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Sample records for arabidopsis protoplast isolation

  1. Isolation of protoplasts from tissues of 14-day-old seedlings of Arabidopsis thaliana.

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    Zhai, Zhiyang; Jung, Ha-Il; Vatamaniuk, Olena K

    2009-08-17

    Protoplasts are plant cells that have had their cell walls enzymatically removed. Isolation of protoplasts from different plant tissues was first reported more than 40 years ago and has since been adapted to study a variety of cellular processes, such as subcellular localization of proteins, isolation of intact organelles and targeted gene-inactivation by double stranded RNA interference (RNAi). Most of the protoplast isolation protocols use leaf tissues of mature Arabidopsis (e.g. 35-day-old plants). We modified existing protocols by employing 14-day-old Arabidopsis seedlings. In this procedure, one gram of 14-day-old seedlings yielded 5 10(6)-10(7) protoplasts that remain intact at least 96 hours. The yield of protoplasts from seedlings is comparable with preparations from leaves of mature Arabidopsis, but instead of 35-36 days, isolation of protoplasts is completed in 15 days. This allows decreasing the time and growth chamber space that are required for isolating protoplasts when mature plants are used, and expedites the downstream studies that require intact protoplasts.

  2. A lower content of de-methylesterified homogalacturonan improves enzymatic cell separation and isolation of mesophyll protoplasts in Arabidopsis.

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    Lionetti, Vincenzo; Cervone, Felice; De Lorenzo, Giulia

    2015-04-01

    Cell adhesion occurs primarily at the level of middle lamella which is mainly composed by pectin polysaccharides. These can be degraded by cell wall degrading enzymes (CWDEs) during developmental processes to allow a controlled separation of plant cells. Extensive cell wall degradation by CWDEs with consequent cell separation is performed when protoplasts are isolated from plant tissues by using mixtures of CWDEs. We have evaluated whether modification of pectin affects cell separation and protoplast isolation. Arabidopsis plants overexpressing the pectin methylesterase inhibitors AtPMEI-1 or AtPMEI-2, and Arabidopsis pme3 plants, mutated in the gene encoding pectin methylesterase 3, showed an increased efficiency of isolation of viable mesophyll protoplasts as compared with Wild Type Columbia-0 plants. The release of protoplasts was correlated with the reduced level of long stretches of de-methylesterified homogalacturonan (HGA) present in these plants. Response to elicitation, cell wall regeneration and efficiency of transfection in protoplasts from transgenic plants was comparable to those of wild type protoplasts.

  3. Cryobehavior of the plasma membrane in protoplasts isolated from cold-acclimated Arabidopsis leaves is related to surface area regulation.

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    Yamazaki, Tomokazu; Kawamura, Yukio; Uemura, Matsuo

    2008-06-01

    Extracellular freezing in plants results in dehydration and mechanical stresses upon the plasma membrane. Plants that acquire enhanced freezing tolerance after cold acclimation can withstand these two physical stresses. To understand the tolerance to freeze-induced physical stresses, the cryobehavior of the plasma membrane was observed using protoplasts isolated from cold-acclimated Arabidopsis thaliana leaves with the combination of a lipophilic fluorescent dye FM 1-43 and cryomicroscopy. We found that many vesicular structures appeared in the cytoplasmic region near the plasma membrane just after extracellular freezing occurred. These structures, referred to as freeze-induced vesicular structures (FIVs), then developed horizontally near the plasma membrane during freezing. There was a strong correlation between the increase in individual FIV size and the decrease in the surface area of the protoplasts during freezing. Some FIVs fused with their neighbors as the temperature decreased. Occasionally, FIVs fused with the plasma membrane, which may be necessary to relax the stress upon the plasma membrane during freezing. Vesicular structures resembling FIVs were also induced when protoplasts were mechanically pressed between a coverslip and slide glass. Fewer FIVs formed when protoplasts were subjected to hyperosmotic solution, suggesting that FIV formation is associated with mechanical stress rather than dehydration. Collectively, these results suggest that cold-acclimated plant cells may balance membrane tension in the plasma membrane by regulating the surface area. This enables plant cells to withstand the direct mechanical stress imposed by extracellular freezing.

  4. In vivo localization in Arabidopsis protoplasts and root tissue.

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    Lee, Myoung Hui; Lee, Yongjik; Hwang, Inhwan

    2013-01-01

    In eukaryotic cells, a large number of proteins are transported to their final destination after translation by a process called intracellular trafficking. Transient gene expression, either in plant protoplasts or in specific plant tissues, is a fast, flexible, and reproducible approach to study the cellular function of proteins, protein subcellular localizations, and protein-protein interactions. Here we describe the general method of protoplast isolation, polyethylene glycol-mediated protoplast transformation and immunostaining of protoplast or intact root tissues for studying the localization of protein in Arabidopsis.

  5. Isolation of Chloroplasts from Plant Protoplasts.

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    Lung, Shiu-Cheung; Smith, Matthew D; Chuong, Simon D X

    2015-10-01

    Chloroplasts can be isolated from higher plants directly following homogenization; however, the resulting yield, purity, and intactness are often low, necessitating a large amount of starting material. This protocol is optimized to produce a high yield of pure chloroplasts from isolated Arabidopsis protoplasts. The two-part method is a simple, scaled-down, and low-cost procedure that readily provides healthy mesophyll protoplasts, which are then ruptured to release intact chloroplasts. Chloroplasts isolated using this method are competent for use in biochemical, cellular, and molecular analyses.

  6. Embryoids derived from isolated protoplasts of carrot.

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    Kameya, T; Uchimiya, H

    1972-12-01

    Protoplasts isolated enzymatically from carrot root tissues developed into cell clusters in a liquid medium containing coconut milk and naphthaleneacetic acid (NAA) or 2,4-dichlorophenoxyacetic acid (2,4-D). Cells of the resulting calluses differentiated into embryoids on an agar medium containing coconut milk or kinetin.

  7. Isolation, culture, and transient transformation of plant protoplasts.

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    Shen, Jinbo; Fu, Jiaxin; Ma, Jin; Wang, Xiangfeng; Gao, Caiji; Zhuang, ChuXiong; Wan, Jianmin; Jiang, Liwen

    2014-06-03

    Transient gene expression in protoplasts, which has been used in several plant species, is an important and versatile tool for rapid functional gene analysis, protein subcellular localization, and biochemical manipulations. This unit describes transient gene expression by electroporation of DNA into protoplasts of Arabidopsis or tobacco suspension-cultured cells and by polyethylene glycol (PEG)-mediated DNA transformation into protoplasts derived from rice leaf sheaths. PEG-mediated DNA transformation for transient gene expression in rice protoplasts in suspension culture is also described as an alternative technique. Methods for collecting intracellular and secreted proteins are also provided.

  8. Dissection of miRNA pathways using arabidopsis mesophyll protoplasts.

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    Martinho, Cláudia; Confraria, Ana; Elias, Carlos Alexandre; Crozet, Pierre; Rubio-Somoza, Ignacio; Weigel, Detlef; Baena-González, Elena

    2015-02-01

    MicroRNAs (miRNAs) control gene expression mostly post-transcriptionally by guiding transcript cleavage and/or translational repression of complementary mRNA targets, thereby regulating developmental processes and stress responses. Despite the remarkable expansion of the field, the mechanisms underlying miRNA activity are not fully understood. In this article, we describe a transient expression system in Arabidopsis mesophyll protoplasts, which is highly amenable for the dissection of miRNA pathways. We show that by transiently overexpressing primary miRNAs and target mimics, we can manipulate miRNA levels and consequently impact on their targets. Furthermore, we developed a set of luciferase-based sensors for quantifying miRNA activity that respond specifically to both endogenous and overexpressed miRNAs and target mimics. We demonstrate that these miRNA sensors can be used to test the impact of putative components of the miRNA pathway on miRNA activity, as well as the impact of specific mutations, by either overexpression or the use of protoplasts from the corresponding mutants. We further show that our miRNA sensors can be used for investigating the effect of chemicals on miRNA activity. Our cell-based transient expression system is fast and easy to set up, and generates quantitative results, being a powerful tool for assaying miRNA activity in vivo.

  9. Isolation of mesophyll protoplasts from mature leaves of soybeans.

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    Lin, W

    1983-12-01

    A procedure based on a combined cellulase-Pectolyase Y-23 enzyme digestion and metrizamide-sorbitol gradient purification protocol was developed for isolating mesophyll protoplasts from mature leaves of soybean (Glycine max L. Merr.). Based on chlorophyll content, this procedure results in a 10 to 15% protoplast yield from fully expanded mature leaves and a 20 to 30% yield from young (expanding) leaves within 3 hours. Isolated protoplasts displayed high rates of HCO(3) (-)-dependent photosynthesis; greater than 75 micromoles O(2) evolved per milligram chlorophyll per hour at 25 degrees C. This photosynthetic rate is comparable to that of mesophyll cells isolated mechanically from the same leaves.

  10. Isolation, culture, and plant regeneration from leaf protoplasts of Passiflora.

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    Davey, Michael R; Anthony, Paul; Power, J Brian; Lowe, Kenneth C

    2006-01-01

    The family Passifloraceae contains many species exploited in the food, pharmaceutical, and ornamental plant industries. The routine culture of isolated protoplasts (naked cells) followed by reproducible plant regeneration, is crucial to the genetic improvement of Passiflora spp. by somatic cell technologies. Such procedures include somatic hybridization by protoplast fusion to generate novel hybrid plants, and gene introduction by transformation. Seedling leaves are a convenient source of totipotent protoplasts. The protoplast-to-plant system developed for Passiflora edulis fv. flavicarpa is summarized in this chapter. The procedure involves enzymatic degradation of leaf tissue using commercially-available Macerozyme R10, Cellulase R10, and Driselase. Isolated protoplasts are cultured in Kao and Michayluk medium, semi-solidified with agarose. The medium containing the suspended protoplasts is dispensed as droplets or thin layers and bathed in liquid medium of the same composition. Shoot regeneration involves transfer of protoplast-derived tissues to Murashige and Skoog-based medium. The protocols developed for P. edulis are applicable to other Passiflora spp. and will underpin the future biotechnological exploitation of a range of species in this important plant family.

  11. Guard cell protoplasts: isolation, culture, and regeneration of plants.

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    Tallman, Gary

    2006-01-01

    Guard cell protoplasts have been used extensively in short-term experiments designed to elucidate the signal transduction mechanisms that regulate stomatal movements. The utility of uard cell protoplasts for other types of longer-term signal transduction experiments is just now being realized. Because highly purified, primary isolates of guard cell protoplasts are synchronous initially, they are uniform in their responses to changes in culture conditions. Such isolates have demonstrated potential to reveal mechanisms that underlie hormonal signalling for plant cell survival, cell cycle re-entry, reprogramming of genes during dedifferentiation to an embryogenic state, and plant cell thermotolerance. Plants have been regenerated from cultured guard cell protoplasts of two species: Nicotiana glauca (Graham), tree tobacco, and Beta vulgaris, sugar beet. Plants genetically engineered for herbicide tolerance have been regenerated from cultured guard cell protoplasts of B. vulgaris. The method for isolating, culturing, and regenerating plants from guard cell protoplasts of N. glauca is described here. A recently developed procedure for large-scale isolation of these cells from as many as nine leaves per experiment is described. Using this protocol, yields of 1.5-2 x 10(7) per isolate may be obtained. Such yields are sufficient for standard methods of molecular, biochemical, and proteomic analysis.

  12. Isolation of Pichia manshurica protoplast from Dahlia sp plant

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    Wijanarka Wijanarka

    2014-02-01

    Full Text Available Isolation of protoplasts is an important step in the fusion process. Protoplasts are cells that have eliminated the cell wall, but the cell membranes and organs can still function properly. Pichia manshurica is one of indogenous yeast that derived from Dahlia €™s plants. The success rate protoplast isolation was determined by various factors, include the age of the culture and the used of lytic enzymes. The purpose of this research is to get the perfect age of yeast culture that is ready to be harvested and also to get the appropriate concentration of Glucanex lytic enzymes which used for protoplast isolation. The yeast of Pichia manshurica grown on YPD broth medium and growth observed in turbidimetry. Observation of the growth of yeasts performed every 6 hours for 42 hours. Glucanex lytic enzyme concentration used for the isolation of protoplasts is 0 mg / mL (L0 = control, 2 mg / mL (L2 and 4 mg / mL (L4. The results showed that the age of the culture is right and ready for harvest at the age of 24 hours and Glucanex lytic enzyme concentration of 4 mg / mL (L4 is able to produce the best of protoplasts at 7.2 x 1010.

  13. Isolation of Protoplasts from Undaria pinnatifida by Alginate Lyase Digestion

    Institute of Scientific and Technical Information of China (English)

    HU Xiaoke; JIANG Xiaolu; GUAN Huashi

    2003-01-01

    The aim of this study is to isolate protoplasts from Undaria pinnatifida. Protoplasts of the alga were isolated enzymatically by using alginate lyase, which was prepared by fermenting culture of a strain Vibrio sp. 510. Monofacterial method was applied for optimizing digestion condition. The optimum condition for protoplast preparation is enzymatic digestion at 28 ℃ for 2 h using alginate lyase at the concentration of 213.36 U (8 mL) every 0.5 g fresh thalline with NaCl 50 and at the shaking speed of 150 r min-1 during digestion. The protoplast yield can reach 2.62 + 0.09 million per 0.5 g fresh leave under the optimum condition. The enzyme activity is inhibited by Ca2+ and slightly enhanced by Fe2+ and Mn2+ at concentrations of 0.05, 0.08 and 0.10 molL-1.

  14. Analysis of a transcription factor using transient assay in Arabidopsis protoplasts.

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    Iwata, Yuji; Lee, Mi-Hyun; Koizumi, Nozomu

    2011-01-01

    Regulation of gene expression by transcription factors is a fundamental mechanism in essentially all aspects of cellular processes. Transient expression assay of a reporter plasmid containing a reporter gene driven by a promoter of interest and an effector plasmid expressing a transcription factor has been a powerful tool for analyzing transcription factors. Here we present a protocol for polyethylene glycol (PEG)-mediated transformation of Arabidopsis protoplasts. It details preparation of protoplasts from Arabidopsis suspension cultured cells or leaves of soil-grown Arabidopsis plants and subsequent PEG-mediated transformation with reporter and effector plasmids. This protocol can be completed within 24 h from protoplast preparation to reporter assay. As an example, analysis of the membrane-bound transcription factor AtbZIP60 and its target BiP3 promoter is shown.

  15. Amino Acid transport in protoplasts isolated from soybean leaves.

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    Vernooy, C D; Lin, W

    1986-05-01

    We isolated large quantities of mesophyll protoplasts from source and sink leaves of soybean plants and examined them for amino acid uptake. Accumulation of amino acids in isolated protoplasts was linear for at least 40 minutes. Uptake kinetics revealed the presence of both saturable and linear components. Increasing external pH decreases the uptake. The uncoupler, carbonyl cyanide p-trifluoromethoxyphenylhydrazone at 15 micromolar inhibited and fusicoccin at 10 micromolar stimulated amino acid uptake. Our data are consistent with a proton-cotransport mechanism for the uptake of l-glutamine and alpha-amino isobutyric acid into soybean mesophyll cells.

  16. Viability of Rhizobium bacteroids isolated from soybean nodule protoplasts.

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    Gresshoff, P M; Rolfe, B G

    1978-01-01

    Bacteriods isolated from protoplasts taken from Rhizobium japonicum induced root nodule of Glycine max L. showed complete viability when plated onto a conventional rhizobial growth medium supplemented with 0.2 M Mannitol. The same medium but without extra mannitol resulted in the absence of colony formation. The protoplast isolation method eliminated the possibility of contaminant bacteria from infection threads to be scored. The redifferentiated bacteroid clones have the same genetical characteristics as the orginal inoculum strain. This and other recent findings of bacteroid viability are discussed in the light of the existing belief that bacteroids are non-viable.

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

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

    2014-11-01

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

  18. An Arabidopsis and tomato mesophyll protoplast system for fast identification of early MAMP-triggered immunity-suppressing effectors.

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    Fraiture, Malou; Zheng, Xiangzi; Brunner, Frédéric

    2014-01-01

    Transient expression in plant mesophyll protoplasts allows rapid characterisation of gene functions in vivo in a simplified and synchronized manner without bias due to the use of bacteria-based gene or protein delivery systems. It offers the possibility to test whether microbial effectors can subvert early events of plant immune signaling that are activated upon recognition of Microbe-Associated Molecular Patterns (MAMPs), the so-called MAMP-triggered immunity (MTI). Here, we describe the isolation and transfection with effector genes of Arabidopsis thaliana and Solanum lycopersicum mesophyll protoplasts, the use of a non-invasive luciferase reporter assay and a simple method to detect activated Mitogen-Activated Protein Kinases (MAPKs) to identify and study, in a medium-throughput manner, new effectors suppressing early signal transduction events of MTI.

  19. [Protoplasts isolation, purification and plant regeneration of Pinellia cordata].

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    Yang, Xian; Ma, Dan-Dan; Jiang, Fu-Sheng; Chen, Ni-Pi; Ding, Bin; Jin, Li-Xia; Qian, Chao-Dong; Ding, Zhi-Shan

    2014-11-01

    The main factors which affected the isolation, purification and cultivation of Pinellia cordata protoplasts from leaves were studied. The results indicated that the optimum enzyme solution for P. cordata leaves was 13% CPW + 1.0% Cellulose +0.1% Pectolase, at pH 6.0, temperature (25-28 degrees C ) for 4 h. The sucrose density gradient centrifugation was adopted to purificate the protoplasts collected, when 25% sucrose was used as mediator, centrifugating at 500 rpm for 10 min. When the protoplasts were shallow liquid and liquid-solid double layer cultured on the medium of MS + 0.5 mg x L(-1) 6-BA + 0.25 mg x L(-1) NAA + 13% mannitol at the density of 2.5 x 104 protoplasts/mL, or fed and nursed cultured at the density of 100-500 protoplasts/mL, cell division could be observed for 3 days; granular calli appeared for 30 days. Calli was proliferated on the medium of MS + 0.5 mg x L(-1) 6-BA + 0.25 mg x L(-1) NAA solidified by 0.55% agar, and differentiated and regenerated after 5-6 months. Plant generation of P. cordata is successfully established.

  20. Isolation, culture, and plant regeneration from Echinacea purpurea protoplasts.

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    Pan, Zeng-guang; Liu, Chun-zhao; Murch, Susan I; Saxena, Praveen K

    2006-01-01

    A plant regeneration system from the isolated protoplasts of Echinacea purpurea L. using an alginate solid/liquid culture is described in the chapter. Viable protoplasts were isolated rom 100 mg of young leaves of 4-wk-old seedlings in an isolation mixture containing 1.0% cellulase Onozuka R-10, 0.5% pectinase, and 0.3 mol/L mannitol. After isolation and purification, the mesophyll protoplasts were embedded into 0.6% Na-alginate at the density 1 x 10(-5) mL and cultured in modified Murashige and Skoog (MS) culture medium supplemented with 0.3 mol/L sucrose, 2.5 micromol/L benzylaminopurine (BA), and 5.0 micromol/L 2,4-dichlorophenoxyacetic acid (2,4-D). The visible colonies were present after 4 wk of culture. The protoplast-derived clones were transferred onto gellan gum-solidified basal medium supplemented with 1.0 micromol/L BA and 2.0 micromol/L indole-3-butyric acid (IBA) and formed compact and green calli. Shoot development was achieved by subculturing the calli onto the same basal medium supplemented with 5.0 micromol/L BA and 2.0 micromol/L IBA. Further subculture onto basal medium resulted in the regeneration of complete plantlets.

  1. Protoplast Isolation in Lupin ( Lupinus mutabilis Sweet): Determination of Optimum Explant Sources and Isolation Conditions

    OpenAIRE

    BABAOĞLU, Mehmet

    2000-01-01

    Effects of cultural factors on the yield, viability and division of protoplasts were investigated in Lupinus mutabilis Sweet containing a high protein content as well as a reasonable oil content which may make this species an alternative crop to soybean in Turkey. Explants from different in vitro seedling parts were evaluated on the suitability of protoplast isolation and viability. Leaf mesophyll was the most suitable tissue as a protoplast source. Pectinases as well as cellulases were es...

  2. Isolation and culture of protoplast from leaves of Lactuca sativa

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    Witool Chaipakdee

    2007-07-01

    Full Text Available Protoplasts were isolated from leaves of lettuce (Lactuca sativa L. seedlings after in vitro germination for 25, 30, 40 and 50 days. The leaves were stripped and incubated in various combinations of cellulase and pectinase. Protoplasts were cultured on MS medium containing various kinds and concentrations of plant growth regulators in different culture systems including liquid media, hanging, drop culture and solid media. Results revealed that the highest number of viable protoplasts, 14.1x105 cells per gram of fresh weight, was obtained from 30 day-old leaves of lettuce seedlings and isolated by using 2% cellulase in combination with 1% pectinase. Liquid MS medium supplemented with 0.5 mg/l NAA and 0.5 mg/l BA promoted the highest cell division up to 17.67%. First division of protoplasts was observed at 4 days after culture and microcolony formation occurred at the 4th week after culturing. Unfortunately, neither callus formation nor plantlet regeneration were obtained.

  3. Glucose-1-phosphate transport into protoplasts and chloroplasts from leaves of Arabidopsis.

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    Fettke, Joerg; Malinova, Irina; Albrecht, Tanja; Hejazi, Mahdi; Steup, Martin

    2011-04-01

    Almost all glucosyl transfer reactions rely on glucose-1-phosphate (Glc-1-P) that either immediately acts as glucosyl donor or as substrate for the synthesis of the more widely used Glc dinucleotides, ADPglucose or UDPglucose. In this communication, we have analyzed two Glc-1-P-related processes: the carbon flux from externally supplied Glc-1-P to starch by either mesophyll protoplasts or intact chloroplasts from Arabidopsis (Arabidopsis thaliana). When intact protoplasts or chloroplasts are incubated with [U-(14)C]Glc-1-P, starch is rapidly labeled. Incorporation into starch is unaffected by the addition of unlabeled Glc-6-P or Glc, indicating a selective flux from Glc-1-P to starch. However, illuminated protoplasts incorporate less (14)C into starch when unlabeled bicarbonate is supplied in addition to the (14)C-labeled Glc-1-P. Mesophyll protoplasts incubated with [U-(14)C]Glc-1-P incorporate (14)C into the plastidial pool of adenosine diphosphoglucose. Protoplasts prepared from leaves of mutants of Arabidopsis that lack either the plastidial phosphorylase or the phosphoglucomutase isozyme incorporate (14)C derived from external Glc-1-P into starch, but incorporation into starch is insignificant when protoplasts from a mutant possessing a highly reduced ADPglucose pyrophosphorylase activity are studied. Thus, the path of assimilatory starch biosynthesis initiated by extraplastidial Glc-1-P leads to the plastidial pool of adenosine diphosphoglucose, and at this intermediate it is fused with the Calvin cycle-driven route. Mutants lacking the plastidial phosphoglucomutase contain a small yet significant amount of transitory starch.

  4. Highly efficient isolation of Populus mesophyll protoplasts and its application in transient expression assays.

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    Jianjun Guo

    Full Text Available BACKGROUND: Populus is a model woody plant and a promising feedstock for lignocellulosic biofuel production. However, its lengthy life cycle impedes rapid characterization of gene function. METHODOLOGY/PRINCIPAL FINDINGS: We optimized a Populus leaf mesophyll protoplast isolation protocol and established a Populus protoplast transient expression system. We demonstrated that Populus protoplasts are able to respond to hormonal stimuli and that a series of organelle markers are correctly localized in the Populus protoplasts. Furthermore, we showed that the Populus protoplast transient expression system is suitable for studying protein-protein interaction, gene activation, and cellular signaling events. CONCLUSIONS/SIGNIFICANCE: This study established a method for efficient isolation of protoplasts from Populus leaf and demonstrated the efficacy of using Populus protoplast transient expression assays as an in vivo system to characterize genes and pathways.

  5. Highly Efficient Isolation of Populus Mesophyll Protoplasts and Its Application in Transient Expression Assays

    Energy Technology Data Exchange (ETDEWEB)

    Guo, Jianjun [ORNL; Morrell-Falvey, Jennifer L [ORNL; Labbe, Jessy L [ORNL; Muchero, Wellington [ORNL; Kalluri, Udaya C [ORNL; Tuskan, Gerald A [ORNL; Chen, Jay [ORNL

    2012-01-01

    Background: Populus is a model woody plant and a promising feedstock for lignocellulosic biofuel production. However, its lengthy life cycle impedes rapid characterization of gene function. Methodology/Principal Findings: We optimized a Populus leaf mesophyll protoplast isolation protocol and established a Populus protoplast transient expression system. We demonstrated that Populus protoplasts are able to respond to hormonal stimuli and that a series of organelle markers are correctly localized in the Populus protoplasts. Furthermore, we showed that the Populus protoplast transient expression system is suitable for studying protein-protein interaction, gene activation, and cellular signaling events. Conclusions/Significance: This study established a method for efficient isolation of protoplasts from Populus leaf and demonstrated the efficacy of using Populus protoplast transient expression assays as an in vivo system to characterize genes and pathways.

  6. Sugar transport into protoplasts isolated from developing soybean cotyledons : I. Protoplast isolation and general characteristics of sugar transport.

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    Lin, W; Schmitt, M R; Hitz, W D; Giaquinta, R T

    1984-08-01

    A procedure is described to isolated functional protoplasts from developing soybean (Glycine max L. Merr. cv Wye) cotyledons. Studies of sucrose and hexose uptake into these protoplasts show that the plasmalemma of cotyledons during the stage of rapid seed growth contains a sucrose-specific carrier which is energetically and kinetically distinct from the system(s) involved in hexose transport. For example, sucrose, but not hexose uptake: (a) is inhibited by alkaline pH and the nonpermeant SH modifier, p-chloromercuribenzene sulfonic acid; (b) is stimulated by fusicoccin; (c) shows both a saturable and a linear component of uptake in response to substrate concentration; and (d) displays a sharp temperature response (high Q(10) value and high activation energies).

  7. Comparative analysis of MAMP-induced calcium influx in Arabidopsis seedlings and protoplasts.

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    Maintz, Jens; Cavdar, Meltem; Tamborski, Janina; Kwaaitaal, Mark; Huisman, Rik; Meesters, Christian; Kombrink, Erich; Panstruga, Ralph

    2014-10-01

    Rapid transient elevation of cytoplasmic calcium (Ca(2+)) levels in plant cells is an early signaling event triggered by many environmental cues including abiotic and biotic stresses. Cellular Ca(2+) levels and their alterations can be monitored by genetically encoded reporter systems such as the bioluminescent protein, aequorin. Employment of proteinaceous Ca(2+) sensors is usually performed in transgenic lines that constitutively express the reporter construct. Such settings limit the usage of these Ca(2+) biosensors to particular reporter variants and plant genetic backgrounds, which can be a severe constraint in genetic pathway analysis. Here we systematically explored the potential of Arabidopsis thaliana leaf mesophyll protoplasts, either derived from a transgenic apoaequorin-expressing line or transfected with apoaequorin reporter constructs, as a complementary biological resource to monitor cytoplasmic changes of Ca(2+) levels in response to various biotic stress elicitors. We tested a range of endogenous and pathogen-derived elicitors in seedlings and protoplasts of the corresponding apoaequorin-expressing reporter line. We found that the protoplast system largely reflects the Ca(2+) signatures seen in intact transgenic seedlings. Results of inhibitor experiments including the calculation of IC50 values indicated that the protoplast system is also suitable for pharmacological studies. Moreover, analyses of Ca(2+)signatures in mutant backgrounds, genetic complementation of the mutant phenotypes and expression of sensor variants targeted to different subcellular localizations can be readily performed. Thus, in addition to the prevalent use of seedlings, the leaf mesophyll protoplast setup represents a versatile and convenient tool for the analysis of Ca(2+) signaling pathways in plant cells.

  8. Improved technique for isolation and culture of protoplasts from young leaves of mangosteen (Garcinia mangostana L.

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    Moosikapala, L.

    2002-04-01

    Full Text Available Improved technique for isolation of protoplasts from young leaves of mangosteen was developed using dark treatment and varying ages of in vitro-grown leaves. In this experiment different kinds and concentrations of cellulase Onozuka R-10, macerozyme R-10 and pectolyase Y-23 were used. One gram fresh weight of leaf tissue was incubated in a 10 ml of enzyme solution and placed on a gyratory shaker at 40-50 rpm under darkness for 12 hours. Yield and viability of protoplasts were compared among those treatments, then the density was adjusted and cultured in MS medium supplemented with different kinds and concentrations of growth regulators. The results showed that 8 week-old leaves (after adding liquid culture medium gave released protoplasts at 1.9 × 105/gram fresh weight (g fr wt. This result was obtained when 2% cellulase Onozuka R-10, 1% macerozyme R-10 and 0.1% pectolyase Y-23 were used. Viability of the protoplasts was 77.63%. Pretreatment the leaves in the dark for 24 hours before being subjected to protoplast isolation resulted in the greatest release of protoplasts at 1 × 106/g fr wt. Viability of the protoplasts was also the highest (91.35%. The protoplasts at density of 5 × 105/ml could promote cell division at 3.41% in a thin layer of liquid MS with 0.5 mg/l BA and 0.5 mg/l TDZ.

  9. Isolation and culture of Celosia cristata L cell suspension protoplasts

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    Retno Mastuti

    2003-06-01

    Full Text Available Developmental competence of Celosia cristata L. cell suspension-derived protoplasts was investigated. The protoplasts were isolatedfrom 3- to 9-d old cultures in enzyme solution containing 2% (w/v Cellulase YC and 0.5% (w/v Macerozyme R-10 which was dissolvedin washing solution (0.4 M mannitol and 10 mM CaCl2 at pH 5.6 for 3 hours. The highest number of viable protoplasts was releasedfrom 5-d old culture of a homogenous cell suspension. Subsequently, three kinds of protoplast culture media were simultaneously examinedwith four kinds of concentration of gelling agent. Culturing the protoplasts on KM8p medium solidified with 1.2% agarose significantlyenhanced plating efficiency as well as microcolony formation. Afterwards, the microcalli actively proliferated into friable watery calluswhen they were subcultured on MS medium supplemented with 0.3 mg/l 2,4-D and 1.0 mg/l kinetin. Although the plant regenerationfrom the protoplasts-derived calli has not yet been obtained, the reproducible developmental step from protoplasts to callus in thisstudy may facilitate the establishment of somatic hybridization using C. cristata as one parent.

  10. Routes to the tonoplast: the sorting of tonoplast transporters in Arabidopsis mesophyll protoplasts.

    Science.gov (United States)

    Wolfenstetter, Susanne; Wirsching, Petra; Dotzauer, Dorina; Schneider, Sabine; Sauer, Norbert

    2012-01-01

    Vacuoles perform a multitude of functions in plant cells, including the storage of amino acids and sugars. Tonoplast-localized transporters catalyze the import and release of these molecules. The mechanisms determining the targeting of these transporters to the tonoplast are largely unknown. Using the paralogous Arabidopsis thaliana inositol transporters INT1 (tonoplast) and INT4 (plasma membrane), we performed domain swapping and mutational analyses and identified a C-terminal di-leucine motif responsible for the sorting of higher plant INT1-type transporters to the tonoplast in Arabidopsis mesophyll protoplasts. We demonstrate that this motif can reroute other proteins, such as INT4, SUCROSE TRANSPORTER2 (SUC2), or SWEET1, to the tonoplast and that the position of the motif relative to the transmembrane helix is critical. Rerouted INT4 is functionally active in the tonoplast and complements the growth phenotype of an int1 mutant. In Arabidopsis plants defective in the β-subunit of the AP-3 adaptor complex, INT1 is correctly localized to the tonoplast, while sorting of the vacuolar sucrose transporter SUC4 is blocked in cis-Golgi stacks. Moreover, we demonstrate that both INT1 and SUC4 trafficking to the tonoplast is sensitive to brefeldin A. Our data show that plants possess at least two different Golgi-dependent targeting mechanisms for newly synthesized transporters to the tonoplast.

  11. Photosynthetic responses of thalli and isolated protoplasts of Bryopsis hypnoides (Bryopsidales,Chlorophyta) during dehydration

    Institute of Scientific and Technical Information of China (English)

    LU Fang; WANG Guangce; JIN Haochen

    2011-01-01

    Bryopsis hypnoides Lamouroux is a unique intertidal siphonous green alga whose extruded protoplasm can aggregate spontaneously in seawater to form numerous new cells that can develop into mature algal thalli. In this study, the photosynthetic responses during dehydration of both the thalli and protoplasts isolated from B. hypnoides were measured using a DuaI-PAM (pulse amplitude modulation)-100 fluorometer. The results show that the photosynthetic rates of B. hypnoides thalli were maintained for an initial period, beyond which continued desiccation resulted in reduced rates of PSI and PSII. However, the photosynthetic performances of the isolated protoplasts dehydrated in air (CO2 concentration 600-700 mg/L) showed a slight increase of Y(Ⅱ) at 20% water loss, but the rates decreased thereafter with declining water content. When protoplasts were dehydrated in CO2 deficient conditions (CO2 concentration 40-80 mg/L), the values of Y(Ⅱ)declined steadily with increased dehydration without an initial rise. These results indicated that the thalli and isolated protoplasts of this alga can utilize CO2 in ambient air effectively, and the photosynthetic performances of the isolated protoplasts were significantly different from that of the thalli during dehydration. Thus the protoplasts may be an excellent system for the study of stress tolerance.

  12. Effects of freezing and cold acclimation on the plasma membrane of isolated protoplasts, progress report

    Energy Technology Data Exchange (ETDEWEB)

    Steponkus, P L

    1993-01-01

    Our goal is to provide a mechanistic understanding of the cellular and molecular aspects of freezing injury and cold acclimation from a perspective of the structural and functional integrity of the plasma membrane -- the primary site of freezing injury in winter cereals. We have utilized protoplasts isolated from leaves of winter rye (Secale cereale L. cv Puma) to study the cryobehavior of the plasma membrane during a freeze/thaw cycle. The focus of our current studies is on lesions in the plasma membrane that result from severe freeze-induced dehydration and result in the alteration of the semipermeable characteristics of the plasma membrane so that the protoplasts are osmotically unresponsive. In protoplasts isolated from non-acclimated rye leaves (NA protoplasts), injury is associated with the formation of aparticulate domains in the plasma membrane, aparticulate lamellae subtending the plasma membrane, and lamellar-to-hexagonal II phase transitions in the plasma membrane and the subtending lamellae. However, lamellar-to-hexagonal II phase transitions are not observed following severe dehydration of protoplasts isolated from cold-acclimated rye leaves (ACC protoplasts). Rather, injury is associated with the fracture-jump lesion,'' which, in freeze-fracture electron microscopy studies, is manifested as localized deviations in the fracture face of the plasma membrane. The fracture plane jumps'' from the plasma membrane to either subtending aparticulate lamellae or aparticulate regions of various endomembranes (predominantly chloroplast envelopes) that are in close apposition with the plasma membrane.

  13. Isolation and regeneration protoplast of an oil palm pathogen, Ganoderma boninense

    Science.gov (United States)

    Irene, Liza Isaac; Bakar, Farah Diba Abu; Idris, Abu Seman; Murad, Abdul Munir Abdul

    2015-09-01

    Ganoderma boninense is a known cause for basal stem rot (BSR) in oil palm. Thus, to curb the infection towards oil palm, the establishment of protoplast isolation and regeneration protocol is crucial to be studied. This will provide information on the functional genes especially those which leads towards infection and pathogenicity. In this study, a method was outlined to isolated protoplast in G. boninense by manipulating parameters such as mycelium age, concentration of lysing enzyme, and duration of mycelia incubation in lytic solution. The results shows that from 0.1 g of wet weight mycelia, the highest protoplast yield obtained was 5.5 × 108 protoplast/ml using 5th day old culture in a lytic mixture containing 2.0 % of lysing enzyme incubated for 4 hours at 30 °C with agitation of 80-100 rpm. The highest percentage of protoplast regeneration obtained from this study was 0.2 % using CYM medium supplemented with 0.6 M sorbitol. To date, this is the first report of protoplast isolation and regeneration for this phytopathogen.

  14. Effect of Different Cellulase and Pectinase Enzyme Treatments on Protoplast Isolation and Viability in Lilium ledebeourii Bioss.

    Directory of Open Access Journals (Sweden)

    Esmaeil CHAMANI

    2012-11-01

    Full Text Available For overcoming interspecific incompatibility, protoplast combination method is a proper procedure for making a new plant withdesired traits. For this purpose, protoplast preparation is a first and important step. Hence, experiments were conducted to evaluatevarious combinations of cellulose, pectinase and their treatment times on protoplast production and protoplast viability in Liliumledebeourii Bioss. The results of experiment revealed that the protoplast yield was significantly affected by different treatment levels.Cellulase at 4% gave the highest numbers of protoplasts at 3.71×105 protoplast/g FW. Pectinase at 1% gave the highest numbers ofprotoplast. For treatment times, the highest yield of protoplast was with leaf explants treated for 24 h. Analysis of variance indicated thatconcentration, time and three-way interaction of cellulase, pectinase and time were significant at p<0.01. Cellulase at 4% and pectinase at0.2% for 24 h gave the highest viability. Interactions of cellulase × pectinase, cellulase × time, pectinase × time and cellulase × pectinase× treatment time were significant at P≤0.05 for protoplast number. The highest and lowest protoplast numbers were produced in mediacontaining 4% cellulase and 1% pectinase for 24 h (6.65×105 protoplast/g FW and 1% cellulase and 0.2% pectinase for 12 h, respectively.It’s concluded that, the best treatment for isolation of Lilium protoplast was 4% cellulase and 1% pectinase for 24 h.

  15. The isolation of coated vesicles from protoplasts of soybean.

    Science.gov (United States)

    Mersey, B G; Griffing, L R; Rennie, P J; Fowke, L C

    1985-03-01

    Fractions enriched in coated vesicles were obtained from protoplasts derived from suspension cultured Glycine max (L.) Merr. cells. Initial enrichment was achieved by isopycnic centrifugation of a protoplast homogenate through a linear sucrose gradient in a vertical rotor. The coated-vesicle fractions from this gradient were pooled and centrifuged through a second linear sucrose gradient in a rate zonal fashion to remove the larger contaminating membrane vesicles. The most prominent polypeptide in the coated-vesicle fractions, plant "clathrin", had a relative molecular mass of approx. 190 kdalton as determined by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. Other enriched polypeptides included bands at 105, 100, 96, 64, 50, 38 and 32 kdalton. This method was compared with a procedure utilizing sucrose step gradients for preparing coated vesicles from soybean protoplasts. The effectiveness of the isopycnic-rate zonal centrifugation procedure was also tested for the preparation of bovine-brain coated vesicles.

  16. Further observations on cell-wall formation around isolated protoplasts of tobacco and tomato.

    Science.gov (United States)

    Willison, J H; Grout, B W

    1978-01-01

    Freeze-etch observations of protoplasts isolated from tobacco (Nicotiana tabacum L.) mesophyll tissue and tomato (Lycopersicum esculentum Mill.) fruit locule tissue are described which clarify earlier observations (Burgess, J., Fleming, E.N., Planta 131, 173-178, 1976; Planta 133, 267-273, 1977), obtained using scanning electron microscopy. of "fibres" associated with "projections" from these cell surfaces. It is demonstrated (1) that the "fibres" consist of bundles of small numbers of microfibrils which have become artifactually thickened by the deposition of coating materials, and (2) that the apparent association between "fibres" and "projections" results from microfibrils being lifted preferentially from protoplast surfaces in regions rich in "projections" (plasmalemmasomes). With the higher resolution available using freeze-etching it can be demonstrated that microfibril deposition does not occur in discontinuous zones on these protoplast surfaces. Globules associated with microfibril termini in radish (Raphanus sativus L.) roots are illustrated and it is proposed that turgor pressure differences between isolated protoplasts and intact tissue may account for the absence of similar globules from isolated protoplast surfaces.

  17. Gravity and light control of the developmental polarity of regenerating protoplasts isolated from prothallial cells of the fern Ceratopteris richardii

    Science.gov (United States)

    Edwards, E. S.; Roux, S. J.

    1998-01-01

    A procedure has been developed for isolating protoplasts from prothalli of Ceratopteris richardii which can be cultured and are capable of regeneration. Protoplasts were isolated from 2-week-old gametophytes in a medium containing wall-digesting enzymes in 0.5 M sucrose, followed by purification of the released protoplasts by floating them up into a 0.5 M sorbitol layer. Regeneration occurred over a period of 10-24 days, and, under optimal osmotic conditions, followed the developmental pattern seen during spore germination, in that the first division gave rise to a primary rhizoid. Thus, prothallial protoplasts are comparable to germinating spores as suitable models for studies of developmental polarity in single cells. As in germinating spores, the polarity of development in regenerating protoplasts is influenced by the vectors of gravity and unilateral light. However, the relative influence of light in fixing this polarity is greater in regenerating protoplasts, while in germinating spores, the influence of gravity is greater.

  18. A method for the isolation of protoplasts from grape berry mesocarp tissue.

    Science.gov (United States)

    Fontes, Natacha; Delrot, Serge; Gerós, Hernâni

    2010-06-01

    As single cell systems, protoplasts have been used in physiological, biochemical and molecular studies aiming towards the investigation, improvement or modification of plants. In grapevine, protoplasts have been isolated from several source tissues but not from grape berry, a major challenge given the uniqueness of grape fruit for human diet and wine production. Also, as the ripe grape berry has long been considered a 'small bag of sugary water' without cell compartmentation and/or membrane integrity, the isolation of intact cells from the mesocarp is of special scientific significance. Protoplasting from grape berry mesocarp cells was achieved with cellulase and pectolyase digestion, followed by differential and gradient centrifugations; however, given the special characteristics of berry tissue, cell wall digestion and protoplast purification were performed in a special environment to maintain their integrity and viability. Light and epifluorescence microscopy revealed the spatial organization of the cytoplasm, where an intricate acidic vacuolar apparatus predominates supporting the idea that berry softening during ripening is not strictly associated with loss in compartmentation and/or membrane integrity. Following the worldwide economical and social importance of wine in modern days, grape berry protoplasts are a major advance for both basic research of fruit ripening and biotechnological applications.

  19. Transport of antimony salts by Arabidopsis thaliana protoplasts over-expressing the human multidrug resistance-associated protein 1 (MRP1/ABCC1).

    Science.gov (United States)

    Gayet, Landry; Picault, Nathalie; Cazalé, Anne-Claire; Beyly, Audrey; Lucas, Philippe; Jacquet, Hélène; Suso, Henri-Pierre; Vavasseur, Alain; Peltier, Gilles; Forestier, Cyrille

    2006-12-22

    ABC transporters from the multidrug resistance-associated protein (MRP) subfamily are glutathione S-conjugate pumps exhibiting a broad substrate specificity illustrated by numerous xenobiotics, such as anticancer drugs, herbicides, pesticides and heavy metals. The engineering of MRP transporters into plants might be interesting either to reduce the quantity of xenobiotics taken up by the plant in the context of "safe-food" strategies or, conversely, in the development of phytoremediation strategies in which xenobiotics are sequestered in the vacuolar compartment. In this report, we obtained Arabidopsis transgenic plants overexpressing human MRP1. In these plants, expression of MRP1 did not increase plant resistance to antimony salts (Sb(III)), a classical glutathione-conjugate substrate of MRP1. However, the transporter was fully translated in roots and shoots, and targeted to the plasma membrane. In order to investigate the functionality of MRP1 in Arabidopsis, mesophyll cell protoplasts (MCPs) were isolated from transgenic plants and transport activities were measured by using calcein or Sb(III) as substrates. Expression of MRP1 at the plasma membrane was correlated with an increase in the MCPs resistance to Sb(III) and a limitation of the metalloid content in the protoplasts due to an improvement in Sb(III) efflux. Moreover, Sb(III) transport was sensitive to classical inhibitors of the human MRP1, such as MK571 or glibenclamide. These results demonstrate that a human ABC transporter can be functionally introduced in Arabidopsis, which might be useful, with the help of stronger promoters, to reduce the accumulation of xenobiotics in plants, such as heavy metals from multi-contaminated soils.

  20. Specific localization and measurement of hydrogen peroxide in Arabidopsis thaliana cell suspensions and protoplasts elicited by COS-OGA.

    Science.gov (United States)

    Ledoux, Quentin; Van Cutsem, Pierre; Markό, Istvan E; Veys, Pascal

    2014-01-01

    H2O2 acts as an important signaling molecule during plant/pathogen interactions but its study remains a challenge due to the current shortcomings in H2O2-responsive probes. In this work, ContPY1, a new molecular probe developed to specifically detect H2O2 was used to study the elicitation of Arabidopsis thaliana cells by a complex of chitosan oligomers (COS) and oligogalacturonides (OGA). The comparison of cell suspensions, protoplasts of cell suspensions and leaf protoplasts treated with different inhibitors gave indications on the potential sources of hydrogen peroxide in plant cells. The relative contribution of the cell wall, of membrane dehydrogenases and of peroxidases depended on cell type and treatment and proved to be variable. Our present protocol can be used to study hydrogen peroxide production in a large variety of plant species by simple protocol adaptation.

  1. The isolation of plasma membrane from protoplasts of soybean suspension cultures.

    Science.gov (United States)

    Galbraith, D W; Northcote, D H

    1977-04-01

    A procedure for the isolation of plasma membranes from protoplasts of suspension-cultured soybean is described. Protoplasts were prepared by enzymic digestion of the cell wall and the plasma membrane was labelled with radioactive diazotized sulphanilic acid. The membrane systems from broken protoplasts were separated by continuous isopycnic sucrose gradient centrifugation. Radioactivity was localized in a band possessing a buoyant density of 1-14 g ml-1. The activities of NADPH- and NADH-cytochrome c reductase, fumarase, Mg2+-ATPase, IDPase and acid phosphodiesterase in the various regions of the density gradient were determined. A plasma membrane fraction was selected which was relatively uncontaminated with membranes derived from endoplasmic reticulum, tonoplasts and mitochondria. The results indicated that Mg2+-ATPase and possibly acid phosphodiesterase were associated with the plasma membrane.

  2. Isolation of protoplast from callus of Populus euphratica and H+ fluxes across plasma membrane under NaCl stress

    Institute of Scientific and Technical Information of China (English)

    Gao Zhun; Dai Song-xiang; Chen Shao-liang; Shen Xin; Wang Rui-gang

    2007-01-01

    We used callus of Populus euphratica Olive to isolate protoplasts, and H+ fluxes across plasma membrane were investigated. The concentration of enzymes for protoplast isolation, e.g. cellulase, pectolyase, macerozyme, hemicellulase, and sorbitol content, incubation time were systemically studied. High yield and viability of protoplast was achieved after 6-8 hours incubation of P. euphratica callus in enzyme solution containing 1.5% (w:v) cellulase R-10, 0.1% (w:v) pectolyase Y-23, 0.2% (w:v) macerozyme membrane of P. euphratica cells. The shift of H+ flux response to NaCl shock and the relevance to salt tolerance were discussed.

  3. Rapid assessment of gene function in the circadian clock using artificial microRNA in Arabidopsis mesophyll protoplasts.

    Science.gov (United States)

    Kim, Jeongsik; Somers, David E

    2010-10-01

    Rapid assessment of the effect of reduced levels of gene products is often a bottleneck in determining how to proceed with an interesting gene candidate. Additionally, gene families with closely related members can confound determination of the role of even a single one of the group. We describe here an in vivo method to rapidly determine gene function using transient expression of artificial microRNAs (amiRNAs) in Arabidopsis (Arabidopsis thaliana) mesophyll protoplasts. We use a luciferase-based reporter of circadian clock activity to optimize and validate this system. Protoplasts transiently cotransfected with promoter-luciferase and gene-specific amiRNA plasmids sustain free-running rhythms of bioluminescence for more than 6 d. Using both amiRNA plasmids available through the Arabidopsis Biological Resource Center, as well as custom design of constructs using the Weigel amiRNA design algorithm, we show that transient knockdown of known clock genes recapitulates the same circadian phenotypes reported in the literature for loss-of-function mutant plants. We additionally show that amiRNA designed to knock down expression of the casein kinase II β-subunit gene family lengthens period, consistent with previous reports of a short period in casein kinase II β-subunit overexpressors. Our results demonstrate that this system can facilitate a much more rapid analysis of gene function by obviating the need to initially establish stably transformed transgenics to assess the phenotype of gene knockdowns. This approach will be useful in a wide range of plant disciplines when an endogenous cell-based phenotype is observable or can be devised, as done here using a luciferase reporter.

  4. Large-scale protein-protein interaction analysis in Arabidopsis mesophyll protoplasts by split firefly luciferase complementation.

    Directory of Open Access Journals (Sweden)

    Jian-Feng Li

    Full Text Available Protein-protein interactions (PPIs constitute the regulatory network that coordinates diverse cellular functions. There are growing needs in plant research for creating protein interaction maps behind complex cellular processes and at a systems biology level. However, only a few approaches have been successfully used for large-scale surveys of PPIs in plants, each having advantages and disadvantages. Here we present split firefly luciferase complementation (SFLC as a highly sensitive and noninvasive technique for in planta PPI investigation. In this assay, the separate halves of a firefly luciferase can come into close proximity and transiently restore its catalytic activity only when their fusion partners, namely the two proteins of interest, interact with each other. This assay was conferred with quantitativeness and high throughput potential when the Arabidopsis mesophyll protoplast system and a microplate luminometer were employed for protein expression and luciferase measurement, respectively. Using the SFLC assay, we could monitor the dynamics of rapamycin-induced and ascomycin-disrupted interaction between Arabidopsis FRB and human FKBP proteins in a near real-time manner. As a proof of concept for large-scale PPI survey, we further applied the SFLC assay to testing 132 binary PPIs among 8 auxin response factors (ARFs and 12 Aux/IAA proteins from Arabidopsis. Our results demonstrated that the SFLC assay is ideal for in vivo quantitative PPI analysis in plant cells and is particularly powerful for large-scale binary PPI screens.

  5. Measuring the osmotic water permeability coefficient (Pf) of spherical cells: isolated plant protoplasts as an example.

    Science.gov (United States)

    Shatil-Cohen, Arava; Sibony, Hadas; Draye, Xavier; Chaumont, François; Moran, Nava; Moshelion, Menachem

    2014-10-08

    Studying AQP regulation mechanisms is crucial for the understanding of water relations at both the cellular and the whole plant levels. Presented here is a simple and very efficient method for the determination of the osmotic water permeability coefficient (P(f)) in plant protoplasts, applicable in principle also to other spherical cells such as frog oocytes. The first step of the assay is the isolation of protoplasts from the plant tissue of interest by enzymatic digestion into a chamber with an appropriate isotonic solution. The second step consists of an osmotic challenge assay: protoplasts immobilized on the bottom of the chamber are submitted to a constant perfusion starting with an isotonic solution and followed by a hypotonic solution. The cell swelling is video recorded. In the third step, the images are processed offline to yield volume changes, and the time course of the volume changes is correlated with the time course of the change in osmolarity of the chamber perfusion medium, using a curve fitting procedure written in Matlab (the 'PfFit'), to yield P(f).

  6. The Detection of Abscisic Acid in Arabidopsis Protoplasts by LC-MS%LC-MS法测定拟南芥原生质体脱落酸的研究

    Institute of Scientific and Technical Information of China (English)

    郭磊; Mohammed Humayun KABIR; 童建华; 黄志刚; 萧浪涛

    2012-01-01

    Abscisic acid plays a very important role in many plant developmental processes and stress response systems. Accurate detection of abscisic acid content on agricultural production and scientific research has important significance. We analyzed the abscisic acid content of protoplasts isolated from soil cultivated Arabidopsis leaves and hydroponically cultivated roots by using high - performance liquid chromatography with mass spectrometry. The results showed that there was a good linear relation between the content of abscisic acid and protoplasts number and the correlation coefficients were 0. 992 3, 0. 993 1 for Arabidopsis leaves and roots protoplasts, respectively. Detection limit for ABA content was 1.07 ng/mL, and the minimum Arabidopsis leaves and roots protoplasts numbers were 30 000 and 20 000, respectively. The material of this study were protoplasts which can avoid the interference of the complex chemicals from intercellular substance as well as improve sensitivity and the precision of phytohormones determination. It can also improve the classical phytohormones determination technologyies.%利用高效液相色谱与质谱联用法(LC-MS法)对从土培拟南芥叶片和水培拟南芥根系中分离的原生质体进行脱落酸含量分析.结果表明,所测原生质体的脱落酸含量与原生质体数目之间具有较好的线性关系,相关系数分别为0.992 3和0.993 1.脱落酸含量的检测下限为1.07 ng/mL,土培拟南芥叶片和水培拟南芥根系原生质体检测下限分别为3万个和2万个.该研究以原生质体为材料,避免了细胞间隙中的复杂化学成干扰,提高了植物激素测定的灵敏度和精度,完善了经典的植物激素测定技术.

  7. An improved protocol for the preparation of protoplasts from an established Arabidopsis thaliana cell suspension culture and infection with RNA of turnip yellow mosaic tymovirus: a simple and reliable method.

    Science.gov (United States)

    Schirawski, J; Planchais, S; Haenni, A L

    2000-04-01

    An improved method for preparation of protoplasts of Arabidopsis thaliana cells grown in suspension culture is presented. This method is fast, reliable and can be used for the production of virtually an unlimited number of protoplasts at any time. These protoplasts can be transformed efficiently with RNA from turnip yellow mosaic tymovirus (TYMV) by polyethyleneglycol-mediated transfection. The simple transfection procedure has been optimized at various steps. Replication of TYMV can be monitored routinely by detection of the coat protein in as few as 2 x 10(4) infected protoplasts.

  8. Inhibition of phenylpropanoid biosynthesis increases cell wall digestibility, protoplast isolation, and facilitates sustained cell division in American elm (Ulmus americana

    Directory of Open Access Journals (Sweden)

    Jones A Maxwell P

    2012-05-01

    Full Text Available Abstract Background Protoplast technologies offer unique opportunities for fundamental research and to develop novel germplasm through somatic hybridization, organelle transfer, protoclonal variation, and direct insertion of DNA. Applying protoplast technologies to develop Dutch elm disease resistant American elms (Ulmus americana L. was proposed over 30 years ago, but has not been achieved. A primary factor restricting protoplast technology to American elm is the resistance of the cell walls to enzymatic degradation and a long lag phase prior to cell wall re-synthesis and cell division. Results This study suggests that resistance to enzymatic degradation in American elm was due to water soluble phenylpropanoids. Incubating tobacco (Nicotiana tabacum L. leaf tissue, an easily digestible species, in aqueous elm extract inhibits cell wall digestion in a dose dependent manner. This can be mimicked by p-coumaric or ferulic acid, phenylpropanoids known to re-enforce cell walls. Culturing American elm tissue in the presence of 2-aminoindane-2-phosphonic acid (AIP; 10-150 μM, an inhibitor of phenylalanine ammonia lyase (PAL, reduced flavonoid content, decreased tissue browning, and increased isolation rates significantly from 11.8% (±3.27 in controls to 65.3% (±4.60. Protoplasts isolated from callus grown in 100 μM AIP developed cell walls by day 2, had a division rate of 28.5% (±3.59 by day 6, and proliferated into callus by day 14. Heterokaryons were successfully produced using electrofusion and fused protoplasts remained viable when embedded in agarose. Conclusions This study describes a novel approach of modifying phenylpropanoid biosynthesis to facilitate efficient protoplast isolation which has historically been problematic for American elm. This isolation system has facilitated recovery of viable protoplasts capable of rapid cell wall re-synthesis and sustained cell division to form callus. Further, isolated protoplasts survived

  9. Direct transfer of synthetic double-stranded RNA into protoplasts of Arabidopsis thaliana.

    Science.gov (United States)

    Jung, Ha-Il; Zhai, Zhiyang; Vatamaniuk, Olena K

    2011-01-01

    Double-stranded (ds) RNA interference (RNAi) is widely used as a reverse genetic approach for functional analysis of plant genes. Constitutive or transient RNAi effects in plants have been achieved via generating stable transformants expressing dsRNAs or artificial microRNAs (amiRNAs) in planta or by viral-induced gene silencing (VIGS). Although these tools provide outstanding resources for functional genomics, they require generation of vectors expressing dsRNAs or amiRNAs against targeted genes, transformation and propagation of transformed plants, or maintenance of multiple VIGS lines and thus impose time, labor, and space requirements. As we showed recently, these limitations can be circumvented by inducing RNAi effects in protoplasts via transfecting them with in vitro-synthesized dsRNAs. In this chapter we detail the procedure for transient gene silencing in protoplasts using synthetic dsRNAs and provide examples of approaches for subsequent functional analyses.

  10. Carbon transitions from either Calvin cycle or transitory starch to heteroglycans as revealed by (14) C-labeling experiments using protoplasts from Arabidopsis.

    Science.gov (United States)

    Malinova, Irina; Steup, Martin; Fettke, Joerg

    2013-09-01

    Plants metabolize transitory starch by precisely coordinated plastidial and cytosolic processes. The latter appear to include the action of water-soluble heteroglycans (SHGin ) whose monosaccharide pattern is similar to that of apoplastic glycans (SHGex ) but, unlike SHGex , SHGin strongly interacts with glucosyl transferases. In this study, we analyzed starch metabolism using mesophyll protoplasts from wild-type plants and two knock-out mutants [deficient in the cytosolic transglucosidase, disproportionating isoenzyme 2 (DPE2) or the plastidial phosphoglucomutase (PGM1)] from Arabidopsis thaliana. Protoplasts prelabeled by photosynthetic (14) CO2 fixation were transferred to an unlabeled medium and were darkened or illuminated. Carbon transitions from the Calvin cycle or from starch to both SHGin and SHGex were analyzed. In illuminated protoplasts, starch turn-over was undetectable but darkened protoplasts continuously degraded starch. During illumination, neither the total (14) C content nor the labeling patterns of the sugar residues of SHGin were significantly altered but both the total amount and the labeling of the constituents of SHGex increased with time. In darkened protoplasts, the (14) C-content of most of the sugar residues of SHGin transiently and strongly increased and then declined. This effect was not observed in any SHGex constituent. In darkened DPE2-deficient protoplasts, none of the SHGin constituents exhibited an essential transient increase in labeling. In contrast, some residues of SHGin from the PGM1 mutant exhibited a transient increase in label but this effect significantly differed from that of the wild type. Two conclusions are reached: first, SHGin and SHGex exert different metabolic functions and second, SHGin is directly involved in starch degradation.

  11. Evaluation of Parameters in the Isolation of Viable Protoplasts from Cultured Tobacco Cells 1

    Science.gov (United States)

    Uchimiya, Hirofumi; Murashige, Toshio

    1974-01-01

    A systematic evaluation disclosed the following conditions to be optimum for the isolation of viable protoplasts from cultured cells of Nicotiana tabacum L. `Bright Yellow' grown in liquid suspensions: (a) the cell culture in the early phase of cell number increase, (b) an enzyme mixture of 1% cellulase “Onozuka” and 0.2% Macerozyme, (c) an enzyme solution pH of 4.7 or 5.7, (d) a 2- to 3-hr incubation period, (e) 5 ml of enzyme solution per 500 mg cells and contained in a 50-ml Delong flask, (f) agitation on a gyrotory shaker at 50 rpm, and (g) 0.3 to 0.8 m mannitol as osmoticum in the cell enzyme mixture. The incubation temperature may be varied from 22 to 37 C. The procedure enabled 30% of the tobacco cells to form protoplasts, 80% of which regenerated cell walls in 4 days and 40% resumed cell division activity when returned to cell culture medium. Images PMID:16659004

  12. Plant regeneration of grapevine (Vitis sp.) protoplasts isolated from embryogenic tissue.

    Science.gov (United States)

    Reustle, G; Harst, M; Alleweldt, G

    1995-12-01

    Protoplasts with high embryogenic competence could be isolated from leaf-disk-derived embryos and embryoids of Vitis sp. cv. Seyval blanc. After a 4-week induction treatment in NN-69 medium supplemented with 4.0mg/l naphthoxyacetic acid (NOA) and 0.9mg/l thidiazuron (TDZ) and subsequent subcultivation in hormone-free medium, 38.5% of the developed microcalluses showed somatic embryogenesis. In contrast, only few formed somatic embryos after induction in CPW-13 medium with either 1.0mg/l 2,4-dichlorophenoxyacetic acid and 0.5mg/l benzylaminopurine treatment (13.8%) or NOA/TDZ treatment (1.4%). Up to 30% of these embryos germinated and about half of them regenerated into typical in vitro grapevines when transferred onto LS-medium in culture tubes.

  13. [Isolation and regeneration of the protoplasts of the streptomycete producers of actinomycins C and X].

    Science.gov (United States)

    Orlova, T I; Masha, G G; Kliueva, N A

    1986-09-01

    Protoplasts of S. michiganensis, S. chrysomallus and Streptomyces sp. 26-115, organisms producing actinomycins C and X form in hypertonic salt solution under the action of 3-4,5 mg/ml of lysozyme on the mycelium suspension. For protoplasting, the streptomycetes were grown on the soybean medium in the presence of 0.2-0.8 per cent of glycine. The mycelium of the streptomycete exponential growth phase was more favourable for protoplast formation. Protoplast regeneration was studied on the medium described by Okanishi et al. The quantitative composition of this medium was not optimal for regeneration of protoplasts of the above streptomycetes. The level of their regeneration depended to various extents on concentration of phosphate, magnesium and calcium ions and sucrose in the regeneration medium.

  14. Protoplast production and isolation from Etlingera elatior=Produção e isolamento de protoplasto de Etlingera elatior

    Directory of Open Access Journals (Sweden)

    Milene Alves de Figueiredo Carvalho

    2012-01-01

    Full Text Available The technique of hybridization using plant protoplasts is widely used in plant breeding programs. The purpose of our study is to further characterize the process of protoplast isolation from the ornamental species Etlingera elatior (Jack R. M. Smith. Protoplasts were isolated from different tissues: in vitro leaves, in vitro pseudostem, and leaves from plants cultivated hydroponically. We tested six enzymatic combinations, four incubation time periods, the rotary system (40 rpm or steady in the dark, and three concentrations of mannitol (0.5, 0.6 and 0.7 M. The diameter and viability of obtained protoplasts were evaluated. The best source of explants used for protoplast isolation was the in vitro leaves, which yielded 22x105 protoplasts g-1 of fresh matter. The optimal incubation period was 15 hours. The in vitro leaves presented a greater viability (96% and larger protoplasts (36.7 µm diameter. Greater yields were obtained using a rotatory system with protoplasts incubated in the dark. The best enzymatic combination was 3% Cellulase “Onozuca” R-10 + 2% Meicelase + 1% Driselase + 1% Dextran + 5 mM MES, followed by the addition of 0.6 M mannitol.Com o objetivo de realizar hibridações que auxiliam em programas de melhoramento genético de flores ornamentais, protoplastos foram isolados a partir de diferentes tecidos (folhas in vitro, pseudocaules in vitro e folhas em sistema hidropônico de Etlnigera elatior (Jack R. M. Smith. Foram testados seis diferentes combinações enzimáticas, quatro períodos de incubação, sistema rotatório (40 rpm ou estacionário no escuro, concentrações de manitol (0,5; 0,6 e 0,7 M, o diâmetro e a viabilidade dos protoplastos isolados. A melhor fonte de explante utilizado no isolamento de protoplastos foi folha in vitro, com rendimento de 22 x105 protoplastos g-1 MF. O melhor tempo de incubação foi 15 horas, pois períodos superiores a este causavam diminuição no rendimento e viabilidade dos

  15. Effects of freezing and cold acclimation on the plasma membrane of isolated protoplasts. Progress report, May 16, 1992--January 9, 1993

    Energy Technology Data Exchange (ETDEWEB)

    Steponkus, P.L.

    1993-05-01

    Our goal is to provide a mechanistic understanding of the cellular and molecular aspects of freezing injury and cold acclimation from a perspective of the structural and functional integrity of the plasma membrane -- the primary site of freezing injury in winter cereals. We have utilized protoplasts isolated from leaves of winter rye (Secale cereale L. cv Puma) to study the cryobehavior of the plasma membrane during a freeze/thaw cycle. The focus of our current studies is on lesions in the plasma membrane that result from severe freeze-induced dehydration and result in the alteration of the semipermeable characteristics of the plasma membrane so that the protoplasts are osmotically unresponsive. In protoplasts isolated from non-acclimated rye leaves (NA protoplasts), injury is associated with the formation of aparticulate domains in the plasma membrane, aparticulate lamellae subtending the plasma membrane, and lamellar-to-hexagonal II phase transitions in the plasma membrane and the subtending lamellae. However, lamellar-to-hexagonal II phase transitions are not observed following severe dehydration of protoplasts isolated from cold-acclimated rye leaves (ACC protoplasts). Rather, injury is associated with the ``fracture-jump lesion,`` which, in freeze-fracture electron microscopy studies, is manifested as localized deviations in the fracture face of the plasma membrane. The fracture plane ``jumps`` from the plasma membrane to either subtending aparticulate lamellae or aparticulate regions of various endomembranes (predominantly chloroplast envelopes) that are in close apposition with the plasma membrane.

  16. Isolation and fusion of protoplasts from the phytopathogenic fungus Sclerotium rolfsii (Sacc.

    Directory of Open Access Journals (Sweden)

    Sikandar Hayat

    2010-03-01

    Full Text Available Sclerotium rolfsii (Sacc. is a serious plant pathogenic fungus and lacks perfect (basidial stage in production. Protoplast fusion technology was employed to reconstruct fusants from this fungus. Two strains designated as A and R were used. Maximum protoplast yields of 3.8x10(5 /g mycelia and 2.8x10(5 /g mycelia were formed in strains A and R respectively. Osmotic stabilizer sucrose 1M gave maximum yield. Lysing enzyme at the rate of 15mg/ml was found best for yield. Fusion of protoplasts from strains A and R was carried out in fusion media containing PEG 4000 30% (w/v with 0.2mM CaCl2. Four fusants F1, F2, F3 and F4 were recovered. Morphological, physiological and pathogenic characters of fusants were compared with parent strains on carrots, beans and tomato.

  17. Establishing RNA interference as a reverse-genetic approach for gene functional analysis in protoplasts.

    Science.gov (United States)

    Zhai, Zhiyang; Sooksa-nguan, Thanwalee; Vatamaniuk, Olena K

    2009-02-01

    Double-stranded (ds)RNA interference (RNAi) is widely used for functional analysis of plant genes and is achieved via generating stable transformants expressing dsRNA in planta. This study demonstrated that RNAi can also be utilized to examine gene functions in protoplasts. Because protoplasts are nongrowing cells, effective RNAi-triggered gene silencing depends not only on a depletion of gene transcripts but also on turnover rates of corresponding polypeptides. Herein, we tested if transient RNAi in protoplasts would result in the depletion of a targeted polypeptide and, because protoplasts have a limited life span, if functional assays of RNAi knockout genes would be feasible in protoplasts. We showed that protoplasts transfection with an in vitro-synthesized dsRNA against Arabidopsis (Arabidopsis thaliana) beta-glutamylcysteine synthase (ECS1), a key enzyme in the synthesis of glutathione, resulted in a 95% depletion of ECS1 transcript, a 72% decrease of ECS1 polypeptide, and a 60% drop in glutathione content. These results were comparable with those obtained upon analysis of Arabidopsis seedlings bearing the cad2-1 mutant allele of ECS1. We also improved the procedure for RNAi inactivation of several genes simultaneously. Finally, because we isolated protoplasts from tissues of 14-d-old seedlings instead of 1-month-old mature plants, the described procedure is rapid (as it only takes 20 d from seed planting to functional studies), suitable for analyzing multiple genes in parallel, and independent of cloning dsRNAs into plant expression vectors. Therefore, RNAi in protoplasts complements existing genetic tools, as it allows rapid, cost- and space-efficient initial screening and selection of genes for subsequent in planta studies.

  18. [Isolation of protoplasts from vegetable tissues using extracellular lytic enzymes from fusarium oxysporum f.sp. melonis].

    Science.gov (United States)

    Alconada, T M; Martínez, M J

    1995-01-01

    Fusarium oxysporum f.sp. melonis, a pathogen of melon (Cucumis melo L.), was grown in shaken cultures at 26 degrees C in a mineral salts medium containing glucose, xylan and apple pectin as carbon sources. The extracellular enzymic complex obtained from these cultures showed lytic activity on plant tissues, causing maceration of melon fruits, potato tubers and carrot roots. Protoplasts were isolated from melon fruits when the maceration was carried out under appropriate osmotic conditions. This fact suggest a possible relationship between the enzymes produced by Fusarium oxysporum f.sp. melonis and their pathogenicity on melon plants.

  19. Isolation of Mesophyll Protoplasts from Mediterranean Woody Plants for the Study of DNA Integrity under Abiotic Stress.

    Science.gov (United States)

    Kuzminsky, Elena; Meschini, Roberta; Terzoli, Serena; Pavani, Liliana; Silvestri, Cristian; Choury, Zineb; Scarascia-Mugnozza, Giuseppe

    2016-01-01

    Abiotic stresses have considerable negative impact on Mediterranean plant ecosystems and better comprehension of the genetic control of response and adaptation of trees to global changes is urgently needed. The single cell gel electrophoresis (SCGE) assay could be considered a good estimator of DNA damage in an individual eukaryotic cell. This method has been mainly employed in animal tissues, because the plant cell wall represents an obstacle for the extraction of nuclei; moreover, in Mediterranean woody species, especially in the sclerophyll plants, this procedure can be quite difficult because of the presence of sclerenchyma and hardened cells. On the other hand, these plants represent an interesting material to be studied because of the ability of these plants to tolerate abiotic stress. For instance, holm oak (Quercus ilex L.) has been selected as the model plant to identify critical levels of O3 for Southern European forests. Consequently, a quantitative method for the evaluation of cell injury of leaf tissues of this species is required. Optimal conditions for high-yield nuclei isolation were obtained by using protoplast technology and a detailed description of the method is provided and discussed. White poplar (Populus alba L.) was used as an internal control for protoplast isolation. Such a method has not been previously reported in newly fully developed leaves of holm oak. This method combined with SCGE assay represents a new tool for testing the DNA integrity of leaf tissues in higher plants under stress conditions.

  20. Isolation and Properties of Deoxyribonucleic Acid from Protoplasts of Cell Suspension Cultures of Ammi visnaga and Carrot (Daucus carota) 1

    Science.gov (United States)

    Ohyama, K.; Gamborg, O. L.; Miller, R. A.

    1972-01-01

    A procedure is described for the isolation of native DNA from protoplasts of ammi (Ammi visnaga) and carrot (Daucus carota) cells. Protoplasts were produced from 40 grams of fresh cells by enzyme hydrolysis and lysed with sodium dodecyl sulfate. The DNA was purified by treatment with pronase and ribonuclease. Final isolation was achieved by sucrose density gradient centrifugation. The melting temperature of ammi and carrot DNA in 0.15 m NaCl and 15 mm trisodium citrate buffer, pH 7.0, was 84.0 C and 84.5 C, respectively. The molecular weight for ammi DNA was 1.43 × 108, and for carrot DNA it was 1.56 × 108. Ammi DNA exhibited a single band at 1.690 grams per cubic centimeter in CsCl, whereas carrot DNA showed two bands, one at 1.693 grams per cubic centimeter and another at 1.706 grams per cubic centimeter. Ammi DNA consisted of a doublestranded form, since denaturation of the DNA caused a complete upward shift of 0.020 grams per cubic centimeter. PMID:16658166

  1. Isolation and Properties of Deoxyribonucleic Acid from Protoplasts of Cell Suspension Cultures of Ammi visnaga and Carrot (Daucus carota).

    Science.gov (United States)

    Ohyama, K; Gamborg, O L; Miller, R A

    1972-09-01

    A procedure is described for the isolation of native DNA from protoplasts of ammi (Ammi visnaga) and carrot (Daucus carota) cells. Protoplasts were produced from 40 grams of fresh cells by enzyme hydrolysis and lysed with sodium dodecyl sulfate. The DNA was purified by treatment with pronase and ribonuclease. Final isolation was achieved by sucrose density gradient centrifugation.The melting temperature of ammi and carrot DNA in 0.15 m NaCl and 15 mm trisodium citrate buffer, pH 7.0, was 84.0 C and 84.5 C, respectively. The molecular weight for ammi DNA was 1.43 x 10(8), and for carrot DNA it was 1.56 x 10(8). Ammi DNA exhibited a single band at 1.690 grams per cubic centimeter in CsCl, whereas carrot DNA showed two bands, one at 1.693 grams per cubic centimeter and another at 1.706 grams per cubic centimeter. Ammi DNA consisted of a doublestranded form, since denaturation of the DNA caused a complete upward shift of 0.020 grams per cubic centimeter.

  2. ISOLATION OF MESOPHYLL PROTOPLASTS FROM MEDITERRANEAN WOODY PLANTS FOR THE STUDY OF DNA INTEGRITY UNDER ABIOTIC STRESS

    Directory of Open Access Journals (Sweden)

    Elena Kuzminsky

    2016-08-01

    Full Text Available Abiotic stresses have considerable negative impact on Mediterranean plant ecosystems and better comprehension of the genetic control of response and adaptation of trees to global changes is urgently needed. The Single Cell Gel Electrophoresis assay could be considered a good estimator of DNA damage in an individual eukaryotic cell. This method has been mainly employed in animal tissues, because the plant cell wall represents an obstacle for the extraction of nuclei; moreover, in Mediterranean woody species, especially in the sclerophyll plants, this procedure can be quite difficult because of the presence of sclerenchyma and hardened cells. On the other hand, these plants represent an interesting material to be studied because of the ability of these plants to tolerate abiotic stress. For instance, holm oak (Quercus ilex L. has been selected as the model plant to identify critical levels of O3 for Southern European forests. Consequently, a quantitative method for the evaluation of cell injury of leaf tissues of this species is required. Optimal conditions for high-yield nuclei isolation were obtained by using protoplast technology and a detailed description of the method is provided and discussed. White poplar (Populus alba L. was used as an internal control for protoplast isolation. Such a method has not been previously reported in newly fully developed leaves of holm oak. This method combined with Single Cell Gel Electrophoresis assay represents a new tool for testing the DNA integrity of leaf tissues in higher plants under stress conditions.

  3. Functional characterization of aromatic amino acid aminotransferase involved in 2-phenylethanol biosynthesis in isolated rose petal protoplasts.

    Science.gov (United States)

    Hirata, Hiroshi; Ohnishi, Toshiyuki; Ishida, Haruka; Tomida, Kensuke; Sakai, Miwa; Hara, Masakazu; Watanabe, Naoharu

    2012-03-15

    In rose flowers, 2-phenylethanol (2PE) is biosynthesized from l-phenylalanine (l-Phe) via phenylacetaldehyde (PAld) by the actions of two enzymes, pyridoxal-5'-phosphate (PLP)-dependent aromatic amino acid decarboxylase (AADC) and phenylacetaldehyde reductase (PAR). We here report that Rosa 'Yves Piaget' aromatic amino acid aminotransferase produced phenylpyruvic acid (PPA) from l-Phe in isolated petal protoplasts. We have cloned three full length cDNAs (RyAAAT1-3) of aromatic amino acid aminotransferase families based on rose EST database and homology regions. The RyAAATs enzymes were heterogeneously expressed in Escherichia coli and characterized biochemically. The recombinant RyAAAT3 showed the highest activity toward l-Phe in comparison with l-tryptophan, l-tyrosine, d-Phe, glycine, and l-alanine, and showed 9.7-fold higher activity with l-Phe rather than PPA as a substrate. RyAAAT3 had an optimal activity at pH 9 and at 45-55°C with α-ketoglutaric acid, and was found to be a PLP dependent enzyme based on the inhibition test using Carbidopa, an inhibitor of PLP-dependent enzymes. The transcript of RyAAAT3 was expressed in flowers as well as other organs of R. 'Yves Piaget'. RNAi suppression of RyAAAT3 decreased 2PE production, revealing the involvement of RyAAAT3 in 2PE biosynthesis in rose protoplasts and indicating that rose protoplasts have potentially two different 2PE biosynthetic pathways, the AADC route and the new route via PPA from l-Phe.

  4. Aislamiento y cultivo de protoplastos en Maracuyá Isolation and cultive of protoplast in passion fruit

    Directory of Open Access Journals (Sweden)

    Rivera Rodríguez Ricardo

    2004-12-01

    Full Text Available En este trabajo se ajustaron las condiciones para la regeneración de plántulas a partir del cultivo de protoplastos, proceso indispensable para avanzar hacia la obtención de híbridos somáticos. Se realizó el aislamiento de protoplastos a partir de cotiledones y hojas de plántulas in vitro de Passiflora edulis var. flavicarpa; estos explantes fueron sumergidos en solución CPW13M para inducir plasmólisis. Posteriormente se ensayaron tres combinaciones enzimáticas, los mayores rendimientos fueron 6,48 x 106 y 4,60 x 106 protoplastos viables /500 mg de tejido, obtenidos respectivamente con la
    combinación de Celulasa R-10 al 1% y Pectolyasa Y-23 al 0,05% a partir de hojas y la solución enzimática Celulasa al 2% y Macerozima al 0,4% para cotiledones. Las mejores densidades de cultivo para los protoplastos fueron 5 x 104 protoplastos/ml para los obtenidos de cotiledones y 1,5 x 105 protoplastos/ml para los aislados de hojas, empleando el sistema de cultivo en gotas de medio KM8p solidificadas con agarosa al 0,6% y recubiertas con medio líquido KM8p con 100 g/l de glucosa y cefotaxim 300 μg/ml. Con las primeras divisiones celulares, se empezó a disminuir el nivel osmótico al renovar el medio líquido con la mezcla de medio KM8p:KM8 en proporción 3:1 y se continuó cada siete días en proporciones 2:1, 1:1 y 1:3 hasta la obtención de colonias y callos. Los callos fueron transferidos a medio MS con 2 mg/l de BAP y 1 mg/l de AIB para inducir la regeneración en condiciones de iluminación; después de seis semanas de cultivo se diferenciaron yemas, posteriormente fueron subcultivadas a medio MS sin reguladores de crecimiento para su enraizamiento.
    In this research we adjust the protoplasts culture and regeneration conditions, necessary to advance into somatic hybrids obtention. Isolation of Passiflora edulis var. flavicarpa protoplasts from in vitro seedlings cotyledons and leaves was carried out. These tissues were

  5. Fluorescence activated cell sorting of plant protoplasts.

    Science.gov (United States)

    Bargmann, Bastiaan O R; Birnbaum, Kenneth D

    2010-02-18

    High-resolution, cell type-specific analysis of gene expression greatly enhances understanding of developmental regulation and responses to environmental stimuli in any multicellular organism. In situ hybridization and reporter gene visualization can to a limited extent be used to this end but for high resolution quantitative RT-PCR or high-throughput transcriptome-wide analysis the isolation of RNA from particular cell types is requisite. Cellular dissociation of tissue expressing a fluorescent protein marker in a specific cell type and subsequent Fluorescence Activated Cell Sorting (FACS) makes it possible to collect sufficient amounts of material for RNA extraction, cDNA synthesis/amplification and microarray analysis. An extensive set of cell type-specific fluorescent reporter lines is available to the plant research community. In this case, two marker lines of the Arabidopsis thaliana root are used: P(SCR;)::GFP (endodermis and quiescent center) and P(WOX5;)::GFP (quiescent center). Large numbers (thousands) of seedlings are grown hydroponically or on agar plates and harvested to obtain enough root material for further analysis. Cellular dissociation of plant material is achieved by enzymatic digestion of the cell wall. This procedure makes use of high osmolarity-induced plasmolysis and commercially available cellulases, pectinases and hemicellulases to release protoplasts into solution. FACS of GFP-positive cells makes use of the visualization of the green versus the red emission spectra of protoplasts excited by a 488 nm laser. GFP-positive protoplasts can be distinguished by their increased ratio of green to red emission. Protoplasts are typically sorted directly into RNA extraction buffer and stored for further processing at a later time. This technique is revealed to be straightforward and practicable. Furthermore, it is shown that it can be used without difficulty to isolate sufficient numbers of cells for transcriptome analysis, even for very scarce

  6. Isolation and Suborganellar Fractionation of Arabidopsis Chloroplasts.

    Science.gov (United States)

    Flores-Pérez, Úrsula; Jarvis, Paul

    2017-01-01

    Chloroplasts are structurally complex organelles containing ~2000-3000 proteins. They are delimited by a double membrane system or envelope, have an inner aqueous compartment called the stroma, and possess a second internal membrane system called the thylakoids. Thus, determining the suborganellar location of a chloroplast protein is vital to understanding or verifying its function. One way in which protein localization can be addressed is through fractionation. Here we present two rapid and simple methods that may be applied sequentially on the same day: (a) The isolation of intact chloroplasts from Arabidopsis thaliana plants that may be used directly (e.g., for functional studies such as protein import analysis), or for further processing as follows; (b) separation of isolated chloroplasts into three suborganellar fractions (envelope membranes, a soluble fraction containing stromal proteins, and the thylakoids). These methods are routinely used in our laboratory, and they provide a good yield of isolated chloroplasts and suborganellar fractions that can be used for various downstream applications.

  7. Plant protoplasts: status and biotechnological perspectives.

    Science.gov (United States)

    Davey, Michael R; Anthony, Paul; Power, J Brian; Lowe, Kenneth C

    2005-03-01

    Plant protoplasts ("naked" cells) provide a unique single cell system to underpin several aspects of modern biotechnology. Major advances in genomics, proteomics, and metabolomics have stimulated renewed interest in these osmotically fragile wall-less cells. Reliable procedures are available to isolate and culture protoplasts from a range of plants, including both monocotyledonous and dicotyledonous crops. Several parameters, particularly the source tissue, culture medium, and environmental factors, influence the ability of protoplasts and protoplast-derived cells to express their totipotency and to develop into fertile plants. Importantly, novel approaches to maximise the efficiency of protoplast-to-plant systems include techniques already well established for animal and microbial cells, such as electrostimulation and exposure of protoplasts to surfactants and respiratory gas carriers, especially perfluorochemicals and hemoglobin. However, despite at least four decades of concerted effort and technology transfer between laboratories worldwide, many species still remain recalcitrant in culture. Nevertheless, isolated protoplasts are unique to a range of experimental procedures. In the context of plant genetic manipulation, somatic hybridisation by protoplast fusion enables nuclear and cytoplasmic genomes to be combined, fully or partially, at the interspecific and intergeneric levels to circumvent naturally occurring sexual incompatibility barriers. Uptake of isolated DNA into protoplasts provides the basis for transient and stable nuclear transformation, and also organelle transformation to generate transplastomic plants. Isolated protoplasts are also exploited in numerous miscellaneous studies involving membrane function, cell structure, synthesis of pharmaceutical products, and toxicological assessments. This review focuses upon the most recent developments in protoplast-based technologies.

  8. PLASMALEMMA PATCH CLAMP EXPERIMENTS IN PLANT-ROOT CELLS - PROCEDURE FOR FAST ISOLATION OF PROTOPLASTS WITH MINIMAL EXPOSURE TO CELL-WALL DEGRADING ENZYMES

    NARCIS (Netherlands)

    VOGELZANG, SA; PRINS, HBA

    1992-01-01

    A convenient and rapid isolation procedure for root cell protoplasts suitable for patch clamp experiments. was developed for root cells of tomato (Lycopersicon esculentum and Plantago species, grown on hydroculture. The procedure is based on a minimal exposure of cells to cell wall degrading enzyme

  9. Cell division and differentiation in protoplasts from cell cultures of Glycine species and leaf tissue of soybean.

    Science.gov (United States)

    Gamborg, O L; Davis, B P; Stahlhut, R W

    1983-08-01

    Protoplasts were isolated from cell cultures of G. soja and G. tabacina, respectively. The isolation procedure employed Percoll for the separation and concentration of protoplasts. The cultured protoplasts formed cells which developed into embryo-like structures. Protoplasts also were isolated from leaf tissue of soybean cv. Williams 82. Upon culture, the protoplasts regenerated cell walls and divided to form cell cultures.

  10. Effects of freezing and cold acclimation on the plasma membrane of isolated protoplasts

    Energy Technology Data Exchange (ETDEWEB)

    Steponkus, P.L.

    1991-01-01

    This project focuses on lesions in the plasma membrane of protoplasts that occur during freezing to temperatures below {minus}5{degrees} which result in changes in the semipermeablity of the plasma membrane. This injury, referred to as loss of osmotic responsiveness, is associated with the formation of large, aparticulate domains in the plasma membrane, aparticulate lamellae subtending the plasma membrane, and lamellar-to-hexagonal{sub II} phase transitions in the plasma membrane and subtending lamellar. The goals of this project are to provide a mechanistic understanding of the mechanism by which freeze-induced dehydration effects the formation of aparticulate domains and lamellar-to-hexagonal{sub II} phase transitions and to determine the mechanisms by which cold acclimation and cryoprotectants preclude or diminish these ultrastructural changes. Our working hypothesis is the formation of aparticulate domains and lamellar-to-hexagon{sub II} phase transitions in the plasma membrane and subtending lamellae are manifestations of hydration-dependent bilayer-bilayer interactions.

  11. Changes in antenna sizes of photosystems during state transitions in granal and stroma-exposed thylakoid membrane of intact chloroplasts in Arabidopsis mesophyll protoplasts.

    Science.gov (United States)

    Kim, Eunchul; Ahn, Tae Kyu; Kumazaki, Shigeichi

    2015-04-01

    In chloroplasts of plants and algae, state transition is an important regulatory mechanism to maintain the excitation balance between PSI and PSII in the thylakoid membrane. Light-harvesting complex II (LHCII) plays a key role as the regulated energy distributor between PSI and PSII. It is widely accepted that LHCII, which is bound to PSII localized mainly in the granal thylakoid, migrates to bind with PSI localized mainly in the stroma-exposed thylakoid under preferential excitation of PSII. The phenomena have been extensively characterized by many methods. However, the exchange of LHCII between PSII and PSI has not been directly observed in vivo at physiological temperatures. Herein we applied fluorescence spectromicroscopy to Arabidopsis mesophyll protoplasts in order to observe in vivo changes in fluorescence spectra of granal and stromal thylakoid regions during the state transition. The microscopic fluorescence spectra obtained from a few sections with different depths were decomposed into PSI and PSII spectra and self-absorption effects were removed. We were able to determine amplitude changes of PSI and PSII in fluorescence spectra solely due to state transition. Subdomain analysis of granal and stromal thylakoid regions clarified variant behaviors in the different regions.

  12. Optimization of Protoplast Isolation Condition in Mulberry%桑树原生质体分离条件的优化

    Institute of Scientific and Technical Information of China (English)

    高丽霞; 蒋冬梅

    2012-01-01

    The factors affecting mulberry protoplast isolation were studied by the enzyme hydrolysis and hemacytometer methods to optimize the mulberry protoplast isolation condition and to provide the theoretical basis for mulberry protoplast fusion, new germplasm and plant genetic improvement. The results showed that enzyme type, different enzyme combination, enzymolysis time and osmotic pressure had significant effect on mulberry protoplast isolation. The higher mulberry protoplast yield could be obtained under the optimum condition including 1.0% cellulase, 0. 5% pectolase, 0. 2% macerozyme, 0. 6 mol/L mannitol, CPW, and 28 "C +2 °C for 6 h.%为了获得桑树原生质体分离的最优条件,为今后桑树原生质体融合、新种质的获取等植物遗传改良提供理论基础,采用酶解法和血球板计数法,对桑树原生质体分离的影响因素进行研究.结果表明,酶、酶液组合、酶解时间和渗透压稳定剂等都对原生质体的制备有显著的影响,较适宜桑树叶片游离的组合条件是1.0%纤维素酶+0.5%果胶酶+0.2%离析酶+0.6 mol/L甘露醇+CPW盐溶液,酶解温度为28℃±2℃,酶解时间为6h.在此条件下可获得高产量的原生质体.

  13. Calcium transport in protoplasts isolated from ml-o barley isolines resistant and susceptible to powdery mildew. [Hordeum vulgare L

    Energy Technology Data Exchange (ETDEWEB)

    Wrona, A.F.; Spanswick, R.M.; Aist, J.R. (Cornell Univ., Ithaca, NY (USA))

    1988-12-01

    Free cytoplasmic calcium has been postulated to play a role in preventing powdery mildew in a series of homozygous ml-o mutants of barley, Hordeum vulgare L. Protoplasts isolated from 7-day-old plants of the ml-o resistant-susceptible (R-S) barley isolines, Riso 5678/3* {times} Carlsberg II R and S, were used to test for differences in fluxes of Ca{sup 2+} across the plasmalemma. Greater influx or lesser efflux might account for a higher free cytosolic Ca{sup 2+} postulated to exist in ml-o R mutants. Uniform patterns of uptake were maintained for 3 hours from solutions of 0.2 and 2 millimolar Ca{sup 2+}. Washout curves of {sup 45}Ca{sup 2+} from R and S protoplasts revealed three compartments - presumed to represent release from the vacuole, organelles, and the cytoplasm (which included bound as well as free Ca{sup 2+}). Uptake and washout did not differ between isolines. On the basis of recent determinations of submicromolar levels of free cytoplasmic Ca{sup 2+} and their initial rates of {sup 45}ca-labeled Ca{sup 2+} uptake, they show that measurement of the unidirectional influx of Ca{sup 2+} across the plasmalemma is not feasible because the specific activity of the pool of free cytoplasmic calcium increases almost instantaneously to a level that would result in a significant, but unknown, efflux of label. Similarly, measurement of the efflux of Ca{sup 2+} across the plasmalemma is not possible since the activity of the pool of free cytoplasmic calcium is a factor of 350 smaller than the most rapid component of the washout experiment. This pool of cytoplasmic free Ca{sup 2+} will wash out too rapidly and be too small to detect under the conditions of these experiments.

  14. Isolation, culture and plantlet regeneration from cotyledon and mesophyll protoplasts of two pickling cucumber (Cucumis sativus L.) genotypes.

    Science.gov (United States)

    Punja, Z K; Tang, F A; Sarmento, G G

    1990-07-01

    Optimal protoplast yields from cotyledons (2.0×10(6) protoplasts/ 0.5 g tissue) and from true leaves (5.0×10(6) protoplasts/g tissue) of two Cucumis sativus genotypes were obtained following a 16 h digestion with, respectively, 1.25% pectinase+0.5% Cellulysin and 0.5 % pectinase+ 1.0% Cellulysin. Enzyme solutions were prepared in modified MS medium containing half-strength major salts, full complement of minor salts and vitamins, 2% sucrose and 0.25 M mannitol. A plating density of 3.5-4.0× 10(4) protoplasts/ml or higher was required for sustained division, with first division occurring in 6-7 days, second-third division in 8-9 days, and minicalli formation by day 13. Embedding in 0.4% agarose provided the highest plating efficiency (proportion that formed minicalli) of mesophyll protoplasts, which was 28.3% for genotype 3672 and 15% for genotype 3676. By comparison, liquid culture and droplet culture gave lower plating efficiencies (10-19%). Cotyledon and mesophyll protoplasts of one genotype formed minicalli on MS medium containing 2,4-D/BA at 1.0/2.5 μM and 5.0/5.0 μM, respectively, within 21 days, while mesophyll protoplasts of the second genotype formed minicalli on MS medium containing NAA/BA at 5.0/5.0 μM within 12 days. Shoot buds or somatic embryos were obtained upon subculture of calli to MS medium containing lower concentrations (0.05-0.01 μM) of 2,4-D/BA or NAA/BA and a few plantlets, ca.18, were recovered on hormone-free medium.

  15. Callus production from photoautotrophic soybean cell culture protoplasts.

    Science.gov (United States)

    Chowhury, V K; Widholm, J M

    1985-10-01

    Protoplasts were prepared from a photoautotrophic (PA) cell line of Glycine max (soybean). A yield of 75 to 90% after two to three hours digestion in a mixture of 1% Cellulase R10, 0.2% Pectolyase Y23 and 2% Driselase was obtained. Cell division and colony formation occurred from approximately 18% of the plated protoplasts. The cultured protoplasts were as sensitive to the herbicide atrazine, a photosynthetic inhibitor, as the original PA cells under the same conditions. Protoplasts and cells of a heterotrophic (HT) soybean culture were not as sensitive to atrazine. The isolated protoplasts retained the PA characteristics of the parental culture in the callus and cell suspension cultures obtained from the protoplasts. The chromosome numbers in the parental cell line and in cells derived from the isolated protoplasts (both PA and HT) were found to be largely (99%) the normal diploid number of 40.

  16. Isolation of plasmodesmata from Arabidopsis suspension culture cells.

    Science.gov (United States)

    Grison, Magali S; Fernandez-Calvino, Lourdes; Mongrand, Sébastien; Bayer, Emmanuelle M F

    2015-01-01

    Due to their position firmly anchored within the plant cell wall, plasmodesmata (PD) are notoriously difficult to isolate from plant tissue. Yet, getting access to isolated PD represents the most straightforward strategy for the identification of their molecular components. Proteomic and lipidomic analyses of such PD fractions have provided and will continue to provide critical information on the functional and structural elements that define these membranous nano-pores. Here, we describe a two-step simple purification procedure that allows isolation of pure PD-derived membranes from Arabidopsis suspension cells. The first step of this procedure consists in isolating cell wall fragments containing intact PD while free of contamination from other cellular compartments. The second step relies on an enzymatic degradation of the wall matrix and the subsequent release of "free" PD. Isolated PD membranes provide a suitable starting material for the analysis of PD-associated proteins and lipids.

  17. [Preparation and vitality detection of protoplast in Salvia miltiorrhiza Bunge].

    Science.gov (United States)

    Zhu, Nan; Liu, Jun; Zhang, Xinyu; Dong, Juan'e

    2014-10-01

    We prepared protoplasts from Salvia miltiorrhiza Bunge suspension culture cells. Then, the protoplasts' vitality and functions were tested by fluorescein diacetate staining method and Fluo-3/AM flourescent probe. The optimal condition of protoplast isolation was Cellulase R-10 1.5%, Pectinase Y-23 0.3%, Macerozyme R-10 0.5%, 40 r/min 12 h, 600 r/min 5 min, and the protoplasts yield was 1.1x10(6) cells/g FW, the vitality was more than 95% by using fluorescein diacetate staining method. It has been confirmed that calcium fluorescent probe Fluo-3/AM can be successfully loaded into protoplasts.

  18. Isolamento e regeneração de protoplastos de Magnaporthe grisea Isolation and regeneration of Magnaporthe grisea protoplasts

    Directory of Open Access Journals (Sweden)

    Carlos Eduardo Marchi

    2006-09-01

    Full Text Available Protoplastos são ferramentas biológicas importantes para pesquisas em fungos filamentosos, sendo empregados intensamente em transformação genética. O isolamento de protoplastos de Magnaporthe grisea foi facilitado com Novozym 234, contudo, este complexo enzimático encontra-se indisponível no mercado. Assim, objetivou-se comparar a eficiência de enzimas líticas disponíveis comercialmente na obtenção de protoplastos de M. grisea. Paralelamente, analisaram-se estabilizadores osmóticos, tempos de digestão e freqüência de regeneração. Maior produção de protoplastos foi obtida com o uso simultâneo de Lysing Enzymes e Cellulase Onozuka R-10. O uso de 10 ou 15 mg de cada complexo enzimático, em 3 mL de estabilizador osmótico, resultou em maior liberação de protoplastos. O melhor estabilizador osmótico foi MgSO4 1,2 M / NaH2PO4 0,01 M, pH 5,8, seguido por MgSO4 0,8 M / NaH2PO4 0,01 M, pH 5,8. O isolamento de protoplastos foi monitorado a cada 60 minutos, atingindo o máximo após incubação por 3 a 6 horas. No entanto, maior freqüência de regeneração (19,4% foi registrada para protoplastos obtidos após 3 horas de hidrólise enzimática.Protoplasts are important biological tools in filamentous fungi research. Fungal protoplasts have been extensively used in experiments with genetic transformation. Protoplastization of Magnaporthe grisea was accomplished with Novozym 234, however, this enzymatic complex is no commercially available for purchase. Thus, the efficiency of several other commercial enzymes in M. grisea protoplasts preparation was investigated. At the same time, osmotic buffer, digestion time and regeneration rate were also analyzed. The highest protoplasts production was obtained with Lysing Enzymes plus Cellulase Onozuka R-10. The use of 10 or 15 mg of each enzymatic complex in 3 mL of osmotic buffer was most effective for the protoplasts yields. The best osmotic buffer was MgSO4 1.2 M / NaH2PO4 0.01 M, pH 5

  19. Eficiência de isolamento e de plaqueamento de protoplastos de laranja-doce Isolation and platting efficiency of sweet orange protoplasts

    Directory of Open Access Journals (Sweden)

    Lívia Mendes de Castro

    2011-06-01

    Full Text Available O isolamento e plaqueamento de protoplastos são fatores fundamentais para o sucesso no cultivo in vitro deste tipo de explante visando a manipulações genéticas. A composição da solução enzimática no isolamento, a densidade de cultivo, bem como o próprio genótipo utilizado são variáveis importantes nestas etapas. Desta forma, o objetivo do trabalho foi avaliar a eficiência de isolamento de protoplastos em função de três soluções enzimáticas e a eficiência de plaqueamento em função de cinco densidades de protoplastos e diferentes composições de meio de cultura em cultivares de laranja-doce. As soluções enzimáticas avaliadas para o isolamento de protoplastos foram: 1. celulase Onozuka RS 1%, macerase R-10 1% e pectoliase 0,2%; 2. celulase Onozuka RS 1%, macerase R-10 1% ; 3. celulase Onozuka R-10 4%, macerase R-10 1%. O plaqueamento dos protoplastos foi realizado nas densidades de 2 x 10(4; 5 x 10(4; 10(5; 2x 10(5 e 3 x 10(5 protoplastos.mL-1, nos meios de cultura EME 0,7M, BH3 0,7M e BH3 + EME 0,7M em ausência de luz, a 25 ± 1 ºC. A solução enzimática 2 proporcionou maior rendimento no isolamento de protoplastos das cultivares 'Hamlin', 'Natal' e 'Pera', e a solução enzimática 1 foi a mais adequada para a laranja 'Westin'. Para a cultivar 'Lima-Verde', a solução enzimática 3 foi a mais eficiente. A eficiência final de plaqueamento, avaliada aos 90 dias de cultivo, foi superior nas densidades de 3 x 10(5 e 2 x 10(5 protoplastos.mL-1 para as cultivares 'Hamlin', 'Natal' e 'Lima-Verde', e nas densidades de 2 x 10(5 e 10(5 protoplastos.mL-1 para a laranja 'Westin'.Protoplast isolation and culture are important factors for adequate in vitro culture of this type of explant to further genetic manipulations. The composition of the enzymatic solution, protoplast platting density, and plant genotype are important variables in these steps. Therefore, this work aimed to evaluate the isolation efficiency of protoplasts

  20. Culture of soybean mesophyll protoplasts in alginate beads.

    Science.gov (United States)

    Tricoli, D M; Hein, M B; Carnes, M G

    1986-10-01

    Mesophyll protoplasts were isolated from leaves of 10 day old aseptically grown soybean seedlings, or from surface disinfested leaves of 3 week old plants grown in environmental chambers. The protoplasts were encapsulated in 2mm diameter Ca alginate beads. Immobilized protoplasts were induced to divide by culturing in shaker flasks containing an actively growing soybean cell suspension. The feeder cell suspension supported the division of protoplasts independent of the protoplast density in the Ca alginate beads. At day 18 after encapsulation, the alginate matrix was dissolved, releasing viable callus colonies. The feeder cell suspension obviated plating of protoplasts at high density which is usually required for subsequent cell division and colony development. Since the protoplasts were embedded at low density, the cell colonies were derived from single cells.

  1. Light-enhanced dark respiration in leaves, isolated cells and protoplasts of various types of C4 plants.

    Science.gov (United States)

    Parys, Eugeniusz; Jastrzebski, Hubert

    2006-04-01

    The rate of respiratory CO2 evolution from the leaves of Zea mays, Panicum miliaceum, and Panicum maximum, representing NADP-ME, NAD-ME, and PEP-CK types of C4 plants, respectively, was increased by approximately two to four times after a period of photosynthesis. This light-enhanced dark respiration (LEDR) was a function of net photosynthetic rate specific to plant species, and was depressed by 1% O2. When malate, aspartate, oxaloacetate or glycine solution at 50 mM concentration was introduced into the leaves instead of water, the rate of LEDR was enhanced, far less in Z. mays (by 10-25%) than in P. miliaceum (by 25-35%) or P. maximum (by 40-75%). The enhancement of LEDR under glycine was relatively stable over a period of 1 h, whereas the remaining metabolites caused its decrease following a transient increase. The metabolites reduced the net photosynthesis rate in the two Panicum species, but not in Z. mays, where this process was stimulated by glycine. The bundle sheath cells from P. miliaceum exhibited a higher rate of LEDR than those of Z. mays and P. maximum. Glycine had no effect on the respiration rate of the cells, but malate increased in cells of Z. mays and P. miliaceum by about 50% and 30%, respectively. With the exception of aspartate, which stimulated both the O2 evolution and O2 uptake in P. maximum, the remaining metabolites reduced photosynthetic O2 evolution from bundle sheath cells in Panicun species. The net O2 exchange in illuminated cells of Z. mays did not respond to CO2 or metabolites. Leaf mesophyll protoplasts of Z. mays and P. miliaceum, and bundle sheath protoplasts of Z. mays, which are unable to fix CO2 photosynthetically, also produced LEDR, but the mesophyll protoplasts, compared with bundle sheath protoplasts, required twice the time of illumination to obtain the maximal rate. The results suggest that the substrates for LEDR in C4 plants are generated during a period of illumination not only via the Calvin cycle reactions, but

  2. Vacuole/extravacuole distribution of soluble protease in Hippeastrum petal and Triticum leaf protoplasts

    Energy Technology Data Exchange (ETDEWEB)

    Wagner, G.J.; Mulready, P.; Cutt, J.

    1981-11-01

    The subcellular distribution of soluble protease in anthesis-stage, anthocyanin-containing Hippeastrum cv. Dutch Red Hybrid petal protoplasts has been reevaluated and that of Triticum aestivum L. var. Red Coat leaf protoplasts determined using /sup 125/I-fibrin as a protease substrate and improved methods for protoplast and vacuole volume estimation. Results indicate that about 20% of the Hippeastrum petal-soluble protease and about 90% of the wheat leaf-soluble protease can be assigned to the vacuole. Protoplast isolation enzyme labeled with /sup 125/I has been used to assess the efficiency of removing isolation enzyme from protoplasts by repeated washing and by separation of protoplasts from debris using density centrifugation. Results of these studies suggest that protoplasts prepared by both methods retain low levels of isolation enzyme. However, when protoplasts prepared by either method were lysed with washing medium lacking osmoticum, little isolation enzyme contaminated the lysates.

  3. Soybean protoplast culture and direct gene uptake and expression by cultured soybean protoplasts.

    Science.gov (United States)

    Lin, W; Odell, J T; Schreiner, R M

    1987-07-01

    A method was developed for culturing protoplasts freshly isolated from developing soybean (Glycine max L.) cotyledons. First cell divisions were observed within 5 days after protoplast isolation and microcalli, consisting of about 20 cells, were formed within 10 days. Thirty days after protoplast isolation, callus tissues were observed without the aid of a microscope. A 30 to 50% plating efficiency was consistently obtained. Using a polyethylene glycol-electroporation technique, DNA was introduced into these protoplasts. The protoplasts were then cultured to form callus. Chloramphenicol acetyltransferase (CAT) activity was detected in protoplast cultures 6 hours after introduction of a 35S-CAT-nopaline synthase 3' chimeric gene. The highest CAT activity was detected in 3-day-old electroporated protoplast cultures, indicating transient expression of the introduced gene. Some CAT activity was detected in 40-day-old callus cultures and in geneticin (G418) selected callus tissues which also received a chimeric neomycin phosphotransferase II gene, indicating the presence of stable transformants. A control chimeric gene with an inverted 35S promoter failed to produce any CAT activity in this system.

  4. Determination of rare earth elements in plant protoplasts by MAA

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    A preliminary study on the speciation of rare earth elements in plant cells has been carried out by molecular activation analysis (MAA). Mesophyll protoplasts of Brassica napus were isolated by enzymatic digestion. After being washed with isosmotic solution containing EDTA for several times, the protoplasts were purified by gradient centrifugation. Then the concentration of rare earth elements (REEs) in the protoplasts was determined by neutron activation analysis. The result shows that REEs can enter the cells of the plant.

  5. GFLV replication in electroporated grapevine protoplasts.

    Science.gov (United States)

    Valat; Toutain; Courtois; Gaire; Decout; Pinck; Mauro; Burrus

    2000-06-29

    Grapevine fanleaf virus (GFLV), responsible for the economically important court-noué disease, is exclusively transmitted to its natural host in the vineyards through Xiphinema nematodes. We have developed direct inoculation of GFLV into grapevine through protoplast electroporation. Protoplasts were isolated from mesophyll of in vitro-grown plants and from embryogenic cell suspensions. Permeation conditions were determined by monitoring calcein uptake. Low salt poration medium was selected. Electrical conditions leading to strong transient gene expression were also tested for GFLV inoculation (isolate F13). GFLV replication was detected with either virus particles (2 µg) or viral RNA (10 ng) in both protoplast populations, as shown by anti-P38 Western blotting. Direct inoculation and replication were also observed with Arabis mosaic virus (ArMV), a closely related nepovirus, as well as with another GFLV isolate. These results will be valuable in grapevine biotechnology, for GFLV replication studies, transgenic plant screening for GFLV resistance, and biorisk evaluation.

  6. Determinação de metodologia para oisolamento de protoplastos de tangerina Cleópatra (Citrus reshni Hort. Methodology choice for protoplast isolation in Cleopatra mandarin (Citrus reshni Hort.

    Directory of Open Access Journals (Sweden)

    R.P. de Oliveira

    1995-04-01

    Full Text Available A hibridação somática via fusão de protoplastos vem sendo utilizada no melhoramento de porta-enxertos de citros em diversos países. Nos Estados Unidos, vários estudos demonstram a eficiência de procedimentos no isolamento e cultivo de protoplastos dessa frutífera. O presente trabalho foi realizado com o objetivo de avaliar o efeito do meio de cultivo de calos embriogênicos do porta-enxerto tangerina Cleópatra (Citrus reshni Hort. sobre o isolamento de protoplastos, bem como sugerir alterações de procedimento. Os resultados mostram a possibilidade do isolamento de 1.4 x 10(6 a 4.7 x 10(6 protoplastos por grama de calos da espécie estudada. Verificou-se que, o subcultivo dos calos de tangerina Cleópatra em meio de cultura, sem reguladores 1 hora, sob condições de escuro a 120 rpm, proporcionou maior eficiência de isolamento de protoplastos (4.7 x 10(6 protoplastos/g de calo.Somatic hybridization has been used for citrus rootstock breeding in many countries. In USA, many reports had proved the efficiency of procedures for the isolation and culture of citrus protoplasts. This research was conducted to evaluate the efficiency of procedures of protoplast isolation using embryogenic callus of the Cleópatra mandarin rootstock. Alterations were proposed to increase protoplast isolation and culture method. Results show the possibility of a protoplast yield of 1.4 to 4.7 x 10(6 pps/g.f.w. for Cleópatra tangerine rootstock callus. Protoplast yield can be raised to 4.7 x 10(6 pps/g.f.w. if the embryogenic callus are grown in a medium supplemented only with 4% sucrose and pre-treated with 1% w/v macerozyme for 1 hour, at 120 rpm, in dark, is applied before protoplast isolation.

  7. Protoplast-to-plant regeneration of American elm (Ulmus americana).

    Science.gov (United States)

    Jones, A M P; Shukla, M R; Biswas, G C G; Saxena, P K

    2015-05-01

    This study describes a protocol for regeneration of plants from cell suspension-derived protoplasts of American elm (Ulmus americana). Efficient protoplast isolation was achieved from a two-phase culture system through the incorporation of 100 μM 2-aminoindan-2-phosphonic acid, with a yield of approximately 2 × 10(6) protoplasts/ml packed cell volume. Isolated protoplasts failed to survive in liquid or alginate bead culture systems but initiated and continued to divide when embedded in low melting point agarose beads. Protoplast-derived callus proliferated and differentiated into shoot buds in response to 10 or 20 μM thidiazuron. Differentiated buds elongated and continued to proliferate on elm shoot medium supplemented with 3.0 μM GA3. The protoplast-derived shoots rooted and acclimatized to greenhouse conditions and continued to grow. This system provides the first protoplast-to-plant regeneration system for American elm and provides a framework for the development of protoplast fusion or genome editing technologies.

  8. Protoplasts: a useful research system for plant cell biology, especially dedifferentiation.

    Science.gov (United States)

    Jiang, Fangwei; Zhu, Jian; Liu, Hai-Liang

    2013-12-01

    As protoplasts have the characteristics of no cell walls, rapid population growth, and synchronicity, they are useful tools for research in many fields, especially cellular biology (Table 1). This article is an overview that focuses on the application of protoplasts to investigate the mechanisms of dedifferentiation, including changes in hormone signals, epigenetic changes, and organelle distribution during the dedifferentiation process. The article also emphasizes the wide range of uses for protoplasts in studying protein positions and signaling during different stresses. The examples provided help to show that protoplast systems, for example the mesophyll protoplast system of Arabidopsis, represent promising tools for studying developmental biology. Meanwhile, specific analysis of protoplast, which comes from different tissue, has specific advantages and limitations (Table 2), and it can provide recommendations to use this system.

  9. ‘富士’苹果花粉原生质体分离初探%Preliminary Study on the Isolation of Mature Pollen Protoplasts in ‘Fuji’ Apple

    Institute of Scientific and Technical Information of China (English)

    张宁; 李威; 顾钊宇; 陈秋菊; 段续伟; 杨清; 李天忠

    2015-01-01

    Little information is available about the research on plant pollen protoplasts,especially the isolation of apple pollen protoplasts.In this study,the protoplasts from mature pollen of Malus × domestica Borkh.‘Fuji’ by the method of ‘germination-enzymatic treatment’ were isolated.To screen the most effective condition of isolation,some key factors were analyzed.The results indicated that:The isolation percentage was up to 6.83% when the pollens germinating for 45 min and then treated with mixed enzyme solution including 1% Cellulase Onozuka R-10,1% Macerozyme R-10 and 18% Mannitol for 6 h.The cell walls of isolated protoplasts were degraded entirely according to the examination of 0.1% calcoflower white staining.0.01% fluorescein diacetate (FDA) staining indicated the protoplasts were viable.Therefore,the proroplasts can be used for further research.%以‘富士’苹果(Malus×domestica Borkh.‘Fuji’)成熟花粉为试材,采用“萌发—酶解二步法”,初步探讨了影响原生质体分离的关键因子,获得了花粉原生质体分离的最佳条件:用萌发后45 min的花粉,转入含1%纤维素酶和1%离析酶的混合酶液中,以18%甘露醇调节渗透压,静置酶解6h,花粉原生质体分离效率可达6.83%,用0.1%荧光增白剂检测表明脱壁完全,用0.01% FDA检测表明其具有生活力,可以作为后续细胞融合等的材料.

  10. [Direct embryogenesis from protoplast of winter wheat].

    Science.gov (United States)

    Ge, T M; Zhang, R D; Qin, F L; Yu, Y J; Xie, Y F

    2000-09-01

    Friable embryogenic calli were obtained on a modified N6 medium (NBD medium) from a winter wheat cultivar "Jinghua No. 1" (Triticum aestivum L. cv. Jinghua No. 1) and were transferred to a modified MS liquid medium (MSDL medium) to initiate embryogenic suspension cultures. Protoplasts were isolated from the suspensions and cultured on a modified MS medium (MSDP medium). The somatic embryoids were formed directly from the protoplasts and germinated into entire plants. The development of the somatic embryoids was very similar to that of zygotic embryos of wheat.

  11. Extracting viral RNAs from plant protoplasts.

    Science.gov (United States)

    Fabian, Marc R; Andrew White, K

    2007-08-01

    The analysis of viral RNA is a fundamental aspect of plant RNA virus research. Studies that focus on viral RNAs often involve virus infections of plant protoplasts (see UNITS 16D.1-16D.4). Protoplast offer the advantage of simultaneous initiation of infections, which allows for superior temporal and quantitative analyses of viral RNAs. The efficient isolation of intact viral RNA is key to any such investigations. This unit describes two basic protocols for extracting viral RNAs from plant protoplasts. An approach for preparing double-stranded viral RNA from total RNA pools is also provided. The viral RNA prepared by using these techniques can be used for further analyses such as primer extension, reverse transcription-PCR, and northern blotting.

  12. Avocado fruit protoplasts: a cellular model system for ripening studies.

    Science.gov (United States)

    Percival, F W; Cass, L G; Bozak, K R; Christoffersen, R E

    1991-12-01

    Mesocarp protoplasts were isolated from mature avocado fruits (Persea americana cv. Hass) at varying stages of propylene-induced ripening. Qualitative changes in the pattern of radiolabel incorporation into polypeptides were observed in cells derived from fruit at the different stages. Many of these differences correlate with those observed during radiolabeling of polypeptides from fresh tissue slices prepared from unripe and ripe fruit. Protoplasts isolated from fruit treated with propylene for one day or more were shown to synthesize cellulase (endo-ß-1,4-glucanase) antigen, similar to the intact propylene-treated fruit. These results suggest that the isolated protoplasts retain at least some biochemical characteristics of the parent tissue. The cells may also be used in transient gene expression assays. Protoplasts isolated from preclimacteric and climacteric fruit were equally competent in expressing a chimeric test gene, composed of the CaMV 35S RNA promoter fused to the bacterial chloramphenicol acetyltransferase gene, which was introduced by electroporation.

  13. Culture and fusion of pollen protoplasts of Brassica oleracea L. var. italica with haploid mesophyll protoplasts of B. rapa L. ssp. pekinensis.

    Science.gov (United States)

    Liu, Fan; Ryschka, U; Marthe, F; Klocke, E; Schumann, G; Zhao, H

    2007-01-01

    Hybrid callus was formed from the successful protoplast fusion between pollen protoplasts of Brassica oleracea var. italica and haploid mesophyll protoplasts of Brassica rapa. The pollen protoplast isolation frequency in broccoli was highly related to the ratio of trinucleate pollens in the male gametophyte population. Large quantities of pollen protoplasts with high vigor could be isolated, and the isolation frequency reached up to 90% in 6.0-7.0 mm long flower buds with about 94.7% trinucleate-stage pollens. Pollen protoplasts could be collected and purified by discontinuous gradient centrifugation. In 1% Na-alginate embedding culture, cell divisions were observed but no further development was found. The haploid mesophyll protoplasts were isolated from in vitro haploid plants of B. rapa. Results strongly showed the variability in culturability of mesophyll protoplasts from different haploid lines. Both pollen protoplasts and haploid mesophyll protoplasts retained a stable round shape in the designed prefusion solution with an osmotic pressure of 0.74 osmol/kg. Polyethylene glycol was used for the protoplast fusion, and 40% polyethylene glycol 4000 enabled the highest fusion frequency of about 20%. Some postfusion protoplasts showed cell divisions up to callus proliferation. Calli were screened by random amplified polymorphic DNA analysis for their hybrid character. Results revealed the existence of the hybrid calli. Some of the hybrid calli grew well with green color and shoot primordia. According to our knowledge, this is the first report about a hybrid formation between two haploid protoplasts. Potential comprehensive applications, as well as problems of this technique, are discussed.

  14. Protoplast isolation and plant regeneration from leaves of Rhodiola sachalinensis%高山红景天叶肉原生质体分离培养与植株再生

    Institute of Scientific and Technical Information of China (English)

    刘剑锋; 程云清; 陈智文

    2009-01-01

    Objective To isolate protoplasts from tube plant leaves of Rhodiola sachalinensis and re-generate plantlets after protoplast culture. Methods Preculture treatment and size of explants, enzyme concentration, mannitol concentration in enzyme mixture related with protoplasts isolation were studied to determine the superior optimized conditions. Results Explants could be used to isolate protoplast without dark preculture, and leaf length should be longer than 1. 5 cm. The best incubating enzyme solution con-mmol/L KH2PO4, and 0.5 mol/L mannitol. The enzyme and explants mixture were shaken for 4 h at 25℃. The protoplasts yield and viability were 39.43×106/g fresh weight and 78.6%, respectively. Purified protoplasts were cultured in medium 1/2 MS+1 mg/L 2, 4-D+0.5 mg/L ZT+0. 5 mol/L mannitol +500 mg/L hydrolysis of casein initially with shallow liquid layers, and calli formed within 40 d. After calli were transferred to MS+ 1 mg/L 6-BA+0. 1 mg/L NAA, adventitious buds were induced from calli. Shoots lon-ger than 2 em rooted within 30 d when they were transferred to 1/2 MS medium. Conclusion The study provides the scientific base for protoplast fusion in polyploidy breeding of R. Sachalinensis.%目的 利用高山红景天组培苗叶片分离得到原生质体,经培养后获得再生植株.方法 研究了外植体前期预处理、外植体大小、酶液配比、酶液中甘露醇浓度等影响原生质分离的相关因素,确定最优分离条件.结果 用于原生质体分离的外植体无需黑暗预培养便可进行原生质体分离,外植体叶片长度大于1.5 cm为宜.获得原生质体的酶液组成为:1.0%纤维素酶Onzuka R-10+0.5%果胶酶Macerozyme R-10+10 mmol/L CaCl2·2H2O+0.1%MES+0.7 mmol/L KH2PO4+0.5 mol/L甘露醇,在25℃条件下酶解4 h,原生质体最高产量为39.43×106个/g鲜质量.原生质体活力为78.6%.原生质体培养基为1/2MS+1 mg/L 2,4-D+0.5 mg/LZT+0.5 mol/L甘露醇+500 mg/L水解酪蛋白.浅层培养40 d时形成

  15. Isolation and Purification of Protoplasts in Roots and Leaves of Soybean Seedlings%大豆幼苗根和叶片原生质的分离与纯化

    Institute of Scientific and Technical Information of China (English)

    张晓可; 於丙军

    2009-01-01

    研究了大豆幼苗根和叶片原生质体的分离、纯化方法及其影响因素.结果表明:适宜大豆根和叶片原生质体分离的酶种类、浓度分别为CPW-13M{CPW(细胞清洗液)+13%(W/V)甘露醇}+3%纤维素酶(cellulose R10)+1.1%果胶酶(macerozyme R-10)+1.0%半纤维素酶(hemicellulase)和0.15%CaCl_2·2H_2O+9%甘露醇+1%cellulase R-10+0.20%pectolase Y-23,pH 5.8,酶解温度为28℃.在根酶解时间为16 h时,原生质体产量可高达1.46×10~5个·g~(-1)FW,活力达57.8%;叶片酶解时间为4 h时,原生质体产量可高达1.74×10~6个·g~(-1)FW,活力达70.3%.对于根而言,从产量和活力两方面考虑,其原生质体用23%蔗糖和CPW-18M混合后的下沉法纯化效果较好,而叶片用25%蔗糖的上浮法纯化效果较好.%The methods for isolation and purification of protoplast in roots and leaves of soybean seedling were investigated together in this study. The results showed that, the suitable enzyme species and concentrations for isolation of root protoplasts were CPW-13M + 13% (W/V) mannotol+3% cellulose R-10 + 1.1% macerozyme R- 10 + 1.0% hemicellulase, pH5.8 ; those for leaf were 0. 15% CaCl_22H_2O+9% mannitol + 1% celhilase R-10 +0.20% pectolase Y-23, pH 5.8, and the temperature was 28℃. The reasonable enzymatic times for isolation of root and leaf protoplasts were 16 and 4 h, respectively; and the higher protoplast yield and viability were accordingly 1. 46 × 10~5 protoplasts · g~(-1) FW and 57. 8% ,1. 74 × 10~6 protoplasts ? g FW and 70. 3% . The better purification for root protoplasts was the sinking method of 23% sucrose mixed with CPW- 18M ,that for leaf was the rising method of 25% sucrose.

  16. Effects of Different Explants on Isolation and Regeneration of Protoplast in Rubber Tree%不同外植体对橡胶树原生质体分离和再生的影响

    Institute of Scientific and Technical Information of China (English)

    戴雪梅; 黄天带; 李季; 杨先锋; 黄华孙

    2014-01-01

    To seek a better approach for plant regeneration from protoplast culture, in this research, anthers and inner integuments of rubber tree, a cultivar of Reyan 7-33-97, were respectively used as initial explants for inducing calli to establish stable embryogenic cell suspensions by suspension culture. Isolating and culturing of protoplast from these two kind of suspension cells were further studied. Protoplast yields and viabilities from different initial explants, protoplast division and growth during feeder layer culture, and subsequently somatic embryogenesis and plant regeneration were statistically analyzed. Results showed that embryogenic cell suspen-sions derived from anthers and inner integuments enabled to yield 7.6í106 protoplasts per mL PCV and 12í106 protoplasts per ml PCV, with mean viabilities of 75.2%and 83.9%, respectively, which was under the inoculation conditions in enzyme solution containing 1.5% cellulase R-10, 0.15% pectolyase y-23 and 0.5% macerozyme R-10 for 12 h. Sustained mitotic divisions were both observed when the two kind of protoplasts were cultured on feeder layer, and 247 and 480 microcalli with the size 2 mm above were respectively formed from 5í105 anther-derived and inner integument-derived protoplasts after 45 d nursing culture, from which 56 and 18 embryos were respectively obtained after 60 d culture on medium for somatic embryo induction. Finally 4.7% embryos developed from anther-derived protoplast were regenerated to plantlets, whereas all embryos obtained from inner integument- derived protoplasts failed to plant regeneration. In conclusion, anther-derived embryonic cell suspension was the optimal material for isolating protoplast bearing regenerated capacity, providing basis and reference for further optimizing protoplast culture system and interspecific somatic hybridization in rubber tree.%为了探寻橡胶树原生质体培养再生植株的较佳途径,本研究以橡胶树热研7-33-97花药和内珠被为起始

  17. Osmotic stress inhibits thymidine incorporation into soybean protoplast DNA.

    Science.gov (United States)

    Cress, D E

    1982-10-01

    DNA synthesis in protoplasts isolated from soybean cell suspension cultures has been investigated by [(3)H] thymidine uptake and incorporation kinetics. Initial rates of incorporation in exponential and 5-fluorodeoxyuridine synchronized protoplasts are inhibited by increased osmolarities of the medium. The inhibition was not readily reversible during 3 h culture in low osmotic medium. Velocity sedimentation analyses of replicating DNA from such protoplasts shows a complex pattern of inhibition. The inhibition probably effects replicon initiation as well as strand elongation and ligation of replication intermediates.

  18. Protoplasts Culture Isolated from Friable Embryogenic Callus of Cassava and Plant Regeneration%木薯脆性胚性愈伤组织原生质体培养与植株再生

    Institute of Scientific and Technical Information of China (English)

    文峰; 肖诗鑫; 聂扬眉; 马秋香; 张鹏; 郭文武

    2012-01-01

    [Objective] The objective of this study is to establish an efficient system of protoplast regeneration for further developing protoplast fusion and transformation in cassava. [ Method ] Protoplasts were isolated from suspension cultures derived from friable embryogenic callus (FEC) of cassava genotype TMS60444. The highest protoplast yield obtained was 3.5x 106 protoplasts/g fresh weight. Viabilities of the protoplasts assessed by the fluorescein diacetate (FDA) were approximately 90%. Protoplasts were cultured in TM2G medium with liquid thin layer culture at densities of 5x105p/mL or 2x105p/mL. During the first 30 d, the medium was refreshed by 0.3 mol-L"1 TM2G fresh medium every 10 d. After that, the medium was refreshed by 0.25 mol-L-1 TM2G fresh medium every 10 d. After cultured for 45 d, calli of 1-2 mm were picked out and separately developed into embryos on MSN medium, into mature embryos on CMM medium, into shoots on CEM medium and into roots on MS medium. [Result] K was showed that all protoplasts cultured at density of 5x105p/mL developed into compact calli (could develop into embryos), protoplasts cultured at density of 2xl05 p/mL developed into compact calli and vacuolar calli (could not develop into embryos). A total of 1 479 compact calli were picked out and developed into 757 cotyledon embryos and regenerated 186 plants in the experiment. [Conclusion] The yield and viability of isolated protoplasts had been greatly increased, the bottleneck of predecessors mentioned was improved, and the efficiency of plant regeneration from protoplasts was promoted.%[目的]建立有效的木薯原生质体再生体系,为原生质体融合以及原生质体转化等研究奠定技术基础.[方法]酶解木薯品种TMS60444的脆性胚性愈伤组织(FEC)的悬浮系,分离原生质体的产量最高达3.5×106个/g,FDA检测其活性约90%.用TM2C培养基以液体浅层法分别在5 ×105个/mL和2× 105个/mL密度下培养,培养过程中前30 d用0.3 mol.L-1

  19. A new tool for plant cell biology: in vivo antibody uptake in plant protoplasts.

    Science.gov (United States)

    Brière, C; Barthou, H; Petitprez, M

    2004-07-01

    We report on the in vivo uptake of antibodies into plant protoplasts. When protoplasts of sunflower, Arabidopsis or tobacco were incubated in vivo with an antibody, this antibody was detected by immunofluorescence in the cytoplasm and/or the nucleus, depending on the location of the target protein. Furthermore, when protoplasts were cultured in the presence of antibodies, specific effects were observed. Incubation with antibodies raised against p34cdc2 led to a strong inhibition of the division rate, and a decrease in the average DNA content of protoplasts. With antibodies against HaWLIM1, a LIM domain protein of the CRP type, a negative effect on actin organisation was observed. We conclude that antibodies can penetrate plant protoplasts in vivo, and thus may be used as powerful tools for the study of protein function.

  20. High affinity RGD-binding sites at the plasma membrane of Arabidopsis thaliana links the cell wall.

    Science.gov (United States)

    Canut, H; Carrasco, A; Galaud, J P; Cassan, C; Bouyssou, H; Vita, N; Ferrara, P; Pont-Lezica, R

    1998-10-01

    The heptapeptide Tyr-Gly-Arg-Gly-Asp-Ser-Pro containing the sequence Arg-Gly-Asp (RGD--the essential structure recognised by animal cells in substrate adhesion molecules) was tested on epidermal cells of onion and cultured cells of Arabidopsis upon plasmolysis. Dramatic changes were observed on both types of cells following treatment: on onion cells, Hechtian strands linking the cell wall to the membrane were lost, while Arabidopsis cells changed from concave to convex plasmolysis. A control heptapeptide Tyr-Gly-Asp-Gly-Arg-Ser-Pro had no effect on the shape of plasmolysed cells. Protoplasts isolated from Arabidopsis cells agglutinate in the presence of ProNectinF, a genetically engineered protein of 72 kDa containing 13 RGD sequences: several protoplasts may adhere to a single molecule of ProNectinF. The addition of the RGD-heptapeptide disrupted the adhesion between the protoplasts. Purified plasma membrane from Arabidopsis cells exhibits specific binding sites for the iodinated RGD-heptapeptide. The binding is saturable, reversible, and two types of high affinity sites (Kd1 approximately 1 nM, and Kd2 approximately 40 nM) can be discerned. Competitive inhibition by several structurally related peptides and proteins noted the specific requirement for the RGD sequence. Thus, the RGD-binding activity of Arabidopsis fulfils the adhesion features of integrins, i.e. peptide specificity, subcellular location, and involvement in plasma membrane-cell wall attachments.

  1. Isolation and identification of Sclerotinia stem rot causal pathogen in Arabidopsis thaliana

    Institute of Scientific and Technical Information of China (English)

    Ai-rong WANG; Wen-wei LIN; Xiao-ting CHEN; Guo-dong LU; Lie ZHOU; Zong-hua WANG

    2008-01-01

    A new stem rot disease is found to occur naturally on Arabidopsis plants in greenhouses of Fuzhou, China. In order to identify its pathogen, we conducted a series of fimgal isolation and purification, plant reinoculation, and ascus and ascospore induction from the sclerotia. The isolate caused typical water-soaked lesions after reinoeulation and produced sclerotia both on Arabidopsis plants and culture medium plates, and the sclerotia could be induced to produce discal apothecia and 8 binucleate ascospores per ascus. These disease symptom and fungal morphology data revealed that the fungus Sclerotinia sclerotiorum (Lib.) de Bary was the pathogen for Arabidopsis stem rot. To confirm this, we further amplified its large subunit ribosomal DNA (LSU rDNA) by polymerase chain reaction (PCR), and compared the sequence with the known LSU rDNA sequences in GenBank. The results show that the sequence shares the highest identities with the LSU rDNAs of different S. sclerotiorum strains. Taking all these data together, we concluded that the fungus that caused the Arabidopsis stem rot is S. sclerotiorum (Lib.) de Bary. This is the first report that Arabidopsis is naturally infected by S. sclerotiorum.

  2. Inverse polymerase chain reaction for rapid gene isolation in Arabidopsis thaliana insertion mutants

    NARCIS (Netherlands)

    Vanderhaeghen, R.; Scheres, B.J.G.; Montagu, M. van; Lijsebetten, M. van

    1992-01-01

    Recently, many mutants have been isolated in the model plant Arabidopsis thaliana by the insertion of the Agrobacterium tumefaciens T-DNA into the plant genome. Instead of applying Southern analysis on these insertion mutants and to avoid the construction of mutant- derived genomic libraries, we pro

  3. 植物叶片原生质体分离的可能机制%Possible mechanism involved with isolation protoplast of leaf

    Institute of Scientific and Technical Information of China (English)

    祖元刚; 于景华; 唐中华; 郭晓瑞; 张宇亮; 孟庆焕

    2005-01-01

    分析了植物叶片在分离液环境中形成原生质体的过程,文中提出,分离液配方中的酸性物质使植物叶片处于酸性环境中并导致植物正常细胞首先发生细胞壁酸性降解,随后出现原生质体脱离细胞壁进入分离液,继而又进一步发生质膜的酸性降解,使细胞核和细胞器进入分离液中,最终分离液中的细胞器以细胞核为中心进行细胞器重组,最后产生外貌形态一致的新的原生质体.植物细胞壁和质膜是植物细胞的包被系统.植物细胞包被系统的酸性降解使植物细胞器重组并产生新的原生质体成为可能.%The process and mechanism of leaf protoplast formation after incubation to cell wall digesting solution was investigated. The acid material in the digesting solution provided the acid environment for leaves and led to the occurrence of acid degradation of cell wall, the separation between cell wall with protoplast and the escape of protoplast from cell wall into solution. The cytoplasm membrane inside the digesting solution was degraded by the acid environment and this accounted for the escape of cell nucleus and organelles from protoplast. In digesting solution, the recombination of organelles surrounding nuclear and subsequent formation of new protoplast with unanimous shape took place. These results showed that plant cell wall and cytoplasm membrane is the coat system of plant cell. The acid degradation of plant cell coat system makes recombination of cell organ and production of new protoplast possible.

  4. Molecular cell biology of male meiotic chromosomes and isolation of male meiocytes in Arabidopsis thaliana.

    Science.gov (United States)

    Wang, Yingxiang; Cheng, Zhihao; Lu, Pingli; Timofejeva, Ljudmilla; Ma, Hong

    2014-01-01

    Plants typically produce numerous flowers whose meiotic chromosomes are relatively easy to observe, making them excellent structures for studying the cellular processes underlying meiosis. In recent years, breakthroughs in light and electron microscopic technologies for small chromosomes, combined with molecular genetic methods, have resulted in major advances in the understanding of meiosis in the model plant Arabidopsis thaliana. In this chapter, we summarize protocols for basic cytology, fluorescence in situ hybridization, immunofluorescence, electron microscopy, and isolation of male meiocytes for the analysis of Arabidopsis meiosis.

  5. Regeneration of plants from mesophyll protoplasts of the wild crucifer Eruca sativa Lam.

    Science.gov (United States)

    Sikdar, S R; Chatterjee, G; Das, S; Sen, S K

    1987-12-01

    Protoplasts isolated from mesophyll cells of Eruca sativa Lam., cultured on suitable medium, underwent sustained cell divisions to form calli. The plating efficiency was found to be 0.4%. The protoplast-derived calli subsequently produced plantlets through organogenesis (15.71%) and somatic embryogenesis (11.25%). Regenerated plants exhibited normal appearance. These results indicate potential to introgress desirable traits from this wild crucifer into important oilseed and cole Brassicas by protoplast fusion and hybrid recovery.

  6. ‘石牌’广藿香叶肉原生质体的分离及纯化%Isolation and Purification of Mesophyll Protoplasts of Pogostem cablin cv. shipaiensis

    Institute of Scientific and Technical Information of China (English)

    莫小路; 邱蔚芬; 黄珊珊; 陈瑜珍; 严振

    2012-01-01

      以‘石牌’广藿香无菌苗叶片为材料,对原生质体分离、纯化方法以及影响因素进行了研究.结果表明:以继代培养12~22 d 的无菌苗顶芽下第3对展开叶片为材料,用0.5%果胶酶、0.2%离析酶和1.5%纤维素酶作用8 h,渗透压调节剂为11%甘露醇,‘石牌’广藿香叶肉原生质体产量达1.85×107个/g fw,活力达89%;原生质体纯化以12%聚蔗糖(Ficoll)漂浮法效果最佳%  Mesophyll protoplasts were isolated and purified from leaves of in vitro propagated Polgostemon cablin (Blanco) Benth. cv. shipaiensis, and different factors affecting protoplasts yield and activity were investigated. The results showed that young leaves of seedling subcultured for 12~22 days in the enzyme solution consisting of 0.5% (w/v) pectolyase Y-23, 0.2%(w/v) Macerozyme R-10 and 1. 5% (w/v) cellulase R-10, in 11% (w/v) mannitol buffered with 0.1% MES and 0.02%CaCl2 for 8 h, the yield was 1.85×107 protoplasts per gram leaves (fresh weight), the viability was above 89%.

  7. Electroporetic transfection of pepper protoplasts with plant potyviruses.

    Science.gov (United States)

    Velasquez, Nubia; Murphy, John F; Suh, Sang-Jin

    2012-01-01

    Potyviruses are a persistent threat to bell pepper (Capsicum annuum L.) production worldwide. Much effort has been expended to study the resistance response of pepper cultivars at whole plant levels but with only limited effort at the cellular level using protoplasts. A pepper protoplast isolation procedure is available but an inoculation procedure is needed that provides consistent and highly efficient infection. An electroporation-based procedure for inoculation of potyviruses was developed using a base procedure developed for Cucumber mosaic virus (CMV). The final parameters identified for efficient potyvirus infection of pepper protoplasts involves two 25ms pulses, 200V each pulse with a 10s interval between pulses. Depending on the method of detection, e.g., ELISA versus RT-PCR, potyvirus RNA inoculum ranged from 10 to 40μg with infection detection occurring with samples of 50,000-100,000 protoplasts.

  8. Ethylene production by apple protoplasts.

    Science.gov (United States)

    Anderson, J D; Lieberman, M; Stewart, R N

    1979-05-01

    Freshly prepared protoplasts from apple tissue that produced ethylene were obtained. Ethylene production was inhibited by osmotic shock, 0.01% Triton X-100, and aminoethoxyvinyl glycine. Protoplasts as well as the ethylene system were not greatly affected by protease treatment.

  9. The Plant Protoplast: A Useful Tool for Plant Research and Student Instruction

    Science.gov (United States)

    Wagner, George J.; And Others

    1978-01-01

    A plant protoplast is basically a plant cell that lacks a cell wall. This article outlines some of the ways in which protoplasts may be used to advance understanding of plant cell biology in research and student instruction. Topics include high efficiency experimental virus infection, organelle isolation, and osmotic effects. (Author/MA)

  10. Prolific plant regeneration from protoplast-derived tissues of Lotus corniculatus L. (birdsfoot trefoil).

    Science.gov (United States)

    Ahuja, P S; Hadiuzzaman, S; Davey, M R; Cocking, E C

    1983-04-01

    Protoplasts isolated enzymatically from seedling roots, hypocotyls and cotyledons of Lotus corniculatus L. produced callus which underwent prolific shoot regeneration. The rapidity and ease of recovering plants from protoplast-derived tissues makes this forage legume an attractive experimental system for genetic manipulation.

  11. Efficient and rapid isolation of early-stage embryos from Arabidopsis thaliana seeds

    OpenAIRE

    Raissig, Michael T; Gagliardini, Valeria; Jaenisch, Johan; Grossniklaus, Ueli; Baroux, Célia

    2013-01-01

    In flowering plants, the embryo develops within a nourishing tissue - the endosperm - surrounded by the maternal seed integuments (or seed coat). As a consequence, the isolation of plant embryos at early stages (1 cell to globular stage) is technically challenging due to their relative inaccessibility. Efficient manual dissection at early stages is strongly impaired by the small size of young Arabidopsis seeds and the adhesiveness of the embryo to the surrounding tissues. Here, we describe a ...

  12. Development of a cell sorting procedure to increase the sensitivity of detection of protein-protein interactions in plant protoplasts.

    Science.gov (United States)

    Zhang, Xin; Wong, Sek Man

    2011-05-01

    To visualize subcellular localization of viral proteins and interactions between viral proteins and host proteins in vivo, transfection of plasmids into protoplasts to over-express transiently fusion proteins with a fluorescent tag is a common method. However, due to the low efficiency (0.1-3.0%) of plasmid transfection into protoplasts, it is difficult to identify protoplasts that emit fluorescence using confocal microscopy. A flow cytometry sorting protocol was developed for separating kenaf protoplasts that emit yellow fluorescence. The sorted protoplasts showed strong fluorescence and the protoplasts were intact. This will improve the use of confocal microscopy for studying subcellular localization and protein interactions in protoplasts isolated from plants with low transfection efficiency.

  13. Shoot regeneration of mesophyll protoplasts transformed by Agrobacterium tumefaciens, not achievable with untransformed protoplasts.

    Science.gov (United States)

    Steffen, A; Eriksson, T; Schieder, O

    1986-04-01

    Alternative methods for shoot regeneration in protoplast derived cultures were developed in Nicotiana paniculata and Physalis minima. In both species protoplast derived callus is not regeneratable to shoots by conventional methods, e.g. hormone treatment. Leaf discs and stem segments of N. paniculata and P. minima were incubated with Agrobacterium tumefaciens "shooter" strains harbouring pGV 2215 or pGV 2298 or wildtype strain B6S3. After 36 h of co-incubation protoplasts were prepared. (Leaf disc and stem segment cloning). Co-cultivation experiments were also undertaken with protoplasts of both species. Transformed clones, characterized by their hormone independent growth and octopine production, could be isolated after about two months. Transformation frequencies of "leaf disc and stem segment cloning" and co-cultivation experiments varied from 5×10(-3) to 5×10(-5). After about one year of cultivation on hormone-free culture medium, shoots could be recovered from colonies of N. paniculata, transformed by the strain harbouring pGV 2298. In protoplast derived colonies of P. minima, shoot induction was obtained only after transformation by bacteria carrying pGV 2215. This demonstrates the importance of the particular "shooter" mutant, as well as the response of the host plant. Transformed shoots of P. minima produced octopine, whereas octopine production in transformed shoots and callus of N. paniculata was undetectable after one year of cultivation, though T-DNA was still present in the plant genome. Transformed shoots of N. paniculata and P. minima do not produce any roots. Shoots of N. paniculata have an especially tumerous phenotype. Shoots of both species were successfully grafted to normal donor plants of N. tabacum.

  14. Plant regeneration from mesophyll and suspension protoplasts of Silybum marianum.

    Science.gov (United States)

    Hetz, E; Perales, E H; Liersch, R; Schieder, O

    1995-12-01

    Mesophyll protoplasts of six lines of Silybum marianum were enzymatically isolated from young leaves, embedded in sodium alginate, and cultivated in KM-medium. Division frequencies observed after ten days were strongly influenced by the protoplast density. When 5 x 10 (4)/ml protoplasts were plated, division frequencies of about 35% were obtained, with a protoplast population density of 1 x 10 (5)/ml division frequencies of about 75% resulted. Plant regeneration experiments undertaken with the protocalluses on medium containing BAP led to shoot formation in only two lines with regeneration frequencies of less than 1% in one (M 24) and up to 7% in a second line (M 2), respectively. However, when the protocalluses from line M 2 were treated with thidiazuron (TDZ) in a first culture step, and with BAP in a second step, the shoot formation frequency rose to 22%. Shoots were rooted on hormone free MS agar medium and transferred into soil where plants grew to maturity. Similar results were obtained when protoplasts of the line M 2, isolated from a suspension culture, were cultivated.

  15. DNA-free RNA isolation protocols for Arabidopsis thaliana, including seeds and siliques

    Directory of Open Access Journals (Sweden)

    Vicente-Carbajosa Jesús

    2008-10-01

    Full Text Available Abstract Background High throughput applications of the reverse transcriptase quantitative PCR (RT-qPCR for quantification of gene expression demand straightforward procedures to isolate and analyze a considerable number of DNA-free RNA samples. Published protocols are labour intensive, use toxic organic chemicals and need a DNase digestion once pure RNAs have been isolated. In addition, for some tissues, the amount of starting material may be limiting. The convenience of commercial kits is often prohibitive when handling large number of samples. Findings We have established protocols to isolate DNA-free RNA from Arabidopsis thaliana tissues ready for RT-qPCR applications. Simple non-toxic buffers were used for RNA isolation from Arabidopsis tissues with the exception of seeds and siliques, which required the use of organic extractions. The protocols were designed to minimize the number of steps, labour time and the amount of starting tissue to as little as 10–20 mg without affecting RNA quality. In both protocols genomic DNA (gDNA can be efficiently removed from RNA samples before the final alcohol precipitation step, saving extra purification steps before cDNA synthesis. The expression kinetics of previously characterized genes confirmed the robustness of the procedures. Conclusion Here, we present two protocols to isolate DNA-free RNA from Arabidopsis tissues ready for RT-qPCR applications that significantly improve existing ones by reducing labour time and the use of organic extractions. Accessibility to these protocols is ensured by its simplicity and the low cost of the materials used.

  16. Effects of freezing and cold acclimation on the plasma membrane of isolated protoplasts. Summary progress report, May 16, 1987--June 1, 1991

    Energy Technology Data Exchange (ETDEWEB)

    Steponkus, P.L.

    1991-12-31

    This project focuses on lesions in the plasma membrane of protoplasts that occur during freezing to temperatures below {minus}5{degrees} which result in changes in the semipermeablity of the plasma membrane. This injury, referred to as loss of osmotic responsiveness, is associated with the formation of large, aparticulate domains in the plasma membrane, aparticulate lamellae subtending the plasma membrane, and lamellar-to-hexagonal{sub II} phase transitions in the plasma membrane and subtending lamellar. The goals of this project are to provide a mechanistic understanding of the mechanism by which freeze-induced dehydration effects the formation of aparticulate domains and lamellar-to-hexagonal{sub II} phase transitions and to determine the mechanisms by which cold acclimation and cryoprotectants preclude or diminish these ultrastructural changes. Our working hypothesis is the formation of aparticulate domains and lamellar-to-hexagon{sub II} phase transitions in the plasma membrane and subtending lamellae are manifestations of hydration-dependent bilayer-bilayer interactions.

  17. Formation kinetics and H2O2 distribution in chloroplasts and protoplasts of photosynthetic leaf cells of higher plants under illumination.

    Science.gov (United States)

    Naydov, I A; Mubarakshina, M M; Ivanov, B N

    2012-02-01

    The dye H(2)DCF-DA, which forms the fluorescent molecule DCF in the reaction with hydrogen peroxide, H(2)O(2), was used to study light-induced H(2)O(2) production in isolated intact chloroplasts and in protoplasts of mesophyll cells of Arabidopsis, pea, and maize. A technique to follow the kinetics of light-induced H(2)O(2) production in the photosynthesizing cells using this dye has been developed. Distribution of DCF fluorescence in these cells in the light has been investigated. It was found that for the first minutes of illumination the intensity of DCF fluorescence increases linearly after a small lag both in isolated chloroplasts and in chloroplasts inside protoplast. In protoplasts of Arabidopsis mutant vtc2-2 with disturbed biosynthesis of ascorbate, the rate of increase in DCF fluorescence intensity in chloroplasts was considerably higher than in protoplasts of the wild type plant. Illumination of protoplasts also led to an increase in DCF fluorescence intensity in mitochondria. Intensity of DCF fluorescence in chloroplasts increased much more rapidly than in cytoplasm. The cessation of cytoplasmic movement under illumination lowered the rate of DCF fluorescence intensity increase in chloroplasts and sharply accelerated it in the cytoplasm. It was revealed that in response to switching off the light, the intensity of fluorescence of both DCF and fluorescent dye FDA increases in the cytoplasm in the vicinity of chloroplasts, while it decreases in the chloroplasts; the opposite changes occur in response to switching on the light again. It was established that these phenomena are connected with proton transport from chloroplasts in the light. In the presence of nigericin, which prevents the establishment of transmembrane proton gradients, the level of DCF fluorescence in cytoplasm was higher and increased more rapidly than in the chloroplasts from the very beginning of illumination. These results imply the presence of H(2)O(2) export from chloroplasts to

  18. The Effects of Weak Combined Magnetic Field on Cell Wall Regeneration and Frequency of Plant Protoplasts Fusion

    Science.gov (United States)

    Nedukha, Olena

    The major purpose of these experiments was to investigate plant protoplast fusion frequency and regeneration of a cell wall by protoplasts at weak combined magnetic field (CMF) with the frequency resonance to the cyclotron frequency of Mg2+, Ca2+ and K+ ions. The protoplasts were isolated from Nicotiana lumbaginifolia and N. silvestris leaf mesophyll and from callus tissues (Nicotiana tabacum and Glycine max). The special extra apparatus with ferromagnetic shield was used for estimate of CMF with the frequency resonance to the cyclotron frequency of Mg2+, Ca2+ and K+ ions. The fusion of protoplasts is realized by using of parent protoplasts isolated from one plant species, as well as from various plant species. Control samples were situated near the apparatus with CMF. The laser confocal microscopy was used for study of cell wall regeneration by single and fused protoplasts. The cytochemical methods with DAPI and calcofluor dye were also applied as the detectors for protoplast fusion and regeneration of cell wall. We have been established that CMF with frequency adjusted to the cyclotron frequency Mg2+ ions have shown the most positive influence on regeneration of cell wall by protoplasts. CMF adjusted to the cyclotron frequency of K+ ions very weakly affected on the frequency of protoplast fusion. Largest frequency of protoplasts fusion is noted in the CMF adjusted to the cyclotron frequency of Ca2+ in comparison with the control samples.

  19. Overproduction of stromal ferredoxin:NADPH oxidoreductase in H2O 2-accumulating Brassica napus leaf protoplasts.

    Science.gov (United States)

    Tewari, Rajesh Kumar; Satoh, Mamoru; Kado, Sayaka; Mishina, Kohei; Anma, Misato; Enami, Kazuhiko; Hanaoka, Mitsumasa; Watanabe, Masami

    2014-12-01

    The isolation of Brassica napus leaf protoplasts induces reactive oxygen species generation and accumulation in the chloroplasts. An activated isoform of NADPH oxidase-like protein was detected in the protoplasts and the protoplast chloroplasts. The purpose of this study is to define the NADH oxidase-like activities in the H2O2-accumulating protoplast chloroplasts. Proteomic analysis of this protein revealed an isoform of ferredoxin:NADPH oxidoreductase (FNR1). While leaves highly expressed the LFNR1 transcript, protoplasts decreased the expression significantly. The protoplast chloroplasts predominantly expressed soluble FNR1 proteins. While the albino leaves of white kale (Brassica oleracea var. acephala f. tricolor cv. white pigeon) expressed FNR1 protein at the same level as B. napus leaves, the protoplasts of albino leaves displayed reduced FNR1 expression. The albino leaf protoplasts of white kale generated and accumulated H2O2 in the cytoplasm and on the plasma membrane. Intracellular pH showed that the chloroplasts were acidic, which suggest that excess H(+) was generated in chloroplast stroma. NADPH content of the protoplast chloroplasts increased by over sixfold during the isolation of protoplasts. This study reports a possibility of mediating electrons to oxygen by an overproduced soluble FNR, and suggests that the FNR has a function in utilizing any excess reducing power of NADPH.

  20. Plant regeneration from protoplasts of Gentiana straminea Maxim

    Directory of Open Access Journals (Sweden)

    Shi Guomin

    2016-04-01

    Full Text Available A protocol is described for plant regeneration from protoplasts of Gentiana straminea Maxim. via somatic embryogenesis. Protoplasts were isolated from embryogenic calli in an enzyme solution composed of 2% Cellulase Onozuka R-10, 0.5% Macerozyme R-10, 0.5% Hemicellulase, and 0.5 M sorbitol with a yield of 3.0 × 106 protoplasts per gram of fresh weight. Liquid, solid-liquid double layer (sLD and agar-pool (aPL culture systems were used for protoplast culture. The aPL culture was the only method that produced embryogenic, regenerative calli. With aPL culture, the highest frequencies of protoplast cell division and colony formation were 39.6% and 16.9%, respectively, on MS medium supplemented with 2 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D and 0.5 mg/L N6-benzylaminopurine (BA. Microcalli were transferred to solid MS medium containing a reduced concentration of 2,4-D (0.5 mg/L to promote the formation of embryogenic calli. Somatic embryos developed into plantlets on MS medium supplemented with 2 mg/L BA at a rate of 43.7%.

  1. Plant regeneration from cultured protoplasts of a glutinous rics

    Institute of Scientific and Technical Information of China (English)

    WangGuangyuan; HsiaChenau; 等

    1990-01-01

    Young embryos of ricy (Oryza sativa L.subsp.japonica var.Guo-xiang No.1) were cultured on MS agar medium(2,4-D 2 mg/l).Calli were formed and subcultured on N6 agar medium (2,4-D 2 mg/l).After selection,the small,grainy and pale yellowish cell clusters with dense cytoplasm were used in protoplast preparation. Isolated protoplasts were cultured in N6 medium (2,4-D 1 mg/l,6-BA 0.2 mg/l)1* with agarose block culture method.The protoplasts grew,divided and formed calli.After inducing differentiation,the regenerated mature plants were obtained.

  2. Protoplast culture and plant regeneration of several species in the genus Dianthus.

    Science.gov (United States)

    Nakano, M; Mii, M

    1992-06-01

    Seventeen cultivars belonging to the genus Dianthus were examined for protoplast isolation, culture and shoot regeneration under the same conditions. These included D. caryophyllus, D. chinensis, D. barbatus, D. plumarius, D. superbus and D. japonicus as well as interspecific hybrid cultivars (D. caryophyllus x D. chinensis and D. chinensis x D. barbatus). In all cultivars, viable protoplasts were isolated at high yields from leaves of axenic shoot cultures and some of these protoplasts divided and formed colonies. However, shoot regeneration frequencies were markedly different among the species. High frequency shoot regeneration was obtained from D. chinensis and interspecific hybrid cultivars, while only low frequency or no shoot regeneration was obtained from other species.

  3. Gibberellin perception at the plasma membrane of Avena fatua aleurone protoplasts.

    Science.gov (United States)

    Hooley, R; Beale, M H; Smith, S J

    1991-01-01

    A functional assay for gibberellin (GA) receptors is described based on the induction of α-amylase gene expression in isolated aleurone protoplasts of Avena fatua L. by GA4 immobilised to Sepharose beads. A 17-thiol derivative of GA4, shown to be biologically active with aleurone protoplasts, has been coupled to epoxy-activated Sepharose 6B. This GA4-17-Sepharose induces high levels of α-amylase when incubated with isolated aleurone protoplasts, while cells of the intact aleurone layer do not respond appreciably to the immobilised GA4. In order to eliminate the possibility that GA4 may be released from the Sepharose when incubated with protoplasts, aleurone layers and isolated aleurone protoplasts have been co-incubated, and their responses to GA4, GA4-17-Sepharose and control Sepharose estimated by determining the relative amounts of α-amylase mRNA induced in each tissue. Evidence from these experiments is consistent with the view that GA417-Sepharose induces α-amylase gene expression in aleurone protoplasts by interacting with the protoplast surface. This indicates that GA receptors may be located at, or near, the external face of the aleurone plasma membrane.

  4. Plant regeneration from protoplast of Brazilian citrus cultivars

    Directory of Open Access Journals (Sweden)

    GLORIA FERNANDA JANUZZI MENDES DA

    2000-01-01

    Full Text Available A procedure is described to regenerate plants from protoplasts of Brazilian citrus cultivars, after isolation, fusion and culture. Protoplasts were isolated from embryogenic cell suspension cultures and from leaf mesophyll of seedlings germinated in vitro. The enzyme solution for protoplast isolation was composed of mannitol (0.7 M, CaCl2 (24.5 mM, NaH2PO4 (0.92 mM, MES (6.15 mM, cellulase (Onozuka RS - Yakult, 1%, macerase (Onozuka R10 - Yakult, 1% and pectolyase Y-23 (Seishin, 0.2%. Protoplast culture in liquid medium after chemical fusion lead to the formation of callus colonies further adapted to solid medium. Somatic embryo formation occurred spontaneously after two subcultures, on modified MT medium supplemented with 500 mg/L of malt extract. Well defined embryos were germinated in modified MT medium with addition of GA3 (2.0 muM and malt extract (500 mg/L. Plant regeneration was also achieved by adventitious shoots obtained through direct organogenesis of not well defined embryos in modified MT medium with addition of malt extract (500 mg/L, BAP (1.32 muM, NAA (1.07 muM and coconut water (10 mL/L. Plantlets were transferred to root medium. Rooted plants were transferred to a greenhouse for further adaptation and development.

  5. Chloroplastic NADPH oxidase-like activity-mediated perpetual hydrogen peroxide generation in the chloroplast induces apoptotic-like death of Brassica napus leaf protoplasts.

    Science.gov (United States)

    Tewari, Rajesh Kumar; Watanabe, Daisuke; Watanabe, Masami

    2012-01-01

    Despite extensive research over the past years, regeneration from protoplasts has been observed in only a limited number of plant species. Protoplasts undergo complex metabolic modification during their isolation. The isolation of protoplasts induces reactive oxygen species (ROS) generation in Brassica napus leaf protoplasts. The present study was conducted to provide new insight into the mechanism of ROS generation in B. napus leaf protoplasts. In vivo localization of H(2)O(2) and enzymes involved in H(2)O(2) generation and detoxification, molecular antioxidant-ascorbate and its redox state and lipid peroxidation were investigated in the leaf and isolated protoplasts. Incubating leaf strips in the macerating enzyme (ME) for different duration (3, 6, and 12 h) induced accumulation of H(2)O(2) and malondialdehyde (lipid peroxidation, an index of membrane damage) in protoplasts. The level of H(2)O(2) was highest just after protoplast isolation and subsequently decreased during culture. Superoxide generating NADPH oxidase (NOX)-like activity was enhanced, whereas superoxide dismutase (SOD) and ascorbate peroxidase (APX) decreased in the protoplasts compared to leaves. Diaminobenzidine peroxidase (DAB-POD) activity was also lower in the protoplasts compared to leaves. Total ascorbate content, ascorbate to dehydroascorbate ratio (redox state), were enhanced in the protoplasts compared to leaves. Higher activity of NOX-like enzyme and weakening in the activity of antioxidant enzymes (SOD, APX, and DAB-POD) in protoplasts resulted in excessive accumulation of H(2)O(2) in chloroplasts of protoplasts. Chloroplastic NADPH oxidase-like activity mediated perpetual H(2)O(2) generation probably induced apoptotic-like cell death of B. napus leaf protoplasts as indicated by parallel DNA laddering and decreased mitochondrial membrane potential.

  6. Progress towards sugar beet improvement through somatic hybridization I. Inactivation of nuclei and cytoplasm in donor and recipient protoplasts

    Directory of Open Access Journals (Sweden)

    Elżbieta Jażdżewska

    2014-02-01

    Full Text Available The isolation and culture of suspension-derived protoplasts from two sugar beet (Beta vulgaris L. genotypes are described. Immobilization of protoplasts in agarose resulted in high frequency divisions and microcallus regeneration, with plating efficiency (PE being clearly genotype-dependent. In further studies towards asymmetric fusion experiments, the effect of different doses of ultraviolet radiation (UV and iodoacetic acid (IA on protoplast physiology was assessed. Viability of both treated (UV, IA and untreated protoplasts (control was determined by FDA staining, and the biological effect was evaluated by testing the ability of protoplasts to divide and to form calli. The results are discussed in terms of the applicability of the methods for the production of asymmetric protoplasts suitable for somatic hybridization within the genus Beta.

  7. [La(3+)-induced fusion of plant protoplasts].

    Science.gov (United States)

    Sheremet'ev, Iu A; Smirnova, D V; Sheremet'eva, A V

    2009-01-01

    The effect of La(3+) on the fusion of plant protoplasts has been studied. It was shown that La(3+) induced the aggregation of plant protoplasts. The incubation of a suspension of aggregated protoplasts at 42 degrees C for 30 min resulted in their fusion.

  8. Isolation of Promoters and Fragments of Genes Controlling Endosperm Development Without Fertilization in Arabidopsis and Engineering of the Antisense Constructions

    Directory of Open Access Journals (Sweden)

    Grigory A. Gerashchenkov

    2015-06-01

    Full Text Available Apomixis is asexual seed reproduction without both meiosis and fertilization based on the complex developmental processes such as apomeiosis, parthenogenesis and specific endosperm development. This investigation is aimed at engineering of apomixis in Arabidopsis thaliana with sexual seed reproduction. The fragments of known genes of endosperm formation MEA, FIE, FIS2 and gene of apomeiosis DYAD (as control were isolated using Q5 high fidelity DNA polymerase. These gene fragments of interest at the antisense orientation were fused with isolated constitutive and meiosis specific promoters of Arabidopsis at NcoI sites. The fused promoter-gene fragment modules were cloned in pCambia1301 at SalI cites. The engineered constructions will be used for the floral dip transformation of Arabidopsis and down regulation of these genes at engineering of apomixis.

  9. Organogenesis of stem and leaf protoplasts of a haploid golden delicious apple clone (Malus Xdomestica Borkh.).

    Science.gov (United States)

    Patat-Ochatt, E M; Boccon-Gibod, J; Duron, M; Ochatt, S J

    1993-01-01

    Highly viable protoplasts were isolated in large numbers from in vitro-grown leaf and stem tissues of a haploid clone of the apple scion cultivar Golden Delicious (Malus Xdomestica Borkh.). Protoplasts from both sources divided rapidly to give microcallus, when cultured in a modified Kao and Michayluk-based medium. Following two successive subcultures for callusing, shoot buds were regenerated from such calli, on half-strength Murashige and Skoog medium with an increased concentration of group B vitamins and containing 5.0 mg.l(-1) 6-benzyl-aminopurine and 0.1 mg.l(-1) l-naphthaleneacetic acid (for the leaf protoplast-derived calli) or 4-indole-3yl-butyric acid (for stem protoplast-derived calli). The mesophyll protoplast-derived shoots were enfeebled and vitrified, in time with their ultimate death. Conversely, for those shoots deriving from the stem protoplasts, in vitro propagation was successfully achieved. This is the first report on the successful isolation, culture and organogenesis from stem protoplasts of a woody plant genotype.

  10. Genetic variability in regenerated Metarhizium flavoviride protoplasts

    Directory of Open Access Journals (Sweden)

    Júlia Kuklinsky-Sobral

    2004-03-01

    Full Text Available Protoplast isolation and regeneration were evaluated in two wild-type and two colour mutant strains of Metarhizium flavoviride. Cultivation in liquid medium, followed by mycelium treatment with Novozym 234 in the presence of KCl 0.7M as osmotic stabilizer, produced 5.05 x 10(6 to 1.15 x 10(7x mL-1 protoplasts. The percentage of regeneration ranged from 6.65 to 27.92%. Following protoplast regeneration, one strain produced spontaneously stable morphological variant colonies. Although colonies with altered morphology have been reported in bacteria following protoplast regeneration, this is the first time that the same is described in a filamentous fungus. The original strain and one derived variant were tested for sensitivity to the fungicides benomyl and captan.A formação e regeneração de protoplastos foram avaliadas em duas linhagens selvagens e duas linhagens mutantes para coloração de conídios em Metarhizium flavoviride. O cultivo em meio líquido seguido do tratamento do micélio com Novozym 234 na presença de KCl 0,7 M como estabilizador osmótico, resultou na produção de 5,05´10(6 a 1,15´10(7 protoplastos´mL-1. A porcentagem de regeneração das diferentes linhagens variou de 6,65 a 27,92%. Após a regeneração, uma das linhagens selvagens produziu espontaneamente variantes estáveis, com morfologia alterada. Embora variantes morfológicos já tenham sido observados após regeneração de protoplastos em bactérias, esta parece ser a primeira vez que tal ocorrência é descrita em fungos filamentosos. Um desses variantes, além da linhagem selvagem da qual ele foi originado, foi testado para sensibilidade aos fungicidas benomil e captano.

  11. Inhibition of lipoxygenase activity in lentil protoplasts by monoclonal antibodies introduced into the cells via electroporation

    OpenAIRE

    J. F. G. Vliegenthart; Maccarrone, M.; Veldink, G.A.

    1992-01-01

    The isolation of lentil protoplasts and the transfer of anti-lipoxygenase monoclonal antibodies into plant protoplasts by electroporation is reported. The dependence of the efficiency of monoclonal antibody incorporation on the field strength is shown as well. The transferred immunoglobulins retained their functional and structural integrity and were able to inhibit the intracellular target enzyme, with a linear relationship between inhibition of lipoxygenase activity and amount of incorporat...

  12. Plants regenerated from mesophyll protoplasts of white mulberry

    Institute of Scientific and Technical Information of China (English)

    WEIZHIMING; ZHIHONGXU; 等

    1994-01-01

    Morus alba(white mulberry) mesophyll protoplasts were isolated from leaves of 30-45 day old sterile shoots,with protoplast yields of 2.5×107 g-1/F.W.after purification.The protoplasts were cultured in a modified K8P liquid medium containing 0.2mg/L 2,4-(2,4-Dichlorophenoxy acetic acid),1mg/L NAA(Naphthyl acetic acid) and 0.5mg/L BA(6-benzylaminopurine).A low plating density(5×104/ml) proved to be favourable to the division of protoplast-derived cells.The first division occurred 4 days after culture,and the division frequency reached 24% at 10 days.A number of cell colonies and microcalli formed in 6 weeks.The microcalli were transferred onto MSB medium with 0.5mg/L NAA and 0.5mg/L BA for further proliferation.Shoot formation was initiated when the calli of 3-4 mm in size were transferred onto MSB differentiation medium with 0.1mg/L NAA and 1mg/L BA.The frequency of shoot formation was 35%.The shoots of 4-5 cm in height were excised from the callus and rooted on half strength MS medium with 0.5 mg/L IBA and 0.1 mg/L BA.After transplantation into pots,the regenerated plants grew vigorously in the phytotron.

  13. Allelopathy in a leguminous mangrove plant, Derris indica: protoplast co-culture bioassay and rotenone effect.

    Science.gov (United States)

    Inoue, Aya; Mori, Daisuke; Minagawa, Reiko; Fujii, Yoshiharu; Sasamoto, Hamako

    2015-05-01

    To investigate allelopathic activity of a leguminous mangrove plant, Derris indica, the 'Protoplasts Co-culture Method' for bioassay of allelopathy was developed using suspension culture. A suspension culture was induced from immature seed and sub-cultured in Murashige and Skoog's (MS) basal medium containing 10 μM each of 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-benzyladenine (BA). The protoplasts were isolated using the separate wells method with 2% each of Cellulase RS, Driselase 20 and Macerozyme R10 in 0.4 M mannitol solution. Protoplast cultures of D. indica revealed that high concentrations of cytokinins, BA and thidiazuron, were effective for cell divisions. The co-cultures of D. indica protoplasts with recipient lettuce protoplasts using 96 multi-well culture plates were performed in MS basal medium containing 0.4 M mannitol solution and 1 μM 2,4-D and 0.1 μM BA. The protoplast density of D. indica used in co-culturing varied from 6 x 10(3) - 10(5) / mL. Very strong inhibitory allelopathic effects of D. indica protoplasts on lettuce protoplast growth were found. A similar strong inhibitory allelopathic activity of dried young leaves on lettuce seedling growth was also observed by using the sandwich method. Rotenone, which is a component of Derris root, dissolved in DMSO, was highly inhibitory on the growth of lettuce protoplasts in culture and this could be one of the causes of the strong allelopathic activity of D. indica.

  14. Electrofusion of tobacco protoplasts in space

    Institute of Scientific and Technical Information of China (English)

    ZHENG Huiqiong; WANG Liufa; CHENG Aidi; LIU Chengxian

    2003-01-01

    Electrofusion of evacuolated (N. Tabacum L. cvs. Gexin no1) and vacuolated (N. Rustic) tobacco mesophyll protoplasts was performed on the Chinese spacecraft (Shenzhou No. 4, from 30th Dec. 2002 to 6th Jan. 2003). The results showed that the frequency of bi-nucleated and multinucleated protoplasts was significantly increased under microgravity. Compared with the control samples incubated on the ground, the viability of protoplasts incubated in space was much higher. In addition, the influence of altered gravity on carbohydrates was also observed. These results confirmed the effect of microgravitation on electrofusion of plant cell protoplasts.

  15. Beneficial Bacteria Isolated from Grapevine Inner Tissues Shape Arabidopsis thaliana Roots.

    Directory of Open Access Journals (Sweden)

    Enrico Baldan

    Full Text Available We investigated the potential plant growth-promoting traits of 377 culturable endophytic bacteria, isolated from Vitis vinifera cv. Glera, as good biofertilizer candidates in vineyard management. Endophyte ability in promoting plant growth was assessed in vitro by testing ammonia production, phosphate solubilization, indole-3-acetic acid (IAA and IAA-like molecule biosynthesis, siderophore and lytic enzyme secretion. Many of the isolates were able to mobilize phosphate (33%, release ammonium (39%, secrete siderophores (38% and a limited part of them synthetized IAA and IAA-like molecules (5%. Effects of each of the 377 grapevine beneficial bacteria on Arabidopsis thaliana root development were also analyzed to discern plant growth-promoting abilities (PGP of the different strains, that often exhibit more than one PGP trait. A supervised model-based clustering analysis highlighted six different classes of PGP effects on root architecture. A. thaliana DR5::GUS plantlets, inoculated with IAA-producing endophytes, resulted in altered root growth and enhanced auxin response. Overall, the results indicate that the Glera PGP endospheric culturable microbiome could contribute, by structural root changes, to obtain water and nutrients increasing plant adaptation and survival. From the complete cultivable collection, twelve promising endophytes mainly belonging to the Bacillus but also to Micrococcus and Pantoea genera, were selected for further investigations in the grapevine host plants towards future application in sustainable management of vineyards.

  16. Beneficial Bacteria Isolated from Grapevine Inner Tissues Shape Arabidopsis thaliana Roots.

    Science.gov (United States)

    Baldan, Enrico; Nigris, Sebastiano; Romualdi, Chiara; D'Alessandro, Stefano; Clocchiatti, Anna; Zottini, Michela; Stevanato, Piergiorgio; Squartini, Andrea; Baldan, Barbara

    2015-01-01

    We investigated the potential plant growth-promoting traits of 377 culturable endophytic bacteria, isolated from Vitis vinifera cv. Glera, as good biofertilizer candidates in vineyard management. Endophyte ability in promoting plant growth was assessed in vitro by testing ammonia production, phosphate solubilization, indole-3-acetic acid (IAA) and IAA-like molecule biosynthesis, siderophore and lytic enzyme secretion. Many of the isolates were able to mobilize phosphate (33%), release ammonium (39%), secrete siderophores (38%) and a limited part of them synthetized IAA and IAA-like molecules (5%). Effects of each of the 377 grapevine beneficial bacteria on Arabidopsis thaliana root development were also analyzed to discern plant growth-promoting abilities (PGP) of the different strains, that often exhibit more than one PGP trait. A supervised model-based clustering analysis highlighted six different classes of PGP effects on root architecture. A. thaliana DR5::GUS plantlets, inoculated with IAA-producing endophytes, resulted in altered root growth and enhanced auxin response. Overall, the results indicate that the Glera PGP endospheric culturable microbiome could contribute, by structural root changes, to obtain water and nutrients increasing plant adaptation and survival. From the complete cultivable collection, twelve promising endophytes mainly belonging to the Bacillus but also to Micrococcus and Pantoea genera, were selected for further investigations in the grapevine host plants towards future application in sustainable management of vineyards.

  17. 热研4号王草原生质体的制备%A Effective Protocol for King Grass Reyan No.4 Protoplasts Preparation of King Grass Reyan No.4

    Institute of Scientific and Technical Information of China (English)

    张继友; 景晓辉; 吴伦英; 刘国道; 吴琳

    2015-01-01

    The prerequisites and foundations of the system for efficient plant regeneration from protoplast via somatic hybridization and transient expression in protoplast for molecular biology research is to build efficient and effective protoplast preparation system. Establishment of efficient isolation protoplasts from King Grass Reyan No.4 have failed thus far. In this paper, enzymatic method was used to isolate high-quality protoplasts from Pennisetumpur pureumíP. americanum cv. Reyan No.4. High-quality protoplast with a viability of 75 % were achieved by incubating the slimmed young leaves in a digestion enzymolic solution comprising 2%cellulase R-10+0.5%pectolyase Y-23+0.5%macerozyme R-10+0.6 mol/L Mannitol+40 mmol/L KCl+6 mmol/L MES, pH 5.7+20 mmol/L CaCl2+0.15% BSA. Arabidopsis gene A TA F1 was further transfected into King Grass Reyan No.4 protoplasts to testify the efficiency of our system. SDS-PAGE electrophoresis and subsequent Western blot detection demonstrate that ATAF1 protein was accumulated in the transformed protoplasts. Together, these results showed that an efficient protoplast isolation system from young leaves was established for King Grass Reyan No.4.%开展原生质体融合育种以及利用原生质体瞬时表达系统进行分子生物学实验的前提和基础是建立高效、有活力的原生质体制备体系。为建立热研4号王草原生质体制备体系,本研究以热研4号王草叶片为原生质体制备的外植体,将切成细丝的热研4号王草幼叶置于含2.0%纤维素酶+0.5%果胶酶Y-23+0.5%崩溃酶+0.6 mol/L甘露醇+40 mmol/L KCl+6 mmol/L MES,pH 5.7+20 mmol/L CaCl2+0.15%BSA的酶液中,在避光条件下置于50 r/min的摇床酶解6~8 h,获得了大量有活力且均匀一致的原生质体。经过荧光素双醋酸酯(fluoresceindiacetate, FDA)染色和Western blot检测目的蛋白表达,结果发现采用酶解法制备的热研4号王草原生质体活力可达75%,且可用于表达拟南芥

  18. Isolation of Persicaria minor sesquiterpene synthase promoter and its deletions for transgenic Arabidopsis thaliana

    Science.gov (United States)

    Omar, Aimi Farehah; Ismail, Ismanizan

    2016-11-01

    Sesquiterpene synthase (SS) catalyzes the formation of sesquiterpenes from farnesyl diphosphate (FDP) via carbocation intermediates. In this study, the promoter region of sesquiterpene synthase was isolated from Persicaria minor to identify possible cis-acting elements in the promoter. The full-length PmSS promoter of P. minor is 1824-bp sequences. The sequence was analyzed and several putative cis-acting regulatory elements were identified. Three cis-acting regulatory elements were selected for deletion analysis which are cis-acting element involved in wound responsiveness (WUN), cis - acting element involved in defense and stress responsiveness (TC) and cis-acting element involved in ABA responsiveness (ABRE). Series of deletions were conducted to assess the promoter activity producing three truncated fragments promoter; Prom 2 1606-bp, Prom 3 1144- bp, and Prom 4 921-bp. The full-length promoter and its deletion series were cloned into the pBGWFS7 vector which contain β-glucuronidase (GUS) gene and green fluorescent protein (GFP) as the reporter gene. All constructs were successfully transformed into Arabidopsis thaliana based on PCR of positive BASTA resistance plants.

  19. Influence of the Nutrient Medium on the Recovery of Dividing Cells from Tobacco Protoplasts 12

    Science.gov (United States)

    Uchimiya, Hirofumi; Murashige, Toshio

    1976-01-01

    Systematic tests resulted in a nutrient solution containing the following, in milligrams per liter, for the culture of protoplasts isolated from Nicotiana tabacum L. callus cells: Murashige and Skoog salts (T. Murashige and F. Skoog, 1962. Physiol. Plant. 15: 473-497); sucrose, 15,000; mannitol, 110,000; α-naphthaleneacetic acid, 0.6; kinetin, 0-0.1; thiamine·HCl, 10; pyridoxine·HCl, 10; nicotinic acid, 5; myo-inositol, 100; and glycine, 2. In this medium, regeneration of cell wall has been observed in 85% and resumption of cell division among 35% of the protoplast isolates. PMID:16659496

  20. Isolamento e regeneração de protoplastos de Penicillium brevicompactum - DOI: 10.4025/actascibiolsci.v26i4.1529 Isolation and regeneration of Penicillium brevicompactum protoplasts - DOI: 10.4025/actascibiolsci.v26i4.1529

    Directory of Open Access Journals (Sweden)

    Jorge Fernando Pereira

    2004-04-01

    Full Text Available O isolamento e regeneração de protoplastos de fungos é um passo fundamental para o estabelecimento de sistemas de transformação, análise do cariótipo molecular e fusão entre linhagens, que são técnicas de ampla aplicação em programas de melhoramento para fungos filamentosos. Neste trabalho foram testados diferentes preparações enzimáticas e estabilizadores osmóticos para estabelecer condições otimizadas de isolamento e regeneração de protoplastos de Penicillium brevicompactum, que é um excelente produtor de pectinases. Protoplastos de P. brevicompactum foram obtidos em maior quantidade quando o micélio foi digerido com 15mg.mL-1 de Glucanex (Novo Nordisk em NaCl 0,8 mol.L-1 como estabilizador osmótico. O melhor estabilizador osmótico para a regeneração dos protoplastos foi KCl 0.8 mol.L–1 apresentando uma freqüência de regeneração de 36,58%. Esse protocolo pode ser utilizado em análises genéticas para essa espécie de Penicillium cujos estudos têm sido pouco reportadosThe isolation and regeneration of fungal protoplasts is a key step for the establishment of transformation systems, electrophoretic karyotype analysis and fusion between strains, all techniques of broad application on improvement programs of filamentous fungi. To establish conditions for the isolation and regeneration of ,em>Penicillium brevicompactum protoplasts, that is an excellent pectinase producer, different lytic enzymes and osmotic stabilizers were tested. P. brevicompactum protoplasts were obtained at a larger scale when their mycelium was digested with 15mg.mL-1 of Glucanex (Novo Nordisk and 0.8 mol.L–1 NaCl as osmotic stabilizer. The best osmotic stabilizer for the regeneration of P. brevicompactum protoplasts was 0.8 mol.L–1 KCl, with a regeneration frequency of 36.58%. This protocol can be applied in genetic analysis of this Penicillium species which, to date has been poorly characterized

  1. In vitro effect of biogenic silver nanoparticles on sterilisation of tobacco leaf explants and for higher yield of protoplasts.

    Science.gov (United States)

    Bansod, Sunita; Bawskar, Manisha; Rai, Mahendra

    2015-08-01

    Isolation of protoplasts from leaves is useful in plant research. The standard reference methods for isolation of protoplasts are tedious, cause cell damage, are low-yield, time consuming and prone to microbial contamination. To overcome this problem, the authors used silver nanoparticles (AgNPs) for the control of microbial contamination and with low concentration of enzyme mixture for rapid release of protoplasts. The leaf explants were sterilised with 95% ethanol for 30 s followed by biologically synthesised AgNPs (1, 5, 10 and 15 mg/l) for 10 to 20 min. The authors found that 10 mg/l concentration of AgNPs treatment on explants showed remarkable inhibitory effect on microbial contamination with high level of tolerance. Moreover, during protoplasts isolation, the addition of 10 mg/l AgNPs in leaf incubation buffer yielded 34% viable protoplasts in 3 h. This is the first report of AgNPs synthesis from waste plant medium, which was applied for the sterilisation of explants and rapid isolation of protoplasts.

  2. Genetic transformation of plants by protoplast electroporation.

    Science.gov (United States)

    Bates, G W

    1994-10-01

    This article describes an optimized protocol for the electroporation of tobacco mesophyll protoplasts together with notes and data on the effects of various parameters and suggestions for work with protoplasts of other species. In this protocol, electroporation is achieved by means of electrical pulses from a high-voltage, capacitive-discharge unit. Procedures are described for measurement of protoplast viability with Evan's blue, the detection of transient expression of CAT and GUS gene plasmid constructs, and for the recovery of stable transformants based on selection for kanamycin resistance.

  3. Isolation and characterization of Arabidopsis halleri and Thlaspi caerulescens phytochelatin synthases.

    Science.gov (United States)

    Meyer, Claire-Lise; Peisker, Daniel; Courbot, Mikael; Craciun, Adrian Radu; Cazalé, Anne-Claire; Desgain, Denis; Schat, Henk; Clemens, Stephan; Verbruggen, Nathalie

    2011-07-01

    The synthesis of phytochelatins (PC) represents a major metal and metalloid detoxification mechanism in various species. PC most likely play a role in the distribution and accumulation of Cd and possibly other metals. However, to date, no studies have investigated the phytochelatin synthase (PCS) genes and their expression in the Cd-hyperaccumulating species. We used functional screens in two yeast species to identify genes expressed by two Cd hyperaccumulators (Arabidopsis halleri and Thlaspi caerulescens) and involved in cellular Cd tolerance. As a result of these screens, PCS genes were identified for both species. PCS1 was in each case the dominating cDNA isolated. The deduced sequences of AhPCS1 and TcPCS1 are very similar to AtPCS1 and their identity is particularly high in the proposed catalytic N-terminal domain. We also identified in A. halleri and T. caerulescens orthologues of AtPCS2 that encode functional PCS. As compared to A. halleri and A. thaliana, T. caerulescens showed the lowest PCS expression. Furthermore, concentrations of PC in Cd-treated roots were the highest in A. thaliana, intermediate in A. halleri and the lowest in T. caerulescens. This mirrors the known capacity of these species to translocate Cd to the shoot, with T. caerulescens being the best translocator. Very low or undetectable concentrations of PC were measured in A. halleri and T. caerulescens shoots, contrary to A. thaliana. These results suggest that extremely efficient alternative Cd sequestration pathways in leaves of Cd hyperaccumulators prevent activation of PC synthase by Cd²⁺ ions.

  4. Reference: 517 [Arabidopsis Phenome Database[Archive

    Lifescience Database Archive (English)

    Full Text Available d isolated aleurone layers of Arabidopsis (Arabidopsis thaliana) were used in experiments designed to iden...tify components of the Arabidopsis seed that contribute to seed dormancy and to lea

  5. Structural and functional organisation of regenerated plant protoplasts exposed to microgravity on Biokosmos 9

    Science.gov (United States)

    Klimchuk, D. A.; Kordyum, E. L.; Danevich, L. A.; Tarnavskaya, E. B.; Tairbekov, M. G.; Iversen, T.-H.; Baggerud, C.; Rasmussen, O.

    Preparatory experiments for the IML-1 mission using plant protoplasts, were flown on a 14-day flight on Biokosmos 9 in September 1989. Thirty-six hours before launch of the biosatellite, protoplasts were isolated from hypocotyl cells of rapeseed (Brassica napus) and suspension cultures of carrot (Daucus carota). Ultrastructural and fluorescence analysis of cell aggregates from these protoplasts, cultured under microgravity conditions, have been performed. In the flight samples as well as in the ground controls, a portion of the total number of protoplasts regenerated cell walls. The processes of cell differentiation and proliferation under micro-g did not differ significantly from those under normal gravity conditions. However, in micro-g differences were observed in the ultrastructure of some organelles such as plastids and mitochondria. There was also an increase in the frequency of the occurrence of folds formed by the plasmalemma together with an increase in the degree of complexity of these folds. In cell cultures developed under micro-g conditions, the calcium content tends to decrease, compared to the ground control. Different aspects of using isolated protoplasts for clarifying the mechanisms of biological effects of microgravity are discussed.

  6. Engineering an enhanced, thermostable, monomeric bacterial luciferase gene as a reporter in plant protoplasts.

    Science.gov (United States)

    Cui, Boyu; Zhang, Lifeng; Song, Yunhong; Wei, Jinsong; Li, Changfu; Wang, Tietao; Wang, Yao; Zhao, Tianyong; Shen, Xihui

    2014-01-01

    The application of the luxCDABE operon of the bioluminescent bacterium Photorhabdus luminescens as a reporter has been published for bacteria, yeast and mammalian cells. We report here the optimization of fused luxAB (the bacterial luciferase heterodimeric enzyme) expression, quantum yield and its application as a reporter gene in plant protoplasts. The fused luxAB gene was mutated by error prone PCR or chemical mutagenesis and screened for enhanced luciferase activity utilizing decanal as substrate. Positive luxAB mutants with superior quantum yield were subsequently shuffled by DNase I digestion and PCR assembly for generation of recombinants with additional increases in luciferase activity in bacteria. The coding sequence of the best recombinant, called eluxAB, was then optimized further to conform to Arabidopsis (Arabidopsis thaliana) codon usage. A plant expression vector of the final, optimized eluxAB gene (opt-eluxAB) was constructed and transformed into protoplasts of Arabidopsis and maize (Zea mays). Luciferase activity was dramatically increased for opt-eluxAB compared to the original luxAB in Arabidopsis and maize cells. The opt-eluxAB driven by two copies of the 35S promoter expresses significantly higher than that driven by a single copy. These results indicate that the eluxAB gene can be used as a reporter in plant protoplasts. To our knowledge, this is the first report to engineer the bacterium Photorhabdus luminescens luciferase luxAB as a reporter by directed evolution which paved the way for further improving the luxAB reporter in the future.

  7. 提高二倍体马铃薯原始栽培种原生质体分离与分裂频率的研究%Studies on improving isolation and division frequency of diploid original cultivar potato protoplast

    Institute of Scientific and Technical Information of China (English)

    陈鹏; 刘卫卫; 魏彩霞; 王清

    2014-01-01

    为获得较高的原生质体分离和分裂频率,试验以马铃薯二倍体原始栽培种‘47-33’‘5-19’试管苗叶片为材料,进行了原生质体游离和培养的研究.结果表明:培养21 d 的两品系试管苗叶片均在含有2.0%纤维素酶+0.5%果胶酶+0.25%离析酶,渗透压为0.35 mol/L 的酶解液中解离效果最好,原生质体产量分别为2.31×106个/gFW和2.52×106个/gFW;在28℃酶解温度条件下缓慢摇动14 h或1 h静置+13 h缓慢摇动的,可促进叶片原生质体的大量释放.此外,1.0 mg/L NAA+0.5 mg/L 2,4-D+0.4 mg/L BAP外源激素组合有利于叶片原生质体的分裂,‘47-33’‘5-19’原生质体一次分裂频率分别达到8.85%和12.56%.在0.35 mol/L渗透压的液体培养基中,‘47-33’叶片原生质体分裂更早,分裂频率达13.85%.研究获得了稳定的原生质体分离体系以及较好的原生质体培养条件,为马铃薯二倍体原始栽培种的体细胞融合奠定了基础.%The potato leaves from 2 kinds of diploid original cultivar ‘47-33’and ‘5-19’were used to study protoplast isolation and division.The optimum combination of enzymes to separate protoplast from 21 d cultured leaves was 2.0% cellulase+0.5% pectinase+0.25% macerozyme.The optimum osmotic pressure was 0.35 mol/L.Under the condition of the above,the protoplast yields of ‘47-33’and ‘5-19’ were 2.52×106/gFW and 2.31 ×106/gFW respectively.The release of leaf protoplasts was significantly promoted by shaking samples for 13 h after 1 h stand or by shaking samples continuously for 14 h,both at 28 ℃ condition.In addition,exogenous hormone combination of 1.0 mg/L NAA+0.5 mg/L 2,4-D+0.4 mg/L BAP obviously enhanced the division of protoplast.The first division frequencies of protoplast from‘47-33’and ‘5-19’were up to 12.56% and 8.85% respectively.Furthermore,the division of protoplast of‘47-33’was earlier in the liquid medium with 0.35 mol/L osmotic pressure than solid or solid and

  8. Conjugated polymer nanoparticles for effective siRNA delivery to tobacco BY-2 protoplasts

    Directory of Open Access Journals (Sweden)

    Verchot Jeanmarie

    2010-12-01

    Full Text Available Abstract Background Post transcriptional gene silencing (PTGS is a mechanism harnessed by plant biologists to knock down gene expression. siRNAs contribute to PTGS that are synthesized from mRNAs or viral RNAs and function to guide cellular endoribonucleases to target mRNAs for degradation. Plant biologists have employed electroporation to deliver artificial siRNAs to plant protoplasts to study gene expression mechanisms at the single cell level. One drawback of electroporation is the extensive loss of viable protoplasts that occurs as a result of the transfection technology. Results We employed fluorescent conjugated polymer nanoparticles (CPNs to deliver siRNAs and knockdown a target gene in plant protoplasts. CPNs are non toxic to protoplasts, having little impact on viability over a 72 h period. Microscopy and flow cytometry reveal that CPNs can penetrate protoplasts within 2 h of delivery. Cellular uptake of CPNs/siRNA complexes were easily monitored using epifluorescence microscopy. We also demonstrate that CPNs can deliver siRNAs targeting specific genes in the cellulose biosynthesis pathway (NtCesA-1a and NtCesA-1b. Conclusions While prior work showed that NtCesA-1 is a factor involved in cell wall synthesis in whole plants, we demonstrate that the same gene plays an essential role in cell wall regeneration in isolated protoplasts. Cell wall biosynthesis is central to cell elongation, plant growth and development. The experiments presented here shows that NtCesA is also a factor in cell viability. We show that CPNs are valuable vehicles for delivering siRNAs to plant protoplasts to study vital cellular pathways at the single cell level.

  9. Citrus asymmetric somatic hybrids produced via fusion of gamma-irradiated and iodoacetamide-treated protoplasts

    Energy Technology Data Exchange (ETDEWEB)

    Bona, Claudine Maria de [Instituto Agronomico do Parana (IAPAR), Curitiba, PR (Brazil)], e-mail: debona@iapar.br; Gould, Jean Howe [Texas A and M University, College Station, TX (United States). Dept. of Ecosystem Science and Management], e-mail: gould@tamu.edu; Miller Junior, J. Creighton [Texas A and M University, College Station, TX (United States). Dept. of Horticultural Sciences], e-mail: jcmillerjr@tamu.edu; Stelly, David [Texas A and M University, College Station, TX (United States). Dept. of Soil and Crop Sciences], e-mail: stelly@tamu.edu; Louzada, Eliezer Silva [Texas A and M University, Kingsville, TX (United States). Citrus Center], e-mail: e-louzada@tamu.edu

    2009-05-15

    The objective of this study was to produce citrus somatic asymmetric hybrids by fusing gamma.irradiated protoplasts with iodoacetamide-treated protoplasts. Protoplasts were isolated from embryogenic suspension cells of grapefruit (Citrus paradisi Macfad.) cultivars Ruby Red and Flame, sweet oranges (C. sinensis Osbeck) 'Itaborai', 'Natal', Valencia', and 'Succari', from 'Satsuma' (C. unshiu Marcow.) and 'Changsha' mandarin (C. reticulata Blanco) and 'Murcott' tangor (C. reticulata x C. sinensis). Donor protoplasts were exposed to gamma rays and receptor protoplasts were treated with 3 mmol L{sup -1} iodoacetamide (IOA), and then they were fused for asymmetric hybridization. Asymmetric embryos were germinated, and the resulting shoots were either grafted onto sour orange, rough lemon or 'Swingle' (C. paradisi x Poncirus trifoliata) x 'Sunki' mandarin rootstock seedlings, or rooted after dipping their bases in indol.butyric acid (IBA) solution. The products were later acclimatized to greenhouse conditions. Ploidy was analyzed by flow cytometry, and hybridity was confirmed by amplified fragment length polymorphism (AFLP) analysis of plantlet DNA samples. The best treatment was the donor-recipient fusion combination of 80 Gy.irradiated 'Ruby Red' protoplasts with 20 min IOA.treated 'Succari' protoplasts. Tetraploid and aneuploid plants were produced. Rooting recalcitrance was solved by dipping shoots' stems in 3,000 mg L{sup -1} IBA solution for 10 min. (author)

  10. Characterization of the pH-induced fusion of liposomes with the plasma membrane of rye protoplasts

    Energy Technology Data Exchange (ETDEWEB)

    Arvinte , T.; Steponkus, P.L.

    1988-07-26

    The authors present evidence that at acidic pH, liposomes composed of soybean lipids fuse with the plasma membrane of protoplasts isolated from rye leaves. Using the resonance energy transfer assay (RET), they determined the rate and extent of liposome and protoplast plasma membrane lipid mixing. The fluorescent donor-acceptor pair was N-(7-nitro-2,1,3-benzoxadiazol-4-yl)phosphatidyl-ethanolamine (N-NBD-PE) and N-(lissamine rhodamine B sulfonyl)phosphatidylethanolamine (N-Rh-PE). Fusion was substantial below pH 5, and the half-time of lipid mixing was fast (t/sub 1/2/ on the order of minutes) and pH, concentration, and temperature dependent. The extent of liposome and protoplast fusion from the total amount of liposomes associated with the protoplasts was also determined by the RET assay. Protoplasts were incubated with fluorescent-labeled liposomes (5 min at 30/sup 0/C) at different pH values and then washed twice by centrifugation. The fluorescence spectra of the protoplast suspension permitted determination of the ratio of N-NBD-PE emission at 530 nm to the N-Rh-PE emission at 590 nm, which is a measure of the degree of lipid mixing. The amount of liposomes associated (fused and unfused) with protoplasts at pH 3.9 was approximately 9 times greater than that at pH 5.6. The transfer of liposome contents to the protoplast interior was studied with a method based on the fluorescence enhancement of a solution of calcein, initially confined in the liposomes at self-quenching concentrations. The kinetics of calcein release were very similar to those of lipid mixing. Fluorescence microscopy showed that after fusion with liposomes containing calcein, the protoplasts exhibited a strong diffuse fluorescence in the interior.

  11. Regeneration of transgenic rice plants from protoplasts following plasmid uptake

    Institute of Scientific and Technical Information of China (English)

    LiWenbin; SUNYongru

    1994-01-01

    Embryogenic cell suspension was obtained from the calli developed from mature seeds of riceRoncarolo (Oryza sativa L., a japonica cultivar from Italy ). The protoplasts were isolated from cell suspension by treatment of enzyme mixture and suspended in the solution containing 0.56%(w/v) MgCl2· 6H2O,0.10%(w/v) MES and 0.4 mol/L, pH 5.6 mannitol to a final density of 2×105/ml.

  12. Plant regeneration from hypocotyl protoplasts of winter oilseed rape (Brassica napus L.

    Directory of Open Access Journals (Sweden)

    Wacław Orczyk

    2014-01-01

    Full Text Available Protoplasts were isolated from hypocotyls of six breeding lines and two cultivars of winter oilseed rape (B. napus L.. Under presented culture conditions almost all of the protoplasts regenerated cell walls. Division frequency depended on the genotype and was from 50% to 64%. Shoot regeneration (also depended on the genotype was induced with the frequency of 3.6% (for cv Bolko on the medium containing IAA (0.1 mg•dm-3, zeatin (0.5 mg•dm-3 and BAP (0.5 mg•dm-3 . All shoots were rooted on MS basal medium supplemented with sucrose 30 g•dm-3.

  13. Cellulose and 1,3-glucan synthesis during the early stages of wall regeneration in soybean protoplasts.

    Science.gov (United States)

    Klein, A S; Montezinos, D; Delmer, D P

    1981-06-01

    Protoplasts isolated from cultured soybean cells (Glycine max (L.) Merr., cv. Mandarin) were used to study polysaccharide biosynthesis during the initial stages of cell wall-regeneration. Within minutes after the protoplasts were transferred to a wall-regeneration medium containing [(14)C]glucose, radioactivity was detected in a product which was chemically characterized as cellulose. The onset and accumulation of radioactivity into cellulose coincided with the appearance fibrils on the surface of protoplasts, as seen under the electron microscope. At these early stages, a variety of polysaccharide-containing polymers other than cellulose were also synthesized. Under conditions where the protoplasts were competent to synthesize cellulose from glucose, uridine diphosphate-[(14)C]glucose and guanosine diphosphate-[(14)C]glucose did not serve as effective substrates for cellulose synthesis. However, substantial amounts of label from uridine diphosphate glucose were incorporated into 1,3-glucan.

  14. Behaviour of the disease resistance gene Asc in protoplasts of Lycopersicon esculentum mill

    NARCIS (Netherlands)

    Moussatos, V.; Witsenboer, H.; Hille, J.; Gilchrist, D.

    1993-01-01

    Action of Asc, a single dominant Mendelian gene controlling disease response at the whole plant level, was detected at the level of individual cells. Protoplasts, freshly isolated from resistant (Asc/Asc) and susceptible (asc/asc) tomato isolines, were differentially sensitive to AAL toxin as observ

  15. Clinostation influence on regeneration of cell wall in Solanum Tuberosum L. protoplasts

    Science.gov (United States)

    Nedukha, Elena M.; Sidorov, V. A.; Samoylov, V. M.

    1994-08-01

    Regeneration of cell walls in protoplasts was investigated using light- and electronmicroscopic methods. The protoplasts were isolated from mesophyll of Solanum tuberosum leaves and were cultivated on the horizontal low rotating clinostat (2 rpm) and in control for 10 days. Using a fluorescent method (with Calcofluor white) it was demonstrated that changes in vector gravity results in an regeneration inhibition of cell wall. With electron-microscopical and electro-cytochemical methods (staining with alcianum blue) dynamics of the regeneration of cell walls in protoplasts was studied; carbohydrate matrix of cell walls is deposited at the earliest stages of this process. The influence of microgravity on the cell wall regeneration is discussed in higher plants.

  16. An En/Spm based transposable element system for gene isolation in Arabidopsis thaliana.

    NARCIS (Netherlands)

    Aarts, M.G.M.

    1996-01-01

    At the start of the research described in this thesis, the main aim was to develop, study and apply an efficient En/Spm-I/dSpm based transposon tagging system in Arabidopsis thaliana to generate tagged mutants and to provide insights in the possibilities for future applications of such a transposon

  17. Single Walled Carbon Nanotubes Exhibit Dual-Phase Regulation to Exposed Arabidopsis Mesophyll Cells

    Science.gov (United States)

    Yuan, Hengguang; Hu, Shanglian; Huang, Peng; Song, Hua; Wang, Kan; Ruan, Jing; He, Rong; Cui, Daxiang

    2011-12-01

    Herein we are the first to report that single-walled carbon nanotubes (SWCNTs) exhibit dual-phase regulation to Arabidopsis mesophyll cells exposed to different concentration of SWCNTs. The mesophyll protoplasts were prepared by enzyme digestion, and incubated with 15, 25, 50, 100 μg/ml SWCNTs for 48 h, and then were observed by optical microscopy and transmission electron microscopy, the reactive oxygen species (ROS) generation was measured. Partial protoplasts were stained with propidium iodide and 4'-6- diamidino-2-phenylindole, partial protoplasts were incubated with fluorescein isothiocyanate-labeled SWCNTs, and observed by fluorescence microscopy. Results showed that SWCNTs could traverse both the plant cell wall and cell membrane, with less than or equal to 50 μg/ml in the culture medium, SWCNTs stimulated plant cells to grow out trichome clusters on their surface, with more than 50 μg/ml SWCNTs in the culture medium, SWCNTs exhibited obvious toxic effects to the protoplasts such as increasing generation of ROS, inducing changes of protoplast morphology, changing green leaves into yellow, and inducing protoplast cells' necrosis and apoptosis. In conclusion, single walled carbon nanotubes can get through Arabidopsis mesophyll cell wall and membrane, and exhibit dose-dependent dual-phase regulation to Arabidopsis mesophyll protoplasts such as low dose stimulating cell growth, and high dose inducing cells' ROS generation, necrosis or apoptosis.

  18. Single Walled Carbon Nanotubes Exhibit Dual-Phase Regulation to Exposed Arabidopsis Mesophyll Cells

    Directory of Open Access Journals (Sweden)

    Huang Peng

    2011-01-01

    Full Text Available Abstract Herein we are the first to report that single-walled carbon nanotubes (SWCNTs exhibit dual-phase regulation to Arabidopsis mesophyll cells exposed to different concentration of SWCNTs. The mesophyll protoplasts were prepared by enzyme digestion, and incubated with 15, 25, 50, 100 μg/ml SWCNTs for 48 h, and then were observed by optical microscopy and transmission electron microscopy, the reactive oxygen species (ROS generation was measured. Partial protoplasts were stained with propidium iodide and 4'-6- diamidino-2-phenylindole, partial protoplasts were incubated with fluorescein isothiocyanate-labeled SWCNTs, and observed by fluorescence microscopy. Results showed that SWCNTs could traverse both the plant cell wall and cell membrane, with less than or equal to 50 μg/ml in the culture medium, SWCNTs stimulated plant cells to grow out trichome clusters on their surface, with more than 50 μg/ml SWCNTs in the culture medium, SWCNTs exhibited obvious toxic effects to the protoplasts such as increasing generation of ROS, inducing changes of protoplast morphology, changing green leaves into yellow, and inducing protoplast cells' necrosis and apoptosis. In conclusion, single walled carbon nanotubes can get through Arabidopsis mesophyll cell wall and membrane, and exhibit dose-dependent dual-phase regulation to Arabidopsis mesophyll protoplasts such as low dose stimulating cell growth, and high dose inducing cells' ROS generation, necrosis or apoptosis.

  19. Novel sporophyte-like plants are regenerated from protoplasts fused between sporophytic and gametophytic protoplasts of Bryopsis plumosa.

    Science.gov (United States)

    Yamagishi, Takahiro; Hishinuma, Tasuku; Kataoka, Hironao

    2004-06-01

    Protoplasts of the marine coenocytic macrophyte Bryopsis plumosa (Hudson) C. Agardh. [Caulerpales] can easily be obtained by cutting gametophytes or sporophytes with sharp scissors. When a protoplast isolated from a gametophyte was fused with a protoplast isolated from a sporophyte of this alga, it germinated and developed into either one of two completely different forms. One plant form, named Type G, appeared quite similar to a gametophyte, and the other, named Type S, looked similar to a sporophyte. While the Type G plant contained many small nuclei of gametophyte origin together with a single giant nucleus of sporophyte origin, the Type S plant contained many large nuclei of uniform size. These large nuclei in the Type S plant had metamorphosed from the gametophytic nuclei, and were not formed through division of the giant nucleus of sporophyte origin. Fragments of the Type S plant, each having such a large nucleus, developed into creeping filaments that look very similar to sporophytes. While cell walls of gametophytes and Type G plants were stained by Congo-red, those of the thalli of regenerated Type S plants and sporophytes were not stained by the dye. This indicated that the large nuclei of the Type S plant did not express genes for xylan synthesis, which are characteristic of gametophytes. Two-dimensional gel electrophoretic analysis revealed that most of the proteins synthesized in the Type S plant were identical to those of sporophytes. These results strongly suggest that in the Type S plant, the gametophytic nuclei are transformed into sporophyte-like nuclei by an unknown factor(s) produced by the giant nucleus of sporophyte origin and that the transformed nuclei express the set of genes characteristic of sporophytes. Despite morphological similarity, however, the regenerated Type S plant could not produce zoospores, because its large nuclei did not divide normally. The transformed large nuclei of gametophyte origin still seemed to be in the haploid

  20. An Effective Strategy for Reliably Isolating Heritable and Cas9-Free Arabidopsis Mutants Generated by CRISPR/Cas9-Mediated Genome Editing.

    Science.gov (United States)

    Gao, Xiuhua; Chen, Jilin; Dai, Xinhua; Zhang, Da; Zhao, Yunde

    2016-07-01

    Mutations generated by CRISPR/Cas9 in Arabidopsis (Arabidopsis thaliana) are often somatic and are rarely heritable. Isolation of mutations in Cas9-free Arabidopsis plants can ensure the stable transmission of the identified mutations to next generations, but the process is laborious and inefficient. Here, we present a simple visual screen for Cas9-free T2 seeds, allowing us to quickly obtain Cas9-free Arabidopsis mutants in the T2 generation. To demonstrate this in principle, we targeted two sites in the AUXIN-BINDING PROTEIN1 (ABP1) gene, whose function as a membrane-associated auxin receptor has been challenged recently. We obtained many T1 plants with detectable mutations near the target sites, but only a small fraction of T1 plants yielded Cas9-free abp1 mutations in the T2 generation. Moreover, the mutations did not segregate in Mendelian fashion in the T2 generation. However, mutations identified in the Cas9-free T2 plants were stably transmitted to the T3 generation following Mendelian genetics. To further simplify the screening procedure, we simultaneously targeted two sites in ABP1 to generate large deletions, which can be easily identified by PCR. We successfully generated two abp1 alleles that contained 1,141- and 711-bp deletions in the ABP1 gene. All of the Cas9-free abp1 alleles we generated were stable and heritable. The method described here allows for effectively isolating Cas9-free heritable CRISPR mutants in Arabidopsis.

  1. Linear sucrose transport in protoplasts from developing soybean cotyledons.

    Science.gov (United States)

    Lin, W

    1985-07-01

    Previous studies with isolated soybean cotyledon protoplasts revealed the presence of a saturable, simple diffusion, and nonsaturating carrier-mediated uptake of sucrose into soybean cotyledon cells. A proton/sucrose cotransport may be involved in the saturable sucrose uptake (Lin et al. 1984 Plant Physiol 75: 936-940 and Schmitt et al. 1984 Plant Physiol 75: 941-946). In this study, we investigated the linear sucrose uptake mechanism by treating isolated protoplasts with 15 micromolar p-trifluoromethoxy-carbonylcyanide phenylhydrazone (FCCP) or 100 micromolar p-chloromecuribenzenesulfonic acid to eliminate the saturable uptake. We found: (a) increasing external pH decreases the linear sucrose uptake; (b) fusicoccin at 20 micromolar stimulates and FCCP at 15 micromolar inhibits this linear sucrose uptake; and (c) the ratio of the initial influx of proton to sucrose is close to one in both saturable and nondiffusive linear (difference between the total linear and diffusive components) uptakes. The results suggest that a proton/sucrose cotransport is also involved in the nondiffusive linear sucrose uptake into soybean cotyledon cells.

  2. Overexpression of Nelumbo nucifera metallothioneins 2a and 3 enhances seed germination vigor in Arabidopsis.

    Science.gov (United States)

    Zhou, Yuliang; Chu, Pu; Chen, Huhui; Li, Yin; Liu, Jun; Ding, Yu; Tsang, Edward W T; Jiang, Liwen; Wu, Keqiang; Huang, Shangzhi

    2012-03-01

    Metallothioneins (MTs) are small, cysteine-rich and metal-binding proteins which are involved in metal homeostasis and scavenging of reactive oxygen species. Although plant MTs have been intensively studied, their roles in seeds remain to be clearly established. Here, we report the isolation and characterization of NnMT2a, NnMT2b and NnMT3 from sacred lotus (Nelumbo nucifera Gaertn.) and their roles in seed germination vigor. The transcripts of NnMT2a, NnMT2b and NnMT3 were highly expressed in developing and germinating sacred lotus seeds, and were dramatically up-regulated in response to high salinity, oxidative stresses and heavy metals. Analysis of transformed Arabidopsis protoplasts showed that NnMT2a-YFP and NnMT3-YFP were localized in cytoplasm and nucleoplasm. Transgenic Arabidopsis seeds overexpressing NnMT2a and NnMT3 displayed improved resistance to accelerated aging (AA) treatment, indicating their significant roles in seed germination vigor. These transgenic seeds also exhibited higher superoxide dismutase activity compared to wild-type seeds after AA treatment. In addition, we showed that NnMT2a and NnMT3 conferred improved germination ability to NaCl and methyl viologen on transgenic Arabidopsis seeds. Taken together, these data demonstrate that overexpression of NnMT2a and NnMT3 in Arabidopsis significantly enhances seed germination vigor after AA treatment and under abiotic stresses.

  3. Regeneration of viable oil palm plants from protoplasts by optimizing media components, growth regulators and cultivation procedures.

    Science.gov (United States)

    Masani, Mat Yunus Abdul; Noll, Gundula; Parveez, Ghulam Kadir Ahmad; Sambanthamurthi, Ravigadevi; Prüfer, Dirk

    2013-09-01

    Oil palm protoplasts are suitable as a starting material for the production of oil palm plants with new traits using approaches such as somatic hybridization, but attempts to regenerate viable plants from protoplasts have failed thus far. Here we demonstrate, for the first time, the regeneration of viable plants from protoplasts isolated from cell suspension cultures. We achieved a protoplast yield of 1.14×10(6) per gram fresh weight with a viability of 82% by incubating the callus in a digestion solution comprising 2% cellulase, 1% pectinase, 0.5% cellulase onuzuka R10, 0.1% pectolyase Y23, 3% KCl, 0.5% CaCl2 and 3.6% mannitol. The regeneration of protoplasts into viable plants required media optimization, the inclusion of plant growth regulators and the correct culture technique. Microcalli derived from protoplasts were obtained by establishing agarose bead cultures using Y3A medium supplemented with 10μM naphthalene acetic acid, 2μM 2,4-dichlorophenoxyacetic acid, 2μM indole-3-butyric acid, 2μM gibberellic acid and 2μM 2-γ-dimethylallylaminopurine. Small plantlets were regenerated from microcalli by somatic embryogenesis after successive subculturing steps in medium with limiting amounts of growth regulators supplemented with 200mg/l ascorbic acid.

  4. Preparation of Biologically Active Arabidopsis Ribosomes and Comparison with Yeast Ribosomes for Binding to a tRNA-Mimic that Enhances Translation of Plant Plus-Strand RNA Viruses

    Directory of Open Access Journals (Sweden)

    Vera Aleksey Stupina

    2013-07-01

    Full Text Available Isolation of biologically active cell components from multicellular eukaryotic organisms often poses difficult challenges such as low yields and inability to retain the integrity and functionality of the purified compound. We previously identified a cap-independent translation enhancer (3’CITE in the 3’UTR of Turnip crinkle virus (TCV that structurally mimics a tRNA and binds to yeast 80S ribosomes and 60S subunits in the P-site. Yeast ribosomes were used for these studies due to the lack of methods for isolation of plant ribosomes with high yields and integrity. To carry out studies with more natural components, a simple and efficient procedure has been developed for the isolation of large quantities of high quality ribosomes and ribosomal subunits from Arabidopsis thaliana protoplasts prepared from seed-derived callus tissue. Attempts to isolate high quality ribosomes from wheat germ, bean sprouts and evacuolated protoplasts were unsuccessful. Addition of purified Arabidopsis 80S plant ribosomes to ribosome-depleted wheat germ lysates resulted in a greater than 1200-fold enhancement in in vitro translation of a luciferase reporter construct. The TCV 3’CITE bound to ribosomes with a 3 to 7-fold higher efficiency when using plant 80S ribosomes compared with yeast ribosomes, indicating that this viral translational enhancer is adapted to interact more efficiently with host plant ribosomes.

  5. Poinsettia protoplasts - a simple, robust and efficient system for transient gene expression studies

    Directory of Open Access Journals (Sweden)

    Pitzschke Andrea

    2012-05-01

    Full Text Available Abstract Background Transient gene expression systems are indispensable tools in molecular biology. Yet, their routine application is limited to few plant species often requiring substantial equipment and facilities. High chloroplast and chlorophyll content may further impede downstream applications of transformed cells from green plant tissue. Results Here, we describe a fast and simple technique for the high-yield isolation and efficient transformation (>70% of mesophyll-derived protoplasts from red leaves of the perennial plant Poinsettia (Euphorbia pulccherrima. In this method no particular growth facilities or expensive equipments are needed. Poinsettia protoplasts display an astonishing robustness and can be employed in a variety of commonly-used downstream applications, such as subcellular localisation (multi-colour fluorescence or promoter activity studies. Due to low abundance of chloroplasts or chromoplasts, problems encountered in other mesophyll-derived protoplast systems (particularly autofluorescence are alleviated. Furthermore, the transgene expression is detectable within 90 minutes of transformation and lasts for several days. Conclusions The simplicity of the isolation and transformation procedure renders Poinsettia protoplasts an attractive system for transient gene expression experiments, including multi-colour fluorescence, subcellular localisation and promoter activity studies. In addition, they offer hitherto unknown possibilities for anthocyan research and industrial applications.

  6. A comparison of Peronospora parasitica (Downy mildew) isolates from Arabidopsis thaliana and Brassica oleracea using amplified fragment length polymorphism and internal transcribed spacer 1 sequence analyses.

    Science.gov (United States)

    Rehmany, A P; Lynn, J R; Tör, M; Holub, E B; Beynon, J L

    2000-07-01

    Amplified fragment length polymorphism (AFLP) fingerprints and internal transcribed spacer 1 (ITS1) sequences from 27 Peronospora parasitica isolates (collected from Arabidopsis thaliana or Brassica oleracea), 5 Albugo candida isolates (from the same hosts and from Capsella bursa-pastoris), and 1 Bremia lactucae isolate (from Lactuca sativa) were compared. The AFLP analysis divided the isolates into five groups that correlated with taxonomic species and, in most cases, with host origin. The only exception was a group consisting of A. candida isolates from both B. oleracea and C. bursa-pastoris. ITS1 sequence analysis divided the isolates into the same five groups, demonstrated the divergence between P. parasitica isolates from A. thaliana and B. oleracea, and, using previously published ITS1 sequences, clearly showed the relationship between A. candida isolates from different hosts.

  7. A step-by-step protocol for formaldehyde-assisted isolation of regulatory elements from Arabidopsis thaliana

    Institute of Scientific and Technical Information of China (English)

    Mohammad Amin Omidbakhshfard; Flavia Vischi Winck; Samuel Arvidsson; Diego M.Riao-Pachn; Bernd Mueller-Roeber

    2014-01-01

    The control of gene expression by transcriptional regulators and other types of functional y relevant DNA transactions such as chromatin remodeling and replication underlie a vast spectrum of biological processes in al organisms. DNA transactions require the control ed interaction of proteins with DNA sequence motifs which are often located in nucleosome-depleted regions (NDRs) of the chromatin. Formaldehyde-assisted isolation of regulatory elements (FAIRE) has been established as an easy-to-implement method for the isolation of NDRs from a number of eukaryotic organisms, and it has been successful y employed for the discovery of new regulatory segments in genomic DNA from, for example, yeast, Drosophila, and humans. Until today, however, FAIRE has only rarely been employed in plant research and currently no detailed FAIRE protocol for plants has been published. Here, we provide a step-by-step FAIRE protocol for NDR discovery in Arabidopsis thaliana. We demonstrate that NDRs isolated from plant chromatin are readily amenable to quantitative polymerase chain reaction and next-generation sequencing. Only minor modification of the FAIRE protocol wil be needed to adapt it to other plants, thus facilitating the global inventory of regulatory regions across species.

  8. Expression of a High Mobility Group Protein Isolated from Cucumis sativus Affects the Germination of Arabidopsis thaliana under Abiotic Stress Conditions

    Institute of Scientific and Technical Information of China (English)

    Ji Young Jang; Kyung Jin Kwak; Hunseung Kang

    2008-01-01

    Although high mobility group B (HMGB) proteins have been identified from a variety of plant species, their importance and functional roles in plant responses to changing environmental conditions are largely unknown. Here, we investigated the functional roles of a CsHMGB isolated from cucumber (Cucurnis sativus L.) in plant responses to environmental stimuli. Under normal growth conditions or when subjected to cold stress, no differences in plant growth were found between the wild.type and transgenic Arabidopsis thaliana overexpressing CsHMGB. By contrast, the transgenic Arabidopsis plants displayed retarded germination compared with the wild-type plants when grown under high salt or dehydration stress conditions. Germination of the transgenic plants was delayed by the addition of abscisic acid (ABA), implying that CsHMGB affects germination through an ABA-dependent way. The expression of CsHMGB had affected only the germination stage, and CsHMGB did not affect the seedling growth of the transgenic plants under the stress conditions. The transcript levels of several germination-responsive genes were modulated by the expression of CsHMGB in Arabidopsis. Taken together, these results suggest that ectopic expression of a CsHMGB in Arabidopsis modulates the expression of several germination-responsive genes, and thereby affects the germination of Arabidopsis plants under different stress conditions.

  9. Laser-induced tobacco protoplast fusion

    Institute of Scientific and Technical Information of China (English)

    李银妹; 关力劼; 楼立人; 崔国强; 姚湲; 王浩威; 操传顺; 鲁润龙; 陈曦

    1999-01-01

    Laser tweezers can manipulate small particles, such as cells and organdies. When coupling them with laser microbeam selective fusion of two tobacco protoplasts containing some chloroplast was achieved. Physical and biological variables that affect laser trapping and laser-induced fusion were also discussed. The results show that the effect of chloroplast content and distribution on the yield of cell fusion is remarkable.

  10. Guard cell chloroplasts are essential for blue light-dependent stomatal opening in Arabidopsis.

    Directory of Open Access Journals (Sweden)

    Noriyuki Suetsugu

    Full Text Available Blue light (BL induces stomatal opening through the activation of H+-ATPases with subsequent ion accumulation in guard cells. In most plant species, red light (RL enhances BL-dependent stomatal opening. This RL effect is attributable to the chloroplasts of guard cell, the only cells in the epidermis possessing this organelle. To clarify the role of chloroplasts in stomatal regulation, we investigated the effects of RL on BL-dependent stomatal opening in isolated epidermis, guard cell protoplasts, and intact leaves of Arabidopsis thaliana. In isolated epidermal tissues and intact leaves, weak BL superimposed on RL enhanced stomatal opening while BL alone was less effective. In guard cell protoplasts, RL enhanced BL-dependent H+-pumping and DCMU, a photosynthetic electron transport inhibitor, eliminated this effect. RL enhanced phosphorylation levels of the H+-ATPase in response to BL, but this RL effect was not suppressed by DCMU. Furthermore, DCMU inhibited both RL-induced and BL-dependent stomatal opening in intact leaves. The photosynthetic rate in leaves correlated positively with BL-dependent stomatal opening in the presence of DCMU. We conclude that guard cell chloroplasts provide ATP and/or reducing equivalents that fuel BL-dependent stomatal opening, and that they indirectly monitor photosynthetic CO2 fixation in mesophyll chloroplasts by absorbing PAR in the epidermis.

  11. Preparation of Epidermal Peels and Guard Cell Protoplasts for Cellular, Electrophysiological, and -Omics Assays of Guard Cell Function.

    Science.gov (United States)

    Zhu, Mengmeng; Jeon, Byeong Wook; Geng, Sisi; Yu, Yunqing; Balmant, Kelly; Chen, Sixue; Assmann, Sarah M

    2016-01-01

    Bioassays are commonly used to study stomatal phenotypes. There are multiple options in the choice of plant materials and species used for observation of stomatal and guard cell responses in vivo. Here, detailed procedures for bioassays of stomatal responses to abscisic acid (ABA) in Arabidopsis thaliana are described, including ABA promotion of stomatal closure, ABA inhibition of stomatal opening, and ABA promotion of reaction oxygen species (ROS) production in guard cells. We also include an example of a stomatal bioassay for the guard cell CO2 response using guard cell-enriched epidermal peels from Brassica napus. Highly pure preparations of guard cell protoplasts can be produced, which are also suitable for studies on guard cell signaling, as well as for studies on guard cell ion transport. Small-scale and large-scale guard cell protoplast preparations are commonly used for electrophysiological and -omics studies, respectively. We provide a procedure for small-scale guard cell protoplasting from A. thaliana. Additionally, a general protocol for large-scale preparation of guard cell protoplasts, with specifications for three different species, A. thaliana, B. napus, and Vicia faba is also provided.

  12. Individual and joint activity of terpenoids, isolated from Calamintha nepeta extract, on Arabidopsis thaliana.

    Science.gov (United States)

    Araniti, Fabrizio; Graña, Elisa; Reigosa, Manuel J; Sánchez-Moreiras, Adela M; Abenavoli, Maria Rosa

    2013-01-01

    Four terpenoids, camphor, pulegone, trans-caryophyllene and farnesene, previously found in Calamintha nepeta (L.) Savi methanolic extract and essential oils were assayed on germination and root growth of Arabidopsis thaliana (L.) Heynh. None of the terpenes, singularly or in combination, was able to inhibit the germination process. Farnesene and trans-caryophyllene caused a strong inhibitory effect on root growth, and pulegone, at the highest concentrations, reduced lateral root formation. Although the mixture of camphor-trans-caryophyllene with or without farnesene did not cause any effect on root growth, the addition of pulegone induced a marked synergistic activity. Moreover, the addition, at low concentration, of farnesene to pulegone-camphor-trans-caryophyllene mixture further increased the inhibitory effect on root elongation. These results suggested that the inhibitory effects caused by C. nepeta methanolic extract may depend on the combined action of different molecules.

  13. Draft Genome Sequence of the Endophytic Strain Rhodococcus kyotonensis KB10, a Potential Biodegrading and Antibacterial Bacterium Isolated from Arabidopsis thaliana

    Science.gov (United States)

    Hong, Chi Eun; Jo, Sung Hee

    2016-01-01

    Rhodococcus kyotonensis KB10 is an endophytic bacterium isolated from Arabidopsis thaliana. The organism showed mild antibacterial activity against the phytopathogen Pseudomonas syringae pv. tomato DC3000. This study reports the genome sequence of R. kyotonensis KB10. This bacterium contains an ectoine biosynthesis gene cluster and has the potential to degrade nitroaromatic compounds. The identified bacterium may be a suitable biocontrol agent and degrader of environmental pollutants. PMID:27389269

  14. Protoplast culture and protoplast symmetric fusion in cotton%棉花原生质体培养和原生质体对称融合研究

    Institute of Scientific and Technical Information of China (English)

    孙玉强

    2011-01-01

    callus, the maturation and germination of somatic embryos,and plant regeneration to some degree. Embryogenic calli of wild species subcultured and conserved on MSB semi-solid medium supplementing with IBA 0. 984 μmol/L,KT 0. 232 mol/L for 4 years still have the capability of differentiation and provide a mass of materials. It is the first report of regeneration of plants via somatic embryogenesis in many wild cotton species.2. Protoplasts were isolated from different explants of 2 species (Coker 201 and YZ1) in Gossypium hirsutum L. (embryogenic cell suspension culture,embryogenic callus,immature somatic embryos,hypo-cotyls,young roots and leaves). Plants regenerated from cultured protoplasts of 6 explants in Coker 201, but the plating frequencies of protoplasts from different explants varied significantly. The plating frequency of suspension culture-protoplast, embryogenic callus-and somatic embryo-protoplast,hypocotyl-young root-and leaf-protoplast was 10%,6%,less than 2%. The plating frequencies of plants regenerated form protoplast cultures isolated from embryogenic suspension cultures, somatic embryos and embryogenic callus in YZ1 were lower (l%-2%) than that of plants regenerated from same explants in Coker 201.Plants regenerated from protoplasts isolated from somatic embryos and embryogenic suspension cultures in wild cotton G. Klotzschianum with the plating frequencies ranging 6% to 8%. RAPD analysis demonstrated that the regenerated plants were genetically homogeneous.This study emphasized on enzyme combinations for protoplast isolation, the influences of culture density and PGR combinations etc for protoplasts sustained division,callus formation,and then a practical protocol for protoplast culture in cotton is established.3. In this research, symmetric fusion including 8 combinations mediated by electricity was carried out. Plants regenerated from Coker 201+G. Klotzschianum, Coker 201+G. Davidsonii, Coker 201 + G. Bickii,Coker 201+G. Stockii, which were

  15. DNA-mediated gene transfer in plant protoplasts

    Energy Technology Data Exchange (ETDEWEB)

    U, Zang Kual; Riu, Key Zung; So, In Sup; Hong, Kyung Ae [Cheju National University, Cheju (Korea, Republic of)

    1994-12-31

    The neomycin phosphotransferase II gene(NPT-II) was introduced into geranium (Pelargonium zonale hybrids) protoplasts by using PEG or electroporation method. The presence of the introduced DNA in the protoplasts and the expressions of the gene in the transformed cells were examined. The presence of the NPT-II DNA in the protoplasts were detected by polymerase chain reaction. The expressions of NPT-II gene in the transformed cells were confirmed by the NPT-II assay. (author)

  16. Isolation of transcription factor complexes from Arabidopsis cell suspension cultures by tandem affinity purification.

    Science.gov (United States)

    Van Leene, Jelle; Eeckhout, Dominique; Persiau, Geert; Van De Slijke, Eveline; Geerinck, Jan; Van Isterdael, Gert; Witters, Erwin; De Jaeger, Geert

    2011-01-01

    Defining protein complexes is critical to virtually all aspects of cell biology because most cellular processes are regulated by stable or more dynamic protein interactions. Elucidation of the protein-protein interaction network around transcription factors is essential to fully understand their function and regulation. In the last decade, new technologies have emerged to study protein-protein interactions under near-physiological conditions. We have developed a high-throughput tandem affinity purification (TAP)/mass spectrometry (MS) platform for cell suspension cultures to analyze protein complexes in Arabidopsis thaliana. This streamlined platform follows an integrated approach comprising generic Gateway-based vectors with high cloning flexibility, the fast generation of transgenic suspension cultures, TAP adapted for plant cells, and tandem matrix-assisted laser desorption ionization MS for the identification of purified proteins. Recently, we evaluated the GS tag, originally developed to study mammalian protein complexes, that combines two IgG-binding domains of protein G with a streptavidin-binding peptide, separated by two tobacco etch virus cleavage sites. We found that this GS tag outperforms the traditional TAP tag in plant cells, regarding both specificity and complex yield. Here, we provide detailed protocols of the GS-based TAP platform that allowed us to characterize transcription factor complexes involved in signaling in response to the plant phytohormone jasmonate.

  17. Beneficial Bacteria Isolated from Grapevine Inner Tissues Shape Arabidopsis thaliana Roots

    OpenAIRE

    Enrico Baldan; Sebastiano Nigris; Chiara Romualdi; Stefano D'Alessandro; Anna Clocchiatti; Michela Zottini; Piergiorgio Stevanato; Andrea Squartini; Barbara Baldan

    2015-01-01

    We investigated the potential plant growth-promoting traits of 377 culturable endophytic bacteria, isolated from Vitis vinifera cv. Glera, as good biofertilizer candidates in vineyard management. Endophyte ability in promoting plant growth was assessed in vitro by testing ammonia production, phosphate solubilization, indole-3-acetic acid (IAA) and IAA-like molecule biosynthesis, siderophore and lytic enzyme secretion. Many of the isolates were able to mobilize phosphate (33%), release ammoniu...

  18. Constitutive Expression of OsIAA9 Affects Starch Granules Accumulation and Root Gravitropic Response in Arabidopsis.

    Science.gov (United States)

    Luo, Sha; Li, Qianqian; Liu, Shanda; Pinas, Nicholaas M; Tian, Hainan; Wang, Shucai

    2015-01-01

    Auxin/Indole-3-Acetic Acid (Aux/IAA) genes are early auxin response genes ecoding short-lived transcriptional repressors, which regulate auxin signaling in plants by interplay with Auxin Response Factors (ARFs). Most of the Aux/IAA proteins contain four different domains, namely Domain I, Domain II, Domain III, and Domain IV. So far all Aux/IAA mutants with auxin-related phenotypes identified in both Arabidopsis and rice (Oryza sativa) are dominant gain-of-function mutants with mutations in Domain II of the corresponding Aux/IAA proteins, suggest that Aux/IAA proteins in both Arabidopsis and rice are largely functional redundantly, and they may have conserved functions. We report here the functional characterization of a rice Aux/IAA gene, OsIAA9. RT-PCR results showed that expression of OsIAA9 was induced by exogenously applied auxin, suggesting that OsIAA9 is an auxin response gene. Bioinformatic analysis showed that OsIAA9 has a repressor motif in Domain I, a degron in Domain II, and the conserved amino acid signatures for protein-protein interactions in Domain III and Domain IV. By generating transgenic plants expressing GFP-OsIAA9 and examining florescence in the transgenic plants, we found that OsIAA9 is localized in the nucleus. When transfected into protoplasts isolated from rosette leaves of Arabidopsis, OsIAA9 repressed reporter gene expression, and the repression was partially released by exogenously IAA. These results suggest that OsIAA9 is a canonical Aux/IAA protein. Protoplast transfection assays showed that OsIAA9 interacted ARF5, but not ARF6, 7, 8 and 19. Transgenic Arabidopsis plants expressing OsIAA9 have increased number of lateral roots, and reduced gravitropic response. Further analysis showed that OsIAA9 transgenic Arabidopsis plants accumulated fewer granules in their root tips and the distribution of granules was also affected. Taken together, our study showed that OsIAA9 is a transcriptional repressor, and it regulates gravitropic

  19. Constitutive expression of OsIAA9 affects starch granules accumulation and root gravitropic response in Arabidopsis

    Directory of Open Access Journals (Sweden)

    Sha eLuo

    2015-12-01

    Full Text Available Auxin/Indole-3-Acetic Acid (Aux/IAA genes are early auxin response genes ecoding short-lived transcriptional repressors, which regulate auxin signaling in plants by interplay with Auxin Response Factors (ARFs. Most of the Aux/IAA proteins contain four different domains, namely Domain I, Domain II, Domain III and Domain IV. So far all Aux/IAA mutants with auxin-related phenotypes identified in both Arabidopsis and rice (Oryza sativa are dominant gain-of-function mutants with mutations in Domain II of the corresponding Aux/IAA proteins, suggest that Aux/IAA proteins in both Arabidopsis and rice are largely functional redundantly, and they may have conserved functions. We report here the functional characterization of a rice Aux/IAA gene, OsIAA9. RT-PCR results showed that expression of OsIAA9 was induced by exogenously applied auxin, suggesting that OsIAA9 is an auxin response gene. Bioinformatic analysis showed that OsIAA9 has a repressor motif in Domain I, a degron in Domain II, and the conserved amino acid signatures for protein-protein interactions in Domain III and Domain IV. By generating transgenic plants expressing GFP-OsIAA9 and examining florescence in the transgenic plants, we found that OsIAA9 is localized in the nucleus. When transfected into protoplasts isolated from rosette leaves of Arabidopsis, OsIAA9 repressed reporter gene expression, and the repression was partially released by exogenously IAA. These results suggest that OsIAA9 is a canonical Aux/IAA protein. Protoplast transfection assays showed that OsIAA9 interacted ARF5, but not ARF6, 7, 8 and 19. Transgenic Arabidopsis plants expressing OsIAA9 have increased number of lateral roots, and reduced gravitropic response. Further analysis showed that OsIAA9 transgenic Arabidopsis plants accumulated fewer granules in their root tips and the distribution of granules was also affected. Taken together, our study showed that OsIAA9 is a transcriptional repressor, and it regulates

  20. A highly efficient miPCR method for isolating FSTs from transgenic Arabidopsis thaliana plants

    Indian Academy of Sciences (India)

    Gennady V. Pogorelko; Oksana V. Fursova

    2008-08-01

    The exact localization of an insertion in the genome of transgenic plants obtained by Agrobacterium-mediated transformation is an integral part of most experiments aimed at studying these types of mutants. There are several methods for isolating unknown nucleotide sequences of genomic DNA which flank the borders of T-DNA integrated in the genome of plants. However, all the methods based on PCR have limitations which in some cases do not permit the desired objective to be achieved. We have developed a new technique for isolating flanking sequence tags (FSTs) via modified inverse PCR. This method is highly efficient and simple, but also retains the advantages of previously well-documented approaches.

  1. A tandem affinity purification tag of TGA2 for isolation of interacting proteins in Arabidopsis thaliana.

    Science.gov (United States)

    Stotz, Henrik U; Findling, Simone; Nukarinen, Ella; Weckwerth, Wolfram; Mueller, Martin J; Berger, Susanne

    2014-01-01

    Tandem affinity purification (TAP) tagging provides a powerful tool for isolating interacting proteins in vivo. TAP-tag purification offers particular advantages for the identification of stimulus-induced protein interactions. Type II bZIP transcription factors (TGA2, TGA5 and TGA6) play key roles in pathways that control salicylic acid, ethylene, xenobiotic and reactive oxylipin signaling. Although proteins interacting with these transcription factors have been identified through genetic and yeast 2-hybrid screening, others are still elusive. We have therefore generated a C-terminal TAP-tag of TGA2 to isolate additional proteins that interact with this transcription factor. Three lines most highly expressing TAP-tagged TGA2 were functional in that they partially complemented reactive oxylipin-responsive gene expression in a tga2 tga5 tga6 triple mutant. TAP-tagged TGA2 in the most strongly overexpressing line was proteolytically less stable than in the other 2 lines. Only this overexpressing line could be used in a 2-step purification process, resulting in isolation of co-purifying bands of larger molecular weight than TGA2. TAP-tagged TGA2 was used to pull down NPR1, a protein known to interact with this transcription factor. Mass spectrometry was used to identify peptides that co-purified with TAP-tagged TGA2. Having generated this TGA2 TAP-tag line will therefore be an asset to researchers interested in stimulus-induced signal transduction processes.

  2. A tandem affinity purification tag of TGA2 for isolation of interacting proteins in Arabidopsis thaliana.

    Science.gov (United States)

    Stotz, Henrik U; Findling, Simone; Nukarinen, Ella; Weckwerth, Wolfram; Mueller, Martin J; Berger, Susanne

    2014-08-19

    Tandem affinity purification (TAP) tagging provides a powerful tool for isolating interacting proteins in vivo. TAP-tag purification offers particular advantages for the identification of stimulus-induced protein interactions. Type II bZIP transcription factors (TGA2, TGA5 and TGA6) play key roles in pathways that control salicylic acid, ethylene, xenobiotic and reactive oxylipin signaling. Although proteins interacting with these transcription factors have been identified through genetic and yeast two-hybrid screening, others are still elusive. We have therefore generated a C-terminal TAP-tag of TGA2 to isolate additional proteins that interact with this transcription factor. Three lines most highly expressing TAP-tagged TGA2 were functional in that they partially complemented reactive oxylipin-responsive gene expression in a tga2 tga5 tga6 triple mutant. TAP-tagged TGA2 in the most strongly overexpressing line was proteolytically less stable than in the other two lines. Only this overexpressing line could be used in a two-step purification process, resulting in isolation of co-purifying bands of larger molecular weight than TGA2. TAP-tagged TGA2 was used to pull down NPR1, a protein known to interact with this transcription factor. Mass spectrometry was used to identify peptides that co-purified with TAP-tagged TGA2. Having generated this TGA2 TAP-tag line will therefore be an asset to researchers interested in stimulus-induced signal transduction processes.

  3. Enhancement of monacolin K production via intergeneric protoplast fusion between Aspergillus terreus and Monascus anka

    Institute of Scientific and Technical Information of China (English)

    Chen Zhi; Lin Wen; Yu Ping; Song Yuan

    2007-01-01

    Intergenric protoplast fusion between Aspergillus terreus CA99 and Monascus anka M-3, the high and low producers of monacolin K respectively, was performed for enhancement of monacolin K production. The 24-hour-old mycelia of A. terreus CA99 and M. anka M-3 were treated with 0.5 % lywallzyme, 0.3 % snailase and 0.3 % cellulase at 34 ℃ for 5 h and at 30 ℃ for 3.5 h, and their protoplasts formation reached 1.76 × 107/mL and 1.68 × 107/mL respectively. Parental protoplasts were irradiated with a 30 W UVlight away from 30 cm for 3 min and then mixed. The mixture was incubated with 30% PEG 6000 for 15 min. The reviving fusants were isolated on the regeneration plates. Of the 363 fusants isolated, over 100 showed enhanced monacolin K production compared with the parental strain M. anka M-3. Ten of them produced monacolin K about 1.6-fold of that M. anka M-3 does and the monacolin K titer of two fusants (F49 and F104) increased by about 1-fold. The monacolin K yields of F49 and F104 were 460 μg/mL and 457 μg/mL respectively. In optimized fermentation medium, the monacolin K titer of F49 reached 1216 μg/mL.

  4. Effect of purine alkaloids on the proliferation of lettuce cells derived from protoplasts.

    Science.gov (United States)

    Sasamoto, Hamako; Fujii, Yoshiharu; Ashihara, Hiroshi

    2015-05-01

    To investigate the ecological role of caffeine, theobromine, theophylline and paraxanthine, which are released from purine alkaloid forming plants, the effects of these purine alkaloids on the division and colony formation of lettuce cells were assessed at concentrations up to 1 mM. Five days after treatment with 500 μM caffeine, theophylline and paraxanthine, division of isolated protoplasts was significantly inhibited. Thirteen days treatment with > 250 μM caffeine had a marked inhibitory effect on the colony formation of cells derived from the protoplasts. Other purine alkaloids also acted as inhibitors. The order of the inhibition was caffeine > theophylline > paraxanthine > theobromine. These observations suggest that a relatively low concentration of caffeine is toxic for proliferation of plant cells. In contrast, theobromine is a weak inhibitor of proliferation. Possible allelopathic roles of purine alkaloids in natural ecosystems are discussed.

  5. [Two interspecific somatic hybrid plants regenerated via protoplast electro-fusion].

    Science.gov (United States)

    Guo, W W; Deng, X X

    2000-03-01

    Protoplasts isolated from cell suspension cultures of 'Bonnaza' navel orange (Citrus sinensis L. Osbeck) were electrically fused with mesophyll protoplasts of rough lemon (Citrus jambhiri Lush) and Goutou orange (Citrus aurantium L.) respectively. Plants regenerated from both fusion combinations. Chromosome counting of randomly selected fifty two globular embryoids as well as all the regenerated seventy four plants from Bonnaza navel + rough lemon revealed that twenty six embryoids were tetraploids, and the rest were diploids while 100% regenerated plants were tetraploids. The results inferred that somatic hybrids were more competitive than parental genotypes in the process of plant regeneration. All the regenerated 14 plants from Bonnaza navel + Goutou orange were tetraploids as revealed by chromosome counting. POX isozyme and RAPD analysis verified that the plants from Bonnaza navel + rough lemon were hybrids, and RAPD analysis confirmed the hybridity of those from Bonnaza navel + Goutou orange.

  6. Protoplast culture of several members of the genus Physalis.

    Science.gov (United States)

    Bapat, V A; Schieder, O

    1981-12-01

    High yields of protoplasts were obtained by enzymic treatment of mesophyll from five different species of the genus Physalis. Subsequent divisions and colony formation were achieved in all the species. However, numerous combinations of phytohormones failed to induce regeneration of shoots from callus tissue developed from protoplasts.

  7. Permeabilizing soybean protoplasts to macromolecules using electroporation and hypotonic shock.

    Science.gov (United States)

    Cutler, A J; Saleem, M

    1987-01-01

    The percentage of soybean cell culture protoplasts permeabilized by electroporation was dependent on the voltage and the number of successive pulses that were applied. Best results were obtained with two 50 milliseconds, 400 volts per centimeter pulses after which 78% of the surviving protoplasts had been permeabilized to the fluorescent dye calcein. Quantitation of the volume of extracellular fluid taken up was performed using radioactive inulin (molecular weight 5000-5500). Typically between 20 and 40 nanoliters of fluid was taken up by 10(6) protoplasts. Electroporation and hypotonic shock treatments (M Saleem, AJ Cutler 1986 J Plant Physiol 124: 11-21) were compared with respect to the volume of fluid taken up under optimum conditions. Electroporation produced 10 times more uptake than hypotonic shock treatment. In all experiments there was a direct relationship between the number of protoplasts lysed and both the amount of fluid taken up and the percentage of surviving protoplasts that were permeabilized.

  8. Kinetics and heterogeneity of energy transfer from light harvesting complex II to photosystem I in the supercomplex isolated from Arabidopsis.

    Science.gov (United States)

    Santabarbara, Stefano; Tibiletti, Tania; Remelli, William; Caffarri, Stefano

    2017-03-29

    State transitions are a phenomenon that maintains the excitation balance between photosystem II (PSII) and photosystem I (PSI-LHCI) by controlling their relative absorption cross-sections. Under light conditions exciting PSII preferentially, a trimeric LHCII antenna moves from PSII to PSI-LHCI to form the PSI-LHCI-LHCII supercomplex. In this work, the excited state dynamics in the PSI-LHCI and PSI-LHCI-LHCII supercomplexes isolated from Arabidopsis have been investigated by picosecond time-resolved fluorescence spectroscopy. The excited state decays were analysed using two approaches based on either (i) a sum of discrete exponentials or (ii) a continuous distribution of lifetimes. The results indicate that the energy transfer from LHCII to the bulk of the PSI antenna occurs with an average macroscopic transfer rate in the 35-65 ns(-1) interval. Yet, the most satisfactory description of the data is obtained when considering a heterogeneous population containing two PSI-LHCI-LHCII supercomplexes characterised by a transfer time of ∼15 and ∼60 ns(-1), likely due to the differences in the strength and orientation of LHCII harboured to PSI. Both these values are of the same order of magnitude of those estimated for the average energy transfer rates from the low energy spectral forms of LHCI to the bulk of the PSI antenna (15-40 ns(-1)), but they are slower than the transfer from the bulk antenna of PSI to the reaction centre (>150 ns(-1)), implying a relatively small kinetics bottleneck for the energy transfer from LHCII. Nevertheless, the kinetic limitation imposed by excited state diffusion has a negligible impact on the photochemical quantum efficiency of the supercomplex, which remains about 98% in the case of PSI-LHCI.

  9. Multiparametric analysis, sorting, and transcriptional profiling of plant protoplasts and nuclei according to cell type.

    Science.gov (United States)

    Galbraith, David W; Janda, Jaroslav; Lambert, Georgina M

    2011-01-01

    Flow cytometry has been employed for the analysis of higher plants for approximately the last 30 years. For the angiosperms, ∼500,000 species, itself a daunting number, parametric measurements enabled through the use of flow cytometers started with basic descriptors of the individual cells and their contents, and have both inspired the development of novel cytometric methods that subsequently have been applied to organisms within other kingdoms of life, and adopted cytometric methods devised for other species, particularly mammals. Higher plants offer unique challenges in terms of flow cytometric analysis, notably the facts that their organs and tissues are complex three-dimensional assemblies of different cell types, and that their individual cells are, in general, larger than those of mammals.This chapter provides an overview of the general types of parametric measurement that have been applied to plants, and provides detailed methods for selected examples based on the plant model Arabidopsis thaliana. These illustrate the use of flow cytometry for the analysis of protoplasts and nuclear DNA contents (genome size and the cell cycle). These are further integrated with measurements focusing on specific cell types, based on transgenic expression of Fluorescent Proteins (FPs), and on analysis of the spectrum of transcripts found within protoplasts and nuclei. These measurements were chosen in particular to illustrate, respectively, the issues encountered in the flow analysis and sorting of large biological cells, typified by protoplasts; how to handle flow analyses under conditions that require processing of large numbers of samples in which the individual samples contain only a very small minority of objects of interest; and how to deal with exceptionally small amounts of RNA within the sorted samples.

  10. Overexpression of a soybean ariadne-like ubiquitin ligase gene GmARI1 enhances aluminum tolerance in Arabidopsis.

    Directory of Open Access Journals (Sweden)

    Xiaolian Zhang

    Full Text Available Ariadne (ARI subfamily of RBR (Ring Between Ring fingers proteins have been found as a group of putative E3 ubiquitin ligases containing RING (Really Interesting New Gene finger domains in fruitfly, mouse, human and Arabidopsis. Recent studies showed several RING-type E3 ubiquitin ligases play important roles in plant response to abiotic stresses, but the function of ARI in plants is largely unknown. In this study, an ariadne-like E3 ubiquitin ligase gene was isolated from soybean, Glycine max (L. Merr., and designated as GmARI1. It encodes a predicted protein of 586 amino acids with a RBR supra-domain. Subcellular localization studies using Arabidopsis protoplast cells indicated GmARI protein was located in nucleus. The expression of GmARI1 in soybean roots was induced as early as 2-4 h after simulated stress treatments such as aluminum, which coincided with the fact of aluminum toxicity firstly and mainly acting on plant roots. In vitro ubiquitination assay showed GmARI1 protein has E3 ligase activity. Overexpression of GmARI1 significantly enhanced the aluminum tolerance of transgenic Arabidopsis. These findings suggest that GmARI1 encodes a RBR type E3 ligase, which may play important roles in plant tolerance to aluminum stress.

  11. Overexpression of a soybean ariadne-like ubiquitin ligase gene GmARI1 enhances aluminum tolerance in Arabidopsis.

    Science.gov (United States)

    Zhang, Xiaolian; Wang, Ning; Chen, Pei; Gao, Mengmeng; Liu, Juge; Wang, Yufeng; Zhao, Tuanjie; Li, Yan; Gai, Junyi

    2014-01-01

    Ariadne (ARI) subfamily of RBR (Ring Between Ring fingers) proteins have been found as a group of putative E3 ubiquitin ligases containing RING (Really Interesting New Gene) finger domains in fruitfly, mouse, human and Arabidopsis. Recent studies showed several RING-type E3 ubiquitin ligases play important roles in plant response to abiotic stresses, but the function of ARI in plants is largely unknown. In this study, an ariadne-like E3 ubiquitin ligase gene was isolated from soybean, Glycine max (L.) Merr., and designated as GmARI1. It encodes a predicted protein of 586 amino acids with a RBR supra-domain. Subcellular localization studies using Arabidopsis protoplast cells indicated GmARI protein was located in nucleus. The expression of GmARI1 in soybean roots was induced as early as 2-4 h after simulated stress treatments such as aluminum, which coincided with the fact of aluminum toxicity firstly and mainly acting on plant roots. In vitro ubiquitination assay showed GmARI1 protein has E3 ligase activity. Overexpression of GmARI1 significantly enhanced the aluminum tolerance of transgenic Arabidopsis. These findings suggest that GmARI1 encodes a RBR type E3 ligase, which may play important roles in plant tolerance to aluminum stress.

  12. ZmSOC1, a MADS-box transcription factor from Zea mays, promotes flowering in Arabidopsis.

    Science.gov (United States)

    Zhao, Suzhou; Luo, Yanzhong; Zhang, Zhanlu; Xu, Miaoyun; Wang, Weibu; Zhao, Yangmin; Zhang, Lan; Fan, Yunliu; Wang, Lei

    2014-11-03

    Zea mays is an economically important crop, but its molecular mechanism of flowering remains largely uncharacterized. The gene, SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1), integrates multiple flowering signals to regulate floral transition in Arabidopsis. In this study, ZmSOC1 was isolated from Zea mays. Sequence alignment and phylogenetic analysis demonstrated that the ZmSOC1 protein contained a highly conserved MADS domain and a typical SOC1 motif. ZmSOC1 protein was localized in the nucleus in protoplasts and showed no transcriptional activation activity in yeast cells. ZmSOC1 was highly expressed in maize reproductive organs, including filaments, ear and endosperm, but expression was very low in embryos; on the other hand, the abiotic stresses could repress ZmSOC1 expression. Overexpression of ZmSOC1 resulted in early flowering in Arabidopsis through increasing the expression of AtLFY and AtAP1. Overall, these results suggest that ZmSOC1 is a flowering promoter in Arabidopsis.

  13. Sequence and characterization of two Arabidopsis thaliana cDNAs isolated by functional complementation of a yeast gln3 gdh1 mutant.

    Science.gov (United States)

    Truong, H N; Caboche, M; Daniel-Vedele, F

    1997-06-30

    We have isolated two Arabidopsis thaliana cDNAs by complementation of a yeast gln3 gdh1 strain that is affected in the regulation of nitrogen metabolism. The two clones (RGA1 and RGA2) are homologous to each other and to the SCARECROW (SCR) gene that is involved in regulating an asymmetric cell division in plants. RGA1, RGA2 and SCR share several structural features and may define a new family of genes. RGA1 and RGA2 have been mapped, respectively, to chromosome II and I, and their expression in plant is constitutive.

  14. Meiosis-specific gene discovery in plants: RNA-Seq applied to isolated Arabidopsis male meiocytes

    Directory of Open Access Journals (Sweden)

    May Gregory D

    2010-12-01

    Full Text Available Abstract Background Meiosis is a critical process in the reproduction and life cycle of flowering plants in which homologous chromosomes pair, synapse, recombine and segregate. Understanding meiosis will not only advance our knowledge of the mechanisms of genetic recombination, but also has substantial applications in crop improvement. Despite the tremendous progress in the past decade in other model organisms (e.g., Saccharomyces cerevisiae and Drosophila melanogaster, the global identification of meiotic genes in flowering plants has remained a challenge due to the lack of efficient methods to collect pure meiocytes for analyzing the temporal and spatial gene expression patterns during meiosis, and for the sensitive identification and quantitation of novel genes. Results A high-throughput approach to identify meiosis-specific genes by combining isolated meiocytes, RNA-Seq, bioinformatic and statistical analysis pipelines was developed. By analyzing the studied genes that have a meiosis function, a pipeline for identifying meiosis-specific genes has been defined. More than 1,000 genes that are specifically or preferentially expressed in meiocytes have been identified as candidate meiosis-specific genes. A group of 55 genes that have mitochondrial genome origins and a significant number of transposable element (TE genes (1,036 were also found to have up-regulated expression levels in meiocytes. Conclusion These findings advance our understanding of meiotic genes, gene expression and regulation, especially the transcript profiles of MGI genes and TE genes, and provide a framework for functional analysis of genes in meiosis.

  15. Modes of exocytotic and endocytotic events in tobacco BY-2 protoplasts.

    Science.gov (United States)

    Bandmann, Vera; Kreft, Marko; Homann, Ulrike

    2011-03-01

    To analyze the kinetics and size of single exo- and endocytotic events in BY-2 protoplasts, we employed cell-attached membrane capacitance measurements. These measurements revealed different modes of fusion and fission of single vesicles. In about half of the observed exocytotic events, fusion occurred transiently, which facilitates rapid recycling of vesicles. In addition, transient sequential or multi-vesicular exocytosis observed in some recordings can contribute to an increase in efficiency of secretory product release. Microscopic analysis of the timescale of cellulose and pectin deposition in protoplasts demonstrates that rebuilding of the cell wall starts soon after isolation of protoplasts and that transient fusion events can fully account for secretion of the required soluble material. The capacitance measurements also allowed us to investigate formation of the fusion pore. We speculate that regulation of secretion may involve control of the length and/or size of fusion pore opening. Together, the different kinetic modes of exo- and endocytosis revealed by capacitance measurements underline the complexity of this process in plants and provide a basis for future research into the underlying mechanisms. The fact that similar fusion/fission kinetics are present in plant and animal cells suggests that many of these mechanisms are highly conserved among eukaryotes.

  16. Modes of Exocytotic and Endocytotic Events in Tobacco BY-2 Protoplasts

    Institute of Scientific and Technical Information of China (English)

    Vera Bandmann; Marko Kreft; Ulrike Homann

    2011-01-01

    To analyze the kinetics and size of single exo- and endocytotic events in BY-2 protoplasts,we employed cell-attached membrane capacitance measurements. These measurements revealed different modes of fusion and fission of single vesicles. In about half of the observed exocytotic events,fusion occurred transiently,which facilitates rapid recycling of vesicles. In addition,transient sequential or multi-vesicular exocytosis observed in some recordings can contribute to an increase in efficiency of secretory product release. Microscopic analysis of the timescale of cellulose and pectin deposition in protoplasts demonstrates that rebuilding of the cell wall starts soon after isolation of protoplasts and that transient fusion events can fully account for secretion of the required soluble material. The capacitance measurements also allowed us to investigate formation of the fusion pore. We speculate that regulation of secretion may involve control of the length and/or size of fusion pore opening. Together,the different kinetic modes of exo- and endocytosis revealed by capacitance measurements underline the complexity of this process in plants and provide a basis for future research into the underlying mechanisms. The fact that similar fusion/fission kinetics are present in plant and animal cells suggests that many of these mechanisms are highly conserved among eukaryotes.

  17. Thidiazuron-induced plant regeneration from protoplasts of Vicia faba cv. Mythos.

    Science.gov (United States)

    Tegeder, M; Gebhardt, D; Schieder, O; Pickardt, T

    1995-12-01

    Protoplasts of 10 cultivars of V. faba were isolated from etiolated shoot-tips and tested for their regeneration capacity. After purification, protoplasts were embedded in sodium alginate and cultivated in the medium of Kao and Michayluk (1975) containing 0.5 mg·1(-1) of each 2,4-dichlorophenoxyacetic acid, naphthylacetic acid and 6-benzylaminopurine. Depending on cultivar, division frequencies of up to 40% were obtained. Six weeks after embedding, protoplast-derived calluses were transferred to Gelrite-solidified media with different combinations of growth regulators. A two step protocol (auxin high/low) was tested for its ability to induce somatic embryogenesis. The formation of globular structures was observed, but no embryo formation could be achieved. In contrast, cultivation of protocalluses on medium supplemented with thidiazuron resulted in shoot development in cultivar Mythos. To generate mature plants, the shoots were grafted onto young seedlings. In order to optimize the in vitro-conditions, different concentrations of thidiazuron alone or in combination with naphthylacetic acid were tested, showing that an increase of thidiazuron and the addition of naphthylacetic acid positively affects both the viability of protocalluses and the regeneration frequency.

  18. Plant regeneration from protoplasts ofVicia narbonensis via somatic embryogenesis and shoot organogenesis.

    Science.gov (United States)

    Tegeder, M; Kohn, H; Nibbe, M; Schieder, O; Pickardt, T

    1996-11-01

    Protoplasts ofVicia narbonensis isolated from epicotyls and shoot tips of etiolated seedlings were embedded in 1.4% sodium-alginate at a final density of 2.5×10(5) protoplasts/ml and cultivated in Kao and Michayluk-medium containing 0.5 mg/I of each of 2,4- dichlorophenoxyacetic acid, naphthylacetic acid and 6 -benzylaminopurine. A division frequency of 36% and a plating efficiency of 0.40-0.5% were obtained. Six weeks after embedding, protoplast-derived calluses were transferred onto gelrite-solidified Murashige and Skoog-media containing various growth regulators. Regeneration of plants was achieved via two morphologically distinguishable pathways. A two step protocol (initially on medium with a high auxin concentration followed by a culture phase with lowered auxin amount) was used to regenerate somatic embryos, whereas cultivation on medium containing thidiazuron and naphthylacetic acid resulted in shoot morphogenesis. Mature plants were recovered from both somatic embryos as well as from thidiazuron-induced shoots.

  19. Regeneration of transformed shoots from electroporated soybean (Glycine max (L.) Merr.) protoplasts.

    Science.gov (United States)

    Dhir, S K; Dhir, S; Sturtevant, A P; Widholm, J M

    1991-06-01

    Stable transformation of soybean (Glycine max (L.) Merr.) protoplasts isolated from immature cotyledons was achieved following electroporation with plasmid DNA carrying chimeric genes encoding ß-glucuronidase (GUS) and hygromycin phosphotransferase (HPT) under the control of the cauliflower mosaic virus (CaMV) 35S promoter. Transformed colonies were stringently selected by growing 15-day-old protoplast-derived cells in the presence of 40 μg/ml of hygromycin-B for 6 weeks. Over 93% of the resistant cells and colonies exhibited GUS activity, indicating that the two marker genes borne on a single plasmid were co-introduced and co-expressed at a very high freguency. This transformation procedure reproducibly yields transformants at frequencies of 2.9-6.8 × 10(-4) (based on the number of protoplasts electroporated) or 23.0% (based on the number of control microcalli formed) counted after 6 weeks of selection. After repeated subculturing on regeneration medium, shoots were induced from 8.0% of the transformed calli. Southern hybridization confirmed the presence of both the GUS and hygromycin genes in the transformed calli and shoots.

  20. Bacterial spore heat resistance correlated with water content, wet density, and protoplast/sporoplast volume ratio.

    Science.gov (United States)

    Beaman, T C; Greenamyre, J T; Corner, T R; Pankratz, H S; Gerhardt, P

    1982-05-01

    Five types of dormant Bacillus spores, between and within species, were selected representing a 600-fold range in moist-heat resistance determined as a D100 value. The wet and dry density and the solids and water content of the entire spore and isolated integument of each type were determined directly from gram masses of material, with correction for interstitial water. The ratio between the volume occupied by the protoplast (the structures bounded by the inner pericytoplasm membrane) and the volume occupied by the sporoplast (the structures bounded by the outer pericortex membrane) was calculated from measurements made on electron micrographs of medially thin-sectioned spores. Among the various spore types, an exponential increase in the heat resistance correlated directly with the wet density and inversely with the water content and with the protoplast/sporoplast volume ratio. Altogether with results supported a hypothesis that the extent of heat resistance is based in whole or in part on the extent of dehydration and diminution of the protoplast in the dormant spore, without implications about physiological mechanisms for attaining this state.

  1. Polyamine metabolism and osmotic stress. II. Improvement of oat protoplasts by an inhibitor of arginine decarboxylase.

    Science.gov (United States)

    Tiburcio, A F; Kaur-Sawhney, R; Galston, A W

    1986-01-01

    We have attempted to improve the viability of cereal mesophyll protoplasts by pretreatment of leaves with DL-alpha-difluoromethylarginine (DFMA), a specific 'suicide' inhibitor of the enzyme (arginine decarboxylase) responsible for their osmotically induced putrescine accumulation. Leaf pretreatment with DFMA before a 6 hour osmotic shock caused a 45% decrease of putrescine and a 2-fold increase of spermine titer. After 136 hours of osmotic stress, putrescine titer in DFMA-pretreated leaves increased by only 50%, but spermidine and spermine titers increased dramatically by 3.2- and 6-fold, respectively. These increases in higher polyamines could account for the reduced chlorophyll loss and enhanced ability of pretreated leaves to incorporate tritiated thymidine, uridine, and leucine into macromolecules. Pretreatment with DFMA significantly improved the overall viability of the protoplasts isolated from these leaves. The results support the view that the osmotically induced rise in putrescine and blockage of its conversion to higher polyamines may contribute to the lack of sustained cell division in cereal mesophyll protoplasts, although other undefined factors must also play a major role.

  2. Isolation of recombinant strains with enhanced pectinase production by protoplast fusion between Penicillium expansum and Penicillium griseoroseum Isolamento de linhagens recombinantes com maior produção de pectinases por meio de fusão de protoplastos entre Penicillium expansum e Penicillium griseoroseum

    Directory of Open Access Journals (Sweden)

    Maurilio Antonio Varavallo

    2007-03-01

    Full Text Available Protoplast fusion between complementary auxotrophic and morphological mutant strains of Penicillium griseoroseum and P. expansum was induced by polyethylene glycol and calcium ions (Ca2+. Fusant strains were obtained in minimal medium and a prototrophic strain, possibly diploid, was chosen for haplodization with the fungicide benomyl. Different recombinant strains were isolated and characterized for occurrence of auxotrophic mutations and pectinolytic enzyme production. The fusant prototrophic did not present higher pectinase production than the parental strains, but among 29 recombinants analyzed, four presented enhanced enzyme activities. The recombinant RGE27, which possesses the same auxotrophic and morphologic mutations as the P. griseoroseum parental strain, presented a considerable increase in polygalacturonase (3-fold and pectin lyase production (1.2-fold.Fusões de protoplastos entre linhagens mutantes auxotróficas e morfológicas complementares de Penicillium griseoroseum e P. expansum foram induzidas por polietilenoglicol e íons cálcio (Ca2+. Fusionantes foram obtidos em meio mínimo e uma linhagem prototrófica, possivelmente diplóide, foi selecionada para a haploidização com o fungicida benomil. Diferentes linhagens recombinantes foram isoladas e caracterizadas quanto à presença de mutações auxotróficas e a produção de enzimas pectinolíticas. O fusionante prototrófico não apresentou maior atividade de pectinases em relação às linhagens parentais, entretanto, entre 29 recombinantes analisados, quatro apresentaram maiores atividades enzimáticas. O recombinante RGE27, o qual possui as mesmas mutações auxotróficas e morfológicas que a linhagem parental de P. griseoroseum, apresentou um aumento considerável na produção de poligalacturonase (3 vezes e de pectina liase (1,2 vezes.

  3. The keratin intermediate filament—like system in maize protoplasts

    Institute of Scientific and Technical Information of China (English)

    SuFei; GuWei; 等

    1990-01-01

    The application of Penman's method of cell fractionation to plant protoplasts leads to our finding of keratin intermediate filament(IF)-like system in maize protoplasts,which was identified by using immunogold labelling with monoclonal antibody of cytokeratin from animal cells.Many gold particles were found to be bound on filaments,linked by 3 nm filaments.After further digestion and extraction with DNase I and ammonium sulphate.IF-like framework-lamina-nuclear matrix system was shown under electron microscope.That IF system exists in plant protoplasts just like in animal cells,and their main component is keratin-like protein.

  4. An inverse relationship between allelopathic activity and salt tolerance in suspension cultures of three mangrove species, Sonneratia alba, S. caseolaris and S. ovata: development of a bioassay method for allelopathy, the protoplast co-culture method.

    Science.gov (United States)

    Hasegawa, Ai; Oyanagi, Tomoya; Minagawa, Reiko; Fujii, Yoshiharu; Sasamoto, Hamako

    2014-11-01

    A bioassay method for allelopathy, the 'protoplast co-culture method' was developed to study the relationship between salt tolerance and allelopathy of three mangrove species, Sonneratia alba, S. caseolaris, and S. ovata. Plants of S. alba grow in the seaward-side high salinity region and plants of the latter two species grow in upstream-side regions of a mangrove forest, respectively. Effects of five sea salts (NaCl, KCl, MgCl2, MgSO4 and CaCl2) on the growth of the suspension cells of the latter two species were first investigated by a small-scale method using 24-well culture plates. S. ovata cells showed higher tolerance than S. caseolaris cells to NaCl and other salts, but were not as halophilic as S. alba cells. Protoplasts isolated from suspension cells were co-cultured with lettuce protoplasts in Murashige and Skoog's (MS) basal medium containing 1 μM 2,4-dichlorophenoxyacetic acid, 0.1 μM benzyladenine, 3% sucrose and 0.6-0.8 M osmoticum. S. caseolaris protoplasts had a higher inhibitory effect on lettuce protoplast cell divisions than S. alba protoplasts at any lettuce protoplast density, and the effect of S. ovata was intermediate between the two. These results were similar to those obtained from a different in vitro bioassay method for allelopathy, the 'sandwich method' with dried leaves. The inverse relationship between allelopathic activity and salt tolerance in suspension cells of Sonneratia mangroves is discussed.

  5. Transient expression assays in tobacco protoplasts.

    Science.gov (United States)

    Vanden Bossche, Robin; Demedts, Brecht; Vanderhaeghen, Rudy; Goossens, Alain

    2013-01-01

    The sequence information generated through genome and transcriptome analysis from plant tissues has reached unprecedented sizes. Sequence homology-based annotations may provide hints for the possible function and roles of particular plant genes, but the functional annotation remains nonexistent or incomplete for many of them. To discover gene functions, transient expression assays are a valuable tool because they can be done more rapidly and at a higher scale than generating stably transformed tissues. Here, we describe a transient expression assay in protoplasts derived from suspension cells of tobacco (Nicotiana tabacum) for the study of the transactivation capacities of transcription factors. To enhance throughput and reproducibility, this method can be automated, allowing medium-throughput screening of interactions between large compendia of potential transcription factors and gene promoters.

  6. Cadmium uptake and interaction with phytochelatins in wheat protoplasts.

    Science.gov (United States)

    Lindberg, Sylvia; Landberg, Tommy; Greger, Maria

    2007-01-01

    In order to investigate the role of phytochelatins in short-time uptake of Cd(2+) into the cytosol of wheat protoplasts, a new method was applied, using fluorescence microscopy and the heavy metal-specific fluorescent dye, 5-nitrobenzothiazole coumarin, BTC-5N. The uptake of Cd(2+) into protoplasts from 5- to 7-day-old wheat seedlings (Triticum aestivum, L. cv. Kadett) was lower in protoplasts from seedlings raised in the presence of 1 microM CdCl(2), than in the absence. Presence of CdCl(2) in the cultivation medium increased the content of phytochelatins (PCs) in the protoplasts. When seedlings were raised in the presence of both Cd(2+) and buthionine sulfoximine (BSO), an inhibitor of glutathione (GSH) synthesis, only little PC was found in the protoplasts. Pre-treatment with BSO alone did not affect the content of PC, but inhibited that of GSH. The inhibition of GSH was independent of pre-treatment with Cd(2+). Unidirectional flux analyses, using (109)Cd(2+), showed approximately the same uptake pattern of Cd(2+) as did the fluorescence experiments showing the cytosolic uptake of Cd(2+). Thus, the diminished uptake of Cd(2+) into protoplasts from cadmium-pre-treated plants was not depending on PCs. Instead, it is likely that pre-treatment with Cd(2+) causes a down-regulation of the short-term Cd(2+) uptake, or an up-regulation of the Cd(2+) extrusion. Moreover, since addition of Cd(2+) to protoplasts from control plants caused a cytosol acidification, it is likely that a Cd(2+/)H(+)-antiport mechanism is involved in the extrusion of Cd(2+) from these protoplasts.

  7. The regulation of gene expression in transformed maize aleurone and endosperm protoplasts. Analysis of promoter activity, intron enhancement, and mRNA untranslated regions on expression.

    Science.gov (United States)

    Gallie, D R; Young, T E

    1994-11-01

    Gene expression in the aleurone and endosperm is highly regulated during both seed development and germination. Studies of alpha-amylase expression in the aleurone of barley (Hordeum vulgare) have generated the current paradigm for hormonal control of gene expression in germinating cereal grain. Gene expression studies in both the aleurone and endosperm tissues of maize (Zea mays) seed have been hampered because of a lack of an efficient transformation system. We report here the rapid isolation of protoplasts from maize aleurone and endosperm tissue, their transformation using polyethylene glycol or electroporation, and the regulation of gene expression in these cells. Adh1 promoter activity was reduced relative to the 35S promoter in aleurone and endosperm protoplasts compared to Black Mexican Sweet suspension cells in which it was nearly as strong as the 35S promoter. Intron-mediated stimulation of expression was substantially higher in transformed aleurone or endosperm protoplasts than in cell-suspension culture protoplasts, and the data suggest that the effect of an intron may be affected by cell type. To examine cytoplasmic regulation, the 5' and 3' untranslated regions from a barley alpha-amylase were fused to the firefly luciferase-coding region, and their effect on translation and mRNA stability was examined following the delivery of in vitro synthesized mRNA to aleurone and endosperm protoplasts. The alpha-amylase untranslated regions regulated translational efficiency in a tissue-specific manner, increasing translation in aleurone or endosperm protoplasts but not in maize or carrot cell-suspension protoplasts, in animal cells, or in in vitro translation lysates.

  8. The effect of external Ca2+ and Ca2+—channel modulators on red—light—induced swelling of protoplasts of Phaseolus radiatus L.

    Institute of Scientific and Technical Information of China (English)

    LONGCHENG; XIAOJINGWANG; 等

    1998-01-01

    Red-light-induced swelling of the protoplasts isolated from hypocotyl of etiolated mung bean(Phaseolus radiatusL.)was observed only when Ca2+ ions were present in the medium.The optimal CaCl2 concentration was 250μM,Swlling response declined when Ca2+ was supplied into the medium after red light irradiation.The Ca2+-chelator EGTA eliminated the red-light-induced swelling and 45Ca2+ accumulation in the protoplasts.In conltrast,A23187,a Ca2+-ionophore,could mimic the effect of red light in darkness.These results indicate that Ca2+ may play a role in light signal transduction.In addition,swelling response was prevented by TFP and CPZ(both are CaM antagonists),implying the involvement of CaM in red-light-induced and Ca2+ -dependent protoplast swelling.

  9. Positive fluorescent selection permits precise, rapid, and in-depth overexpression analysis in plant protoplasts.

    Science.gov (United States)

    Bargmann, Bastiaan O R; Birnbaum, Kenneth D

    2009-03-01

    Transient genetic modification of plant protoplasts is a straightforward and rapid technique for the study of numerous aspects of plant biology. Recent studies in metazoan systems have utilized cell-based assays to interrogate signal transduction pathways using high-throughput methods. Plant biologists could benefit from new tools that expand the use of cell culture for large-scale analysis of gene function. We have developed a system that employs fluorescent positive selection in combination with flow cytometric analysis and fluorescence-activated cell sorting to isolate responses in the transformed protoplasts exclusively. The system overcomes the drawback that transfected protoplast suspensions are often a heterogeneous mix of cells that have and have not been successfully transformed. This Gateway-compatible system enables high-throughput screening of genetic circuitry using overexpression. The incorporation of a red fluorescent protein selection marker enables combined utilization with widely available green fluorescent protein (GFP) tools. For instance, such a dual labeling approach allows cytometric analysis of GFP reporter gene activation expressly in the transformed cells or fluorescence-activated cell sorting-mediated isolation and downstream examination of overexpression effects in a specific GFP-marked cell population. Here, as an example, novel uses of this system are applied to the study of auxin signaling, exploiting the red fluorescent protein/GFP dual labeling capability. In response to manipulation of the auxin response network through overexpression of dominant negative auxin signaling components, we quantify effects on auxin-responsive DR5::GFP reporter gene activation as well as profile genome-wide transcriptional changes specifically in cells expressing a root epidermal marker.

  10. Isolation of protoplast from Kappaphycus and Eucheuma using crude extracts of Siganus fuscessens viscus%篮子鱼内脏粗提液制备长心卡帕藻和细齿麒麟菜原生质体的初步研究

    Institute of Scientific and Technical Information of China (English)

    李俊鹏; 刘建国; 庞通; 李虎

    2014-01-01

    Protoplast of Kappaphycus and Eucheuma, two main traditional carrageenan-producing seaweeds, were prepared using crude enzyme extracts of Siganus fuscessens viscus from Sep, 2011 to Jan, 2013 at our tropical seaweed experimental station in Lingshui, Hainan. Both the stomach and liver of S. fuscessens viscus as well as the young branches of Kappaphycus and Eucheuma were pre-homogenized. Filtrate of S. fuscessens was used as the crude enzymatic extracts to digest the sendimented pellets of Kappaphycus and Eucheuma. Then, the total protoplast output and protoplast yield per gram of the algal homogenate pellets exposed to gradients of pH, temeprature and enzymic extracts were compared. The results showed that the protoplasts from Eucheuma were easily prepared compared to Kappaphycus, and that the amount of protoplast obtained depended on the digestion time, dosage of enzyme extract and the algal pellets. The more the algal homogenate pellets were added in the tested range (0.1-0.4 g fresh weight), the higher the total protoplast output and the lower protoplast yield per gram biomass were obtained. Meanwhile, the more enzyme extract was added, the higher the total protoplast output and the protoplast yield per gram biomass were harvested. Prolonging the enzymatic digestion time could linearly improve the total protoplast output and the protoplast yield per gram. The pH and temperature also significantly affected the protoplast production. The optimal pH and temperature for protoplast preparation were pH 6.0 and 25℃, respectively. Based on the above studies, a optmized mode for Kappaphycus and Eucheuma protoplast preparation was suggested as below:0.1 g homogenized algal pellets, 3 ml crude enzyme extract from 0.15 g homogenate of S. fuscessens stomach and liver with 50 mM phosphate buffer (pH 6.0), then adjusted the mixture pH to 6.0 and incubated at 25℃ for≥48 h.%于2011年9月至2013年1月,在海南陵水热带海藻实

  11. Genetic analysis of a host determination mechanism of bromoviruses in Arabidopsis thaliana.

    Science.gov (United States)

    Fujisaki, Koki; Iwahashi, Fukumatsu; Kaido, Masanori; Okuno, Tetsuro; Mise, Kazuyuki

    2009-03-01

    Brome mosaic virus (BMV) and Spring beauty latent virus (SBLV) are closely related, tripartite RNA plant viruses. In Arabidopsis thaliana, BMV shows limited multiplication whereas SBLV efficiently multiplies. Such distinct multiplication abilities have been observed commonly in all Arabidopsis accessions tested. We used this model system to analyze the molecular mechanism of viral resistance in plants at the species level. Unlike SBLV, BMV multiplication was limited even in protoplasts and a reassortment assay indicated that at least viral RNA1 and/or RNA2 determine such distinct infectivities. By screening Arabidopsis mutants with altered defense responses, we found that BMV multiplies efficiently in cpr5-2 mutant plants. This mutation specifically enhanced BMV multiplication in protoplasts, which depended on the functions of RNA1 and RNA2. In the experiment using DNA vectors to express BMV replication proteins encoded by RNA1 and RNA2, BMV RNA3 accumulation in cpr5-2 protoplasts was similar to that in wild-type Col-0 protoplasts, despite significant reduction of accumulation levels of replication proteins, suggesting that cpr5-2 mutation could enhance BMV multiplication independently of increased accumulation, therefore enhanced translation and stabilization, of the replication proteins.

  12. Ethylene antagonizes salt-induced growth retardation and cell death process via transcriptional controlling of ethylene-, BAG- and senescence-associated genes in Arabidopsis

    Directory of Open Access Journals (Sweden)

    YaJie ePan

    2016-05-01

    Full Text Available The existing question whether ethylene is involved in the modulation of salt-induced cell death to mediate plant salt tolerance is important for understanding the salt tolerance mechanisms. Here, we employed Arabidopsis plants to study the possible role of ethylene in salt-induced growth inhibition and programmed cell death (PCD profiles. The root length, DNA ladder and cell death indicated by Evan’s blue detection were measured by compared to the control or salt-stressed seedlings. Secondly, the protoplasts isolated from plant leaves and dyed with Annexin V-FITC were subjected to flow cytometric (FCM assay. Our results showed that ethylene works effectively in seedling protoplasts, antagonizing salt-included root retardation and restraining cell death both in seedlings or protoplasts. Due to salinity, the entire or partial insensitivity of ethylene signaling resulted in an elevated levels of cell death in ein2-5 and ein3-1 plants and the event were amended in ctr1-1 plants after salt treatment. The subsequent experiment with exogenous ACC further corroborated that ethylene could modulate salt-induced PCD process actively. Plant Bcl-2-associated athanogene (BAG family genes are recently identified to play an extensive role in plant PCD processes ranging from growth, development to stress responses and even cell death. Our result showed that salinity alone significantly suppressed the transcripts of BAG6, BAG7 and addition of ACC in the saline solution could obviously re-activate BAG6 and BAG7 expressions, which might play a key role to inhibit the salt-induced cell death. In summary, our research implies that ethylene and salinity antagonistically control BAG family-, ethylene-, and senescence-related genes to alleviate the salt-induced cell death.

  13. Isolation and RNA gel blot analysis of genes that could serve as potential molecular markers for leaf senescence in Arabidopsis thaliana.

    Science.gov (United States)

    Yoshida, S; Ito, M; Nishida, I; Watanabe, A

    2001-02-01

    Nine cDNAs, representing genes in which the transcripts accumulated in senescent leaves of Arabidopsis thaliana, were isolated by differential display reverse transcription polymerase chain reaction (DDRT-PCR) and the genes were designated yellow-leaf-specific gene 1 to 9 (YLS1-YLS9). Sequence analysis revealed that none of the YLS genes, except YLS6, had been reported as senescence-up-regulated genes. RNA gel blot analysis revealed that the transcripts of YLS3 accumulated at the highest level at an early senescence stage, whereas the transcripts from the other YLS genes reached their maximum levels in late senescence stages. Transcripts of YLS genes showed various accumulation patterns under natural senescence, and under artificial senescence induced by darkness, ethylene or ABA. These expression characteristics of YLS genes will be useful as potential molecular markers, which will enhance our understanding of natural and artificial senescence processes.

  14. Elastic constant of Dendrobium protoplasts in AC electric fields

    Directory of Open Access Journals (Sweden)

    Pikul Wanichapichart

    2002-11-01

    Full Text Available This work reports elongation of Dendrobium protoplasts in an ac electric field between two cylindrical electrodes. A protoplast firstly was translated towards an electrode by dielectrophoretic force in 17 kV.m-1 field strength at 1 MHz, and secondly it was elongated due to an interaction between an induced electric dipole (μ and the electric field (E. Protoplast elongation was observed by varying both the field strength at 30, 45, 60, and 85 kV.m-1 and field frequency at 0.5, 1, 5, and 10 MHz. For a given field frequency and field strength, a parameter a/b (major/minor axis was measured as the protoplast elongation.Two-step elongation and restoration phases were observed. The former was completed within 2 minutes of field exposure, and the latter was completed within 15 seconds regardless of the field exposure time between 3 and 20 minutes. The evidence of a complete restoration indicated that the elasticity of the protoplast membrane obeyed Hooke’s law. This study also found that elastic constant k of the membrane varied non-linearly with the field strength. It was found to be from 0.04 to 0.08 mN.m-1, dependent on the field frequency.

  15. Callus formation from leaf mesophyll protoplasts of Malus Xdomestica Borkh. cv. Greensleeves.

    Science.gov (United States)

    Doughty, S; Power, J B

    1988-05-01

    Large yields (1.85 × 10(7)/g.f.wt.) of viable protoplasts were obtained from leaves of axenic shoot cultures of Malus Xdomestica Borkh. cv. 'Greensleeves'. Protoplasts cultured in liquid or agarose semi-solidified KM8P medium underwent cell wall regeneration and colony formation.Protoplast-derived cell colonies developed to callus on semi-solid KM8 medium. This is the first report of callus formation from mesophyll protoplasts of apple.

  16. Development of plant protoplasts during the IML-1 mission

    Science.gov (United States)

    Rasmussen, O.; Bondar, R. L.; Baggerud, C.; Iversen, T.-H.

    1994-08-01

    During the 8 day IML-1 mission, regeneration of cell walls and cell divisions in rapeseed protoplasts were studied using the Biorack microscope onboard the Space Shuttle ``Discovery''. Samples from μ-g and 1g protoplast cultures were loaded on microscope slides. Visual microscopic observations were reported by the payload specialist Roberta Bondar, by down-link video transmission and by use of a microscope camera. Protoplasts grown under μ-g conditions do regenerate cell walls but to a lesser extent than under 1 g. Cell divisions are delayed under μ-g. Few cell aggregates with maximum 4-6 cells per aggregate are formed under μ-g conditions, indicating that microgravity may have a profound influence on plant cell differentiation.

  17. Simulated microgravity inhibits cell wall regeneration of Penicillium decumbens protoplasts

    Science.gov (United States)

    Zhao, C.; Sun, Y.; Yi, Z. C.; Rong, L.; Zhuang, F. Y.; Fan, Y. B.

    2010-09-01

    This work compares cell wall regeneration from protoplasts of the fungus Penicillium decumbens under rotary culture (simulated microgravity) and stationary cultures. Using an optimized lytic enzyme mixture, protoplasts were successfully released with a yield of 5.3 × 10 5 cells/mL. Under simulated microgravity conditions, the protoplast regeneration efficiency was 33.8%, lower than 44.9% under stationary conditions. Laser scanning confocal microscopy gave direct evidence for reduced formation of polysaccharides under simulated conditions. Scanning electron microscopy showed the delayed process of cell wall regeneration by simulated microgravity. The delayed regeneration of P. decumbens cell wall under simulated microgravity was likely caused by the inhibition of polysaccharide synthesis. This research contributes to the understanding of how gravitational loads affect morphological and physiological processes of fungi.

  18. Benzoylsalicylic acid isolated from seed coats of Givotia rottleriformis induces systemic acquired resistance in tobacco and Arabidopsis.

    Science.gov (United States)

    Kamatham, Samuel; Neela, Kishore Babu; Pasupulati, Anil Kumar; Pallu, Reddanna; Singh, Surya Satyanarayana; Gudipalli, Padmaja

    2016-06-01

    Systemic acquired resistance (SAR), a whole plant defense response to a broad spectrum of pathogens, is characterized by a coordinated expression of a large number of defense genes. Plants synthesize a variety of secondary metabolites to protect themselves from the invading microbial pathogens. Several studies have shown that salicylic acid (SA) is a key endogenous component of local and systemic disease resistance in plants. Although SA is a critical signal for SAR, accumulation of endogenous SA levels alone is insufficient to establish SAR. Here, we have identified a new acyl derivative of SA, the benzoylsalicylic acid (BzSA) also known as 2-(benzoyloxy) benzoic acid from the seed coats of Givotia rottleriformis and investigated its role in inducing SAR in tobacco and Arabidopsis. Interestingly, exogenous BzSA treatment induced the expression of NPR1 (Non-expressor of pathogenesis-related gene-1) and pathogenesis related (PR) genes. BzSA enhanced the expression of hypersensitivity related (HSR), mitogen activated protein kinase (MAPK) and WRKY genes in tobacco. Moreover, Arabidopsis NahG plants that were treated with BzSA showed enhanced resistance to tobacco mosaic virus (TMV) as evidenced by reduced leaf necrosis and TMV-coat protein levels in systemic leaves. We, therefore, conclude that BzSA, hitherto unknown natural plant product, is a new SAR inducer in plants.

  19. Review of Plant Protoplast Recalcitrance and Its Physiological and Genetic Bases%植物原生质体顽拗现象及其生理和遗传基础研究进展

    Institute of Scientific and Technical Information of China (English)

    曹文娟; 蔡小东

    2012-01-01

    Regeneration ability of protoplast is the prerequisite for biotechnology breeding using protoplasts as initial materials. However, protoplast culture of some plant genotypes has not been established successfully at present. This review focused on the phenomenon of plant protoplast recalcitrance and relevant physiological and genetic bases from the aspects of the phenomenon of oxidative stress during the process of protoplast isolation and culture as well as the molecular mechanisms of the difference of regeneration ability of plant protoplasts.%利用原生质体进行生物技术育种的先决条件是其能再生完整植株,但目前一些基因型植物原生质体的培养仍然未获得成功.综述了植物原生质体顽拗现象,并从原生质体分离和培养过程中的氧化胁迫、原生质体再生能力差异的分子机制方面对与该现象有关的生理和遗传基础研究进展进行了综述.

  20. Environmentally induced programmed cell death in leaf protoplasts of Aponogeton madagascariensis.

    Science.gov (United States)

    Lord, Christina E N; Gunawardena, Arunika H L A N

    2011-02-01

    Within plant systems, two main forms of programmed cell death (PCD) exist: developmentally regulated and environmentally induced. The lace plant (Aponogeton madagascariensis) naturally undergoes developmentally regulated PCD to form perforations between longitudinal and transverse veins over its leaf surface. Developmental PCD in the lace plant has been well characterized; however, environmental PCD has never before been studied in this plant species. The results presented here portray heat shock (HS) treatment at 55 °C for 20 min as a promising inducer of environmental PCD within lace plant protoplasts originally isolated from non-PCD areas of the plant. HS treatment produces cells displaying many characteristics of developmental PCD, including blebbing of the plasma membrane, increased number of hydrolytic vesicles and transvacuolar strands, nuclear condensation, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling positive nuclei, as well as increased Brownian motion within the vacuole. Results presented here for the first time provide evidence of chloroplasts in the vacuole of living protoplasts undergoing environmentally induced PCD. Findings suggest that the mitochondria play a critical role in the cell death process. Changes in mitochondrial dynamics were visualized in HS-treated cells, including loss of mitochondrial mobility, reduction in ΔΨ(m), as well as the proximal association with chloroplasts. The role of the mitochondrial permeability transition pore (PTP) was examined by pre-treatment with the PTP agonist cyclosporine A. Overall, HS is depicted as a reliable method to induce PCD within lace plant protoplasts, and proves to be a reliable technique to enable comparisons between environmentally induced and developmentally regulated PCD within one species of plant.

  1. Direct somatic embryogenesis from protoplasts of Foeniculum vulgare.

    Science.gov (United States)

    Miura, Y; Tabata, M

    1986-08-01

    Protoplasts prepared from an embryogenic cell suspension culture of fennel gave rise to somatic embryoids directly through unequal cell divisions of enlarged, ellipsoidal cells, when embedded in hormone-free LS agarose medium. On the other hand, protoplasts embedded in LS agarose medium containing 2,4-D and kinetin proliferated through unpolarized cell divisions to form calli, which gave somatic embryoids on the surface upon transfer onto the same medium. In either case, somatic embryoids germinated to develop into normal plantlets when cultured on hormone-free LS agar medium under illumination.

  2. 一种筛选拟南芥突变体的有效方法%A Efficient Method for Isolation of Arabidopsis Mutants

    Institute of Scientific and Technical Information of China (English)

    赵淑清

    2001-01-01

    his paper introduces a root-bending assay for isol ation of Arabidopsis mutants tolerant to nutrition stress. Seeds of wild-ty pe Arabidopsis thaliana (ecotype Landersberg erecta) were mutagenized wi th ethyl methyl sulfide (EMS),and M2 populations were screened for mutants. Fo ur-day-old seedlings with 1-to 1.5-cm-long roots were transferred from the vertical agar plates onto to a second agar medium that was supplemented with det erminate stress. The seedlings were arranged in rows, and the plates were orient ed vertically with the roots pointing upward. After another 4 days, the root be nding seedlings were selected for putative mutants and transferred to soil to gr ow to maturity.Seeds from the putative mutants were screened again to determine the true mutants.By using this root-bending assay we have isolated a low-K+ -tolerant (lkt1) mutant which is caused by single recessive nuclear mutation. F or lkt1 mutant screening,K+concentration of the medium was 100μmol/L because root growth of wild type seedlings was completely inhibited at or below this con centration.This root-bending assay is also applicable to other type of Arabid opsis mutant isolation.%经甲基磺酸乙酯(EMS)诱变处理的拟南芥种子,接种于MS培养基上,垂直放置培养4天后,将幼苗转移至胁迫培养基中,以倒置幼苗180°所形成的弯曲生长根作为指标筛选拟南芥耐营养胁迫突变体。利用这种方法,成功地筛选到一个耐低钾的隐性单基因拟南芥突变体。本方法同样适用于其他类型突变体的筛选。

  3. Area Expansivity Moduli of Regenerating Plant Protoplast Cell Walls Exposed to Shear Flows

    Science.gov (United States)

    Fujimura, Yuu; Iino, Masaaki; Watanabe, Ugai

    2005-05-01

    To control the elasticity of the plant cell wall, protoplasts isolated from cultured Catharanthus roseus cells were regenerated in shear flows of 115 s-1 (high shear) and 19.2 s-1 (low shear, as a control). The surface area expansivity modulus and the surface breaking strength of these regenerating protoplasts were measured by a micropipette aspiration technique. Cell wall synthesis was also measured using a cell wall-specific fluorescent dye. High shear exposure for 3 h doubled both the surface area modulus and breaking strength observed under low shear, significantly decreased cell wall synthesis, and roughly quadrupled the moduli of the cell wall. Based on the cell wall synthesis data, we estimated the three-dimensional modulus of the cell wall to be 4.1± 1.2 GPa for the high shear, and 0.35± 0.2 GPa for the low shear condition, using the surface area expansivity modulus divided by the cell wall thickness, which is identical with the Young’s modulus divided by 2(1-σ), where σ is Poisson's ratio. We concluded that high shear exposure considerably strengthens the newly synthesized cell wall.

  4. Transformation of soybean protoplasts from permanent suspension cultures by cocultivation with cells of Agrobacterium tumefaciens.

    Science.gov (United States)

    Baldes, R; Moos, M; Geider, K

    1987-03-01

    Cell wall regenerating protoplasts from soybean cells kept in suspension culture were cocultivated with bacteria which were derived from the nopaline strain C58 of Agrobacterium tumefaciens. When the bacteria carried an oncogenic Ti-plasmid, about 5% of the surviving protoplasts were able to form calli on hormone-free agar in contrast to controls, where bacteria without Ti-plasmid were applied, and where no calli were formed. After isolation of DNA from hormone-independently growing cells further evidence for transformation was obtained by hybridization to Ti-plasmid specific RNA and by rescue of a segment with a bacterial resistance gene which had been inserted before into the T-DNA. Transfer of T-DNA harboring a neomycin-resistance gene activated by the nos-promoter resulted in calli growing on kanamycin. Verification of segments located at the left and the right part of the T-DNA indicated the presence of its entire length in transformed soybean cells. Expression of T-DNA genes was measured by the assay of nopaline-synthase. Cells cultured on agar had a much higher level of nopaline-synthase than fast growing cells in suspension culture. Transferring them to agar or treatment with azacytidine strongly increased synthesis of nopaline-synthase indicating a reversible repression presumably via a methylation mechanism.

  5. The location of aluminium in protoplasts and suspension cells taken from Coffea arabica L. with different tolerance of Al.

    Science.gov (United States)

    Ramírez-Benítez, J Efraín; Hernández-Sotomayor, S M Teresa; Muñoz-Sánchez, J Armando

    2009-11-01

    Biotechnological advances in coffee research (in vitro manipulation, multiplication, generation and development of transgenic coffee plants with specific traits like high yield and good quality) have contributed to description of the metabolic pathways involved in the response mechanisms to environmental factors like abiotic stress. Coffea arabica L. plants grow in acidic soils, and therefore aluminium (Al) toxicity is a major negative impact on crop productivity. To understand Al toxicity mechanisms in cells via the Al absorption kinetic, we isolated protoplasts from two C. arabica L. suspension cell lines: Al-sensitive (L2) and Al-tolerant (LAMt). Protoplasts of LAMt line exhibited lower Al absorption levels than protoplasts of the L2 line. Use of two fluorescent tracers (morin and calcofluor white) indicated that Al interacts with internal cell structures, such as the plasma membrane and nucleus, with differences in both cell lines. Al-tolerance in the LAMt is probably associated with the cell wall as well as intracellular structures. These data will help to better understand Al toxicity in C. arabica, and Al toxicity mechanisms in plant cells.

  6. Polyamine levels as related to growth, differentiation and senescence in protoplast-derived cultures of Vigna aconitifolia and Avena sativa

    Science.gov (United States)

    Kaur Sawhney, R.; Shekhawat, N. S.; Galston, A. W.

    1985-01-01

    We have previously reported that aseptically cultured mesophyll protoplasts of Vigna divide rapidly and regenerate into complete plants, while mesophyll protoplasts of Avena divide only sporadically and senesce rapidly after isolation. We measured polyamine titers in such cultures of Vigna and Avena, to study possible correlations between polyamines and cellular behavior. We also deliberately altered polyamine titer by the use of selective inhibitors of polyamine biosynthesis, noting the effects on internal polyamine titer, cell division activity and regenerative events. In Vigna cultures, levels of free and bound putrescine and spermidine increased dramatically as cell division and differentiation progressed. The increase in bound polyamines was largest in embryoid-forming callus tissue while free polyamine titer was highest in root-forming callus. In Avena cultures, the levels of total polyamines decreased as the protoplast senesced. The presence of the inhibitors alpha-difluoromethyl-arginine (specific inhibitor of arginine decarboxylase), alpha-difluoromethylornithine (specific inhibitor of ornithine decarboxylase) and dicyclohexylamine (inhibitor of spermidine synthase) reduced cell division and organogenesis in Vigna cultures. Addition of low concentration of polyamines to such cultures containing inhibitors or removal of inhibitors from the culture medium restored the progress of growth and differentiation with concomitant increase in polyamine levels.

  7. Purification of Intact Plant Protoplasts by Flotation at 1g

    Directory of Open Access Journals (Sweden)

    John Graham

    2002-01-01

    Full Text Available From a standard plant tissue digest adjusted to a density of 1.07 g/ml, protoplasts can be harvested by flotation through a low density barrier (1.03 g/ml. The delicate nature of these bodies is suited to this flotation strategy which can be carried out at 1g.

  8. Transformation of undomesticated strains of Bacillus subtilis by protoplast electroporation.

    Science.gov (United States)

    Romero, Diego; Pérez-García, Alejandro; Veening, Jan-Willem; de Vicente, Antonio; Kuipers, Oscar P

    2006-09-01

    A rapid method combining the use of protoplasts and electroporation was developed to transform recalcitrant wild strains of Bacillus subtilis. The method described here allows transformation with both replicative and integrative plasmids, as well as with chromosomal DNA, and provides a valuable tool for molecular genetic analysis of interesting Bacillus strains, which are hard to transform by conventional methods.

  9. Transformation of undomesticated strains of Bacillus subtilis by protoplast electroporation

    NARCIS (Netherlands)

    Romero, Diego; Perez-Garcia, Alejandro; Veening, Jan-Willem; de Vicente, Antonio; Kuipers, Oscar P.; de, Vicente A.

    2006-01-01

    A rapid method combining the use of protoplasts and electroporation was developed to transform recalcitrant wild strains of Bacillus subtilis. The method described here allows transformation with both replicative and integrative plasmids, as well as with chromosomal DNA, and provides a valuable tool

  10. Dynamic organization of actin cytoskeleton during the polarity formation and germination of pollen protoplasts

    Institute of Scientific and Technical Information of China (English)

    XU Xia; Zl Huijun; SUN Yina; REN Haiyun

    2004-01-01

    The formation of the polarity of pollen protoplast and the dynamics of actin cytoskeleton were observed by non-fixation, Alexa-Phalloidin probing and confocal laser scanning microscopy. Our results showed that the protoplast obtained from stored pollen contained numerous crystalline fusiform bodies to constitute a storage form of actin. When dormant pollen was hydrated, the actin cytoskeleton forms a fine network spreading uniformly in the protoplast. In the process of polarity formation and germination of pollen protoplast, actin filaments marshaled slowly to the brim, and then formed multilayer continuous actin filament bundles surrounding the cortical of the protoplast. When the protoplast was exposed to actin filament-disrupting drugs, such as Latrunculin A and Cytochalasin D, continuously arranged actin bundles were disturbed and in this condition, the protoplast could not germinate. But when exposed to actin filament stabiling drug-phalliodin, the dynamics of actin filaments in the protoplasts behaved normally and the protoplasts could germinate normally. These results were also confirmed by the pharmacology experiments on pollen grains. And when Latrunculin A or Cytochalasin D was washed off, the ratio of pollen germination was resumed partly. All the results above show that the dynamic organization of the actin cytoskeleton are critical in the cell polarity formation and germination of pollen protoplast, and that the reorganization of actin cytoskeleton is mainly due to the rearrangement of actin filament arrays.

  11. Isolation of T—DNA flanking plant DNA from T—DNA insertional embryo—lethal mutants of Arabidopsis thaliana by plasmid rescue technique

    Institute of Scientific and Technical Information of China (English)

    YAOXIAOLI; JIANGESUN; 等

    1996-01-01

    Three T-DNA insertional embryonic lethal mutants from NASC(The Nottingham Arabidopsis Stock Center) were first checked with their segregation ratio of abortive and normal seeds and the copy number of T-DNA insertion.The N4081 mutant has a segregation ratio of 1:3.04 in average and one T-DNA insertion site according to our assay.It was therefore chosen for further analysis.To isolate the joint fragment of T-DNA and plant DNA,the plasmid rescue technique was used.pEL-7,one of plasmids from left border of T-DNA,which contained pBR322 was selected from ampicillin plate.The T-DNA fragment of pEL-7 was checked by restriction enzyme analysis and Southern Blot.Restriction analysis confirmed the presence of known sites of EcoRI,PstI and PvuII on it.For confirming the presence of flanking plant DNA in this plasmid,pEL-7 DNA was labeled and hybridized with wild type and mutant plant DNA.The Southern Blot indicated the hybridization band in both of them.Furthermore,the junction of T-DNA/plant DNA was subcloned into bluescript SK+ and sequenced by Applied Biosystem 373A sequencer.The results showed the 822 bp fragment contained a 274 bp sequence,which is 99.6%homolog(273bp/274bp) to Ti plasmid pTi 15955,DNA.The bp of left 25 bp border repeat were also found in the juction of T-DNA and Plant DNA. Taken together,pEL-7 should coutain a joint fragment of T-DNA and flanking plant DNA.This plasmid DNA could be used for the isolation of plant gene,which will be helpful to elucidate the relationship between gene function and plant embryo development.

  12. Plant regeneration via somatic embryogenesis from protoplast of Clausena harmandiana (Engl. Swing and M. Kell

    Directory of Open Access Journals (Sweden)

    Hasan Basri JUMIN

    2013-05-01

    Full Text Available Protoplasts isolated from embryogenic callus of Clausena harmandiana (Engl. 'Swing. and M. Kell. were cultured in MT (Murashige and Tucker 1969 basal medium containing 5% sucrose supplemented with bezyladenine (BA, malt extract (ME and 0.6 M sorbitol. The highest plating efficiency was obtained on MT basal medium containing 5% sucrose supplemented with 0.01 mg 1-1 BA and 600 mg 1-1 ME, MT basal medium containing 5% sucrose and supplemented with 0.01 mg 1-1 6(-y,y-dimethylallylamino-purine was found to be a medium suitable for the development somatic embryos into heart-shaped somatic embryos. The highest percentage of shoot formation- was obtained using 0.1 mg 1-1 gibberellic acid (GA3 + 0.1 mg 1-1 zeatin. In this investigation 25 plants were survived and grew normally in the soil.

  13. Localization of acid hydrolases in protoplasts. Examination of the proposed lysosomal function of the mature vacuole

    Energy Technology Data Exchange (ETDEWEB)

    Butcher, H.C.; Wagner, G.J.; Siegelman, H.W.

    1977-06-01

    The development of techniques to isolate and purify relatively large quantities of intact vacuoles from mature tissues permits direct biochemical analysis of this ubiquitous mature plant cell organelle. Vacuoles and a fraction enriched in soluble cytoplasmic constituents were quantitatively prepared from Hippeastrum flower petal protoplasts. Vacuolar lysate and soluble cytoplasmic fractions were examined for acid hydrolase activities commonly associated with animal lysosomes, and pH optima were determined. Esterase, protease, carboxypeptidase, ..beta..-galactosidase, ..cap alpha..-glycosidase and ..beta..-glycosidase, not found in the vacuole lysate fraction, were components of the soluble cytoplasmic fraction. Acid phosphatase, RNase and DNase were present in both fractions. Vacuolar enzyme activities were also examined as a function of flower development from bud through senescent stages. The data obtained are not consistent with the concept that the mature plant cell vacuole functions as a generalized lysosome.

  14. Screening, mutagenesis and protoplast fusion of Aspergillus niger for the enhancement of extracellular glucose oxidase production.

    Science.gov (United States)

    Khattab, A A; Bazaraa, W A

    2005-07-01

    Various strains of Aspergillus niger were screened for extracellular glucose oxidase (GOD) activity. The most effective producer, strain FS-3 (15.9 U mL(-1)), was mutagenized using UV-irradiation or ethyl methane sulfonate. Of the 400 mutants obtained, 32 were found to be resistant to 2-deoxy D: -glucose, and 17 of these exhibited higher GOD activities (from 114.5 to 332.1%) than the original FS-3 strain. Following determination of antifungal resistance of the highest producing mutants, four mutants were selected and used in protoplast fusions in three different intraspecific crosses. All fusants showed higher activities (from 285.5 to 394.2%) than the original strain. Moreover, of the 30 fusants isolated, 19 showed higher GOD activity than their corresponding higher-producing parent strain.

  15. The Arabidopsis a zinc finger domain protein ARS1 is essential for seed germination and ROS homeostasis in response to ABA and oxidative stress

    Directory of Open Access Journals (Sweden)

    Dongwon eBaek

    2015-11-01

    Full Text Available The phytohormone abscisic acid (ABA induces accumulation of reactive oxygen species (ROS, which can disrupt seed dormancy and plant development. Here, we report the isolation and characterization of an Arabidopsis thaliana mutant called ars1 (aba and ros sensitive 1 that showed hypersensitivity to ABA during seed germination and to methyl viologen (MV at the seedling stage. ARS1 encodes a nuclear protein with one zinc finger domain, two nuclear localization signal (NLS domains, and one nuclear export signal (NES. The ars1 mutants showed reduced expression of a gene for superoxide dismutase (CSD3 and enhanced accumulation of ROS after ABA treatment. Transient expression of ARS1 in Arabidopsis protoplasts strongly suppressed ABA-mediated ROS production. Interestingly, nuclear-localized ARS1 translocated to the cytoplasm in response to treatment with ABA, H2O2, or MV. Taken together, these results suggest that ARS1 modulates seed germination and ROS homeostasis in response to ABA and oxidative stress in plants.

  16. De novo pyrimidine nucleotide synthesis mainly occurs outside of plastids, but a previously undiscovered nucleobase importer provides substrates for the essential salvage pathway in Arabidopsis.

    Science.gov (United States)

    Witz, Sandra; Jung, Benjamin; Fürst, Sarah; Möhlmann, Torsten

    2012-04-01

    Nucleotide de novo synthesis is highly conserved among organisms and represents an essential biochemical pathway. In plants, the two initial enzymatic reactions of de novo pyrimidine synthesis occur in the plastids. By use of green fluorescent protein fusions, clear support is provided for a localization of the remaining reactions in the cytosol and mitochondria. This implies that carbamoyl aspartate, an intermediate of this pathway, must be exported and precursors of pyrimidine salvage (i.e., nucleobases or nucleosides) are imported into plastids. A corresponding uracil transport activity could be measured in intact plastids isolated from cauliflower (Brassica oleracea) buds. PLUTO (for plastidic nucleobase transporter) was identified as a member of the Nucleobase:Cation-Symporter1 protein family from Arabidopsis thaliana, capable of transporting purine and pyrimidine nucleobases. A PLUTO green fluorescent protein fusion was shown to reside in the plastid envelope after expression in Arabidopsis protoplasts. Heterologous expression of PLUTO in an Escherichia coli mutant lacking the bacterial uracil permease uraA allowed a detailed biochemical characterization. PLUTO transports uracil, adenine, and guanine with apparent affinities of 16.4, 0.4, and 6.3 μM, respectively. Transport was markedly inhibited by low concentrations of a proton uncoupler, indicating that PLUTO functions as a proton-substrate symporter. Thus, a protein for the absolutely required import of pyrimidine nucleobases into plastids was identified.

  17. Endocytosis of cationized ferritin by coated vesicles of soybean protoplasts.

    Science.gov (United States)

    Tanchak, M A; Griffing, L R; Mersey, B G; Fowke, L C

    1984-12-01

    Soybean (Glycine max (L.) Merr.) protoplasts have been surface-labelled with cationized ferritin, and the fate of the label has been followed ultrastructurally. Endocytosis of the label occurs via the coated-membrane system. The pathway followed by the label, once it has been taken into the interior of the protoplast, appears to be similar to that found during receptor-mediated endocytosis in animal cells. Cationized ferritin is first seen in coated vesicles but rapidly appears in smooth vesicles. Labelled, partially coated vesicles are occasionally observed, indicating that the smooth vesicles may have arisen by the uncoating of coated vesicles. Structures which eventually become labelled with cationized ferritin include multivesicular bodies, dictyosomes, large smooth vesicles, and a system of partially coated reticula.

  18. Transformation of undomesticated strains of Bacillus subtilis by protoplast electroporation

    OpenAIRE

    Romero, Diego; Perez-Garcia, Alejandro; Veening, Jan-Willem; Vicente, Antonio; Oscar P. Kuipers; Vicente A.

    2006-01-01

    A rapid method combining the use of protoplasts and electroporation was developed to transform recalcitrant wild strains of Bacillus subtilis. The method described here allows transformation with both replicative and integrative plasmids, as well as with chromosomal DNA, and provides a valuable tool for molecular genetic analysis of interesting Bacillus strains, which are hard to transform by conventional methods. (c) 2006 Elsevier B.V. All rights reserved.

  19. Plant protoplast fusion and growth of intergeneric hybrid cells.

    Science.gov (United States)

    Kao, K N; Constabel, F; Michayluk, M R; Gamborg, O L

    1974-01-01

    Interspecific and intergeneric fusions of plant protoplasts were induced by polyethylene glycol (PEG) 1540 or 4000. The frequency of heterokaryocyte formation (or rate of fusion) was much higher when PEG was eluted with a high pH-high Ca(2+) solution or a salt solution than when it was eluted with a protoplast culture medium. The frequency of heterokaryocyte formation was also affected by the types of enzymes used for wall degradation, duration of enzyme incubation and molality of the PEG solutions.The maximum frequency of heterokaryocyte formation was 23% for V. hajastana Grossh.-soybean (Glycine max L.) and barley (Hordeum vulgare L.)-soybean, 35% for pea (Pisum sativum L.)-soybean, 20% for pea-V. hajastana, 14% for corn (Zea mays L.)-soybean and 10% for V. villosa Roth-V. hajastana.40% of the barley-soybean, corn-soybean and pea-soybean heterokaryocytes divided at least once. Some divided many times and formed clusters of up to 100 cells in 2 weeks. The heterokaryocytes of soybean-V. hajastana, V. villosa-V. hajastana also divided. Of the PEG-treated protoplasts of N. langsdorffii and N. glauca 13.5% developed into tumor-like calli. The morphology of these calli was very much like that of the tumors produced on amphidiploid plants of N. langsdorffii x glauca.Nuclear staining indicated that heterokaryocytes of V. hajastana-soybean, pea-soybean, corn-soybean and barley-soybean could undergo mitosis. Nuclear divisions in a heterokaryocyte were usually synchronized or almost synchronized. Nuclear fusion and true hybrid formation usually occurred during the first mitotic division after protoplast fusion. A hybrid of barley-soybean in third cell division was observed. The frequency of heterokaryocytes which underwent nuclear fusion has not been determined. Multipole formation and chimeral cell colonies were also observed.

  20. The influence of combined magnetic field on the fusion of plant protoplasts.

    Science.gov (United States)

    Nedukha, O; Kordyum, E; Bogatina, N; Sobol, M; Vorobyeva, T; Ovcharenko, Yu

    2007-07-01

    The study of the influence of weak, alternating magnetic field, which was adjusted to the cyclotron frequency of Ca2+ and K+ ions, on the fusion of tobacco and soya protoplasts was carried out using the extra apparatus with ferromagnetic shield. An increase in the frequency of protoplasts fusion in 2-3 times and participation of calcium ions in the induction of protoplast fusion in weak alternating magnetic field have been established.

  1. Regeneration of Transgenic Soybean (Glycine max) Plants from Electroporated Protoplasts.

    Science.gov (United States)

    Dhir, S K; Dhir, S; Savka, M A; Belanger, F; Kriz, A L; Farrand, S K; Widholm, J M

    1992-05-01

    Transgenic soybean (Glycine max [L.] Merr.) plants were regenerated from calli derived from protoplasts electroporated with plasmid DNA-carrying genes for a selectable marker, neomycin phosphotransferase (NPTII), under the control of the cauliflower mosaic virus 35-Svedberg unit promoter, linked with a nonselectable mannityl opine synthesis marker. Following electroporation and culture, the protoplast-derived colonies were subjected to kanamycin selection (50 micrograms per milliliter) beginning on day 15 for 6 weeks. Approximately, 370 to 460 resistant colonies were recovered from 1 x 10(6) electroporated protoplasts, giving an absolute transformation frequency of 3.7 to 4.6 x 10(-4). More than 80% of the kanamycin-resistant colonies showed NPTII activity, and about 90% of these also synthesized opines. This indicates that the linked marker genes were co-introduced and co-expressed at a very high frequency. Plants were regenerated from the transformed cell lines. Southern blot analysis of the transformed callus and leaf DNA demonstrated the integration of both genes. Single-plant assays performed with different plant parts showed that both shoot and root tissues express NPTII activity and accumulate opines. Experiments with NPTII and mannityl opine synthesis marker genes on separate plasmids resulted in a co-expression rate of 66%. These results indicate that electroporation can be used to introduce both linked and unlinked genes into the soybean to produce transformed plants.

  2. Reference: 507 [Arabidopsis Phenome Database[Archive

    Lifescience Database Archive (English)

    Full Text Available een them. However, little is known about the mechanisms that regulate the two pathways and the metabolic cro...ss-talk. To identify such regulatory mechanisms, we isolated and characterized the Arabidopsis T-DNA inserti

  3. The absence of eukaryotic initiation factor eIF(iso)4E affects the systemic spread of a Tobacco etch virus isolate in Arabidopsis thaliana.

    Science.gov (United States)

    Contreras-Paredes, Carlos A; Silva-Rosales, Laura; Daròs, José-Antonio; Alejandri-Ramírez, Naholi D; Dinkova, Tzvetanka D

    2013-04-01

    Translation initiation factor eIF4E exerts an important role during infection of viral species in the family Potyviridae. Particularly, a eIF(iso)4E family member is required for Arabidopsis thaliana susceptibility to Turnip mosaic virus, Lettuce mosaic virus, and Tobacco etch virus (TEV). In addition, a resistance mechanism named restriction of TEV movement (RTM) in A. thaliana controls the systemic spread of TEV in Col-0 ecotype. Here, we describe that TEV-TAMPS, a Mexican isolate, overcomes the RTM resistance mechanism reported for TEV-7DA infection of the Col-0 ecotype but depends on eIF(iso)4E for its systemic spread. To understand at which level eIF(iso)4E participates in A. thaliana TEV-TAMPS infection, the viral RNA replication and translation were measured. The absence or overexpression of eIF(iso)4E did not affect viral translation, and replication was still observed in the absence of eIF(iso)4E. However, the TEV-TAMPS systemic spread was completely abolished in the null mutant. The viral protein genome-linked (VPg) precursor NIa was found in coimmunoprecipitated complexes with both, eIF(iso)4E and eIF4E. However, the viral coat protein (CP) was only present in the eIF(iso)4E complexes. Since both the VPg and the CP proteins are needed for systemic spread, we propose a role of A. thaliana eIF(iso)4E in the movement of TEV-TAMPS within this host.

  4. Methods of staining and visualization of sphingolipid enriched and non-enriched plasma membrane regions of Arabidopsis thaliana with fluorescent dyes and lipid analogues

    Directory of Open Access Journals (Sweden)

    Blachutzik Jörg O

    2012-08-01

    Full Text Available Abstract Background Sterols and Sphingolipids form lipid clusters in the plasma membranes of cell types throughout the animal and plant kingdoms. These lipid domains provide a medium for protein signaling complexes at the plasma membrane and are also observed to be principal regions of membrane contact at the inception of infection. We visualized different specific fluorescent lipophilic stains of the both sphingolipid enriched and non-sphingolipid enriched regions in the plasma membranes of live protoplasts of Arabidopsis thaliana. Results Lipid staining protocols for several fluorescent lipid analogues in plants are presented. The most emphasis was placed on successful protocols for the single and dual staining of sphingolipid enriched regions and exclusion of sphingolipid enriched regions on the plasma membrane of Arabidopsis thaliana protoplasts. A secondary focus was placed to ensure that these staining protocols presented still maintain cell viability. Furthermore, the protocols were successfully tested with the spectrally sensitive dye Laurdan. Conclusion Almost all existing staining procedures of the plasma membrane with fluorescent lipid analogues are specified for animal cells and tissues. In order to develop lipid staining protocols for plants, procedures were established with critical steps for the plasma membrane staining of Arabidopsis leaf tissue and protoplasts. The success of the plasma membrane staining protocols was additionally verified by measurements of lipid dynamics by the fluorescence recovery after photobleaching technique and by the observation of new phenomena such as time dependent lipid polarization events in living protoplasts, for which a putative physiological relevance is suggested.

  5. Plant regeneration from protoplasts of immature Vigna sinensis cotyledons via somatic embryogenesis.

    Science.gov (United States)

    Li, X B; Xu, Z H; Wei, Z M

    1995-12-01

    Protoplasts were isolated from immature cotyledons of Vigna sinensis and cultured in a modified MS Liquid medium containing 0. 2 mg/l 2, 4-dichlorophenoxyacetic acid (2, 4-D), 1 mg/l naphthaleneacetic acid (NAA) and 0. 5 mg/l 6-benzylaminopurine (BAP) in the dark at a density of 1 × 10(5)/ml. The protoplasts began to divide in 3-5 days. Sustained cell division resulted in formation of cell clusters and small calli, with the cell division frequency and plating efficiency of cell colonies reaching 27. 7% and 1. 7% respectively. When calli of 2 mm in size were transferred onto MSB medium (MS salts and B5 vitamins) containing 500mg/l NaCl, 500 mg/ 1 casein hydrolysate (CH), 2 mg/l 2,4-D and 0. 5 mg/l BAP for further growth, approximately 5% of the calli developed embryogenically. The embryogenic calli were selected and subcultured on the same composition of MSB medium and were able to maintain somatic embryogenesis capacity in subculture for a long time. When the calli were moved to MSB medium with 0. 1 mg/l indole-3-acetic acid (IAA), 0. 5mg/l kinetin(KT), 3-5% mannitol and 2% sucrose in the light, many somatic embryos formed from the calli. Only part of the embryoids developed further to the cotyledonary stage, and the others died at the globular, heart-shaped or torpedo stages. Finally, some cotyledonary embryoids germinated and developed into plants or shoots. The shoots were readily rooted on 1/2 strength MS medium with 0. 1-0.3 mg/l indole-3-butyric acid (IBA). The plants grew well in soil and were fertile.

  6. Phytosulfokine-α controls hypocotyl length and cell expansion in Arabidopsis thaliana through phytosulfokine receptor 1.

    Directory of Open Access Journals (Sweden)

    Nils Stührwohldt

    Full Text Available The disulfated peptide growth factor phytosulfokine-α (PSK-α is perceived by LRR receptor kinases. In this study, a role for PSK signaling through PSK receptor PSKR1 in Arabidopsis thaliana hypocotyl cell elongation is established. Hypocotyls of etiolated pskr1-2 and pskr1-3 seedlings, but not of pskr2-1 seedlings were shorter than wt due to reduced cell elongation. Treatment with PSK-α did not promote hypocotyl growth indicating that PSK levels were saturating. Tyrosylprotein sulfotransferase (TPST is responsible for sulfation and hence activation of the PSK precursor. The tpst-1 mutant displayed shorter hypocotyls with shorter cells than wt. Treatment of tpst-1 seedlings with PSK-α partially restored elongation growth in a dose-dependent manner. Hypocotyl elongation was significantly enhanced in tpst-1 seedlings at nanomolar PSK-α concentrations. Cell expansion was studied in hypocotyl protoplasts. WT and pskr2-1 protoplasts expanded in the presence of PSK-α in a dose-dependent manner. By contrast, pskr1-2 and pskr1-3 protoplasts were unresponsive to PSK-α. Protoplast swelling in response to PSK-α was unaffected by ortho-vanadate, which inhibits the plasma membrane H(+-ATPase. In maize (Zea mays L., coleoptile protoplast expansion was similarly induced by PSK-α in a dose-dependent manner and was dependent on the presence of K(+ in the media. In conclusion, PSK-α signaling of hypocotyl elongation and protoplast expansion occurs through PSKR1 and likely involves K(+ uptake, but does not require extracellular acidification by the plasma membrane H(+-ATPase.

  7. Stringent control of cytoplasmic Ca2+ in guard cells of intact plants compared to their counterparts in epidermal strips or guard cell protoplasts.

    Science.gov (United States)

    Levchenko, V; Guinot, D R; Klein, M; Roelfsema, M R G; Hedrich, R; Dietrich, P

    2008-01-01

    Cytoplasmic calcium elevations, transients, and oscillations are thought to encode information that triggers a variety of physiological responses in plant cells. Yet Ca(2+) signals induced by a single stimulus vary, depending on the physiological state of the cell and experimental conditions. We compared Ca(2+) homeostasis and stimulus-induced Ca(2+) signals in guard cells of intact plants, epidermal strips, and isolated protoplasts. Single-cell ratiometric imaging with the Ca(2+)-sensitive dye Fura 2 was applied in combination with electrophysiological recordings. Guard cell protoplasts were loaded with Fura 2 via a patch pipette, revealing a cytoplasmic free Ca(2+) concentration of around 80 nM at -47 mV. Upon hyperpolarization of the plasma membrane to -107 mV, the Ca(2+) concentration increased to levels exceeding 400 nM. Intact guard cells were able to maintain much lower cytoplasmic free Ca(2+) concentrations at hyperpolarized potentials, the average concentration at -100 mV was 183 and 90 nM in epidermal strips and intact plants, respectively. Further hyperpolarization of the plasma membrane to -160 mV induced a sustained rise of the guard cell cytoplasmic Ca(2+) concentration, which slowly returned to the prestimulus level in intact plants but not in epidermal strips. Our results show that cytoplasmic Ca(2+) concentrations are stringently controlled in guard cells of intact plants but become increasingly more sensitive to changes in the plasma membrane potential in epidermal strips and isolated protoplasts.

  8. Cell-fractionation analysis of glucan synthase I and II distribution and polysaccharide secretion in soybean protoplasts : Evidence for the involvement of coated vesicles in wall biogenesis.

    Science.gov (United States)

    Griffing, L R; Mersey, B G; Fowke, L C

    1986-02-01

    The organelles of soybean (Glycine max (L.) Merr.) protoplasts were separated using a recently developed procedure which allows rapid (3-h) recovery of a fraction enriched for coated vesicles (CVs). As determined by marker-enzyme enrichment and ultrastructural analysis of isolated membrane fractions, endoplasmic reticulum, Golgi membranes, glucan-synthase-II (EC 2.4.1.34)-containing membranes (putative plasma membrane), mitochondria, and CVs were enriched in separate fractions in a sucrose density gradient. Glucan synthase I (EC 2.4.1.12) had the highest specific activity in the Golgi-enriched and CV-enriched fractions and was found to comigrate with CVs upon rate-zonal centrifugation of a CV-enriched fraction. For further elucidation of the role of these latter organelles in cell-wall regeneration, freshly isolated protoplasts were pulsed with [(3)H]glucose for 20 min, and the disappearance of label from the organelles was followed for the ensuing 1 h. Although a CV-enriched fraction contained glucan synthase I, it contained very small amounts of labelled polysaccharide during the period of study. Pulse-chase experiments with [(3)H]glucose helped to confirm the role of the Golgi apparatus in secretion of matrix polysaccharides by protoplasts.

  9. Improvement of regeneration of Lycopersicon pennellii protoplasts by decreasing ethylene production

    NARCIS (Netherlands)

    Rethmeier, N.O.M.; Jansen, C.E.; Snel, E.A.M.; Nijkamp, H.J.J.; Hille, J.

    1991-01-01

    Lycopersicon pennellii shoots, cultured in vitro for more than a year (type I plants) produced few viable protoplasts in contrast to shoots cultured in vitro for less than five months (type II plants). Ethylene production of both plant types was compared. The low viability of plant type I protoplast

  10. A method for high-frequency intergeneric fusion of plant protoplasts.

    Science.gov (United States)

    Kao, K N; Michayluk, M R

    1974-12-01

    Protoplasts of Vicia hajastana Grossh. obtained from suspension-culture cells and Pisum sativum L. obtained from leaves adhered tightly to each other in concentrated solutions of high-molecular-weight polyethylene glycol (PEG). The adhesion occurred non-specifically between the free protoplasts from the same species as well as from the different species and genus. It was enhanced by enrichment of the PEG solution with calcium. Very few heteroplasmic fusions occurred during the period when the protoplasts were incubated in the PEG solution. However, many heterokaryons (up to 10%) were formed soon after the PEG solution was diluted out. The same phenomena were also observed in protoplasts from suspension-culture cells of Glycine max L. and from leaves of Hordeum vulgare L. Vicia and soybean protoplasts obtained from cultured cells regenerated cell walls and underwent sustained cell division after such treatment. Some Vicia-pea heterokaryons divided once. Over 10% of the soybean-barley hybrids divided in 7 days. Some divided 4-5 times and formed small clusters of cells in 10 days. The hybrids were recognizable because they contained chloroplasts from the leaf protoplast and exhibited morphological characters typical of the chlorophyll-less cells. None of the protoplasts from pea and barley leaves, either with or without PEG treatment, underwent cell division during the period of observation. The mechanism of adhesion and fusion of the protoplasts has been discussed.

  11. Application of optical tweezers and excimer laser to study protoplast fusion

    Science.gov (United States)

    Kantawang, Titirat; Samipak, Sompid; Limtrakul, Jumras; Chattham, Nattaporn

    2015-07-01

    Protoplast fusion is a physical phenomenon that two protoplasts come in contact and fuse together. Doing so, it is possible to combine specific genes from one protoplast to another during fusion such as drought resistance and disease resistance. There are a few possible methods to induce protoplast fusion, for example, electrofusion and chemical fusion. In this study, chemical fusion was performed with laser applied as an external force to enhance rate of fusion and observed under a microscope. Optical tweezers (1064 nm with 100X objective N.A. 1.3) and excimer laser (308 nm LMU-40X-UVB objective) were set with a Nikon Ti-U inverted microscope. Samples were prepared by soaking in hypertonic solution in order to induce cell plasmolysis. Elodea Canadensis and Allium cepa plasmolysed leaves were cut and observed under microscope. Concentration of solution was varied to induce difference turgor pressures on protoplasts pushing at cell wall. Free protoplasts in solution were trapped by optical tweezers to study the effect of Polyethylene glycol (PEG) solution. PEG was diluted by Ca+ solution during the process to induced protoplast cell contact and fusion. Possibility of protoplast fusion by excimer laser was investigated and found possible. Here we report a novel tool for plant cell fusion using excimer laser. Plant growth after cell fusion is currently conducted.

  12. A simple and effective method to encapsulate tobacco mesophyll protoplasts to maintain cell viability

    Directory of Open Access Journals (Sweden)

    Rong Lei

    2015-01-01

    • It is very convenient to change or collect the solution without mechanically disturbing the protoplasts. This simple and effective silica sol–gel/alginate two-step immobilization of protoplasts in Transwell has great potential for applications in genetic transformation, metabolite production, and migration assays.

  13. Efficient production of Aschersonia placenta protoplasts for transformation using optimization algorithms.

    Science.gov (United States)

    Wei, Xiuyan; Song, Xinyue; Dong, Dong; Keyhani, Nemat O; Yao, Lindan; Zang, Xiangyun; Dong, Lili; Gu, Zijian; Fu, Delai; Liu, Xingzhong; Qiu, Junzhi; Guan, Xiong

    2016-07-01

    The insect pathogenic fungus Aschersonia placenta is a highly effective pathogen of whiteflies and scale insects. However, few genetic tools are currently available for studying this organism. Here we report on the conditions for the production of transformable A. placenta protoplasts using an optimized protocol based on the response surface method (RSM). Critical parameters for protoplast production were modelled by using a Box-Behnken design (BBD) involving 3 levels of 3 variables that was subsequently tested to verify its ability to predict protoplast production (R(2) = 0.9465). The optimized conditions resulted in the highest yield of protoplasts ((4.41 ± 0.02) × 10(7) cells/mL of culture, mean ± SE) when fungal cells were treated with 26.1 mg/mL of lywallzyme for 4 h of digestion, and subsequently allowed to recover for 64.6 h in 0.7 mol/L NaCl-Tris buffer. The latter was used as an osmotic stabilizer. The yield of protoplasts was approximately 10-fold higher than that of the nonoptimized conditions. Generated protoplasts were transformed with vector PbarGPE containing the bar gene as the selection marker. Transformation efficiency was 300 colonies/(μg DNA·10(7) protoplasts), and integration of the vector DNA was confirmed by PCR. The results show that rational design strategies (RSM and BBD methods) are useful to increase the production of fungal protoplasts for a variety of downstream applications.

  14. Enhancement of yellow pigment production by intraspecific protoplast fusion of Monascus spp. yellow mutant (ade(-)) and white mutant (prototroph).

    Science.gov (United States)

    Klinsupa, Worawan; Phansiri, Salak; Thongpradis, Panida; Yongsmith, Busaba; Pothiratana, Chetsada

    2016-01-10

    To breed industrially useful strains of a slow-growing, yellow pigment producing strain of Monascus sp., protoplasts of Monascus purpureus yellow mutant (ade(-)) and rapid-growing M. purpureus white mutant (prototroph) were fused and fusants were selected on minimal medium (MM). Preliminary conventional protoplast fusion of the two strains was performed and the result showed that only white colonies were detected on MM. It was not able to differentiate the fusants from the white parental prototroph. To solve this problem, the white parental prototroph was thus pretreated with 20mM iodoacetamide (IOA) for cytoplasm inactivation and subsequently taken into protoplast fusion with slow-growing Monascus yellow mutant. Under this development technique, only the fusants, with viable cytoplasm from Monascus yellow mutant (ade(-)), could thus grow on MM, whereas neither IOA pretreated white parental prototroph nor yellow auxotroph (ade(-)) could survive. Fifty-three fusants isolated from yellow colonies obtained through this developed technique were subsequently inoculated on complete medium (MY agar). Fifteen distinguished yellow colonies from their parental yellow mutant were then selected for biochemical, morphological and fermentative properties in cassava starch and soybean flour (SS) broth. Finally, three most stable fusants (F7, F10 and F43) were then selected and compared in rice solid culture. Enhancement of yellow pigment production over the parental yellow auxotroph was found in F7 and F10, while enhanced glucoamylase activity was found in F43. The formation of fusants was further confirmed by monacolin K content, which was intermediate between the two parents (monacolin K-producing yellow auxotroph and non-monacolin K producing white prototroph).

  15. Electron-microscope observations of mitosis and cytokinesis in multinucleate protoplasts of soybean.

    Science.gov (United States)

    Fowke, L C; Bech-Hansen, C W; Gamborg, O L; Constabel, F

    1975-08-01

    Multinucleate soybean protoplasts produced by spontaneous fusion during enzyme digestion of the cell wall initiated cell division after approximately 40 h in culture. The structure of these protoplasts during mitosis and cytokinesis was studied with both light and electron microscopes. Most nuclei did not fuse but divided synchronously. Interphase nuclei was commonly connected by short narrow nuclear bridges. At prophase and metaphase the nuclei appeared typical of those in most higher plants; technical difficulties prevented an adequate examination of protoplasts at anaphase. Telophase was characterized by cytokinesis involving phragmoplast and cell plate formation; however, complete partitioning of the cytoplasm by cell plants was not observed. Numerous coated vesicles were present near to or continuous with the cell plate and plasmalemma. The presence of a few dividing protoplasts with at least double the normal chromosome number suggests that some nuclear fusion occurred prior to mitosis. Very little cell wall material was detected at the margin of the dividing protoplasts.

  16. Functional characterization and reconstitution of ABA signaling components using transient gene expression in rice protoplasts

    Directory of Open Access Journals (Sweden)

    Namhyo eKim

    2015-08-01

    Full Text Available The core component of ABA-dependent gene expression signaling have been identified in Arabidopsis and rice. This signaling pathway consists of four major components; group A OsbZIPs, SAPKs, subclass A OsPP2Cs and OsPYL/RCARs in rice. These might be able to make thousands of combinations through interaction networks resulting in diverse signaling responses. We tried to characterize those gene functions using transient gene expression for rice protoplasts (TGERP because it is instantaneous and convenient system. Firstly, in order to monitor the ABA signaling output, we developed reporter system named pRab16A-fLUC which consists of Rab16A promoter of rice and luciferase gene. It responses more rapidly and sensitively to ABA than pABRC3-fLUC that consists of ABRC3 of HVA1 promoter in TGERP. We screened the reporter responses for over-expression of each signaling components from group A OsbZIPs to OsPYL/RCARs with or without ABA in TGERP. OsbZIP46 induced reporter most strongly among OsbZIPs tested in the presence of ABA. SAPKs could activate the OsbZIP46 even in the ABA independence. Subclass A OsPP2C6 and -8 almost completely inhibited the OsbZIP46 activity in the different degree through the SAPK9. Lastly, OsPYL/RCAR2 and -5 rescued the OsbZIP46 activity in the presence of SAPK9 and OsPP2C6 dependent on ABA concentration and expression level. By using TGERP, we could characterize successfully the effects of ABA dependent gene expression signaling components in rice. In conclusion, TGERP represents very useful technology to study systemic functional genomics in rice or other monocots.

  17. A WD40-repeat gene from Malus x domestica is a functional homologue of Arabidopsis thaliana TRANSPARENT TESTA GLABRA1.

    Science.gov (United States)

    Brueggemann, Julian; Weisshaar, Bernd; Sagasser, Martin

    2010-03-01

    The WD40 repeat protein TRANSPARENT TESTA GLABRA1 (TTG1) is involved in a multitude of developmental and biochemical reactions in Arabidopsis thaliana such as the production of seed coat colour and mucilage, pigmentation by anthocyanins as well as the formation of trichomes and root hairs. In this study, a putative TTG1 homologue was isolated from apple (Malus x domestica Borkh.) showing 80.2% identity to A. thaliana TTG1 on nucleotide and 90.7% similarity on amino acid level. The MdTTG1 candidate was able to activate the AtBAN promoter in cooperation with the A. thaliana transcription factors TT2 and TT8 in A. thaliana protoplasts. This indicates that the encoded protein can be integrated into the complex that activates BAN in A. thaliana, and that a similar complex might also be present in apple. When transformed into ttg1 mutants of A. thaliana, the apple sequence was able to restore trichome growth, anthocyanin production in young seedlings as well as proanthocyanidin production in seeds. Additionally, roots of complemented mutant plants showed root hair formation resembling wild type. These results show that the studied apple WD40 gene is a functional homologue of AtTTG1 and we refer to this gene as MdTTG1.

  18. Chromosomal proteins of Arabidopsis thaliana.

    Science.gov (United States)

    Moehs, C P; McElwain, E F; Spiker, S

    1988-07-01

    In plants with large genomes, each of the classes of the histones (H1, H2A, H2B, H3 and H4) are not unique polypeptides, but rather families of closely related proteins that are called histone variants. The small genome and preponderance of single-copy DNA in Arabidopsis thaliana has led us to ask if this plant has such families of histone variants. We have thus isolated histones from Arabidopsis and analyzed them on four polyacrylamide gel electrophoretic systems: an SDS system; an acetic acid-urea system; a Triton transverse gradient system; and a two-dimensional system combining SDS and Triton-acetic acid-urea systems. This approach has allowed us to identify all four of the nucleosomal core histones in Arabidopsis and to establish the existence of a set of H2A and H2B variants. Arabidopsis has at least four H2A variants and three H2B variants of distinct molecular weights as assessed by electrophoretic mobility on SDS-polyacrylamide gels. Thus, Arabidopsis displays a diversity in these histones similar to the diversity displayed by plants with larger genomes such as wheat.The high mobility group (HMG) non-histone chromatin proteins have attracted considerable attention because of the evidence implicating them as structural proteins of transcriptionally active chromatin. We have isolated a group of non-histone chromatin proteins from Arabidopsis that meet the operational criteria to be classed as HMG proteins and that cross-react with antisera to HMG proteins of wheat.

  19. Preparation and Regeneration of Ascosphaera apis Protoplasts%蜜蜂球囊菌(A scosphaera apis)原生质体制备及再生

    Institute of Scientific and Technical Information of China (English)

    何祥凤; 李薇; Abebe Jenberie Wubie; 薛菲; 国占宝; 周婷; 徐书法

    2013-01-01

    为建立高效稳定的蜜蜂球囊菌(A scosphaera apis)遗传转化体系,构建具有不同表型特征的球囊菌转化子,本实验对影响蜜蜂球囊菌菌株原生质体制备及再生的因子进行了系统的研究,同时对蜜蜂球囊菌原生质体的释放及再生过程进行了显微观察。结果表明,采用液体培养基进行24 h培养的蜜蜂球囊菌菌体,在28℃条件下,应用50 mg/mL的崩溃酶,经4 h酶解所制备的原生质体释放量最大,达到34.00×105/mL。在上述条件下,采用0.8 mol/L柠檬酸与NaCl的混合液作为稳渗剂,原生质体的再生率也较高,达到53.06%。蜜蜂球囊菌以菌丝断裂方式释放原生质体,原生质体再生时表现为原生质体先是突出,其后延长,并最终发育成为正常菌丝。本实验首次优化了蜜蜂球囊菌原生质体制备实验流程及原生质体再生最佳条件,为深入研究蜜蜂球囊菌的致病机理及寻找蜜蜂球囊菌致病相关基因奠定基础。%In order to develop an efficient and reproducible protoplast isolation and regeneration protocol targeting to make A scosphaera apis amenable to genetic studies and transformation, different enzymolysis and osmotic pressure stabilizing agents along with different growth mediums, incubation periods, and temperature variants have been utilized . The fungus of A scosphaera apis has demonstrated varying responses in terms of protoplast yield and regeneration rates to different factors tested . More specifically , incubation in liquid growth medium for 24 hours has yielded the highest number of protoplasts (34.00í105/mL). The use of 50 mg/mL driselase was the best enzymolysis agent at 28℃, yielded the highest number of isolated protoplasts. Furthermore, 0.8 mol/L citric acid-monohydrate with NaCl as an osmotic stabilizer and 4 hours of enzymolysis time has supported 53.06%proto-plast regeneration. From observation by microscope, we understood that

  20. The phosphoproteome in regenerating protoplasts from Physcomitrella patens protonemata shows changes paralleling postembryonic development in higher plants.

    Science.gov (United States)

    Wang, Xiaoqin; Qi, Meiyan; Li, Jingyun; Ji, Zhongzhong; Hu, Yong; Bao, Fang; Mahalingam, Ramamurthy; He, Yikun

    2014-05-01

    The moss Physcomitrella patens is an ideal model plant to study plant developmental processes. To better understand the mechanism of protoplast regeneration, a phosphoproteome analysis was performed. Protoplasts were prepared from protonemata. By 4 d of protoplast regeneration, the first cell divisions had ensued. Through a highly selective titanium dioxide (TiO2)-based phosphopeptide enrichment method and mass spectrometric technology, more than 300 phosphoproteins were identified as protoplast regeneration responsive. Of these, 108 phosphoproteins were present on day 4 but not in fresh protoplasts or those cultured for 2 d. These proteins are catalogued here. They were involved in cell-wall metabolism, transcription, signal transduction, cell growth/division, and cell structure. These protein functions are related to cell morphogenesis, organogenesis, and development adjustment. This study presents a comprehensive analysis of phosphoproteome involved in protoplast regeneration and indicates that the mechanism of plant protoplast regeneration is similar to that of postembryonic development.

  1. Methods for suspension culture, protoplast extraction, and transformation of high-biomass yielding perennial grass Arundo donax.

    Science.gov (United States)

    Pigna, Gaia; Dhillon, Taniya; Dlugosz, Elizabeth M; Yuan, Joshua S; Gorman, Connor; Morandini, Piero; Lenaghan, Scott C; Stewart, C Neal

    2016-12-01

    Arundo donax L. is a promising biofuel feedstock in the Mediterranean region. Despite considerable interest in its genetic improvement, Arundo tissue culture and transformation remains arduous. The authors developed methodologies for cell- and tissue culture and genetic engineering in Arundo. A media screen was conducted, and a suspension culture was established using callus induced from stem axillary bud explants. DBAP medium, containing 9 µM 2,4-D and 4.4 µM BAP, was found to be the most effective medium among those tested for inducing cell suspension cultures, which resulted in a five-fold increase in tissue mass over 14 days. In contrast, CIM medium containing 13 µM 2,4-D, resulted in just a 1.4-fold increase in mass over the same period. Optimized suspension cultures were superior to previously-described solidified medium-based callus culture methods for tissue mass increase. Suspension cultures proved to be very effective for subsequent protoplast isolation. Protoplast electroporation resulted in a 3.3 ± 1.5% transformation efficiency. A dual fluorescent reporter gene vector enabled the direct comparison of the CAMV 35S promoter with the switchgrass ubi2 promoter in single cells of Arundo. The switchgrass ubi2 promoter resulted in noticeably higher reporter gene expression compared with that conferred by the 35S promoter in Arundo.

  2. Efficient transformation of oil palm protoplasts by PEG-mediated transfection and DNA microinjection.

    Directory of Open Access Journals (Sweden)

    Mat Yunus Abdul Masani

    Full Text Available BACKGROUND: Genetic engineering remains a major challenge in oil palm (Elaeis guineensis because particle bombardment and Agrobacterium-mediated transformation are laborious and/or inefficient in this species, often producing chimeric plants and escapes. Protoplasts are beneficial as a starting material for genetic engineering because they are totipotent, and chimeras are avoided by regenerating transgenic plants from single cells. Novel approaches for the transformation of oil palm protoplasts could therefore offer a new and efficient strategy for the development of transgenic oil palm plants. METHODOLOGY/PRINCIPAL FINDINGS: We recently achieved the regeneration of healthy and fertile oil palms from protoplasts. Therefore, we focused on the development of a reliable PEG-mediated transformation protocol for oil palm protoplasts by establishing and validating optimal heat shock conditions, concentrations of DNA, PEG and magnesium chloride, and the transfection procedure. We also investigated the transformation of oil palm protoplasts by DNA microinjection and successfully regenerated transgenic microcalli expressing green fluorescent protein as a visible marker to determine the efficiency of transformation. CONCLUSIONS/SIGNIFICANCE: We have established the first successful protocols for the transformation of oil palm protoplasts by PEG-mediated transfection and DNA microinjection. These novel protocols allow the rapid and efficient generation of non-chimeric transgenic callus and represent a significant milestone in the use of protoplasts as a starting material for the development of genetically-engineered oil palm plants.

  3. Formation and regeneration of protoplasts for protease production in Streptomyces rimosus.

    Science.gov (United States)

    Yang, S S; Lei, C M

    2001-03-01

    To improve the formation and regeneration frequency of protoplasts for protease production, experiments were performed using a cultivation of Streptomyces rimosus TM-55 (CCRC 940061) in a Tryptic-soy broth (TSB) containing 2% of glycine for 2 days. It was found that the protoplast formation decreased with increased incubation temperature and increased ratio of culture broth to vessel volume. The optimal incubation temperature was 28 degreesC and the ratio of culture broth to vessel volume was 2:5. The hypertonic medium containing 10 mM MgCl2, 25 mM CaCl2 and 500 mM sucrose provided high stability for protoplasts. Supplementation with MgSO4, KCl and NaNO3 improved the regeneration frequency of protoplasts. The smear method had a higher protoplast regeneration frequency than the pour plate method. Protoplasts had protease productivity which was similar to that obtained with fresh mycelia, with each milliliter of culture broth yielding 141 units of protease with 3.5 x 10(8) protoplasts and 148 units of protease with 14.25 mg fresh mycelia respectively in a shaking culture, while the values were 15 and eight units of protease in a static culture.

  4. Formation and Growth of Bryopsis hypnoides Lamouroux Regenerated from Its Protoplasts

    Institute of Scientific and Technical Information of China (English)

    Nai-Hao YE; Guang-Ce WANG; Fa-Zuo WANG; Cheng-Kui ZENG

    2005-01-01

    Tissue culture, SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and spectra analysis were used for studying the aggregation mechanism of protoplasts from Bryopsis hypnoides Lamouroux and the discrepancy between the protoplast-regenerated plants and the wild type. The aggregation of protoplasts from B. hypnoides was observed in natural seawater and artificial seawater with different pH values, and the location and mechanism of the materials causing the aggregation were also studied. Results showed that the protoplasts could aggregate into some viable spheres in natural seawater and subsequently grow into mature individuals. Aggregation of the protoplasts depended exclusively upon the pH value (6-11), and the protoplasts aggregated best at pH 8-9. Some of the extruded protoplasts were separated into two parts by centrifugation: the pellet (PO) and the supernatant (PL). The PO could aggregate in artificial seawater (pH 8.3) but not in PL. No aggregation was found in PO cultured in natural seawater containing nigericin, which can dissipate the proton gradients across the membrane. These experiments suggest that the aggregation of protoplasts is proton-gradient dependent and the materials causing the aggregation were not in the vacuolar sap, but located on the surface or inside the organelles. Furthermore, the transfer of the materials across the membrane was similar to △pH-based translocation (△pH/TAT) pathway that occurs in the chloroplasts of higher plants and bacteria. Obvious discrepancies in both the total soluble proteins and the ratio of chlorophyll a to chlorophyll b between the regenerated B. hypnoides and the wild type were found, which may be related to the exchange of genetic material during aggregation of the organelles. In the process of development, diatom Amphora coffeaeformis Agardh attached to the protoplast aggregations, retarding their further development, and once they were removed, the aggregations immediately germinated, which showed that

  5. Content and vacuole extravacuole distribution of neutral sugars, free amino acids, and anthocyanin in protoplasts

    Energy Technology Data Exchange (ETDEWEB)

    Wagner, G.J.

    1979-07-01

    Neutral sugar, free amino acid, and anthocyanin levels and vacuole/extravacuole distribution were determined for Hippeastrum and Tulipa petal and Tulipa leaf protoplasts. Glucose and fructose, the predominant neutral monosaccharides observed, were primarily vacuolar in location. Glutamine, the predominant free amino acid found, was primarily extravacuolar. ..gamma..-methyleneglutamate was identified as a major constituent of Tulipa protoplasts. Qualitative characterization of Hippeastrum petal and vacuole organic acids indicated the presence of oxalic, malic, citric, and isocitric acids. Data are presented which indicate that vacuoles obtained by gentle osmotic shock of protoplasts in dibasic phosphate have good purity and retain their contents.

  6. Isolation and characterization of the Arabidopsis heat-intolerant 2 (hit2) mutant reveal the essential role of the nuclear export receptor EXPORTIN1A (XPO1A) in plant heat tolerance.

    Science.gov (United States)

    Wu, Shin-Jye; Wang, Lian-Chin; Yeh, Ching-Hui; Lu, Chun-An; Wu, Shaw-Jye

    2010-06-01

    *The Arabidopsis heat-intolerant 2 (hit2) mutant was isolated on the basis of its impaired ability to withstand moderate heat stress (37 degrees C). Determination of the genetic mutation that underlies the hit2 thermosensitive phenotype allowed better understanding of the mechanisms by which plants cope with heat stress. *Genetic analysis revealed that hit2 is a single recessive mutation. Map-based cloning was used to identify the hit2 locus. The response of hit2 to other types of heat stress was also investigated to characterize the protective role of HIT2. *hit2 was defective in basal but not in acquired thermotolerance. hit2 was sensitive to methyl viologen-induced oxidative stress, and the survival of hit2 seedlings in response to heat stress was affected by light conditions. The mutated locus was located at the EXPORTIN1A (XPO1A) gene, which encodes a nuclear transport receptor. Two T-DNA insertion lines, xpo1a-1 and xpo1a-3, exhibited the same phenotypes as hit2. *The results provide evidence that Arabidopsis XPO1A is dispensable for normal plant growth and development but is essential for thermotolerance, in part by mediating the protection of plants against heat-induced oxidative stress.

  7. Inhibition of brome mosaic virus (BMV) amplification in protoplasts from transgenic tobacco plants expressing replicable BMV RNAs.

    Science.gov (United States)

    Kaido, M; Mori, M; Mise, K; Okuno, T; Furusawa, I

    1995-11-01

    Transgenic tobacco plants (V123 plants) expressing a set of full-length brome mosaic virus (BMV) genomic RNAs from the cauliflower mosaic virus 35S promoter were produced. The accumulation level of BMV RNAs in V123 plant cells was approximately 1% of that in nontransgenic tobacco protoplasts inoculated with BMV RNAs. The level of BMV RNA in V123 protoplasts did not increase after inoculating the protoplasts with BMV RNAs, whereas V123 protoplasts supported the accumulation of cucumber mosaic virus (CMV) RNAs to a level similar to that in non-transgenic tobacco protoplasts after inoculation with CMV RNA. Such BMV-specific resistance was also observed in protoplasts from V12 plants expressing full-length BMV RNA1 and RNA2, both of which are required and sufficient for BMV RNA replication. On the other hand, protoplasts from M12 plants, expressing truncated BMV RNA1 and RNA2 in which the 3' 200 nucleotides required for BMV RNA replication were deleted, exhibited weaker resistance to infection with BMV RNA than V12 protoplasts, although the accumulation level of truncated BMV RNA1 and RNA2 in M12 protoplasts was higher than that of BMV RNA1 and RNA2 in V12 protoplasts. These results suggest that expression of BMV RNA replicons is involved in the induction of resistance, rather than high-level accumulation of BMV RNAs and/or their encoded proteins.

  8. A comparison of different Gracilariopsis lemaneiformis (Rhodophyta) parts in biochemical characteristics, protoplast formation and regeneration

    Science.gov (United States)

    Wang, Zhongxia; Sui, Zhenghong; Hu, Yiyi; Zhang, Si; Pan, Yulong; Ju, Hongri

    2014-08-01

    Gracilariopsis lemaneiformis is a commercially exploited alga. Its filaceous thallus can be divided into three parts, holdfast, middle segment and tip. The growth and branch forming trend and agar content of these three parts were analyzed, respectively, in this study. The results showed that the tip had the highest growth rate and branched most, although it was the last part with branch forming ability. The holdfast formed branches earliest but slowly. Holdfast had the highest agar content. We also assessed the difference in protoplast formation and regeneration among three parts. The middle segment displayed the shortest enzymolysis time and the highest protoplast yield; whereas the tip had the strongest vitality of protoplasts formation. Juvenile plants were only obtained from the protoplasts generated from the tip. These results suggested that the differentiation and function of G. lemaneiformis was different.

  9. Hybridisation experiments with protoplasts from chlorophyll-deficient mutants of some Solanaceous species.

    Science.gov (United States)

    Schieder, O

    1977-01-01

    Following fusion between protoplasts from two different chlorophyll-deficient diploid mutants of Datura innoxia Mill. it was possible to select 33 green hybrid calli on agar culture medium. Half of the somatic hybrids gave rise to leaves and some to shoots. The chromosome number of 20 somatic hybrids was determined: five were tetraploid, eight hexaploid, three octoploid, and four showed an aneuploid chromosome number. After transfer of the shoots of the five tetraploid hybrids to soil they developed roots. In control experiments in which protoplasts of the two mutants were cultured either as a mixture without being treated with the fusion agent, or cultured separately, no green callus could be obtained. Similar experiments involving protoplasts from one chlorophyll-deficient mutant of Datura innoxia, on the one hand, and those from similar mutants of Nicotiana sylvestris Spegazz. et Comes and Petunia hybrida, on the other, yielded no green somatic hybrid although hybrid protoplasts could be detected.

  10. Regeneration of intergeneric somatic hybrids by protoplast fusion between Onobrychis viciaefolia and Medicago sativa

    Institute of Scientific and Technical Information of China (English)

    徐子勤; 贾敬芬

    1997-01-01

    Protoplast fusion was induced between sainfoin and alfalfa by an improved polyethyleneglycol (PEG) method. The intergeneric somatic calluses were selected based on complementation of hydroxyproline-resistance of sainfoin and hormone autonomy growth of alfalfa transformation cell line. 17 somatic hybrid plantlets were regenerat-ed. PEG could induce the tight agglutination of protoplasts. During diluting and washing process, cyclization of the linked membrane and formation of vesicle-like structures were observed, resulting in protoplast fusion. 5%-10% glycerol supplemented in the fusion inducing solution markedly increased the frequency of heterogeneous fusion. Better fusion results were obtained when mixed protoplast suspension was dripped in petri dishes in which PEG solution was previously placed. Chromosome number of regenerated hybrid buds varied from 30 to 60. The genome of hybrids in-cluded the small chromosome from sainfoin and two chromosomes with two clear constrictions from alfalfa. The hybrid

  11. LYSIS OF BACTERIAL PROTOPLASTS AND SPHEROPLASTS BY STAPHYLOCOCCAL ALPHA-TOXIN AND STREPTOLYSIN S.

    Science.gov (United States)

    BERNHEIMER, A W; SCHWARTZ, L L

    1965-05-01

    Bernheimer, Alan W. (New York University School of Medicine, New York, N.Y.), and Lois L. Schwartz. Lysis of bacterial protoplasts and spheroplasts by staphylococcal alpha-toxin and streptolysin S. J. Bacteriol. 89:1387-1392. 1965.-Protoplasts of Bacillus megaterium, Sarcina lutea, and Streptococcus pyogenes, and spheroplasts of Escherichia coli were lysed by staphylococcal alpha-toxin, whereas spheroplasts of Vibrio metschnikovii and V. comma were not. In the spectrum of its lytic action, streptolysin S qualitatively resembled staphylococcal alpha-toxin except for failure to lyse S. pyogenes protoplasts. In contrast to the two foregoing agents, streptolysin O did not lyse protoplasts and spheroplasts. The observations are interpreted in relation to similarities and differences in lipid composition of bacterial and mammalian cell membranes.

  12. Polypeptide synthesis induced in Nicotiana clevelandii protoplasts by infection with raspberry ringspot nepovirus.

    Science.gov (United States)

    Acosta, O; Mayo, M A

    1993-01-01

    Infection of Nicotiana clevelandii protoplasts by raspberry ringspot nepovirus resulted in the accumulation of about 24 polypeptides that differed in M(r) and pI from polypeptides accumulating in mock-inoculated protoplasts. Similar polypeptides accumulated in protoplasts infected with the S and E strains of RRV but different infection-specific polypeptides were detected in protoplasts infected with tobacco ringspot nepovirus. The M(r) of RRV-specific polypeptides ranged from 210,000 to 18,000 and most are presumed to be derived from others by proteolytic cleavage. No evidence was found for marked changes in polypeptide abundance with time after inoculation or for any virus-specific polypeptide becoming disproportionately abundant in the medium during culture.

  13. Incorporation of radiolabeled polyamines and methionine into turnip yellow mosaic virus in protoplasts from infected plants

    Energy Technology Data Exchange (ETDEWEB)

    Balint, R.; Cohen, S.S.

    1985-07-15

    Turnip yellow mosaic virus contains large amounts of nonexchangeable spermidine and induces an accumulation of spermidine in infected Chinese cabbage. By 7 days after inoculation, a majority of protoplasts isolated from newly emerging leaves stain with fluorescent antibody to the virus. (/sup 14/C)Spermidine (10 microM) was taken up by these cells in amounts comparable to the original endogenous pool within 24 hr. However, after an initial rise, the spermidine content of the cell returned to its original level, implying considerable regulation of the endogenous pool(s). Putrescine and spermine were major products of the metabolism of exogenous spermidine. Radioactivity from exogenous (/sup 14/C)spermidine was also readily incorporated into the ribonucleoprotein component(s) of the virus, where it appeared as both spermidine and spermine. The specific radioactivities of the viral polyamines were approximately twice those of spermidine and spermine extracted from the whole cell. Radioactivity from (2-/sup 14/C)methionine was readily incorporated into the protein, spermidine, and spermine of the virus. Again, the specific activities of these amines were substantially higher in the virus than in the whole cell. Thus, newly formed virus contained predominantly newly synthesized spermidine and spermine. However, inhibition of spermidine synthesis by dicyclohexylamine led to incorporation of preexisting spermidine and increased amounts of spermine into newly formed virus.

  14. Enhancement and selective production of avermectin B by recombinants of Streptomyces avermitilis via intraspecific protoplast fusion

    Institute of Scientific and Technical Information of China (English)

    CHEN Zhi; WEN Jia; SONG Yuan; WEN Ying; LI JiLun

    2007-01-01

    Among eight components of avermectin, B1 fractions have the most effective antiparasitic activities and the lowest level of toxic side-effects and are used widely in veterinary and agricultural fields. Intraspecific protoplast fusion between two strains of Streptomyces avermitilis, one an avermectin high producer (strain 76-05) and the other a genetically engineered strain containing the mutations aveD- and olmA- (strain 73-12) was performed for enhancement and selective production of avermectin B in the absence of oligomycin. Two recombinant strains (F23 and F29) were isolated and characterized with regards to the parental merits. F23 and F29 produced only the four avermectin B components with high yield and produced no oligomycin. The avermectin production of F23 and F29 was about 84.20% and 103.45% of the parental strain 76-05, respectively, and increased about 2.66-fold and 3.50-fold, respectively, compared to that of parental strain 73-12. F23 and F29 were genetically stable prototrophic recombinants and F29 was quite tolerant of fermentation conditions compared to avermectin high producer parental strain 76-05. The ability to produce avermectin B with high yield without the production of other avermectin components and oligomycin will make F23 and F29 useful strains for avermectin production. Strain F29's tolerance of fermentation conditions will also make it suitable for industrial applications.

  15. Effect of various irradiation treatments of plant protoplasts on the transformation rates after direct gene transfer.

    Science.gov (United States)

    Köhler, F; Benediktsson, I; Cardon, G; Andreo, C S; Schieder, O

    1990-05-01

    In P. hybrida and B. nigra an enhancement of transformation rates (direct gene transfer) of about six to seven-fold was obtained after irradiation of protoplasts with 12.5 Gy (X-ray). The effect of protoplast irradiation was similar in experiments where protoplasts were irradiated 1h before transformation (X-ray/DNA) or 1h after completion of the transformation procedure (DNA/X-ray). Increased X-ray doses up to 62.5 Gy resulted in further enhancement of percentages of transformed colonies, indicating a correlation between relative transformation frequencies (RTF) and the doses applied. Estimation of degradation rates of plasmid sequences in plant protoplasts yielded a reduction of plasmid concentration to 50% 8-12 h after transformation. In 1-day-old protoplasts, the level of plasmid fragments dropped to 0%-10% compared to 1h after transformation. The results demonstrate that the integration rates of plasmid sequences into the plant genome may in part be governed by DNA repair mechanisms. This could be an explanation for the observed genotypic dependence of transformation rates in different plant species and plant genotypes. Gene copy number reconstructions revealed enhanced integration rates of plasmid sequences in transformed colonies derived from irradiated protoplasts.

  16. Experiments on tissue culture in the genus Lycopersicon miller : Shoot formation from protoplasts of tomato long-term cell cultures.

    Science.gov (United States)

    Koblitz, H; Koblitz, D

    1982-06-01

    Callus cultures from cotyledon explants were established and maintained in culture for more than two years. After several months callus cultures were transferred into liquid medium and cultured as cell suspensions. Protoplasts were isolated from these cell suspension cultures and cultured in a liquid medium. After formation of new cell walls the cells were further cultured in liquid medium and afterwards transferred to an agar-solidified medium to give a vigorously growing callus culture. In the case of the cultivar 'Lukullus' shoots were recovered from callus. All efforts to root these shoots failed and this, in addition to variations in appearence, suggests that the shoots are changed genetically possibly due to the prolonged culture period.

  17. Expression and Protein Interaction Analyses Reveal Combinatorial Interactions of LBD Transcription Factors During Arabidopsis Pollen Development.

    Science.gov (United States)

    Kim, Mirim; Kim, Min-Jung; Pandey, Shashank; Kim, Jungmook

    2016-11-01

    LATERAL ORGAN BOUNDARIES DOMAIN (LBD) transcription factor gene family members play key roles in diverse aspects of plant development. LBD10 and LBD27 have been shown to be essential for pollen development in Arabidopsis thaliana. From the previous RNA sequencing (RNA-Seq) data set of Arabidopsis pollen, we identified the mRNAs of LBD22, LBD25 and LBD36 in addition to LBD10 and LBD27 in Arabidopsis pollen. Here we conducted expression and cellular analysis using GFP:GUS (green fluorescent protein:β-glucuronidase) reporter gene and subcellular localization assays using LBD:GFP fusion proteins expressed under the control of their own promoters in Arabidopsis. We found that these LBD proteins display spatially and temporally distinct and overlapping expression patterns during pollen development. Bimolecular fluorescence complementation and GST (glutathione S-transferase) pull-down assays demonstrated that protein-protein interactions occur among the LBDs exhibiting overlapping expression during pollen development. We further showed that LBD10, LBD22, LBD25, LBD27 and LBD36 interact with each other to form heterodimers, which are localized to the nucleus in Arabidopsis protoplasts. Taken together, these results suggest that combinatorial interactions among LBD proteins may be important for their function in pollen development in Arabidopsis.

  18. Surface galactolipids of wheat protoplasts as receptors for soybean agglutinin and their possible relevance to host-parasite interaction.

    Science.gov (United States)

    Kogel, K H; Ehrlich-Rogozinski, S; Reisener, H J; Sharon, N

    1984-12-01

    Soybean agglutinin, a lectin specific for N-acetyl-d-galactosamine and d-galactose, was previously shown to agglutinate wheat leaf protoplasts (Larkin 1978 Plant Physiol 61: 626-629). We investigated the receptors for soybean agglutinin on the plasma membrane of these protoplasts. After treatment of the protoplasts with galactose oxidase, they were no longer agglutinated by the lectin, whereas upon reduction of the galactose oxidase-treated protoplasts with sodium borohydride the susceptibility to agglutination was restored. Analysis of the glycolipids of protoplasts surface labeled by the galactose oxidase-borotritide method, revealed that the radioactivity was mainly present in monogalactosyldiglyceride and digalactosyldiglyceride. The same galactolipids were identified as the only receptors for soybean agglutinin by direct binding of the (125)I-labeled lectin to a thin layer chromatogram of the glycolipids of wheat leaf protoplasts.

  19. The intrinsically disordered protein LEA7 from Arabidopsis thaliana protects the isolated enzyme lactate dehydrogenase and enzymes in a soluble leaf proteome during freezing and drying.

    Science.gov (United States)

    Popova, Antoaneta V; Rausch, Saskia; Hundertmark, Michaela; Gibon, Yves; Hincha, Dirk K

    2015-10-01

    The accumulation of Late Embryogenesis Abundant (LEA) proteins in plants is associated with tolerance against stresses such as freezing and desiccation. Two main functions have been attributed to LEA proteins: membrane stabilization and enzyme protection. We have hypothesized previously that LEA7 from Arabidopsis thaliana may stabilize membranes because it interacts with liposomes in the dry state. Here we show that LEA7, contrary to this expectation, did not stabilize liposomes during drying and rehydration. Instead, it partially preserved the activity of the enzyme lactate dehydrogenase (LDH) during drying and freezing. Fourier-transform infrared (FTIR) spectroscopy showed no evidence of aggregation of LDH in the dry or rehydrated state under conditions that lead to complete loss of activity. To approximate the complex influence of intracellular conditions on the protective effects of a LEA protein in a convenient in-vitro assay, we measured the activity of two Arabidopsis enzymes (glucose-6-P dehydrogenase and ADP-glucose pyrophosphorylase) in total soluble leaf protein extract (Arabidopsis soluble proteome, ASP) after drying and rehydration or freezing and thawing. LEA7 partially preserved the activity of both enzymes under these conditions, suggesting its role as an enzyme protectant in vivo. Further FTIR analyses indicated the partial reversibility of protein aggregation in the dry ASP during rehydration. Similarly, aggregation in the dry ASP was strongly reduced by LEA7. In addition, mixtures of LEA7 with sucrose or verbascose reduced aggregation more than the single additives, presumably through the effects of the protein on the H-bonding network of the sugar glasses.

  20. Plant regeneration from mesophyll protoplast of indica rice Qiugui'ai 11 (Oryza sativa L.)

    Institute of Scientific and Technical Information of China (English)

    JIANYuyu; JintaanankulSuwan

    1998-01-01

    In the recent decade, plant regeneration from protoplast has been obtained through embryogenic cell suspension cultures of rice. However, not only the establishment of embryogenic call suspension cultures of rice was difficult, but also the protoplasts became less and less regenerable and the genetic change was gradu ally accumulated during the prolonged culture.Since 1976 (Deka.), extensive efforts have been made to induce sustained division and regenerate plants from rnesophyll protoplasts of rice, but not successful.

  1. Ability of Bacillus subtilis protoplasts to repair irradiated bacteriophage deoxyribonucleic acid via acquired and natural enzymatic systems.

    OpenAIRE

    Yasbin, R E; Andersen, B J; Sutherland, B M

    1981-01-01

    A novel form of "enzyme therapy" was achieved by utilizing protoplasts of Bacillus subtilis. Photoreactivating enzyme of Escherichia coli was successfully inserted into the protoplasts of B. subtilis treated with polyethylene glycol. This enzyme was used to photoreactivate ultraviolet-damaged bacteriophage deoxyribonucleic acid (DNA). Furthermore, in polyethylene glycol-treated protoplasts, ultraviolet-irradiated transfecting bacteriophage DNA was shown to be a functional substrate for the ho...

  2. Isolation of a Mutant of Fer1 Gene, Acting Synergistically with the ARF8 Gene to Control Development of the Anther and Filament in Arabidopsis

    Institute of Scientific and Technical Information of China (English)

    Chang-En TIAN; Yu-Ping ZHOU; Shun-Zhi LIU; Kotaro YAMAMOTO

    2005-01-01

    Auxin response factors (ARFs) play a central role in plants as transcriptional factors in response to auxin. The Arabidopsis ARF8 gene is a light-inducible gene and ARF8 protein might control auxin homeostasis in a negative feed-back fashion through regulation of GH3 gene expression. In a double mutant designated infertile line including arf8-1 (a T-DNA insertion mutant of ARF8), we isolatedfertility1-1 (fer1-1), a mutant of Fer1, which acts synergistically with ARF8 to control the development of the anther and filament in Arabidopsis. Genetics analysis has demonstrated thatfer1-1 is a T-DNA insertion line,indicating that Fer1 might be cloned by inverse polymerase chain reaction (PCR) or the TAIL-PCR approach.Phenotypic identification and molecular analysis offer1-1 and the infertile line will be helpful to characterize the function of Fer1, to further study the function of ARF8, and to reveal the molecular mechanism underlying the interaction of Fer1 and ARF8 in controlling development of the anther and filament.

  3. Dynamic actin controls polarity induction de novo in protoplasts.

    Science.gov (United States)

    Zaban, Beatrix; Maisch, Jan; Nick, Peter

    2013-02-01

    Cell polarity and axes are central for plant morphogenesis. To study how polarity and axes are induced de novo, we investigated protoplasts of tobacco Nicotiana tabacum cv. BY-2 expressing fluorescently-tagged cytoskeletal markers. We standardized the system to such a degree that we were able to generate quantitative data on the temporal patterns of regeneration stages. The synthesis of a new cell wall marks the transition to the first stage of regeneration, and proceeds after a long preparatory phase within a few minutes. During this preparatory phase, the nucleus migrates actively, and cytoplasmic strands remodel vigorously. We probed this system for the effect of anti-cytoskeletal compounds, inducible bundling of actin, RGD-peptides, and temperature. Suppression of actin dynamics at an early stage leads to aberrant tripolar cells, whereas suppression of microtubule dynamics produces aberrant sausage-like cells with asymmetric cell walls. We integrated these data into a model, where the microtubular cytoskeleton conveys positional information between the nucleus and the membrane controlling the release or activation of components required for cell wall synthesis. Cell wall formation is followed by the induction of a new cell pole requiring dynamic actin filaments, and the new cell axis is manifested as elongation growth perpendicular to the orientation of the aligned cortical microtubules.

  4. Dynamic Actin Controls Polarity Induction de novo in Protoplasts

    Institute of Scientific and Technical Information of China (English)

    Beatrix Zaban; Jan Maisch; Peter Nick

    2013-01-01

    Cell polarity and axes are central for plant morphogenesis.To study how polarity and axes are induced de novo,we investigated protoplasts of tobacco Nicotiana tabacum cv.BY-2 expressing fluorescentlytagged cytoskeletal markers.We standardized the system to such a degree that we were able to generate quantitative data on the temporal patterns of regeneration stages.The synthesis of a new cell wall marks the transition to the first stage of regeneration,and proceeds after a long preparatory phase within a few minutes.During this preparatory phase,the nucleus migrates actively,and cytoplasmic strands remodel vigorously.We probed this system for the effect of anti-cytoskeletal compounds,inducible bundling of actin,RGD-peptides,and temperature.Suppression of actin dynamics at an early stage leads to aberrant tripolar cells,whereas suppression of microtubule dynamics produces aberrant sausagelike cells with asymmetric cell walls.We integrated these data into a model,where the microtubular cytoskeleton conveys positional information between the nucleus and the membrane controlling the release or activation of components required for cell wall synthesis.Cell wall formation is followed by the induction of a new cell pole requiring dynamic actin filaments,and the new cell axis is manifested as elongation growth perpendicular to the orientation of the aligned cortical microtubules.

  5. Quantitative Analysis of the Fate of Exogenous DNA in Nicotiana Protoplasts 1

    Science.gov (United States)

    Uchimiya, Hirofumi; Murashige, Toshio

    1977-01-01

    After a 5-hour incubation of protoplasts of Nicotiana tabacum L. `Xanthi' with 3H-DNA (7.26 μg/ml) from N. tabacum L. `Xanthi nc' 3.5% of the initial radioactivity was found in acid-insoluble substances of the protoplasts. The addition of DEAE-dextran and poly-l-lysine to the incubation medium nearly doubled radioactivity adsorption. The absorption was inhibited by 2,4-dinitrophenol, KCN, and low temperature (0 C); this inhibition could not be reversed by exogenous ATP. About 500 tobacco plants established from protoplasts of a normally tobacco-mosaic virus-susceptible cultivar that had been allowed to absorb DNA prepared from a resistant cultivar did not show transfer of the virus-resistant gene. A detailed analysis was performed of the disposition of exogenous DNA in plant protoplasts, by employing Escherichia coli3H-DNA and Nicotiana glutinosa protoplasts. In 5 to 20 hours, about 10% of the 3H-DNA entered the protoplasts. Competition experiments between the 3H-DNA and unlabeled DNA or thymidine showed that the entry occurred as undegraded 3H-DNA. Examination of intraprotoplast fractions revealed that 60 to 80% of the absorbed radioactivity resided in the “soluble” fraction of the cytoplasm and 20% in the nuclear fraction. The mitochondrion fraction also contained measurable radioactivity. Sizing on sucrose density gradients showed that the bulk of the absorbed E. coli DNA had been depolymerized. Of the incorporated radioactivity, 15% was accountable as DNA, exogenous as well as resynthesized, and 15% as RNA, protein, and other cell constituents. DNA/DNA hybridization test indicated that 17.6% of the re-extractable 3H-DNA retained homology with the E. coli DNA; this was equivalent to 2.6% of the absorbed radioactivity. Resynthesized receptor protoplast DNA was represented by a fraction at least 1.7% of the total absorbed radioactivity. The amount of bacterial DNA remaining in protoplasts suggests that each protoplast retained 2.3 × 10−15g donor DNA, or

  6. Regeneration of fertile plants from protoplasts of soybean (Glycine max L. Merr.): genotypic differences in culture response.

    Science.gov (United States)

    Dhir, S K; Dhir, S; Widholm, J M

    1992-06-01

    Fourteen soybean (Glycine max L.) genotypes were evaluated for their regenerability from protoplasts using a procedure previously descibed for the cultivar Clark 63. Protoplasts were isolated from immature cotyledon tissue and were cultured in liquid or agarose gelled KP8, MS or B5 medium with different sugars. Significant differences were observed in plating efficiency, which was as high as 63% in Jack and A-2396, and as low as 38% in X-3337. Upon regular dilution with K8 medium, 1-2 mm diameter colonies were formed in 5-6 weeks with all the genotypes tested. These colonies were then transferred onto MSB (MS salts; Murashige & Skoog, 1962 + B5 organics; Gamborg et al., 1968) medium with 0.5 mg l(-1) each of 2,4-D, BA and KN and 500 mg l(-1) CH for further growth. Once the colonies had become green, compact and nodular, and were 8-10 mm in size, they were transferred to regeneration medium. Upon regular subculturing, calli of six genotypes; A-2396, Chamberlain, Heilong-26, Jack, Resnick and XP-3015 developed shoots, with the regeneration frequency being highest 27% in Jack (52 calli out of 192 produced 8-12 shoots). The regenerated shoots from different genotypes were elongated and rooted. So far, sixty three complete plants have been obtained, including twelve A-2396, nineteen Chamberlain, fifteen Jack, nine Resnick and eight XP-3015. A total of thirty five plants have been transplanted into pots in the greenhouse. Sixteen have set seeds and others are producing flowers and pods.

  7. Effects of simulated microgravity on the regeneration of P. decumbens protoplasts

    Science.gov (United States)

    Sun, Yan; Zhao, Chen; Yi, Zong-Chun; He, Jingwen; Rong, Long; Zhuang, Fengyuan

    Introduction: It is known that growth and differentiation of cells during long periods of microgravity take place normally. Structural and functional changes are observed at the cellular level, and those changes take place not only within the nucleus and the cytoplasmic organelles, but also in the cell walls. Like the plant cells, cultured under microgravity, the protoplasts' regeneration rates have some changes. This experiment attempt to make out the related changes of the P. decumbens protoplasts under the simulated microgravity. Methods: Protoplasts of P. decumbens was produced at first. Simulated microgravity was obtained by rotary cultivation of 15 RPM., the regeneration rate was calculated by the clone formation. Polysaccharide of cell wall was measured by flow cytometry. Results: For the morphologic study, there is no distinct different between the colonies in the impact of the microgravity and the normal condition. The regeneration rate of the P. decumbens protoplasts is lower under the effect of microgravity than the normal condition (33.8% vs. 44.9%). Polysaccharide of protoplast after rotary cultivation decreased 13.8% compare to the control. The conclusion seems that the regeneration of protoplasm is lower under the condition of simulated microgravity. This result may give rise a question that: Did cell wall do something with sensing microgravity? More works should be done to answer this question. *This work was supported by the fund of 'National Natural Science Foundation of China, No. 10502004'

  8. Assembly of the Protoplasm of Codium fragile (Bryopsidales, Chlorophyta) into New Protoplasts

    Institute of Scientific and Technical Information of China (English)

    Demao Li; Fang Lü; Guangce Wang; Baicheng Zhou

    2008-01-01

    The cell organelles of the coenocytic alga Codium fragile (Sun) Hariot aggregated rapidly and protoplasts were formed when its protoplasm was extruded out in seawater. Continuous observation showed that there were long and gelatinous threads connecting the cell organelles. The threads contracted, and thus the cell organelles aggregated into protoplasmic masses. The enzyme digestion experiments and Coomassie Brilliant Blue and Anthrone stainings showed that the long and gelatinous threads involved in the formation of the protoplasts might Include protein and saccharides as structure components. Nile Red staining Indicated that the protoplast primary envelope was non-lipid at first, and then lipid materials Integrated Into its surface gradually. The fluorescent brightener staining Indicated that the cell wall did not regenerate in the newly formed protoplasts and they all disintegrated within 72 h after formation. Transmission electron microscopy of the cell wall of wild C. fragile showed electron-dense material embedded in the whole cell wall at regular intervals. The experiments indicated that C. fragile would be a suitable model alga for studying the formation of protoplasts.

  9. Preliminary Study on Protoplast Culture of Lilium oriental Hybrids‘Sor-bonne’%东方百合‘Sorbonne’原生质体培养初步研究

    Institute of Scientific and Technical Information of China (English)

    秦晓杰; 段华金; 朱永平; 王小巧; 李琼洁; 赵兴富; 和凤美

    2013-01-01

    百合原生质体培养对百合远源杂交育种具有重要意义。本研究以东方百合‘Sorbonne’花托为外植体,诱导胚性愈伤组织,并对其原生质体提取和培养进行初步研究。结果表明:MS+Picloram 3 mg/L为胚性愈伤组织诱导最佳培养基;胚性愈伤组织在酶解液为2%纤维素酶R-10+0.5%离析酶R-10+0.05%果胶酶Y23+147 mg/L二水合氯化钙+976 mg/L 2-吗啉乙磺酸+0.6 mol/L甘露醇,酶解12 h,制备的原生质体产量最高达4×105个/mL,活性达52%;在胚性愈伤组织诱导培养基中进行原生质体的悬滴培养、液体培养、固体培养、固液结合培养和看护培养。结果表明,看护培养是最佳培养方法,并在改良MS+Picloram 2 mg/L的培养基中培养2~3 d观察到长细胞壁的原生质体,培养4~6 d,可见原生质体的第一次分裂,培养40~45 d,观察到原生质体分化成的细胞团。%It is sign ifican t to culture the protoplast for wide cross breeding of Lily. In this study, the embryonic callus of receptacle of lilium oriental hybrids‘Sorbonne’ were induced and protoplasts were isolated and cultiv-ated. The results showed that feasible culture medium of inductive embryonic callus was MS+Picloram 3 mg/L;The optimal enzyme solution used for protoplasts isolation with 12 hours enzymolysis was 2%Cellulase R-10+0.5%Macerozyme R-10+0.05%Pectolyase Y23+147 mg/L CaCl2·2H2O+976 mg/L MES+6 mol/L Sorbitol, and yield reached its highest, which was up to 4×105/mL, and the activity was up to 52%. Comparing with the hanging drop culture, liquid culture, solid culture, liquid and solid culture, the nurse culture was the best way to cultivate protoplast in feasible culture medium of inductive embryonic callus. Some changes can be observed in the process of protoplast culture with improved medium that is MS+Picloram 2 mg/L by the nurse culture, such as cell wall for 2~3 days, the first division of protoplast for 4~6 days, cell mass for

  10. Plastid transformation in lettuce (Lactuca sativa L.) by polyethylene glycol treatment of protoplasts.

    Science.gov (United States)

    Lelivelt, Cilia L C; van Dun, Kees M P; de Snoo, C Bastiaan; McCabe, Matthew S; Hogg, Bridget V; Nugent, Jacqueline M

    2014-01-01

    A detailed protocol for PEG-mediated plastid transformation of Lactuca sativa cv. Flora, using leaf protoplasts, is described. Successful plastid transformation using protoplasts requires a large number of viable cells, high plating densities, and an efficient regeneration system. Transformation was achieved using a vector that targets genes to the trnI/trnA intergenic region of the lettuce plastid genome. The aadA gene, encoding an adenylyltransferase enzyme that confers spectinomycin resistance, was used as a selectable marker. With the current method, the expected transformation frequency is 1-2 spectinomycin-resistant cell lines per 10(6) viable protoplasts. Fertile, diploid, homoplasmic, plastid-transformed lines were obtained. Transmission of the plastid-encoded transgene to the T1 generation was demonstrated.

  11. Plant regeneration from protoplasts of hydroxyproline resistant cell line in Onobrychis viciaefolia

    Institute of Scientific and Technical Information of China (English)

    XUZIQIN; JINGFENJIA

    1995-01-01

    An efficient protocol for plant regeneration from protoplasts of hydroxyproline(HYP)resistant cell line of Onobrychis viciaefolia was established.In SH medium supplemented with 1mg/L2,4-dichlorophenoxy-acetic acid(2,4-D),0.5mg/L kinetin(KT)and 0.2mg/L naphthalene acetic acid(NAA),the division frequency of protoplastderived cells reached up to over 60%,and microcalli were obtained in 5-6wk.Upon transferring them on agar solidified MS medium plus 2mg/L indole-3-acetic acid (IAA),shoots were induced.After cultivating them on MS medium with or without IAA,roots were regenerated.Chromosome number of all protoplast-regenerated plants examined were normal(2n=28).The protoplast-derived calli and plants grew vigorously on the medium containing 10 mmol/L HYP.

  12. Protein-protein interactions visualized by bimolecular fluorescence complementation in tobacco protoplasts and leaves.

    Science.gov (United States)

    Schweiger, Regina; Schwenkert, Serena

    2014-03-09

    Many proteins interact transiently with other proteins or are integrated into multi-protein complexes to perform their biological function. Bimolecular fluorescence complementation (BiFC) is an in vivo method to monitor such interactions in plant cells. In the presented protocol the investigated candidate proteins are fused to complementary halves of fluorescent proteins and the respective constructs are introduced into plant cells via agrobacterium-mediated transformation. Subsequently, the proteins are transiently expressed in tobacco leaves and the restored fluorescent signals can be detected with a confocal laser scanning microscope in the intact cells. This allows not only visualization of the interaction itself, but also the subcellular localization of the protein complexes can be determined. For this purpose, marker genes containing a fluorescent tag can be coexpressed along with the BiFC constructs, thus visualizing cellular structures such as the endoplasmic reticulum, mitochondria, the Golgi apparatus or the plasma membrane. The fluorescent signal can be monitored either directly in epidermal leaf cells or in single protoplasts, which can be easily isolated from the transformed tobacco leaves. BiFC is ideally suited to study protein-protein interactions in their natural surroundings within the living cell. However, it has to be considered that the expression has to be driven by strong promoters and that the interaction partners are modified due to fusion of the relatively large fluorescence tags, which might interfere with the interaction mechanism. Nevertheless, BiFC is an excellent complementary approach to other commonly applied methods investigating protein-protein interactions, such as coimmunoprecipitation, in vitro pull-down assays or yeast-two-hybrid experiments.

  13. Determinação da viabilidade de protoplastos irradiados de laranja 'pêra' Determination of viability of irradiated 'pera' orange protoplasts

    Directory of Open Access Journals (Sweden)

    Mariângela Cristofani

    1993-01-01

    Full Text Available Estudou-se a viabilidade de protoplastos de laranja 'Pêra' (Citrus sinensis Osbeck, submetidos a diferentes doses de radiação gama, com a finalidade de determinar a dose letal (DL 50 - dose que causa 50% de letalidade. Empregou-se a análise por fluorescência, utilizando-se o corante diacetato de fluoresceína (DAF: suas diluições testadas - 1:50; 1:100 e 1:150 - não mostraram diferenças significativas entre si, tendo sido possível o uso da maior diluição para a determinação da viabilidade dos protoplastos. A viabilidade mostrou-se inversamente proporcional às doses de radiação gama e a DL 50 foi cerca de 41 Gy. Os protoplastos não irradiados apresentaram até 84% de viabilidade, quando esta foi estudada logo após o isolamento daqueles.The viability of 'Pera' orange (Citrus sinensis Osbeck protoplasts, submitted to different dosages of Gamma radiation, was studied to determine the lethal dose (DL 50. The analysis by fluorescence was employed using Fluorescein diacetate (FDA. The dilutions of FDA (1:50; 1:100 and 1:150 did not show any statistical difference: Then it was possible to use the 1:150 dilution in order to determine the protoplasts viability. The viability was inversaly proportional to Gamma radiation and the DL 50 was about 41 Gy. The non-irradiated protoplasts had their viability up to 84% when tested as soon after their isolation.

  14. Ultrastructure of fusion products from soybean cell culture and sweet clover leaf protoplasts.

    Science.gov (United States)

    Fowke, L C; Rennie, P J; Kirkpatrick, J W; Constabel, F

    1976-01-01

    Protoplasts from cultured cells of soybean (Glycine max L.) and from sweet clover (Melilotus officinalis L.) mesophyll cells were fused with polyethylene glycol and subsequently cultured for six days. The resulting fusion products as well as unfused protoplasts of each parental species regenerated cell walls and divided. The fusion products were characterized by the presence of soybean leucoplasts and sweet clover chloroplasts. The chloroplasts appeared to be degenerating but other cytoplasmic organelles were typical of actively growing plant cells. The fate of individual nuclei could not be determined.

  15. Subcellular Compartmentation of the 4-Aminobutyrate Shunt in Protoplasts from Developing Soybean Cotyledons.

    Science.gov (United States)

    Breitkreuz, K. E.; Shelp, B. J.

    1995-05-01

    The subcellular localization of enzymes involved in the 4-ami-nobutyrate shunt was investigated in protoplasts prepared from developing soybean [Glycine max (L.) Merrill cv Maple Arrow] cotyledons. Protoplast lysate was fractionated by differential and continuous Percoll-gradient centrifugation to separate organelle fractions. Glutamate decarboxylase (EC 4.1.1.15) was found exclusively in the cytosol, whereas 4-aminobutyrate:pyruvate transami-nase (EC 2.6.1.19) and succinic semialdehyde dehydrogenase (EC 1.2.1.16) were associated exclusively with the mitochondrial fractions. Mitochondrial fractions also catabolized [U-14C]4-aminobu-tyrate to labeled succinate.

  16. Reference: 59 [Arabidopsis Phenome Database[Archive

    Lifescience Database Archive (English)

    Full Text Available 59 http://metadb.riken.jp/db/SciNetS_ria224i/cria224u4ria224u14563930i Kaczorowski Kare...naling network in Arabidopsis, we used a sensitized genetic screen for deetiolation-defective seedlings. Two allelic mutants were... isolated that exhibited reduced sensitivity to both continuous red and far-re...d light, suggesting involvement in both phyA and phyB signaling. The molecular lesions res...ponsible for the phenotype were shown to be mutations in the Arabidopsis PSEUDO-RESPONSE REGULATOR7 (PRR7) g

  17. Analysis of a Partial Male-Sterile Mutant of Arabidopsis thaliana Isolated from a Low-Energy Argon Ion Beam Mutagenized Pool

    Institute of Scientific and Technical Information of China (English)

    XU Min; BIAN Po; WU Yuejin; YU Zengliang

    2008-01-01

    A screen for Arabidopsis fertility mutants, mutagenized by low-energy argon ion beam, yielded two partial male-sterile mutants tc243-1 and tc243-2 which have similar phenotypes. tc243-2 was investigated in detail. The segregation ratio of the mutant phenotypes in the M2 pools suggested that mutation behaved as single Mendelian recessive mutations, tc243 showed a series of mutant phenotypes, among which partial male-sterile was its striking mutant characteristic. Phenotype analysis indicates that there are four factors leading to male sterility, a. Floral organs normally develop inside the closed bud, but the anther filaments do not elongate sufficiently to position the locules above the stigma at anthesis, b. The anther locules do not dehisce at the time of flower opening (although limited dehiscence occurs later), c. Pollens of mutant plants develop into several types of pollens at the trinucleated stage, as determined by staining with DAPI (4',6-diamidino-2-phenylindole), which shows a variable size, shape and number of nucleus. d. The viability of pollens is lower than that of the wild type on the germination test in vivo and vitro.

  18. Regulation of WRKY46 transcription factor function by mitogen-activated protein kinases in Arabidopsis thaliana.

    Directory of Open Access Journals (Sweden)

    Arsheed Hussain Sheikh

    2016-02-01

    Full Text Available AbstractMitogen-activated protein kinase (MAPK cascades are central signalling pathways activated in plants after sensing internal developmental and external stress cues. Knowledge about the downstream substrate proteins of MAPKs is still limited in plants. We screened Arabidopsis WRKY transcription factors as potential targets downstream of MAPKs, and concentrated on characterizing WRKY46 as a substrate of the MAPK, MPK3. Mass spectrometry revealed in vitro phosphorylation of WRKY46 at amino acid position S168 by MPK3. However, mutagenesis studies showed that a second phosphosite, S250, can also be phosphorylated. Elicitation with pathogen-associated molecular patterns (PAMPs, such as the bacterial flagellin-derived flg22 peptide led to in vivo destabilization of WRKY46 in Arabidopsis protoplasts. Mutation of either phosphorylation site reduced the PAMP-induced degradation of WRKY46. Furthermore, the protein for the double phosphosite mutant is expressed at higher levels compared to wild-type proteins or single phosphosite mutants. In line with its nuclear localization and predicted function as a transcriptional activator, overexpression of WRKY46 in protoplasts raised basal plant defence as reflected by the increase in promoter activity of the PAMP-responsive gene, NHL10, in a MAPK-dependent manner. Thus, MAPK-mediated regulation of WRKY46 is a mechanism to control plant defence.

  19. Arabidopsis hybrid speciation processes.

    Science.gov (United States)

    Schmickl, Roswitha; Koch, Marcus A

    2011-08-23

    The genus Arabidopsis provides a unique opportunity to study fundamental biological questions in plant sciences using the diploid model species Arabidopsis thaliana and Arabidopsis lyrata. However, only a few studies have focused on introgression and hybrid speciation in Arabidopsis, although polyploidy is a common phenomenon within this genus. More recently, there is growing evidence of significant gene flow between the various Arabidopsis species. So far, we know Arabidopsis suecica and Arabidopsis kamchatica as fully stabilized allopolyploid species. Both species evolved during Pleistocene glaciation and deglaciation cycles in Fennoscandinavia and the amphi-Beringian region, respectively. These hybrid studies were conducted either on a phylogeographic scale or reconstructed experimentally in the laboratory. In our study we focus at a regional and population level. Our research area is located in the foothills of the eastern Austrian Alps, where two Arabidopsis species, Arabidopsis arenosa and A. lyrata ssp. petraea, are sympatrically distributed. Our hypothesis of genetic introgression, migration, and adaptation to the changing environment during the Pleistocene has been confirmed: We observed significant, mainly unidirectional gene flow between the two species, which has given rise to the tetraploid A. lyrata. This cytotype was able to escape from the narrow ecological niche occupied by diploid A. lyrata ssp. petraea on limestone outcrops by migrating northward into siliceous areas, leaving behind a trail of genetic differentiation.

  20. Construction of Potent Recombinant Strain Through Intergeneric Protoplast Fusion in Endophytic Fungi for Anticancerous Enzymes Production Using Rice Straw.

    Science.gov (United States)

    El-Gendy, Mervat Morsy Abbas Ahmed; Al-Zahrani, Salha Hassan Mastour; El-Bondkly, Ahmed Mohamed Ahmed

    2017-02-15

    Among all fungal endophytes isolates derived from different ethno-medical plants, the hyper-yield L-asparaginase and L-glutaminase wild strains Trichoderma sp. Gen 9 and Cladosporium sp. Gen 20 using rice straw under solid-state fermentation (SSF) were selected. The selected strains were used as parents for the intergeneric protoplast fusion program to construct recombinant strain for prompt improvement production of these enzymes in one recombinant strain. Among 21 fusants obtained, the recombinant strain AYA 20-1, with 2.11-fold and 2.58-fold increase in L-asparaginase and L-glutaminase activities more than the parental isolates Trichoderma sp. Gen 9 and Cladosporium sp. Gen 20, respectively, was achieved using rice straw under SSF. Both therapeutic enzymes L-asparaginase and L-glutaminase were purified and characterized from the culture supernatant of the recombinant AYA 20-1 strain with molecular weights of 50.6 and 83.2 kDa, respectively. Both enzymes were not metalloenzymes. Whereas thiol group blocking reagents such as p-chloromercurybenzoate and iodoacetamide totally inhibited L-asparaginase activity, which refer to sulfhydryl groups and cysteine residues involved in its catalytic activity, they have no effect toward L-glutaminase activity. Interestingly, potent anticancer, antioxidant, and antimicrobial activities were detected for both enzymes.

  1. Isolation of intact and pure chloroplasts from leaves of Arabidopsis thaliana plants acclimated to low irradiance for studies on Rubisco regulation

    Directory of Open Access Journals (Sweden)

    Magda Grabsztunowicz

    2012-11-01

    Full Text Available A protocol is presented for low-cost and fast isolation of intact and pure chloroplasts from leaves of plants acclimated to low irradiance. The protocol is based on a differential centrifugation of cleared leaf homogenate and omits a centrifugation on Percoll gradient step. The intactness and purity of the chloroplasts isolated from leaves of low irradiance-acclimated plants by using this protocol (confirmed by phase contrast microscopy as well as enzymatic and immunological approaches allows plausible studies on low irradiance-dependent Rubisco regulation.

  2. GENE-REGULATION IN INTERTYPIC HETEROKARYONS OF SOLANUM-TUBEROSUM AND NICOTIANA-TABACUM TISSUE PROTOPLASTS

    NARCIS (Netherlands)

    VANKESTEREN, WJP; BIJMOLT, EW; TEMPELAAR, MJ

    1994-01-01

    Activities of the beta-glucuronidase (GUS) reporter enzyme were evaluated in transgenic plants, protoplasts, and intertypic heterokaryons of Solanum tuberosum and Nicotiana tabacum. With GUS under control of the promoter of the cauliflower-mosaicvirus 35S RNA gene (CaMV), activities of the enzyme we

  3. [Efficient transient expression to analyze miRNA targets in rice protoplasts].

    Science.gov (United States)

    Guo, Ping; Wu, Yao; Li, Jia; Fang, Rongxiang; Jia, Yantao

    2014-11-01

    Compared with the transgenic approach, transient assays provide a convenient alternative to analyze gene expression. To analyze the relationship between miRNAs and their target genes, a rice protoplast system to detect target gene activity was established. The MIRNA and GFP-fused target sequence (or GFP-fused mutated sequence as a non-target control) were constructed into the same plasmid, and then delivered into rice protoplasts. The GFP expression level decreased significantly when the protoplasts were transfected with the plasmid containing GFP-fused target compared to that of the plasmid with non-target sequence either by fluorescence microscopy or qRT-PCR method. Two microRNA genes, osaMIR156 and osaMIR397, and their target sequences were used to prove the feasibility of the rice protoplast transient assay system. This method will facilitate large-scale screening of rice miRNA target in vivo, and may be suitable for functional analysis of miRNAs of other monocot plants that might share the evolutionarily conserved small RNA processing system with rice.

  4. Induction of Apoptosis in Protoplasts and Suspension Cultures of Plant Cells

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Many studies have showed that apoptosis exists in plants. Our study shows that (1) menadione(VK3) induces apoptosis in suspension cultures of carrot cells; (2) heat shock induces apoptosis in suspension cultures of tobacco cells; and (3) ethrel induces apoptosis in carrot protoplasts. Some important indications of apoptosis were observed, including DNA laddering, TUNEL-positive reaction, condensation and degradation of nuclei.

  5. Expression of the tomato ringspot nepovirus movement and coat proteins in protoplasts

    NARCIS (Netherlands)

    Sanfacon, H.; Wieczorek, A.; Hans, F.

    1995-01-01

    Tomato ringspot nepovirus (TomRSV) produces a 45 kDa movement protein and a 58 kDa coat protein in infected plants. Accumulation of the movement protein in relation to that of the coat protein was studied in infected protoplasts using a monoclonal antibody against the movement protein and polyclonal

  6. [Promotion of transformation frequency of soybean (Glycine max L.) protoplasts using poly-L-ornithine].

    Science.gov (United States)

    Nan, X R; Wei, Z M

    1999-12-01

    The foreign Bt gene was transferred into protoplasts of soybean using PEG and PLO methods, respectively. The result indicated that the transformation frequency of PLO method was about 0.1% higher than PEG method. The PCR and Southern blotting analysis of the regeneration plants confirmed the integration of foreign gene into the genome of soybean.

  7. Tomato protoplast DNA transformation : physical linkage and recombination of exogenous DNA sequences

    NARCIS (Netherlands)

    Jongsma, Maarten; Koornneef, Maarten; Zabel, Pim; Hille, Jacques

    1987-01-01

    Tomato protoplasts have been transformed with plasmid DNA's, containing a chimeric kanamycin resistance gene and putative tomato origins of replication. A calcium phosphate-DNA mediated transformation procedure was employed in combination with either polyethylene glycol or polyvinyl alcohol. There w

  8. AGGREGATION AND FUSION OF PLANT-PROTOPLASTS AFTER SURFACE-LABELING WITH BIOTIN AND AVIDIN

    NARCIS (Netherlands)

    VANKESTEREN, WJP; MOLEMA, E; TEMPELAAR, MJ

    1993-01-01

    In mass electrofusion systems with aggregation of protoplasts by alignment, the yield and composition of fusion products can be predicted by a simple model. Through computer simulation, upper limits were found for the yield of binary and multi fusions. To overcome constraints on binary products, sur

  9. Improvement of polysaccharide and triterpenoid production of Ganoderma lucidum through mutagenesis of protoplasts

    Directory of Open Access Journals (Sweden)

    Ren Peng

    2016-03-01

    Full Text Available Ganoderma lucidum is a traditional medicinal macrofungus in China, which has two kinds of key bioactive compounds -- polysaccharides and triterpenoids. To improve the polysaccharide and triterpenoid production from G. lucidum, the preparation and regeneration conditions of protoplasts were optimized. This was done by systematic trials with various parameters, and protoplast mutation was subsequently performed. A mycelium that was cultivated for seven days and treated with 0.33 mL of 1% snailase and 0.66 mL of 0.5% cellulase solution for 2.5 h at 30 °C in the presence of osmotic pressure stabilizer mannitol (0.5 mol/L, had the best conditions, in which the resultant protoplasts were 6.40 × 105/mL and the regeneration rate was 6.25%. The resultant protoplasts were subjected to subsequent mutation by lithium chloride or by the combination of lithium chloride and Triton X-100. The highest yields of intracellular polysaccharide and triterpenoid in two mutant strains were 37.50 and 40.81 mg/g, which were increased with 568.45% and 373.43%, respectively, as compared to the original strain. Furthermore, the yields of intracellular polysaccharides and triterpenoids in the second generation and the third generation of the mutants were comparable to that of the first generation, which showed genetic stability of the mutants for the production of polysaccharides and triterpenoids.

  10. OXYGEN DEPENDENCE OF PHOTOINHIBITION AT LOW-TEMPERATURE IN INTACT PROTOPLASTS OF VALERIANELLA-LOCUSTA L

    NARCIS (Netherlands)

    VANWIJK, KJ; KRAUSE, GH

    1991-01-01

    Photoinhibition of photosynthesis in vivo is shown to be considerably promoted by O2 under circumstances where energy turnover by photorespiration and photosynthetic carbon metabolism are low. Intact protoplasts of Valerianella locusta L. were photoinhibited by 30 min irradiation with 3000-mu-mol ph

  11. Overexpression of several Arabidopsis histone genes increases agrobacterium-mediated transformation and transgene expression in plants.

    Science.gov (United States)

    Tenea, Gabriela N; Spantzel, Joerg; Lee, Lan-Ying; Zhu, Yanmin; Lin, Kui; Johnson, Susan J; Gelvin, Stanton B

    2009-10-01

    The Arabidopsis thaliana histone H2A-1 is important for Agrobacterium tumefaciens-mediated plant transformation. Mutation of HTA1, the gene encoding histone H2A-1, results in decreased T-DNA integration into the genome of Arabidopsis roots, whereas overexpression of HTA1 increases transformation frequency. To understand the mechanism by which HTA1 enhances transformation, we investigated the effects of overexpression of numerous Arabidopsis histones on transformation and transgene expression. Transgenic Arabidopsis containing cDNAs encoding histone H2A (HTA), histone H4 (HFO), and histone H3-11 (HTR11) displayed increased transformation susceptibility, whereas histone H2B (HTB) and most histone H3 (HTR) cDNAs did not increase transformation. A parallel increase in transient gene expression was observed when histone HTA, HFO, or HTR11 overexpression constructs were cotransfected with double- or single-stranded forms of a gusA gene into tobacco (Nicotiana tabacum) protoplasts. However, these cDNAs did not increase expression of a previously integrated transgene. We identified the N-terminal 39 amino acids of H2A-1 as sufficient to increase transient transgene expression in plants. After transfection, transgene DNA accumulates more rapidly in the presence of HTA1 than with a control construction. Our results suggest that certain histones enhance transgene expression, protect incoming transgene DNA during the initial stages of transformation, and subsequently increase the efficiency of Agrobacterium-mediated transformation.

  12. Isolation and gene expression analysis of Arabidopsis thaliana mutants with constitutive expression of ATL2, an early elicitor-response RING-H2 zinc-finger gene.

    OpenAIRE

    2004-01-01

    Genes with unstable transcripts often encode proteins that play important regulatory roles. ATL2 is a member of a multigene family coding highly related RING-H2 zinc-finger proteins that may function as E3 ubiquitin ligases. ATL2 mRNA accumulation occurs rapidly and transiently after incubation with elicitors of pathogen response. We screened 50,000 M(2) families from a line that carries a fusion of pATL2 to the GUS reporter gene and isolated five mutants, which we named eca (expresión consti...

  13. Cytohistological analysis of somatic embryogenesis in cucumber (Cucumis sativus L. II. Natural fluorescence and direct somatic embryogenesis from protoplasts

    Directory of Open Access Journals (Sweden)

    W. Burza

    2014-02-01

    Full Text Available The development of protoplast derived from somatic embryos and some of their characteristics were compared with embryos from suspension and in vivo in the same B line. Embryos formed in a protoplast culture differed from others that their younger stages contained vacuolated cells, and older ones had altered morphological and histological structure. Somatic embryogenesis is more regular from suspension then from protoplasts. No distinct differences were observed in the rate of embryo development in vivo and in vitro, and in vitro embryos show a larger variation in size at the same stage. Embryos in vitro with fluorescence are generally larger than zygotic ones at each stage. The use of fluorescence is suggested for the selection of heterokariocytes after protoplast fusion.

  14. Anoxia-induced elevation of cytosolic Ca2+ concentration depends on different Ca2+ sources in rice and wheat protoplasts.

    Science.gov (United States)

    Yemelyanov, Vladislav V; Shishova, Maria F; Chirkova, Tamara V; Lindberg, Sylvia M

    2011-08-01

    The anoxia-dependent elevation of cytosolic Ca(2+) concentration, [Ca(2+)](cyt), was investigated in plants differing in tolerance to hypoxia. The [Ca(2+)](cyt) was measured by fluorescence microscopy in single protoplasts loaded with the calcium-fluoroprobe Fura 2-AM. Imposition of anoxia led to a fast (within 3 min) significant elevation of [Ca(2+)](cyt) in rice leaf protoplasts. A tenfold drop in the external Ca(2+) concentration (to 0.1 mM) resulted in considerable decrease of the [Ca(2+)](cyt) shift. Rice root protoplasts reacted upon anoxia with higher amplitude. Addition of plasma membrane (verapamil, La(3+) and EGTA) and intracellular membrane Ca(2+)-channel antagonists (Li(+), ruthenium red and cyclosporine A) reduced the anoxic Ca(2+)-accumulation in rice. Wheat protoplasts responded to anoxia by smaller changes of [Ca(2+)](cyt). In wheat leaf protoplasts, the amplitude of the Ca(2+)-shift little depended on the external level of Ca(2+). Wheat root protoplasts were characterized by a small shift of [Ca(2+)](cyt) under anoxia. Plasmalemma Ca(2+)-channel blockers had little effect on the elevation of cytosolic Ca(2+) in wheat protoplasts. Intact rice seedlings absorbed Ca(2+) from the external medium under anoxic treatment. On the contrary, wheat seedlings were characterized by leakage of Ca(2+). Verapamil abolished the Ca(2+) influx in rice roots and Ca(2+) efflux from wheat roots. Anoxia-induced [Ca(2+)](cyt) elevation was high particularly in rice, a hypoxia-tolerant species. In conclusion, both external and internal Ca(2+) stores are important for anoxic [Ca(2+)](cyt) elevation in rice, whereas the hypoxia-intolerant wheat does not require external sources for [Ca(2+)](cyt) rise. Leaf and root protoplasts similarly responded to anoxia, independent of their organ origin.

  15. In vitro synthesis of cellulose microfibrils by a membrane protein from protoplasts of the non-vascular plant Physcomitrella patens.

    Science.gov (United States)

    Cho, Sung Hyun; Du, Juan; Sines, Ian; Poosarla, Venkata Giridhar; Vepachedu, Venkata; Kafle, Kabindra; Park, Yong Bum; Kim, Seong H; Kumar, Manish; Nixon, B Tracy

    2015-09-01

    Plant cellulose synthases (CesAs) form a family of membrane proteins that are associated with hexagonal structures in the plasma membrane called CesA complexes (CSCs). It has been difficult to purify plant CesA proteins for biochemical and structural studies. We describe CesA activity in a membrane protein preparation isolated from protoplasts of Physcomitrella patens overexpressing haemagglutinin (HA)-tagged PpCesA5. Incubating the membrane preparation with UDP-glucose predominantly produced cellulose. Negative-stain EM revealed microfibrils. Cellulase bound to and degraded these microfibrils. Vibrational sum frequency generation (SFG) spectroscopic analysis detected the presence of crystalline cellulose in the microfibrils. Putative CesA proteins were frequently observed attached to the microfibril ends. Combined cross-linking and gradient centrifugation showed bundles of cellulose microfibrils with larger particle aggregates, possibly CSCs. These results suggest that P. patens is a useful model system for biochemical and structural characterization of plant CSCs and their components.

  16. A comparison of the phenotypic and genetic stability of recombinant Trichoderma spp. generated by protoplast- and Agrobacterium-mediated transformation.

    Science.gov (United States)

    Cardoza, Rosa Elena; Vizcaino, Juan Antonio; Hermosa, Maria Rosa; Monte, Enrique; Gutiérrez, Santiago

    2006-08-01

    Four different Trichoderma strains, T. harzianum CECT 2413, T. asperellum T53, T. atroviride T11 and T. longibrachiatum T52, which represent three of the four sections contained in this genus, were transformed by two different techniques: a protocol based on the isolation of protoplasts and a protocol based on Agrobacterium-mediated transformation. Both methods were set up using hygromycin B or phleomycin resistance as the selection markers. Using these techniques, we obtained phenotypically stable transformants of these four different strains. The highest transformation efficiencies were obtained with the T. longibrachiatum T52 strain: 65-70 transformants/microg DNA when transformed with the plasmid pAN7-1 (hygromycin B resistance) and 280 transformants/107 spores when the Agrobacterium-mediated transformation was performed with the plasmid pUR5750 (hygromycin B resistance). Overall, the genetic analysis of the transformants showed that some of the strains integrated and maintained the transforming DNA in their genome throughout the entire transformation and selection process. In other cases, the integrated DNA was lost.

  17. Reference: 2 [Arabidopsis Phenome Database[Archive

    Lifescience Database Archive (English)

    Full Text Available at share 60 to 80% protein sequence identity. Gene disruptions of the yeast (Saccharomyces cerevisiae) ortho... that these syntaxins are not essential for growth in yeast. However, we have isolated and characterized gene disruption...s in two genes from each family, finding that disruption of individual syntaxins from these fami...lies is lethal in the male gametophyte of Arabidopsis. Complementation of the syp21-1 gene disruption

  18. Reference: 341 [Arabidopsis Phenome Database[Archive

    Lifescience Database Archive (English)

    Full Text Available owth. Despite the physiological importance of this process, the molecular mechanism is unknown. Here..., a genetic screen has been used to identify Arabidopsis thaliana mutants that exhibit a ...postgerminative growth arrest phenotype, which can be rescued by providing sugar. Seventeen sugar-dependent (sdp) mutants were... isolated, and six represent new loci. Triacylglycerol hydrolas...e assays showed that sdp1, sdp2, and sdp3 seedlings are deficient specifically in the lipase activity that i

  19. [Analysis of tobacco regeneration plants from the protoplasts produced by electrofusion [corrected] in space].

    Science.gov (United States)

    Li, Xiu-Gen; Chen, Ai-Di; Wang, Liu-Fa; Zheng, Hui-Qiong

    2007-10-01

    Vacuolated mesophyll protoplasts of Nicotiana rustica L. were electrically fused with evacuolated protoplasts of the same genus (N. tabacum cv. 'Gexin No.1') during a 7-day space flight in the Chinese spacecraft "SZ-4". The initial cell division leading to micro-callus formation took place after landing (Fig.1). Higher plating efficiencies were observed in the flight samples than the control culture, but the frequency of plantlets regeneration reduced by about 20% of the control (Table 1). The hybrid characters were tested by chromosome counting, isozyme analysis and comparison of morphological characteristics (Figs.2-4). About 32% of the regenerates showed hybrid character. Leaf morphological modifications were found in 3 hybrids, i.e., H23, H25 and H27. After backcrossing with N. rustica, alterations in flower color and leaf shape occurred in the somatic hybrid H23 (Fig.5). These results demonstrate that the hybrids formed under microgravity condition could regenerate fertile plants.

  20. Quantitative analysis of protein-protein interactions by split firefly luciferase complementation in plant protoplasts.

    Science.gov (United States)

    Li, Jian-Feng; Zhang, Dandan

    2014-07-01

    This unit describes the split firefly luciferase complementation (SFLC) assay, a high-throughput quantitative method that can be used to investigate protein-protein interactions (PPIs) in plant mesophyll protoplasts. In SFLC, the two proteins to be tested for interaction are expressed as chimeric proteins, each fused to a different half of firefly luciferase. If the proteins interact, a functional luciferase can be transitorily reconstituted, and is detected using the cell-permeable substrate D-luciferin. An advantage of the SFLC assay is that dynamic changes in PPIs in a cell can be detected in a near real-time manner. Another advantage is the unusually high DNA co-transfection and protein expression efficiencies that can be achieved in plant protoplasts, thereby enhancing the throughput of the method.

  1. Selection of somatic hybrids after fusion of protoplasts from Datura innoxia Mill. and Atropa belladonna L.

    Science.gov (United States)

    Krumbiegel, G; Schieder, O

    1979-01-01

    After fusion of protoplasts from a diploid (2n=24) and a tetraploid (4n=48) chlorophyll-deficient mutant of Datura innoxia Mill. with diploid (2n=72) green wild-type protoplasts of Atropa belladonna L. thirteen somatic hybrids could be selected, most of which had already started to produce leaves and shoots. Hybrid calli were recognizable by the production of hairs, typical for Datura innoxia, and the green colour, derived from Atropa belladonna. Further proof for the hybrid nature was furnished by cytological investigations. The metaphase chromosomes of both species are easily distinguishable in their size: chromosomes of Datura innoxia are about twice as large as those of Atropa belladonna. The chromosome numbers of the hybrids varied from ca. 84 to ca. 175.

  2. Enhanced sinefungin production by medium improvement, mutagenesis and protoplast regeneration of Streptomyces incarnatus NRRL 8089.

    Science.gov (United States)

    Malina, H; Tempete, C; Robert-Gero, M

    1985-09-01

    Increased production of sinefungin, a very potent antifungal and antiparasitic nucleoside antibiotic was achieved by medium and strain improvement. When soybean-meal, dextrin and yeast extract were added as carbon and nitrogen sources to the fermentation medium, instead of corn steep liquor, soya-oil and glucose; the antibiotic yield increased from 40 micrograms/ml to 126 micrograms/ml with low biomass production. Strain improvement was attempted by two methods. The mean antibiotic yield of the variants after multistep mutagenesis by N-methyl-N'-nitro-N-nitrosoguanidine and ethyleneimine was 466 micrograms/ml. Protoplasts of the parental strain were prepared by lysozyme digestion from mycelia grown in a medium containing 0.7% glycine. The mean activity of the regenerated protoplasts was 664 micrograms/ml. Thus, the overall sinefungin production could be increased 16-fold.

  3. Rapid optimization of electroporation conditions for plant cells, protoplasts, and pollen.

    Science.gov (United States)

    Saunders, J A; Lin, C H; Hou, B H; Cheng, J; Tsengwa, N; Lin, J J; Smith, C R; McIntosh, M S; Van Wert, S

    1995-06-01

    The optimization of electroporation conditions for maximal uptake of DNA during direct gene transfer experiments is critical to achieve high levels of gene expression in transformed plant cells. Two stains, trypan blue and fluorescein diacetate, have been applied to optimize electroporation conditions for three plant cell types, using different square wave and exponential wave electroporation devices. The different cell types included protoplasts from tobacco, a stable mixotrophic suspension cell culture from soybean with intact cell walls, and germinating pollen from alfalfa and tobacco. Successful electroporation of each of these cell types was obtained, even in the presence of an intact cell wall when conditions were optimized for the electroporation pulse. The optimal field strength for each of these cells differs, protoplasts having the lowest optimal pulse field strength, followed by suspension cells and finally germinating pollen requiring the strongest electroporation pulse. A rapid procedure is described for optimizing electroporation parameters using different types of cells from different plant sources.

  4. Fine structure of fusion products from soybean cell culture and pea leaf protoplasts.

    Science.gov (United States)

    Fowke, L C; Constabel, F; Gamborg, O L

    1977-01-01

    Protoplasts from pea (Pisum sativum L.) leaves and cultured soybean (Glycine max L.) cells were fused by means of polyethylene glycol and subsequently cultured for one week. Both agglutinated protoplasts and cultured fusion products were examined by electron microscopy. Agglutination occurred over large areas of the plasma membranes. The membrane contanct was discontinuous and irregularly spaced. Many cultured fusion products regenerated cell walls and divided to form cell clusters. Fusion of pea and soybean interphase nuclei occurred in some cells. The detection of heterochromatin typical of pea in the synkaryon, even after division, suggests the cells were hybrids. The cytoplasm of the cells from the fusion products contained both soybean leucoplasts and pea chloroplasts. The chloroplasts had apparently ceased dividing and some showed signs of degenerating. Large multinucleate fusion products developed cell walls but failed to divide.

  5. 植物原生质体全能性表达及其在甘蓝类蔬菜育种上的应用%Totipotency Expression of Plant Protoplast and Its Application in Brassica oleracea L.Vegetable Breeding

    Institute of Scientific and Technical Information of China (English)

    盛小光; 顾宏辉; 赵振卿; 虞慧芳; 王建升; 张晓辉

    2011-01-01

    The isolated protoplasts, exposed cells without coated cell wall, own the totipotency to regenerate a whole plant under appropriate culture conditions. Separation, culture and reproduction technology of plant protoplast is a platform for plant breeding, gene engineering and cell physiology study. Therefore it is of important significance. This paper expounds the obtain, culture and regeneration etc key technology in the process of protoplast totipotency expression, and their application in Brassica oleracea vegetables breeding. Existing problems in protoplast technology were discussed. Suggestions were proposed for improving plant protoplast culture system and for this work in the near future.%植物原生质体指植物除去细胞壁后被质膜包被的裸露细胞,在适当培养条件下具有再生成完整植株的全能性.植物原生质体分离、培养及植株再生技术是进行植物育种、基因工程、遗传理论及细胞生理特性研究的平台,具有重要的研究意义.本文概述了植物原生质体全能性表达过程中原生质体的获得、培养和再生等关键技术环节及其在甘蓝类蔬菜育种上的应用,同时讨论了原生质体培养技术中存在的问题,并且对完善植物原生质体培养体系及今后的工作方向提出了建议.

  6. Callus formation and plant regeneration from protoplasts of sunflower calli and hypocotyls

    Directory of Open Access Journals (Sweden)

    Conceição Santos

    2014-02-01

    Full Text Available Sunflower (cv. Girapac SH222 protoplasts were obtained from 4-7 day-old hypocotyls and cotyledons and from two-month old calli. Higher yields of protoplasts were achieved with medium El (KCl 25g dm-3, CaCl2 2g dm-3, MES 0.7 g• dm-3, pH 5.5 and the combination of Driselase Fluka 0.2%, Macerozyme Onozuka 0.2% and Cellulase Onozuka R10 0.2%. Hypocotyls gave the highest yields of protoplasts, followed by cotyledons and calli. Protoplasts were cultivated in liquid and on solid media using both L4M (Burrus et al., 1991 and V-KM (Bokelman and Roest, 1983 media. Culture on solid M1 medium (L4M medium supplemented with NAA 3.0 mg•dm- 3, 2,4-D 0.1 mg•dm-3 and BA 1.0 mg•dm -3 gave a good planting efficiency with the development of many white-green colonies. These colonies gave rise to small calli which were transferred to MSmod medium (MS medium supplemented with KCI 5 g•dm-', and polyvinylpyrrolidone (PVP, 4 g• dm-3 containing benziladenine (BA, 0.5 mg•dm-3, naphtaleneacetic acid (NAA, 0.5 mg•dm-3 and giberelic acid (GA3 0.1 mg•dm-3. After two weeks, calli were transferred to MSmod medium containing BA 1.0 mg•dm-3, NAA 0.1 mg•dm-3, and GA3 0.1 mg•dm-3 for shoot formation. Shoots were excised and induced to root in MSmod supplemented with BA 0.1 mg•dm-3, NAA 1.0 mg•dm-3, and GA3 0.1 mg•dm-3. Plantlets were then transferred to sterilised vermiculite for greenhouse acclimation.

  7. The effect of microgravity on the development of plant protoplasts flown on Biokosmos 9

    Science.gov (United States)

    Iversen, T.-H.; Rasmussen, O.; Gmünder, F.; Baggerud, C.; Kordyum, E. L.; Lozovaya, V. V.; Tairbekov, M.

    An experiment using plant protoplasts has been accepted for the IML-1 Space Shuttle mission scheduled for 1991. Preparatory experiments have been performed using both fast and slow rotating clinostats and in orbit to study the effect of simulated and real weightlessness on protoplast regeneration. Late access to the space vehicles before launch has required special attention since it is important to delay cell wall regeneration until the samples are in orbit. On a flight on Biokosmos 9 (``Kosmos-2044'') in September 1989 some preliminary results were obtained. Compared to the ground control, the growth of both carrot and rapeseed protoplasts was decreased by 18% and 44% respectively, after 14 days in orbit. The results also indicated that there is less cell wall regeneration under micro-g conditions. Compared to the ground controls the production of cellulose in rapeseed and carrot flight samples was only 46% and 29% respectively. The production of hemicellulose in the flight samples was 63% and 67% respectively of that of the ground controls. In both cases all samples reached the stage of callus development. The peroxidase activity was also found to be lower in the flight samples than in the ground controls, and the number of different isoenzymes was decreased in the flight samples. In general, the regeneration processes were retarded in the flight samples with respect to the ground controls. From a simulation experiment for IML-1 performed in January 1990 at ESTEC, Holland, regenerated plants have been obtained. These results are discussed and compared to the results obtained on Biokosmos 9. Protoplast regeneration did not develop beyond the callus stage in either the flight or the ground control samples from the Biokosmos 9 experiment.

  8. Alternative translational initiation of ATP sulfurylase underlying dual localization of sulfate assimilation pathways in plastids and cytosol in Arabidopsis thaliana

    Directory of Open Access Journals (Sweden)

    Anne-Sophie eBohrer

    2015-01-01

    Full Text Available Plants assimilate inorganic sulfate into sulfur-containing vital metabolites. ATP sulfurylase (ATPS is the enzyme catalyzing the key entry step of the sulfate assimilation pathway in both plastids and cytosol in plants. Arabidopsis thaliana has four ATPS genes (ATPS1, -2, -3 and -4 encoding ATPS pre-proteins containing N-terminal transit peptide sequences for plastid targeting, however, the genetic identity of the cytosolic ATPS has remained unverified. Here we show that Arabidopsis ATPS2 dually encodes plastidic and cytosolic ATPS isoforms, differentiating their subcellular localizations by initiating translation at AUGMet1 to produce plastid-targeted ATPS2 pre-proteins or at AUGMet52 or AUGMet58 within the transit peptide to have ATPS2 stay in cytosol. Translational initiation of ATPS2 at AUGMet52 or AUGMet58 was verified by expressing a tandem-fused synthetic gene, ATPS2(5’UTR-His12:Renilla luciferase:ATPS2(Ile13-Val77:firefly luciferase, under a single constitutively active CaMV 35S promoter in Arabidopsis protoplasts and examining the activities of two different luciferases translated in-frame with split N-terminal portions of ATPS2. Introducing missense mutations at AUGMet52 and AUGMet58 significantly reduced the firefly luciferase activity, while AUGMet52 was a relatively preferred site for the alternative translational initiation. The activity of luciferase fusion protein starting at AUGMet52 or AUGMet58 was not modulated by changes in sulfate conditions. The dual localizations of ATPS2 in plastids and cytosol were further evidenced by expression of ATPS2-GFP fusion proteins in Arabidopsis protoplasts and transgenic lines, while they were also under control of tissue-specific ATPS2 promoter activity found predominantly in leaf epidermal cells, guard cells, vascular tissues and roots.

  9. Effect of tobacco mosaic virus infection on host and virus-specific protein synthesis in protoplasts

    Energy Technology Data Exchange (ETDEWEB)

    Siegel, A.; Hari, V.; Kolacz, K.

    1978-04-01

    The nature and rate of virus-specific protein synthesis were determined in tobacco mosaic virus-infected protoplasts as a function of time after inoculation. Samples of infected and mock-infected protoplasts were exposed to radioactive amino acid for relatively short sequential time periods and the consequent labeled proteins were assessed following SDS-polyacrylamide gel electrophoresis and fluorography. The synthesis of three virus-specific proteins of molecular weights 160,000, 135,000, and 17,500 was confirmed. Synthesis of all three proteins was first detected during the 5- to 7-hr postinoculation period at which time the synthetic rate of the 135,000-dalton protein was greatest.This was soon overtaken by the 17,500-dalton capsid protein, the synthetic rate of which kept increasing until it accounted for a major portion of total protoplast protein synthesis. At 1 day postinoculation, it accounted for 50% and, at not quite 2 days, 70% of the total protein synthesis. Evidence is presented to suggest that virus-specific protein synthesis occurs in addition to, rather than at the expense of, normal cellular protein synthesis.

  10. Screening and characterization of a high taxol producing fungus by protoplast mutagenesis

    Institute of Scientific and Technical Information of China (English)

    Zhao Kai; Sun Qingshen; Zhang Yanjun; Ping Wenxiang; Jin Tao; Zhou Dongpo

    2009-01-01

    The preparation, regeneration and mutagenesis of the taxol-producing fungus UV40-19 protoplasts were discussed in the experiment. Totally 42 strains displayed hygromycin resistance. Six strains were found to be positive mutants when screened on plate containing 90μg/mL hygromycin. One hereditarily stable strain UN05-6 was obtained, which raised the taxol yield from (376.38±8.41)μg/L to (493.12±11.36)μg/L. The optimal conditions for the preparation, regeneration and mutagenesis of the taxol producing fungus UV40-19 were as follows: 1)enzymolysis in a solution containing 3% lywallzyme, 4% snailase, 1% lysozyme and 3% cellulose at 30℃ water bath, pH5.5~6.0 for 5h; 2) The prepared protoplasts were regenerated by using bilayer plate culturing method; 3)To mutagenize the fungus UV40-19, the protoplast suspension was treated with 0.8mg/mL NTG for 15min, followed by UV irradiation (30W, 30cm distance)for 40s under magnetic stirring. The purified products of the fungus UN05-6 fermented extracts have significant inhibitive effects on SMMC-7721 cell.

  11. Analysis of plants regenerated from protoplast fusions between Brassica napus and Eruca sativa.

    Science.gov (United States)

    Fahleson, J; Råhlén, L; Glimelius, K

    1988-10-01

    Protoplasts from etiolated hypocotyls of Brassica napus stained with carboxyfluorescein were fused with mesophyll protoplasts from Eruca sativa. Hybrid cells could be identified under the light microscope by (1) fully developed chloroplasts derived from E. sativa and (2) the cytoplasmic strands of the B. napus hypocotyl protoplasts, or (3) by the presence of both red and green fluorescence when investigated under UV light. The heterokaryons were selected using either a micro-manipulator or a flow sorter. On average, 5.4% of the calli obtained after selection differentiated into shoots. Regenerated shoots were subjected to isozyme analysis for verification of their hybrid character. Of the 23 hybrids successfully transferred to the greenhouse, 11 were asymmetric according to isozyme analysis. The nuclear DNA content of the hybrids was determined by flow cytometry, which gives an estimate of chromosome number. Most of the hybrids had a DNA content, and thus a chromosome number, that deviated from the expected sum of the parents. Almost all of the hybrids had some degree of fertility and produced seeds. Seed set, expressed as seeds per pollinated flower, was on average 7% of that of B. napus in the case of self-pollination and 26% of that of B. napus when backcrossed to B. napus. The chloroplast genotype was investigated in 13 hybrids. Of these, 11 had chloroplasts derived from B. napus, while only 2 had chloroplasts of E. sativa origin.

  12. DNA-Free Genetically Edited Grapevine and Apple Protoplast Using CRISPR/Cas9 Ribonucleoproteins

    Science.gov (United States)

    Malnoy, Mickael; Viola, Roberto; Jung, Min-Hee; Koo, Ok-Jae; Kim, Seokjoong; Kim, Jin-Soo; Velasco, Riccardo; Nagamangala Kanchiswamy, Chidananda

    2016-01-01

    The combined availability of whole genome sequences and genome editing tools is set to revolutionize the field of fruit biotechnology by enabling the introduction of targeted genetic changes with unprecedented control and accuracy, both to explore emergent phenotypes and to introduce new functionalities. Although plasmid-mediated delivery of genome editing components to plant cells is very efficient, it also presents some drawbacks, such as possible random integration of plasmid sequences in the host genome. Additionally, it may well be intercepted by current process-based GMO regulations, complicating the path to commercialization of improved varieties. Here, we explore direct delivery of purified CRISPR/Cas9 ribonucleoproteins (RNPs) to the protoplast of grape cultivar Chardonnay and apple cultivar such as Golden delicious fruit crop plants for efficient targeted mutagenesis. We targeted MLO-7, a susceptible gene in order to increase resistance to powdery mildew in grape cultivar and DIPM-1, DIPM-2, and DIPM-4 in the apple to increase resistance to fire blight disease. Furthermore, efficient protoplast transformation, the molar ratio of Cas9 and sgRNAs were optimized for each grape and apple cultivar. The targeted mutagenesis insertion and deletion rate was analyzed using targeted deep sequencing. Our results demonstrate that direct delivery of CRISPR/Cas9 RNPs to the protoplast system enables targeted gene editing and paves the way to the generation of DNA-free genome edited grapevine and apple plants. PMID:28066464

  13. Arabidopsis gene expression patterns during spaceflight

    Science.gov (United States)

    Paul, A.-L.; Ferl, R. J.

    The exposure of Arabidopsis thaliana (Arabidopsis) plants to spaceflight environments resulted in the differential expression of hundreds of genes. A 5 day mission on orbiter Columbia in 1999 (STS-93) carried transgenic Arabidopsis plants engineered with a transgene composed of the alcohol dehydrogenase (Adh) gene promoter linked to the β -Glucuronidase (GUS) reporter gene. The plants were used to evaluate the effects of spaceflight on two fronts. First, expression patterns visualized with the Adh/GUS transgene were used to address specifically the possibility that spaceflight induces a hypoxic stress response, and to assess whether any spaceflight response was similar to control terrestrial hypoxia-induced gene expression patterns. (Paul et al., Plant Physiol. 2001, 126:613). Second, genome-wide patterns of native gene expression were evaluated utilizing the Affymetrix ATH1 GeneChip? array of 8,000 Arabidopsis genes. As a control for the veracity of the array analyses, a selection of genes identified with the arrays was further characterized with quantitative Real-Time RT PCR (ABI - TaqmanTM). Comparison of the patterns of expression for arrays of hybridized with RNA isolated from plants exposed to spaceflight compared to the control arrays revealed hundreds of genes that were differentially expressed in response to spaceflight, yet most genes that are hallmarks of hypoxic stress were unaffected. These results will be discussed in light of current models for plant responses to the spaceflight environment, and with regard to potential future flight opportunities.

  14. Membrane-bound ATPase of intact vacuoles and tonoplasts isolated from mature plant tissue

    Energy Technology Data Exchange (ETDEWEB)

    Lin, W.; Wagner, G.J.; Siegelman, H.W.; Hind, G.

    1977-01-01

    Intact vacuoles were isolated from petals of Hippeastrum and Tulipa (Wagner, G. J. and Siegelman, H. W. (1975) Science 190, 1298 to 1299). The ATPase activity of fresh vacuole suspensions was found to be 2 to 3 times that of protoplasts from the same tissue. 70 to 80% of the ATPase activity of intact vacuoles was recovered in tonoplast preparations. The antibiotic Dio-9 at 6 ..mu..g/10/sup 6/ vacuoles or protoplasts causes 40% inhibition. However, only the protoplast ATPase is sensitive to oligomycin. N,N'-dicyclohexylcarbondiimide (DCCD) slightly stimulates ATPase activity in both vacuole and protoplast suspensions, whereas ethyl-3-(3-dimethylaminopropyl carbodiimide) (EDAC) strongly inhibits. Spectrophotometric studies show that in the petal the vacuolar contents have a pH of 4.0 for Tulipa and 4.3 for Hippeastrum, whereas the intact isolated vacuole has an internal pH of 7.0 (in pH 8.0 buffer) for Tulipa and about 7.3 for Hippeastrum. Internal ion concentrations of 150, 46, 30, 30 and 6 mM were found for K/sup +/, Na/sup +/, Mg/sup 2 +/, Cl/sup -/, and Ca/sup 2 +/ respectively, which are about the same as those in protoplasts.

  15. Localization of the Arabidopsis Senescence- and Cell Death-Associated BFN1 Nuclease: From the ER to Fragmented Nuclei

    Institute of Scientific and Technical Information of China (English)

    Sarit Farage-Barhom; Shaul Burd; Lilian Sonego; Ana Mett; Eduard Belausov; David Gidoni; Amnon Lers

    2011-01-01

    Plant senescence- or PCD-associated nucleases share significant homology with nucleases from different organisms.However,knowledge of their function is limited.Intracellular localization of the Arabidopsis senescenceand PCD-associated nuclease BFN1 was investigated.Analysis of BFN1-GFP localization in transiently transformed tobacco protoplasts revealed initial localization in filamentous structures spread throughout the cytoplasm,which then clustered around the nuclei as the protoplasts senesced.These filamentous structures were identified as being of ER origin.In BFN1GFP-transgenic Arabidopsis plants,similar localization of BFN1-GFP was observed in young leaves,that is,in filamentous structures that reorganized around the nuclei only in senescing cells.In late senescence,BFN1-GFP was localized with fragmented nuclei in membrane-wrapped vesicles.BFN1's postulated function as a nucleic acid-degrading enzyme in senescence and PCD is supported by its localization pattern.Our results suggest the existence of a dedicated compartment mediating nucleic acid degradation in senescence and PCD processes.

  16. Integrin-like Protein Is Involved in the Osmotic Stress-induced Abscisic Acid Biosynthesis in Arabidopsis thaliana

    Institute of Scientific and Technical Information of China (English)

    Bing Lü; Feng Chen; Zhong-Hua Gong; Hong Xie; Jian-Sheng Liang

    2007-01-01

    We studied the perception of plant cells to osmotic stress that leads to the accumulation of abscisic acid (ABA) in stressed Arabidopsis thaliana L. cells. A significant difference was found between protoplasts and cells in terms of their responses to osmotic stress and ABA biosynthesis, implying that cell wall and/or cell wall-plasma membrane interaction are essential in identifying osmotic stress. Western blotting and immunofluorescence localization experiments, using polyclonal antibody against human integrin β1, revealed the existence of a protein similar to the integrin protein of animals in the suspension-cultured cells located in the plasma membrane fraction.Treatment with a synthetic pentapeptide, Gly-Arg-Gly-Asp-Ser (GRGDS), which contains an RGD domain and interacts specifically with integrin protein and thus blocks the cell wall-plasma membrane interaction, significantly inhibited osmotic stress-induced ABA biosynthesis in cells, but not in protoplasts. These results demonstrate that cell wall and/or cell wall-plasma membrane interaction mediated by integrin-like proteins played important roles in osmotic stress-induced ABA biosynthesis in Arabidopsis thaliana.

  17. Quantitative Proteomic Analysis of the Response to Zinc, Magnesium, and Calcium Deficiency in Specific Cell Types of Arabidopsis Roots

    Directory of Open Access Journals (Sweden)

    Yoichiro Fukao

    2016-01-01

    Full Text Available The proteome profiles of specific cell types have recently been investigated using techniques such as fluorescence activated cell sorting and laser capture microdissection. However, quantitative proteomic analysis of specific cell types has not yet been performed. In this study, to investigate the response of the proteome to zinc, magnesium, and calcium deficiency in specific cell types of Arabidopsis thaliana roots, we performed isobaric tags for relative and absolute quantification (iTRAQ-based quantitative proteomics using GFP-expressing protoplasts collected by fluorescence-activated cell sorting. Protoplasts were collected from the pGL2-GFPer and pMGP-GFPer marker lines for epidermis or inner cell lines (pericycle, endodermis, and cortex, respectively. To increase the number of proteins identified, iTRAQ-labeled peptides were separated into 24 fractions by OFFGFEL electrophoresis prior to high-performance liquid chromatography coupled with mass spectrometry analysis. Overall, 1039 and 737 proteins were identified and quantified in the epidermal and inner cell lines, respectively. Interestingly, the expression of many proteins was decreased in the epidermis by mineral deficiency, although a weaker effect was observed in inner cell lines such as the pericycle, endodermis, and cortex. Here, we report for the first time the quantitative proteomics of specific cell types in Arabidopsis roots.

  18. 宽礁膜原生质体的分化与发育%Differentiation and development of protoplasts of Monostroma latissimum

    Institute of Scientific and Technical Information of China (English)

    谢恩义; 花卫华; 马家海

    2011-01-01

    为缩短传统育苗时间,开发室内种质保存技术,满足大规模栽培苗种需求,实验用4%的果胶酶和2%纤维素酶混合,将来自于不同月份和不同藻体部位的宽礁膜切段,分别在相同条件下解离成原生质体,详细研究了这些不同部位和不同生长时期细胞的再生、分化和发育途径.根据体细胞后代的外形、有无假根、有无公共膜、细胞大小和排列方式,以及它们最终的发育分化趋势,将宽礁膜原生质体发育方式分为8种结果,既可形成体细胞,也可形成生殖细胞.形成体细胞的发育途径又分3种类型7种结果,即细胞团、畸形苗和正常苗3种类型,其中发育成细胞团的类型又分为规则细胞团和不规则细胞团2种结果,发育成畸形苗的类型又分为假根为主的畸形苗、有类假根的畸形苗、无假根的畸形苗和管状畸形苗4种结果.各种发育类型与藻体的日龄、大小及部位有关,其原生质体直接发育形成正常形态藻体的发育方式只是8种发育结果的一种.%Monostrorna latissimum is one edible Chlorophyta species, and is a potential new species for economic seaweed cultivation by fishermen in China, for viable protoplasts could be potentially used as a source for seed material of macrophytic marine algae cultivation and for other applied phycological research,so the aim of this study was to develop a new breeding method by culturing the protoplasts of M. latissimum,to form seedlings quickly, to shorten the traditional period of germlings cultivation, to maintain a stock of seedlings in the laboratory for longer periods, and to provide abundant germlings for large-scale commercial cultivation. Protoplasts were isolated respectively from different parts of the thalli of M. latissimum which are collected at different time by enzymatic method( optimal enzyme composition consisted of 4% pectinase and 2% cellulase ). And the regeneration, differentiation and development

  19. Peptomics, identification of novel cationic Arabidopsis peptides with conserved sequence motifs

    DEFF Research Database (Denmark)

    Olsen, Addie Nina; Mundy, John; Skriver, Karen

    2002-01-01

    Few plant peptides involved in intercellular communication have been experimentally isolated. Sequence analysis of the Arabidopsis thaliana genome has revealed numerous transmembrane receptors predicted to bind proteinacious ligands, emphasizing the importance of identifying peptides with signali...

  20. Production and regeneration of protoplasts from orchid Mycorrhizal Fungi Epulorhiza repens and Ceratorhiza sp.

    Directory of Open Access Journals (Sweden)

    Irene da Silva Coelho

    2010-02-01

    Full Text Available The aim of this work was to study the standardization of conditions to obtain and regenerate Epulorhiza repens and Ceratorhiza sp. protoplasts. For E. repens, the largest number of protoplasts (8.0 × 10(6 protoplasts/mL was obtained in 0.6 M KCl, using 15 mg/mL of Lysing Enzymes, and 2-day-old fungal mycelium. When 0.5 M sucrose was used as osmotic stabilizer, the highest frequency of regeneration was achieved (8.5 %; 80.0 % of protoplasts were nucleated, and 20.0 % anucleated. For Ceratorhiza sp., the largest number of protoplasts (4.0 × 10(7 protoplasts/mL was achieved in 0.6 M NaCl, when 15 mg/mL of Lysing Enzymes and 15mg/mL of Glucanex, with 2-day-old fungal mycelium were used. The highest frequency of regeneration was 6.7 % using 0.5 M sucrose as osmotic stabilizer; 88.8 % of protoplasts were nucleated, and 11.2 % anucleated.O objetivo deste trabalho foi padronizar as condições de obtenção e regeneração de protoplastos de Epulorhiza repens e Ceratorhiza sp. Para o fungo E. repens, a maior produção de protoplastos, 8.0 x 10(6 protoplastos/mL, foi obtida em KCl 0.6 M, na presença de 15 mg/mL de "Lysing Enzymes" e micélio fúngico com 2 dias de idade. A maior freqüência de regeneração obtida foi de 8,5 % quando sacarose 0.5 M foi utilizada como estabilizador osmótico. Do total de protoplastos obtidos, 80 % eram nucleados e 20 % anucleados. Para Ceratorhiza sp., a maior produção de protoplastos, 4,0 x 10(7 protoplastos/mL, foi obtida em NaCl 0.6 M, na presença de 15 mg/mL de "Lysing Enzymes" e 15mg/mL de Glucanex, e micélio fúngico com 2 dias de idade. A maior freqüência de regeneração obtida foi de 6.7 % utilizando sacarose 0.5 M como estabilizador osmótico. Do total de protoplastos obtidos, 88.8 % eram nucleados e 1.2 % anucleados. O estabelecimento de protocolo otimizado para obtenção e regeneração de protoplastos dos fungos E. repens e Ceratorhiza sp. é importante, permitindo o estabelecimento de t

  1. Plant Regeneration from Stem-Derived Protoplasts of Solanum lycopersicoides%类番茄茄茎段原生质体再生成株的研究

    Institute of Scientific and Technical Information of China (English)

    张长远; 吴定华

    2001-01-01

    利用类番茄茄(Solamum lycopersicoides Dun.)无菌苗幼嫩茎段酶解获得大量原生质体(个)(2×106/g),将原生质体密度稀释至1×105/mL于HMA培养基中培养,则10d左右出现小细胞团,38~40d形成肉眼可见的小愈伤组织(1~1.5mm),转移至MC增殖培养基2周后,再转移到15#分化培养基诱导出芽,切取芽体转入50P培养基诱发根原基,再转入50#发根培养基形成发达根系,成为再生植株,整个再生周期80~90d,再生植株移植至土壤中均能正常生长、开花。%A procedure for protoplast isolation, culture and plant regeneration has been developed for solanum lycopersicoides. Stem-protoplasts were diluted to 1×105/mL and cultured in HMA medium, cell colonies (20~30 cells) appeared after 10 days, micro calli (1~1.5mm) formed after 38~40 days from initially culturing. The micro calli were transferred to MC greenig medium for 2 weeks. The green calli were transferred to 15# medium for shoot inducting. The shoots which excised from the callus were transferred to test tubes with 50p medium first for root promoting about 3~4 days and then transferred to 50# rooting medium. Plants regenerated after 80~90 days after initially culturing. All the plants which potted in soil could vigorously grow and blossom.

  2. Protoplast Culture and Plantlet Regeneration from Cell Line of Agrobacterium rhizogenes A4-transformed Alhagi pseudalhagi Desv%骆驼刺发根农杆菌转化系的原生质体培养和植株再生

    Institute of Scientific and Technical Information of China (English)

    张改娜; 贾敬芬

    2009-01-01

    The protoplasts were isolated from calli which were induced from hairy root segments of Agrobacterium rhizogenes A4-transformed Alhagi pseudalhagi. After cultured in the DPD medium supplemented with 1.5 mg·L~(-1) 2,4-D, 0.2 mg·L~(-1) 6-BA, 0.3 mol·L~(-1) mannitol, 500 mg·L~(-1) casein hydrolysate (CH) and 2% (W/V) sucrose, the protoplasts underwent sustained divisions and formed calli. The protoplast density of 4×10~5 mL~(-1) and (450±3) mOsm·kg~(-1) osmotic pressure in culture medium were proved to be appropriate for obtaining higher division frequency of protoplasts. A lot of protoplasts could be obtained by the enzymatic hydrolysis of yellowish subcultured calli after cultured on MS medium supplemented with 1.5 mg·L~(-1) NAA, 1.0 mg·L~(-1) 6-BA, 500 mg·L~(-1) CH and 2% (W/V) sucrose for 7-10 d. Lower temperature (4 ℃) pretreatment of subcultured calli enhanced ratios of protoplast isolation and subsequent divisions. The division frequency of protoplasts was about 50%. After transferred on the MS medium added with 1-2 mg·L~(-1) 6-BA (or KT) and 0.2 mg·L~(-1) NAA, the protoplast-derived calli differentiated and formed the regenerated plantlets. Paper electrophoresis analysis indicated that the protoplast-derived calli and regenerated plantlets still contained special product-opine in transgenic root hairs.%从发根农杆菌A4转化的荒漠植物-骆驼刺毛状根愈伤组织中分离的原生质体培养的结果表明,酶解新转代7~10 d的淡黄色松软愈伤组织,可获得大量有活力的原生质体.原生质体在附加有1.5 mg·L~(-1) 2,4-D、0.2 mg·L~(-1) 6-BA、0.3 mol·L~(-1)甘露醇、2%(W/V)蔗糖和500 mg·L~(-1)水解酪蛋白的DPD培养基中进行液体浅层培养可持续分裂.培养基的最适渗透压为(450±3)mOsm·kg~(-1),原生质体的最适植板密度为4×10~5个·mL~(-1).制备原生质体的愈伤组织以低温(4℃)预处理后,原生质体的产率和分裂频率均提高,分裂频率最高可达50%.

  3. 烟草原生质体的制备和分析%Preparation and Analysis of Tobacco Protoplasts

    Institute of Scientific and Technical Information of China (English)

    尚飞; 李咪咪; 李莉; 梁卫红

    2013-01-01

    植物原生质体作为一个良好的实验系统,广泛应用于植物分子及细胞学研究中.本研究以烟草NC89叶片为材料,采用酶解法制备烟草原生质体,经台盼蓝染色鉴定活性,结果显示,分离纯化后的原生质体产量为(12.7±1.6)×106 g-1,表明该方法能成功地制备高产率、高活力的原生质体,建立了一种烟草原生质体的简易制备方法.%Plant protoplast provides us a unique single cell system in biology study.To explore an effective method of protoplast preparation,we take tobacco leaves as material and digest it in enzyme solution,finally use trypan blue staining to identify the activities of protoplast.The results showed that the yield of protoplasts were up to (12.7 ± 1.6)× 106/g,higher than most of the recent reports.It suggest that a method had been established for high-yield and high-activity protoplast preparation in this research which has a certain reference value.

  4. Factors affecting the production and regeneration of protoplasts from Colletotrichum lindemuthianum Fatores que afetam a produção e regeneração de protoplastos de Colletotrichum lindemuthianum

    Directory of Open Access Journals (Sweden)

    Francine Hiromi Ishikawa

    2010-02-01

    Full Text Available The present work reports factors affecting the production and regeneration of protoplasts from Colletotrichum lindemuthianum. The usefulness of protoplast isolation is relevant for many different applications and has been principally used in procedures involving genetic manipulation. Osmotic stabilizers, lytic enzymes, incubation time and mycelial age were evaluated in terms of their effects on protoplast yield. The optimal condition for protoplast production included the incubation of young mycelia (48 h in 0.6 mol l-1 NaCl as the osmotic stabilizer, with 30 mg ml-1 Lysing Enzymes from Trichoderma harzianum for 3 h of incubation. In these conditions protoplasts production was higher than 10(6 protoplatos ml-1 in the digestion mixture, number suitable enough for experiments of transformation in fungi. Sucrose concentrations of 1.2 mol l-1 and 1 mol l-1 were the most suitable osmotic stabilizers for the regeneration after 48 h, with rates of 16.35% and 14.54%, respectively. This study produced an efficient method for protoplast production and reverted them into a typical mycelial morphology using a Colletotrichum lindemuthianum LV115 isolate.O presente trabalho apresenta os fatores que afetam a produção e regeneração de protoplastos de Colletotrichum lindemuthianum. O isolamento de protoplastos é muito relevante para diferentes aplicações, principalmente, em procedimentos que envolvem a manipulação genética. Estabilizadores osmóticos, enzimas líticas, tempo de incubação e idade micelial foram testados com relação ao efeito na liberação de protoplastos. As condições otimizadas para produção de protoplastos foram incubação de micélio jovem (48 h em estabilizador osmótico NaCl 0.6 mol l-1, acrescido de 30 mg ml-1 da enzima Lysing Enzymes de Trichoderma harzianum incubado, durante 3 h. Nessas condições, a obtenção de protoplastos foi maior que 10(6 protoplatos ml-1 na mistura de digestão, número suficientemente

  5. Exploring the use of cDNA-AFLP with leaf protoplasts as a tool to study primary cell wall biosynthesis in potato

    NARCIS (Netherlands)

    Oomen, R.J.F.J.; Bergervoet-van Deelen, J.E.M.; Bachem, C.W.B.; Visser, R.G.F.; Vincken, J.P.

    2003-01-01

    An RNA fingerprinting study of potato leaf protoplasts was performed to explore its suitability for identifying candidate genes involved in primary cell wall biosynthesis. Microscopic analysis, using calcofluor white to stain cellulose, showed that the protoplasts generated a new cell wall in the fi

  6. Identification and characterization of inward K ~+-channels in plasma membranes of Arabidopsis root cortex cells

    Institute of Scientific and Technical Information of China (English)

    于川江; 武维华

    1999-01-01

    Patch clamping whole-cell reeording techniques were apphed to study the inward K+ channels in Arabidopsis root cortex cells. The inward K+-channels in the plasma membranes of the root cortex cell protoplasts were activated by hyperpolarized membrane potentials. The channels were highly selective tor K+ ions over Na+ ions. The channel activity was significantly inbibited by the external TEA(?) or Ba(?) The changes in cytoplasmic Ca2+ concentrations did not affect the whole-cell inward K+-currents. The possible asso(?)ation betw(?)en the channel selectivity to K+ and Na(?) ions and plant salt-tolerance was also discussed.

  7. Fusion of protoplasts with irradiated micro protoplasts as a tool for radiation hybrid panel in citrus;Fusao de protoplastos com microprotoplastos irradiados como ferramenta para painel hibrido de radiacao em citros

    Energy Technology Data Exchange (ETDEWEB)

    Bona, Claudine Maria de, E-mail: debona@iapar.b [Instituto Agronomico do Parana (IAPAR), Curitiba, PR (Brazil). Centro Administrativo do Governo do Estado; Stelly, David, E-mail: stelly@tamu.ed [Texas A and M University (Tamu), College Station, TX (United States). Dept. of Soil and Crop Sciences; Miller Junior, J. Creighton, E-mail: jcmillerjr@tamu.ed [Texas A and M University (Tamu), College Station, TX (United States). Dept. of Horticultural Sciences; Louzada, Eliezer Silva, E-mail: elouzada@ag.tamu.ed [Texas A and M University, (Tamuk), Weslaco, TX (United States)

    2009-12-15

    The objective of this work was to combine asymmetric somatic hybridization (donor-recipient fusion or gamma fusion) to microprotoplast-mediated chromosome transfer, as a tool to be used for chromosome mapping in Citrus. Swinglea glutinosa micro protoplasts were irradiated either with 50, 70, 100 or 200 gamma rays and fused to cv. Ruby Red grapefruit or Murcott tangor protoplasts. Cell colonies were successfully formed and AFLP analyses confirmed presence of S. glutinosa in both 'Murcott' tangor and 'Ruby Red' grapefruit genomes. (author)

  8. Inhibition of Blue Light-Dependent H+ Pumping by Abscisic Acid in Vicia Guard-Cell Protoplasts.

    Science.gov (United States)

    Goh, C. H.; Kinoshita, T.; Oku, T.; Shimazaki, Ki.

    1996-06-01

    Blue-light (BL)-dependent H+ pumping in guard-cell protoplasts (GCPs) from Vicia faba was inhibited by 65% in the presence of abscisic acid (ABA). The inhibition increased with the time after application of ABA and was concentration dependent with a saturating concentration of 1 [mu]M at pH 6.2. The inhibition was nearly independent of the pH of the medium in the range 5.4 to 7.2 when ABA was applied at 10 [mu]M, whereas it was dependent on pH when the ABA concentration was decreased. The protonated form of ABA was saturating at 40 nM in inhibiting BL-dependent H+ pumping under various experimental conditions, whereas the dissociated form at 500 nM had no inhibitory effect on the pumping, suggesting that the protonated form of ABA is the form active in inhibiting the pumping. Fusicoccin (10 [mu]M), an activator of plasma membrane H+-ATPase, induced H+ pumping from GCPs, and the rate of H+ pumping was decreased to 70% by ABA. In contrast, ABA did not inhibit H+ pumping in isolated microsome vesicles from GCPs. These results suggest that the inhibition of BL-dependent H+ pumping by ABA in GCPs may be due to indirect inactivation of plasma membrane H+-ATPase and/or inhibition of the BL-signaling pathway. The pump inhibition by ABA causes membrane depolarization and can be an initial step to induce stomatal closure and reduces the transpirational water loss under drought stress in the daytime.

  9. Calcium-Mediated Mitochondrial Permeability Transition Involved in Hydrogen Peroxide-Induced Apoptosis in Tobacco Protoplasts

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    In the present study, we focused on whether intracellular free Ca2+ ([Ca2+]i) regulates the formation of mitochondrial permeability transition pore (MPTP) in H2O2-induced apoptosis in tobacco protoplasts. It was shown that the decrease in mitochondrial membrane potential (△Ψm) preceded the appearance of H2O2-induced apoptosis;pretreatment with the specific MPTP inhibitor cyclosporine A, which also inhibits Ca2+ cycling by the mitochondria,effectively retarded apoptosis and the decrease in △Ψm. Apoptosis and decreased △Ψm were exacerbated by CaCl2, whereas the plasma membrane voltage-dependent Ca2+ channel blocker lanthanum chloride (LaCl3)attentuated these responses. Chelation of extracellular Ca2+ with EGTA almost totally inhibited apoptosis and the decrease in △Ψm induced by H2O2. The time-course of changes in [Ca2+]i in apoptosis was detected using the Ca2+ probe Fluo-3 AM. These studies showed that [Ca2+]i was increased at the very early stage of H2O2-induced apoptosis. The EGTA evidently inhibited the increase in [Ca2+]i induced by H2O2, whereas it was only partially inhibited by LaCl3. The results suggest that H2O2 may elevate cytoplasmic free Ca2+ concentrations in tobacco protoplasts, which mainly results from the entry of extracellular Ca2+, to regulate mitochondrial permeability transition. The signaling pathway of [Ca2+]i-mediated mitochondrial permeability transition was associated with H2O2-induced apoptosis in tobacco protoplasts.

  10. Polyamines inhibit biosynthesis of ethylene in higher plant tissue and fruit protoplasts.

    Science.gov (United States)

    Apelbaum, A; Burgoon, A C; Anderson, J D; Lieberman, M

    1981-08-01

    Ethylene production in apple fruit and protoplasts and in leaf tissue was inhibited by spermidine or spermine. These polyamines, as well as putrescine, inhibited auxin-induced ethylene production and the conversion of methionine and 1-aminocyclopropane-1-carboxylic acid to ethylene. Polyamines were more effective as inhibitors of ethylene synthesis at the early, rather than at the late, stages of fruit ripening. Ca(2+) in the incubation medium reduced the inhibitory effect caused by the amines. A possible mode of action by which polyamines inhibit ethylene production is discussed.

  11. Deciphering the Molecular Mechanisms Underpinning the Transcriptional Control of Gene Expression by Master Transcriptional Regulators in Arabidopsis Seed.

    Science.gov (United States)

    Baud, Sébastien; Kelemen, Zsolt; Thévenin, Johanne; Boulard, Céline; Blanchet, Sandrine; To, Alexandra; Payre, Manon; Berger, Nathalie; Effroy-Cuzzi, Delphine; Franco-Zorrilla, Jose Manuel; Godoy, Marta; Solano, Roberto; Thevenon, Emmanuel; Parcy, François; Lepiniec, Loïc; Dubreucq, Bertrand

    2016-06-01

    In Arabidopsis (Arabidopsis thaliana), transcriptional control of seed maturation involves three related regulators with a B3 domain, namely LEAFY COTYLEDON2 (LEC2), ABSCISIC ACID INSENSITIVE3 (ABI3), and FUSCA3 (ABI3/FUS3/LEC2 [AFLs]). Although genetic analyses have demonstrated partially overlapping functions of these regulators, the underlying molecular mechanisms remained elusive. The results presented here confirmed that the three proteins bind RY DNA elements (with a 5'-CATG-3' core sequence) but with different specificities for flanking nucleotides. In planta as in the moss Physcomitrella patens protoplasts, the presence of RY-like (RYL) elements is necessary but not sufficient for the regulation of the OLEOSIN1 (OLE1) promoter by the B3 AFLs. G box-like domains, located in the vicinity of the RYL elements, also are required for proper activation of the promoter, suggesting that several proteins are involved. Consistent with this idea, LEC2 and ABI3 showed synergistic effects on the activation of the OLE1 promoter. What is more, LEC1 (a homolog of the NF-YB subunit of the CCAAT-binding complex) further enhanced the activation of this target promoter in the presence of LEC2 and ABI3. Finally, recombinant LEC1 and LEC2 proteins produced in Arabidopsis protoplasts could form a ternary complex with NF-YC2 in vitro, providing a molecular explanation for their functional interactions. Taken together, these results allow us to propose a molecular model for the transcriptional regulation of seed genes by the L-AFL proteins, based on the formation of regulatory multiprotein complexes between NF-YBs, which carry a specific aspartate-55 residue, and B3 transcription factors.

  12. UV-fusion Between Protoplasts of Common Wheat and Haynaldia villosa%普通小麦与簇毛麦原生质体的紫外线融合

    Institute of Scientific and Technical Information of China (English)

    周爱芬; 陈秀玲; 夏光敏; 陈惠民

    2002-01-01

    从来源于普通小麦品种济南177(Triticum aestivum cv. Jinan 177)悬浮细胞系的原生质体与来源于簇毛麦(Haynaldia villosa)胚性愈伤组织的原生质体融合获得体细胞杂种.供体簇毛麦原生质体在融合之前用紫外线照射30 s或1 min,紫外线剂量为360 μW/cm2.仅由紫外线照射30 s的组合获得再生愈伤组织克隆.细胞学、生物化学及PCR分析结果证实了再生克隆的杂种性质.用线粒体基因特异的探针进行的RFLP分析的结果表明,杂种中含有融合双亲的线粒体并且发生了重组.由杂种愈伤组织再生得到白化苗.讨论了紫外线对融合产物的影响.%Intergeneric somatic hybrids were obtained by fusion between suspension cell-derived protoplasts of Triticum aestivum cv. Jinan 177 and protoplasts of Haynaldia villosa isolated from embryogeneric calli. Protoplasts of H. villosa were exposed to UV (360 μW/cm2) for 30 s and 1 min before fusion. Regenerated colonies were obtained only from the fusion combination in which the protoplasts of donor H. villosa were exposed to UV for 30 s. Results of cytological, biochemical and polymerase chain reaction (PCR) analysis of the 5S rDNA spacer sequence confirmed the hybrid nature of the regenerated colonies. Restriction fragment length polymorphism (RFLP) analysis with gene-specific mitochondrial probe showed that mitochondria from both parents existed and recombined in the hybrid clones. Albinos instead of normal green plants were regenerated from hybrid colonies. The effects of UV on fusion products were discussed.

  13. Cas9-based genome editing in Arabidopsis and tobacco.

    Science.gov (United States)

    Li, Jian-Feng; Zhang, Dandan; Sheen, Jen

    2014-01-01

    Targeted modification of plant genome is key to elucidating and manipulating gene functions in plant research and biotechnology. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) technology is emerging as a powerful genome-editing method in diverse plants that traditionally lacked facile and versatile tools for targeted genetic engineering. This technology utilizes easily reprogrammable guide RNAs (sgRNAs) to direct Streptococcus pyogenes Cas9 endonuclease to generate DNA double-stranded breaks in targeted genome sequences, which facilitates efficient mutagenesis by error-prone nonhomologous end-joining (NHEJ) or sequence replacement by homology-directed repair (HDR). In this chapter, we describe the procedure to design and evaluate dual sgRNAs for plant codon-optimized Cas9-mediated genome editing using mesophyll protoplasts as model cell systems in Arabidopsis thaliana and Nicotiana benthamiana. We also discuss future directions in sgRNA/Cas9 applications for generating targeted genome modifications and gene regulations in plants.

  14. Ultrastructure of the partially coated reticulum and dictyosomes during endocytosis by soybean protoplasts.

    Science.gov (United States)

    Tanchak, M A; Rennie, P J; Fowke, L C

    1988-10-01

    Individual and serial sections were used to obtain detailed information regarding the morphology and distribution of the partially coated reticulum (PCR) and to determine its relationship with dictyosomes in endocytotically active soybean (Glycine max. (L.) Merr.) protoplasts. The results confirm and extend the description of the PCR provided by T.C. Pesacreta and W.J. Lucas (1985, Protoplasma 125, 173-184) from whole cells of selected angiosperms. The PCR of soybean protoplasts consists of a set of interconnected tubular membranes bearing a clathrin-like coat over part of their cytoplasmic surface. A dilation, sometimes containing small vesicles, is frequently seen in this organelle. The PCR often appears associated with dictyosomes but also occurs independent of other cell organelles. Only one example of a direct connection between the PCR and dictyosomes was observed.Following adsorptive endocytosis of cationized ferritin, the label appears in the PCR within 2 min and accumulates with time. It is never observed in the membrane dilations. Serial sectioning established that dictyosomes are labelled with cationized ferritin around the periphery of several cisternae, including those on both sides of the same dictyosome.

  15. Transmission of Fusarium boothii mycovirus via protoplast fusion causes hypovirulence in other phytopathogenic fungi.

    Directory of Open Access Journals (Sweden)

    Kyung-Mi Lee

    Full Text Available There is increasing concern regarding the use of fungicides to control plant diseases, whereby interest has increased in the biological control of phytopathogenic fungi by the application of hypovirulent mycoviruses as a possible alternative to fungicides. Transmission of hypovirulence-associated double-stranded RNA (dsRNA viruses between mycelia, however, is prevented by the vegetative incompatibility barrier that often exists between different species or strains of filamentous fungi. We determined whether protoplast fusion could be used to transmit FgV1-DK21 virus, which is associated with hypovirulence on F. boothii (formerly F. graminearum strain DK21, to F. graminearum, F. asiaticum, F. oxysporum f. sp. lycopersici, and Cryphonectria parasitica. Relative to virus-free strains, the FgV1-DK21 recipient strains had reduced growth rates, altered pigmentation, and reduced virulence. These results indicate that protoplast fusion can be used to introduce FgV1-DK21 dsRNA into other Fusarium species and into C. parasitica and that FgV1-DK21 can be used as a hypovirulence factor and thus as a biological control agent.

  16. Expression of wild-type PtrIAA14.1, a poplar Aux/IAA gene causes morphological changes in Arabidopsis

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    Shanda eLiu

    2015-06-01

    Full Text Available Aux/IAA proteins are transcriptional repressors that control auxin signaling by interacting with Auxin Response Factors (ARFs. So far all of the identified Aux/IAA mutants with auxin-related phenotypes in Arabidopsis and rice (Oryza sativa are dominant gain-of-function mutants, with mutantions in Domain II that affected stability of the corresponding Aux/IAA proteins. On the other hand, morphological changes were observed in knock-down mutants of Aux/IAA genes in tomato (Solanum lycopersicum, suggesting that functions of Aux/IAA proteins may be specific for certain plant species. We report here the characterization of PtrIAA14.1, a poplar (Populus trichocarpa homologue of IAA7. Bioinformatics analysis showed that PtrIAA14.1 is a classic Aux/IAA protein. It contains four conserved domains with the repressor motif in Domain I, the degron in Domain II, and the conserved amino acid signatures for protein-protein interactions in Domain III and Domain IV. Protoplast transfection assays showed that PtrIAA14.1 is localized in nucleus. It is unable in the presence of auxin, and it represses auxin response reporter gene expression. Expression of wild type PtrIAA14.1 in Arabidopsis resulted in auxin-related phenotypes including down-curling leaves, semi-draft with increased number of branches, and greatly reduced fertility, but expression of the Arabidopsis Aux/IAA genes tested remain largely unchanged in the transgenic plants. Protein-protein interaction assays in yeast and protoplasts showed that PtrIAA14.1 interacted with ARF5, but not other ARFs. Consistent with this observation, vascular patterning was altered in the transgenic plants, and the expression of AtHB8 (Arabidopsis thaliana Homeobox Gene 8 was reduced in transgenic plants.

  17. UV-MUTAGENESIS OF PROTOPLASTS OF Pleurotus eryngii%紫外线对杏鲍菇原生质体的诱变作用

    Institute of Scientific and Technical Information of China (English)

    刘海英; 张运峰; 范永山; 李科南; 刘先拉

    2011-01-01

    对杏鲍菇(Pleurotus eryngii)菌株PL7的原生质体进行了紫外线诱变的研究。结果发现,用20W紫外灯(245nm),在垂直距灯管30cm处照射90s,PL7菌株的原生质体致死率达74%。获得再生菌株234株,其中20株与亲本菌株PL7有明显的拮抗反应,6个菌株菌落生长速度提高11.0%~17.8%,液体培养生物量提高19.3%~32.4%,Rep-PCR分析表现为新的基因型。通过出菇试验,6个菌株较PL7菌株长满培养料天数缩短3~6d,出现原基的时间提前5~8d,第一茬平均产量提高12.5%~40.6%,总生物学效率提高11.1%~32.4%。结果表明原生质体诱变是快速选育食用菌高产新菌株和改善品种性状的重要方法之一。%The UV-mutagenesis of protoplast was conducted on Pleurotus eryngii strain PL7.The results showed that the lethality rate of protoplast reached to 74% by using a 20W germicidal lamp with emitting wavelength of 254nm under irradiation condition of vertical distance of 30cm from lamp to culture media for an irradiation duration of 90s.Twenty mutants were selected from 234 regenerating colonies by antagonistic test.6 isolates exhibiting 11.0%~17.8% increase of colony growth speed,19.3%~32.4% increase of mycelium biomass in liquid medium,and new Rep-PCR genetypic pattern were screened from the 20 mutants.The assay of mushroom cultivation revealed that the 6 isolates shortened the overgrowth time of mycelium on culture medium by 3~6 days,advanced the appearance time of mushroom anlage by 5~8 days,and increased the mushroom yield of the first harvest by 12.5%~40.6% and the total biological efficiency by 11.1%~32.4%.The results indicated that the protoplast UV-mutagenesis was a better approach for novel high-yield isolate selection and strain improvement of edible fungi.

  18. Reference: 101 [Arabidopsis Phenome Database[Archive

    Lifescience Database Archive (English)

    Full Text Available 101 http://metadb.riken.jp/db/SciNetS_ria224i/cria224u4ria224u15295074i Sorensen A...nna-Marie et al. 2004 Jul. Plant Cell Physiol. 45(7):905-13. Screening a T-DNA mutagenized population of Arabidopsis thaliana for re...duced seed set and segregation distortion led to the isolation of the ABNORMAL GAMET...OPHYTES (AGM) mutant. Homozygous plants were never recovered, but heterozygous pl...ants showed mitotic defects during gametogenesis resulting in approximately 50% abortion of both the male an

  19. Reference: 666 [Arabidopsis Phenome Database[Archive

    Lifescience Database Archive (English)

    Full Text Available clarified. The cyo1 mutant in Arabidopsis thaliana has albino cotyledons but normal gre...en true leaves. Chloroplasts develop abnormally in cyo1 mutant plants grown in the light, but etioplasts are... normal in mutants grown in the dark. We isolated CYO1 by T-DNA tagging and verified that the mutant allele was re... has a C(4)-type zinc finger domain similar to that of Escherichia coli DnaJ. CYO1 is expressed mainly in yo...1 mutation, but the level of photosynthetic proteins is decreased in cyo1 mutants. Recombinant CYO1 accelerates disulfide bond re

  20. Reference: 3 [Arabidopsis Phenome Database[Archive

    Lifescience Database Archive (English)

    Full Text Available t al. 2001 Jul. Plant J. 27(2):89-99. We isolated an Arabidopsis lesion initiation 2 (lin2) mutant, which develops lesion...droxylase (nahG) gene. This suggests that the lesion formation triggered in lin2 plants is determined prior ...to or independently of the accumulation of SA but that the accumulation is required to limit the spread of lesion...s in lin2 plants. A deficiency of coproporphyrinogen III oxidase causes lesion...s, usually activated by pathogen infection. These results demonstrate that a porphyrin pathway impairment is responsible for the lesi

  1. Reference: 706 [Arabidopsis Phenome Database[Archive

    Lifescience Database Archive (English)

    Full Text Available et al. 2008 Jan. Plant Cell Physiol. 49(1):2-10. To understand better the plant response to ozone, we isola...ted and characterized an ozone-sensitive (ozs1) mutant strain from a set of T-DNA-tagged Arabidopsis thalian...a ecotype Columbia. The mutant plants show enhanced sensitivity to ozone, desicca...vels. The T-DNA was inserted at a single locus which is linked to ozone sensitivity. Identification of the g...h either of two different T-DNA insertions in this gene were also sensitive to ozone, and these plants faile

  2. Reference: 186 [Arabidopsis Phenome Database[Archive

    Lifescience Database Archive (English)

    Full Text Available nce rooting during propagation. To better understand the role of IBA, we isolated Arabidopsis IBA-response (ibr) mutants that display...t, whereas acx1 acx3 and acx1 acx5 double mutants display enhanced IBA resistance...cx1 acx2 double mutants display enhanced IBA resistance and are sucrose dependent during seedling developmen...tive in ACX1, ACX3, or ACX4 have reduced fatty acyl-CoA oxidase activity on specific substrates. Moreover, a

  3. OBTENÇÃO DE PLANTAS DE LIMÃO CRAVO (Citrus limonia Osbeck E TANGERINA CLEÓPATRA (Citrus reshni Hort. A PARTIR DO CULTIVO DE PROTOPLASTOS DE SUSPENSÃO CELULAR PLANT REGENERATION OF 'RANGPUR' LIME (Citrus limonia Osbeck AND 'CLEÓPATRA' MANDARIN (Citrus reshni Hort. THROUGH PROTOPLASTS OF CELL SUSPENSION

    Directory of Open Access Journals (Sweden)

    Rodrigo Rocha Latado

    1999-01-01

    Full Text Available Este trabalho descreve uma metodologia para a regeneração de plantas de tangerina 'Cleópatra' e limão 'Cravo', a partir do cultivo de protoplastos de suspensão celular. Para tal, calos nucelares foram induzidos em meio contendo BAP e cultivados em meio sem reguladores de crescimento. Protoplastos foram isolados de suspensões celulares e cultivados em gotas de agarose, com densidade de 2 X 105 protoplastos.ml-1. O meio MT, contendo ácido giberélico e água de coco, foi eficiente na germinação de embriões somáticos. Os métodos de aclimatação de plantas testados apresentaram baixa eficiência. Como resultado final, 17 plantas adaptadas de tangerina e 8 de limão foram obtidas.The present research describes the regeneration of 'Cleópatra' mandarin and 'Rangpur' lime plants from cell suspension protoplasts. Nucelar calli were induced on a medium containing BAP and maintained on growth regulator free medium. Protoplasts were isolated from embryogenic suspension and plated at a concentration of 2 X 105 protoplasts.ml-1, on agarose droplets. The MT medium with gibberellic acid and coconut water was efficient to stimulate somatic embryo conversion. Rooted plants acclimation had low efficiency. Seventeen mandarin plants and eight lime plants were obtained.

  4. Efficient gusA Transient Expression in Porphyra yezoensis Protoplasts Mediated by Endogenous Beta-tubulin Flanking Sequences

    Institute of Scientific and Technical Information of China (English)

    GONG Qianhong; YU Wengong; DAI Jixun; LIU Hongquan; XU Rifu; GUAN Huashi; PAN Kehou

    2007-01-01

    Endogenous tubulin promoter has been widely used for expressing foreign genes in green algae, but the efficiency and feasibility of endogenous tubulin promoter in the economically important Porphyra yezoensis (Rhodophyta) are tmknown. In this study, the flanking sequences of beta-tubulin gene from P. yezoensis were amplified and two transient expression vectors were constructed to determine their transcription promoting feasibility for foreign gene gusA. The testing vector pATubGUS was constructed by inserting 5'- and 3'-flanking regions (Tub5'and Tub3') up- and down-stream of β-glucuronidase (GUS) gene (gusA), respectively,into pA, a derivative of pCAT(R)3-enhancer vector. The control construct, pAGUSTub3, contains only gusA and Tub3 '. These constructs were electroporated into P. yezoensis protoplasts and the GUS activities were quantitatively analyzed by spectrometry. The results demonstrated that gusA gene was efficiently expressed in P. yezoensis protoplasts under the regulation of 5'-flanking sequence of the beta-tubulin gene. More interestingly, the pATubGUS produced stronger GUS activity in P. yezoensis protoplasts when compared to the result from pBI221, in which the gusA gene was directed by a constitutive CaMV 35 S promoter. The data suggest that the integration of P. yezoensis protoplast and its endogenous beta-tubulin flanking sequences is a potential novel system for foreign gene expression.

  5. Incorporation of hygromycin resistance in Brassica nigra and its transfer to B. napus through asymmetric protoplast fusion.

    Science.gov (United States)

    Sacristán, M D; Gerdemann-Knörck, M; Schieder, O

    1989-08-01

    With the idea to develop a selection system for asymmetric somatic hybrids between oilseed rape (Brassica napus) and black mustard (B. nigra), the marker gene hygromycin resistance was introduced in this last species by protoplast transformation with the disarmed Agrobacterium tumefaciens strain C58 pGV 3850 HPT. The B. nigra lines used for transformation had been previously selected for resistance to two important rape pathogens (Phoma lingam, Plasmodiophora brassicae). Asymmetric somatic hybrids were obtained through fusion of X-ray irradiated (mitotically inactivated) B. nigra protoplasts from transformed lines as donor with intact protoplasts of B. napus, using the hygromycin resistance as selection marker for fusion products. The somatic hybrids hitherto obtained expressed both hygromycin phosphotransferase and nopaline synthase genes. Previous experience with other plant species had demonstrated that besides the T-DNA, other genes of the donor genome can be co-transferred. In this way, the produced hybrids constitute a valuable material for studying the possibility to transfer agronomically relevant characters - in our case, diseases resistances - through asymmetric protoplast fusion.

  6. Studies on protein synthesis by protoplasts of Saccharomyces carlsbergensis I. The effect of ribonuclease on protein synthesis

    NARCIS (Netherlands)

    Kloet, S.R. de; Wermeskerken, R.K.A. van; Koningsberger, V.V.

    1961-01-01

    Ribonuclease was found to inhibit the protein synthesis in the naked yeast protoplast for nearly 100%. Even small concentrations (5 μg/ml) were found inhibitory. The cause of this inhibition can be attributed at least in part to a 90% inhibition of the respiration. Amino acid uptake was found to be

  7. Early activation of lipoxygenase in lentil (Lens culinaris) root protoplasts by oxidative stress induces programmed cell death

    NARCIS (Netherlands)

    Vliegenthart, J.F.G.; Maccarrone, M.; Zadelhoff, G. van; Veldink, G.A.; Finazzi Agrò, A.

    2000-01-01

    Oxidative stress caused by hydrogen peroxide (H2O2) triggers the hypersensitive response of plants to pathogens. Here, short pulses of H2O2 are shown to cause death of lentil (Lens culinaris) root protoplasts. Dead cells showed DNA fragmentation and ladder formation, typical hallmarks of apoptosis (

  8. Arabidopsis gene expression patterns are altered during spaceflight

    Science.gov (United States)

    Paul, Anna-Lisa; Popp, Michael P.; Gurley, William B.; Guy, Charles; Norwood, Kelly L.; Ferl, Robert J.

    The exposure of Arabidopsis thaliana (Arabidopsis) plants to spaceflight environments results in differential gene expression. A 5-day mission on orbiter Columbia in 1999 (STS-93) carried transgenic Arabidopsis plants engineered with a transgene composed of the alcohol dehydrogenase (Adh) gene promoter linked to the β-Glucuronidase (GUS) reporter gene. The plants were used to evaluate the effects of spaceflight on gene expression patterns initially by using the Adh/GUS transgene to address specifically the possibility that spaceflight induces a hypoxic stress response (Paul, A.L., Daugherty, C.J., Bihn, E.A., Chapman, D.K., Norwood, K.L., Ferl, R.J., 2001. Transgene expression patterns indicate that spaceflight affects stress signal perception and transduction in arabidopsis, Plant Physiol. 126, 613-621). As a follow-on to the reporter gene analysis, we report here the evaluation of genome-wide patterns of native gene expression within Arabidopsis shoots utilizing the Agilent DNA array of 21,000 Arabidopsis genes. As a control for the veracity of the array analyses, a selection of genes was further characterized with quantitative Real-Time RT PCR (ABI - Taqman®). Comparison of the patterns of expression for arrays probed with RNA isolated from plants exposed to spaceflight compared to RNA isolated from ground control plants revealed 182 genes that were differentially expressed in response to the spaceflight mission by more than 4-fold, and of those only 50 genes were expressed at levels chosen to support a conservative change call. None of the genes that are hallmarks of hypoxic stress were induced to this level. However, genes related to heat shock were dramatically induced - but in a pattern and under growth conditions that are not easily explained by elevated temperatures. These gene expression data are discussed in light of current models for plant responses to the spaceflight environment and with regard to potential future spaceflight experiment

  9. Identification of the Raptor-binding motif on Arabidopsis S6 kinase and its use as a TOR signaling suppressor.

    Science.gov (United States)

    Son, Ora; Kim, Sunghan; Hur, Yoon-Sun; Cheon, Choong-Ill

    2016-03-25

    TOR (target of rapamycin) kinase signaling plays central role as a regulator of growth and proliferation in all eukaryotic cells and its key signaling components and effectors are also conserved in plants. Unlike the mammalian and yeast counterparts, however, we found through yeast two-hybrid analysis that multiple regions of the Arabidopsis Raptor (regulatory associated protein of TOR) are required for binding to its substrate. We also identified that a 44-amino acid region at the N-terminal end of Arabidopsis ribosomal S6 kinase 1 (AtS6K1) specifically interacted with AtRaptor1, indicating that this region may contain a functional equivalent of the TOS (TOR-Signaling) motif present in the mammalian TOR substrates. Transient over-expression of this 44-amino acid fragment in Arabidopsis protoplasts resulted in significant decrease in rDNA transcription, demonstrating a feasibility of developing a new plant-specific TOR signaling inhibitor based upon perturbation of the Raptor-substrate interaction.

  10. Cell Wall Regeneration by Protoplasts in the Weak Combined Magnetic Field

    Science.gov (United States)

    Nedukha, Olena; Bogatina, Nina; Kordyum, Elizabeth; Ovcharenko, Yu.; Vorobyeva, T.

    2008-06-01

    Role of gravity on growth of high plants has been studied for many years, but many questions on biogenesis of plant cell wall are investigated insufficiently, and require new experiments. We have studied regeneration of cell wall in the fused and separate protoplasts of tobacco and soyabean in the presence of the weak, alternating magnetic field that consisted of frequency of 32 Hz (for Ca2+ ; F=40 μT) or 75 Hz (for Mg2+; F=60 μT) in side μ-metal shield. We discovered that the combined magnetic field that was adjusted to the cyclotron frequency of Ca2+ or Mg2+ is changed the rate of cell wall regeneration. Light and confocal laser microscopy were used for the investigations.

  11. Factors influencing electroporation-mediated gene transfer to Stylosanthes guianensis (Aubl. Sw. protoplasts

    Directory of Open Access Journals (Sweden)

    Quecini V.M.

    2002-01-01

    Full Text Available In order to develop a high-efficiency and reproducible transformation protocol for Stylosanthes guianensis we assessed the biological and physical parameters affecting plant electroporation protoplasts. Energy input, as combinations of electric field strengths discharged by different capacitors, electroporation buffer and DNA form were evaluated. Transformation efficiency was assayed in vivo as transient reporter gene expression, using the GFP-coding gene mgfp5 driven by a CaMV 35S constitutive promoter. Energy input and electric field strength had a critical influence on transgene expression with higher transformation levels being achieved with 250 V.cm-1 discharged by 900 and 1000 muF capacitors. Linear plasmid DNA, the absence of chloride and the presence of calcium ions also increased transient gene expression, albeit not significantly.

  12. Break of symmetry in regenerating tobacco protoplasts is independent of nuclear positioning

    Institute of Scientific and Technical Information of China (English)

    Linda Brochhausen; Jan Maisch; Peter Nick

    2016-01-01

    Nuclear migration and positioning are crucial for the morphogenesis of plant cells. We addressed the potential role of nuclear positioning for polarity induction using an experimental system based on regenerating protoplasts, where the induction of a cell axis de novo can be followed by quantification of specific regeneration stages. Using overexpression of fluorescently tagged extranuclear (perinu-clear actin basket, kinesins with a calponin homology domain (KCH)) as well as intranuclear (histone H2B) factors of nuclear positioning and time-lapse series of the early stages of regeneration, we found that nuclear position is no prerequi-site for polarity formation. However, polarity formation and nuclear migration were both modulated in the transgenic lines, indicating that both phenomena depend on factors affecting cytoskeletal tensegrity and chromatin structure. We integrated these findings into a model where retrograde signals are required for polarity induction. These signals travel via the cytoskeleton from the nucleus toward targets at the plasma membrane.

  13. Improved inhibitor tolerance in xylose-fermenting yeast Spathaspora passalidarum by mutagenesis and protoplast fusion

    DEFF Research Database (Denmark)

    Hou, Xiaoru; Yao, Shuo

    2012-01-01

    The xylose-fermenting yeast Spathaspora passalidarum showed excellent fermentation performance utilizing glucose and xylose under anaerobic conditions. But this yeast is highly sensitive to the inhibitors such as furfural present in the pretreated lignocellulosic biomass. In order to improve...... final ethanol than the wild-type strain in a synthetic xylose medium containing 2 g/l furfural. However, this mutant was unable to grow in a medium containing 75% liquid fraction of pretreated wheat straw (WSLQ), in which furfural and many other inhibitors were present. Hybrid yeast strains, obtained...... from fusion of the protoplasts of S. passalidarum M7 and a robust yeast, Saccharomyces cerevisiae ATCC 96581, were able to grow in 75% WSLQ and produce around 0.4 g ethanol/g consumed xylose. Among the selected hybrid strains, the hybrid FS22 showed the best fermentation capacity in 75% WSLQ...

  14. Diuretics Prime Plant Immunity in Arabidopsis thaliana

    Science.gov (United States)

    Noutoshi, Yoshiteru; Ikeda, Mika; Shirasu, Ken

    2012-01-01

    Plant activators are agrochemicals that activate the plant immune system, thereby enhancing disease resistance. Due to their prophylactic and durable effects on a wide spectrum of diseases, plant activators can provide synergistic crop protection when used in combination with traditional pest controls. Although plant activators have achieved great success in wet-rice farming practices in Asia, their use is still limited. To isolate novel plant activators applicable to other crops, we screened a chemical library using a method that can selectively identify immune-priming compounds. Here, we report the isolation and characterization of three diuretics, bumetanide, bendroflumethiazide and clopamide, as immune-priming compounds. These drugs upregulate the immunity-related cell death of Arabidopsis suspension-cultured cells induced with an avirulent strain of Pseudomonas syringae pv. tomato in a concentration-dependent manner. The application of these compounds to Arabidopsis plants confers disease resistance to not only the avirulent but also a virulent strain of the pathogen. Unlike salicylic acid, an endogenous phytohormone that governs disease resistance in response to biotrophic pathogens, the three diuretic compounds analyzed here do not induce PR1 or inhibit plant growth, showing potential as lead compounds in a practical application. PMID:23144763

  15. Diuretics prime plant immunity in Arabidopsis thaliana.

    Directory of Open Access Journals (Sweden)

    Yoshiteru Noutoshi

    Full Text Available Plant activators are agrochemicals that activate the plant immune system, thereby enhancing disease resistance. Due to their prophylactic and durable effects on a wide spectrum of diseases, plant activators can provide synergistic crop protection when used in combination with traditional pest controls. Although plant activators have achieved great success in wet-rice farming practices in Asia, their use is still limited. To isolate novel plant activators applicable to other crops, we screened a chemical library using a method that can selectively identify immune-priming compounds. Here, we report the isolation and characterization of three diuretics, bumetanide, bendroflumethiazide and clopamide, as immune-priming compounds. These drugs upregulate the immunity-related cell death of Arabidopsis suspension-cultured cells induced with an avirulent strain of Pseudomonas syringae pv. tomato in a concentration-dependent manner. The application of these compounds to Arabidopsis plants confers disease resistance to not only the avirulent but also a virulent strain of the pathogen. Unlike salicylic acid, an endogenous phytohormone that governs disease resistance in response to biotrophic pathogens, the three diuretic compounds analyzed here do not induce PR1 or inhibit plant growth, showing potential as lead compounds in a practical application.

  16. A novel procedure for the localization of viral RNAs in protoplasts and whole plants.

    Science.gov (United States)

    Zhang, Fengli; Simon, Anne E

    2003-09-01

    Analysis of virus spread using co-expressed reporter proteins has provided important details on cell-to-cell and long-distance movement of viruses in plants. However, most viruses cannot tolerate insertion of large non-viral segments or loss of any open-reading frames, procedures required to detect viruses non-evasively. A technique used to localize mRNAs intracellularly in yeast has been modified for detection of viral RNAs in whole plants. The technique makes use of the binding of the coat protein of MS2 bacteriophage (CPMS2) to a 19 base hairpin (hp). A fusion protein, consisting of the CPMS2, green fluorescent protein (GFP), and a nuclear localization signal (NLS), was nuclear-localized upon transient expression in protoplasts. However, addition of the hp to the 3' untranslated region of Turnip crinkle virus (TCV-hp) and co-transfection of the virus and fusion protein construct into protoplasts resulted in the re-location of GFP to the cytoplasm. Neither the insertion of the hp nor the interaction with the fusion protein impaired any viral functions. Transgenic plants expressing the GFP-NLS-CPMS2 fusion protein were generated, and GFP was detected in nuclei of young plant cells. Foci of GFP cytoplasmic fluorescence were detected in TCV-hp-inoculated leaves at 2 days post-inoculation. Later, GFP was detected in young leaves near the midvein and in the base (support) cells of trichomes in the vicinity of secondary and tertiary veins. In older leaves, cytoplasmic GFP could be visualized throughout many of the leaves. This technique should be amenable for detection of any virus with a transformable plant (or animal) host and may also prove useful for localizing properly engineered host RNAs.

  17. An Arabidopsis callose synthase

    DEFF Research Database (Denmark)

    Ostergaard, Lars; Petersen, Morten; Mattsson, Ole

    2002-01-01

    in the Arabidopsis mpk4 mutant which exhibits systemic acquired resistance (SAR), elevated beta-1,3-glucan synthase activity, and increased callose levels. In addition, AtGsl5 is a likely target of salicylic acid (SA)-dependent SAR, since AtGsl5 mRNA accumulation is induced by SA in wild-type plants, while...... expression of the nahG salicylate hydroxylase reduces AtGsl5 mRNA levels in the mpk4 mutant. These results indicate that AtGsl5 is likely involved in callose synthesis in flowering tissues and in the mpk4 mutant....

  18. Identification, isolation, and expression analysis of heat shock transcription factors in the diploid woodland strawberry Fragaria vesca.

    Science.gov (United States)

    Hu, Yang; Han, Yong-Tao; Wei, Wei; Li, Ya-Juan; Zhang, Kai; Gao, Yu-Rong; Zhao, Feng-Li; Feng, Jia-Yue

    2015-01-01

    Heat shock transcription factors (Hsfs) are known to play dominant roles in plant responses to heat, as well as other abiotic or biotic stress stimuli. While the strawberry is an economically important fruit plant, little is known about the Hsf family in the strawberry. To explore the functions of strawberry Hsfs in abiotic and biotic stress responses, this study identified 17 Hsf genes (FvHsfs) in a wild diploid woodland strawberry (Fragaria vesca, 2n = 2x = 14) and isolated 14 of these genes. Phylogenetic analysis divided the strawberry FvHsfs genes into three main groups. The evolutionary and structural analyses revealed that the FvHsf family is conserved. The promoter sequences of the FvHsf genes contain upstream regulatory elements corresponding to different stress stimuli. In addition, 14 FvHsf-GFP fusion proteins showed differential subcellular localization in Arabidopsis mesophyll protoplasts. Furthermore, we examined the expression of the 17 FvHsf genes in wild diploid woodland strawberries under various conditions, including abiotic stresses (heat, cold, drought, and salt), biotic stress (powdery mildew infection), and hormone treatments (abscisic acid, ethephon, methyl jasmonate, and salicylic acid). Fifteen of the seventeen FvHsf genes exhibited distinct changes on the transcriptional level during heat treatment. Of these 15 FvHsfs, 8 FvHsfs also exhibited distinct responses to other stimuli on the transcriptional level, indicating versatile roles in the response to abiotic and biotic stresses. Taken together, the present work may provide the basis for further studies to dissect FvHsf function in response to stress stimuli.

  19. Identification, Isolation, and Expression Analysis of Heat Shock Transcription Factors in the Diploid Woodland Strawberry Fragaria vesca

    Directory of Open Access Journals (Sweden)

    Yang eHu

    2015-09-01

    Full Text Available Heat shock transcription factors (Hsfs are known to play dominant roles in plant responses to heat, as well as other abiotic or biotic stress stimuli. While the strawberry is an economically important fruit plant, little is known about the Hsf family in the strawberry. To explore the functions of strawberry Hsfs in abiotic and biotic stress responses, this study identified 17 Hsf genes (FvHsfs in a wild diploid woodland strawberry (Fragaria vesca, 2n = 2x = 14 and isolated 14 of these genes. Phylogenetic analysis divided the strawberry FvHsfs genes into three main groups. The evolutionary and structural analyses revealed that the FvHsf family is conserved. The promoter sequences of the FvHsf genes contain upstream regulatory elements corresponding to different stress stimuli. In addition, 14 FvHsf-GFP fusion proteins showed differential subcellular localization in Arabidopsis mesophyll protoplasts. Furthermore, we examined the expression of the 17 FvHsf genes in wild diploid woodland strawberries under various conditions, including abiotic stresses (heat, cold, drought, and salt, biotic stress (powdery mildew infection, and hormone treatments (abscisic acid, ethephon, methyl jasmonate, and salicylic acid. Fifteen of the 17 FvHsf genes exhibited distinct changes on the transcriptional level during heat treatment. Of these 15 FvHsfs, 8 FvHsfs also exhibited distinct responses to other stimuli on the transcriptional level, indicating versatile roles in the response to abiotic and biotic stresses. Taken together, the present work may provide the basis for further studies to dissect FvHsf function in response to stress stimuli.

  20. Functional overlap of the Arabidopsis leaf and root microbiota.

    Science.gov (United States)

    Bai, Yang; Müller, Daniel B; Srinivas, Girish; Garrido-Oter, Ruben; Potthoff, Eva; Rott, Matthias; Dombrowski, Nina; Münch, Philipp C; Spaepen, Stijn; Remus-Emsermann, Mitja; Hüttel, Bruno; McHardy, Alice C; Vorholt, Julia A; Schulze-Lefert, Paul

    2015-12-17

    Roots and leaves of healthy plants host taxonomically structured bacterial assemblies, and members of these communities contribute to plant growth and health. We established Arabidopsis leaf- and root-derived microbiota culture collections representing the majority of bacterial species that are reproducibly detectable by culture-independent community sequencing. We found an extensive taxonomic overlap between the leaf and root microbiota. Genome drafts of 400 isolates revealed a large overlap of genome-encoded functional capabilities between leaf- and root-derived bacteria with few significant differences at the level of individual functional categories. Using defined bacterial communities and a gnotobiotic Arabidopsis plant system we show that the isolates form assemblies resembling natural microbiota on their cognate host organs, but are also capable of ectopic leaf or root colonization. While this raises the possibility of reciprocal relocation between root and leaf microbiota members, genome information and recolonization experiments also provide evidence for microbiota specialization to their respective niche.

  1. Protoplast formation and regeneration from Streptomyces clavuligerus NRRL 3585 and clavulanic acid production Formação e regeneração de protoplastos de Streptomyces clavuligerus NRRL 3585 e produção de ácido clavulânico

    Directory of Open Access Journals (Sweden)

    Maria das Graças Carneiro-da-Cunha

    2002-12-01

    Full Text Available Protoplasts of the wild type Streptomyces clavuligerus NRRL 3585 (ATCC 27064 were formed from spores cultures obtained in the lag, exponential and stationary growth phases by using 0.5% glycine in the culture medium. The protoplasts were obtained by treatment of the cells with lysozyme (EC-3.2.1.17 40,000 U (1mg/mL, in an osmotic solution for 90 min at 28ºC. The frequency of regenerated protoplasts in the lag phase was 1.7x10³ CFU/mL (28.97%, in the beginning of the exponential phase 0.4x10² CFU/mL (31.67%, in the exponential growth phase 2.5x10³ CFU/mL (46.30% and 1.0x10(5 CFU/mL in stationary phase (48.45%. Antibiotic production and activity of regenerated protoplasts were observed in all phases, except in the lag phase. The protoplast formation and regeneration techniques resulted in a new isolate strain of Streptomyces clavuligerus that produced approximately 2.5 fold more clavulanic acid.Protoplastos foram formados a partir de esporos da amostra selvagem de Streptomyces clavuligerus durante a fase lag, exponencial e estacionária de crescimento, utilizando glicina a 0.5% como meio de cultura. Os protoplastos foram obtidos pelo tratamento das células com lisozima (EC-3.2.1.17 40.000 U (1mg/mL em solução osmótica de sorbitol e TES, por 90 min a 28ºC. A freqüência de protoplastos regenerados na fase lag foi de 1,7x10³ UFC/mL (28,97%, no início da fase exponencial correspondeu a 0,4x10² UFC/mL (31,67%, no final da fase exponencial observou-se 2,5x10³ UFC/mL (46,30% e para a fase estacionária de crescimento apresentou 1,0x10(5 UFC/mL (48,45%. A produção do antibiótico e a atividade antibiótica dos protoplastos regenerados foram observadas em todas as fases de crescimento, exceto na fase lag. As técnicas de formação de protoplastos e regeneração resultaram em uma nova linhagem de Streptomyces clavuligerus produzindo 2,5 vezes mais ácido clavulânico.

  2. The Arabidopsis thaliana aleurone layer responds to nitric oxide, gibberellin, and abscisic acid and is sufficient and necessary for seed dormancy

    Science.gov (United States)

    Seed dormancy is a common phase of the plant life cycle and several parts of the seed can contribute to dormancy. Whole seeds, seeds lacking the testa, embryos, and isolated aleurone layers of Arabidopsis thaliana were used in experiments designed to identify components of the arabidopsis seed that ...

  3. Effects of dicyclohexylamine on polyamine biosynthesis and incorporation into turnip yellow mosaic virus in Chinese cabbage protoplasts infected in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Balint, R.; Cohen, S.S.

    1985-07-15

    The authors have reported that protoplasts from plants infected with turnip yellow mosaic virus (TYMV) continue to produce virus in culture and that newly formed virus particles contained predominantly newly synthesized spermidine and spermine. They now report similar results with healthy protoplasts infected in vitro, in which essentially all of the virus is newly formed. Again, newly synthesized spermidine and spermine were preferentially incorporated into virus. DCHA inhibited spermidine synthesis by 85%, leading in 20 hr to a 60% depletion of the cellular spermidine and a 30% reduction in the amount of spermidine per virion. Spermine synthesis increased, however, producing a 40% increase in cellular spermine and 50-100% increase in the amount of spermine per virion. Thus, in spite of spermidine depletion, the total positive charge contributed by polyamines to the virus was essentially conserved.

  4. "Erussica", the intergeneric fertile somatic hybrid developed through protoplast fusion between Eruca sativa Lam. and Brassica juncea (L.) Czern.

    Science.gov (United States)

    Sikdar, S R; Chatterjee, G; Das, S; Sen, S K

    1990-04-01

    Hypocotyl calli-derived protoplasts of two cultivars of Brassica juncea (2n=36), a major oil-seed crop, were fused with normal as well as γ-irradiated mesophyll protoplasts of Eruca sativa (2n=22). The irradiation of the Eruca fusion partner increased the plating efficiency as well as the morphogenic potentiality of the fusion products over the normal fusion. Fertile plants could be regenerated from such fusion products. Analysis of 63 out of 181 plants regenerated showed that, indeed, 11 somatic hybrids (2n=58) and 10 partial somatic hybrids (chromosome number ranged between 50 and 56) had been obtained. Pollen viability (0%-82.9%) and seed set (0%-50%) of the hybrids indicated them to be useful for future studies.

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

    Science.gov (United States)

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

    2014-03-01

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

  6. Real-time monitoring of oxidative burst from single plant protoplasts using microelectrochemical sensors modified by platinum nanoparticles.

    Science.gov (United States)

    Ai, Feng; Chen, Hong; Zhang, Shu-Hui; Liu, Sheng-Yi; Wei, Fang; Dong, Xu-Yan; Cheng, Jie-Ke; Huang, Wei-Hua

    2009-10-15

    Oxidative bursts from plants play significant roles in plant disease defense and signal transduction; however, it has not hitherto been investigated on individual living plant cells. In this article, we fabricated a novel sensitive electrochemical sensor based on electrochemical deposition of Pt nanoparticles on the surface of carbon fiber microdisk electrodes via a nanopores containing polymer matrix, Nafion. The numerous hydrophilic nanochannels in the Nafion clusters coated on the electrode surface served as the molecular template for the deposition and dispersion of Pt, which resulted in the uniform construction of small Pt nanoparticles. The novel sensor displayed a high sensitivity for detection of H(2)O(2) with a detection limit of 5.0 x 10(-9) M. With the use of this microelectrochemical sensor, the oxidative burst from individual living plant protoplasts have been real-time monitored for the first time. The results showed that oxidative burst from single protoplasts triggered by a pathogen analogue were characterized by quanta release with a large number of "transient oxidative microburst" events, and protoplasts from the transgenic plants biologically displayed better disease-resistance and showed a distinguished elevation and longer-lasting oxidative burst.

  7. Synergistic interaction between the Potyvirus, Turnip mosaic virus and the Crinivirus, Lettuce infectious yellows virus in plants and protoplasts.

    Science.gov (United States)

    Wang, Jinbo; Turina, Massimo; Medina, Vicente; Falk, Bryce W

    2009-09-01

    Lettuce infectious yellows virus (LIYV), the type member of the genus Crinivirus in the family Closteroviridae, is specifically transmitted by the sweet potato whitefly (Bemisia tabaci) in a semipersistent manner. LIYV infections result in a low virus titer in plants and protoplasts, impeding reverse genetic efforts to analyze LIYV gene/protein functions. We found that synergistic interactions occurred in mixed infections of LIYV and Turnip mosaic virus (TuMV) in Nicotiana benthamiana plants, and these resulted in enhanced accumulation of LIYV. Furthermore, we examined the ability of transgenic plants and protoplasts expressing only the TuMV P1/HC-Pro sequence to enhance the accumulation of LIYV. LIYV RNA and protein titers increased by as much as 8-fold in these plants and protoplasts relative to control plants. LIYV infections remained phloem-limited in P1/HC-Pro transgenic plants, suggesting that enhanced accumulation of LIYV in these plants was due primarily to increased replication efficiency, not to greater spread.

  8. Regulating role of acetylcholine and its antagonists in inward rectified K~+ channels from guard cell protoplasts of Vicia faba

    Institute of Scientific and Technical Information of China (English)

    冷强; 花宝光; 郭玉海; 娄成后

    2000-01-01

    The inward rectified potassium current of Vicia faba guard cell protoplasts treated with acetylcholine (ACh) or the antagonists of its receptors were recorded by employing the patch clamp technique. The results show that ACh at lower concentrations increases the inward K+ current, in contrast, ACh at higher concentrations inhibits it. Treated with d-Tubocurarine (d-Tub), an antagonist of the nicotine ACh receptor (nAChR) inhibits the inward K+ current by 30%. Treated with atropine (Atr), an antagonist of the muscarine (Mus) ACh receptor (mAChR) also inhibits it by 36%. However, if guard cell protoplasts are treated with d-Tub and Atr together, the inward K+ current is inhibited by 60%-75%. Tetraethylammonium chloride (TEA), a strong inhibitor of K+ channels has no effect on the inward K+ current regulated by ACh, suggesting that there are inward K+ channels modulated by AChRs on the membrane of the guard cell protoplasts. These data demonstrate an ACh-regulated mechanism for stomatal movement.

  9. Regulating role of acetylcholine and its antagonists in inward rectified K+ channels from guard cell protoplasts of Vicia faba

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    The inward rectified potassium current of Vicia faba guard cell protoplasts treated with acetylcholine (ACh) or the antagonists of its receptors were recorded by employing the patch clamp technique. The results show that ACh at lower concentrations increases the inward K+ current, in contrast, ACh at higher concentrations inhibits it. Treated with d-Tubocurarine (d-Tub), an antagonist of the nicotine ACh receptor (nAChR) inhibits the inward K+ current by 30%. Treated with atropine (Atr), an antagonist of the muscarine (Mus) ACh receptor (mAChR) also inhibits it by 36%.However,if guard cell protoplasts are treated with d-Tub and Atr together, the inward K+ current is inhibited by 60%-75%. Tetraethylammonium chloride (TEA), a strong inhibitor of K+ channels has no effect on the inward K+ current regulated by ACh, suggesting that there are inward K+ channels modulated by AChRs on the membrane of the guard cell protoplasts. These data demonstrate an ACh-regulated mechanism for stomatal movement.

  10. Fission yeast HMT1 lowers seed cadmium through phytochelatin-dependent vacuolar sequestration in Arabidopsis.

    Science.gov (United States)

    Huang, Jing; Zhang, Yu; Peng, Jia-Shi; Zhong, Chen; Yi, Hong-Ying; Ow, David W; Gong, Ji-Ming

    2012-04-01

    Much of our dietary uptake of heavy metals is through the consumption of plants. A long-sought strategy to reduce chronic exposure to heavy metals is to develop plant varieties with reduced accumulation in edible tissues. Here, we describe that the fission yeast (Schizosaccharomyces pombe) phytochelatin (PC)-cadmium (Cd) transporter SpHMT1 produced in Arabidopsis (Arabidopsis thaliana) was localized to tonoplast, and enhanced tolerance to and accumulation of Cd2+, copper, arsenic, and zinc. The action of SpHMT1 requires PC substrates, and failed to confer Cd2+ tolerance and accumulation when glutathione and PC synthesis was blocked by L-buthionine sulfoximine, or only PC synthesis is blocked in the cad1-3 mutant, which is deficient in PC synthase. SpHMT1 expression enhanced vacuolar Cd2+ accumulation in wild-type Columbia-0, but not in cad1-3, where only approximately 35% of the Cd2+ in protoplasts was localized in vacuoles, in contrast to the near 100% found in wild-type vacuoles and approximately 25% in those of cad2-1 that synthesizes very low amounts of glutathione and PCs. Interestingly, constitutive SpHMT1 expression delayed root-to-shoot metal transport, and root-targeted expression confirmed that roots can serve as a sink to reduce metal contents in shoots and seeds. These findings suggest that SpHMT1 function requires PCs in Arabidopsis, and it is feasible to promote food safety by engineering plants using SpHMT1 to decrease metal accumulation in edible tissues.

  11. A membrane protein / signaling protein interaction network for Arabidopsis version AMPv2

    Directory of Open Access Journals (Sweden)

    Sylvie Lalonde

    2010-09-01

    Full Text Available Interactions between membrane proteins and the soluble fraction are essential for signal transduction and for regulating nutrient transport. To gain insights into the membrane-based interactome, 3,852 open reading frames (ORFs out of a target list of 8,383 representing membrane and signaling proteins from Arabidopsis thaliana were cloned into a Gateway compatible vector. The mating-based split-ubiquitin system was used to screen for potential protein-protein interactions (pPPIs among 490 Arabidopsis ORFs. A binary robotic screen between 142 receptor-like kinases, 72 transporters, 57 soluble protein kinases and phosphatases, 40 glycosyltransferases, 95 proteins of various functions and 89 proteins with unknown function detected 387 out of 90,370 possible PPIs. A secondary screen confirmed 343 (of 387 pPPIs between 179 proteins, yielding a scale-free network (r2=0.863. Eighty of 142 transmembrane receptor-like kinases (RLK tested positive, identifying three homomers, 63 heteromers and 80 pPPIs with other proteins. Thirty-one out of 142 RLK interactors (including RLKs had previously been found to be phosphorylated; thus interactors may be substrates for respective RLKs. None of the pPPIs described here had been reported in the major interactome databases, including potential interactors of G protein-coupled receptors, phospholipase C, and AMT ammonium transporters. Two RLKs found as putative interactors of AMT1;1 were independently confirmed using a split luciferase assay in Arabidopsis protoplasts. These RLKs may be involved in ammonium-dependent phosphorylation of the C-terminus and regulation of ammonium uptake activity. The robotic screening method established here will enable a systematic analysis of membrane protein interactions in fungi, plants and metazoa.

  12. Mutant analysis, protein-protein interactions and subcellular localization of the Arabidopsis B sister (ABS) protein.

    Science.gov (United States)

    Kaufmann, Kerstin; Anfang, Nicole; Saedler, Heinz; Theissen, Günter

    2005-09-01

    Recently, close relatives of class B floral homeotic genes, termed B(sister) genes, have been identified in both angiosperms and gymnosperms. In contrast to the B genes themselves, B(sister) genes are exclusively expressed in female reproductive organs, especially in the envelopes or integuments surrounding the ovules. This suggests an important ancient function in ovule or seed development for B(sister) genes, which has been conserved for about 300 million years. However, investigation of the first loss-of-function mutant for a B(sister) gene (ABS/TT16 from Arabidopsis) revealed only a weak phenotype affecting endothelium formation. Here, we present an analysis of two additional mutant alleles, which corroborates this weak phenotype. Transgenic plants that ectopically express ABS show changes in the growth and identity of floral organs, suggesting that ABS can interact with floral homeotic proteins. Yeast-two-hybrid and three-hybrid analyses indicated that ABS can form dimers with SEPALLATA (SEP) floral homeotic proteins and multimeric complexes that also include the AGAMOUS-like proteins SEEDSTICK (STK) or SHATTERPROOF1/2 (SHP1, SHP2). These data suggest that the formation of multimeric transcription factor complexes might be a general phenomenon among MIKC-type MADS-domain proteins in angiosperms. Heterodimerization of ABS with SEP3 was confirmed by gel retardation assays. Fusion proteins tagged with CFP (Cyan Fluorescent Protein) and YFP (Yellow Fluorescent Protein) in Arabidopsis protoplasts showed that ABS is localized in the nucleus. Phylogenetic analysis revealed the presence of a structurally deviant, but closely related, paralogue of ABS in the Arabidopsis genome. Thus the evolutionary developmental genetics of B(sister) genes can probably only be understood as part of a complex and redundant gene network that may govern ovule formation in a conserved manner, which has yet to be fully explored.

  13. Study on the Protoplast Fusion and Plant Regeneration System between Brassica napus and Isatis tinctoria%甘蓝型油菜与菘蓝原生质体融合及植株再生体系的研究

    Institute of Scientific and Technical Information of China (English)

    杜雪竹; 李再云

    2012-01-01

    Protoplast was isolated from leaves of Brassira napus and hatis linctoria and fused by PEG-high pH and high calcium method. Callus was then induced by solid-liquid double layer culture method. Effects of factors including enzyme type and concentration on protoplast isolation, type and dose of hormone on callus induction and differentiation were studied to improve the plant regeneration efficiency. The results suggested that the enzyme combination suitable for protoplast isolation was 5 mg/L cellulase+3 mg/L macerozyme. The concentration of hormone for protoplasts induction, proliferation and differentiation were 1.0 mg/L 6-BA+l.0mg/L NAA+0.25 mg/L 2,4-D, 2.0 mg/L 6-BA+0.4 mg/L NAA+0.1 mg/L 2,4-D and 2.0 mg/L 6-BA +0.1 mg/L NAA +0.02 mg/L 2,4-D. A total of 15 somatic hybrids were obtained. The leaves of the hybrids seedling was dark green and thick, covered with wax layer. The adult plants were shorter and flowered later than B. Napus. Their stamens were ateleiosis; and the chromosomes number was 48-66 in somatic cells.%以甘蓝型油菜(Brassica napus)和菘蓝(Isatis tinctoria)叶片为材料提取原生质体,采用PEG-高钙高pH法融合亲本原生质体,采用固液双层培养法诱导愈伤组织的形成.研究适宜原生质体分离的酶种类及浓度,并考察了激素种类和用量对愈伤组织诱导和分化的影响.结果表明,5 mg/L纤维素酶+3 mg/L离析酶适合甘蓝型油菜和菘蓝的酶解.各培养基中适宜的激素浓度分别为,诱导培养基PellB 1.0 mg/L6-BA+1.0 mg/L NAA+0.25 mg/L 2,4-D;增殖培养基PellC 2.0 mg/L 6-BA+0.1 mg/L NAA+0.1 mg/L2,4-D;分化培养基PellE 2.0 mg/L 6-BA +0.1 mg/L NAA+0.02 mg/L 2,4-D.获得了15株再生植株,杂种幼苗叶片均呈深绿色,叶表面覆有厚的蜡质,叶片肥厚,植株在成熟期时株高比甘蓝型油菜矮,开花较晚,雄蕊发育不完全,杂种体细胞染色体数目为48~66.

  14. Lateral diffusion of phospholipids in the plasma membrane of soybean protoplasts: Evidence for membrane lipid domains.

    Science.gov (United States)

    Metcalf, T N; Wang, J L; Schindler, M

    1986-01-01

    Fluorescent lipid and phospholipid probes were incorporated at 4 degrees C into soybean protoplasts prepared from cultured soybean (SB-1) cells. Fluorescence microscopy showed that the plasma membrane as well as the nucleus were labeled. Fluorescence redistribution after photobleaching (FRAP) analysis was performed on these cells at 18 degrees C to monitor the lateral mobility of the incorporated probes. After labeling at low concentrations (40 mug/ml) of phosphatidyl-N-(4-nitrobenzo-2-oxa-1,3-diazolyl)ethanolamine (NBD-PtdEtn), a single mobile component was observed with a diffusion coefficient (D) of approximately 3 x 10(-9) cm(2)/sec. After labeling at higher probe concentrations (>/=100 mug/ml), two diffusing species were observed, with diffusion coefficients of approximately 3 x 10(-9) cm(2)/sec ("fast") and approximately 5 x 10(-10) cm(2)/sec ("slow"). Similar results were observed with fluorescent derivatives of phosphatidylcholine and fatty acids. In contrast to these results, parallel analysis of 3T3 fibroblasts, using the same probes and conditions, yielded only a single diffusion component. These results suggest that the soybean plasma membrane may contain two distinct lipid domains in terms of lipid mobility. Consistent with this idea, experiments with soybean protoplasts yielded a single diffusion component under the following conditions: (i) labeling with NBD-PtdEtn (100 mug/ml), FRAP analysis at 37 degrees C (D = 1.1 x 10(-8) cm(2)/sec); (ii) labeling with NBD-PtdEtn (100 mug/ml), FRAP analysis at 18 degrees C in the presence of 2 mM EGTA (D = 4.2 x 10(-9) cm(2)/sec); (iii) labeling with 5-(N-dodecanoyl)aminofluorescein (a short-chain lipid probe), FRAP analysis at 18 degrees C or 37 degrees C (D = 2.5 x 10(-8) cm(2)/sec). These results suggest that the plasma membrane of soybean cells may contain stable immiscible domains of fluid and gel-like lipids.

  15. Citrus asymmetric somatic hybrids produced via fusion of gamma-irradiated and iodoacetamide-treated protoplasts Híbridos somáticos assimétricos de citros produzidos pela fusão de protoplastos irradiados e tratados com iodoacetamida

    Directory of Open Access Journals (Sweden)

    Claudine Maria de Bona

    2009-05-01

    Full Text Available The objective of this study was to produce citrus somatic asymmetric hybrids by fusing gamma-irradiated protoplasts with iodoacetamide-treated protoplasts. Protoplasts were isolated from embryogenic suspension cells of grapefruit (Citrus paradisi Macfad. cultivars Ruby Red and Flame, sweet oranges (C. sinensis Osbeck 'Itaboraí', 'Natal', Valencia', and 'Succari', from 'Satsuma' (C. unshiu Marcow. and 'Changsha' mandarin (C. reticulata Blanco and 'Murcott' tangor (C. reticulata x C. sinensis. Donor protoplasts were exposed to gamma rays and receptor protoplasts were treated with 3 mmol L-1 iodoacetamide (IOA, and then they were fused for asymmetric hybridization. Asymmetric embryos were germinated, and the resulting shoots were either grafted onto sour orange, rough lemon or 'Swingle' (C. paradisi x Poncirus trifoliata x 'Sunki' mandarin rootstock seedlings, or rooted after dipping their bases in indol-butyric acid (IBA solution. The products were later acclimatized to greenhouse conditions. Ploidy was analyzed by flow cytometry, and hybridity was confirmed by amplified fragment length polymorphism (AFLP analysis of plantlet DNAsamples. The best treatment was the donor-recipient fusion combination of 80 Gy-irradiated 'Ruby Red' protoplasts with 20 min IOA-treated 'Succari' protoplasts. Tetraploid and aneuploid plants were produced. Rooting recalcitrance was solved by dipping shoots' stems in 3,000 mg L-1 IBA solution for 10 min.O objetivo deste trabalho foi produzir híbridos somáticos assimétricos de citros pela fusão de protoplastos irradiados com raios gama e protoplastos tratados com iodoacetamida. Protoplastos foram isolados de suspensões celulares embriogênicas de pomelo (Citrus paradisi Macfad., cultivares Ruby Red e Flame, de laranja doce (C. sinensis Osbeck 'Itaboraí', 'Natal', Valencia' e 'Succari', de tangerinas 'Satsuma' (C. unshiu Marcow. e 'Changsha' (C. reticulata Blanco e de tangor 'Murcott' (C. reticulata x C. sinensis

  16. 氯化锂诱变黑曲霉原生质体选育高产植酸酶菌株%Breeding of Phytase High-producing Aspergillus niger Using Protoplasts by Lithium Chloride Mutagenesis

    Institute of Scientific and Technical Information of China (English)

    李文; 王陶; 李同祥

    2012-01-01

    采用氯化锂诱变黑曲霉原生质体,筛选高产植酸酶菌株。获得制备黑曲霉原生质体的最适条件:纤维素酶1.0%,蜗牛酶0.5%,菌龄24h,酶解温度30℃,酶解时间2h,渗透压稳定剂0.7mol/LNaCl。采用氯化锂对制得的原生质体进行诱变,结果表明:经0.15%LiCl诱变后,原生质体存活率为23.37%,此时,获得一株植酸酶活最高的突变株,为19.24U/mL,比出发菌株提高54.41%,该菌株具有良好的遗传稳定性。%Lithium chloride was used to induce protoplasts of Aspergillus niger. Mutation strain with higher phytase content was screened in this paper. The optimized isolation method for protoplasts from Aspergillus niger was obtained. The results showed that the suitable conditions were cellulose 1% , snailase 0.5% , mycelium age 24h, en- zymatic hydrolysis temperature 30℃ , time 2h and osmotic pressure stabilizer 0.7 mol/L sodium chloride. Lithium chloride was used to induce the protoplasts. The results showed that the survival rate of strains was 23.3% induced with 0.15% Lithium chloride. And one strain with the highest enzyme activity of 19.24 U/mL was obtained, and the enzyme activity has a 54.41% increase compared with the original strain. Heredity of induced strains was stable.

  17. Bimolecular fluorescence complementation as a tool to study interactions of regulatory proteins in plant protoplasts.

    Science.gov (United States)

    Pattanaik, Sitakanta; Werkman, Joshua R; Yuan, Ling

    2011-01-01

    Protein-protein interactions are an important aspect of the gene regulation process. The expression of a gene in response to certain stimuli, within a specific cell type or at a particular developmental stage, involves a complex network of interactions between different regulatory proteins and the cis-regulatory elements present in the promoter of the gene. A number of methods have been developed to study protein-protein interactions in vitro and in vivo in plant cells, one of which is bimolecular fluorescence complementation (BiFC). BiFC is a relatively simple technique based upon the reconstitution of a fluorescent protein. The interacting protein complex can be visualized directly in a living plant cell when two non-fluorescent fragments, of an otherwise fluorescent protein, are fused to proteins found within that complex. Interaction of tagged proteins brings the two non-fluorescent fragments into close proximity and reconstitutes the fluorescent protein. In addition, the subcellular location of an interacting protein complex in the cell can be simultaneously determined. Using this approach, we have successfully demonstrated a protein-protein interaction between a R2R3 MYB and a basic helix-loop-helix MYC transcription factor related to flavonoid biosynthetic pathway in tobacco protoplasts.

  18. Subcellular compartmentalization in protoplasts from Artemisia annua cell cultures: engineering attempts using a modified SNARE protein.

    Science.gov (United States)

    Di Sansebastiano, Gian Pietro; Rizzello, Francesca; Durante, Miriana; Caretto, Sofia; Nisi, Rossella; De Paolis, Angelo; Faraco, Marianna; Montefusco, Anna; Piro, Gabriella; Mita, Giovanni

    2015-05-20

    Plants are ideal bioreactors for the production of macromolecules but transport mechanisms are not fully understood and cannot be easily manipulated. Several attempts to overproduce recombinant proteins or secondary metabolites failed. Because of an independent regulation of the storage compartment, the product may be rapidly degraded or cause self-intoxication. The case of the anti-malarial compound artemisinin produced by Artemisia annua plants is emblematic. The accumulation of artemisinin naturally occurs in the apoplast of glandular trichomes probably involving autophagy and unconventional secretion thus its production by undifferentiated tissues such as cell suspension cultures can be challenging. Here we characterize the subcellular compartmentalization of several known fluorescent markers in protoplasts derived from Artemisia suspension cultures and explore the possibility to modify compartmentalization using a modified SNARE protein as molecular tool to be used in future biotechnological applications. We focused on the observation of the vacuolar organization in vivo and the truncated form of AtSYP51, 51H3, was used to induce a compartment generated by the contribution of membrane from endocytosis and from endoplasmic reticulum to vacuole trafficking. The artificial compartment crossing exocytosis and endocytosis may trap artemisinin stabilizing it until extraction; indeed, it is able to increase total enzymatic activity of a vacuolar marker (RGUSChi), probably increasing its stability. Exploring the 51H3-induced compartment we gained new insights on the function of the SNARE SYP51, recently shown to be an interfering-SNARE, and new hints to engineer eukaryote endomembranes for future biotechnological applications.

  19. [Senescence and apoptosis of protoplasts from flax fibers: an ultrastructural analysis].

    Science.gov (United States)

    Ageeva, M V; Chernova, T E; Gorshkova, T A

    2012-01-01

    Plant fibers represent specialized cells that perform a mechanical function. Their development includes the following phases, typical for the most plant cells: anlage, extension growth, specialization, senescence, and apoptosis. Ultrastructural analysis of these cells has been carried out at the late phases of their development (senescence and apoptosis) using flax phloem fibers, a classical object for the analysis of sclerenchyma fiber formation. The results of the performed analysis show that flax fiber protoplasts remain viable until the end ofa vegetation season. The ultrastructural analysis of flax phloem fibers has not revealed any typical apoptosis manifestations. Gradual degradation of the cytoplasm starts during the active thickening of a secondary cell wall and manifests via the intensification of autolytic processes, causing a partial loss of cell content. The final stage represents the breaking of tonoplast integrity. The obtained data allow us to suppose that the apoptosis of flax fibers occurs during their senescence, and its program is similar to the cell death program realized in the xylem fibers of woody plants.

  20. Methods for in vitro propagation of Pelargonium x Hortorum and others: from meristems to protoplasts.

    Science.gov (United States)

    Dorion, Noëlle; Ben Jouira, Hatem; Gallard, Anthony; Hassanein, Anber; Nassour, Mazen; Grapin, Agnès

    2010-01-01

    Geraniums (Pelargonium spp.) are among the most popular bedding and pot plants (25% of the French domestic market). On one hand, as vegetatively propagated plants, Pelargonium are submitted to pathogen pressure. On the other hand, innovation via interspecific hybridisation faces some difficulties. In this chapter, the two first protocols (from seeds and meristems) explain how in vitro plants free of virus could be obtained. The development of this technique is the long-term preservation of genetic resources via meristem cryopreservation. The third protocol describes propagation of Pelargonium with limited risks of variation. This technique also allows the constitution and the maintenance of a plant-stock from which explants can be taken for other studies. The two last protocols describe plant regenerations from leaf discs and mesophyll protoplasts, used for gene transfer and somatic hybridisation. These protocols were established mainly with Pelargonium x hortorum cultivars, but we propose possible solutions for the other species: P. x peltatum, P. x domesticum, P. capitatum and P. graveolens.

  1. Water consumption and biomass production of protoplast fusion lines of poplar hybrids under drought stress

    Directory of Open Access Journals (Sweden)

    Anne eHennig

    2015-05-01

    Full Text Available Woody crops such as poplars (Populus can contribute to meet the increasing energy demand of a growing human population and can therefore enhance the security of energy supply. Using energy from biomass increases ecological sustainability as biomass is considered to play a pivotal role in abating climate change. Because areas for establishing poplar plantations are often confined to marginal sites drought tolerance is one important trait for poplar genotypes cultivated in short rotation coppice. We tested nine-month-old plants of four tetraploid Populus tremula (L. x P. tremuloides (Michx. lines that were generated by protoplast fusion and their diploid counterpart for water consumption and drought stress responses in a greenhouse experiment. The fusion lines showed equivalent or decreased height growth, stem biomass and total leaf area compared to the diploid line. The relative height increment of the fusion lines was not reduced compared to the diploid line when the plants were exposed to drought. The fusion lines were distinguished from the diploid counterpart by stomatal characteristics such as increased size and lower density. The changes in the stomatal apparatus did not affect the stomatal conductance. When exposed to drought the carbohydrate concentrations increased more strongly in the fusion lines than in the diploid line. Two fusion lines consumed significantly less water with regard to height growth, producing equivalent or increased relative stem biomass under drought compared to their diploid relative. Therefore, these tetraploid fusion lines are interesting candidates for short rotation biomass plantation on dry sites.

  2. Characterization of a new lectin involved in the protoplast regeneration of Bryopsis hypnoides

    Institute of Scientific and Technical Information of China (English)

    NIU Jianfeng; WANG Guangce; L(U) Fang; ZHOU Baicheng; PENG Guang

    2009-01-01

    A group of coenocytic marine algae differs from higher plants, whose totipotency depends on an intact cell (or protoplast). Instead, this alga is able to aggregate its extruded protoplasm in sea water and generate new mature individuals. It is thought that lectins play a key role in the aggregation process. We purified a lectin associated with the aggregation of cell organelles in Bryopsis hypnoides. The lectin was ca. 27 kDa with a pI between pH 5 and pH 6. The absence of carbohydrate suggested that the lectin was not a glycoprotein. The hemagglutinating activity (HA) of the lectin was not dependent on the presence of divalent cations and was inhibited by N-Acetylgalactosamine, N-Acetylglucosamine, and the glycoprotein bovine submaxillary mucin. The lectin preferentially agglutinated Gram-negative bacterium. The HA of this lectin was stable between pH 4 to pH 10. Cell organelles outside the cytoplasm were agglutinated by the addition of lectin solution (0.5 mg ml-1). Our results suggest that the regeneration of B. hypnoides is mediated by this lectin. We also demonstrated that the formation of cell organelle aggregates was inhibited by nigericin in natural seawater (pH 8.0). Given that nigericin dissipates proton gradients across the membrane, we hypothesize that the aggregation of cell organelles was proton-gradient dependent.

  3. Reference: 774 [Arabidopsis Phenome Database[Archive

    Lifescience Database Archive (English)

    Full Text Available an essential gene, the disruption of which causes embryonic lethality. Plants carrying a hypomorphic smg7 mu...e progression from anaphase to telophase in the second meiotic division in Arabidopsis. Arabidopsis SMG7 is

  4. Reference: 398 [Arabidopsis Phenome Database[Archive

    Lifescience Database Archive (English)

    Full Text Available plays attenuated chloroplast movements under intermediate and high light intensitie...hese movements. In this work, we describe plastid movement impaired 2 (pmi2), a mutant in Arabidopsis (Arabidopsis thaliana) that dis

  5. Reference: 173 [Arabidopsis Phenome Database[Archive

    Lifescience Database Archive (English)

    Full Text Available mical approaches to elucidate the action mechanisms of sirtinol in Arabidopsis. A...tic and chemical analyses of the action mechanisms of sirtinol in Arabidopsis. 8 3129-34 15710899 2005 Feb P

  6. Reference: 718 [Arabidopsis Phenome Database[Archive

    Lifescience Database Archive (English)

    Full Text Available displayed a moderate but significant decrease in germination in the presence of D...NA damage. This report links Ubc13-Uev with functions in DNA damage response in Arabidopsis. Arabidopsis UEV

  7. Arabidopsis CDS blastp result: AK068856 [KOME

    Lifescience Database Archive (English)

    Full Text Available eme oxygenase (HY1) [Arabidopsis thaliana] GI:4877362, heme oxygenase 1 [Arabidopsis thaliana] GI:4530591 GB:AF132475; annotation upd...ated per Seth J. Davis at University of Wisconsin-Madison 3e-90 ...

  8. Arabidopsis CDS blastp result: AK104955 [KOME

    Lifescience Database Archive (English)

    Full Text Available B:AF132475; annotation updated per Seth J. Davis at University of Wisconsin-Madison 3e-90 ... ...heme oxygenase (HY1) [Arabidopsis thaliana] GI:4877362, heme oxygenase 1 [Arabidopsis thaliana] GI:4530591 G

  9. Reference: 110 [Arabidopsis Phenome Database[Archive

    Lifescience Database Archive (English)

    Full Text Available on process. Our study shows that an Arabidopsis SNM protein, although structurally closer to the SNM1/PSO2 members, shares some prope...rties with ARTEMIS but also has novel characteristics. Arabidopsis plants defective

  10. 木本植物原生质体制备体系的研究进展%Advances in preparation system of woody plants protoplast

    Institute of Scientific and Technical Information of China (English)

    张良波; 李培旺; 黄振; 李昌珠

    2011-01-01

    对木本植物原生质体制备国内外研究发展历程和现状进行了回顾,详细阐述了构成木本植物原生质体制备体系的各个环节和注意事项.分析了木本植物原生质体研究早期缓慢和后期发展迅速的原因,主要是由于原生质体制备体系技术的进步.分析结果对利用木本植物原生质体进行木本植物遗传改良具有重要意义.%The development history and present situation of woody plants protoplast preparation system at home and abroad were reviewed. The processes and announcements of woody plants protoplast preparation were given in detail. The reasons of developing slowly at earlier and rapidly later of the research on woody plants protoplast are the realization of preparation system of woody plants protoplast. The findings provide a beneficial guidance for utilizing woody plants protoplast to improve woody plant's hereditary character.

  11. Monoclonal antibodies directed against protoplasts of soybean cells : Generation of hybridomas and characterization of a monoclonal antibody reactive with the cell surface.

    Science.gov (United States)

    Villanueva, M A; Metcalf, T N; Wang, J L

    1986-09-01

    Splenocytes, derived from mice that had been immunized with protoplasts prepared from suspension cultures of root cells of Glycine max (L.) Merr. (SB-1 cell line), were fused with a murine myeloma cell line. The resulting hybridoma cultures were screened for the production of antibodies directed against the soybean protoplasts and were then cloned. One monoclonal antibody, designated MVS-1, was found to bind to the outer surface of the plasma membrane on the basis of several criteria: (a) agglutination of the protoplasts; (b) binding of fluorescence-labeled immunoglobulin on protoplasts yielding a ring staining pattern with prominent intensity at the edges; and (c) saturable binding by protoplasts of (125)I-labeled Antibody MVS-1. The antigenic target of Antibody MVS-1, identified by immunoblotting techniques, contained a polypeptide of relative molecular mass (Mr) approx. 400000 under both reducing and non-reducing conditions. When the antigenic target of Antibody MVS-1 was chromatographed in potassium phosphate buffer, the position of elution corresponded to that of a high-molecular-weight species (Mr 400000). These results provide the protein characterization required for the analysis of the mobility of Antibody MVS-1 bound to the plasma membrane of SB-1 cells.

  12. Direct gene transfer study and transgenic plant regeneration after electroporation into mesophyll protoplasts of Pelargonium x hortorum, 'Panaché Sud'.

    Science.gov (United States)

    Hassanein, Anber; Hamama, Latifa; Loridon, Karine; Dorion, Noëlle

    2009-10-01

    Direct genetic transformation of mesophyll protoplasts was studied in Pelargonium x hortorum. Calcein and green-fluorescent protein (GFP) gene were used to set up the process. Electroporation (three electric pulses from a 33-microF capacitor in a 250-V cm(-1) electric field) was more efficient than PEG 6000 for membrane permeation, protoplast survival and cell division. Transient expression of GFP was detected in 33-36% of electroporated protoplasts after 2 days and further in colonies. A protoplast suspension conductivity of >1,500 microS cm(-1) allowed high colony formation and plant regeneration. Stable transformation was obtained using the plasmid FAJ3000 containing uidA and nptII genes. When selection (50 mg l(-1) kanamycin) was achieved 6 weeks after electroporation, regenerated shoots were able to grow and root on 100 mg l(-1) kanamycin. The maximum transformation efficiency was 4.5%, based on the number of colonies producing kanamycin-resistant rooted plants or 0.7% based on the number of cultured protoplasts. Polymerase chain reaction (PCR) analysis on in vitro micropropagated plants showed that 18 clones out of 20 contained the nptII gene, while the uidA gene was absent. These results were confirmed after PCR analyses of five glasshouse-acclimatized clones.

  13. Cadmium-inducible expression of the ABC-type transporter AtABCC3 increases phytochelatin-mediated cadmium tolerance in Arabidopsis.

    Science.gov (United States)

    Brunetti, Patrizia; Zanella, Letizia; De Paolis, Angelo; Di Litta, Davide; Cecchetti, Valentina; Falasca, Giuseppina; Barbieri, Maurizio; Altamura, Maria Maddalena; Costantino, Paolo; Cardarelli, Maura

    2015-07-01

    The heavy metal cadmium (Cd) is a widespread environmental contaminant with harmful effects on living cells. In plants, phytochelatin (PC)-dependent Cd detoxification requires that PC-Cd complexes are transported into vacuoles. Here, it is shown that Arabidopsis thaliana seedlings defective in the ABCC transporter AtABCC3 (abcc3) have an increased sensitivity to different Cd concentrations, and that seedlings overexpressing AtABCC3 (AtABCC3ox) have an increased Cd tolerance. The cellular distribution of Cd was analysed in protoplasts from abcc3 mutants and AtABCC3 overexpressors grown in the presence of Cd, by means of the Cd-specific fluorochromes 5-nitrobenzothiazole coumarin (BTC-5N) and Leadmium™ Green AM dye. This analysis revealed that Cd is mostly localized in the cytosol of abcc3 mutant protoplasts whereas there is an increase in vacuolar Cd in protoplasts from AtABCC3ox plants. Overexpression of AtABCC3 in cad1-3 mutant seedlings defective in PC production and in plants treated with l-buthionine sulphoximine (BSO), an inhibitor of PC biosynthesis, had no effect on Cd tolerance, suggesting that AtABCC3 acts via PCs. In addition, overexpression of AtABCC3 in atabcc1 atabcc2 mutant seedlings defective in the Cd transporters AtABCC1 and AtABCC2 complements the Cd sensitivity of double mutants, but not in the presence of BSO. Accordingly, the level of AtABCC3 transcript in wild type seedlings was lower than that of AtABCC1 and AtABCC2 in the absence of Cd but higher after Cd exposure, and even higher in atabcc1 atabcc2 mutants. The results point to AtABCC3 as a transporter of PC-Cd complexes, and suggest that its activity is regulated by Cd and is co-ordinated with the activity of AtABCC1/AtABCC2.

  14. The quantitative basis of the Arabidopsis innate immune system to endemic pathogens depends on pathogen genetics

    DEFF Research Database (Denmark)

    Corwin, Jason A; Copeland, Daniel; Feusier, Julie;

    2016-01-01

    The most established model of the eukaryotic innate immune system is derived from examples of large effect monogenic quantitative resistance to pathogens. However, many host-pathogen interactions involve many genes of small to medium effect and exhibit quantitative resistance. We used...... the Arabidopsis-Botrytis pathosystem to explore the quantitative genetic architecture underlying host innate immune system in a population of Arabidopsis thaliana. By infecting a diverse panel of Arabidopsis accessions with four phenotypically and genotypically distinct isolates of the fungal necrotroph B....... cinerea, we identified a total of 2,982 genes associated with quantitative resistance using lesion area and 3,354 genes associated with camalexin production as measures of the interaction. Most genes were associated with resistance to a specific Botrytis isolate, which demonstrates the influence...

  15. Post-Translational Modification and Secretion of Azelaic Acid Induced 1 (AZI1, a Hybrid Proline-Rich Protein from Arabidopsis

    Directory of Open Access Journals (Sweden)

    Andrea Pitzschke

    2016-01-01

    Full Text Available Arabidopsis EARLI-type hybrid proline-rich proteins (HyPRPs consist of a putative N-terminal secretion signal, a proline-rich domain (PRD, and a characteristic eight-cysteine-motif (8-CM. They have been implicated in biotic and abiotic stress responses. AZI1 is required for systemic acquired resistance and it has recently been identified as a target of the stress-induced mitogen-activated protein kinase MPK3. AZI1 gel migration properties strongly indicate AZI1 to undergo major post-translational modifications. These occur in a stress-independent manner and are unrelated to phosphorylation by MAPKs. As revealed by transient expression of AZI1 in Nicotiana benthamiana and Tropaeolum majus, the Arabidopsis protein is similarly modified in heterologous plant species. Proline-rich regions, resembling arabinogalactan proteins point to a possible proline hydroxylation and subsequent O-glycosylation of AZI1. Consistently, inhibition of prolyl hydroxylase reduces its apparent protein size. AZI1 secretion was examined using Arabidopsis protoplasts and seedling exudates. Employing Agrobacterium-mediated leaf infiltration of N. benthamiana, we attempted to assess long-distance movement of AZI1. In summary, the data point to AZI1 being a partially secreted protein and a likely new member of the group of hydroxyproline-rich glycoproteins. Its dual location suggests AZI1 to exert both intra- and extracellular functions.

  16. Regulation of secondary cell wall biosynthesis by poplar R2R3 MYB transcription factor PtrMYB152 in Arabidopsis.

    Science.gov (United States)

    Wang, Shucai; Li, Eryang; Porth, Ilga; Chen, Jin-Gui; Mansfield, Shawn D; Douglas, Carl J

    2014-05-23

    Poplar has 192 annotated R2R3 MYB genes, of which only three have been shown to play a role in the regulation of secondary cell wall formation. Here we report the characterization of PtrMYB152, a poplar homolog of the Arabidopsis R2R3 MYB transcription factor AtMYB43, in the regulation of secondary cell wall biosynthesis. The expression of PtrMYB152 in secondary xylem is about 18 times of that in phloem. When expressed in Arabidopsis under the control of either 35S or PtrCesA8 promoters, PtrMYB152 increased secondary cell wall thickness, which is likely caused by increased lignification. Accordingly, elevated expression of genes encoding sets of enzymes in secondary wall biosynthesis were observed in transgenic plants expressing PtrMYB152. Arabidopsis protoplast transfection assays suggested that PtrMYB152 functions as a transcriptional activator. Taken together, our results suggest that PtrMYB152 may be part of a regulatory network activating expression of discrete sets of secondary cell wall biosynthesis genes.

  17. Regulation of secondary cell wall biosynthesis by poplar R2R3 MYB transcription factor PtrMYB152 in Arabidopsis

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Shucai [Northeast Normal Univ., Changchun (China); Univ. of British Columbia, Vancouver, BC (Canada); Li, Eryang [Univ. of British Columbia, Vancouver, BC (Canada); Porth, Ilga [Univ. of British Columbia, Vancouver, BC (Canada); Chen, Jin-Gui [Univ. of British Columbia, Vancouver, BC (Canada); Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); Mansfield, Shawn D. [Univ. of British Columbia, Vancouver, BC (Canada); Douglas, Carl [Univ. of British Columbia, Vancouver, BC (Canada)

    2014-05-23

    Poplar has 192 annotated R2R3 MYB genes, of which only three have been shown to play a role in the regulation of secondary cell wall formation. Here we report the characterization of PtrMYB152, a poplar homolog of the Arabidopsis R2R3 MYB transcription factor AtMYB43, in the regulation of secondary cell wall biosynthesis. The expression of PtrMYB152 in secondary xylem is about 18 times of that in phloem. When expressed in Arabidopsis under the control of either 35S or PtrCesA8 promoters, PtrMYB152 increased secondary cell wall thickness, which is likely caused by increased lignification. Accordingly, elevated expression of genes encoding sets of enzymes in secondary wall biosynthesis were observed in transgenic plants expressing PtrMYB152. Arabidopsis protoplast transfection assays suggested that PtrMYB152 functions as a transcriptional activator. Taken together, our results suggest that PtrMYB152 may be part of a regulatory network activating expression of discrete sets of secondary cell wall biosynthesis genes.

  18. Molecular cloning of cryptochrome 1 from apple and its functional characterization in Arabidopsis.

    Science.gov (United States)

    Li, Yuan-Yuan; Mao, Ke; Zhao, Cheng; Zhang, Rui-Fen; Zhao, Xian-Yan; Zhang, Hua-Lei; Shu, Huai-Rui; Zhao, Yu-Jin

    2013-06-01

    Cryptochromes are blue-light photoreceptors involved in regulating many aspects of plant growth and development. Investigations of cryptochromes in plants have largely focused on Arabidopsis (Arabidopsis thaliana), tomato (Solanum lycopersicum), rice (Oryza sativa) and pea (Pisum sativum). Here, we isolated the cryptochrome 1 gene from apple (Malus domestica) (MdCRY1) and analyzed its function in transgenic Arabidopsis. The predicted MdCRY1 protein was most closely homologous to strawberry CRY1. In terms of transcript levels, MdCRY1 expression was up-regulated by light. The function of MdCRY1 was analyzed through heterologous expression in Arabidopsis. Overexpression of MdCRY1 in Arabidopsis is able to rescue the cry1 mutant phenotype, inhibit hypocotyl elongation, promote root growth, and enhance anthocyanin accumulation in wild-type seedlings under blue light. These data provide functional evidence for a role of MdCRY1 in controlling photomorphogenesis under blue light and indicate that CRY1 function is conserved between Arabidopsis and apple. Furthermore, we found that MdCRY1 interacts with AtCOP1 in both yeast and onion cells. This interaction may represent an important regulatory mechanism in blue-light signaling pathway in apple.

  19. Novel symbiotic protoplasts formed by endophytic fungi explain their hidden existence, lifestyle switching, and diversity within the plant kingdom.

    Directory of Open Access Journals (Sweden)

    Peter R Atsatt

    Full Text Available Diverse fungi live all or part of their life cycle inside plants as asymptomatic endophytes. While endophytic fungi are increasingly recognized as significant components of plant fitness, it is unclear how they interact with plant cells; why they occur throughout the fungal kingdom; and why they are associated with most fungal lifestyles. Here we evaluate the diversity of endophytic fungi that are able to form novel protoplasts called mycosomes. We found that mycosomes cultured from plants and phylogenetically diverse endophytic fungi have common morphological characteristics, express similar developmental patterns, and can revert back to the free-living walled state. Observed with electron microscopy, mycosome ontogeny within Aureobasidium pullulans may involve two organelles: double membrane-bounded promycosome organelles (PMOs that form mycosomes, and multivesicular bodies that may form plastid-infecting vesicles. Cultured mycosomes also contain a double membrane-bounded organelle, which may be homologous to the A. pullulans PMO. The mycosome PMO is often expressed as a vacuole-like organelle, which alternatively may contain a lipoid body or a starch grain. Mycosome reversion to walled cells occurs within the PMO, and by budding from lipid or starch-containing mycosomes. Mycosomes discovered in chicken egg yolk provided a plant-independent source for analysis: they formed typical protoplast stages, contained fungal ITS sequences and reverted to walled cells, suggesting mycosome symbiosis with animals as well as plants. Our results suggest that diverse endophytic fungi express a novel protoplast phase that can explain their hidden existence, lifestyle switching, and diversity within the plant kingdom. Importantly, our findings outline "what, where, when and how", opening the way for cell and organelle-specific tests using in situ DNA hybridization and fluorescent labels. We discuss developmental, ecological and evolutionary contexts that

  20. Novel symbiotic protoplasts formed by endophytic fungi explain their hidden existence, lifestyle switching, and diversity within the plant kingdom.

    Science.gov (United States)

    Atsatt, Peter R; Whiteside, Matthew D

    2014-01-01

    Diverse fungi live all or part of their life cycle inside plants as asymptomatic endophytes. While endophytic fungi are increasingly recognized as significant components of plant fitness, it is unclear how they interact with plant cells; why they occur throughout the fungal kingdom; and why they are associated with most fungal lifestyles. Here we evaluate the diversity of endophytic fungi that are able to form novel protoplasts called mycosomes. We found that mycosomes cultured from plants and phylogenetically diverse endophytic fungi have common morphological characteristics, express similar developmental patterns, and can revert back to the free-living walled state. Observed with electron microscopy, mycosome ontogeny within Aureobasidium pullulans may involve two organelles: double membrane-bounded promycosome organelles (PMOs) that form mycosomes, and multivesicular bodies that may form plastid-infecting vesicles. Cultured mycosomes also contain a double membrane-bounded organelle, which may be homologous to the A. pullulans PMO. The mycosome PMO is often expressed as a vacuole-like organelle, which alternatively may contain a lipoid body or a starch grain. Mycosome reversion to walled cells occurs within the PMO, and by budding from lipid or starch-containing mycosomes. Mycosomes discovered in chicken egg yolk provided a plant-independent source for analysis: they formed typical protoplast stages, contained fungal ITS sequences and reverted to walled cells, suggesting mycosome symbiosis with animals as well as plants. Our results suggest that diverse endophytic fungi express a novel protoplast phase that can explain their hidden existence, lifestyle switching, and diversity within the plant kingdom. Importantly, our findings outline "what, where, when and how", opening the way for cell and organelle-specific tests using in situ DNA hybridization and fluorescent labels. We discuss developmental, ecological and evolutionary contexts that provide a robust

  1. The ACR11 encodes a novel type of chloroplastic ACT domain repeat protein that is coordinately expressed with GLN2 in Arabidopsis

    Directory of Open Access Journals (Sweden)

    Hsu Chih-Ping

    2011-08-01

    Full Text Available Abstract Background The ACT domain, named after bacterial aspartate kinase, chorismate mutase and TyrA (prephenate dehydrogenase, is a regulatory domain that serves as an amino acid-binding site in feedback-regulated amino acid metabolic enzymes. We have previously identified a novel type of ACT domain-containing protein family, the ACT domain repeat (ACR protein family, in Arabidopsis. Members of the ACR family, ACR1 to ACR8, contain four copies of the ACT domain that extend throughout the entire polypeptide. Here, we describe the identification of four novel ACT domain-containing proteins, namely ACR9 to ACR12, in Arabidopsis. The ACR9 and ACR10 proteins contain three copies of the ACT domain, whereas the ACR11 and ACR12 proteins have a putative transit peptide followed by two copies of the ACT domain. The functions of these plant ACR proteins are largely unknown. Results The ACR11 and ACR12 proteins are predicted to target to chloroplasts. We used protoplast transient expression assay to demonstrate that the Arabidopsis ACR11- and ACR12-green fluorescent fusion proteins are localized to the chloroplast. Analysis of an ACR11 promoter-β-glucuronidase (GUS fusion in transgenic Arabidopsis revealed that the GUS activity was mainly detected in mature leaves and sepals. Interestingly, coexpression analysis revealed that the GLN2, which encodes a chloroplastic glutamine synthetase, has the highest mutual rank in the coexpressed gene network connected to ACR11. We used RNA gel blot analysis to confirm that the expression pattern of ACR11 is similar to that of GLN2 in various organs from 6-week-old Arabidopsis. Moreover, the expression of ACR11 and GLN2 is highly co-regulated by sucrose and light/dark treatments in 2-week-old Arabidopsis seedlings. Conclusions This study reports the identification of four novel ACT domain repeat proteins, ACR9 to ACR12, in Arabidopsis. The ACR11 and ACR12 proteins are localized to the chloroplast, and the expression

  2. Arabidopsis thaliana peroxidase N

    DEFF Research Database (Denmark)

    Mirza, Osman Asghar; Henriksen, A; Ostergaard, L

    2000-01-01

    The structure of the neutral peroxidase from Arabidopsis thaliana (ATP N) has been determined to a resolution of 1.9 A and a free R value of 20.5%. ATP N has the expected characteristic fold of the class III peroxidases, with a C(alpha) r.m.s.d. of 0.82 A when compared with horseradish peroxidase C...... (HRP C). HRP C is 54% identical to ATP N in sequence. When the structures of four class III plant peroxidases are superimposed, the regions with structural differences are non-randomly distributed; all are located in one half of the molecule. The architecture of the haem pocket of ATP N is very similar...... to that of HRP C, in agreement with the low small-molecule substrate specificity of all class III peroxidases. The structure of ATP N suggests that the pH dependence of the substrate turnover will differ from that of HRP C owing to differences in polarity of the residues in the substrate-access channel. Since...

  3. Research Progress of Protoplast Culture in Woody Plants%木本植物原生质体培养体系研究进展

    Institute of Scientific and Technical Information of China (English)

    彭邵锋; 陆佳; 陈永忠; 王瑞; 陈隆升; 马力; 王湘南

    2013-01-01

    为了给木本植物原生质体培养技术体系的建立提供理论依据,推动木本植物快速繁殖和品种遗传改良工作的开展,从原生质体分离、纯化、培养方式等方面对木本植物原生质体培养体系进行了详细阐述,对比分析了原生质体不同分离方法和培养方式的优缺点,研究总结出影响原生质体培养的主要因素,并提出了木本植物原生质体培养今后的研究重点.%In order to provide theoretical basis for establishment of protoplast culture system and promote the work of rapid propagation and genetic improvement of woody plants, a detailed description about protoplast culture system in woody plants from three aspects such as separation, purification, culture method and medium composition was made in the article. Advantages and disadvantages in different types of separation and culture method of protoplast were analyzed. The main influence factors on protoplast culture in woody plants were summerized. Significance for distant hybridization, new variety creation and genetic improvement by the way of protoplast culture in woody plants was brought out. Research emphasis on protoplast culture in woody plants was also proposed.

  4. Identification of genes affecting the response of tomato and Arabidopsis upon powdery mildew infection

    NARCIS (Netherlands)

    Gao, D.

    2014-01-01

      Many plant species are hosts of powdery mildew fungi, including Arabidopsis and economically important crops such as wheat, barley and tomato. Resistance has been explored using induced mutagenesis and natural variation in the plant species. The isolated genes encompass loss-of-function susc

  5. The genetics of some planthormones and photoreceptors in Arabidopsis thaliana (L.) Heynh

    NARCIS (Netherlands)

    Koornneef, M.

    1982-01-01

    This thesis describes the isolation and characterization in Arabidopsis thaliana (L.) Heynh. of induced mutants, deficient for gibberellins (GA's), abscisic acid (ABA) and photoreceptors.These compounds are known to regulate various facets of plant growth and differentiation, so mutants lacking one

  6. Enhanced Arabidopsis pattern-triggered immunity by overexpression of cysteine-rich receptor-like kinases

    Directory of Open Access Journals (Sweden)

    Yu-Hung eYeh

    2015-05-01

    Full Text Available Upon recognition of microbe-associated molecular patterns (MAMPs such as the bacterial flagellin (or the derived peptide flg22 by pattern-recognition receptors (PRRs such as the FLAGELLIN SENSING2 (FLS2, plants activate the pattern-triggered immunity (PTI response. The L-type lectin receptor kinase-VI.2 (LecRK-VI.2 is a positive regulator of Arabidopsis thaliana PTI. Cysteine-rich receptor-like kinases (CRKs possess two copies of the C-X8-C-X2-C (DUF26 motif in their extracellular domains and are thought to be involved in plant stress resistance, but data about CRK functions are scarce. Here we show that Arabidopsis overexpressing the LecRK-VI.2-responsive CRK4, CRK6 and CRK36 demonstrated an enhanced PTI response and were resistant to virulent bacteria Pseudomonas syringae pv. tomato DC3000. Notably, the flg22-triggered oxidative burst was primed in CRK4, CRK6, and CRK36 transgenics and up-regulation of the PTI-responsive gene FLG22-INDUCED RECEPTOR-LIKE 1 (FRK1 was potentiated upon flg22 treatment in CRK4 and CRK6 overexpression lines or constitutively increased by CRK36 overexpression. PTI-mediated callose deposition was not affected by overexpression of CRK4 and CRK6, while CRK36 overexpression lines demonstrated constitutive accumulation of callose. In addition, Pst DC3000-mediated stomatal reopening was blocked in CRK4 and CRK36 overexpression lines, while overexpression of CRK6 induced constitutive stomatal closure suggesting a strengthening of stomatal immunity. Finally, bimolecular fluorescence complementation and co-immunoprecipitation analyses in Arabidopsis protoplasts suggested that the plasma membrane localized CRK4, CRK6 and CRK36 associate with the PRR FLS2. Association with FLS2 and the observation that overexpression of CRK4, CRK6, and CRK36 boosts specific PTI outputs and resistance to bacteria suggest a role for these CRKs in Arabidopsis innate immunity.

  7. Control of trichome formation in Arabidopsis by poplar single-repeat R3 MYB transcription factors

    Directory of Open Access Journals (Sweden)

    Limei eZhou

    2014-06-01

    Full Text Available In Arabidopsis, trichome formation is regulated by the interplay of R3 MYBs and several others transcription factors including the WD40-repeat protein TRANSPARENT TESTA GLABRA1 (TTG1, the R2R3 MYB transcription factor GLABRA1 (GL1, the bHLH transcription factor GLABRA3 (GL3 or ENHANCER OF GLABRA3 (EGL3, and the homeodomain protein GLABRA2 (GL2. R3 MYBs including TRICHOMELESS1 (TCL1, TRYPTICHON (TRY, CAPRICE (CPC, ENHANCER OF TRY AND CPC1 (ETC1, ETC2 and ETC3 negatively regulate trichome formation by competing with GL1 for binding GL3 or EGL3, thus blocking the formation of TTG1-GL3/EGL3-GL1, an activator complex required for the activation of the trichome positive regulator gene GL2. However, it is largely unknown if R3 MYBs in other plant species especially woody plants have similar functions. By BLASTing the Populus trichocarpa protein database using the entire amino acid sequence of TCL1, an Arabidopsis R3 MYB transcription factor, we identified a total of eight R3 MYB transcription factor genes in poplar, namely Populus trichocarpa TRICHOMELESS1through 8 (PtrTCL1-PtrTCL8. The amino acid signature required for interacting with bHLH transcription factors and the amino acids required for cell-to-cell movement of R3 MYBs are not fully conserved in all PtrTCLs. When tested in Arabidopsis protoplasts, however, all PtrTCL interacted with GL3. Expressing each of the eight PtrTCLs genes in Arabidopsis resulted in either glabrous phenotypes or plants with reduced trichome numbers, and expression levels of GL2 in all transgenic plants tested were greatly reduced. Expression of PtrTCL1 under the control of TCL1 native promoter almost completely complemented the mutant phenotype of tcl. In contrast, expression of PtrTCL1 under the control of TRY native promoter in the try mutant, or under the control of CPC native promoter in the cpc mutant resulted in glabrous phenotypes, suggesting that PtrTCL1 functions similarly to TCL1, but not TRY and CPC.

  8. Apoplastic polyesters in Arabidopsis surface tissues--a typical suberin and a particular cutin.

    Science.gov (United States)

    Franke, Rochus; Briesen, Isabel; Wojciechowski, Tobias; Faust, Andrea; Yephremov, Alexander; Nawrath, Christiane; Schreiber, Lukas

    2005-11-01

    Cutinized and suberized cell walls form physiological important plant-environment interfaces as they act as barriers limiting water and nutrient loss and protect from radiation and invasion by pathogens. Due to the lack of protocols for the isolation and analysis of cutin and suberin in Arabidopsis, the model plant for molecular biology, mutants and transgenic plants with a defined altered cutin or suberin composition are unavailable, causing that structure and function of these apoplastic barriers are still poorly understood. Transmission electron microscopy (TEM) revealed that Arabidopsis leaf cuticle thickness ranges from only 22 nm in leaf blades to 45 nm on petioles, causing the difficulty in cuticular membrane isolation. We report the use of polysaccharide hydrolases to isolate Arabidopsis cuticular membranes, suitable for depolymerization and subsequent compositional analysis. Although cutin characteristic omega-hydroxy acids (7%) and mid-chain hydroxylated fatty acids (8%) were detected, the discovery of alpha,omega-diacids (40%) and 2-hydroxy acids (14%) as major depolymerization products reveals a so far novel monomer composition in Arabidopsis cutin, but with chemical analogy to root suberin. Histochemical and TEM analysis revealed that suberin depositions were localized to the cell walls in the endodermis of primary roots and the periderm of mature roots of Arabidopsis. Enzyme digested and solvent extracted root cell walls when subjected to suberin depolymerization conditions released omega-hydroxy acids (43%) and alpha,omega-diacids (24%) as major components together with carboxylic acids (9%), alcohols (6%) and 2-hydroxyacids (0.1%). This similarity to suberin of other species indicates that Arabidopsis roots can serve as a model for suberized tissue in general.

  9. An intergenic region shared by At4g35985 and At4g35987 in Arabidopsis thaliana is a tissue specific and stress inducible bidirectional promoter analyzed in transgenic arabidopsis and tobacco plants.

    Directory of Open Access Journals (Sweden)

    Joydeep Banerjee

    Full Text Available On chromosome 4 in the Arabidopsis genome, two neighboring genes (calmodulin methyl transferase At4g35987 and senescence associated gene At4g35985 are located in a head-to-head divergent orientation sharing a putative bidirectional promoter. This 1258 bp intergenic region contains a number of environmental stress responsive and tissue specific cis-regulatory elements. Transcript analysis of At4g35985 and At4g35987 genes by quantitative real time PCR showed tissue specific and stress inducible expression profiles. We tested the bidirectional promoter-function of the intergenic region shared by the divergent genes At4g35985 and At4g35987 using two reporter genes (GFP and GUS in both orientations in transient tobacco protoplast and Agro-infiltration assays, as well as in stably transformed transgenic Arabidopsis and tobacco plants. In transient assays with GFP and GUS reporter genes the At4g35985 promoter (P85 showed stronger expression (about 3.5 fold compared to the At4g35987 promoter (P87. The tissue specific as well as stress responsive functional nature of the bidirectional promoter was evaluated in independent transgenic Arabidopsis and tobacco lines. Expression of P85 activity was detected in the midrib of leaves, leaf trichomes, apical meristemic regions, throughout the root, lateral roots and flowers. The expression of P87 was observed in leaf-tip, hydathodes, apical meristem, root tips, emerging lateral root tips, root stele region and in floral tissues. The bidirectional promoter in both orientations shows differential up-regulation (2.5 to 3 fold under salt stress. Use of such regulatory elements of bidirectional promoters showing spatial and stress inducible promoter-functions in heterologous system might be an important tool for plant biotechnology and gene stacking applications.

  10. Localization of Seed Oil Body Proteins in Tobacco Protoplasts Reveals Specific Mechanisms of Protein Targeting to Leaf Lipid Droplets

    Institute of Scientific and Technical Information of China (English)

    Stefania De Domenico; Stefania Bonsegna; Marcello Salvatore Lenucci; Palmiro Poltronieri; Gian Pietro Di Sansebastiano; Angelo Santino

    2011-01-01

    Oleosin,caleosin and steroleosin are normally expressed in developing seed cells and are targeted to oil bodies.In the present work,the cDNA of each gene tagged with fluorescent proteins was transiently expressed into tobacco protoplasts and the fluorescent patterns observed by confocal laser scanning microscopy.Our results indicated clear differences in the endocellular localization of the three proteins.Oleosin and caleosin both share a common structure consisting of a central hydrophobic domain flanked by two hydrophilic domains and were correctly targeted to lipid droplets (LD),whereas steroleosin,characterized by an N-terminal oil body anchoring domain,was mainly retained in the endoplasmic reticulum (ER).Protoplast fractionation on sucrose gradients indicated that both oleosin and caleosingreen fluorescent protein (GFP) peaked at different fractions than where steroleosin-GFP or the ER marker binding immunoglobulin protein (BiP),were recovered.Chemical analysis confirmed the presence of triacylglycerols in one of the fractions where oleosin-GFP was recovered.Finally,only oleosinand caleosin-GFP were able to reconstitute artificial oil bodies in the presence of triacylglycerols and phospholipids.Taken together,our results pointed out for the first time that leaf LDs can be separated by the ER and both oleosin or caleosin are selectively targeted due to the existence of selective mechanisms controlling protein association with these organelles.

  11. High-affinity binding of fungal beta-glucan fragments to soybean (Glycine max L.) microsomal fractions and protoplasts.

    Science.gov (United States)

    Cosio, E G; Pöpperl, H; Schmidt, W E; Ebel, J

    1988-08-01

    We have recently reported the existence of binding sites in soybean membranes for a beta-glucan fraction derived from the fungal pathogen Phytophthora megasperma f. sp. glycinea, which may play a role in the elicitor-mediated phytoalexin response of this plant [Schmidt, W. E. & Ebel, J. (1987) Proc. Natl Acad. Sci. USA 84, 4117-4121]. The specificity of beta-glucan binding to soybean membranes has now been investigated using a variety of competing polyglucans and oligoglucans of fungal origin. P. megasperma beta-glucan binding showed high apparent affinity for branched glucans with degrees of polymerization greater than 12. Binding affinity showed good correlation with elicitor activity as measured in a soybean cotyledon bioassay. Modification of the glucans at the reducing end with phenylalkylamine reagents had no effect on binding affinity. This characteristic was used to synthesize an oligoglucosyl tyramine derivative suitable for radioiodination. The 125I-glucan (15-30 Ci/mmol) provided higher sensitivity and lower detection limits for the binding assays while behaving in a manner identical to the [3H]glucan used previously. More accurate determinations of the Kd value for glucan binding indicated a higher affinity than previously shown (37 nM versus 200 nM). The 125I-glucan was used to provide the first reported evidence of specific binding of a fungal beta-glucan fraction in vivo to soybean protoplasts. The binding affinity to protoplasts proved identical to that found in microsomal fractions.

  12. Exploiting Natural Variation in Arabidopsis

    NARCIS (Netherlands)

    Molenaar, J.A.; Keurentjes, J.J.B.

    2014-01-01

    Natural variation for many traits is present within the species Arabidopsis thaliana . This chapter describes the use of natural variation to elucidate genes underlying the regulation of quantitative traits. It deals with the development and use of mapping populations, the detection and handling of

  13. Exploiting natural variation in Arabidopsis

    NARCIS (Netherlands)

    J.A. Molenaar; J.J.B. Keurentjes

    2014-01-01

    Natural variation for many traits is present within the species Arabidopsis thaliana. This chapter describes the use of natural variation to elucidate genes underlying the regulation of quantitative traits. It deals with the development and use of mapping populations, the detection and handling of g

  14. The salty tale of Arabidopsis.

    Science.gov (United States)

    Sanders, D

    2000-06-29

    High concentrations of sodium chloride are toxic to most plant species. New insights into the mechanisms by which plants tolerate salt have emerged from the identification of genes in Arabidopsis thaliana that play a critical part in physiological resistance to salt.

  15. Arabidopsis ABCB21 is a facultative auxin importer/exporter regulated by cytoplasmic auxin concentration.

    Science.gov (United States)

    Kamimoto, Yoshihisa; Terasaka, Kazuyoshi; Hamamoto, Masafumi; Takanashi, Kojiro; Fukuda, Shoju; Shitan, Nobukazu; Sugiyama, Akifumi; Suzuki, Hideyuki; Shibata, Daisuke; Wang, Bangjun; Pollmann, Stephan; Geisler, Markus; Yazaki, Kazufumi

    2012-12-01

    The phytohormone auxin is critical for plant growth and many developmental processes. Members of the P-glycoprotein (PGP/ABCB) subfamily of ATP-binding cassette (ABC) transporters have been shown to function in the polar movement of auxin by transporting auxin over the plasma membrane in both monocots and dicots. Here, we characterize a new Arabidopsis member of the ABCB subfamily, ABCB21/PGP21, a close homolog of ABCB4, for which conflicting transport directionalities have been reported. ABCB21 is strongly expressed in the abaxial side of cotyledons and in junctions of lateral organs in the aerial part, whereas in roots it is specifically expressed in pericycle cells. Membrane fractionation by sucrose density gradient centrifugation followed by Western blot showed that ABCB21 is a plasma membrane-localized ABC transporter. A transport assay with Arabidopsis protoplasts suggested that ABCB21 was involved in IAA transport in an outward direction, while naphthalene acetic acid (NAA) was a less preferable substrate for ABCB21. Further functional analysis of ABCB21 using yeast import and export assays showed that ABCB21 mediates the 1-N-naphthylphthalamic acid (NPA)-sensitive translocation of auxin in an inward direction when the cytoplasmic IAA concentration is low, whereas this transporter mediates outward transport under high internal IAA. An increase in the cytoplasmic IAA concentration by pre-loading of IAA into yeast cells abolished the IAA uptake activity by ABCB21 as well as ABCB4. These findings suggest that ABCB21 functions as a facultative importer/exporter controlling auxin concentrations in plant cells.

  16. Structure and organ specificity of an anionic peroxidase from Arabidopsis thaliana cell suspension culture

    DEFF Research Database (Denmark)

    Ostergaard, L; Abelskov, A K; Mattsson, O

    1996-01-01

    The predominant peroxidase (pI 3.5) (E.C. 1.11.1.7) of an Arabidopsis thaliana cell suspension culture was purified and partially sequenced. Oligonucleotides were designed and a specific probe was obtained. A cDNA clone was isolated from an Arabidopsis cell suspension cDNA library and completely...... sequenced. The cDNA clone comprised 1194 bp and encodes a 30 residue signal peptide and a 305 residue mature protein (Mr 31,966). The sequence of the mature protein is 95% identical to the well-characterized horseradish peroxidase HRP A2 and has therefore been designated ATP A2. Three introns at positions...

  17. Soybean GmPHD-type transcription regulators improve stress tolerance in transgenic Arabidopsis plants.

    Directory of Open Access Journals (Sweden)

    Wei Wei

    Full Text Available BACKGROUND: Soybean [Glycine max (L. Merr.] is one of the most important crops for oil and protein resource. Improvement of stress tolerance will be beneficial for soybean seed production. PRINCIPAL FINDINGS: Six GmPHD genes encoding Alfin1-type PHD finger protein were identified and their expressions differentially responded to drought, salt, cold and ABA treatments. The six GmPHDs were nuclear proteins and showed ability to bind the cis-element "GTGGAG". The N-terminal domain of GmPHD played a major role in DNA binding. Using a protoplast assay system, we find that GmPHD1 to GmPHD5 had transcriptional suppression activity whereas GmPHD6 did not have. In yeast assay, the GmPHD6 can form homodimer and heterodimer with the other GmPHDs except GmPHD2. The N-terminal plus the variable regions but not the PHD-finger is required for the dimerization. Transgenic Arabidopsis plants overexpressing the GmPHD2 showed salt tolerance when compared with the wild type plants. This tolerance was likely achieved by diminishing the oxidative stress through regulation of downstream genes. SIGNIFICANCE: These results provide important clues for soybean stress tolerance through manipulation of PHD-type transcription regulator.

  18. β-amylase-like proteins function as transcription factors in Arabidopsis, controlling shoot growth and development.

    Science.gov (United States)

    Reinhold, Heike; Soyk, Sebastian; Simková, Klára; Hostettler, Carmen; Marafino, John; Mainiero, Samantha; Vaughan, Cara K; Monroe, Jonathan D; Zeeman, Samuel C

    2011-04-01

    Plants contain β-amylase-like proteins (BAMs; enzymes usually associated with starch breakdown) present in the nucleus rather than targeted to the chloroplast. They possess BRASSINAZOLE RESISTANT1 (BZR1)-type DNA binding domains--also found in transcription factors mediating brassinosteroid (BR) responses. The two Arabidopsis thaliana BZR1-BAM proteins (BAM7 and BAM8) bind a cis-regulatory element that both contains a G box and resembles a BR-responsive element. In protoplast transactivation assays, these BZR1-BAMs activate gene expression. Structural modeling suggests that the BAM domain's glucan binding cleft is intact, but the recombinant proteins are at least 1000 times less active than chloroplastic β-amylases. Deregulation of BZR1-BAMs (the bam7bam8 double mutant and BAM8-overexpressing plants) causes altered leaf growth and development. Of the genes upregulated in plants overexpressing BAM8 and downregulated in bam7bam8 plants, many carry the cis-regulatory element in their promoters. Many genes that respond to BRs are inversely regulated by BZR1-BAMs. We propose a role for BZR1-BAMs in controlling plant growth and development through crosstalk with BR signaling. Furthermore, we speculate that BZR1-BAMs may transmit metabolic signals by binding a ligand in their BAM domain, although diurnal changes in the concentration of maltose, a candidate ligand produced by chloroplastic β-amylases, do not influence their transcription factor function.

  19. Comparative expression profiling reveals gene functions in female meiosis and gametophyte development in Arabidopsis.

    Science.gov (United States)

    Zhao, Lihua; He, Jiangman; Cai, Hanyang; Lin, Haiyan; Li, Yanqiang; Liu, Renyi; Yang, Zhenbiao; Qin, Yuan

    2014-11-01

    Megasporogenesis is essential for female fertility, and requires the accomplishment of meiosis and the formation of functional megaspores. The inaccessibility and low abundance of female meiocytes make it particularly difficult to elucidate the molecular basis underlying megasporogenesis. We used high-throughput tag-sequencing analysis to identify genes expressed in female meiocytes (FMs) by comparing gene expression profiles from wild-type ovules undergoing megasporogenesis with those from the spl mutant ovules, which lack megasporogenesis. A total of 862 genes were identified as FMs, with levels that are consistently reduced in spl ovules in two biological replicates. Fluorescence-assisted cell sorting followed by RNA-seq analysis of DMC1:GFP-labeled female meiocytes confirmed that 90% of the FMs are indeed detected in the female meiocyte protoplast profiling. We performed reverse genetic analysis of 120 candidate genes and identified four FM genes with a function in female meiosis progression in Arabidopsis. We further revealed that KLU, a putative cytochrome P450 monooxygenase, is involved in chromosome pairing during female meiosis, most likely by affecting the normal expression pattern of DMC1 in ovules during female meiosis. Our studies provide valuable information for functional genomic analyses of plant germline development as well as insights into meiosis.

  20. Exploring the neutral invertase-oxidative stress defence connection in Arabidopsis thaliana.

    Science.gov (United States)

    Xiang, Li; Le Roy, Katrien; Bolouri-Moghaddam, Mohammad-Reza; Vanhaecke, Mieke; Lammens, Willem; Rolland, Filip; Van den Ende, Wim

    2011-07-01

    Over the past decades, considerable advances have been made in understanding the crucial role and the regulation of sucrose metabolism in plants. Among the various sucrose-catabolizing enzymes, alkaline/neutral invertases (A/N-Invs) have long remained poorly studied. However, recent findings have demonstrated the presence of A/N-Invs in various organelles in addition to the cytosol, and their importance for plant development and stress tolerance. A cytosolic (At-A/N-InvG, At1g35580) and a mitochondrial (At-A/N-InvA, At1g56560) member of the A/N-Invs have been analysed in more detail in Arabidopsis and it was found that At-A/N-InvA knockout plants show an even more severe growth phenotype than At-A/N-InvG knockout plants. The absence of either A/N-Inv was associated with higher oxidative stress defence gene expression, while transient overexpression of At-A/N-InvA and At-A/N-InvG in leaf mesophyll protoplasts down-regulated the oxidative stress-responsive ascorbate peroxidase 2 (APX2) promoter. Moreover, up-regulation of the APX2 promoter by hydrogen peroxide or abscisic acid could be blocked by adding metabolizable sugars or ascorbate. A hypothetical model is proposed in which both mitochondrial and cytosolic A/N-Invs can generate glucose as a substrate for mitochondria-associated hexokinase, contributing to mitochondrial reactive oxygen species homeostasis.

  1. Comparative transcriptomics of early meiosis in Arabidopsis and maize.

    Science.gov (United States)

    Dukowic-Schulze, Stefanie; Harris, Anthony; Li, Junhua; Sundararajan, Anitha; Mudge, Joann; Retzel, Ernest F; Pawlowski, Wojciech P; Chen, Changbin

    2014-03-20

    Though sexually reproductive plants share the same principle and most processes in meiosis, there are distinct features detectable. To address the similarities and differences of early meiosis transcriptomes from the dicot model system Arabidopsis and monocot model system maize, we performed comparative analyses of RNA-seq data of isolated meiocytes, anthers and seedlings from both species separately and via orthologous genes. Overall gene expression showed similarities, such as an increased number of reads mapping to unannotated features, and differences, such as the amount of differentially expressed genes. We detected major similarities and differences in functional annotations of genes up-regulated in meiocytes, which point to conserved features as well as unique features. Transcriptional regulation seems to be quite similar in Arabidopsis and maize, and we could reveal known and novel transcription factors and cis-regulatory elements acting in early meiosis. Taken together, meiosis between Arabidopsis and maize is conserved in many ways, but displays key distinctions that lie in the patterns of gene expression.

  2. 木薯原生质体培养的影响因子研究%Factors affecting Protoplast Culture of Cassava

    Institute of Scientific and Technical Information of China (English)

    付莉莉; 谭德冠; 韩冰莹; 孙雪飘; 张家明

    2014-01-01

    原生质体技术是细胞工程的核心部分,是进行作物改良和种质创新的重要方法。外植体来源及预处理、酶液组成和浓度、酶解时间、酶液渗透压、培养基组成、培养方法、培养密度等对原生质体培养均有很大影响。对影响木薯(Manihot esculenta Crantz.)原生质体培养再生植株的相关因素进行了综合分析,探讨了原生质体培养在植物育种上的应用潜力。%Protoplast technology, the core of cell engineering, is the important method of crop improvement and germplasm innovation. Source and pretreatment of explant, solution composition and concentration of enzyme, enzymatic duration,osmotic pressure of enzyme solution, composition of the medium, culture methods, culture density and other factors had a great in-fluence on protoplast culture. The factors affecting plant regeneration from protoplast of cassava (Manihot esculenta Crantz.) were analysized comprehensively. The potential of apply protoplast culture in plant breeding were discussed.

  3. Free-flow electrophoresis for fractionation of Arabidopsis thaliana membranes.

    Science.gov (United States)

    Bardy, N; Carrasco, A; Galaud, J P; Pont-Lezica, R; Canut, H

    1998-06-01

    Highly purified tonoplast and plasma membrane vesicles were isolated from microsomes of Arabidopsis thaliana by preparative free-flow electrophoresis. The most electronegative fractions were identified as tonoplast using nitrate-inhibited Mg2+-ATPase as enzyme marker. The least electronegative fractions were identified as plasma membrane using glucan-synthase II, UDPG: sterol-glucosyl-transferase, and vanadate-inhibited Mg2+-ATPase as enzyme markers. Other membrane markers, latent inosine-5'-diphosphatase (Golgi), NADPH-cytochrome-c reductase (endoplasmic reticulum) and cytochrome-c oxidase (mitochondria) were recovered in the fractions intermediate between tonoplast and plasma membrane. Immunoblot analysis of membrane fractions by antibodies directed against tonoplast and plasma membrane proteins confirmed the nature and the purity of the isolated membranes. The cytoskeletal protein actin, which was also identified by immunoblotting, was found to be specifically attached to the plasma membrane vesicles. The structural and functional integrity of the isolated membranes from Arabidopsis thaliana is discussed in the light of results obtained for the location of receptors and enzymes, or for the determination of ligand binding activity.

  4. Major latex protein-like protein 43 (MLP43) functions as a positive regulator during abscisic acid responses and confers drought tolerance in Arabidopsis thaliana.

    Science.gov (United States)

    Wang, Yanping; Yang, Li; Chen, Xi; Ye, Tiantian; Zhong, Bao; Liu, Ruijie; Wu, Yan; Chan, Zhulong

    2016-01-01

    Drought stress is one of the disadvantageous environmental conditions for plant growth and reproduction. Given the importance of abscisic acid (ABA) to plant growth and abiotic stress responses, identification of novel components involved in ABA signalling transduction is critical. In this study, we screened numerous Arabidopsis thaliana mutants by seed germination assay and identified a mutant mlp43 (major latex protein-like 43) with decreased ABA sensitivity in seed germination. The mlp43 mutant was sensitive to drought stress while the MLP43-overexpressed transgenic plants were drought tolerant. The tissue-specific expression pattern analysis showed that MLP43 was predominantly expressed in cotyledons, primary roots and apical meristems, and a subcellular localization study indicated that MLP43 was localized in the nucleus and cytoplasm. Physiological and biochemical analyses indicated that MLP43 functioned as a positive regulator in ABA- and drought-stress responses in Arabidopsis through regulating water loss efficiency, electrolyte leakage, ROS levels, and as well as ABA-responsive gene expression. Moreover, metabolite profiling analysis indicated that MLP43 could modulate the production of primary metabolites under drought stress conditions. Reconstitution of ABA signalling components in Arabidopsis protoplasts indicated that MLP43 was involved in ABA signalling transduction and acted upstream of SnRK2s by directly interacting with SnRK2.6 and ABF1 in a yeast two-hybrid assay. Moreover, ABA and drought stress down-regulated MLP43 expression as a negative feedback loop regulation to the performance of MLP43 in ABA and drought stress responses. Therefore, this study provided new insights for interpretation of physiological and molecular mechanisms of Arabidopsis MLP43 mediating ABA signalling transduction and drought stress responses.

  5. The quantitative basis of the Arabidopsis innate immune system to endemic pathogens depends on pathogen genetics

    DEFF Research Database (Denmark)

    Corwin, Jason A; Copeland, Daniel; Feusier, Julie

    2016-01-01

    the Arabidopsis-Botrytis pathosystem to explore the quantitative genetic architecture underlying host innate immune system in a population of Arabidopsis thaliana. By infecting a diverse panel of Arabidopsis accessions with four phenotypically and genotypically distinct isolates of the fungal necrotroph B...... of pathogen genetic variation in analyzing host quantitative resistance. While known resistance genes, such as receptor-like kinases (RLKs) and nucleotide-binding site leucine-rich repeat proteins (NLRs), were found to be enriched among associated genes, they only account for a small fraction of the total......, including defense hormone signaling and ROS production, as well as novel processes, such as leaf development. Validation of single gene T-DNA knockouts in a Col-0 background demonstrate a high success rate (60%) when accounting for differences in environmental and Botrytis genetic variation. This study...

  6. Reference: 710 [Arabidopsis Phenome Database[Archive

    Lifescience Database Archive (English)

    Full Text Available n factor family in Arabidopsis (Arabidopsis thaliana). Treatment with abscisic acid (ABA) induced AtMYB44 tr...anscript accumulation within 30 min. The gene was also activated under various abiotic stre...sses, such as dehydration, low temperature, and salinity. In transgenic Arabidopsis carrying an At...MYB44 promoter-driven beta-glucuronidase (GUS) construct, strong GUS activity was observed in the vasculature... and leaf epidermal guard cells. Transgenic Arabidopsis overexpressing AtMYB44 is more

  7. Ultrastructure of the endocytotic pathway in glutaraldehyde-fixed and high-pressure frozen/freeze-substituted protoplasts of white spruce (Picea glauca).

    Science.gov (United States)

    Galway, M E; Rennie, P J; Fowke, L C

    1993-11-01

    An ultrastructural study of endocytosis has been made for the first time in protoplasts of a gymnosperm, white spruce (Picea glauca), fixed by high-pressure freezing and freeze substitution. Protoplasts derived from the WS1 line of suspension-cultured embryogenic white spruce were labelled with cationized ferritin, a non-specific marker of the plasma membrane. The timing of cationized ferritin uptake and its subcellular distribution were determined by fixing protoplasts at various intervals after labelling. To address concerns about using chemical fixation to study the membrane-bound transport of cationized ferritin, protoplasts were fixed both by conventional glutaraldehyde fixation and by rapid freezing in a Balzers high-pressure freezing apparatus (followed by freeze substitution). Cationized ferritin appeared rapidly in coated pits and coated vesicles after labelling. Later it was present in uncoated vesicles, and in Golgi bodies, trans-Golgi membranes and partially coated reticula, then subsequently in multivesicular bodies, which may ultimately fuse with and deliver their contents to lytic vacuoles. The results show that the time course and pathway of cationized ferritin uptake in the gymnosperm white spruce is very similar to the time course and pathway elucidated for cationized ferritin uptake in the angiosperm soybean. High-pressure freezing yielded much better preservation of intracellular membranes and organelles, although plasma membranes appeared ruffled. Protoplasts fixed by both methods possessed numerous smooth vesicles in the cortex and smooth invaginations of the plasma membrane. These became labelled with cationized ferritin, but apparently did not contribute directly to the internalization of cationized ferritin, except via the formation of coated pits and vesicles from their surfaces.

  8. 榆钱菠菜脯氨酸转运蛋白基因的克隆及转基因拟南芥的耐盐性%Overexpression of Proline Transporter Gene Isolated from Halophyte Confers Salt Tolerance in Arabidopsis

    Institute of Scientific and Technical Information of China (English)

    沈义国; 张万科; 阎冬青; 杜保兴; 张劲松; 陈受宜

    2002-01-01

    脯氨酸是自然界中分布最广泛,作用最重要的渗透保护剂之一,同时又是高等植物中一类重要的碳源和氮源物质.为了解环境胁迫下脯氨酸的转运调节,从一个典型的盐生植物榆钱菠菜( Atriplex hortensis L.)中通过cDNA文库筛选和5′-RACE的方法获得了一个全长的cDNA (AhProT1),其编码蛋白与脯氨酸转运蛋白有60%~69%的同源性,含有11个跨膜结构域.聚类分析表明,微生物和高等植物的脯氨酸转运蛋白同源程度相对高于哺乳动物.为进一步分析脯氨酸转运蛋白在植物中的功能,将AhProT1置于35S启动子下转入拟南芥(Arabidopsis thaliana).通过同位素示踪法发现, 与对照植物相比, 转基因植物在根中积累更多的脯氨酸;在一系列不同浓度的盐胁迫试验中,转基因植株最高可耐受200 mmol/L NaCl,并可持续生长,而对照植株在150 mmol/L NaCl下即已死亡.%Proline is one of the most important and widespread osmolyte which functions in adaptation to adverse environmental stresses in many organisms. Also it is an important carbon and nitrogen resource in higher plants. Metabolism of proline has been elucidated in many plant species. However, transport of proline was poorly characterized although transport system plays an important role in proline distribution in different tissues. We isolated one full-length cDNA encoding proline transporter from the typical halophyte: Atriplex hortensis L. through cDNA library screening and 5′-RACE. The deduced amino acid sequence had eleven transmembrane domains, showed 60%-69% similarities to other ProTs and the gene was designated AhProT1. In the phylogenetic tree, higher plants' ProTs, e.g. AhProT1, showed more similar to ProP from microorganisms than ProT from mammalians. AhProT1 gene was transformed into Arabidopsis thaliana under 35S promoter. In MS medium containing [U-14C] proline, AhProT1+ plants were able to accumulate much more radiolabeled proline in the

  9. Transformation of haploid Datura innoxia protoplasts and analysis of the plasmid integration pattern in regenerated transgenic plants.

    Science.gov (United States)

    Schmidt-Rogge, T; Meixner, M; Srivastava, V; Guha-Mukherjee, S; Schieder, O

    1993-05-01

    We developed a highly efficient transformation protocol for the PEG-mediated direct transfer of plasmid DNA into protoplasts of haploid Datura innoxia. Vectors harbouring a neomycin phosphotransferase II gene or a hygromycin B phosphotransferase gene under the control of different promoters were used in the transformation experiments. Various amounts of plasmid DNA were applied without any carrier DNA to show the direct influence of the plasmid DNA concentration on the transformation efficiency. Approximately 95% of the selected calli were regenerated to plants; 20% of them remained haploid. Total DNA of different transgenic plants was analysed with regard to the integration pattern of the plasmid DNA. Plants carrying only one or two copies of the vector DNA were observed as well as individuals with multi-copy integration (up to ten or more copies).

  10. Rice planthopper resistance of interspecific protoplast fusin line "pf9279" between O. sative and O.officinalis

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    @@ An interspecific hybrid line pf9279 was obtained by protoplast fusion between 02428(japonica, with a wide compatibility gene) and CNW240 (O. officinalis, from Malaysia) at CNRRI in 1992. Possible introgression of planthopper resistance from O.officinalis into pf9279 was investigated by field and laboratory experiments during 1998-1999 at CNRRI. Thirty-day-old seedlings of pf9279 and other rice varieties were individually transplanted with a spacing of 18× 24 cm in each plot (ca 7× 20 m) on Jun 15, 1999. Population trends of brown planthopper(BPH), Nilaparvata lugens, and whitebacked planthopper(WBPH),Sogatella furcifera were examined weekly by visual counting of adult females on 50-100 hills for each variety.

  11. Growth of Avena Coleoptiles and pH Drop of Protoplast Suspensions Induced by Chlorinated Indoleacetic Acids

    DEFF Research Database (Denmark)

    Engvild, Kjeld Christensen; Doll, Hans; Böttger, M.

    1978-01-01

    -auxins. Some of the derivatives were compared for their effect on pH decline in stem protoplast suspensions of Helianthus annuus L. and Pisum sativum L. The change of pH occurs without a lag period or with only a very short one. Derivatives which are very active in the Avena straight growth assay cause......Several indoleacetic acids, substituted in the benzene ring, were compared in the Avena straight growth bioassay. 4-Chloroindoleacetic acid, a naturally occurring plant hormone, is one of the strongest hormones in this bioassay. With an optimum at 10-6 mol l-1, it is more active than indoleacetic...... acid, 2,4-dichlorphenoxyacetic acid and naphthaleneacetic acid. 5-Chloro- and 6-chloroindoleacetic acids are very strong auxins as well. Other derivatives tested have a lower activity. 5,7-Dichloro- and 5-hydroxyindoleacetic acids have very low auxin activity at 10-4 mol l-1 and may be anti...

  12. [Construction and analysis of transgenic plants of Nicotiana tabacum L. expressing a bacterial gene for beta-1,3-glucanase. I. Construction of vector plasmids for transfer into plants and expression of a modified gene for beta-1,3-glucanase from Clostridium thermocellum in tobacco protoplasts].

    Science.gov (United States)

    Darbinian, N S; Popov, Iu G; Mochul'skiĭ, A V; Volkova, L V; Piruzian, E S; Vasilevko, V T

    1996-02-01

    We constructed two vectors, pC27-glc and pC29-glc, that allow expression of the beta-1,3-glucanase gene (glc) in plant cells. The glc gene was previously cloned from anaerobic thermophilous bacterium Clostridium thermocellum. To increase the efficiency of expression, the N-terminal fragment of the glc gene encoding bacterial transient peptide was deleted, and hybrid variants of lacZ-glc were obtained. Analysis of expression of the hybrid genes in Escherichia coli showed that deletion of the fragment corresponding to 31 amino acids (a.a.) of beta-glucanase affected neither activity nor thermostability of the enzyme. The modified gene was subcloned into two vectors, pC27 and pC29, in which its expression was controlled by the TR2' promoter of the 2' gene of T-DNA and the rbcS promoter from Arabidopsis, respectively. Each of the resulting plasmids, pC27-glc and pC29-glc, was transfected into protoplasts of Nicotiana plumbaginifolia. Both the plasmids were shown to allow a high level of activity of the thermostable beta-1,3-glucanase. We plan to use the vectors obtained for transformation of agrobacteria and construction of transgenic plants.

  13. An International Bioinformatics Infrastructure to Underpin the Arabidopsis Community

    Science.gov (United States)

    The future bioinformatics needs of the Arabidopsis community as well as those of other scientific communities that depend on Arabidopsis resources were discussed at a pair of recent meetings held by the Multinational Arabidopsis Steering Committee (MASC) and the North American Arabidopsis Steering C...

  14. Arabidopsis CDS blastp result: AK240652 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK240652 J023098G11 At5g63090.2 68418.m07919 LOB domain protein / lateral organ boundaries... protein (LOB) identical to LOBa [Arabidopsis thaliana] GI:17484100, SP|Q9FML4 LATERAL ORGAN BOUNDARIES protein {Arabidopsis thaliana} 1e-13 ...

  15. Arabidopsis CDS blastp result: AK241761 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK241761 J065205C18 At5g63090.1 68418.m07918 LOB domain protein / lateral organ boundaries... protein (LOB) identical to LOBa [Arabidopsis thaliana] GI:17484100, SP|Q9FML4 LATERAL ORGAN BOUNDARIES protein {Arabidopsis thaliana} 5e-32 ...

  16. Arabidopsis CDS blastp result: AK240652 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK240652 J023098G11 At5g63090.1 68418.m07918 LOB domain protein / lateral organ boundaries... protein (LOB) identical to LOBa [Arabidopsis thaliana] GI:17484100, SP|Q9FML4 LATERAL ORGAN BOUNDARIES protein {Arabidopsis thaliana} 1e-13 ...

  17. Arabidopsis CDS blastp result: AK240652 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK240652 J023098G11 At5g63090.4 68418.m07921 LOB domain protein / lateral organ boundaries... protein (LOB) identical to LOBa [Arabidopsis thaliana] GI:17484100, SP|Q9FML4 LATERAL ORGAN BOUNDARIES protein {Arabidopsis thaliana} 1e-13 ...

  18. Arabidopsis CDS blastp result: AK241761 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK241761 J065205C18 At5g63090.3 68418.m07920 LOB domain protein / lateral organ boundaries... protein (LOB) identical to LOBa [Arabidopsis thaliana] GI:17484100, SP|Q9FML4 LATERAL ORGAN BOUNDARIES protein {Arabidopsis thaliana} 5e-32 ...

  19. Arabidopsis CDS blastp result: AK241761 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK241761 J065205C18 At5g63090.2 68418.m07919 LOB domain protein / lateral organ boundaries... protein (LOB) identical to LOBa [Arabidopsis thaliana] GI:17484100, SP|Q9FML4 LATERAL ORGAN BOUNDARIES protein {Arabidopsis thaliana} 5e-32 ...

  20. Arabidopsis CDS blastp result: AK241761 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK241761 J065205C18 At5g63090.4 68418.m07921 LOB domain protein / lateral organ boundaries... protein (LOB) identical to LOBa [Arabidopsis thaliana] GI:17484100, SP|Q9FML4 LATERAL ORGAN BOUNDARIES protein {Arabidopsis thaliana} 5e-32 ...

  1. Arabidopsis CDS blastp result: AK240652 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK240652 J023098G11 At5g63090.3 68418.m07920 LOB domain protein / lateral organ boundaries... protein (LOB) identical to LOBa [Arabidopsis thaliana] GI:17484100, SP|Q9FML4 LATERAL ORGAN BOUNDARIES protein {Arabidopsis thaliana} 1e-13 ...

  2. Arabidopsis CDS blastp result: AK105527 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK105527 001-127-G05 At5g63090.4 LOB domain protein / lateral organ boundaries prot...ein (LOB) identical to LOBa [Arabidopsis thaliana] GI:17484100, SP|Q9FML4 LATERAL ORGAN BOUNDARIES protein {Arabidopsis thaliana} 3e-52 ...

  3. Using "Arabidopsis" Genetic Sequences to Teach Bioinformatics

    Science.gov (United States)

    Zhang, Xiaorong

    2009-01-01

    This article describes a new approach to teaching bioinformatics using "Arabidopsis" genetic sequences. Several open-ended and inquiry-based laboratory exercises have been designed to help students grasp key concepts and gain practical skills in bioinformatics, using "Arabidopsis" leucine-rich repeat receptor-like kinase (LRR…

  4. Arabidopsis CDS blastp result: AK240730 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK240730 J043030K09 At2g32440.1 68415.m03963 ent-kaurenoic acid hydroxylase, putati...ve / cytochrome P450, putative identical to ent-kaurenoic acid hydroxylase / cytochrome P450 CYP88A (GI:1302...1856) [Arabidopsis thaliana]; similar to ent-kaurenoic acid hydroxylase [Arabidopsis thaliana] GI:13021853 2e-11 ...

  5. Arabidopsis CDS blastp result: AK288052 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK288052 J075151I09 At2g32440.1 68415.m03963 ent-kaurenoic acid hydroxylase, putati...ve / cytochrome P450, putative identical to ent-kaurenoic acid hydroxylase / cytochrome P450 CYP88A (GI:1302...1856) [Arabidopsis thaliana]; similar to ent-kaurenoic acid hydroxylase [Arabidopsis thaliana] GI:13021853 6e-14 ...

  6. Arabidopsis CDS blastp result: AK240911 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK240911 J065037E05 At2g32440.1 68415.m03963 ent-kaurenoic acid hydroxylase, putati...ve / cytochrome P450, putative identical to ent-kaurenoic acid hydroxylase / cytochrome P450 CYP88A (GI:1302...1856) [Arabidopsis thaliana]; similar to ent-kaurenoic acid hydroxylase [Arabidopsis thaliana] GI:13021853 4e-22 ...

  7. Arabidopsis CDS blastp result: AK241119 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK241119 J065094C22 At2g32440.1 68415.m03963 ent-kaurenoic acid hydroxylase, putati...ve / cytochrome P450, putative identical to ent-kaurenoic acid hydroxylase / cytochrome P450 CYP88A (GI:1302...1856) [Arabidopsis thaliana]; similar to ent-kaurenoic acid hydroxylase [Arabidopsis thaliana] GI:13021853 2e-13 ...

  8. Arabidopsis CDS blastp result: AK243149 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK243149 J100032I21 At2g32440.1 68415.m03963 ent-kaurenoic acid hydroxylase, putati...ve / cytochrome P450, putative identical to ent-kaurenoic acid hydroxylase / cytochrome P450 CYP88A (GI:1302...1856) [Arabidopsis thaliana]; similar to ent-kaurenoic acid hydroxylase [Arabidopsis thaliana] GI:13021853 7e-12 ...

  9. Arabidopsis CDS blastp result: AK241581 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK241581 J065181K09 At2g32440.1 68415.m03963 ent-kaurenoic acid hydroxylase, putati...ve / cytochrome P450, putative identical to ent-kaurenoic acid hydroxylase / cytochrome P450 CYP88A (GI:1302...1856) [Arabidopsis thaliana]; similar to ent-kaurenoic acid hydroxylase [Arabidopsis thaliana] GI:13021853 4e-15 ...

  10. Arabidopsis CDS blastp result: AK287479 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK287479 J043023O14 At2g32440.1 68415.m03963 ent-kaurenoic acid hydroxylase, putati...ve / cytochrome P450, putative identical to ent-kaurenoic acid hydroxylase / cytochrome P450 CYP88A (GI:1302...1856) [Arabidopsis thaliana]; similar to ent-kaurenoic acid hydroxylase [Arabidopsis thaliana] GI:13021853 1e-17 ...

  11. Reference: 631 [Arabidopsis Phenome Database[Archive

    Lifescience Database Archive (English)

    Full Text Available ggest that atRZ-1a has a negative impact on seed germination and seedling growth of Arabidopsis under salt o...binding protein, atRZ-1a, has a negative impact on seed germination and seedling growth of Arabidopsis thali

  12. Interspecific and interploidal gene flow in Central European Arabidopsis (Brassicaceae

    Directory of Open Access Journals (Sweden)

    Jørgensen Marte H

    2011-11-01

    Full Text Available Abstract Background Effects of polyploidisation on gene flow between natural populations are little known. Central European diploid and tetraploid populations of Arabidopsis arenosa and A. lyrata are here used to study interspecific and interploidal gene flow, using a combination of nuclear and plastid markers. Results Ploidal levels were confirmed by flow cytometry. Network analyses clearly separated diploids according to species. Tetraploids and diploids were highly intermingled within species, and some tetraploids intermingled with the other species, as well. Isolation with migration analyses suggested interspecific introgression from tetraploid A. arenosa to tetraploid A. lyrata and vice versa, and some interploidal gene flow, which was unidirectional from diploid to tetraploid in A. arenosa and bidirectional in A. lyrata. Conclusions Interspecific genetic isolation at diploid level combined with introgression at tetraploid level indicates that polyploidy may buffer against negative consequences of interspecific hybridisation. The role of introgression in polyploid systems may, however, differ between plant species, and even within the small genus Arabidopsis, we find very different evolutionary fates when it comes to introgression.

  13. The pattern of polymorphism in Arabidopsis thaliana.

    Directory of Open Access Journals (Sweden)

    2005-07-01

    Full Text Available We resequenced 876 short fragments in a sample of 96 individuals of Arabidopsis thaliana that included stock center accessions as well as a hierarchical sample from natural populations. Although A. thaliana is a selfing weed, the pattern of polymorphism in general agrees with what is expected for a widely distributed, sexually reproducing species. Linkage disequilibrium decays rapidly, within 50 kb. Variation is shared worldwide, although population structure and isolation by distance are evident. The data fail to fit standard neutral models in several ways. There is a genome-wide excess of rare alleles, at least partially due to selection. There is too much variation between genomic regions in the level of polymorphism. The local level of polymorphism is negatively correlated with gene density and positively correlated with segmental duplications. Because the data do not fit theoretical null distributions, attempts to infer natural selection from polymorphism data will require genome-wide surveys of polymorphism in order to identify anomalous regions. Despite this, our data support the utility of A. thaliana as a model for evolutionary functional genomics.

  14. Integration of bioinformatics and synthetic promoters leads to the discovery of novel elicitor-responsive cis-regulatory sequences in Arabidopsis.

    Science.gov (United States)

    Koschmann, Jeannette; Machens, Fabian; Becker, Marlies; Niemeyer, Julia; Schulze, Jutta; Bülow, Lorenz; Stahl, Dietmar J; Hehl, Reinhard

    2012-09-01

    A combination of bioinformatic tools, high-throughput gene expression profiles, and the use of synthetic promoters is a powerful approach to discover and evaluate novel cis-sequences in response to specific stimuli. With Arabidopsis (Arabidopsis thaliana) microarray data annotated to the PathoPlant database, 732 different queries with a focus on fungal and oomycete pathogens were performed, leading to 510 up-regulated gene groups. Using the binding site estimation suite of tools, BEST, 407 conserved sequence motifs were identified in promoter regions of these coregulated gene sets. Motif similarities were determined with STAMP, classifying the 407 sequence motifs into 37 families. A comparative analysis of these 37 families with the AthaMap, PLACE, and AGRIS databases revealed similarities to known cis-elements but also led to the discovery of cis-sequences not yet implicated in pathogen response. Using a parsley (Petroselinum crispum) protoplast system and a modified reporter gene vector with an internal transformation control, 25 elicitor-responsive cis-sequences from 10 different motif families were identified. Many of the elicitor-responsive cis-sequences also drive reporter gene expression in an Agrobacterium tumefaciens infection assay in Nicotiana benthamiana. This work significantly increases the number of known elicitor-responsive cis-sequences and demonstrates the successful integration of a diverse set of bioinformatic resources combined with synthetic promoter analysis for data mining and functional screening in plant-pathogen interaction.

  15. Regeneração de plantas após fusão de protoplastos de tangelo 'Page' e toranja 'Lau Tau' Plant regeneration after protoplast fusion of 'Page' tangelo and 'Lau Tau' pummelo

    Directory of Open Access Journals (Sweden)

    Dayse Cristina de Carvalho

    2007-08-01

    Full Text Available Buscou-se a hibridação somática entre tangelo 'Page' e toranja 'Lau Tau' visando à produção de porta-enxerto semelhante à laranja-azeda, por esta espécie ser considerada um provável híbrido entre C. reticulata e C. grandis. Após isolamento, fusão e cultivo de protoplastos, obtiveram-se brotações que foram enxertadas in vitro, em laranja 'Hamlin'. Dezessete plantas foram aclimatizadas em casa de vegetação. A análise de citometria de fluxo confirmou a constituição diplóide dessas plantas. Marcadores moleculares RAPD das plantas regeneradas apresentaram padrão de bandas similar ao de tangelo 'Page'. Entretanto, todas as plantas apresentaram conformação fenotípica diferente dos genitores.This work aimed to produce somatic hybrid between 'Page' tangelo and 'Lau Tau' pummelo in an attempt to regenerate a similar rootstock to sour orange, because this species is considered a probable hybrid between C. reticulata and C. grandis. After protoplast isolation, fusion and culture regenerated shoots were in vitro grafted on 'Hamlin' sweet orange. Seventeen plants were acclimatized in a greenhouse. Citometric flow analyses revealed that all plants are diploid. RAPD molecular markers of regenerated plants had the same pattern as compared to 'Page' tangelo. However, all plants had phenotypic traits different from both genitors.

  16. Adenosine-5'-phosphosulfate kinase is essential for Arabidopsis viability.

    Science.gov (United States)

    Mugford, Sarah G; Matthewman, Colette A; Hill, Lionel; Kopriva, Stanislav

    2010-01-04

    In Arabidopsis thaliana, adenosine-5'-phosphosulfate kinase (APK) provides activated sulfate for sulfation of secondary metabolites, including the glucosinolates. We have successfully isolated three of the four possible triple homozygous mutant combinations of this family. The APK1 isoform alone was sufficient to maintain WT levels of growth and development. Analysis of apk1 apk2 apk3 and apk1 apk3 apk4 mutants suggests that APK3 and APK4 are functionally redundant, despite being located in cytosol and plastids, respectively. We were, however, unable to isolate apk1 apk3 apk4 mutants, most probably because the apk1 apk3 apk4 triple mutant combination is pollen lethal. Therefore, we conclude that APS kinase is essential for plant reproduction and viability.

  17. Selection of Arabidopsis mutants overexpressing genes driven by the promoter of an auxin-inducible glutathione S-transferase gene

    NARCIS (Netherlands)

    Kop, D.A.M. van der; Schuyer, M.; Pinas, J.E.; Zaal, B.J. van der; Hooykaas, P.J.J.

    1999-01-01

    Transgenic arabidopsis plants were isolated that contained a T-DNA construct in which the promoter of an auxin-inducible glutathione S-transferase (GST) gene from tobacco was fused to the kanamycin resistance (nptII) as well as to the β-glucuronidase (gusA) reporter gene. Subsequently, seeds were tr

  18. Sulfinylated azadecalins act as functional mimics of a pollen germination stimulant in Arabidopsis pistils.

    Science.gov (United States)

    Qin, Yuan; Wysocki, Ronald J; Somogyi, Arpad; Feinstein, Yelena; Franco, Jessica Y; Tsukamoto, Tatsuya; Dunatunga, Damayanthi; Levy, Clara; Smith, Steven; Simpson, Robert; Gang, David; Johnson, Mark A; Palanivelu, Ravishankar

    2011-12-01

    Polarized cell elongation is triggered by small molecule cues during development of diverse organisms. During plant reproduction, pollen interactions with the stigma result in the polar outgrowth of a pollen tube, which delivers sperm cells to the female gametophyte to effect double fertilization. In many plants, pistils stimulate pollen germination. However, in Arabidopsis, the effect of pistils on pollen germination and the pistil factors that stimulate pollen germination remain poorly characterized. Here, we demonstrate that stigma, style, and ovules in Arabidopsis pistils stimulate pollen germination. We isolated an Arabidopsis pistil extract fraction that stimulates Arabidopsis pollen germination, and employed ultra-high resolution electrospray ionization (ESI), Fourier-transform ion cyclotron resonance (FT-ICR) and MS/MS techniques to accurately determine the mass (202.126 Da) of a compound that is specifically present in this pistil extract fraction. Using the molecular formula (C10H19NOS) and tandem mass spectral fragmentation patterns of the m/z (mass to charge ratio) 202.126 ion, we postulated chemical structures, devised protocols, synthesized N-methanesulfinyl 1- and 2-azadecalins that are close structural mimics of the m/z 202.126 ion, and showed that they are sufficient to stimulate Arabidopsis pollen germination in vitro (30 μm stimulated approximately 50% germination) and elicit accession-specific response. Although N-methanesulfinyl 2-azadecalin stimulated pollen germination in three species of Lineage I of Brassicaceae, it did not induce a germination response in Sisymbrium irio (Lineage II of Brassicaceae) and tobacco, indicating that activity of the compound is not random. Our results show that Arabidopsis pistils promote germination by producing azadecalin-like molecules to ensure rapid fertilization by the appropriate pollen.

  19. Jasmonate Signal Pathway in Arabidopsis

    Institute of Scientific and Technical Information of China (English)

    Xiao-Yi Shan; Zhi-Long Wang; Daoxin Xie

    2007-01-01

    Jasmonates (JAs), which include jasmonic acid and its cyclopentane derivatives are synthesized from the octadecanoid pathway and widely distributed throughout the plant kingdom. JAs modulate the expression of numerous genes and mediate responses to stress, wounding, insect attack, pathogen infection, and UV damage. They also affect a variety of processes in many plant developmental processes. The JA signal pathway involves two important events: the biosynthesis of JA and the transduction of JA signal. Several important Arabidopsis mutants in jasmonate signal pathway were described in this review.

  20. Preparation Methods for Protoplast during Breeding of Streptomyces avermitilis%阿维链霉菌原生质体制备方法的研究

    Institute of Scientific and Technical Information of China (English)

    张毅

    2012-01-01

    This research aimed to study the preparation for protoplast of Streptomyces auermitilis. The results showed that the optimum conditions for preparation of protoplast are as follows: at first, inoculation of spore suspending liquid to YEME culture medium which containing 35 mL of 0.5% glycin, and then cultured at 28t with 200 r/min for 40 h; then, collecting mycelia with 3 000 r/min, and then the collected mycelia were washed by 10.3% sucrose solution for two times; finally, the cleaned mycelia were suspended in P buffer with 4 mg/mL of lysozyme at 30X. For 50 min to finish the preparation of protoplast. When 50% PEG 1 000 was used as fusion promoting agent, the fusion rate of protoplasts was the highest.%对阿维链霉菌原生质体制备进行了研究,结果表明:阿维链霉菌原生质体制备的最佳条件为在35 mL含0.5%甘氨酸的YEME培养基中接种孢子悬液,28℃、200 r/min培养40h;3 000 r/min收集菌丝体,用10.3%蔗糖溶液清洗2次;溶菌酶浓度为4mg/mL的P缓冲液悬浮菌丝体,30℃下处理50 min完成原生质体制备.以50% PEG1000为促融剂,原生质体融合率最高.

  1. Monoclonal antibodies directed against protoplasts of soybean cells: analysis of the lateral mobility of plasma membrane-bound antibody MVS-1.

    Science.gov (United States)

    Metcalf, T N; Villanueva, M A; Schindler, M; Wang, J L

    1986-04-01

    A monoclonal antibody (MVS-1) was used to monitor the lateral mobility of a defined component (Mr approximately 400,000) of the plasma membrane of soybean protoplasts prepared from suspension cultures of Glycine max (SB-1 cell line). The diffusion coefficient (D) of antibody MVS-1 bound to its target was determined (D = 3.2 X 10(-10) cm2/s) by fluorescence redistribution after photobleaching. Pretreatment of the protoplasts with soybean agglutinin (SBA) resulted in a 10-fold reduction of the lateral mobility of antibody MVS-1 (D = 4.1 X 10(-11) cm2/s). This lectin-induced modulation could be partially reversed by prior treatment of the protoplasts with either colchicine or cytochalasin B. When used together, these drugs completely reversed the modulation effect induced by SBA. These results have refined our previous analysis of the effect of SBA on receptor mobility to the level of a defined receptor and suggest that the binding of SBA to the plasma membrane results in alterations in the plasma membrane such that the lateral diffusion of other receptors is restricted. These effects are most likely mediated by the cytoskeletal components of the plant cell.

  2. The nucleocytoplasmic microfilament network in protoplasts from cultured soybean cells is a plastic entity that pervades the cytoplasm except the central vacuole.

    Science.gov (United States)

    Villanueva, Marco A; Schindler, Melvin; Wang, John L

    2005-11-01

    The microfilament network of cultured Glycine max cells (SB-1 line), and protoplasts was visualized with rhodamine-phalloidin under conditions that lysed the protoplast and changed the cell shape. The whole cell had the typical microfilament distribution of a "cage" around the nucleus, from which the large subcortical cables and transvacuolar strands radiated towards the cortex until it reached the cortical microfilament network. Upon cell wall removal, the network conserved its compartmentalization. Thus, the redistribution of the shape where the vacuole becomes a central entity, made the cytoplasm displace peripherally, but the network distribution was conserved. When protoplasts were lysed in a low osmotic medium, the vacuoles were gradually released intact. Under these conditions, the F-actin staining remained within the ghost of the cell, but none was detected in either emerging or almost completely released vacuoles. Most of the released F-actin was found in debris from the cell lysate in the form of microfilaments. When the ghosts were constrained in a coverslip with an air bubble, the shape of the ghost changed accordingly, but the microfilament network distribution remained constant. These results provide further evidence that the vacuole of plant cells does not have detectable associated F-actin. In addition, we demonstrate that the actin microfilament network is a moldable entity that can change its shape but keeps its distribution under constant conditions, in these cultured cells.

  3. In Vitro Synthesized RNA Generated from cDNA Clones of Both Genomic Components of Cucurbit yellow stunting disorder virus Replicates in Cucumber Protoplasts

    Directory of Open Access Journals (Sweden)

    Carolyn A. Owen

    2016-06-01

    Full Text Available Cucurbit yellow stunting disorder virus (CYSDV, a bipartite whitefly-transmitted virus, constitutes a major threat to commercial cucurbit production worldwide. Here, construction of full-length CYSDV RNA1 and RNA2 cDNA clones allowed the in vitro synthesis of RNA transcripts able to replicate in cucumber protoplasts. CYSDV RNA1 proved competent for replication; transcription of both polarities of the genomic RNA was detectable 24 h post inoculation. Hybridization of total RNA extracted from transfected protoplasts or from naturally CYSDV-infected cucurbits revealed high-level transcription of the p22 subgenomic RNA species. Replication of CYSDV RNA2 following co-transfection with RNA1 was also observed, with similar transcription kinetics. A CYSDV RNA2 cDNA clone (T3CM8Δ comprising the 5′- and 3′-UTRs plus the 3′-terminal gene, generated a 2.8 kb RNA able to replicate to high levels in protoplasts in the presence of CYSDV RNA1. The clone T3CM8Δ will facilitate reverse genetics studies of CYSDV gene function and RNA replication determinants.

  4. In Vitro Synthesized RNA Generated from cDNA Clones of Both Genomic Components of Cucurbit yellow stunting disorder virus Replicates in Cucumber Protoplasts

    Science.gov (United States)

    Owen, Carolyn A.; Moukarzel, Romy; Huang, Xiao; Kassem, Mona A.; Eliasco, Eleonora; Aranda, Miguel A.; Coutts, Robert H. A.; Livieratos, Ioannis C.

    2016-01-01

    Cucurbit yellow stunting disorder virus (CYSDV), a bipartite whitefly-transmitted virus, constitutes a major threat to commercial cucurbit production worldwide. Here, construction of full-length CYSDV RNA1 and RNA2 cDNA clones allowed the in vitro synthesis of RNA transcripts able to replicate in cucumber protoplasts. CYSDV RNA1 proved competent for replication; transcription of both polarities of the genomic RNA was detectable 24 h post inoculation. Hybridization of total RNA extracted from transfected protoplasts or from naturally CYSDV-infected cucurbits revealed high-level transcription of the p22 subgenomic RNA species. Replication of CYSDV RNA2 following co-transfection with RNA1 was also observed, with similar transcription kinetics. A CYSDV RNA2 cDNA clone (T3CM8Δ) comprising the 5′- and 3′-UTRs plus the 3′-terminal gene, generated a 2.8 kb RNA able to replicate to high levels in protoplasts in the presence of CYSDV RNA1. The clone T3CM8Δ will facilitate reverse genetics studies of CYSDV gene function and RNA replication determinants. PMID:27314380

  5. Reference: 572 [Arabidopsis Phenome Database[Archive

    Lifescience Database Archive (English)

    Full Text Available et al. 2007 May. Plant J. 50(3):439-51. Although glycine-rich RNA-binding protein 2 (GRP2) has been implicated in plant re...sponses to environmental stresses, the function and importance of GRP2 in stress responses are largely unknown. Here...haliana under high-salinity, cold or osmotic stress. GRP2 affects seed germination of Arabidopsis plants under salt stre...ss, but does not influence seed germination and seedling growth of Arabidopsis plants under osmotic stre...ss. GRP2 accelerates seed germination and seedling growth in Arabidopsis plants under cold stre

  6. Reference: 446 [Arabidopsis Phenome Database[Archive

    Lifescience Database Archive (English)

    Full Text Available rk E et al. 2006 Nov. Plant Physiol. 142(3):1004-13. Arabidopsis (Arabidopsis thaliana) QUARTET (QRT) genes are require...d for pollen separation during normal floral development. In qrt mutants, the four products of microsporogenesis re...main fused and pollen grains are released as tetrads. In Arabid...opsis, tetrad analysis in qrt mutants has been used to map all five centromeres, easily distinguish sporophy...tic from gametophytic mutations, and accurately assess crossover interference. Using a combination of forward and re

  7. Variation in the Subcellular Localization and Protein Folding Activity among Arabidopsis thaliana Homologs of Protein Disulfide Isomerase

    Directory of Open Access Journals (Sweden)

    Christen Y. L. Yuen

    2013-10-01

    Full Text Available Protein disulfide isomerases (PDIs catalyze the formation, breakage, and rearrangement of disulfide bonds to properly fold nascent polypeptides within the endoplasmic reticulum (ER. Classical animal and yeast PDIs possess two catalytic thioredoxin-like domains (a, a′ and two non-catalytic domains (b, b′, in the order a-b-b′-a′. The model plant, Arabidopsis thaliana, encodes 12 PDI-like proteins, six of which possess the classical PDI domain arrangement (AtPDI1 through AtPDI6. Three additional AtPDIs (AtPDI9, AtPDI10, AtPDI11 possess two thioredoxin domains, but without intervening b-b′ domains. C-terminal green fluorescent protein (GFP fusions to each of the nine dual-thioredoxin PDI homologs localized predominantly to the ER lumen when transiently expressed in protoplasts. Additionally, expression of AtPDI9:GFP-KDEL and AtPDI10: GFP-KDDL was associated with the formation of ER bodies. AtPDI9, AtPDI10, and AtPDI11 mediated the oxidative folding of alkaline phosphatase when heterologously expressed in the Escherichia coli protein folding mutant, dsbA−. However, only three classical AtPDIs (AtPDI2, AtPDI5, AtPDI6 functionally complemented dsbA−. Interestingly, chemical inducers of the ER unfolded protein response were previously shown to upregulate most of the AtPDIs that complemented dsbA−. The results indicate that Arabidopsis PDIs differ in their localization and protein folding activities to fulfill distinct molecular functions in the ER.

  8. Reference: 411 [Arabidopsis Phenome Database[Archive

    Lifescience Database Archive (English)

    Full Text Available id permease (AAP) subfamily genes are preferentially expressed in the vascular tissue, suggesting roles in l...ter LYSINE HISTIDINE TRANSPORTER1 (LHT1), an AAP homolog, is expressed in both th...en sources because of the severe inhibition of amino acid uptake from the medium, and uptake of amino acids ...into mesophyll protoplasts is inhibited. Interestingly, lht1 mutants, which show ...growth defects on fertilized soil, can be rescued when LHT1 is reexpressed in green tissue. These findings are

  9. Localization and secretory pathways of a 58K-like protein in multi-vesicular bodies in callus of Arabidopsis thaliana

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    Multi-vesicular bodies in endocytosis and protoplasts are special cellular structures that are consid-ered to be originated from invagination of plasma membranes. However, the genesis and function of multi-vesicular bodies, the relationship with Golgi bodies and cell walls, and their secretory pathways remain controversial and ambiguous. Using a monoclonal antibody against an animal 58K protein, we have detected, by Western blotting and confocal microscopy, that a 58K-like protein is present in the calli of Arabidopsis thaliana and Hypericum perforatum. The results of immuno-electron microscopy showed that the 58K-like protein was located in the cisternae of Golgi bodies, secretory vesicles, multi-vesicular bodies, cell walls and vacuoles in callus of Arabidopsis thaliana, suggesting that the multi-vesicular bodies may be originated from Golgi bodies and function as a transporter carrying substances synthesized in Golgi bodies to cell walls and vacuoles. It seems that multi-vesicular bodies have a close relationship with the development of the cell wall and vacuole. The possible secretory pathways of multi-vesicular bodies might be in exocytosis, in which multi-vesicular bodies carry sub-stances to the cell wall for its construction, and in endocytosis, in which multi-vesicular bodies carry substances to the vacuole for its development, depending on what they carry and where the materials are transported. We hence propose that there is more than one pathway for the secretion of multi-vesicular bodies. In addition, our results provided a paradigm that a plant molecule, such as the 58k-like protein in callus of Arabidopsis thaliana, can be detected using a cross-reactive monoclonal antibody induced by an animal protein, and illustrate the existence of analog molecules in both animal and plant kingdoms.

  10. FIT interacts with AtbHLH38 and AtbHLH39 in regulating iron uptake gene expression for iron homeostasis in Arabidopsis

    Institute of Scientific and Technical Information of China (English)

    Youxi Yuan; Huilan Wu; Ning Wang; Jie Li; Weina Zhao; Juan Du; Daowen Wang; Hong-Qing Ling

    2008-01-01

    Iron is an essential element for plant growth and development. Iron homeostasis in plants is tightly regulated at both transcriptional and posttranscriptional level. Several bHLH transcription factors involved in iron homeostasis have been identified recently. However, their regulatory mechanisms remain unknown. In this work, we demonstrate that the transcription factor FIT interacted with AtbHLH38 and AtbHLH39 and directly conferred the expression regulation of iron uptake genes for iron homeostasis in Arabidopsis. Yeast two-hybrid analysis and transient expression in Arabidopsis protoplasts showed that AtbH LH38 or AtbHLH39 interacted with FIT, a central transcription factor involved in iron homeostasis in Arabidopsis. Expression of FIT/AtbHLH38 or FIT/AtbHLH39 in yeast cells activated GUS expression driven by ferric chelate reductase (FRO2) and ferrous transporter (IRT1) promoters. Overexpression of FIT with either AtbHLH38 or AtbHLH39 in plants converted the expression of the iron uptake genes FRO2 and IRT1 from induced to constitutive. Further analysis revealed that FR02 and IRT1 were not regulated at the posttranscriptional level in these plants because IRT1 protein accumulation and high ferric chelate reductase activity were detected in the overexpression plants under both iron deficiency and iron sufficiency. The double overexpression plants accumulated more iron in their shoots than wild type or the plants overexpressing either AtbHLH38,AtbHLH39 or FIT. Our data support that ferric-chelate reductase FRO2 and ferrous-transporter IRT1 are the targets of the three transcription factors and the transcription of FRO2 and IRT1 is directly regulated by a complex of FIT/AtbHLH38 or FIT/AtbHLH39.

  11. Suppressor of Overexpression of CO 1 Negatively Regulates Dark-Induced Leaf Degreening and Senescence by Directly Repressing Pheophytinase and Other Senescence-Associated Genes in Arabidopsis.

    Science.gov (United States)

    Chen, Junyi; Zhu, Xiaoyu; Ren, Jun; Qiu, Kai; Li, Zhongpeng; Xie, Zuokun; Gao, Jiong; Zhou, Xin; Kuai, Benke

    2017-03-01

    Although the biochemical pathway of chlorophyll (Chl) degradation has been largely elucidated, how Chl is rapidly yet coordinately degraded during leaf senescence remains elusive. Pheophytinase (PPH) is the enzyme for catalyzing the removal of the phytol group from pheophytin a, and PPH expression is significantly induced during leaf senescence. To elucidate the transcriptional regulation of PPH, we used a yeast (Saccharomyces cerevisiae) one-hybrid system to screen for its trans-regulators. SUPPRESSOR OF OVEREXPRESSION OF CO 1 (SOC1), a key flowering pathway integrator, was initially identified as one of the putative trans-regulators of PPH After dark treatment, leaves of an SOC1 knockdown mutant (soc1-6) showed an accelerated yellowing phenotype, whereas those of SOC1-overexpressing lines exhibited a partial stay-green phenotype. SOC1 and PPH expression showed a negative correlation during leaf senescence. Substantially, SOC1 protein could bind specifically to the CArG box of the PPH promoter in vitro and in vivo, and overexpression of SOC1 significantly inhibited the transcriptional activity of the PPH promoter in Arabidopsis (Arabidopsis thaliana) protoplasts. Importantly, soc1-6 pph-1 (a PPH knockout mutant) double mutant displayed a stay-green phenotype similar to that of pph-1 during dark treatment. These results demonstrated that SOC1 inhibits Chl degradation via negatively regulating PPH expression. In addition, measurement of the Chl content and the maximum photochemical efficiency of photosystem II of soc1-6 and SOC1-OE leaves after dark treatment suggested that SOC1 also negatively regulates the general senescence process. Seven SENESCENCE-ASSOCIATED GENES (SAGs) were thereafter identified as its potential target genes, and NONYELLOWING1 and SAG113 were experimentally confirmed. Together, we reveal that SOC1 represses dark-induced leaf Chl degradation and senescence in general in Arabidopsis.

  12. New Arabidopsis thaliana cytochrome c partners: a look into the elusive role of cytochrome c in programmed cell death in plants.

    Science.gov (United States)

    Martínez-Fábregas, Jonathan; Díaz-Moreno, Irene; González-Arzola, Katiuska; Janocha, Simon; Navarro, José A; Hervás, Manuel; Bernhardt, Rita; Díaz-Quintana, Antonio; De la Rosa, Miguel Á

    2013-12-01

    Programmed cell death is an event displayed by many different organisms along the evolutionary scale. In plants, programmed cell death is necessary for development and the hypersensitive response to stress or pathogenic infection. A common feature in programmed cell death across organisms is the translocation of cytochrome c from mitochondria to the cytosol. To better understand the role of cytochrome c in the onset of programmed cell death in plants, a proteomic approach was developed based on affinity chromatography and using Arabidopsis thaliana cytochrome c as bait. Using this approach, ten putative new cytochrome c partners were identified. Of these putative partners and as indicated by bimolecular fluorescence complementation, nine of them bind the heme protein in plant protoplasts and human cells as a heterologous system. The in vitro interaction between cytochrome c and such soluble cytochrome c-targets was further corroborated using surface plasmon resonance. Taken together, the results obtained in the study indicate that Arabidopsis thaliana cytochrome c interacts with several distinct proteins involved in protein folding, translational regulation, cell death, oxidative stress, DNA damage, energetic metabolism, and mRNA metabolism. Interestingly, some of these novel Arabidopsis thaliana cytochrome c-targets are closely related to those for Homo sapiens cytochrome c (Martínez-Fábregas et al., unpublished). These results indicate that the evolutionarily well-conserved cytosolic cytochrome c, appearing in organisms from plants to mammals, interacts with a wide range of targets on programmed cell death. The data have been deposited to the ProteomeXchange with identifier PXD000280.

  13. METACASPASE9 modulates autophagy to confine cell death to the target cells during Arabidopsis vascular xylem differentiation

    Directory of Open Access Journals (Sweden)

    Sacha Escamez

    2016-02-01

    Full Text Available We uncovered that the level of autophagy in plant cells undergoing programmed cell death determines the fate of the surrounding cells. Our approach consisted of using Arabidopsis thaliana cell cultures capable of differentiating into two different cell types: vascular tracheary elements (TEs that undergo programmed cell death (PCD and protoplast autolysis, and parenchymatic non-TEs that remain alive. The TE cell type displayed higher levels of autophagy when expression of the TE-specific METACASPASE9 (MC9 was reduced using RNAi (MC9-RNAi. Misregulation of autophagy in the MC9-RNAi TEs coincided with ectopic death of the non-TEs, implying the existence of an autophagy-dependent intercellular signalling from within the TEs towards the non-TEs. Viability of the non-TEs was restored when AUTOPHAGY2 (ATG2 was downregulated specifically in MC9-RNAi TEs, demonstrating the importance of autophagy in the spatial confinement of cell death. Our results suggest that other eukaryotic cells undergoing PCD might also need to tightly regulate their level of autophagy to avoid detrimental consequences for the surrounding cells.

  14. Variation in molybdenum content across broadly distributed populations of Arabidopsis thaliana is controlled by a mitochondrial molybdenum transporter (MOT1.

    Directory of Open Access Journals (Sweden)

    Ivan Baxter

    2008-02-01

    Full Text Available Molybdenum (Mo is an essential micronutrient for plants, serving as a cofactor for enzymes involved in nitrate assimilation, sulfite detoxification, abscisic acid biosynthesis, and purine degradation. Here we show that natural variation in shoot Mo content across 92 Arabidopsis thaliana accessions is controlled by variation in a mitochondrially localized transporter (Molybdenum Transporter 1 - MOT1 that belongs to the sulfate transporter superfamily. A deletion in the MOT1 promoter is strongly associated with low shoot Mo, occurring in seven of the accessions with the lowest shoot content of Mo. Consistent with the low Mo phenotype, MOT1 expression in low Mo accessions is reduced. Reciprocal grafting experiments demonstrate that the roots of Ler-0 are responsible for the low Mo accumulation in shoot, and GUS localization demonstrates that MOT1 is expressed strongly in the roots. MOT1 contains an N-terminal mitochondrial targeting sequence and expression of MOT1 tagged with GFP in protoplasts and transgenic plants, establishing the mitochondrial localization of this protein. Furthermore, expression of MOT1 specifically enhances Mo accumulation in yeast by 5-fold, consistent with MOT1 functioning as a molybdate transporter. This work provides the first molecular insight into the processes that regulate Mo accumulation in plants and shows that novel loci can be detected by association mapping.

  15. 桦褐孔菌原生质体制备与再生%Preparation and Regeneration of Inonotus obliquus Protoplasts

    Institute of Scientific and Technical Information of China (English)

    孙勇; 曹小迎; 陈永强; 缪倩; 蒋继宏

    2011-01-01

    研究桦褐孔菌原生质体制备与再生条件。采用正交试验法确定复合酶比例,用单因素试验测定不同等渗液、酶解时间、桦褐孔菌菌龄、酶解温度、再生培养基对桦褐孔菌原生质体制备与再生的影响。结果表明:混合酶(纤维素酶5mg/mL、蜗牛酶15mg/mL、溶壁酶15mg/mL、崩溃酶15mg/mL)、菌龄为7d的菌丝,采用0.6mol/LMgSO4为渗透压稳定液,酶解温度35℃,酶解时间5.5h,有利于原生质体的制备。就原生质体再生而言,酶解时间为3.5h制得的原生质体,以MgSO4作为渗透压稳定液,以完全再生培养基(CYM%The conditions for protoplast preparation and regeneration from the mycelia of Inonotus obliquus were investigated.The optimal amounts of four enzymes were explored by orthogonal array design and other conditions were studied by single factor method.The results showed that the mycelium harvesting after 7-day culture,adding 5 mg/mL cellulase,15 mg/mL snailase,15 mg/mL lywallzyme and 15 mg/mL drislease,using 0.6 mol/L MgSO4 as the osmotic stabilizer,digestion at 35 ℃ for 5.5 h were found to be best for the preparation of protoplasts.The optimal conditions for protoplast regeneration of Inonotus obliquus were CYM or GYM as the medium,0.6 mol/L mannitol as the osmotic stabilizer using and digestion time of 3.5 h.No significant difference in protoplast regeneration rate was observed between CYM and GYM media.The highest protoplast regeneration rate was 0.13%.

  16. Arabidopsis CDS blastp result: AK065259 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK065259 J013002J18 At5g16910.1 cellulose synthase family protein similar to gi:2827143 cellulose... synthase catalytic subunit, Arabidopsis thaliana, gi:9622886 cellulose synthase-7 from Zea mays 0.0 ...

  17. Arabidopsis CDS blastp result: AK102134 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK102134 J033085F12 At5g16910.1 cellulose synthase family protein similar to gi:2827143 cellulose... synthase catalytic subunit, Arabidopsis thaliana, gi:9622886 cellulose synthase-7 from Zea mays 0.0 ...

  18. Arabidopsis CDS blastp result: AK066835 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK066835 J013087I16 At5g16910.1 cellulose synthase family protein similar to gi:2827143 cellulose... synthase catalytic subunit, Arabidopsis thaliana, gi:9622886 cellulose synthase-7 from Zea mays 1e-171 ...

  19. Arabidopsis CDS blastp result: AK100523 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK100523 J023100P04 At5g16910.1 cellulose synthase family protein similar to gi:2827143 cellulose... synthase catalytic subunit, Arabidopsis thaliana, gi:9622886 cellulose synthase-7 from Zea mays 0.0 ...

  20. Arabidopsis CDS blastp result: AK102695 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK102695 J033103F21 At5g16910.1 cellulose synthase family protein similar to gi:2827143 cellulose... synthase catalytic subunit, Arabidopsis thaliana, gi:9622886 cellulose synthase-7 from Zea mays 0.0 ...