.; Omholt, T.E.; Pace, G.M.; Bouthyette, P.Y.

    1987-01-01

    Nitrate regulation of protein synthesis and RNA translation in maize (Zea mays L. var B73) roots was examined, using in vivo labeling with [ 35 S]methionine and in vitro translation. Nitrate enhanced the synthesis of a 31 kilodalton membrane polypeptide which was localized in a fraction enriched in tonoplast and/or endoplasmic reticulum membrane vesicles. The nitrate-enhanced synthesis was correlated with an acceleration of net nitrate uptake by seedlings during initial exposure to nitrate. Nitrate did not consistently enhance protein synthesis in other membrane fractions. Synthesis of up to four soluble polypeptides (21, 40, 90, and 168 kilodaltons) was also enhanced by nitrate. The most consistent enhancement was that of the 40 kilodalton polypeptide. No consistent nitrate-induced changes were noted in the organellar fraction (14,000g pellet of root homogenates). When roots were treated with nitrate, the amount of [ 35 S]methionine increased in six in vitro translation products (21, 24, 41, 56, 66, and 90 kilodaltons). Nitrate treatment did not enhance accumulation of label in translation products with a molecular weight of 31,000 (corresponding to the identified nitrate-inducible membrane polypeptide). Incubation of in vitro translation products with root membranes caused changes in the SDS-PAGE profiles in the vecinity of 31 kilodaltons. The results suggest that the nitrate-inducible, 31 kilodalton polypeptide from a fraction enriched in tonoplast and/or endoplasmic reticulum may be involved in regulating nitrate accumulation by maize roots

  20. Characterization of the vacuolar H sup + -ATPase of higher plants

    Energy Technology Data Exchange (ETDEWEB)

    Manolson, M F

    1988-01-01

    The tonoplast H{sup +}-ATPase of Beta vulgaris L. was partially purified by Triton X-100 solubilization and Sepharose 4B chromatography resulting in the enrichment of two polypeptides. Kinetic analysis of ({alpha}-{sup 32}P) BzATP labeling identified the 57 kDa polypeptide as a nucleotide-binding subunit with a possible regulatory function. In addition, ({sup 14}C) DCCD-labeling identified a 16 kDa polypeptide as a putative transmembrane proton channel. It is concluded that the tonoplast H{sup +}-ATPase is a multimer composed of at least three polypeptides. Anti-57 and anti-67 kDa sera reacted with polypeptides of the corresponding size in bovine chromaffin granules, bovine clathrin-coated vesicles, and yeast vacuolar membranes, suggesting common structural features and common ancestry for endomembrane H{sup +}-ATPase of different organelles and different phyla. Anti-57 serum was used to isolate a cDNA encoding the corresponding subunit from Arabidopsis. Protein sequence analysis revealed homologies between endomembrane, F{sub 0}F{sub 1} and archaebacterial ATPases, suggesting that these different classes of ATPases have evolved from a common ancestor.

  1. Regulation by nitrate of protein synthesis and translation of RNA in maize roots

    International Nuclear Information System (INIS)

    McClure, P.R.; Bouthyette, P.Y.

    1986-01-01

    Roots of maize seedlings were exposed to 35 S-methionine in the presence or absence of nitrate. Using SDS-PAGE, nitrate-induced changes in labeled polypeptides were noted in the soluble (at 92, 63 and 21kD) and organellar(at 14kD) fractions, as well as in a membrane fraction of putative tonoplast origin (at 31kD). No nitrate-induced changes were noted in a plasmamembrane-enriched fraction or in a membrane fraction of mixed origin. Total RNA from nitrate-treated and control roots was translated in a rabbit reticulocyte system. Five translation products (94, 63, 41, 39 and 21kD) were identified as nitrate-inducible by comparative gel electrophoresis. Changes in protein synthesis and translation of mRNA were apparent within 2-3 h after introduction of nitrate. Within 4-6 h after removal of nitrate, the level of nitrate-inducible translation products diminished to that of control roots. In contrast, the 31kD tonoplast polypeptide was still labeled 26 h after removal of external nitrate and 35 S-methionine. The results will be discussed in relation to the nitrate induction of nitrate reductase, nitrite reductase, and the nitrate uptake system

  2. Exchangers man the pumps

    Science.gov (United States)

    Barkla, Bronwyn J; Hirschi, Kendal D

    2008-01-01

    Tonoplast-localised proton-coupled Ca2+ transporters encoded by cation/H+ exchanger (CAX) genes play a critical role in sequestering Ca2+ into the vacuole. These transporters may function in coordination with Ca2+ release channels, to shape stimulus-induced cytosolic Ca2+ elevations. Recent analysis of Arabidopsis CAX knockout mutants, particularly cax1 and cax3, identified a variety of phenotypes including sensitivity to abiotic stresses, which indicated that these transporters might play a role in mediating the plant's stress response. A common feature of these mutants was the perturbation of H+-ATPase activity at both the tonoplast and the plasma membrane, suggesting a tight interplay between the Ca2+/H+ exchangers and H+ pumps. We speculate that indirect regulation of proton flux by the exchangers may be as important as the direct regulation of Ca2+ flux. These results suggest cautious interpretation of mutant Ca2+/H+ exchanger phenotypes that may be due to either perturbed Ca2+ or H+ transport. PMID:19841670

  3. Exchangers man the pumps: Functional interplay between proton pumps and proton-coupled Ca exchangers.

    Science.gov (United States)

    Barkla, Bronwyn J; Hirschi, Kendal D; Pittman, Jon K

    2008-05-01

    Tonoplast-localised proton-coupled Ca(2+) transporters encoded by cation/H(+)exchanger (CAX) genes play a critical role in sequestering Ca(2+) into the vacuole. These transporters may function in coordination with Ca(2+) release channels, to shape stimulus-induced cytosolic Ca(2+) elevations. Recent analysis of Arabidopsis CAX knockout mutants, particularly cax1 and cax3, identified a variety of phenotypes including sensitivity to abiotic stresses, which indicated that these transporters might play a role in mediating the plant's stress response. A common feature of these mutants was the perturbation of H(+)-ATPase activity at both the tonoplast and the plasma membrane, suggesting a tight interplay between the Ca(2+)/H(+) exchangers and H(+) pumps. We speculate that indirect regulation of proton flux by the exchangers may be as important as the direct regulation of Ca(2+) flux. These results suggest cautious interpretation of mutant Ca(2+)/H(+) exchanger phenotypes that may be due to either perturbed Ca(2+) or H(+) transport.

  4. Stress regulated members of the plant organic cation transporter family are localized to the vacuolar membrane

    Directory of Open Access Journals (Sweden)

    Koch Wolfgang

    2008-07-01

    Full Text Available Abstract Background In Arabidopsis six genes group into the gene family of the organic cation transporters (OCTs. In animals the members of the OCT-family are mostly characterized as polyspecific transporters involved in the homeostasis of solutes, the transport of monoamine neurotransmitters and the transport of choline and carnitine. In plants little is known about function, localisation and regulation of this gene family. Only one protein has been characterized as a carnitine transporter at the plasma membrane so far. Findings We localized the five uncharacterized members of the Arabidopsis OCT family, designated OCT2-OCT6, via GFP fusions and protoplast transformation to the tonoplast. Expression analysis with RNA Gel Blots showed a distinct, organ-specific expression pattern of the individual genes. With reporter gene fusion of four members we analyzed the tissue specific distribution of OCT2, 3, 4, and 6. In experiments with salt, drought and cold stress, we could show that AtOCT4, 5 and 6 are up-regulated during drought stress, AtOCT3 and 5 during cold stress and AtOCT 5 and 6 during salt stress treatments. Conclusion Localisation of the proteins at the tonoplast and regulation of the gene expression under stress conditions suggests a specific role for the transporters in plant adaptation to environmental stress.

  5. Functional size of vacuolar H+ pumps: Estimates from radiation inactivation studies

    International Nuclear Information System (INIS)

    Sarafian, V.; Poole, R.J.

    1991-01-01

    The PPase and the ATPase from red beet (Beta vulgaris) vacuolar membranes were subjected to radiation inactivation by a 60 Co source in both the native tonoplast and detergent-solubilized states, in order to determine their target molecular sizes. Analysis of the residual phosphohydrolytic and proton transport activities, after exposure to varying doses of radiation, yielded exponential relationships between the activities and radiation doses. The deduced target molecular sizes for PPase activity in native and solubilized membranes were 125kD and 259kD respectively and 327kD for H + -transport. This suggests that the minimum number of subunits of 67kD for PPi hydrolysis is two in the native state and four after Triton X-100 solubilization. At least four subunits would be required for H + -translocation. Analysis of the ATPase inactivation patterns revealed target sizes of 384kD and 495kD for ATP hydrolysis in native and solubilized tonoplast respectively and 430kD for H + -transport. These results suggest that the minimum size for hydrolytic or transport functions is relatively constant for the ATPase

  6. Differential Activity of Plasma and Vacuolar Membrane Transporters Contributes to Genotypic Differences in Salinity Tolerance in a Halophyte Species, Chenopodium quinoa

    Directory of Open Access Journals (Sweden)

    Edgar Bonales-Alatorre

    2013-04-01

    Full Text Available Halophytes species can be used as a highly convenient model system to reveal key ionic and molecular mechanisms that confer salinity tolerance in plants. Earlier, we reported that quinoa (Chenopodium quinoa Willd., a facultative C3 halophyte species, can efficiently control the activity of slow (SV and fast (FV tonoplast channels to match specific growth conditions by ensuring that most of accumulated Na+ is safely locked in the vacuole (Bonales-Alatorre et al. (2013 Plant Physiology. This work extends these finding by comparing the properties of tonoplast FV and SV channels in two quinoa genotypes contrasting in their salinity tolerance. The work is complemented by studies of the kinetics of net ion fluxes across the plasma membrane of quinoa leaf mesophyll tissue. Our results suggest that multiple mechanisms contribute towards genotypic differences in salinity tolerance in quinoa. These include: (i a higher rate of Na+ exclusion from leaf mesophyll; (ii maintenance of low cytosolic Na+ levels; (iii better K+ retention in the leaf mesophyll; (iv a high rate of H+ pumping, which increases the ability of mesophyll cells to restore their membrane potential; and (v the ability to reduce the activity of SV and FV channels under saline conditions. These mechanisms appear to be highly orchestrated, thus enabling the remarkable overall salinity tolerance of quinoa species.

  7. Differential activity of plasma and vacuolar membrane transporters contributes to genotypic differences in salinity tolerance in a Halophyte Species, Chenopodium quinoa.

    Science.gov (United States)

    Bonales-Alatorre, Edgar; Pottosin, Igor; Shabala, Lana; Chen, Zhong-Hua; Zeng, Fanrong; Jacobsen, Sven-Erik; Shabala, Sergey

    2013-04-29

    Halophytes species can be used as a highly convenient model system to reveal key ionic and molecular mechanisms that confer salinity tolerance in plants. Earlier, we reported that quinoa (Chenopodium quinoa Willd.), a facultative C3 halophyte species, can efficiently control the activity of slow (SV) and fast (FV) tonoplast channels to match specific growth conditions by ensuring that most of accumulated Na+ is safely locked in the vacuole (Bonales-Alatorre et al. (2013) Plant Physiology). This work extends these finding by comparing the properties of tonoplast FV and SV channels in two quinoa genotypes contrasting in their salinity tolerance. The work is complemented by studies of the kinetics of net ion fluxes across the plasma membrane of quinoa leaf mesophyll tissue. Our results suggest that multiple mechanisms contribute towards genotypic differences in salinity tolerance in quinoa. These include: (i) a higher rate of Na+ exclusion from leaf mesophyll; (ii) maintenance of low cytosolic Na+ levels; (iii) better K+ retention in the leaf mesophyll; (iv) a high rate of H+ pumping, which increases the ability of mesophyll cells to restore their membrane potential; and (v) the ability to reduce the activity of SV and FV channels under saline conditions. These mechanisms appear to be highly orchestrated, thus enabling the remarkable overall salinity tolerance of quinoa species.

  8. 'Elaioplasts' identified as lipotubuloids in Althaea rosea, Funkia sieboldiana and Vanilla planifolia contain lipid bodies connected with microtubules

    Directory of Open Access Journals (Sweden)

    Maria Kwiatkowska

    2011-09-01

    Full Text Available "Elaioplasts" observed in Vanilla planifolia, Funkia Sieboldiana and Althaea rosea exhibit all the features characteristic of lipotubuloids earlier described in Ornithogalum umbellatum. They are cytoplasmic domains containing aggregates of lipid bodies connected with microtubules. The immunogold technique confirmed the presence of tubulin in this domain. These structures do not have their own membranes but they are surrounded by a tonoplast at the side of a vacuole since they invaginate into it. In cytoplasm of this domain among lipid bodies there are numerous ribosomes, ER cisternae and vesicles as well as few mitochondria, Golgi structures and microbodies while at older developmental stages there are also autolytic vacuoles. The fact that they are so similar to O. umbellatum lipotubuloids suggest that "elaioplasts" of V. planifolia, F. Sieboldiana and A. rosea can also be named lipotubuloids.

  9. Free Flow Zonal Electrophoresis for Fractionation of Plant Membrane Compartments Prior to Proteomic Analysis.

    Science.gov (United States)

    Barkla, Bronwyn J

    2018-01-01

    Free flow zonal electrophoresis (FFZE) is a versatile, reproducible, and potentially high-throughput technique for the separation of plant organelles and membranes by differences in membrane surface charge. It offers considerable benefits over traditional fractionation techniques, such as density gradient centrifugation and two-phase partitioning, as it is relatively fast, sample recovery is high, and the method provides unparalleled sample purity. It has been used to successfully purify chloroplasts and mitochondria from plants but also, to obtain highly pure fractions of plasma membrane, tonoplast, ER, Golgi, and thylakoid membranes. Application of the technique can significantly improve protein coverage in large-scale proteomics studies by decreasing sample complexity. Here, we describe the method for the fractionation of plant cellular membranes from leaves by FFZE.

  10. Use of DMACA to visualise flavan-3-ols in grape berry skins (Vitis vinifera L., CV Cabernet Franc

    Directory of Open Access Journals (Sweden)

    Michel Chevalier

    2003-09-01

    Full Text Available Phenolic compounds are of great importance in Enology. They play a major role in the elaboration of red wines and during wine preservation, and contribute greatly to their organoleptic properties. Among them, flavonoïds constitute the major group present in berry skins. Their histological study is based classically on the use of Toluidine blue O as a staining agent, which is not specific of any type of phenolics. The use of DMACA (p-dimethylaminocinnamaldehyde, which is specific of tannins (flavan-3-ols, permitted to precise the localisation of these compounds without any risk of error ; it allowed also for the visualisation of tannins, stuck to the tonoplast, which had not been identified with Toluidine blue O.

  11. Differential compartmentation of sucrose and gentianose in the cytosol and vacuoles of storage root protoplasts from Gentiana Lutea L.

    Science.gov (United States)

    Keller, F; Wiemken, A

    1982-12-01

    The storage roots of perennial Gentiana lutea L.plants contain several sugars. The predominant carbohydrate reserve is gentianose (β-D-glucopyranosyl-(1 → 6)-α-D-glucopyranosyl-(1 ↔ 2)-β-D-fructofuranoside). Vacuoles were isolated from root protoplasts and purified through a betaine density gradient. The yield was about 75%. Gentianose and gentiobiose were localized to 100% in the vacuoles, fructose and glucose to about 80%, and sucrose to only about 50%. Taking the volumes of the vacuolar and extravacuolar (cytosolic) compartments into account it is inferred that gentianose is located exclusively in the vacuoles, whilst sucrose is much more concentrated in the cytosol where it may play a role as a cryoprotectant. The concentration of fructose and glucose appeared to be similar on both sides of the tonoplast.

  12. Many rivers to cross

    DEFF Research Database (Denmark)

    Olsen, Lene Irene; Palmgren, Michael Broberg

    2014-01-01

    An important goal of micronutrient biofortification is to enhance the amount of bioavailable zinc in the edible seed of cereals and more specifically in the endosperm. The picture is starting to emerge for how zinc is translocated from the soil through the mother plant to the developing seed...... especially interesting as potential transport bottlenecks. Inside the cell, zinc can be imported into or exported out of organelles by other transporters. The function of several membrane proteins involved in the transport of zinc across the tonoplast, chloroplast or plasma membranes are currently known....... These include members of the ZIP (ZRT-IRT-like Protein), and MTP (Metal Tolerance Protein) and heavy metal ATPase (HMA) families. An important player in the transport process is the ligand nicotianamine that binds zinc to increase its solubility in living cells and in this way buffers the intracellular zinc...

  13. Ectopic expression of Arabidopsis ABC transporter MRP7 modifies cadmium root-to-shoot transport and accumulation

    International Nuclear Information System (INIS)

    Wojas, Sylwia; Hennig, Jacek; Plaza, Sonia; Geisler, Markus; Siemianowski, Oskar; Sklodowska, Aleksandra; Ruszczynska, Anna; Bulska, Ewa; Antosiewicz, Danuta M.

    2009-01-01

    Arabidopsis MRPs/ABCCs have been shown to remove various organic and inorganic substrates from the cytosol to other subcellular compartments. Here we first demonstrate that heterologous expression of AtMRP7 in tobacco (Nicotiana tabacum var. Xanthi) modifies cadmium accumulation, distribution and tolerance. Arabidopsis MRP7 was localized both in the tonoplast and in the plasma membrane when expressed in tobacco. Its overexpression increased tobacco Cd-tolerance and resulted in enhanced cadmium concentration in leaf vacuoles, indicating more efficient detoxification by means of vacuolar storage. Heterologous AtMRP7 expression also led to more efficient retention of Cd in roots, suggesting a contribution to the control of cadmium root-to-shoot translocation. The results underscore the use of AtMRP7 in plant genetic engineering to modify the heavy-metal accumulation pattern for a broad range of applications. - AtMRP7 expression in tobacco enhances Cd-tolerance and increases Cd storage in vacuoles

  14. Choline but not its derivative betaine blocks slow vacuolar channels in the halophyte Chenopodium quinoa: implications for salinity stress responses.

    Science.gov (United States)

    Pottosin, Igor; Bonales-Alatorre, Edgar; Shabala, Sergey

    2014-11-03

    Activity of tonoplast slow vacuolar (SV, or TPC1) channels has to be under a tight control, to avoid undesirable leak of cations stored in the vacuole. This is particularly important for salt-grown plants, to ensure efficient vacuolar Na(+) sequestration. In this study we show that choline, a cationic precursor of glycine betaine, efficiently blocks SV channels in leaf and root vacuoles of the two chenopods, Chenopodium quinoa (halophyte) and Beta vulgaris (glycophyte). At the same time, betaine and proline, two major cytosolic organic osmolytes, have no significant effect on SV channel activity. Physiological implications of these findings are discussed. Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  15. The varied functions of aluminium-activated malate transporters–much more than aluminium resistance

    Science.gov (United States)

    Palmer, Antony J.; Baker, Alison; Muench, Stephen P.

    2016-01-01

    The ALMT (aluminium-activated malate transporter) family comprises a functionally diverse but structurally similar group of ion channels. They are found ubiquitously in plant species, expressed throughout different tissues, and located in either the plasma membrane or tonoplast. The first family member identified was TaALMT1, discovered in wheat root tips, which was found to be involved in aluminium resistance by means of malate exudation into the soil. However, since this discovery other family members have been shown to have many other functions such as roles in stomatal opening, general anionic homoeostasis, and in economically valuable traits such as fruit flavour. Recent evidence has also shown that ALMT proteins can act as key molecular actors in GABA (γ-aminobutyric acid) signalling, the first evidence that GABA can act as a signal transducer in plants. PMID:27284052

  16. The varied functions of aluminium-activated malate transporters-much more than aluminium resistance.

    Science.gov (United States)

    Palmer, Antony J; Baker, Alison; Muench, Stephen P

    2016-06-15

    The ALMT (aluminium-activated malate transporter) family comprises a functionally diverse but structurally similar group of ion channels. They are found ubiquitously in plant species, expressed throughout different tissues, and located in either the plasma membrane or tonoplast. The first family member identified was TaALMT1, discovered in wheat root tips, which was found to be involved in aluminium resistance by means of malate exudation into the soil. However, since this discovery other family members have been shown to have many other functions such as roles in stomatal opening, general anionic homoeostasis, and in economically valuable traits such as fruit flavour. Recent evidence has also shown that ALMT proteins can act as key molecular actors in GABA (γ-aminobutyric acid) signalling, the first evidence that GABA can act as a signal transducer in plants. © 2016 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

  17. Density gradient localization of vanadate- and NO-3-sensitive ATPase from sterile cultures of Spirodela polyrrhiza (L. Schleiden

    Directory of Open Access Journals (Sweden)

    Józef Buczek

    2014-01-01

    Full Text Available The present work deals with the separation and some characteristics of ATPase activities bound with plant membanes prepared from sterile cultures of Spirodela polyrrhiza. The membrane-bound ATPases were separated on sucrose gradients and distinguished by membrane density and sensitivity to several inhibitors. The results showed that N0-3-sensitive ATPase activity associated with the tonoplast was localized at a sucrose density between 1.095-1.117 g•cm-3. The vanadate-sensitive ATPase activity bound with the plasma membrane showed a density between 1.127-1.151 g•cm-3. Both ATPases were insensitive to azide and oligomycin and were separable from markers for mitochondria.

  18. Arabinogalactan Proteins Are Involved in Salt-Adaptation and Vesicle Trafficking in Tobacco by-2 Cell Cultures.

    Science.gov (United States)

    Olmos, Enrique; García De La Garma, Jesús; Gomez-Jimenez, Maria C; Fernandez-Garcia, Nieves

    2017-01-01

    Arabinogalactan proteins (AGPs) are a highly diverse family of glycoproteins that are commonly found in most plant species. However, little is known about the physiological and molecular mechanisms of their function. AGPs are involved in different biological processes such as cell differentiation, cell expansion, tissue development and somatic embryogenesis. AGPs are also involved in abiotic stress response such as salinity modulating cell wall expansion. In this study, we describe how salt-adaptation in tobacco BY-2 cell cultures induces important changes in arabinogalactan proteins distribution and contents. Using the immuno-dot blot technique with different anti-AGP antibodies (JIM13, JIM15, and others), we observed that AGPs were highly accumulated in the culture medium of salt-adapted tobacco cells, probably due to the action of phospholipases. We located these AGP epitopes using immunogold labeling in the cytoplasm associated to the endoplasmic reticulum, the golgi apparatus, and vesicles, plasma membrane and tonoplast. Our results show that salt-adaptation induced a significant reduction of the cytoplasm, plasma membrane and tonoplast content of these epitopes. Yariv reagent was added to the control and salt-adapted tobacco cell cultures, leading to cell death induction in control cells but not in salt-adapted cells. Ultrastructural and immunogold labeling revealed that cell death induced by Yariv reagent in control cells was due to the interaction of Yariv reagent with the AGPs linked to the plasma membranes. Finally, we propose a new function of AGPs as a possible sodium carrier through the mechanism of vesicle trafficking from the apoplast to the vacuoles in salt-adapted tobacco BY-2 cells. This mechanism may contribute to sodium homeostasis during salt-adaptation to high saline concentrations.

  19. Mycorrhizal fungi influence on silver uptake and membrane protein gene expression following silver nanoparticle exposure

    Energy Technology Data Exchange (ETDEWEB)

    Noori, Azam [State University of New York, College of Environmental Science and Forestry (United States); White, Jason C. [Connecticut Agricultural Experiment Station (United States); Newman, Lee A., E-mail: lanewman@esf.edu [State University of New York, College of Environmental Science and Forestry (United States)

    2017-02-15

    The rapid growth of nanotechnology and the high demand for nanomaterial use have greatly increased the risk of particle release into the environment. Understanding nanomaterial interactions with crop species and their associated microorganisms is critical to food safety and security. In the current study, tomato was inoculated with mycorrhizal fungi and subsequently exposed to 12, 24, or 36 mg/kg of 2- or 15-nm silver nanoparticles (Ag-NPs). Mycorrhizal (M) and non-mycorrhizal (NM) tomatoes exposed to 36 mg/kg of 2-nm Ag-NPs accumulated 1300 and 1600 μg/g silver in their tissues, respectively. Mycorrhizal plants accumulated 14% less silver compared to non-mycorrhizal plants. To begin to understand the mechanisms by which plants accumulate NPs, the expression of two aquaporin channel genes, the plasma membrane intrinsic protein (PIP) and the tonoplast membrane intrinsic protein (TIP), and one potassium channel (KC) gene were studied. In non-mycorrhizal plants, the expression of KC, PIP, and TIP was eight, five, and nine times higher than the control, respectively. These expressions for mycorrhizal plants were 5.8, 3.5, and 2 times higher than controls, respectively. The expression of KC and PIP, which are located on the plasma membrane, was 3.5 and 2.5, respectively, times higher than TIP, which is located on the tonoplast. PIP expression was significantly higher in NM tomatoes exposed to 12 mg/kg of 2-nm Ag-NPs compared to M plants. These results show that mycorrhizal colonization decreases Ag accumulation in NP-exposed plants and also moderates changes in expression level of membrane transport proteins.

  20. Mycorrhizal fungi influence on silver uptake and membrane protein gene expression following silver nanoparticle exposure

    International Nuclear Information System (INIS)

    Noori, Azam; White, Jason C.; Newman, Lee A.

    2017-01-01

    The rapid growth of nanotechnology and the high demand for nanomaterial use have greatly increased the risk of particle release into the environment. Understanding nanomaterial interactions with crop species and their associated microorganisms is critical to food safety and security. In the current study, tomato was inoculated with mycorrhizal fungi and subsequently exposed to 12, 24, or 36 mg/kg of 2- or 15-nm silver nanoparticles (Ag-NPs). Mycorrhizal (M) and non-mycorrhizal (NM) tomatoes exposed to 36 mg/kg of 2-nm Ag-NPs accumulated 1300 and 1600 μg/g silver in their tissues, respectively. Mycorrhizal plants accumulated 14% less silver compared to non-mycorrhizal plants. To begin to understand the mechanisms by which plants accumulate NPs, the expression of two aquaporin channel genes, the plasma membrane intrinsic protein (PIP) and the tonoplast membrane intrinsic protein (TIP), and one potassium channel (KC) gene were studied. In non-mycorrhizal plants, the expression of KC, PIP, and TIP was eight, five, and nine times higher than the control, respectively. These expressions for mycorrhizal plants were 5.8, 3.5, and 2 times higher than controls, respectively. The expression of KC and PIP, which are located on the plasma membrane, was 3.5 and 2.5, respectively, times higher than TIP, which is located on the tonoplast. PIP expression was significantly higher in NM tomatoes exposed to 12 mg/kg of 2-nm Ag-NPs compared to M plants. These results show that mycorrhizal colonization decreases Ag accumulation in NP-exposed plants and also moderates changes in expression level of membrane transport proteins.

  1. Exploring emergent properties in cellular homeostasis using OnGuard to model K+ and other ion transport in guard cells.

    Science.gov (United States)

    Blatt, Michael R; Wang, Yizhou; Leonhardt, Nathalie; Hills, Adrian

    2014-05-15

    It is widely recognized that the nature and characteristics of transport across eukaryotic membranes are so complex as to defy intuitive understanding. In these circumstances, quantitative mathematical modeling is an essential tool, both to integrate detailed knowledge of individual transporters and to extract the properties emergent from their interactions. As the first, fully integrated and quantitative modeling environment for the study of ion transport dynamics in a plant cell, OnGuard offers a unique tool for exploring homeostatic properties emerging from the interactions of ion transport, both at the plasma membrane and tonoplast in the guard cell. OnGuard has already yielded detail sufficient to guide phenotypic and mutational studies, and it represents a key step toward 'reverse engineering' of stomatal guard cell physiology, based on rational design and testing in simulation, to improve water use efficiency and carbon assimilation. Its construction from the HoTSig libraries enables translation of the software to other cell types, including growing root hairs and pollen. The problems inherent to transport are nonetheless challenging, and are compounded for those unfamiliar with conceptual 'mindset' of the modeler. Here we set out guidelines for the use of OnGuard and outline a standardized approach that will enable users to advance quickly to its application both in the classroom and laboratory. We also highlight the uncanny and emergent property of OnGuard models to reproduce the 'communication' evident between the plasma membrane and tonoplast of the guard cell. Copyright © 2014 The Authors. Published by Elsevier GmbH.. All rights reserved.

  2. Cloning, localization and expression analysis of vacuolar sugar transporters in the CAM plant Ananas comosus (pineapple).

    Science.gov (United States)

    Antony, Edna; Taybi, Tahar; Courbot, Mikaël; Mugford, Sam T; Smith, J Andrew C; Borland, Anne M

    2008-01-01

    In photosynthetic tissues of the CAM plant pineapple (Ananas comosus), storage of soluble sugars in the central vacuole during the daytime and their remobilization at night is required to provide carbon skeletons for nocturnal CO(2) fixation. However, soluble sugars produced photosynthetically must also be exported to support growth processes in heterotrophic tissues. To begin to address how vacuolar sugar storage and assimilate partitioning are regulated in A. comosus, degenerate PCR and cDNA library screening were used to clone three candidate sugar transporters from the leaves of this species. Subcellular localization of the three transporters was investigated via expression of YFP-fusion proteins in tobacco epidermal cells and their co-localization with subcellular markers by confocal microscopy. Using this strategy, a putative hexose transporter (AcMST1) and a putative inositol transporter (AcINT1) were identified that both localized to the tonoplast, whereas a putative sucrose transporter (AcSUT1) was found to localize to prevacuolar compartments. A cDNA (AcMST2) with high similarity to a recently characterized tonoplast hexose transporter in Arabidopsis was also identified from an A. comosus fruit EST database. Analyses of transcript abundance indicated that AcMST1 was more highly expressed in fruits compared to leaves of A. comosus, whilst transcripts of AcINT1, AcSUT1, and AcMST2 were more abundant in leaves. Transcript abundance of AcINT1, the putative inositol transporter, showed day-night changes comparable to those of other CAM-related transcripts described in Mesembryanthemum crystallinum. The results are discussed in terms of the role of vacuolar sugar transporters in regulating carbon flow during the diel cycle in CAM plants.

  3. Day–Night Changes of Energy-rich Compounds in Crassulacean Acid Metabolism (CAM) Species Utilizing Hexose and Starch

    Science.gov (United States)

    CHEN, LI-SONG; NOSE, AKIHIRO

    2004-01-01

    • Background and Aims Plants with crassulacean acid metabolism (CAM) can be divided into two groups according to the major carbohydrates used for malic acid synthesis, either polysaccharide (starch) or monosaccharide (hexose). This is related to the mechanism and affects energy metabolism in the two groups. In Kalanchoë pinnata and K. daigremontiana, which utilize starch, ATP-dependent phosphofructokinase (tonoplast inorganic pyrophosphatase) activity is greater than inorganic pyrophosphate-dependent phosphofructokinase (tonoplast adenosine triphosphatase) activity, but the reverse is the case in pineapple (Ananas comosus) utilizing hexose. To test the hypothesis that the energy metabolism of the two groups differs, day-night changes in the contents of ATP, ADP, AMP, inorganic phosphate (Pi), phosphoenolpyruvate (PEP) and inorganic pyrophosphate (PPi) in K. pinnata and K. daigremontiana leaves and in pineapple chlorenchyma were analysed. • Methods The contents of energy-rich compounds were measured spectrophotometrically in extracts of tissue sampled in the light and dark, using potted plants, kept for 15 d before the experiments in a growth chamber. • Key Results In the three species, ATP content and adenylate energy charge (AEC) increased in the dark and decreased in the light, in contrast to ADP and AMP. Changes in ATP and AEC were greater in Kalanchoë leaves than in pineapple chlorenchyma. PPi content in the three species increased in the dark, but on illumination it decreased rapidly and substantially, remaining little changed through the rest of the light period. Pi content of Kalanchoë leaves did not change between dark and light, whereas Pi in pineapple chlorenchyma increased in the dark and decreased in the light, and the changes were far greater than in Kalanchoë leaves. Light-dark changes in PEP content in the three species were similar. • Conclusions These results corroborate our hypothesis that day–night changes in the contents of energy

  4. Day-night changes of energy-rich compounds in crassulacean acid metabolism (CAM) species utilizing hexose and starch.

    Science.gov (United States)

    Chen, Li-Song; Nose, Akihiro

    2004-09-01

    Plants with crassulacean acid metabolism (CAM) can be divided into two groups according to the major carbohydrates used for malic acid synthesis, either polysaccharide (starch) or monosaccharide (hexose). This is related to the mechanism and affects energy metabolism in the two groups. In Kalanchoë pinnata and K. daigremontiana, which utilize starch, ATP-dependent phosphofructokinase (tonoplast inorganic pyrophosphatase) activity is greater than inorganic pyrophosphate-dependent phosphofructokinase (tonoplast adenosine triphosphatase) activity, but the reverse is the case in pineapple (Ananas comosus) utilizing hexose. To test the hypothesis that the energy metabolism of the two groups differs, day-night changes in the contents of ATP, ADP, AMP, inorganic phosphate (Pi), phosphoenolpyruvate (PEP) and inorganic pyrophosphate (PPi) in K. pinnata and K. daigremontiana leaves and in pineapple chlorenchyma were analysed. The contents of energy-rich compounds were measured spectrophotometrically in extracts of tissue sampled in the light and dark, using potted plants, kept for 15 d before the experiments in a growth chamber. In the three species, ATP content and adenylate energy charge (AEC) increased in the dark and decreased in the light, in contrast to ADP and AMP. Changes in ATP and AEC were greater in Kalanchoë leaves than in pineapple chlorenchyma. PPi content in the three species increased in the dark, but on illumination it decreased rapidly and substantially, remaining little changed through the rest of the light period. Pi content of Kalanchoë leaves did not change between dark and light, whereas Pi in pineapple chlorenchyma increased in the dark and decreased in the light, and the changes were far greater than in Kalanchoë leaves. Light-dark changes in PEP content in the three species were similar. These results corroborate our hypothesis that day-night changes in the contents of energy-rich compounds differ between CAM species and are related to the

  5. Evolutionary relationships and functional diversity of plant sulfate transporters.

    Science.gov (United States)

    Takahashi, Hideki; Buchner, Peter; Yoshimoto, Naoko; Hawkesford, Malcolm J; Shiu, Shin-Han

    2011-01-01

    Sulfate is an essential nutrient cycled in nature. Ion transporters that specifically facilitate the transport of sulfate across the membranes are found ubiquitously in living organisms. The phylogenetic analysis of known sulfate transporters and their homologous proteins from eukaryotic organisms indicate two evolutionarily distinct groups of sulfate transport systems. One major group named Tribe 1 represents yeast and fungal SUL, plant SULTR, and animal SLC26 families. The evolutionary origin of SULTR family members in land plants and green algae is suggested to be common with yeast and fungal SUL and animal anion exchangers (SLC26). The lineage of plant SULTR family is expanded into four subfamilies (SULTR1-SULTR4) in land plant species. By contrast, the putative SULTR homologs from Chlorophyte green algae are in two separate lineages; one with the subfamily of plant tonoplast-localized sulfate transporters (SULTR4), and the other diverged before the appearance of lineages for SUL, SULTR, and SLC26. There also was a group of yet undefined members of putative sulfate transporters in yeast and fungi divergent from these major lineages in Tribe 1. The other distinct group is Tribe 2, primarily composed of animal sodium-dependent sulfate/carboxylate transporters (SLC13) and plant tonoplast-localized dicarboxylate transporters (TDT). The putative sulfur-sensing protein (SAC1) and SAC1-like transporters (SLT) of Chlorophyte green algae, bryophyte, and lycophyte show low degrees of sequence similarities with SLC13 and TDT. However, the phylogenetic relationship between SAC1/SLT and the other two families, SLC13 and TDT in Tribe 2, is not clearly supported. In addition, the SAC1/SLT family is absent in the angiosperm species analyzed. The present study suggests distinct evolutionary trajectories of sulfate transport systems for land plants and green algae.

  6. The Arabidopsis vacuolar malate channel is a member of the ALMT family.

    Science.gov (United States)

    Kovermann, Peter; Meyer, Stefan; Hörtensteiner, Stefan; Picco, Cristiana; Scholz-Starke, Joachim; Ravera, Silvia; Lee, Youngsook; Martinoia, Enrico

    2007-12-01

    In plants, malate is a central metabolite and fulfills a large number of functions. Vacuolar malate may reach very high concentrations and fluctuate rapidly, whereas cytosolic malate is kept at a constant level allowing optimal metabolism. Recently, a vacuolar malate transporter (Arabidopsis thaliana tonoplast dicarboxylate transporter, AttDT) was identified that did not correspond to the well-characterized vacuolar malate channel. We therefore hypothesized that a member of the aluminum-activated malate transporter (ALMT) gene family could code for a vacuolar malate channel. Using GFP fusion constructs, we could show that AtALMT9 (A. thaliana ALMT9) is targeted to the vacuole. Promoter-GUS fusion constructs demonstrated that this gene is expressed in all organs, but is cell-type specific as GUS activity in leaves was detected nearly exclusively in mesophyll cells. Patch-clamp analysis of an Atalmt9 T-DNA insertion mutant exhibited strongly reduced vacuolar malate channel activity. In order to functionally characterize AtALMT9 as a malate channel, we heterologously expressed this gene in tobacco and in oocytes. Overexpression of AtALMT9-GFP in Nicotiana benthamiana leaves strongly enhanced the malate current densities across the mesophyll tonoplasts. Functional expression of AtALMT9 in Xenopus oocytes induced anion currents, which were clearly distinguishable from endogenous oocyte currents. Our results demonstrate that AtALMT9 is a vacuolar malate channel. Deletion mutants for AtALMT9 exhibit only slightly reduced malate content in mesophyll protoplasts and no visible phenotype, indicating that AttDT and the residual malate channel activity are sufficient to sustain the transport activity necessary to regulate the cytosolic malate homeostasis.

  7. Evolutionary relationships and functional diversity of plant sulfate transporters

    Directory of Open Access Journals (Sweden)

    Hideki eTakahashi

    2012-01-01

    Full Text Available Sulfate is an essential nutrient cycled in nature. Ion transporters that specifically facilitate the transport of sulfate across the membranes are found ubiquitously in living organisms. The phylogenetic analysis of known sulfate transporters and their homologous proteins from eukaryotic organisms indicate two evolutionarily distinct groups of sulfate transport systems. One major group named Tribe 1 represents yeast and fungal SUL, plant SULTR and animal SLC26 families. The evolutionary origin of SULTR family members in land plants and green algae is suggested to be common with yeast and fungal sulfate transporters (SUL and animal anion exchangers (SLC26. The lineage of plant SULTR family is expanded into four subfamilies (SULTR1 to SULTR4 in land plant species. By contrast, the putative SULTR homologues from Chlorophyte green algae are in two separate lineages; one with the subfamily of plant tonoplast-localized sulfate transporters (SULTR4, and the other diverged before the appearance of lineages for SUL, SULTR and SLC26. There also was a group of yet undefined members of putative sulfate transporters in yeast and fungi divergent from these major lineages in Tribe 1. The other distinct group is Tribe 2, primarily composed of animal sodium-dependent sulfate/carboxylate transporters (SLC13 and plant tonoplast-localized dicarboxylate transporters (TDT. The putative sulfur-sensing protein (SAC1 and SAC1-like transporters (SLT of Chlorophyte green algae, bryophyte and lycophyte show low degrees of sequence similarities with SLC13 and TDT. However, the phylogenetic relationship between SAC1/SLT and the other two families, SLC13 and TDT in Tribe 2, is not clearly supported. In addition, the SAC1/SLT family is completely absent in the angiosperm species analyzed. The present study suggests distinct evolutionary trajectories of sulfate transport systems for land plants and green algae.

  8. Chloroplast behaviour and interactions with other organelles in Arabidopsis thaliana pavement cells.

    Science.gov (United States)

    Barton, Kiah A; Wozny, Michael R; Mathur, Neeta; Jaipargas, Erica-Ashley; Mathur, Jaideep

    2018-01-29

    Chloroplasts are a characteristic feature of green plants. Mesophyll cells possess the majority of chloroplasts and it is widely believed that, with the exception of guard cells, the epidermal layer in most higher plants does not contain chloroplasts. However, recent observations on Arabidopsis thaliana have shown a population of chloroplasts in pavement cells that are smaller than mesophyll chloroplasts and have a high stroma to grana ratio. Here, using stable transgenic lines expressing fluorescent proteins targeted to the plastid stroma, plasma membrane, endoplasmic reticulum, tonoplast, nucleus, mitochondria, peroxisomes, F-actin and microtubules, we characterize the spatiotemporal relationships between the pavement cell chloroplasts (PCCs) and their subcellular environment. Observations on the PCCs suggest a source-sink relationship between the epidermal and the mesophyll layers, and experiments with the Arabidopsis mutants glabra2 ( gl2 ) and immutans ( im ), which show altered epidermal plastid development, underscored their developmental plasticity. Our findings lay down the foundation for further investigations aimed at understanding the precise role and contributions of PCCs in plant interactions with the environment. © 2018. Published by The Company of Biologists Ltd.

  9. Programmes of cell death and autolysis in tracheary elements: when a suicidal cell arranges its own corpse removal.

    Science.gov (United States)

    Escamez, Sacha; Tuominen, Hannele

    2014-03-01

    Tracheary element (TE) differentiation represents a unique system to study plant developmental programmed cell death (PCD). TE PCD occurs after deposition of the secondary cell walls when an unknown signal induces tonoplast rupture and the arrest of cytoplasmic streaming. TE PCD is tightly followed by autolysis of the protoplast and partial hydrolysis of the primary cell walls. This review integrates TE differentiation, programmed cell death (PCD), and autolysis in a biological and evolutionary context. The collective evidence from the evolutionary and molecular studies suggests that TE differentiation consists primarily of a programme for cell death and autolysis under the direct control of the transcriptional master switches VASCULAR NAC DOMAIN 6 (VND6) and VND7. In this scenario, secondary cell walls represent a later innovation to improve the water transport capacity of TEs which necessitates transcriptional regulators downstream of VND6 and VND7. One of the most fascinating features of TEs is that they need to prepare their own corpse removal by expression and accumulation of hydrolases that are released from the vacuole after TE cell death. Therefore, TE differentiation involves, in addition to PCD, a programmed autolysis which is initiated before cell death and executed post-mortem. It has recently become clear that TE PCD and autolysis are separate processes with separate molecular regulation. Therefore, the importance of distinguishing between the cell death programme per se and autolysis in all plant PCD research and of careful description of the morphological, biochemical, and molecular sequences in each of these processes, is advocated.

  10. Function and regulation of plant major intrinsic proteins

    DEFF Research Database (Denmark)

    Popovic, Milan

    ;1 in Arabidopsis. That led to the discovery that tip4;1 is gametophytic lethal- gene essential for normal seed set. ICP-MS analyses of the elemental composition of tip4;1 heterozygous T-DNA insert mutant plants and 35S::TIP4;1 over-expression plants indicate that AtTIP4;1 has a role in arsenic distribution...... inorganic forms of arsenic in the environment, can be taken up by plants and thus enter the food chain. Once inside the root cells, As(V) is reduced to As(III) which is then extruded to the soil solution or bound to phytochelatins (PCs) and transported to the vacuole in an effort to accomplish...... detoxification. Plant Noduline 26-like Intrinsic Proteins (NIPs) can channel As(III) and consequently influence the detoxification process. The role of the Tonoplast Intrinsic Proteins (TIPs) in As(III) detoxification remains to be clarified, yet TIPs could have an impact on As(III) accumulation in plant cell...

  11. You Shall Not Pass: Root Vacuoles as a Symplastic Checkpoint for Metal Translocation to Shoots and Possible Application to Grain Nutritional Quality

    Directory of Open Access Journals (Sweden)

    Felipe K. Ricachenevsky

    2018-04-01

    Full Text Available Plant nutrient uptake is performed mostly by roots, which have to acquire nutrients while avoiding excessive amounts of essential and toxic elements. Apoplastic barriers such as the casparian strip and suberin deposition block free diffusion from the rhizosphere into the xylem, making selective plasma membrane transporters able to control elemental influx into the root symplast, efflux into the xylem and therefore shoot translocation. Additionally, transporters localized to the tonoplast of root cells have been demonstrated to regulate the shoot ionome, and may be important for seed elemental translocation. Here we review the role of vacuolar transporters in the detoxification of elements such as zinc (Zn, manganese (Mn, cadmium (Cd, cobalt (Co and nickel (Ni that are co-transported with iron (Fe during the Fe deficiency response in Arabidopsis thaliana, and the possible conservation of this mechanism in rice (Oryza sativa. We also discuss the evidence that vacuolar transporters are linked to natural variation in shoot ionome in Arabidopsis and rice, indicating that vacuolar storage might be amenable to genetic engineering without strong phenotypical changes. Finally, we discuss the possible use of root’s vacuolar transporters to increase the nutritional quality of crop grains.

  12. Effect of Hf on the fine structure of mesophyll cells from Glycine max, Merr

    Energy Technology Data Exchange (ETDEWEB)

    Wei, L.; Miller, G.W.

    1972-04-01

    A series of ultrastructural changes were observed in soybean leaves fumigated with 40 to 50 ppb of hydrogen fluoride. In the cytoplasm the presence of small vacuoles was the first noticeable initial change. The fragmentation of the vacuolar membrane occurred either simultaneously or followed immediately. Lipid-droplet-like globules and numerous vesicles occurred subsequently in the cytoplasm and increased as the injury became more severe. There was a decrease in polysomes and a detachment of ribosome from the rough endoplasmic reticulum. Free ribosome concentration also decreased as the injury became severe. Mitochondrial modifications involving dilation of outer and cristae membranes followed by reduction of both cristae number and matrix electron density and the disappearance of mitochondrial granules were observed in the chlorotic leaves. Electron dense inclusions accumulated in some mitochondria as well. The first noticeable change observed in the chloroplast was the presence of clusters of phytoferritin granules within the stoma after only 2 days of fumigation. Alterations in nuclear structures were observed in later stages of injury. Numerous small electron dense particles were found on various types of membranes in cells of severely chlorotic leaves. They were distributed on outer mitochondrial membranes, endoplasmic reticula, dictyosomes, tonoplasts, plasmalemma, nuclear envelopes, and disintegrating organelles and vesicles, but were never observed on membranes of chloroplasts and microbodies. The presence of fluoride has attracted the attention of many workers primarily in certain industrial areas where the emitted atmospheric fluoride concentrates and is accumulated by plants initiating injury. 6 references.

  13. Light-induced, dark-reversible colour shifts in petals of Phlox

    International Nuclear Information System (INIS)

    Bjön, G.S.; Braune, W.; Bjön, L.O.

    1985-01-01

    Flowers of some Phlox (Phlox x paniculata L.) varieties undergo daily colour shifts, being blue in the early morning, turning red during the day, and returning to blue in the evening. The colour shift, which occurs only in the upper (adaxial) petal surfaces, is due to the daily changes in ambient light. In the laboratory, colour shifts could be induced by 2.5 h of ultraviolet, visible or far-red light and recorded by reflectance spectrophotometry. There are indications that irradiations with different kinds of light cause qualitatively different colour shifts, and that thus more than one photoreceptor pigment and more than one primary light reaction may be involved. The presence of phytochrome was demonstrated in petals of white Phlox flowers by in vivo transmission spectrophotometry. It is therefore possible that colour shifts in coloured Phlox flowers are mediated by phytochrome. Possibly the movement of ions (e.g. hydrogen ions) into or out of the vacuole (where the visible pigments are located) is affected by light absorption in a pigment in the tonoplast

  14. Job Sharing in the Endomembrane System: Vacuolar Acidification Requires the Combined Activity of V-ATPase and V-PPase.

    Science.gov (United States)

    Kriegel, Anne; Andrés, Zaida; Medzihradszky, Anna; Krüger, Falco; Scholl, Stefan; Delang, Simon; Patir-Nebioglu, M Görkem; Gute, Gezahegn; Yang, Haibing; Murphy, Angus S; Peer, Wendy Ann; Pfeiffer, Anne; Krebs, Melanie; Lohmann, Jan U; Schumacher, Karin

    2015-12-01

    The presence of a large central vacuole is one of the hallmarks of a prototypical plant cell, and the multiple functions of this compartment require massive fluxes of molecules across its limiting membrane, the tonoplast. Transport is assumed to be energized by the membrane potential and the proton gradient established by the combined activity of two proton pumps, the vacuolar H(+)-pyrophosphatase (V-PPase) and the vacuolar H(+)-ATPase (V-ATPase). Exactly how labor is divided between these two enzymes has remained elusive. Here, we provide evidence using gain- and loss-of-function approaches that lack of the V-ATPase cannot be compensated for by increased V-PPase activity. Moreover, we show that increased V-ATPase activity during cold acclimation requires the presence of the V-PPase. Most importantly, we demonstrate that a mutant lacking both of these proton pumps is conditionally viable and retains significant vacuolar acidification, pointing to a so far undetected contribution of the trans-Golgi network/early endosome-localized V-ATPase to vacuolar pH. © 2015 American Society of Plant Biologists. All rights reserved.

  15. Relations between fluxes and concentrations of Na in cell suspensions of Acer pseudoplatanus

    International Nuclear Information System (INIS)

    Pennarun, A.-M.

    1978-01-01

    Taking in account the data provided by preliminary compartmental analysis, the net influxes of 24 Na measured in Acer cells after a short loading period (45 minutes) followed by a short wash (1 minute) represent the influx across the plasmalemma (phi sub(0c)) and, after a long loading period (4 hours) followed by a long wash (2 hours) represent the quasi-steady influx from the external solution to the vacuole (phi sub(0v). At flux equilibrium and when the external Na concentration is high enough, the other unidirectional fluxes - phi sub(c0), phi sub(cv) and phi sub(vc) - can be determined from these measurements. This method was used to study the variation of Na flux in terms of the external concentrations and the resulting internal concentrations. The kinetics obtained confirm the active nature of the efflux phi sub(vc) across the tonoplast according to the conclusions given by the application of the USSING-TEORELL criterion to the results of compartmental analysis. On the contrary, they suggest a passive character for the efflux phi sub(c0) accross the plasmalemma which could be considered as active according to the USSING-TEORELL criterion. The contradiction could be eliminated by taking into consideration the important underestimation of the Na activity coefficient in the cytoplasm, due to the neglecting of water binding [fr

  16. Genome-wide identification, characterization, and expression profile of aquaporin gene family in flax (Linum usitatissimum).

    Science.gov (United States)

    Shivaraj, S M; Deshmukh, Rupesh K; Rai, Rhitu; Bélanger, Richard; Agrawal, Pawan K; Dash, Prasanta K

    2017-04-27

    Membrane intrinsic proteins (MIPs) form transmembrane channels and facilitate transport of myriad substrates across the cell membrane in many organisms. Majority of plant MIPs have water transporting ability and are commonly referred as aquaporins (AQPs). In the present study, we identified aquaporin coding genes in flax by genome-wide analysis, their structure, function and expression pattern by pan-genome exploration. Cross-genera phylogenetic analysis with known aquaporins from rice, arabidopsis, and poplar showed five subgroups of flax aquaporins representing 16 plasma membrane intrinsic proteins (PIPs), 17 tonoplast intrinsic proteins (TIPs), 13 NOD26-like intrinsic proteins (NIPs), 2 small basic intrinsic proteins (SIPs), and 3 uncharacterized intrinsic proteins (XIPs). Amongst aquaporins, PIPs contained hydrophilic aromatic arginine (ar/R) selective filter but TIP, NIP, SIP and XIP subfamilies mostly contained hydrophobic ar/R selective filter. Analysis of RNA-seq and microarray data revealed high expression of PIPs in multiple tissues, low expression of NIPs, and seed specific expression of TIP3 in flax. Exploration of aquaporin homologs in three closely related Linum species bienne, grandiflorum and leonii revealed presence of 49, 39 and 19 AQPs, respectively. The genome-wide identification of aquaporins, first in flax, provides insight to elucidate their physiological and developmental roles in flax.

  17. The role of water channel proteins in facilitating recovery of leaf hydraulic conductance from water stress in Populus trichocarpa.

    Directory of Open Access Journals (Sweden)

    Joan Laur

    Full Text Available Gas exchange is constrained by the whole-plant hydraulic conductance (Kplant. Leaves account for an important fraction of Kplant and may therefore represent a major determinant of plant productivity. Leaf hydraulic conductance (Kleaf decreases with increasing water stress, which is due to xylem embolism in leaf veins and/or the properties of the extra-xylary pathway. Water flow through living tissues is facilitated and regulated by water channel proteins called aquaporins (AQPs. Here we assessed changes in the hydraulic conductance of Populus trichocarpa leaves during a dehydration-rewatering episode. While leaves were highly sensitive to drought, Kleaf recovered only 2 hours after plants were rewatered. Recovery of Kleaf was absent when excised leaves were bench-dried and subsequently xylem-perfused with a solution containing AQP inhibitors. We examined the expression patterns of 12 highly expressed AQP genes during a dehydration-rehydration episode to identify isoforms that may be involved in leaf hydraulic adjustments. Among the AQPs tested, several genes encoding tonoplast intrinsic proteins (TIPs showed large increases in expression in rehydrated leaves, suggesting that TIPs contribute to reversing drought-induced reductions in Kleaf. TIPs were localized in xylem parenchyma, consistent with a role in facilitating water exchange between xylem vessels and adjacent living cells. Dye uptake experiments suggested that reversible embolism formation in minor leaf veins contributed to the observed changes in Kleaf.

  18. The role of water channel proteins in facilitating recovery of leaf hydraulic conductance from water stress in Populus trichocarpa.

    Science.gov (United States)

    Laur, Joan; Hacke, Uwe G

    2014-01-01

    Gas exchange is constrained by the whole-plant hydraulic conductance (Kplant). Leaves account for an important fraction of Kplant and may therefore represent a major determinant of plant productivity. Leaf hydraulic conductance (Kleaf) decreases with increasing water stress, which is due to xylem embolism in leaf veins and/or the properties of the extra-xylary pathway. Water flow through living tissues is facilitated and regulated by water channel proteins called aquaporins (AQPs). Here we assessed changes in the hydraulic conductance of Populus trichocarpa leaves during a dehydration-rewatering episode. While leaves were highly sensitive to drought, Kleaf recovered only 2 hours after plants were rewatered. Recovery of Kleaf was absent when excised leaves were bench-dried and subsequently xylem-perfused with a solution containing AQP inhibitors. We examined the expression patterns of 12 highly expressed AQP genes during a dehydration-rehydration episode to identify isoforms that may be involved in leaf hydraulic adjustments. Among the AQPs tested, several genes encoding tonoplast intrinsic proteins (TIPs) showed large increases in expression in rehydrated leaves, suggesting that TIPs contribute to reversing drought-induced reductions in Kleaf. TIPs were localized in xylem parenchyma, consistent with a role in facilitating water exchange between xylem vessels and adjacent living cells. Dye uptake experiments suggested that reversible embolism formation in minor leaf veins contributed to the observed changes in Kleaf.

  19. Parallel Sequencing of Expressed Sequence Tags from Two Complementary DNA Libraries for High and Low Phosphorus Adaptation in Common Beans

    Directory of Open Access Journals (Sweden)

    Matthew W. Blair

    2011-11-01

    Full Text Available Expressed sequence tags (ESTs have proven useful for gene discovery in many crops. In this work, our objective was to construct complementary DNA (cDNA libraries from root tissues of common beans ( L. grown under low and high P hydroponic conditions and to conduct EST sequencing and comparative analyses of the libraries. Expressed sequence tag analysis of 3648 clones identified 2372 unigenes, of which 1591 were annotated as known genes while a total of 465 unigenes were not associated with any known gene. Unigenes with hits were categorized according to biological processes, molecular function, and cellular compartmentalization. Given the young tissue used to make the root libraries, genes for catalytic activity and binding were highly expressed. Comparisons with previous root EST sequencing and between the two libraries made here resulted in a set of genes to study further for differential gene expression and adaptation to low P, such as a 14 kDa praline-rich protein, a metallopeptidase, tonoplast intrinsic protein, adenosine triphosphate (ATP citrate synthase, and cell proliferation genes expressed in the low P treated plants. Given that common beans are often grown on acid soils of the tropics and subtropics that are usually low in P these genes and the two parallel libraries will be useful for selection for better uptake of this essential macronutrient. The importance of EST generation for common bean root tissues under low P and other abiotic soil stresses is also discussed.

  20. [Effects of desulfurization waste on calcium distribution, Ca(2+)-ATPase activity, and antioxidant characteristics of rice leaf under alkali stress].

    Science.gov (United States)

    Mao, Gui-Lian; Xu, Xing; Zeng, Jin; Yue, Zi-Hui; Yang, Shu-Juan

    2012-02-01

    To approach the action mechanisms of desulfurization waste on alleviating alkali stress-induced injury of rice, a pot experiment was conducted to study the variations of leaf total calcium content, calcium distribution, plasma membrane Ca(2+)-ATPase activity, and reactive oxygen content of rice seedlings under alkali stress after the application of desulfurization waste. In the control, a few calcium particulates scattered in the cell wall and chloroplasts, while applying desulfurization waste or CaSO4 increased the calcium particulates in the plasma membrane, intercellular space, cell wall, and vacuole significantly. With the increasing application rate of desulfurization waste or CaSO4, the leaf total calcium content increased, Ca(2+)-ATPase activity in plasma membrane and tonoplast presented an increasing trend, plasma membrane relative permeability, MDA content, and O2 production rate decreased, and SOD and POD activities increased. The desulfurization waste could relieve the alkali stress to rice in some extent, and the main reactive compound in the waste could be CaSO4.

  1. Autophagy-Related Direct Membrane Import from ER/Cytoplasm into the Vacuole or Apoplast: A Hidden Gateway also for Secondary Metabolites and Phytohormones?

    Directory of Open Access Journals (Sweden)

    Ivan Kulich

    2014-04-01

    Full Text Available Transportation of low molecular weight cargoes into the plant vacuole represents an essential plant cell function. Several lines of evidence indicate that autophagy-related direct endoplasmic reticulum (ER to vacuole (and also, apoplast transport plays here a more general role than expected. This route is regulated by autophagy proteins, including recently discovered involvement of the exocyst subcomplex. Traffic from ER into the vacuole bypassing Golgi apparatus (GA acts not only in stress-related cytoplasm recycling or detoxification, but also in developmentally-regulated biopolymer and secondary metabolite import into the vacuole (or apoplast, exemplified by storage proteins and anthocyanins. We propose that this pathway is relevant also for some phytohormones’ (e.g., auxin, abscisic acid (ABA and salicylic acid (SA degradation. We hypothesize that SA is not only an autophagy inducer, but also a cargo for autophagy-related ER to vacuole membrane container delivery and catabolism. ER membrane localized enzymes will potentially enhance the area of biosynthetic reactive surfaces, and also, abundant ER localized membrane importers (e.g., ABC transporters will internalize specific molecular species into the autophagosome biogenesis domain of ER. Such active ER domains may create tubular invaginations of tonoplast into the vacuoles as import intermediates. Packaging of cargos into the ER-derived autophagosome-like containers might be an important mechanism of vacuole and exosome biogenesis and cytoplasm protection against toxic metabolites. A new perspective on metabolic transformations intimately linked to membrane trafficking in plants is emerging.

  2. Abscisic acid induction of vacuolar H+-ATPase activity in mesembryanthemum crystallinum is developmentally regulated

    Science.gov (United States)

    Barkla; Vera-Estrella; Maldonado-Gama; Pantoja

    1999-07-01

    Abscisic acid (ABA) has been implicated as a key component in water-deficit-induced responses, including those triggered by drought, NaCl, and low- temperature stress. In this study a role for ABA in mediating the NaCl-stress-induced increases in tonoplast H+-translocating ATPase (V-ATPase) and Na+/H+ antiport activity in Mesembryanthemum crystallinum, leading to vacuolar Na+ sequestration, were investigated. NaCl or ABA treatment of adult M. crystallinum plants induced V-ATPase H+ transport activity, and when applied in combination, an additive effect on V-ATPase stimulation was observed. In contrast, treatment of juvenile plants with ABA did not induce V-ATPase activity, whereas NaCl treatment resulted in a similar response to that observed in adult plants. Na+/H+ antiport activity was induced in both juvenile and adult plants by NaCl, but ABA had no effect at either developmental stage. Results indicate that ABA-induced changes in V-ATPase activity are dependent on the plant reaching its adult phase, whereas NaCl-induced increases in V-ATPase and Na+/H+ antiport activity are independent of plant age. This suggests that ABA-induced V-ATPase activity may be linked to the stress-induced, developmentally programmed switch from C3 metabolism to Crassulacean acid metabolism in adult plants, whereas, vacuolar Na+ sequestration, mediated by the V-ATPase and Na+/H+ antiport, is regulated through ABA-independent pathways.

  3. Day/night regulation of aquaporins during the CAM cycle in Mesembryanthemum crystallinum.

    Science.gov (United States)

    Vera-Estrella, Rosario; Barkla, Bronwyn J; Amezcua-Romero, Julio C; Pantoja, Omar

    2012-03-01

    Mesembryanthemum crystallinum exhibits induction of Crassulacean acid metabolism (CAM) after a threshold stage of development, by exposure to long days with high light intensities or by water and salt stress. During the CAM cycle, fluctuations in carbon partitioning within the cell lead to transient drops in osmotic potential, which are likely stabilized/balanced by passive movement of water via aquaporins (AQPs). Protoplast swelling assays were used to detect changes in water permeability during the day/night cycle of CAM. To assess the role of AQPs during the same period, we followed transcript accumulation and protein abundance of four plasma membrane intrinsic proteins (PIPs) and one tonoplast intrinsic protein (TIP). CAM plants showed a persistent rhythm of specific AQP protein abundance changes throughout the day/night cycle, including changes in amount of McPIP2;1, McTIP1;2, McPIP1;4 and McPIP1;5, while the abundance of McPIP1;2 was unchanged. These protein changes did not appear to be coordinated with transcript levels for any of the AQPs analysed; however, they did occur in parrallel to alterations in water permeability, as well as variations in cell osmolarity, pinitol, glucose, fructose and phosphoenolpyruvate carboxylase (PEPc) levels measured throughout the day/night CAM cycle. Results suggest a role for AQPs in maintaining water balance during CAM and highlight the complexity of protein expression during the CAM cycle. © 2011 Blackwell Publishing Ltd.

  4. Novel Regulation of Aquaporins during Osmotic Stress1

    Science.gov (United States)

    Vera-Estrella, Rosario; Barkla, Bronwyn J.; Bohnert, Hans J.; Pantoja, Omar

    2004-01-01

    Aquaporin protein regulation and redistribution in response to osmotic stress was investigated. Ice plant (Mesembryanthemum crystallinum) McTIP1;2 (McMIPF) mediated water flux when expressed in Xenopus leavis oocytes. Mannitol-induced water imbalance resulted in increased protein amounts in tonoplast fractions and a shift in protein distribution to other membrane fractions, suggesting aquaporin relocalization. Indirect immunofluorescence labeling also supports a change in membrane distribution for McTIP1;2 and the appearance of a unique compartment where McTIP1;2 is expressed. Mannitol-induced redistribution of McTIP1;2 was arrested by pretreatment with brefeldin A, wortmannin, and cytochalasin D, inhibitors of vesicle trafficking-related processes. Evidence suggests a role for glycosylation and involvement of a cAMP-dependent signaling pathway in McTIP1;2 redistribution. McTIP1;2 redistribution to endosomal compartments may be part of a homeostatic process to restore and maintain cellular osmolarity under osmotic-stress conditions. PMID:15299122

  5. Novel regulation of aquaporins during osmotic stress.

    Science.gov (United States)

    Vera-Estrella, Rosario; Barkla, Bronwyn J; Bohnert, Hans J; Pantoja, Omar

    2004-08-01

    Aquaporin protein regulation and redistribution in response to osmotic stress was investigated. Ice plant (Mesembryanthemum crystallinum) McTIP1;2 (McMIPF) mediated water flux when expressed in Xenopus leavis oocytes. Mannitol-induced water imbalance resulted in increased protein amounts in tonoplast fractions and a shift in protein distribution to other membrane fractions, suggesting aquaporin relocalization. Indirect immunofluorescence labeling also supports a change in membrane distribution for McTIP1;2 and the appearance of a unique compartment where McTIP1;2 is expressed. Mannitol-induced redistribution of McTIP1;2 was arrested by pretreatment with brefeldin A, wortmannin, and cytochalasin D, inhibitors of vesicle trafficking-related processes. Evidence suggests a role for glycosylation and involvement of a cAMP-dependent signaling pathway in McTIP1;2 redistribution. McTIP1;2 redistribution to endosomal compartments may be part of a homeostatic process to restore and maintain cellular osmolarity under osmotic-stress conditions.

  6. Gene Structures, Evolution, Classification and Expression Profiles of the Aquaporin Gene Family in Castor Bean (Ricinus communis L..

    Directory of Open Access Journals (Sweden)

    Zhi Zou

    Full Text Available Aquaporins (AQPs are a class of integral membrane proteins that facilitate the passive transport of water and other small solutes across biological membranes. Castor bean (Ricinus communis L., Euphobiaceae, an important non-edible oilseed crop, is widely cultivated for industrial, medicinal and cosmetic purposes. Its recently available genome provides an opportunity to analyze specific gene families. In this study, a total of 37 full-length AQP genes were identified from the castor bean genome, which were assigned to five subfamilies, including 10 plasma membrane intrinsic proteins (PIPs, 9 tonoplast intrinsic proteins (TIPs, 8 NOD26-like intrinsic proteins (NIPs, 6 X intrinsic proteins (XIPs and 4 small basic intrinsic proteins (SIPs on the basis of sequence similarities. Functional prediction based on the analysis of the aromatic/arginine (ar/R selectivity filter, Froger's positions and specificity-determining positions (SDPs showed a remarkable difference in substrate specificity among subfamilies. Homology analysis supported the expression of all 37 RcAQP genes in at least one of examined tissues, e.g., root, leaf, flower, seed and endosperm. Furthermore, global expression profiles with deep transcriptome sequencing data revealed diverse expression patterns among various tissues. The current study presents the first genome-wide analysis of the AQP gene family in castor bean. Results obtained from this study provide valuable information for future functional analysis and utilization.

  7. Identification of PEG-induced water stress responsive transcripts using co-expression network in Eucalyptus grandis.

    Science.gov (United States)

    Ghosh Dasgupta, Modhumita; Dharanishanthi, Veeramuthu

    2017-09-05

    Ecophysiological studies in Eucalyptus have shown that water is the principal factor limiting stem growth. Effect of water deficit conditions on physiological and biochemical parameters has been extensively reported in Eucalyptus. The present study was conducted to identify major polyethylene glycol induced water stress responsive transcripts in Eucalyptus grandis using gene co-expression network. A customized array representing 3359 water stress responsive genes was designed to document their expression in leaves of E. grandis cuttings subjected to -0.225MPa of PEG treatment. The differentially expressed transcripts were documented and significantly co-expressed transcripts were used for construction of network. The co-expression network was constructed with 915 nodes and 3454 edges with degree ranging from 2 to 45. Ninety four GO categories and 117 functional pathways were identified in the network. MCODE analysis generated 27 modules and module 6 with 479 nodes and 1005 edges was identified as the biologically relevant network. The major water responsive transcripts represented in the module included dehydrin, osmotin, LEA protein, expansin, arabinogalactans, heat shock proteins, major facilitator proteins, ARM repeat proteins, raffinose synthase, tonoplast intrinsic protein and transcription factors like DREB2A, ARF9, AGL24, UNE12, WLIM1 and MYB66, MYB70, MYB 55, MYB 16 and MYB 103. The coordinated analysis of gene expression patterns and coexpression networks developed in this study identified an array of transcripts that may regulate PEG induced water stress responses in E. grandis. Copyright © 2017 Elsevier B.V. All rights reserved.

  8. The Wheat E Subunit of V-Type H+-ATPase Is Involved in the Plant Response to Osmotic Stress

    Directory of Open Access Journals (Sweden)

    Xiao-Hong Zhang

    2014-09-01

    Full Text Available The vacuolar type H+-ATPase (V-type H+-ATPase plays important roles in establishing an electrochemical H+-gradient across tonoplast, energizing Na+ sequestration into the central vacuole, and enhancing salt stress tolerance in plants. In this paper, a putative E subunit of the V-type H+-ATPase gene, W36 was isolated from stress-induced wheat de novo transcriptome sequencing combining with 5'-RACE and RT-PCR methods. The full-length of W36 gene was 1097 bp, which contained a 681 bp open reading frame (ORF and encoded 227 amino acids. Southern blot analysis indicated that W36 was a single-copy gene. The quantitative real-time PCR (qRT-PCR analysis revealed that the expression level of W36 could be upregulated by drought, cold, salt, and exogenous ABA treatment. A subcellular localization assay showed that the W36 protein accumulated in the cytoplasm. Isolation of the W36 promoter revealed some cis-acting elements responding to abiotic stresses. Transgenic Arabidopsis plants overexpressing W36 were enhanced salt and mannitol tolerance. These results indicate that W36 is involved in the plant response to osmotic stress.

  9. Evidence for the presence of a sucrose carrier in immature sugar-beet roots

    International Nuclear Information System (INIS)

    Lemoine, R.; Daie, J.; Wyse, R.

    1987-01-01

    Unlike in mature sugar-beet roots, sucrose is assumed to be hydrolyzed by a wall-bound invertase prior to uptake by immature roots. To test this hypothesis, they used a sucrose analog, 1'fluorosucrose which is recognized by the carrier but is a poor substrate for invertases. Asymmetrically labeled sucrose ( 3 H-fructose) 1'fluorosucrose ( 14 C-glucose) were applied at 10 mM ( 3 H/ 14 C=1) to an attached source leaf. After 6 h, sugars from plant parts in the translocation path were separated on HPLC. 14 C-1'fluorosucrose was translocated and accumulated in the root at a higher rate than 3 H-sucrose due to greater metabolism of 3 H-sucrose in the shoot (indicated by the presence of 3 H in hexose fractions and loss of asymmetry). In the root 25% of the 3 H-sucrose was hydrolyzed to hexoses whereas no 14 C was detected in hexose fractions. The data indicate that despite high cell-wall invertase and cytoplasmic sucrose synthase activities, young sugar-beet roots import and store sucrose without hydrolysis. Therefore, the function of a group translocator at the tonoplast is unclear

  10. Rapid Ammonia Gas Transport Accounts for Futile Transmembrane Cycling under NH3/NH4+ Toxicity in Plant Roots1[C][W

    Science.gov (United States)

    Coskun, Devrim; Britto, Dev T.; Li, Mingyuan; Becker, Alexander; Kronzucker, Herbert J.

    2013-01-01

    Futile transmembrane NH3/NH4+ cycling in plant root cells, characterized by extremely rapid fluxes and high efflux to influx ratios, has been successfully linked to NH3/NH4+ toxicity. Surprisingly, the fundamental question of which species of the conjugate pair (NH3 or NH4+) participates in such fluxes is unresolved. Using flux analyses with the short-lived radioisotope 13N and electrophysiological, respiratory, and histochemical measurements, we show that futile cycling in roots of barley (Hordeum vulgare) seedlings is predominately of the gaseous NH3 species, rather than the NH4+ ion. Influx of 13NH3/13NH4+, which exceeded 200 µmol g–1 h–1, was not commensurate with membrane depolarization or increases in root respiration, suggesting electroneutral NH3 transport. Influx followed Michaelis-Menten kinetics for NH3 (but not NH4+), as a function of external concentration (Km = 152 µm, Vmax = 205 µmol g–1 h–1). Efflux of 13NH3/13NH4+ responded with a nearly identical Km. Pharmacological characterization of influx and efflux suggests mediation by aquaporins. Our study fundamentally revises the futile-cycling model by demonstrating that NH3 is the major permeating species across both plasmalemma and tonoplast of root cells under toxicity conditions. PMID:24134887

  11. Rapid ammonia gas transport accounts for futile transmembrane cycling under NH3/NH4+ toxicity in plant roots.

    Science.gov (United States)

    Coskun, Devrim; Britto, Dev T; Li, Mingyuan; Becker, Alexander; Kronzucker, Herbert J

    2013-12-01

    Futile transmembrane NH3/NH4(+) cycling in plant root cells, characterized by extremely rapid fluxes and high efflux to influx ratios, has been successfully linked to NH3/NH4(+) toxicity. Surprisingly, the fundamental question of which species of the conjugate pair (NH3 or NH4(+)) participates in such fluxes is unresolved. Using flux analyses with the short-lived radioisotope (13)N and electrophysiological, respiratory, and histochemical measurements, we show that futile cycling in roots of barley (Hordeum vulgare) seedlings is predominately of the gaseous NH3 species, rather than the NH4(+) ion. Influx of (13)NH3/(13)NH4(+), which exceeded 200 µmol g(-1) h(-1), was not commensurate with membrane depolarization or increases in root respiration, suggesting electroneutral NH3 transport. Influx followed Michaelis-Menten kinetics for NH3 (but not NH4(+)), as a function of external concentration (Km = 152 µm, Vmax = 205 µmol g(-1) h(-1)). Efflux of (13)NH3/(13)NH4(+) responded with a nearly identical Km. Pharmacological characterization of influx and efflux suggests mediation by aquaporins. Our study fundamentally revises the futile-cycling model by demonstrating that NH3 is the major permeating species across both plasmalemma and tonoplast of root cells under toxicity conditions.

  12. Cell-specific vacuolar calcium storage mediated by CAX1 regulates apoplastic calcium concentration, gas exchange, and plant productivity in Arabidopsis.

    Science.gov (United States)

    Conn, Simon J; Gilliham, Matthew; Athman, Asmini; Schreiber, Andreas W; Baumann, Ute; Moller, Isabel; Cheng, Ning-Hui; Stancombe, Matthew A; Hirschi, Kendal D; Webb, Alex A R; Burton, Rachel; Kaiser, Brent N; Tyerman, Stephen D; Leigh, Roger A

    2011-01-01

    The physiological role and mechanism of nutrient storage within vacuoles of specific cell types is poorly understood. Transcript profiles from Arabidopsis thaliana leaf cells differing in calcium concentration ([Ca], epidermis 60 mM) were compared using a microarray screen and single-cell quantitative PCR. Three tonoplast-localized Ca(2+) transporters, CAX1 (Ca(2+)/H(+)-antiporter), ACA4, and ACA11 (Ca(2+)-ATPases), were identified as preferentially expressed in Ca-rich mesophyll. Analysis of respective loss-of-function mutants demonstrated that only a mutant that lacked expression of both CAX1 and CAX3, a gene ectopically expressed in leaves upon knockout of CAX1, had reduced mesophyll [Ca]. Reduced capacity for mesophyll Ca accumulation resulted in reduced cell wall extensibility, stomatal aperture, transpiration, CO(2) assimilation, and leaf growth rate; increased transcript abundance of other Ca(2+) transporter genes; altered expression of cell wall-modifying proteins, including members of the pectinmethylesterase, expansin, cellulose synthase, and polygalacturonase families; and higher pectin concentrations and thicker cell walls. We demonstrate that these phenotypes result from altered apoplastic free [Ca(2+)], which is threefold greater in cax1/cax3 than in wild-type plants. We establish CAX1 as a key regulator of apoplastic [Ca(2+)] through compartmentation into mesophyll vacuoles, a mechanism essential for optimal plant function and productivity.

  13. Two Silene vulgaris copper transporters residing in different cellular compartments confer copper hypertolerance by distinct mechanisms when expressed in Arabidopsis thaliana.

    Science.gov (United States)

    Li, Yanbang; Iqbal, Mazhar; Zhang, Qianqian; Spelt, Cornelis; Bliek, Mattijs; Hakvoort, Henk W J; Quattrocchio, Francesca M; Koes, Ronald; Schat, Henk

    2017-08-01

    Silene vulgaris is a metallophyte of calamine, cupriferous and serpentine soils all over Europe. Its metallicolous populations are hypertolerant to zinc (Zn), cadmium (Cd), copper (Cu) or nickel (Ni), compared with conspecific nonmetallicolous populations. These hypertolerances are metal-specific, but the underlying mechanisms are poorly understood. We investigated the role of HMA5 copper transporters in Cu-hypertolerance of a S. vulgaris copper mine population. Cu-hypertolerance in Silene is correlated and genetically linked with enhanced expression of two HMA5 paralogs, SvHMA5I and SvHMA5II, each of which increases Cu tolerance when expressed in Arabidopsis thaliana. Most Spermatophytes, except Brassicaceae, possess homologs of SvHMA5I and SvHMA5II, which originate from an ancient duplication predating the appearance of spermatophytes. SvHMA5II and the A. thaliana homolog AtHMA5 localize in the endoplasmic reticulum and upon Cu exposure move to the plasma membrane, from where they are internalized and degraded in the vacuole. This resembles trafficking of mammalian homologs and is apparently an extremely ancient mechanism. SvHMA5I, instead, neofunctionalized and always resides on the tonoplast, likely sequestering Cu in the vacuole. Adaption of Silene to a Cu-polluted soil is at least in part due to upregulation of two distinct HMA5 transporters, which contribute to Cu hypertolerance by distinct mechanisms. © 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

  14. Association Genetics of Wood Physical Traits in the Conifer White Spruce and Relationships With Gene Expression

    Science.gov (United States)

    Beaulieu, Jean; Doerksen, Trevor; Boyle, Brian; Clément, Sébastien; Deslauriers, Marie; Beauseigle, Stéphanie; Blais, Sylvie; Poulin, Pier-Luc; Lenz, Patrick; Caron, Sébastien; Rigault, Philippe; Bicho, Paul; Bousquet, Jean; MacKay, John

    2011-01-01

    Marker-assisted selection holds promise for highly influencing tree breeding, especially for wood traits, by considerably reducing breeding cycles and increasing selection accuracy. In this study, we used a candidate gene approach to test for associations between 944 single-nucleotide polymorphism markers from 549 candidate genes and 25 wood quality traits in white spruce. A mixed-linear model approach, including a weak but nonsignificant population structure, was implemented for each marker–trait combination. Relatedness among individuals was controlled using a kinship matrix estimated either from the known half-sib structure or from the markers. Both additive and dominance effect models were tested. Between 8 and 21 single-nucleotide polymorphisms (SNPs) were found to be significantly associated (P ≤ 0.01) with each of earlywood, latewood, or total wood traits. After controlling for multiple testing (Q ≤ 0.10), 13 SNPs were still significant across as many genes belonging to different families, each accounting for between 3 and 5% of the phenotypic variance in 10 wood characters. Transcript accumulation was determined for genes containing SNPs associated with these traits. Significantly different transcript levels (P ≤ 0.05) were found among the SNP genotypes of a 1-aminocyclopropane-1-carboxylate oxidase, a β-tonoplast intrinsic protein, and a long-chain acyl-CoA synthetase 9. These results should contribute toward the development of efficient marker-assisted selection in an economically important tree species. PMID:21385726

  15. Many rivers to cross: the journey of zinc from soil to seed

    Directory of Open Access Journals (Sweden)

    Lene Irene Olsen

    2014-02-01

    Full Text Available An important goal of micronutrient biofortification is to enhance the amount of bioavailable zinc in the edible seed of cereals and more specifically in the endosperm. The picture is starting to emerge for how zinc is translocated from the soil through the mother plant to the developing seed. On this journey, zinc is transported from symplast to symplast via multiple apoplastic spaces. During each step, zinc is imported into a symplast before it is exported again. Cellular import and export of zinc requires passage through biological membranes, which makes membrane-bound transporters of zinc especially interesting as potential transport bottlenecks. Inside the cell, zinc can be imported into or exported out of organelles by other transporters. The function of several membrane proteins involved in the transport of zinc across the tonoplast, chloroplast or plasma membranes are currently known. These include members of the ZIP (ZRT-IRT-like Protein, and MTP (Metal Tolerance Protein and Heavy Metal ATPase (HMA families. An important player in the transport process is the ligand nicotianamine that binds zinc to increase its solubility in living cells and in this way buffers the intracellular zinc concentration.

  16. Two showy traits, scent emission and pigmentation, are finely coregulated by the MYB transcription factor PH4 in petunia flowers.

    Science.gov (United States)

    Cna'ani, Alon; Spitzer-Rimon, Ben; Ravid, Jasmin; Farhi, Moran; Masci, Tania; Aravena-Calvo, Javiera; Ovadis, Marianna; Vainstein, Alexander

    2015-11-01

    The mechanism underlying the emission of phenylpropanoid volatiles is poorly understood. Here, we reveal the involvement of PH4, a petunia MYB-R2R3 transcription factor previously studied for its role in vacuolar acidification, in floral volatile emission. We used the virus-induced gene silencing (VIGS) approach to knock down PH4 expression in petunia, measured volatile emission and internal pool sizes by GC-MS, and analyzed transcript abundances of scent-related phenylpropanoid genes in flowers. Silencing of PH4 resulted in a marked decrease in floral phenylpropanoid volatile emission, with a concurrent increase in internal pool levels. Expression of scent-related phenylpropanoid genes was not affected. To identify putative scent-related targets of PH4, we silenced PH5, a tonoplast-localized H(+) -ATPase that maintains vacuolar pH homeostasis. Suppression of PH5 did not yield the reduced-emission phenotype, suggesting that PH4 does not operate in the context of floral scent through regulation of vacuolar pH. We conclude that PH4 is a key floral regulator that integrates volatile production and emission processes and interconnects two essential floral traits - color and scent. © 2015 The Authors. New Phytologist © 2015 New Phytologist Trust.

  17. The plant homolog to the human sodium/dicarboxylic cotransporter is the vacuolar malate carrier.

    Science.gov (United States)

    Emmerlich, Vera; Linka, Nicole; Reinhold, Thomas; Hurth, Marco A; Traub, Michaela; Martinoia, Enrico; Neuhaus, H Ekkehard

    2003-09-16

    Malate plays a central role in plant metabolism. It is an intermediate in the Krebs and glyoxylate cycles, it is the store for CO2 in C4 and crassulacean acid metabolism plants, it protects plants from aluminum toxicity, it is essential for maintaining the osmotic pressure and charge balance, and it is therefore involved in regulation of stomatal aperture. To fulfil many of these roles, malate has to be accumulated within the large central vacuole. Many unsuccessful efforts have been made in the past to identify the vacuolar malate transporter; here, we describe the identification of the vacuolar malate transporter [A. thaliana tonoplast dicarboxylate transporter (AttDT)]. This transporter exhibits highest sequence similarity to the human sodium/dicarboxylate cotransporter. Independent T-DNA [portion of the Ti (tumor-inducing) plasmid that is transferred to plant cells] Arabidopsis mutants exhibit substantially reduced levels of leaf malate, but respire exogenously applied [14C]malate faster than the WT. An AttDT-GFP fusion protein was localized to vacuole. Vacuoles isolated from Arabidopsis WT leaves exhibited carbonylcyanide m-chlorophenylhydrazone and citrate inhibitable malate transport, which was not stimulated by sodium. Vacuoles isolated from mutant plants import [14C]-malate at strongly reduced rates, confirming that this protein is the vacuolar malate transporter.

  18. Genes Encoding Aluminum-Activated Malate Transporter II and their Association with Fruit Acidity in Apple

    Directory of Open Access Journals (Sweden)

    Baiquan Ma

    2015-11-01

    Full Text Available A gene encoding aluminum-activated malate transporter (ALMT was previously reported as a candidate for the locus controlling acidity in apple ( × Borkh.. In this study, we found that apple genes can be divided into three families and the gene belongs to the family. Duplication of genes in apple is related to the polyploid origin of the apple genome. Divergence in expression has occurred between the gene and its homologs in the family and only the gene is significantly associated with malic acid content. The locus consists of two alleles, and . resides in the tonoplast and its ectopic expression in yeast was found to increase the influx of malic acid into yeast cells significantly, suggesting it may function as a vacuolar malate channel. In contrast, encodes a truncated protein because of a single nucleotide substitution of G with A in the last exon. As this truncated protein resides within the cell membrane, it is deemed to be nonfunctional as a vacuolar malate channel. The frequency of the genotype is very low in apple cultivars but is high in wild relatives, which suggests that apple domestication may be accompanied by selection for the gene. In addition, variations in the malic acid content of mature fruits were also observed between accessions with the same genotype in the locus. This suggests that the gene is not the only genetic determinant of fruit acidity in apple.

  19. Ultrastructural changes in aster yellows phytoplasma affected Limonium sinuatum Mill. plants II. Pathology of cortex parenchyma cells

    Directory of Open Access Journals (Sweden)

    Anna Rudzińska-Langwald

    2014-01-01

    Full Text Available In Limonium sinuatum Mill, plants with severe symptoms of aster yellows infection phytoplasmas were present not only in the phloem but also in some cortex parenchymas cells. These parenchyma cells were situated at some distance from the conducting bundles. The phytoplasmas were observed directly in parenchyma cells cytoplasm. The number of phytoplasmas present in each selected cell varies. The cells with a small number of phytoplasmas show little pathological changes compared with the unaffected cells of the same zone of the stem as well with the cells of healthy plants. The cells filled with a number of phytoplasmas had their protoplast very much changed. The vacuole was reduced and in the cytoplasm a reduction of the number of ribosomes was noted and regions of homogenous structure appeared. Mitochondria were moved in the direction of the tonoplast and plasma membrane. Compared to the cells unaffected by phytoplasma, the mitochondria were smaller and had an enlarged cristae internal space. The chloroplasts from affected cells had a very significant reduction in size and the tylacoids system had disappeared. The role of these changes for creating phytoplasma friendly enviroment is discused.

  20. Role of aquaporins in determining transpiration and photosynthesis in water-stressed plants: crop water-use efficiency, growth and yield.

    Science.gov (United States)

    Moshelion, Menachem; Halperin, Ofer; Wallach, Rony; Oren, Ram; Way, Danielle A

    2015-09-01

    The global shortage of fresh water is one of our most severe agricultural problems, leading to dry and saline lands that reduce plant growth and crop yield. Here we review recent work highlighting the molecular mechanisms allowing some plant species and genotypes to maintain productivity under water stress conditions, and suggest molecular modifications to equip plants for greater production in water-limited environments. Aquaporins (AQPs) are thought to be the main transporters of water, small and uncharged solutes, and CO2 through plant cell membranes, thus linking leaf CO2 uptake from the intercellular airspaces to the chloroplast with water loss pathways. AQPs appear to play a role in regulating dynamic changes of root, stem and leaf hydraulic conductivity, especially in response to environmental changes, opening the door to using AQP expression to regulate plant water-use efficiency. We highlight the role of vascular AQPs in regulating leaf hydraulic conductivity and raise questions regarding their role (as well as tonoplast AQPs) in determining the plant isohydric threshold, growth rate, fruit yield production and harvest index. The tissue- or cell-specific expression of AQPs is discussed as a tool to increase yield relative to control plants under both normal and water-stressed conditions. © 2014 John Wiley & Sons Ltd.

  1. Application of plant biotechnology to address water and salt stress in developing countries (abstract)

    International Nuclear Information System (INIS)

    Masmoudi, K.

    2005-01-01

    Drought and salinity are major constraints on crop production and food security, and have adverse impact especially on socio-economic aspect in the Middle East and North Africa region. Studies of the physiological response of wheat to salt stress indicate that sequestering sodium that enters the leaf away from the cell cytosol, and enhancing osmotic adjustment capability, can ameliorate the negative impact of soil water salinity on plant growth. Sodium at high millimolar levels in the cytoplasm is toxic to plant and yeast cells, Sequestration of Na/sup +/ ions into the vacuole through the action of tonoplast proton pumps (an H/sup +/-ATPase in the case of yeast, and either an H/sup +/-pyrophosphatase (H/sup +/-PPase) or H/sup +/-ATPase in the case of plants) and an Na/sup +//H/sup +/ anti porter is one mechanism that confers salt tolerance to these organisms. The cloning and characterization of genes encoding these tonoplast transport proteins from crop plants may contribute to our understanding of how to enhance crop plant response to saline stress. We cloned wheat ortho logs of the Arabidopsis genes AtNHXI and AVP I using a wheat cDNA library, The full length sequence for the wheat Na/sup +//H/sup +/ anti porter (TNHX3) and the vacuolar H/sup +/-pyrophosphatase (TVP I) were deposited in Genbank database under the accession number AY296910 and AY296911, respectively. The deduced amino acid sequence of TNHXj is l homologous to the sequences of other NHX gene products cloned from wheat as well as barley and Arabidopsis. The vacuolar H/sup +/-PPase pump we cloned, TVP I is the first member of this gene family cloned from wheat. Function of TNHXj as a cation/proton antiporter was demonstrated using the nhxl yeast mutant. TNHXj was capable of suppressing the hygromycin sensitivity of nhxl. Functional characterization of the wheat H/sup +/-PPase TVP I was demonstrated using the yeast enal (plasma membrane Na/sup +/-efflux transporter) mutant. Expression of TVP I in enal

  2. A system dynamics model integrating physiology and biochemical regulation predicts extent of crassulacean acid metabolism (CAM) phases.

    Science.gov (United States)

    Owen, Nick A; Griffiths, Howard

    2013-12-01

    A system dynamics (SD) approach was taken to model crassulacean acid metabolism (CAM) expression from measured biochemical and physiological constants. SD emphasizes state-dependent feedback interaction to describe the emergent properties of a complex system. These mechanisms maintain biological systems with homeostatic limits on a temporal basis. Previous empirical studies on CAM have correlated biological constants (e.g. enzyme kinetic parameters) with expression over the CAM diel cycle. The SD model integrates these constants within the architecture of the CAM 'system'. This allowed quantitative causal connections to be established between biological inputs and the four distinct phases of CAM delineated by gas exchange and malic acid accumulation traits. Regulation at flow junctions (e.g. stomatal and mesophyll conductance, and malic acid transport across the tonoplast) that are subject to feedback control (e.g. stomatal aperture, malic acid inhibition of phosphoenolpyruvate carboxylase, and enzyme kinetics) was simulated. Simulated expression for the leaf-succulent Kalanchoë daigremontiana and more succulent tissues of Agave tequilana showed strong correlation with measured gas exchange and malic acid accumulation (R(2)  = 0.912 and 0.937, respectively, for K. daigremontiana and R(2)  = 0.928 and 0.942, respectively, for A. tequilana). Sensitivity analyses were conducted to quantitatively identify determinants of diel CO2 uptake. The transition in CAM expression from low to high volume/area tissues (elimination of phase II-IV carbon-uptake signatures) was achieved largely by the manipulation three input parameters. © 2013 The Authors. New Phytologist © 2013 New Phytologist Trust.

  3. Molecular cloning, functional expression and subcellular localization of two putative vacuolar voltage-gated chloride channels in rice (Oryza sativa L.).

    Science.gov (United States)

    Nakamura, Atsuko; Fukuda, Atsunori; Sakai, Shingo; Tanaka, Yoshiyuki

    2006-01-01

    We isolated two cDNA clones (OsCLC-1 and OsCLC-2) homologous to tobacco CLC-Nt1, which encodes a voltage-gated chloride channel, from rice (Oryza sativa L. ssp. japonica, cv. Nipponbare). The deduced amino acid sequences were highly conserved (87.9% identity with each other). Southern blot analysis of the rice genomic DNA revealed that OsCLC-1 and OsCLC-2 were single-copy genes on chromosomes 4 and 2, respectively. OsCLC-1 was expressed in most tissues, whereas OsCLC-2 was expressed only in the roots, nodes, internodes and leaf sheaths. The level of expression of OsCLC-1, but not of OsCLC-2, was increased by treatment with NaCl. Both genes could partly substitute for GEF1, which encodes the sole chloride channel in yeast, by restoring growth under ionic stress. These results indicate that both genes are chloride channel genes. The proteins from both genes were immunochemically detected in the tonoplast fraction. Tagged synthetic green fluorescent protein which was fused to OsCLC-1 or OsCLC-2 localized in the vacuolar membranes. These results indicate that the proteins may play a role in the transport of chloride ions across the vacuolar membrane. We isolated loss-of-function mutants of both genes from a panel of rice mutants produced by the insertion of a retrotransposon, Tos17, in the exon region, and found inhibition of growth at all life stages.

  4. Subcellular localization of H(+)-ATPase from pumpkin hypocotyls (Cucurbita maxima L.) by membrane fractionation.

    Science.gov (United States)

    Scherer, G F

    1984-03-01

    A new method of preparing sealed vesicles from membrane fractions of pumpkin hypocotyls in ethanolamine-containing buffers was used to investigate the subcellular localization of H(+)-ATPase measured as nigericin-stimulated ATPase. In a fluorescence-quench assay, the H(+) pump was directly demonstrated. The H(+) pump was substrate-specific for Mg·ATP and 0.1 mM diethylstilbestrol completely prevented the development of a Δ pH. The presence of unsupecific phosphatase hampered the detection of nigericin-stimulated ATPase. Unspecific phosphatases could be demonstrated by comparing the broad substrate specificity of the hydrolytic activities of the fractions with the clear preference for Mg·ATP as the substrate for the proton pump. Inhibitor studies showed that neither orthovanadate nor molybdate are absolutely specific for ATPase or acid phosphatase, respectively. Diethylstilbestrol seemed to be a specific inhibitor of ATPase activity in fractions containing nigericin-stimulated ATPase, but it stimulated acid phosphatase which tended to obscure its effect on ATPase activity. Nigericin-stimulated ATPase had its optimum at pH 6.0 and the nigericin effect was K(+)-dependent. The combination of valinomycin and carbonylcyanide m-chlorophenylhydrazone had a similar effect to nigericin, but singly these ionophores were much less stimulatory. After prolonged centrifugation on linear sucrose gradients, nigericin-stimulated ATPase correlated in dense fractions with plasma membrane markers but a part of it remained at the interphase. This lessdense part of the nigericin-stimulated ATPase could be derived from tonoplast vesicles because α-mannosidase, an enzyme of the vacuolar sap, remained in the upper part of the gradient. Nigericinstimulated ATPase did not correlate with the mitochondrial marker, cytochrome c oxidase, whereas azide inhibition of ATPase activity did.

  5. Saccharose action on chrysanthemum cut inflorescences, Dendranthema grandiflorum Kitamura, after exposition to gamma radiation

    International Nuclear Information System (INIS)

    Kikuchi, Olivia Kimiko

    1998-01-01

    During the last years the Brazilian floriculture expanded and it is expected to achieve a prominent role in near future. The national territory has many favorable regions, with adequate climate for the development of commercially important cultures. Flower exportation is not expressive yet, but the country has conditions to become a great exotic tropical plants exporter. On the other hand, developed countries, as USA and Japan, have a rigorous phyto sanitary inspection to prevent the introduction of new plagues and diseases through fresh products. Ionizing radiation is considered a reliable disinfestation method to control numerous fruit and flower plagues. The aim of of this work was to verify the tolerance of some Compositae family flowers to ionizing radiation and the effects to sucrose action in protecting the flowers against the gamma-radiation induced damages. This was done by measuring biochemical and physiological parameters as a function of time after irradiation. The chrysanthemum flowers were sensitive to gamma radiation, but when the inflorescences were supplied after irradiation with preservative solution containing 2% sucrose, protection against the damaging effects of radiation was achieved. The dose of 750 Gy, considered appropriate for disinfestation purpose, did not modify the protein and lipid levels, nor plasma, tonoplast and mitochondria membrane ATPase or mitochondria cytochrome-c oxidase activities. The ethylene and carbonic gas rate production increased soon after the irradiation, but decreased one day later. The decrease of the microsomal membrane fluidity and the increase of the chrysanthemum were the most sensitive parameters to measure the irradiation treatment changes. The sucrose supply was able to maintain the irradiated flowers membrane fluidity level close to the unirradiated control. Rhodante manglesii Lindl and Helichrysum bracteatum Andr. were tolerant up to 1 KGy, thus being adequate to be disinfected by gamma radiation. (author)

  6. Annotation of Selaginella moellendorffii major intrinsic proteins and the evolution of the protein family in terrestrial plants

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    Hanna Isa Anderberg

    2012-02-01

    Full Text Available Major intrinsic proteins (MIPs also called aquaporins form pores in membranes to facilitate the permeation of water and certain small polar solutes across membranes. MIPs are present in virtually every organism but are uniquely abundant in land plants. To elucidate the evolution and function of MIPs in terrestrial plants, the MIPs encoded in the genome of the spikemoss Selaginella moellendorffii were identified and analyzed. In total 19 MIPs were found in S. moellendorffii belonging to six of the seven MIP subfamilies previously identified in the moss Physcomitrella patens. Only three of the MIPs were classified as members of the conserved water specific plasma membrane intrinsic protein (PIP subfamily whereas almost half were found to belong to the diverse NOD26-like intrinsic protein (NIP subfamily permeating various solutes. The small number of PIPs in S. moellendorffii is striking compared to all other land plants and no other species has more NIPs than PIPs. Similar to moss, S. moellendorffii only has one type of tonoplast intrinsic protein (TIP. Based on ESTs from non-angiosperms we conclude that the specialized groups of TIPs present in higher plants are not found in primitive vascular plants but evolved later in a common ancestor of seed plants. We also note that the silicic acid permeable NIP2 group that has been reported from angiosperms appears at the same time. We suggest that the expansion of the number MIP isoforms in higher plants is primarily associated with an increase in the different types of specialized tissues rather than the emergence of vascular tissue per se and that the loss of subfamilies has been possible due to a functional overlap between some subfamilies.

  7. Live imaging of intra- and extracellular pH in plants using pHusion, a novel genetically encoded biosensor

    Science.gov (United States)

    Gjetting, Kisten Sisse Krag; Ytting, Cecilie Karkov; Schulz, Alexander; Fuglsang, Anja Thoe

    2012-01-01

    Changes in pH are now widely accepted as a signalling mechanism in cells. In plants, proton pumps in the plasma membrane and tonoplast play a key role in regulation of intracellular pH homeostasis and maintenance of transmembrane proton gradients. Proton transport in response to external stimuli can be expected to be finely regulated spatially and temporally. With the ambition to follow such changes live, a new genetically encoded sensor, pHusion, has been developed. pHusion is especially designed for apoplastic pH measurements. It was constitutively expressed in Arabidopsis and targeted for expression in either the cytosol or the apoplast including intracellular compartments. pHusion consists of the tandem concatenation of enhanced green fluorescent protein (EGFP) and monomeric red fluorescent protein (mRFP1), and works as a ratiometric pH sensor. Live microscopy at high spatial and temporal resolution is highly dependent on appropriate immobilization of the specimen for microscopy. Medical adhesive often used in such experiments destroys cell viability in roots. Here a novel system for immobilizing Arabidopsis seedling roots for perfusion experiments is presented which does not impair cell viability. With appropriate immobilization, it was possible to follow changes of the apoplastic and cytosolic pH in mesophyll and root tissue. Rapid pH homeostasis upon external pH changes was reflected by negligible cytosolic pH fluctuations, while the apoplastic pH changed drastically. The great potential for analysing pH regulation in a whole-tissue, physiological context is demonstrated by the immediate alkalinization of the subepidermal apoplast upon external indole-3-acetic acid administration. This change is highly significant in the elongation zone compared with the root hair zone and control roots. PMID:22407646

  8. A physiological and genetic approach to the improvement of tomato (Lycopersicon esculentum Mill.) fruit soluble solids

    International Nuclear Information System (INIS)

    Damon, S.E.

    1989-01-01

    Physiological processes and the genetic basis determining soluble solids content (SSC) of processing tomato fruit were addressed. Analysis of [ 3 H]-(fructosyl)-sucrose translocation in tomato indicates that phloem unloading in the fruit occurs, at least in part, to the apoplast. Apoplastic sucrose, glucose and fructose concentrations were estimated as 1 to 7, 12 to 49 and 8 to 63 millimolar, respectively in tomato fruit pericarp. Short-term uptake of [ 14 C]sucrose, -glucose and -fructose in tomato pericarp discs showes first order kinetics over the physiologically relevant concentration range. The uptake of [ 14 C]-(glycosyl)-1'fluorosucrose was identical to the rate of [ 14 C] sucrose uptake suggesting sucrose may be taken up directly without prior extracellular hydrolysis. Short-term uptake of all three sugars was insensitive to 10 micromolar carbonyl cyanide m-chlorophenylhydrazone and to 10 micromolar p-chloromercuribenzene sulfonic acid. However, long-term accumulation of glucose was sensitive to carbonyl cyanide m-chlorophenylhydrazone. Sugar uptake across the plasmamembrane does not appear to be energy dependent, suggesting that sugar accumulation in the tomato is driven by subsequent intracellular metabolism and/or active uptake at the tonoplast. Fourteen genomic DNA probes and ten restriction endonucleases were used to identify restriction fragment length polymorphisms (RFLPs) useful in the linkage analysis of quantitative trait loci controlling the expression of SSC in a segregating F 2 population from a cross between L. esculentum (UC204B) and L. cheesmanii f. minor, a wild species with high fruit soluble solids. RFLPs were detected between the DNAs of the two tomato species with all 14 probes

  9. Ion Frequency Landscape in Growing Plants.

    Directory of Open Access Journals (Sweden)

    Mariusz Pietruszka

    Full Text Available It has been interesting that nearly all of the ion activities that have been analysed thus far have exhibited oscillations that are tightly coupled to growth. Here, we present discrete Fourier transform (DFT spectra with a finite sampling of tip-growing cells and organs that were obtained from voltage measurements of the elongating coleoptiles of maize in situ. The electromotive force (EMF oscillations (~ 0.1 μV were measured in a simple but highly sensitive resistor-inductor circuit (RL circuit, in which the solenoid was initially placed at the tip of the specimen and then was moved thus changing its position in relation to growth (EMF can be measured first at the tip, then at the sub-apical part and finally at the shank. The influx- and efflux-induced oscillations of Ca2+, along with H+, K+ and Cl- were densely sampled (preserving the Nyquist theorem in order to 'grasp the structure' of the pulse, the logarithmic amplitude of pulse spectrum was calculated, and the detected frequencies, which displayed a periodic sequence of pulses, were compared with the literature data. A band of life vital individual pulses was obtained in a single run of the experiment, which not only allowed the fundamental frequencies (and intensities of the processes to be determined but also permitted the phase relations of the various transport processes in the plasma membrane and tonoplast to be established. A discrete (quantised frequency spectrum was achieved for a growing plant for the first time, while all of the metabolic and enzymatic functions of the life cell cycle were preserved using this totally non-invasive treatment.

  10. Aquaporin Expression and Water Transport Pathways inside Leaves Are Affected by Nitrogen Supply through Transpiration in Rice Plants

    Directory of Open Access Journals (Sweden)

    Lei Ding

    2018-01-01

    Full Text Available The photosynthetic rate increases under high-N supply, resulting in a large CO2 transport conductance in mesophyll cells. It is less known that water movement is affected by nitrogen supply in leaves. This study investigated whether the expression of aquaporin and water transport were affected by low-N (0.7 mM and high-N (7 mM concentrations in the hydroponic culture of four rice varieties: (1 Shanyou 63 (SY63, a hybrid variant of the indica species; (2 Yangdao 6 (YD6, a variant of indica species; (3 Zhendao 11 (ZD11, a hybrid variant of japonica species; and (4 Jiuyou 418 (JY418, another hybrid of the japonica species. Both the photosynthetic and transpiration rate were increased by the high-N supply in the four varieties. The expressions of aquaporins, plasma membrane intrinsic proteins (PIPs, and tonoplast membrane intrinsic protein (TIP were higher in high-N than low-N leaves, except in SY63. Leaf hydraulic conductance (Kleaf was lower in high-N than low-N leaves in SY63, while Kleaf increased under high-N supply in the YD6 variant. Negative correlations were observed between the expression of aquaporin and the transpiration rate in different varieties. Moreover, there was a significant negative correlation between transpiration rate and intercellular air space. In conclusion, the change in expression of aquaporins could affect Kleaf and transpiration. A feedback effect of transpiration would regulate aquaporin expression. The present results imply a coordination of gas exchange with leaf hydraulic conductance.

  11. The mycorrhiza-dependent defensin MtDefMd1 of Medicago truncatula acts during the late restructuring stages of arbuscule-containing cells.

    Directory of Open Access Journals (Sweden)

    Marian Uhe

    Full Text Available Different symbiotic and pathogenic plant-microbe interactions involve the production of cysteine-rich antimicrobial defensins. In Medicago truncatula, the expression of four MtDefMd genes, encoding arbuscular mycorrhiza-dependent defensins containing an N-terminal signal peptide and exhibiting some differences to non-symbiotic defensins, raised over the time of fungal colonization. Whereas the MtDefMd1 and MtDefMd2 promoters were inactive in cells containing young arbuscules, cells with fully developed arbuscules displayed different levels of promoter activities, indicating an up-regulation towards later stages of arbuscule formation. MtDefMd1 and MtDefMd2 expression was absent or strongly down-regulated in mycorrhized ram1-1 and pt4-2 mutants, known for defects in arbuscule branching or premature arbuscule degeneration, respectively. A ~97% knock-down of MtDefMd1/MtDefMd2 expression did not significantly affect arbuscule size. Although overexpression of MtDefMd1 in arbuscule-containing cells led to an up-regulation of MtRam1, encoding a key transcriptional regulator of arbuscule formation, no morphological changes were evident. Co-localization of an MtDefMd1-mGFP6 fusion with additional, subcellular markers revealed that this defensin is associated with arbuscules in later stages of their life-cycle. MtDefMd1-mGFP6 was detected in cells with older arbuscules about to collapse, and ultimately in vacuolar compartments. Comparisons with mycorrhized roots expressing a tonoplast marker indicated that MtDefMd1 acts during late restructuring processes of arbuscule-containing cells, upon their transition into a post-symbiotic state.

  12. Salt Stress in Thellungiella halophila Activates Na+ Transport Mechanisms Required for Salinity Tolerance1

    Science.gov (United States)

    Vera-Estrella, Rosario; Barkla, Bronwyn J.; García-Ramírez, Liliana; Pantoja, Omar

    2005-01-01

    Salinity is considered one of the major limiting factors for plant growth and agricultural productivity. We are using salt cress (Thellungiella halophila) to identify biochemical mechanisms that enable plants to grow in saline conditions. Under salt stress, the major site of Na+ accumulation occurred in old leaves, followed by young leaves and taproots, with the least accumulation occurring in lateral roots. Salt treatment increased both the H+ transport and hydrolytic activity of salt cress tonoplast (TP) and plasma membrane (PM) H+-ATPases from leaves and roots. TP Na+/H+ exchange was greatly stimulated by growth of the plants in NaCl, both in leaves and roots. Expression of the PM H+-ATPase isoform AHA3, the Na+ transporter HKT1, and the Na+/H+ exchanger SOS1 were examined in PMs isolated from control and salt-treated salt cress roots and leaves. An increased expression of SOS1, but no changes in levels of AHA3 and HKT1, was observed. NHX1 was only detected in PM fractions of roots, and a salt-induced increase in protein expression was observed. Analysis of the levels of expression of vacuolar H+-translocating ATPase subunits showed no major changes in protein expression of subunits VHA-A or VHA-B with salt treatment; however, VHA-E showed an increased expression in leaf tissue, but not in roots, when the plants were treated with NaCl. Salt cress plants were able to distribute and store Na+ by a very strict control of ion movement across both the TP and PM. PMID:16244148

  13. Cadmium and zinc activate adaptive mechanisms in Nicotiana tabacum similar to those observed in metal tolerant plants.

    Science.gov (United States)

    Vera-Estrella, Rosario; Gómez-Méndez, María F; Amezcua-Romero, Julio C; Barkla, Bronwyn J; Rosas-Santiago, Paul; Pantoja, Omar

    2017-09-01

    Tobacco germinated and grew in the presence of high concentrations of cadmium and zinc without toxic symptoms. Evidence suggests that these ions are sequestered into the vacuole by heavy metal/H + exchanger mechanisms. Heavy metal hyperaccumulation and hypertolerance are traits shared by a small set of plants which show specialized physiological and molecular adaptations allowing them to accumulate and sequester toxic metal ions. Nicotiana tabacum was used to test its potential as a metal-accumulator in a glass house experiment. Seed germination was not affected in the presence of increasing concentrations of zinc and cadmium. Juvenile and adult plants could concentrate CdCl 2 and ZnSO 4 to levels exceeding those in the hydroponic growth medium and maintained or increased their leaf dry weight when treated with 0.5- or 1-mM CdCl 2 or 1-mM ZnSO 4 for 5 days. Accumulation of heavy metals did not affect the chlorophyll and carotenoid levels, while variable effects were observed in cell sap osmolarity. Heavy metal-dependent H + transport across the vacuole membrane was monitored using quinacrine fluorescence quenching. Cadmium- or zinc-dependent fluorescence recovery revealed that increasing concentrations of heavy metals stimulated the activities of the tonoplast Cd 2+ or Zn 2+ /H + exchangers. Immunodetection of the V-ATPase subunits showed that the increased proton transport by zinc was not due to changes in protein amount. MTP1 and MTP4 immunodetection and semiquantitative RT-PCR of NtMTP1, NtNRAMP1, and NtZIP1 helped to identify the genes that are likely involved in sequestration of cadmium and zinc in the leaf and root tissue. Finally, we demonstrated that cadmium and zinc treatments induced an accumulation of zinc in leaf tissues. This study shows that N. tabacum possesses a hyperaccumulation response, and thus could be used for phytoremediation purposes.

  14. Abscisic Acid Induction of Vacuolar H+-ATPase Activity in Mesembryanthemum crystallinum Is Developmentally Regulated1

    Science.gov (United States)

    Barkla, Bronwyn J.; Vera-Estrella, Rosario; Maldonado-Gama, Minerva; Pantoja, Omar

    1999-01-01

    Abscisic acid (ABA) has been implicated as a key component in water-deficit-induced responses, including those triggered by drought, NaCl, and low- temperature stress. In this study a role for ABA in mediating the NaCl-stress-induced increases in tonoplast H+-translocating ATPase (V-ATPase) and Na+/H+ antiport activity in Mesembryanthemum crystallinum, leading to vacuolar Na+ sequestration, were investigated. NaCl or ABA treatment of adult M. crystallinum plants induced V-ATPase H+ transport activity, and when applied in combination, an additive effect on V-ATPase stimulation was observed. In contrast, treatment of juvenile plants with ABA did not induce V-ATPase activity, whereas NaCl treatment resulted in a similar response to that observed in adult plants. Na+/H+ antiport activity was induced in both juvenile and adult plants by NaCl, but ABA had no effect at either developmental stage. Results indicate that ABA-induced changes in V-ATPase activity are dependent on the plant reaching its adult phase, whereas NaCl-induced increases in V-ATPase and Na+/H+ antiport activity are independent of plant age. This suggests that ABA-induced V-ATPase activity may be linked to the stress-induced, developmentally programmed switch from C3 metabolism to Crassulacean acid metabolism in adult plants, whereas, vacuolar Na+ sequestration, mediated by the V-ATPase and Na+/H+ antiport, is regulated through ABA-independent pathways. PMID:10398716

  15. Salt stress in Thellungiella halophila activates Na+ transport mechanisms required for salinity tolerance.

    Science.gov (United States)

    Vera-Estrella, Rosario; Barkla, Bronwyn J; García-Ramírez, Liliana; Pantoja, Omar

    2005-11-01

    Salinity is considered one of the major limiting factors for plant growth and agricultural productivity. We are using salt cress (Thellungiella halophila) to identify biochemical mechanisms that enable plants to grow in saline conditions. Under salt stress, the major site of Na+ accumulation occurred in old leaves, followed by young leaves and taproots, with the least accumulation occurring in lateral roots. Salt treatment increased both the H+ transport and hydrolytic activity of salt cress tonoplast (TP) and plasma membrane (PM) H(+)-ATPases from leaves and roots. TP Na(+)/H+ exchange was greatly stimulated by growth of the plants in NaCl, both in leaves and roots. Expression of the PM H(+)-ATPase isoform AHA3, the Na+ transporter HKT1, and the Na(+)/H+ exchanger SOS1 were examined in PMs isolated from control and salt-treated salt cress roots and leaves. An increased expression of SOS1, but no changes in levels of AHA3 and HKT1, was observed. NHX1 was only detected in PM fractions of roots, and a salt-induced increase in protein expression was observed. Analysis of the levels of expression of vacuolar H(+)-translocating ATPase subunits showed no major changes in protein expression of subunits VHA-A or VHA-B with salt treatment; however, VHA-E showed an increased expression in leaf tissue, but not in roots, when the plants were treated with NaCl. Salt cress plants were able to distribute and store Na+ by a very strict control of ion movement across both the TP and PM.

  16. Expression of FlHMA3, a P1B2-ATPase from Festulolium loliaceum, correlates with response to cadmium stress.

    Science.gov (United States)

    Guo, Qiang; Meng, Lin; Humphreys, Mike W; Scullion, John; Mur, Luis A J

    2017-03-01

    Heavy metal ATPase 3 (HMA3), a P 1B2 -ATPase, is a key tonoplast transporter involved in mediating the vacuolar sequestration of cadmium (Cd) to detoxify the intake of this element by plants. HMA3 expression in response to Cd stress has not been previously examined in the grass hybrid species Festulolium loliaceum (Huds.) P. Fourn. In this study, FlHMA3 isolated from F. loliaceum was found to comprise 833 amino acid residues with 77% homology to the rice OsHMA3. Transient expression of FlHMA3 fused to enhanced green fluorescent protein in Arabidopsis protoplasts suggested its localization to vacuolar membranes. Quantitative real-time RT-PCR analysis of F. loliaceum revealed that FlHMA3 is expressed predominantly within roots and up-regulated by excess Cd. Over the 168 h treatment, Cd content of F. loliaceum roots was significantly higher than that of shoots, regardless of external CdCl 2 concentrations. A significant positive correlation was found between FlHMA3 expression and Cd accumulation in roots of F. loliaceum seedlings subjected to 10-100 mg L -1 CdCl 2 for 168 h or, in a separate experiment, to 25 or 100 mg L -1 CdCl 2 for the same duration. These findings provide evidence that FlHMA3 encodes a vacuolar P 1B2 -ATPase that may play an important role in Cd 2+ sequestration into root cell vacuoles, thereby limiting the entry of Cd 2+ into the cytoplasm and reducing Cd 2+ toxicity. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  17. Heterologous Expression of Two Jatropha Aquaporins Imparts Drought and Salt Tolerance and Improves Seed Viability in Transgenic Arabidopsis thaliana.

    Directory of Open Access Journals (Sweden)

    Kasim Khan

    Full Text Available Drought and high salinity are environmental conditions that cause adverse effects on the growth and productivity of crops. Aquaporins are small integral membrane proteins that belong to the family of the major intrinsic proteins (MIPs, with members in animals, plants and microbes, where they facilitate the transport of water and/or small neutral solutes thereby affecting water balance. In this study we characterized two aquaporin genes namely, plasma membrane intrinsic protein (PIP2;7 and tonoplast intrinsic protein TIP1;3 from Jatropha curcas that are localised to the plasma membrane and vacuole respectively. Transgenic Arabidopsis thaliana lines over-expressing JcPIP2;7 and JcTIP1;3 under a constitutive promoter show improved germination under high salt and mannitol compared to control seeds. These transgenic plants also show increased root length under abiotic stress conditions compared to wild type Col-0 plants. Transgenic lines exposed to drought conditions by withholding water for 20 days, were able to withstand water stress and attained normal growth after re-watering unlike control plants which could not survive. Transgenic lines also had better seed yield than control under salt stress. Importantly, seed viability of transgenic plants grown under high salt concentration was 35%-45% compared to less than 5% for control seeds obtained from plants growing under salt stress. The effect of JcPIP2;7 and JcTIP1;3 on improving germination and seed viability in drought and salinity make these important candidates for genetic manipulation of plants for growth in saline soils.

  18. The grapevine VvCAX3 is a cation/H+ exchanger involved in vacuolar Ca2+ homeostasis.

    Science.gov (United States)

    Martins, Viviana; Carneiro, Filipa; Conde, Carlos; Sottomayor, Mariana; Gerós, Hernâni

    2017-12-01

    The grapevine VvCAX3 mediates calcium transport in the vacuole and is mostly expressed in green grape berries and upregulated by Ca 2+ , Na + and methyl jasmonate. Calcium is an essential plant nutrient with important regulatory and structural roles in the berries of grapevine (Vitis vinifera L.). On the other hand, the proton-cation exchanger CAX proteins have been shown to impact Ca 2+ homeostasis with important consequences for fruit integrity and resistance to biotic/abiotic stress. Here, the CAX gene found in transcriptomic databases as having one of the highest expressions in grapevine tissues, VvCAX3, was cloned and functionally characterized. Heterologous expression in yeast showed that a truncated version of VvCAX3 lacking its NNR autoinhibitory domain (sCAX3) restored the ability of the yeast strain to grow in 100-200 mM Ca 2+ , demonstrating a role in Ca 2+ transport. The truncated VvCAX3 was further shown to be involved in the transport of Na + , Li + , Mn 2+ and Cu 2+ in yeast cells. Subcellular localization studies using fluorescently tagged proteins confirmed VvCAX3 as a tonoplast transporter. VvCAX3 is expressed in grapevine stems, leaves, roots, and berries, especially at pea size, decreasing gradually throughout development, in parallel with the pattern of calcium accumulation in the fruit. The transcript abundance of VvCAX3 was shown to be regulated by methyl jasmonate (MeJA), Ca 2+ , and Na + in grape cell suspensions, and the VvCAX3 promotor contains several predicted cis-acting elements related to developmental and stress response processes. As a whole, the results obtained add new insights on the mechanisms involved in calcium homeostasis and intracellular compartmentation in grapevine, and indicate that VvCAX3 may be an interesting target towards the development of strategies for enhancement of grape berry properties.

  19. Characterization of Gene Candidates for Vacuolar Sodium Transport from Hordeum Vulgare

    KAUST Repository

    Scheu, Arne Hagen August

    2017-05-01

    Soil salinity is a major abiotic stress for land plants, and multiple mechanisms of salt tolerance have evolved. Tissue tolerance is one of these mechanisms, which involves the sequestration of sodium into the vacuole to retain low cytosolic sodium concentrations. This enables the plant to maintain cellular functions, and ultimately maintain growth and yield. However, the molecular components involved in tissue tolerance remain elusive. Several candidate genes for vacuolar sodium sequestration have recently been identified by proteome analysis of vacuolar membranes purified from the salt-tolerant cereal Hordeum vulgare (barley). In this study, I aimed to characterize these candidates in more detail. I successfully cloned coding sequences for the majority of candidate genes with primers designed based on the barley reference genome sequence. During the course of this study a newer genome sequence with improved annotations was published, to which I also compared my observations. To study the candidate genes, I used the heterologous expression system Saccharomyces cerevisiae (yeast). I used several salt sensitive yeast strains (deficient in intrinsic sodium transporters) to test whether the candidate genes would affect their salt tolerance by mediating the sequestration of sodium into the yeast vacuole. I observed a reduction in growth upon expression for several of the gene candidate under salt-stress conditions. However, confocal microscopy suggests that most gene products are subject to degradation, and did not localize to the vacuolar membrane (tonoplast). Therefore, growth effects cannot be linked to protein function without further evidence. Various potential causes are discussed, including inaccuracies in the genome resource used as reference for primer design and issues inherent to the model system. Finally, I make suggestions on how to proceed to further characterize the candidate genes and hopefully identify novel sodium transporters from barley.

  20. Genome-Wide Characterization and Expression Analysis of Major Intrinsic Proteins during Abiotic and Biotic Stresses in Sweet Orange (Citrus sinensis L. Osb.).

    Science.gov (United States)

    Martins, Cristina de Paula Santos; Pedrosa, Andresa Muniz; Du, Dongliang; Gonçalves, Luana Pereira; Yu, Qibin; Gmitter, Frederick G; Costa, Marcio Gilberto Cardoso

    2015-01-01

    The family of aquaporins (AQPs), or major intrinsic proteins (MIPs), includes integral membrane proteins that function as transmembrane channels for water and other small molecules of physiological significance. MIPs are classified into five subfamilies in higher plants, including plasma membrane (PIPs), tonoplast (TIPs), NOD26-like (NIPs), small basic (SIPs) and unclassified X (XIPs) intrinsic proteins. This study reports a genome-wide survey of MIP encoding genes in sweet orange (Citrus sinensis L. Osb.), the most widely cultivated Citrus spp. A total of 34 different genes encoding C. sinensis MIPs (CsMIPs) were identified and assigned into five subfamilies (CsPIPs, CsTIPs, CsNIPs, CsSIPs and CsXIPs) based on sequence analysis and also on their phylogenetic relationships with clearly classified MIPs of Arabidopsis thaliana. Analysis of key amino acid residues allowed the assessment of the substrate specificity of each CsMIP. Gene structure analysis revealed that the CsMIPs possess an exon-intron organization that is highly conserved within each subfamily. CsMIP loci were precisely mapped on every sweet orange chromosome, indicating a wide distribution of the gene family in the sweet orange genome. Investigation of their expression patterns in different tissues and upon drought and salt stress treatments, as well as with 'Candidatus Liberibacter asiaticus' infection, revealed a tissue-specific and coordinated regulation of the different CsMIP isoforms, consistent with the organization of the stress-responsive cis-acting regulatory elements observed in their promoter regions. A special role in regulating the flow of water and nutrients is proposed for CsTIPs and CsXIPs during drought stress, and for most CsMIPs during salt stress and the development of HLB disease. These results provide a valuable reference for further exploration of the CsMIPs functions and applications to the genetic improvement of both abiotic and biotic stress tolerance in citrus.

  1. Genome-Wide Characterization and Expression Analysis of Major Intrinsic Proteins during Abiotic and Biotic Stresses in Sweet Orange (Citrus sinensis L. Osb..

    Directory of Open Access Journals (Sweden)

    Cristina de Paula Santos Martins

    Full Text Available The family of aquaporins (AQPs, or major intrinsic proteins (MIPs, includes integral membrane proteins that function as transmembrane channels for water and other small molecules of physiological significance. MIPs are classified into five subfamilies in higher plants, including plasma membrane (PIPs, tonoplast (TIPs, NOD26-like (NIPs, small basic (SIPs and unclassified X (XIPs intrinsic proteins. This study reports a genome-wide survey of MIP encoding genes in sweet orange (Citrus sinensis L. Osb., the most widely cultivated Citrus spp. A total of 34 different genes encoding C. sinensis MIPs (CsMIPs were identified and assigned into five subfamilies (CsPIPs, CsTIPs, CsNIPs, CsSIPs and CsXIPs based on sequence analysis and also on their phylogenetic relationships with clearly classified MIPs of Arabidopsis thaliana. Analysis of key amino acid residues allowed the assessment of the substrate specificity of each CsMIP. Gene structure analysis revealed that the CsMIPs possess an exon-intron organization that is highly conserved within each subfamily. CsMIP loci were precisely mapped on every sweet orange chromosome, indicating a wide distribution of the gene family in the sweet orange genome. Investigation of their expression patterns in different tissues and upon drought and salt stress treatments, as well as with 'Candidatus Liberibacter asiaticus' infection, revealed a tissue-specific and coordinated regulation of the different CsMIP isoforms, consistent with the organization of the stress-responsive cis-acting regulatory elements observed in their promoter regions. A special role in regulating the flow of water and nutrients is proposed for CsTIPs and CsXIPs during drought stress, and for most CsMIPs during salt stress and the development of HLB disease. These results provide a valuable reference for further exploration of the CsMIPs functions and applications to the genetic improvement of both abiotic and biotic stress tolerance in citrus.

  2. Differential activation of genes related to aluminium tolerance in two contrasting rice cultivars.

    Science.gov (United States)

    Roselló, Maite; Poschenrieder, Charlotte; Gunsé, Benet; Barceló, Juan; Llugany, Mercè

    2015-11-01

    Rice (Oryza sativa) is a highly Al-tolerant crop. Among other mechanisms, a higher expression of STAR1/STAR2 (sensitive to Al rhizotoxicity1/2) genes and of Nrat1 (NRAMP Aluminium Transporter 1), and ALS1 (Aluminium sensitive 1) can at least in part be responsible for the inducible Al tolerance in this species. Here we analysed the responses to Al in two contrasting rice varieties. All analysed toxicity/tolerance markers (root elongation, Evans blue, morin and haematoxylin staining) indicated higher Al-tolerance in variety Nipponbare, than in variety Modan. Nipponbare accumulated much less Al in the roots than Modan. Aluminium supply caused stronger expression of STAR1 in Nipponbare than in Modan. A distinctively higher increase of Al-induced abscisic acid (ABA) accumulation was found in the roots of Nipponbare than in Modan. Highest ABA levels were observed in Nipponbare after 48 h exposure to Al. This ABA peak was coincident in time with the highest expression level of STAR1. It is proposed that ABA may be required for cell wall remodulation facilitated by the enhanced UDP-glucose transport to the walls through STAR1/STAR2. Contrastingly, in the roots of Modan the expression of both Nrat1 coding for a plasma membrane Al-transporter and of ALS1 coding for a tonoplast-localized Al transporter was considerably enhanced. Moreover, Modan had a higher Al-induced expression of ASR1 a gene that has been proposed to code for a reactive oxygen scavenging protein. In conclusion, the Al-exclusion strategy of Nipponbare, at least in part mediated by STAR1 and probably regulated by ABA, provided better protection against Al toxicity than the accumulation and internal detoxification strategy of Modan mediated by Nrat1, ALS1 and ARS1. Copyright © 2015 Elsevier Inc. All rights reserved.

  3. Physiological and Molecular Analysis of Aluminium-Induced Organic Acid Anion Secretion from Grain Amaranth (Amaranthus hypochondriacus L.) Roots

    Science.gov (United States)

    Fan, Wei; Xu, Jia-Meng; Lou, He-Qiang; Xiao, Chuan; Chen, Wei-Wei; Yang, Jian-Li

    2016-01-01

    Grain amaranth (Amaranthus hypochondriacus L.) is abundant in oxalate and can secrete oxalate under aluminium (Al) stress. However, the features of Al-induced secretion of organic acid anions (OA) and potential genes responsible for OA secretion are poorly understood. Here, Al-induced OA secretion in grain amaranth roots was characterized by ion charomatography and enzymology methods, and suppression subtractive hybridization (SSH) together with quantitative real-time PCR (qRT-PCR) was used to identify up-regulated genes that are potentially involved in OA secretion. The results showed that grain amaranth roots secrete both oxalate and citrate in response to Al stress. The secretion pattern, however, differs between oxalate and citrate. Neither lanthanum chloride (La) nor cadmium chloride (Cd) induced OA secretion. A total of 84 genes were identified as up-regulated by Al, in which six genes were considered as being potentially involved in OA secretion. The expression pattern of a gene belonging to multidrug and toxic compound extrusion (MATE) family, AhMATE1, was in close agreement with that of citrate secretion. The expression of a gene encoding tonoplast dicarboxylate transporter and four genes encoding ATP-binding cassette transporters was differentially regulated by Al stress, but the expression pattern was not correlated well with that of oxalate secretion. Our results not only reveal the secretion pattern of oxalate and citrate from grain amaranth roots under Al stress, but also provide some genetic information that will be useful for further characterization of genes involved in Al toxicity and tolerance mechanisms. PMID:27144562

  4. Chromium-Induced Ultrastructural Changes and Oxidative Stress in Roots of Arabidopsis thaliana

    Directory of Open Access Journals (Sweden)

    Eleftherios P. Eleftheriou

    2015-07-01

    Full Text Available Chromium (Cr is an abundant heavy metal in nature, toxic to living organisms. As it is widely used in industry and leather tanning, it may accumulate locally at high concentrations, raising concerns for human health hazards. Though Cr effects have extensively been investigated in animals and mammals, in plants they are poorly understood. The present study was then undertaken to determine the ultrastructural malformations induced by hexavalent chromium [Cr(VI], the most toxic form provided as 100 μM potassium dichromate (K2Cr2O7, in the root tip cells of the model plant Arabidopsis thaliana. A concentration-dependent decrease of root growth and a time-dependent increase of dead cells, callose deposition, hydrogen peroxide (H2O2 production and peroxidase activity were found in Cr(VI-treated seedlings, mostly at the transition root zone. In the same zone, nuclei remained ultrastructurally unaffected, but in the meristematic zone some nuclei displayed bulbous outgrowths or contained tubular structures. Endoplasmic reticulum (ER was less affected under Cr(VI stress, but Golgi bodies appeared severely disintegrated. Moreover, mitochondria and plastids became spherical and displayed translucent stroma with diminished internal membranes, but noteworthy is that their double-membrane envelopes remained structurally intact. Starch grains and electron dense deposits occurred in the plastids. Amorphous material was also deposited in the cell walls, the middle lamella and the vacuoles. Some vacuoles were collapsed, but the tonoplast appeared integral. The plasma membrane was structurally unaffected and the cytoplasm contained opaque lipid droplets and dense electron deposits. All electron dense deposits presumably consisted of Cr that is sequestered from sensitive sites, thus contributing to metal tolerance. It is concluded that the ultrastructural changes are reactive oxygen species (ROS-correlated and the malformations observed are organelle specific.

  5. Response of Ca2+-ATPase to clinorotaion of pea seedlings. O. M. Nedukha and E. L. Kordyum

    Science.gov (United States)

    Nedukha, Olena

    2016-07-01

    The present study was aimed to reveal of response of Ca2+-ATPase activity of cortex cells in distal elongation zone of Pisum sativum root to slow clinorotation. Pea seedlings were grown on a horizontal clinostat (2 rpm) and in the stationary control for 6 days. The electron-cytochemical method was used to examine the effects of imitated microgravity on the distribution of Ca2+-ATPase in outer layers of root cortex. The quantitative analysis of the density of cytochemical reaction products was measured using the Image J program. Electron microscopy showed the presence of electron-dense lead phosphate precipitated grains, the enzymatic activity reaction products on the plasma membrane, membranes of vesicular structures, endoplasmic reticulum (ER) and on organelles envelope in both of samples of the stationary control and clinorotated seedlings. We revealed the sensitivity of Ca2+-ATPase to clinorotation. The quantitative analysis of the area and density of enzymatic activity reaction products revealed that clinorotation led to the decrease of 3.4 times the density of reaction products on the plasma membrane and the increase of reaction products density on endomembranes and organelles membranes, in particular: in 2.2 times on mitochondria membranes; in 1.3 times - on membranes of ER; in 2.5 times - on tonoplast; by an order of magnitude greater - on contacting membranes of organelles with plasma membrane in comparison with such in cells of control samples. The data analysis can indicate an intensification of calcium pump on endomembranes, on envelopes of cytoplasmic organelles and nucleus. The obtained data suggest that the redistribution of Ca2+-ATPase activity in cells can be mediated by the activation of certain isoforms of enzyme or/and by an activation of Ca2+/H+ antiporter in plasma membrane that helps to maintain optimal calcium balance in plant cells under imitated microgravity.

  6. Membrane Targeting of P-type ATPases in Plant Cells

    International Nuclear Information System (INIS)

    Harper, Jeffrey F.

    2004-01-01

    How membrane proteins are targeted to specific subcellular locations is a very complex and poorly understood area of research. Our long-term goal is to use P-type ATPases (ion pumps), in a model plant system Arabidopsis, as a paradigm to understand how members of a family of closely related membrane proteins can be targeted to different subcellular locations. The research is divided into two specific aims. The first aim is focused on determining the targeting destination of all 10 ACA-type calcium pumps (Arabidopsis Calcium ATPase) in Arabidopsis. ACAs represent a plant specific-subfamily of plasma membrane-type calcium pumps. In contrast to animals, the plant homologs have been found in multiple membrane systems, including the ER (ACA2), tonoplast (ACA4) and plasma membrane (ACA8). Their high degree of similarity provides a unique opportunity to use a comparative approach to delineate the membrane specific targeting information for each pump. One hypothesis to be tested is that an endomembrane located ACA can be re-directed to the plasma membrane by including targeting information from a plasma membrane isoform, ACA8. Our approach is to engineer domain swaps between pumps and monitor the targeting of chimeric proteins in plant cells using a Green Fluorescence Protein (GFP) as a tag. The second aim is to test the hypothesis that heterologous transporters can be engineered into plants and targeted to the plasma membrane by fusing them to a plasma membrane proton pump. As a test case we are evaluating the targeting properties of fusions made between a yeast sodium/proton exchanger (Sod2) and a proton pump (AHA2). This fusion may potentially lead to a new strategy for engineering salt resistant plants. Together these aims are designed to provide fundamental insights into the biogenesis and function of plant cell membrane systems

  7. When is a cell not a cell? A theory relating coenocytic structure to the unusual electrophysiology of Ventricaria ventricosa (Valonia ventricosa).

    Science.gov (United States)

    Shepherd, V A; Beilby, M J; Bisson, M A

    2004-06-01

    Ventricaria ventricosa and its relatives have intrigued cell biologists and electrophysiologists for over a hundred years. Historically, electrophysiologists have regarded V. ventricosa as a large single plant cell with unusual characteristics including a small and positive vacuole-to-outside membrane potential difference. However, V. ventricosa has a coenocytic construction, with an alveolate cytoplasm interpenetrated by a complex vacuole containing sulphated polysaccharides. We present a theory relating the coenocytic structure to the unusual electrophysiology of V. ventricosa. The alveolate cytoplasm of V. ventricosa consists of a collective of uninucleate cytoplasmic domains interconnected by fine cytoplasmic strands containing microtubules. The cytoplasm is capable of disassociating into single cytoplasmic domains or aggregations of domains that can regenerate new coenocytes. The cytoplasmic domains are enclosed by outer (apical) and inner (basolateral) faces of a communal membrane with polarised K(+)-transporting functions, stabilised by microtubules and resembling a tissue such as a polarised epithelium. There is evidence for membrane trafficking through endocytosis and exocytosis and so "plasmalemma" and "tonoplast" do not have fixed identities. Intra- and extracellular polysaccharide mucilage has effects on electrophysiology through reducing the activity of water and through ion exchange. The vacuole-to-outside potential difference, at which the cell membrane conductance is maximal, reverses its sign from positive under hypertonic conditions to negative under hypotonic conditions. The marked mirror symmetry of the characteristics of current as a function of voltage and conductance as a function of voltage is interpreted as a feature of the communal membrane with polarised K(+) transport. The complex inhomogeneous structure of the cytoplasm places in doubt previous measurements of cytoplasm-to-outside potential difference.

  8. Control of the Water Transport Activity of Barley HvTIP3;1 Specifically Expressed in Seeds.

    Science.gov (United States)

    Utsugi, Shigeko; Shibasaka, Mineo; Maekawa, Masahiko; Katsuhara, Maki

    2015-09-01

    Tonoplast intrinsic proteins (TIPs) are involved in the transport and storage of water, and control intracellular osmotic pressure by transporting material related to the water potential of cells. In the present study, we focused on HvTIP3;1 during the periods of seed development and desiccation in barley. HvTIP3;1 was specifically expressed in seeds. An immunochemical analysis showed that HvTIP3;1 strongly accumulated in the aleurone layers and outer layers of barley seeds. The water transport activities of HvTIP3;1 and HvTIP1;2, which also accumulated in seeds, were measured in the heterologous expression system of Xenopus oocytes. When they were expressed individually, HvTIP1;2 transported water, whereas HvTIP3;1 did not. However, HvTIP3;1 exhibited water transport activity when co-expressed with HvTIP1;2 in oocytes, and this activity was higher than when HvTIP1;2 was expressed alone. This is the first report to demonstrate that the water permeability of a TIP aquaporin was activated when co-expressed with another TIP. The split-yellow fluorescent protein (YFP) system in onion cells revealed that HvTIP3;1 interacted with HvTIP1;2 to form a heterotetramer in plants. These results suggest that HvTIP3;1 functions as an active water channel to regulate water movement through tissues during the periods of seed development and desiccation. © The Author 2015. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  9. Cadmium-induced ultramorphological and physiological changes in leaves of two transgenic cotton cultivars and their wild relative

    International Nuclear Information System (INIS)

    Daud, M.K.; Variath, M.T.; Ali, Shafaqat; Najeeb, U.; Jamil, Muhammad; Hayat, Y.; Dawood, M.; Khan, Muhammad Imran; Zaffar, M.; Cheema, Sardar Alam; Tong, X.H.; Zhu Shuijin

    2009-01-01

    The present study describes cadmium-induced alterations in the leaves as well as at the whole plant level in two transgenic cotton cultivars (BR001 and GK30) and their wild relative (Coker 312) using both ultramorphological and physiological indices. With elevated levels of Cd (i.e. 10, 100, 1000 μM), the mean lengths of root, stem and leaf and leaf width as well as their fresh and dry biomasses linearly decreased over their respective controls. Moreover, root, stem and leaf water absorption capacities progressively stimulated, which were high in leaves followed by roots and stems. BR001 accumulated more cadmium followed by GK30 and Coker 312. Root and shoot cadmium uptakes were significantly and directly correlated with each other as well as with leaf, stem and root water absorption capacities. The ultrastructural modifications in leaf mesophyll cells were triggered with increase in Cd stress regime. They were more obvious in BR001 followed by GK30 and Coker 312. Changes in morphology of chloroplast, increase in number and size of starch grains as well as increase in number of plastoglobuli were the noticed qualitative effects of Cd on photosynthetic organ. Cd in the form of electron dense granules could be seen inside the vacuoles and attached to the cell walls in all these cultivars. From the present experiment, it can be well established that both apoplastic and symplastic bindings are involved in Cd detoxification in these cultivars. Absence of tonoplast invagination reveals that Cd toxic levels did not cause water stress in any cultivars. Additionally, these cultivars possess differential capabilities towards Cd accumulation and its sequestration.

  10. Physiological and Molecular Analysis of Aluminium-Induced Organic Acid Anion Secretion from Grain Amaranth (Amaranthus hypochondriacus L.) Roots.

    Science.gov (United States)

    Fan, Wei; Xu, Jia-Meng; Lou, He-Qiang; Xiao, Chuan; Chen, Wei-Wei; Yang, Jian-Li

    2016-04-30

    Grain amaranth (Amaranthus hypochondriacus L.) is abundant in oxalate and can secrete oxalate under aluminium (Al) stress. However, the features of Al-induced secretion of organic acid anions (OA) and potential genes responsible for OA secretion are poorly understood. Here, Al-induced OA secretion in grain amaranth roots was characterized by ion charomatography and enzymology methods, and suppression subtractive hybridization (SSH) together with quantitative real-time PCR (qRT-PCR) was used to identify up-regulated genes that are potentially involved in OA secretion. The results showed that grain amaranth roots secrete both oxalate and citrate in response to Al stress. The secretion pattern, however, differs between oxalate and citrate. Neither lanthanum chloride (La) nor cadmium chloride (Cd) induced OA secretion. A total of 84 genes were identified as up-regulated by Al, in which six genes were considered as being potentially involved in OA secretion. The expression pattern of a gene belonging to multidrug and toxic compound extrusion (MATE) family, AhMATE1, was in close agreement with that of citrate secretion. The expression of a gene encoding tonoplast dicarboxylate transporter and four genes encoding ATP-binding cassette transporters was differentially regulated by Al stress, but the expression pattern was not correlated well with that of oxalate secretion. Our results not only reveal the secretion pattern of oxalate and citrate from grain amaranth roots under Al stress, but also provide some genetic information that will be useful for further characterization of genes involved in Al toxicity and tolerance mechanisms.

  11. Physiological and Molecular Analysis of Aluminium-Induced Organic Acid Anion Secretion from Grain Amaranth (Amaranthus hypochondriacus L. Roots

    Directory of Open Access Journals (Sweden)

    Wei Fan

    2016-04-01

    Full Text Available Grain amaranth (Amaranthus hypochondriacus L. is abundant in oxalate and can secrete oxalate under aluminium (Al stress. However, the features of Al-induced secretion of organic acid anions (OA and potential genes responsible for OA secretion are poorly understood. Here, Al-induced OA secretion in grain amaranth roots was characterized by ion charomatography and enzymology methods, and suppression subtractive hybridization (SSH together with quantitative real-time PCR (qRT-PCR was used to identify up-regulated genes that are potentially involved in OA secretion. The results showed that grain amaranth roots secrete both oxalate and citrate in response to Al stress. The secretion pattern, however, differs between oxalate and citrate. Neither lanthanum chloride (La nor cadmium chloride (Cd induced OA secretion. A total of 84 genes were identified as up-regulated by Al, in which six genes were considered as being potentially involved in OA secretion. The expression pattern of a gene belonging to multidrug and toxic compound extrusion (MATE family, AhMATE1, was in close agreement with that of citrate secretion. The expression of a gene encoding tonoplast dicarboxylate transporter and four genes encoding ATP-binding cassette transporters was differentially regulated by Al stress, but the expression pattern was not correlated well with that of oxalate secretion. Our results not only reveal the secretion pattern of oxalate and citrate from grain amaranth roots under Al stress, but also provide some genetic information that will be useful for further characterization of genes involved in Al toxicity and tolerance mechanisms.

  12. The vacuolar channel VvALMT9 mediates malate and tartrate accumulation in berries of Vitis vinifera.

    Science.gov (United States)

    De Angeli, Alexis; Baetz, Ulrike; Francisco, Rita; Zhang, Jingbo; Chaves, Maria Manuela; Regalado, Ana

    2013-08-01

    Vitis vinifera L. represents an economically important fruit species. Grape and wine flavour is made from a complex set of compounds. The acidity of berries is a major parameter in determining grape berry quality for wine making and fruit consumption. Despite the importance of malic and tartaric acid (TA) storage and transport for grape berry acidity, no vacuolar transporter for malate or tartrate has been identified so far. Some members of the aluminium-activated malate transporter (ALMT) anion channel family from Arabidopsis thaliana have been shown to be involved in mediating malate fluxes across the tonoplast. Therefore, we hypothesised that a homologue of these channels could have a similar role in V. vinifera grape berries. We identified homologues of the Arabidopsis vacuolar anion channel AtALMT9 through a TBLASTX search on the V. vinifera genome database. We cloned the closest homologue of AtALMT9 from grape berry cDNA and designated it VvALMT9. The expression profile revealed that VvALMT9 is constitutively expressed in berry mesocarp tissue and that its transcription level increases during fruit maturation. Moreover, we found that VvALMT9 is targeted to the vacuolar membrane. Using patch-clamp analysis, we could show that, besides malate, VvALMT9 mediates tartrate currents which are higher than in its Arabidopsis homologue. In summary, in the present study we provide evidence that VvALMT9 is a vacuolar malate channel expressed in grape berries. Interestingly, in V. vinifera, a tartrate-producing plant, the permeability of the channel is apparently adjusted to TA.

  13. Variation in soil aluminium tolerance genes is associated with local adaptation to soils at the Park Grass Experiment.

    Science.gov (United States)

    Gould, Billie; McCouch, Susan; Geber, Monica

    2014-12-01

    Studies of the wild grass Anthoxanthum odoratum at the long-term Park Grass Experiment (PGE, Harpenden, UK) document a well-known example of rapid plant evolution in response to environmental change. Repeated fertilizer applications have acidified the soil in some experimental plots over the past 150+ years, and Anthoxanthum subpopulations have quickly become locally adapted. Early reciprocal transplants showed subpopulation differentiation specifically in response to soil aluminium (Al) toxicity across the experiment, even at small (30 m) spatial scales. Almost 40 years after its original measurement, we reassessed the degree of local adaptation to soil Al at the PGE using updated phenotyping methods and identified genes with variation linked to the tolerance trait. Root growth assays show that plants are locally adapted to soil Al at both the seedling and adult growth stages, but to a smaller extent than previously inferred. Among a large suite of candidate loci that were previously shown to have Al-sensitive expression differences between sensitive and tolerant plants, three loci contained SNPs that are associated with both Al tolerance and soil acidity: an Al-sensitive malate transporter (ALMT), a tonoplast intrinsic protein (TIP) and the putative homolog of the rice cell-wall modification gene STAR1. Natural genetic variation at these loci is likely to have contributed to the recent rapid evolution at PGE. Continued study of Al tolerance variants in Anthoxanthum will allow us to test hypotheses about the nature and source of genetic variation that enables some species to adapt to soil acidification and other types of rapid environmental change. © 2014 John Wiley & Sons Ltd.

  14. Live imaging of intra- and extracellular pH in plants using pHusion, a novel genetically encoded biosensor.

    Science.gov (United States)

    Gjetting, Kisten Sisse Krag; Ytting, Cecilie Karkov; Schulz, Alexander; Fuglsang, Anja Thoe

    2012-05-01

    Changes in pH are now widely accepted as a signalling mechanism in cells. In plants, proton pumps in the plasma membrane and tonoplast play a key role in regulation of intracellular pH homeostasis and maintenance of transmembrane proton gradients. Proton transport in response to external stimuli can be expected to be finely regulated spatially and temporally. With the ambition to follow such changes live, a new genetically encoded sensor, pHusion, has been developed. pHusion is especially designed for apoplastic pH measurements. It was constitutively expressed in Arabidopsis and targeted for expression in either the cytosol or the apoplast including intracellular compartments. pHusion consists of the tandem concatenation of enhanced green fluorescent protein (EGFP) and monomeric red fluorescent protein (mRFP1), and works as a ratiometric pH sensor. Live microscopy at high spatial and temporal resolution is highly dependent on appropriate immobilization of the specimen for microscopy. Medical adhesive often used in such experiments destroys cell viability in roots. Here a novel system for immobilizing Arabidopsis seedling roots for perfusion experiments is presented which does not impair cell viability. With appropriate immobilization, it was possible to follow changes of the apoplastic and cytosolic pH in mesophyll and root tissue. Rapid pH homeostasis upon external pH changes was reflected by negligible cytosolic pH fluctuations, while the apoplastic pH changed drastically. The great potential for analysing pH regulation in a whole-tissue, physiological context is demonstrated by the immediate alkalinization of the subepidermal apoplast upon external indole-3-acetic acid administration. This change is highly significant in the elongation zone compared with the root hair zone and control roots.

  15. RNAi-directed downregulation of vacuolar H(+ -ATPase subunit a results in enhanced stomatal aperture and density in rice.

    Directory of Open Access Journals (Sweden)

    Huiying Zhang

    Full Text Available Stomatal movement plays a key role in plant development and response to drought and salt stress by regulating gas exchange and water loss. A number of genes have been demonstrated to be involved in the regulation of this process. Using inverse genetics approach, we characterized the function of a rice (Oryza sativa L. vacuolar H(+-ATPase subunit A (OsVHA-A gene in stomatal conductance regulation and physiological response to salt and osmotic stress. OsVHA-A was constitutively expressed in different rice tissues, and the fusion protein of GFP-OsVHA-A was exclusively targeted to tonoplast when transiently expressed in the onion epidermal cells. Heterologous expression of OsVHA-A was able to rescue the yeast mutant vma1Δ (lacking subunit A activity phenotype, suggesting that it partially restores the activity of V-ATPase. Meanwhile, RNAi-directed knockdown of OsVHA-A led to a reduction of vacuolar H(+-ATPase activity and an enhancement of plasma membrane H(+-ATPase activity, thereby increasing the concentrations of extracellular H(+ and intracellular K(+ and Na(+ under stress conditions. Knockdown of OsVHA-A also resulted in the upregulation of PAM3 (plasma membrane H(+-ATPase 3 and downregulation of CAM1 (calmodulin 1, CAM3 (calmodulin 3 and YDA1 (YODA, a MAPKK gene. Altered level of the ion concentration and the gene expression by knockdown of OsVHA-A probably resulted in expanded aperture of stomatal pores and increased stomatal density. In addition, OsVHA-A RNAi plants displayed significant growth inhibition under salt and osmotic stress conditions. Taken together, our results suggest that OsVHA-A takes part in regulating stomatal density and opening via interfering with pH value and ionic equilibrium in guard cells and thereby affects the growth of rice plants.

  16. Saccharose action on chrysanthemum cut inflorescences, Dendranthema grandiflorum Kitamura, after exposition to gamma radiation; Acao da sacarose na manutencao de inflorescencias cortadas de crisantemo, Dendranthema grandiflorum Kitamura, apos exposicao a radiacao gama

    Energy Technology Data Exchange (ETDEWEB)

    Kikuchi, Olivia Kimiko

    1998-07-01

    During the last years the Brazilian floriculture expanded and it is expected to achieve a prominent role in near future. The national territory has many favorable regions, with adequate climate for the development of commercially important cultures. Flower exportation is not expressive yet, but the country has conditions to become a great exotic tropical plants exporter. On the other hand, developed countries, as USA and Japan, have a rigorous phyto sanitary inspection to prevent the introduction of new plagues and diseases through fresh products. Ionizing radiation is considered a reliable disinfestation method to control numerous fruit and flower plagues. The aim of of this work was to verify the tolerance of some Compositae family flowers to ionizing radiation and the effects to sucrose action in protecting the flowers against the gamma-radiation induced damages. This was done by measuring biochemical and physiological parameters as a function of time after irradiation. The chrysanthemum flowers were sensitive to gamma radiation, but when the inflorescences were supplied after irradiation with preservative solution containing 2% sucrose, protection against the damaging effects of radiation was achieved. The dose of 750 Gy, considered appropriate for disinfestation purpose, did not modify the protein and lipid levels, nor plasma, tonoplast and mitochondria membrane ATPase or mitochondria cytochrome-c oxidase activities. The ethylene and carbonic gas rate production increased soon after the irradiation, but decreased one day later. The decrease of the microsomal membrane fluidity and the increase of the chrysanthemum were the most sensitive parameters to measure the irradiation treatment changes. The sucrose supply was able to maintain the irradiated flowers membrane fluidity level close to the unirradiated control. Rhodante manglesii Lindl and Helichrysum bracteatum Andr. were tolerant up to 1 KGy, thus being adequate to be disinfected by gamma radiation. (author)

  17. Organ and Tissue-specific Sucrose Transporters. Important Hubs in Gene and Metabolite Networks Regulating Carbon Use in Wood-forming Tissues of Populus

    Energy Technology Data Exchange (ETDEWEB)

    Harding, Scott A. [Univ. of Georgia, Athens, GA (United States); Tsai, Chung-Jui [Univ. of Georgia, Athens, GA (United States)

    2016-01-04

    The overall project objective was to probe the relationship between sucrose transporters and plant productivity in the biomass for biofuels woody perennial, Populus. At the time the proposal was written, sucrose transporters had already been investigated in many plant model systems, primarily with respect to the export of photosynthate sucrose from source leaves, and the uptake of sucrose in storage organs and seeds. Preliminary findings by the PI found that in Populus, sucrose transporter genes (SUTs) were well expressed in wood-forming tissues that comprise the feedstock for biofuels production. Because sucrose comprises by far the predominant form in which photosynthate is delivered from source organs to sink organs like roots and wood-forming tissues, SUTs control a gate that nominally at least could impact the allocation or partitioning of sucrose for potentially competing end uses like growth (stem biomass) and storage. In addition, water use might be conditioned by the way in which sucrose is distributed throughout the plant, and/or by the way in which sucrose is partitioned intracellularly. Several dozen transgenic lines were produced in year 1 of the project to perturb the expression ratio of multiple plasma membrane (PM) SUTs (intercellular trafficking), versus the single tonoplast membrane (TM) sucrose transporter that effectively regulates intracellular trafficking of sucrose. It was possible to obtain transgenic lines with dual SUT gene knockdown using the 35S promoter, but not the wood-specific TUA1 promoter. By the end of project year 2, a decision was made to work with the 35S plants while archiving the TUA1 plants. The PhD candidate charged with producing the transgenic lines abandoned the project during its second year, substantially contributing to the decision to operate with just the 35S lines. That student’s interests ranged more toward evolutionary topics, and a report on SUT gene evolution was published (Peng et al 2014).

  18. Expression profiles of aquaporin homologues and petal movement during petal development in Tulipa gesneriana.

    Science.gov (United States)

    Azad, Abul Kalam; Hanawa, Ryosuke; Ishikawa, Takahiro; Sawa, Yoshihiro; Shibata, Hitoshi

    2013-07-01

    Previously, we have characterized two tonoplast intrinsic proteins (TIPs) and four plasma membrane intrinsic proteins (PIPs) from the 2-day-old petals of tulip (Tulipa gesneriana). In this study, we analyzed the development of tulip petals and stems, temperature-dependent petal movement, the amount of ³H₂O transported into petals and stems during petal movement, and the transcript levels of two TIP (TgTIP1;1 and TgTIP1;2) and four TgPIP genes in petals and stems, from the first day of petal opening to day 12. The development of the petals and stems was completed by days 6 and 9, respectively, after the first day of petal opening. Temperature-dependent petal movement and the amount of ³H₂O that was transported into petals could be detected at significant levels up to day 6 with petal movement reaching a peak at day 3. Real-time reverse transcription-polymerase chain reaction analysis revealed that TgTIP1;1 and TgTIP1;2 were expressed ubiquitously in petals, stems, leaves, bulbs and roots. However, the expression level of TgTIP1;2 was very low in bulbs. The expression of both TgTIP1 genes was upregulated in close association with the development of petals but not with that of the stem. The four TgPIP genes were expressed at almost the same level during the development of the petals and the stem. However, the levels of the TgTIP1 and TgPIP transcripts in petals decreased during the course of petal wilting from day 9 onwards. These results suggest that TgTIP1;1 and TgTIP1;2 may contribute to petal development. Copyright © Physiologia Plantarum 2012.

  19. Genome-wide identification of Jatropha curcas aquaporin genes and the comparative analysis provides insights into the gene family expansion and evolution in Hevea brasiliensis

    Directory of Open Access Journals (Sweden)

    Zhi eZou

    2016-03-01

    Full Text Available Aquaporins (AQPs are channel-forming integral membrane proteins that transport water and other small solutes across biological membranes. Despite the vital role of AQPs, to date, little is known in physic nut (Jatropha curcas L., Euphorbiaceae, an important non-edible oilseed crop with great potential for the production of biodiesel. In this study, 32 AQP genes were identified from the physic nut genome and the family number is relatively small in comparison to 51 in another Euphorbiaceae plant, rubber tree (Hevea brasiliensis Muell. Arg.. Based on the phylogenetic analysis, the JcAQPs were assigned to five subfamilies, i.e., 9 plasma membrane intrinsic proteins (PIPs, 9 tonoplast intrinsic proteins (TIPs, 8 NOD26-like intrinsic proteins (NIPs, 2 X intrinsic proteins (XIPs and 4 small basic intrinsic proteins (SIPs. Like rubber tree and other plant species, functional prediction based on the aromatic/arginine selectivity filter, Froger’s positions and specificity-determining positions showed a remarkable difference in substrate specificity among subfamilies of JcAQPs. Genome-wide comparative analysis revealed the specific expansion of PIP and TIP subfamilies in rubber tree and the specific gene loss of the XIP subfamily in physic nut. Furthermore, by analyzing deep transcriptome sequencing data, the expression evolution especially the expression divergence of duplicated HbAQP genes was also investigated and discussed. Results obtained from this study not only provide valuable information for future functional analysis and utilization of Jc/HbAQP genes, but also provide a useful reference to survey the gene family expansion and evolution in Euphorbiaceae plants and other plant species.

  20. Electrophysiological and growing aspects of ultraviolet (UV-C) radiation action

    International Nuclear Information System (INIS)

    Karcz, W.

    1992-01-01

    Effects of UV-C (254 nm) radiation on electrical parameters and growth processes in plant cells were studied. It was found in Nitellopsis obtusa cells the UV-C radiation caused transient depolarization of plasmalemma and tonoplast and simultaneous increase in electric conductance. These effects were partly reversible and the degree of the recovery depended on the duration of the exposure, temperature of the medium and area of the irradiation. Exposure of Nitellopsis obtusa cells with large potential difference between vacuole and external medium (more negative than - 140 mV) brought about the generation of an action potential, whose shape depended on the duration of irradiation. In the cells pretreated with IAA in the dark or exposed to visible light, the UV-C irradiation not only abolished the hyper polarization induced by IAA or visible light, but caused a further depolarization. Similar effects of IAA and UV-C on membrane potential were demonstrated in cells of Zea mays L. coleoptile segments. The hyper polarized state created by visible light in Sagittaria leaf cells was also fully suppressed by the radiation. The growth experiments were based on elongation growth of Zea mays L. coleoptile segments and simultaneously measured changes of pH of the incubation medium. It was shown that for high doses of irradiation (1170, 3900, and 5850 J m -2 ) UV-C inhibited elongation growth, whereas at 195 J m -2 stimulation of growth was observed. The administration of IAA (10 -5 M) and FC (10 -6 M) to the incubation medium of coleoptile segments partially abolished the inhibitory effect of UV-C. The pH of the incubation medium showed that the exposure of the segments to UV-C caused inhibition H + -extrusion (or stimulation of H + uptake). The presence of IAA (10 -5 M) or FC (10 -6 M) in the incubation medium of irradiated coleoptile segments promoted H + -extrusion to a level comparable with that produced by IAA or FC in non-irradiated coleoptile segments. It is suggested

  1. Distribution of some pectic and arabinogalactan protein epitopes during Solanum lycopersicum (L.) adventitious root development.

    Science.gov (United States)

    Sala, Katarzyna; Malarz, Katarzyna; Barlow, Peter W; Kurczyńska, Ewa U

    2017-01-25

    The adventitious roots (AR) of plants share the same function as primary and lateral roots (LR), although their development is mainly an adaptive reaction to stress conditions. Regeneration of grafted plants is often accompanied by AR formation thus making the grafting technique a good model for studying AR initiation and development and their means of emergence. Pectins and arabinogalactan proteins (AGP) are helpful markers of particular cellular events, such as programmed cell death (PCD), elongation, proliferation or other differentiation events that accompany AR development. However, little is known about the distribution of pectins and AGPs during AR ontogeny, either in the primordium or stem tissues from which AR arise or their correspondence with these events during LR formation. AR were developed from different stem tissues such as parenchyma, xylem rays and the cambium, depending on the stem age and treatment (grafting versus cutting) of the parental tissue. Immunochemical analysis of the presence of pectic (LM8, LM19, LM20) and AGP (JIM8, JIM13, JIM16) epitopes in AR and AR-associated tissues showed differential, tissue-specific distributions of these epitopes. Two pectic epitopes (LM19, LM20) were developmentally regulated and the occurrence of the LM8 xylogalacturonan epitope in the root cap of the AR differed from other species described so far. AGP epitopes were abundantly present in the cytoplasmic compartments (mainly the tonoplast) and were correlated with the degree of cell vacuolisation. JIM8 and JIM13 epitopes were detected in the more advanced stages of primordium development, whereas the JIM16 epitope was present from the earliest division events of the initial AR cells. The comparison between AR and LR showed quantitative (AGP,) and qualitative (pectins) differences. The chemical compositions of adventitious and lateral root cells show differences that correlate with the different origins of these cells. In AR, developmental changes in the

  2. Physiological and Molecular Responses to Excess Boron in Citrus macrophylla W.

    Science.gov (United States)

    Martínez-Cuenca, Mary-Rus; Martínez-Alcántara, Belén; Quiñones, Ana; Ruiz, Marta; Iglesias, Domingo J; Primo-Millo, Eduardo; Forner-Giner, M Ángeles

    2015-01-01

    This work provides insight into several mechanisms involved in boron (B) regulation pathway in response to high B conditions in Citrus. The study was carried out in Citrus macrophylla W. (Cm) seedlings cultured "in vitro" in media with 50 or 400 μM H3BO3 (control, Ct, and B-excess, +B, plants, respectively). Growth parameters, B concentration, leaf chlorophyll (Chl) concentration, the expression of the main putative genes involved in B transport and distribution, and leaf and root proline and malonaldehyde (MDA) concentrations, were assessed. Excess B led to high B concentration in +B plants (3.8- and 1.4-fold in leaves and roots, respectively) when compared with Ct ones. However, a minor effect was recorded in the plant (incipient visual symptoms, less than 27% reduction in root growth and 26% decrease in Chl b concentration). B toxicity down-regulated by half the expression level of putative B transporter genes NIP5 and PIP1. CmBOR1 gene was not repressed in +B plants and B accumulated in the shoots. High B level increased the transcripts of putative gene TIP5, involved in B transport across the tonoplast, by 3.3- and 2.4-fold in leaves and roots, respectively. The activity of V-PPiase proton pump, related with the electrochemical gradient in the vacuole, was also enhanced in +B organs. B toxicity up-regulated putative BOR4 gene (2.1- and 2.7-fold in roots and leaves, respectively), which codifies for an active efflux B transporter. Accordingly, B was located in +B plants preferently in an insoluble form on cell walls. Finally, excess B caused a significant rise in proline concentration (51% and 34% in roots and leaves, respectively), while the MDA level did not exceed 20%. In conclusion, Cm tolerance to a high B level is likely based on the synergism of several specific mechanisms against B toxicity, including: 1/ down-regulation of NIP5 and PIP1 boron transporters; 2/ activation of B efflux from cells due to the up-regulation of putative BOR4 gene; 3

  3. Physiological and Molecular Responses to Excess Boron in Citrus macrophylla W.

    Directory of Open Access Journals (Sweden)

    Mary-Rus Martínez-Cuenca

    Full Text Available This work provides insight into several mechanisms involved in boron (B regulation pathway in response to high B conditions in Citrus. The study was carried out in Citrus macrophylla W. (Cm seedlings cultured "in vitro" in media with 50 or 400 μM H3BO3 (control, Ct, and B-excess, +B, plants, respectively. Growth parameters, B concentration, leaf chlorophyll (Chl concentration, the expression of the main putative genes involved in B transport and distribution, and leaf and root proline and malonaldehyde (MDA concentrations, were assessed. Excess B led to high B concentration in +B plants (3.8- and 1.4-fold in leaves and roots, respectively when compared with Ct ones. However, a minor effect was recorded in the plant (incipient visual symptoms, less than 27% reduction in root growth and 26% decrease in Chl b concentration. B toxicity down-regulated by half the expression level of putative B transporter genes NIP5 and PIP1. CmBOR1 gene was not repressed in +B plants and B accumulated in the shoots. High B level increased the transcripts of putative gene TIP5, involved in B transport across the tonoplast, by 3.3- and 2.4-fold in leaves and roots, respectively. The activity of V-PPiase proton pump, related with the electrochemical gradient in the vacuole, was also enhanced in +B organs. B toxicity up-regulated putative BOR4 gene (2.1- and 2.7-fold in roots and leaves, respectively, which codifies for an active efflux B transporter. Accordingly, B was located in +B plants preferently in an insoluble form on cell walls. Finally, excess B caused a significant rise in proline concentration (51% and 34% in roots and leaves, respectively, while the MDA level did not exceed 20%. In conclusion, Cm tolerance to a high B level is likely based on the synergism of several specific mechanisms against B toxicity, including: 1/ down-regulation of NIP5 and PIP1 boron transporters; 2/ activation of B efflux from cells due to the up-regulation of putative BOR4 gene

  4. Colza cell autophagy induced of high dose of industrial sewage sludge

    Science.gov (United States)

    Lasoued, Najla; Guenole Bilal, Issam; Rejeb, Saloua; Bilal, Essaid; Rejeb, Nejib

    2013-04-01

    This preliminary study is to evaluate the effects on colza of land application of industrial sludge containing heavy metals especially lead and chromium. We are interested in high doses spreading 100t/ha to better observe the phenomena of induced transformations on colza by the absorption of heavy metals. We used the technique for ultrastructural observation in a transmission electron microscope. The colza cells show a compaction and marginalization of nuclear chromatin, nuclear membrane and cytoplasmic convolution and condensation of cytoplasm. The kernel then fragments, each fragment are surrounded by a jacket. Some cytoplasmic and nuclear elements are released and are phagocytized by neighboring cells. We observed vacuolation of the cytoplasm and the formation of autophagic vesicles. The two main ways to cell death are apoptosis and autophagy. Apoptosis was not seen in plant yet. At the nucleus level cell death main characteristics are the nuclear blebbing and fragmentation. At the molecular level, caspases activity (VPE for plants, or metacaspases I and II), chromatin condensation, degradation of DNA detected by TUNEL assay and DNA laddering detected by comet test are the main events. Autophagy is the major degradation and recycling process in cells. Its aim is to address part of the cytoplasm or organelles to the proteasome. In macro-Autophagy a specific feature is the double membrane structure that we can see in electron microscopy. This membrane is known to fusion with the lysosome/vacuole where this is in process. As a rule, the vacuole grows more and more until no organelles remains. Small lytic vacuoles appear in increasing quantity also. Autophagosomes tend to be pushed against the membrane and wall of the cell. Sometime in the literature it was describe a permeabilization or a tonoplast disruption; this is the last stage called mega-autophagy. The stress generated by heavy metals in industrial sludge spreading, produces in colza cells programmed death

  5. Nitrate transporters in leaves and their potential roles in foliar uptake of nitrogen dioxide

    Directory of Open Access Journals (Sweden)

    Yanbo eHu

    2014-07-01

    Full Text Available While plant roots are specialized organs for the uptake and transport of water and nutrients, the absorption of gaseous or liquid mineral elements by aerial plant parts has been recognized since more than one century. Nitrogen (N is an essential macronutrient which generally absorbed either as nitrate (NO3- or ammonium (NH4+ by plant roots. Gaseous nitrogen pollutants like N dioxide (NO2 can also be absorbed by plant surfaces and assimilated via the NO3– assimilation pathway. The subsequent NO3– flux may induce or repress the expression of various NO3–-responsive genes encoding for instance, the transmembrane transporters, NO3–/NO2– (nitrite reductase, or assimilatory enzymes involved in N metabolism. Based on the existing information, the aim of this review was to theoretically analyze the potential link between foliar NO2 absorption and N transport and metabolism. For such purpose, an overview of the state of knowledge on the NO3– transporter genes identified in leaves or shoots of various species and their roles for NO3– transport across the tonoplast and plasma membrane, in addition to the process of phloem loading is briefly provided. It is assumed that a NO2-induced ac-cumulation of NO3–/NO2– may alter the expression of such genes, hence linking transmembrane NO3– transporters and foliar uptake of NO2. It is likely that NRT1/NRT2 gene expression and spe-cies-dependent apoplastic buffer capacity may be also related to the species-specific foliar NO2 uptake process. It is concluded that further work focusing on the expression of NRT1 (NRT1.1, NRT1.7, NRT1.11 and NRT1.12, NRT2 (NRT2.1, NRT2.4 and NRT2.5 and chloride channel family genes (CLCa and CLCd may help us elucidate the physiological and metabolic response of plants fumigated with NO2.

  6. Salinity tolerance in plants. Quantitative approach to ion transport starting from halophytes and stepping to genetic and protein engineering for manipulating ion fluxes.

    Science.gov (United States)

    Volkov, Vadim

    2015-01-01

    Ion transport is the fundamental factor determining salinity tolerance in plants. The Review starts from differences in ion transport between salt tolerant halophytes and salt-sensitive plants with an emphasis on transport of potassium and sodium via plasma membranes. The comparison provides introductory information for increasing salinity tolerance. Effects of salt stress on ion transport properties of membranes show huge opportunities for manipulating ion fluxes. Further steps require knowledge about mechanisms of ion transport and individual genes of ion transport proteins. Initially, the Review describes methods to measure ion fluxes, the independent set of techniques ensures robust and reliable basement for quantitative approach. The Review briefly summarizes current data concerning Na(+) and K(+) concentrations in cells, refers to primary thermodynamics of ion transport and gives special attention to individual ion channels and transporters. Simplified scheme of a plant cell with known transport systems at the plasma membrane and tonoplast helps to imagine the complexity of ion transport and allows choosing specific transporters for modulating ion transport. The complexity is enhanced by the influence of cell size and cell wall on ion transport. Special attention is given to ion transporters and to potassium and sodium transport by HKT, HAK, NHX, and SOS1 proteins. Comparison between non-selective cation channels and ion transporters reveals potential importance of ion transporters and the balance between the two pathways of ion transport. Further on the Review describes in detail several successful attempts to overexpress or knockout ion transporters for changing salinity tolerance. Future perspectives are questioned with more attention given to promising candidate ion channels and transporters for altered expression. Potential direction of increasing salinity tolerance by modifying ion channels and transporters using single point mutations is discussed and

  7. Identification and Characterization of Novel Plant Adenylate Cyclases – The Arabidopsis Thaliana Potassium Uptake Permeases

    KAUST Repository

    Al-Younis, Inas M.

    2018-05-01

    Adenylyl Cyclases (ACs) catalyze the formation of the key universal second messenger adenosine 3’, 5’-cyclic monophosphate (cAMP) from adenosine 5’- triphosphate. Cyclic AMP participates in several signal transduction pathways and is present in bacteria and higher and lower eukaryotes including higher plants. Previous studies in plants have shown a role for cAMP in signal transduction during e.g. the cell cycle, elongation of the pollen tube and stimulation of protein kinase activity. More recently cAMP has been shown to play a role in stress responses. Interestingly, cAMP has also been shown to regulate ion transport in plant cells. Here we used a similar strategy that led to the discovery of the first guanylyl cyclase in plants that was based on the alignment of conserved and functionally assigned amino acids in the catalytic centre of annotated nucleotide cyclases from lower and higher eukaryotes, to identify a novel candidate ACs in Arabidopsis (Arabidopsis thaliana K+ Uptake 5 and 7). ATKUP5 and 7 are homologous to K+ uptake permeases (KUPs) from bacteria and high-affinity K+ transporters (HAKs) from fungi. The AC activity was investigated by recombinantly expressing the ATKUP5 and 7 AC domain in vitro and by complementation of an E. coli AC mutant (cyaA). Furthermore, ATKUP5 was tested for its ability to functionally complement a yeast mutant deficient in Trk1 and Trk2 high affinity potassium uptake transporters. Site-mutagenesis in the AC domain was used to test the effect of both functions in each other. Furthermore, ATKUP5 was characterized electrophysiologically in HEK-293 cells to characterize the nature of this transporter. The localization of the ATKUP5 in Arabidopsis was examined using a Green Fluorescent Protein (GFP) fusion with the ATKUP5 to determine whether ATKUP5 is expressed at the plasma or tonoplast membrane. Arabiodpsis thaliana of the wild type, overexpressing ATKUP5 and atkup5 mutant lines were used to examine phenotypic differences.

  8. Newly Identified Wild Rice Accessions Conferring High Salt Tolerance Might Use a Tissue Tolerance Mechanism in Leaf

    Science.gov (United States)

    Prusty, Manas R.; Kim, Sung-Ryul; Vinarao, Ricky; Entila, Frederickson; Egdane, James; Diaz, Maria G. Q.; Jena, Kshirod K.

    2018-01-01

    + sequestration in leaf might be occurring independent of tonoplast-localized OsNHX1. The newly isolated wild rice accessions will be valuable materials for both rice improvement to salinity stress and the study of salt tolerance mechanism in plants. PMID:29740456

  9. Role of sodium ion transporters and osmotic adjustments in stress alleviation of Cynodon dactylon under NaCl treatment: a parallel investigation with rice.

    Science.gov (United States)

    Roy, Swarnendu; Chakraborty, Usha

    2018-01-01

    Comparative analyses of the responses to NaCl in Cynodon dactylon and a sensitive crop species like rice could effectively unravel the salt tolerance mechanism in the former. C. dactylon, a wild perennial chloridoid grass having a wide range of ecological distribution is generally adaptable to varying degrees of salinity stress. The role of salt exclusion mechanism present exclusively in the wild grass was one of the major factors contributing to its tolerance. Salt exclusion was found to be induced at 4 days when the plants were treated with a minimum conc. of 200 mM NaCl. The structural peculiarities of the salt exuding glands were elucidated by the SEM and TEM studies, which clearly revealed the presence of a bicellular salt gland actively functioning under NaCl stress to remove the excess amount of Na + ion from the mesophyll tissues. Moreover, the intracellular effect of NaCl on the photosynthetic apparatus was found to be lower in C. dactylon in comparison to rice; at the same time, the vacuolization process increased in the former. Accumulation of osmolytes like proline and glycine betaine also increased significantly in C. dactylon with a concurrent check on the H 2 O 2 levels, electrolyte leakage and membrane lipid peroxidation. This accounted for the proper functioning of the Na + ion transporters in the salt glands and also in the vacuoles for the exudation and loading of excess salts, respectively, to maintain the osmotic balance of the protoplasm. In real-time PCR analyses, CdSOS1 expression was found to increase by 2.5- and 5-fold, respectively, and CdNHX expression increased by 1.5- and 2-fold, respectively, in plants subjected to 100 and 200 mM NaCl treatment for 72 h. Thus, the comparative analyses of the expression pattern of the plasma membrane and tonoplast Na + ion transporters, SOS1 and NHX in both the plants revealed the significant role of these two ion transporters in conferring salinity tolerance in Cynodon.

  10. Validation of reference genes for gene expression analysis in olive (Olea europaea) mesocarp tissue by quantitative real-time RT-PCR

    Science.gov (United States)

    2014-01-01

    Background Gene expression analysis using quantitative reverse transcription PCR (qRT-PCR) is a robust method wherein the expression levels of target genes are normalised using internal control genes, known as reference genes, to derive changes in gene expression levels. Although reference genes have recently been suggested for olive tissues, combined/independent analysis on different cultivars has not yet been tested. Therefore, an assessment of reference genes was required to validate the recent findings and select stably expressed genes across different olive cultivars. Results A total of eight candidate reference genes [glyceraldehyde 3-phosphate dehydrogenase (GAPDH), serine/threonine-protein phosphatase catalytic subunit (PP2A), elongation factor 1 alpha (EF1-alpha), polyubiquitin (OUB2), aquaporin tonoplast intrinsic protein (TIP2), tubulin alpha (TUBA), 60S ribosomal protein L18-3 (60S RBP L18-3) and polypyrimidine tract-binding protein homolog 3 (PTB)] were chosen based on their stability in olive tissues as well as in other plants. Expression stability was examined by qRT-PCR across 12 biological samples, representing mesocarp tissues at various developmental stages in three different olive cultivars, Barnea, Frantoio and Picual, independently and together during the 2009 season with two software programs, GeNorm and BestKeeper. Both software packages identified GAPDH, EF1-alpha and PP2A as the three most stable reference genes across the three cultivars and in the cultivar, Barnea. GAPDH, EF1-alpha and 60S RBP L18-3 were found to be most stable reference genes in the cultivar Frantoio while 60S RBP L18-3, OUB2 and PP2A were found to be most stable reference genes in the cultivar Picual. Conclusions The analyses of expression stability of reference genes using qRT-PCR revealed that GAPDH, EF1-alpha, PP2A, 60S RBP L18-3 and OUB2 are suitable reference genes for expression analysis in developing Olea europaea mesocarp tissues, displaying the highest level

  11. Rice hypersensitive induced reaction protein 1 (OsHIR1) associates with plasma membrane and triggers hypersensitive cell death.

    Science.gov (United States)

    Zhou, Liang; Cheung, Ming-Yan; Li, Man-Wah; Fu, Yaping; Sun, Zongxiu; Sun, Sai-Ming; Lam, Hon-Ming

    2010-12-30

    In plants, HIR (Hypersensitive Induced Reaction) proteins, members of the PID (Proliferation, Ion and Death) superfamily, have been shown to play a part in the development of spontaneous hypersensitive response lesions in leaves, in reaction to pathogen attacks. The levels of HIR proteins were shown to correlate with localized host cell deaths and defense responses in maize and barley. However, not much was known about the HIR proteins in rice. Since rice is an important cereal crop consumed by more than 50% of the populations in Asia and Africa, it is crucial to understand the mechanisms of disease responses in this plant. We previously identified the rice HIR1 (OsHIR1) as an interacting partner of the OsLRR1 (rice Leucine-Rich Repeat protein 1). Here we show that OsHIR1 triggers hypersensitive cell death and its localization to the plasma membrane is enhanced by OsLRR1. Through electron microscopy studies using wild type rice plants, OsHIR1 was found to mainly localize to the plasma membrane, with a minor portion localized to the tonoplast. Moreover, the plasma membrane localization of OsHIR1 was enhanced in transgenic rice plants overexpressing its interacting protein partner, OsLRR1. Co-localization of OsHIR1 and OsLRR1 to the plasma membrane was confirmed by double-labeling electron microscopy. Pathogen inoculation studies using transgenic Arabidopsis thaliana expressing either OsHIR1 or OsLRR1 showed that both transgenic lines exhibited increased resistance toward the bacterial pathogen Pseudomonas syringae pv. tomato DC3000. However, OsHIR1 transgenic plants produced more extensive spontaneous hypersensitive response lesions and contained lower titers of the invading pathogen, when compared to OsLRR1 transgenic plants. The OsHIR1 protein is mainly localized to the plasma membrane, and its subcellular localization in that compartment is enhanced by OsLRR1. The expression of OsHIR1 may sensitize the plant so that it is more prone to HR and hence can react more

  12. Salinity tolerance in plants. Quantitative approach to ion transport starting from halophytes and stepping to genetic and protein engineering for manipulating ion fluxes

    Directory of Open Access Journals (Sweden)

    Vadim eVolkov

    2015-10-01

    Full Text Available Ion transport is the fundamental factor determining salinity tolerance in plants. The Review starts from differences in ion transport between salt tolerant halophytes and salt-sensitive plants with an emphasis on transport of potassium and sodium via plasma membranes. The comparison provides introductory information for increasing salinity tolerance. Effects of salt stress on ion transport properties of membranes show huge opportunities for manipulating ion fluxes. Further steps require knowledge about mechanisms of ion transport and individual genes of ion transport proteins. Initially, the Review describes methods to measure ion fluxes, the independent set of techniques ensures robust and reliable basement for quantitative approach. The Review briefly summarises current data concerning Na+ and K+ concentrations in cells, refers to primary thermodynamics of ion transport and gives special attention to individual ion channels and transporters. Simplified scheme of a plant cell with known transport systems at the plasma membrane and tonoplast helps to imagine the complexity of ion transport and allows to choose specific transporters for modulating ion transport. The complexity is enhanced by the influence of cell size and cell wall on ion transport. Special attention is given to ion transporters and to potassium and sodium transport by HKT, HAK, NHX and SOS1 proteins. Comparison between nonselective cation channels and ion transporters reveals potential importance of ion transporters and the balance between the two pathways of ion transport. Further on the Review describes in detail several successful attempts to overexpress or knockout ion transporters for changing salinity tolerance. Future perspectives are questioned with more attention given to promising candidate ion channels and transporters for altered expression. Potential direction of increasing salinity tolerance by modifying ion channels and transporters using single point mutations is

  13. Genome-Wide Analysis of the Aquaporin Gene Family in Chickpea (Cicer arietinum L.).

    Science.gov (United States)

    Deokar, Amit A; Tar'an, Bunyamin

    2016-01-01

    Aquaporins (AQPs) are essential membrane proteins that play critical role in the transport of water and many other solutes across cell membranes. In this study, a comprehensive genome-wide analysis identified 40 AQP genes in chickpea ( Cicer arietinum L.). A complete overview of the chickpea AQP (CaAQP) gene family is presented, including their chromosomal locations, gene structure, phylogeny, gene duplication, conserved functional motifs, gene expression, and conserved promoter motifs. To understand AQP's evolution, a comparative analysis of chickpea AQPs with AQP orthologs from soybean, Medicago, common bean, and Arabidopsis was performed. The chickpea AQP genes were found on all of the chickpea chromosomes, except chromosome 7, with a maximum of six genes on chromosome 6, and a minimum of one gene on chromosome 5. Gene duplication analysis indicated that the expansion of chickpea AQP gene family might have been due to segmental and tandem duplications. CaAQPs were grouped into four subfamilies including 15 NOD26-like intrinsic proteins (NIPs), 13 tonoplast intrinsic proteins (TIPs), eight plasma membrane intrinsic proteins (PIPs), and four small basic intrinsic proteins (SIPs) based on sequence similarities and phylogenetic position. Gene structure analysis revealed a highly conserved exon-intron pattern within CaAQP subfamilies supporting the CaAQP family classification. Functional prediction based on conserved Ar/R selectivity filters, Froger's residues, and specificity-determining positions suggested wide differences in substrate specificity among the subfamilies of CaAQPs. Expression analysis of the AQP genes indicated that some of the genes are tissue-specific, whereas few other AQP genes showed differential expression in response to biotic and abiotic stresses. Promoter profiling of CaAQP genes for conserved cis -acting regulatory elements revealed enrichment of cis -elements involved in circadian control, light response, defense and stress responsiveness

  14. The Vitis vinifera sugar transporter gene family: phylogenetic overview and macroarray expression profiling

    Directory of Open Access Journals (Sweden)

    Atanassova Rossitza

    2010-11-01

    Full Text Available Abstract Background In higher plants, sugars are not only nutrients but also important signal molecules. They are distributed through the plant via sugar transporters, which are involved not only in sugar long-distance transport via the loading and the unloading of the conducting complex, but also in sugar allocation into source and sink cells. The availability of the recently released grapevine genome sequence offers the opportunity to identify sucrose and monosaccharide transporter gene families in a woody species and to compare them with those of the herbaceous Arabidopsis thaliana using a phylogenetic analysis. Results In grapevine, one of the most economically important fruit crop in the world, it appeared that sucrose and monosaccharide transporter genes are present in 4 and 59 loci, respectively and that the monosaccharide transporter family can be divided into 7 subfamilies. Phylogenetic analysis of protein sequences has indicated that orthologs exist between Vitis and Arabidospis. A search for cis-regulatory elements in the promoter sequences of the most characterized transporter gene families (sucrose, hexoses and polyols transporters, has revealed that some of them might probably be regulated by sugars. To profile several genes simultaneously, we created a macroarray bearing cDNA fragments specific to 20 sugar transporter genes. This macroarray analysis has revealed that two hexose (VvHT1, VvHT3, one polyol (VvPMT5 and one sucrose (VvSUC27 transporter genes, are highly expressed in most vegetative organs. The expression of one hexose transporter (VvHT2 and two tonoplastic monosaccharide transporter (VvTMT1, VvTMT2 genes are regulated during berry development. Finally, three putative hexose transporter genes show a preferential organ specificity being highly expressed in seeds (VvHT3, VvHT5, in roots (VvHT2 or in mature leaves (VvHT5. Conclusions This study provides an exhaustive survey of sugar transporter genes in Vitis vinifera and

  15. Calcium in plant cells

    Directory of Open Access Journals (Sweden)

    V. V. Schwartau

    2014-04-01

    Full Text Available The paper gives the review on the role of calcium in many physiological processes of plant organisms, including growth and development, protection from pathogenic influences, response to changing environmental factors, and many other aspects of plant physiology. Initial intake of calcium ions is carried out by Ca2+-channels of plasma membrane and they are further transported by the xylem owing to auxins’ attractive ability. The level of intake and selectivity of calcium transport to ove-ground parts of the plant is controlled by a symplast. Ca2+enters to the cytoplasm of endoderm cells through calcium channels on the cortical side of Kaspary bands, and is redistributed inside the stele by the symplast, with the use of Ca2+-АТPases and Ca2+/Н+-antiports. Owing to regulated expression and activity of these calcium transporters, calclum can be selectively delivered to the xylem. Important role in supporting calcium homeostasis is given to the vacuole which is the largest depo of calcium. Regulated quantity of calcium movement through the tonoplast is provided by a number of potential-, ligand-gated active transporters and channels, like Ca2+-ATPase and Ca2+/H+ exchanger. They are actively involved in the inactivation of the calcium signal by pumping Ca2+ to the depo of cells. Calcium ATPases are high affinity pumps that efficiently transfer calcium ions against the concentration gradient in their presence in the solution in nanomolar concentrations. Calcium exchangers are low affinity, high capacity Ca2+ transporters that are effectively transporting calcium after raising its concentration in the cell cytosol through the use of protons gradients. Maintaining constant concentration and participation in the response to stimuli of different types also involves EPR, plastids, mitochondria, and cell wall. Calcium binding proteins contain several conserved sequences that provide sensitivity to changes in the concentration of Ca2+ and when you