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Sample records for arabidopsis protoplast isolation

  1. Analyzing Synthetic Promoters Using Arabidopsis Protoplasts.

    Science.gov (United States)

    Stracke, Ralf; Thiedig, Katharina; Kuhlmann, Melanie; Weisshaar, Bernd

    2016-01-01

    This chapter describes a transient protoplast co-transfection method that can be used to quantitatively study in vivo the activity and function of promoters and promoter elements (reporters), and their induction or repression by transcription factors (effectors), stresses, hormones, or metabolites. A detailed protocol for carrying out transient co-transfection assays with Arabidopsis At7 protoplasts and calculating the promoter activity is provided. PMID:27557761

  2. Influence of IAA Treatment on Isolation of Protoplasts in Leaf of Arabidopsis thaliana%IAA处理对拟南芥叶原生质体分离的影响

    Institute of Scientific and Technical Information of China (English)

    赵严伟; 黄志刚; 李合松

    2011-01-01

    By treated leaves of Arabidopsis thaliana with different concentrations of IAA, the changes of amount and activity of protoplasts under different enzymolysis methods with different enzymatic solution combinations and different enzymolysis time were compared to analyze influence of IAA on isolation of protoplast. The results showed that 10-7 mol/L of IAA can effectively improve amount and activity of protoplasts, and the activity of protoplast was the highest when combining 0.6% cellulase R-10 with 0.2% macerozyme R-10, which was 87.44%. IAA also can enhance isolation speed of protoplasts.%通过对拟南芥叶片进行不同浓度的IAA处理,比较其在不同的酶解方式、酶液组合、酶解时间下原生质体数量与活力的变化,探讨了IAA对原生质体分离的影响.结果表明,10-7 mol/L的IAA能有效增加活力原生质体数量,且在与0.6%纤维素酶R-10与0.2%离析酶R-10组合时活力最高,为87.44%.材料外施IAA可提高原生质体分离速率.

  3. Regeneration from leaf protoplasts of Arabidopsis thaliana ecotype estland.

    Science.gov (United States)

    Gandhi, R; Khurana, P

    2001-07-01

    Protoplasts (2 x 10(7)/g fresh wt) were isolated from leaves of A. thaliana ecotype estland, with a viability of more than 90%. Protoplasts cultured in calcium alginate beads or layers showed division while culture in liquid or agarose beads failed to elicit any division. Effect of culture density showed highest frequency of division occurring at 5 x 10(5) while no division was seen when cultured at a density of 5 x 10(4). Culture in MS medium resulted in higher division frequency and better sustenance of microcolonies as compared to B5 medium. Under optimized conditions, macrocolonies were formed at a frequency of 1.8%. Shoot regeneration was seen in 50% of microcalli transferred to shoot induction medium for regeneration. Shoots were rooted and plantlets transferred to pots. The plants produced flowers and were fertile. PMID:12019766

  4. 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.

  5. Isolation of protoplasts from undaria pinnatifida by alginate lyase digestion

    Science.gov (United States)

    Xiaoke, Hu; Xiaolu, Jiang; Huashi, Guan

    2003-04-01

    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°C for 2h using alginate lyase at the concentration of 213.36 U (8 mL) every 0.5g 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 mol L-1.

  6. 洗液对拟南芥叶原生质体分离的影响%The Influence of Washing Buffer on Isolation of Arabidopsis thaliana Mesophyll Protoplasts

    Institute of Scientific and Technical Information of China (English)

    赵严伟; 黄志刚; 李合松

    2011-01-01

    Washing buffer plays an important role in protoplasts purification and storing when protoplasts isolated. The study compared yiled and viability change of protoplasts in washing buffer of WI, W5 and MMg in 30 h, analyzed the influence of CaCl2 with different concentration on protoplasts in WI, W5 and MMg to get the suitable washing buffer for the highest viable protoplasts. The results indicated that WI was better at maintaining plasma membrane, while W5 was better on keeping viability, after 30 h W5 got the highest viable protoplasts population which was good for storing protoplasts in a short period; CaCl2 treatment showed little influence on protoplasts viability and its population improving effect depended on its concentration and the washing buffer type, there was a uptrend of viable protoplasts in MMg when treated with 0-50 mM CaCl2.%原生质体分离过程中洗液对原生质体的纯化和保存起着重要的作用.本试验通过比较30 h内WI、W5、MMg 3种洗液中原生质体数量与活力的变化以及分析不同浓度CaCl2处理WI、W5、MMg后对原生质体的影响,选择出获得最多活性原生质体数量的洗液.结果表明,WI在维持质膜稳定性上较有优势,而W5则更有利于保持原生质体活力且30 h后得到的活性原生质体最多,较适宜在短期内保存原生质体;CaCl2处理洗液对原生质体活力无显著影响,且其对原生质体数量的影响取决于CaCl2处理浓度及洗液种类,MMg洗液在补充0~50 mM CaCl2后原生质体数量呈上升趋势.

  7. Isolation and culture of suspension protoplasts of vetiver

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    Somporn Prasertsongskun

    2005-05-01

    Full Text Available In this research, protoplasts were isolated from cell suspension derived from inflorescence of vetiver (Vetiveria zizanioides Nash Surat Thani germplasm. The optimum condition for protoplast isolation was established by using 2% cellulase Onozuka R10, 2% macerozyme R10, 0.5% pectinase in 0.4 M mannitol and 7 mM CaCl2.2H2O at pH 5.8 and incubated for 10 hours in the dark on the rotary shaker at 50 rpm. Maximum protoplast yields were 8.4 × 104 protoplasts/ml PCV. Division of protoplasts was observed only in liquid medium. The first cell division was observed after 3 days of culture initiation, and the average division was 5.0% in the N6 medium supplemented with 1.0 mg/l 2,4-D (2,4-dichlorophenoxyacetic acid and 0.5 mg/l BA (Benzyladenine. An optimal density for culture division was 1 × 105 protoplasts/ml.

  8. 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...

  9. 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.

  10. Effects of environmental preconditioning, donor tissue and isolation conditions on tomato (Lycopersicon esculentum Mill. protoplast yield

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    Elżbieta Kuźniak

    2013-12-01

    Full Text Available The effects of soil or in vitro grown plants, pretreatment conditions, donor tissue and isolation procedure on protoplast yield from cotyledons and leaves of tomato cv. 'Perkoz' and 'Zorza' were studied. The highest protoplast yield of 1.5 x 107/g FW was obtained from leaves of in vitro grown plants. Low light intensity during donor plants in vitro culture and dark pretreatment were essential for successful protoplast isolation while cold pretreatment was not. Tissue preplasmolysis prior to transfer to enzyme mixture increased 4-fold the number of isolated protoplasts. Glycine and bovine serum albumin in the isolation medium did not significantly influence the protoplast yield.

  11. Isolation and Fusion of Protoplasts from Basella rubra Leaf and Stem Cultures

    OpenAIRE

    Okamura, Tokumitsu; Matsuo, Shiho; Miyashige, Kayoko

    1999-01-01

    Isolation and fusion of protoplasts from Basella rubra leaf and stem were examined. In preparation of protoplasts, the enzyme solution gave a high yield of protoplasts. The diameter of protoplasts induced ranged from 30μm to 120μm. Cell division into 2 cells was observed after 24 hours of culture, then micro colonies formed after 7 days, followed by colony formation within 2 weeks

  12. 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.

  13. 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.

  14. Isolation and characterization of protoplasts and vacuoles from sugar beet leaf mesophyll

    International Nuclear Information System (INIS)

    The present paper describes methods for isolation of protoplasts and vacuoles from sugar beet (Beta vulgaris L.) leaf mesophyll. Protoplasts were isolated by the enzymatic method in two stages. The yield of protoplasts in the crude suspension attained 3-10 units from 1g of fresh tissue mass. Two methods of purifying the crude protoplast suspension are compared in the paper, the indicated methods employing gradients of Percoll (method 1) and Ficoll (method 2). The final yield comprised 4.5-9.0-10.5 protoplasts from 1g of fresh tissue mass after purification method 1 and 6.0-10.5-1.2-10 protoplasts after method 2. The photosynthesis rate in such protoplasts under optimal conditions comprised 75-100 μmoles of CO2h per mg of chlorophyll as compared with 100-130 μmoles in leaf blade disks. The two methods were used to obtain vacuoles, method 1 involving osmotic lysis of protoplasts (the yield constituting 6-15% of vacuoles of the protoplasts taken) and method 2 consisting of ultracentrifugation in a Ficoll gradient (giving a yield of 25-45%). As was monitored microscopically and from the absence of activity of extravacuolar enzymes (NADH-cytochrome-c reductase and cytochrome-c oxidase), vacuoles free of foreign impurities were obtained in both cases. The time needed to obtain protoplasts from leaf tissue comprised 2-3 h, whereas 1.5-2 h was needed to obtain vacuoles from protoplasts

  15. Protoplast isolation from Ulmus americana l. Pollen mother cells, tetrads, and microspores

    Energy Technology Data Exchange (ETDEWEB)

    Redenbaugh, M.K.; Westfall, R.D.; Karnosky, D.F.

    1980-01-01

    Meiotic protoplasts of U. amerciana are potentially valuable for producing interspecific elm hybrids through protoplast fusion. Meiotic cells(pollen mother cells, tetrads, and microspores) were incubated in either a cellulase, hemicellylase and pectinase enzyme solution of a beta-1,3-glucanase (lainarinase) solution. Respective protoplast isolation frequencies for the three meiotic cell types were 100, 50, and 10%. Exclusion staining with 0.2% Evans blue and 0.1% methyl blue suggested protoplast viability. Some of the microspore protoplasts were vacuolated, which is an important condition for cell division. Although attempts of regenerating cell walls and inducing cell division were unsuccessful, these two problems may be superceded by protoplast fusion with more regenerative protoplasts.

  16. Influences of explant type and enzyme incubation on isolated protoplast density and viability in two garlic cultivars

    International Nuclear Information System (INIS)

    The present study reports on optimizing protoplast isolation and fusion in two garlic cultivars Balady and Seds 40. Protoplast density and viability were investigated in four different explants (etiolated and green parts of the pseudostem and lower and upper parts of the leaves) under enzyme incubation for 1, 2, 3 and 4 h. Among different explants, used for protoplast isolation in Balady cultivar, the upper and lower parts of the leaves produced the highest number of total protoplasts (70 and 66 pps/0.1 ml) at 4 and 3 h enzyme incubation, respectively. However, the etiolated part of pseudostem produced the highest number of viable protoplast in which 52.5 pps/0.1 ml were obtained at 3 h enzyme incubation. For protoplast isolation in Seds 40 cultivar, the highest number of total protoplasts (125 and 107.5 pps/0.1 ml) as well as viable protoplasts (105 and 107.5 pps/0.1 ml) was obtained from the etiolated and the green parts of pseudostem, respectively. The cultivar Seds 40 yielded higher total and viable protoplasts than Balady cultivar. Isolated protoplasts of Seds 40 and Balady were fused successfully at a protoplast density of 1 * 105 using either physical and/or electrical method. Optimization of the source of plant material as well as protoplast isolation conditions for garlic is a crucial step towards a successful protoplast fusion and subsequent colony formation. (author)

  17. RNA synthesis in newly isolated and cultivated mesophyll protoplasts

    Energy Technology Data Exchange (ETDEWEB)

    Paszkowski, J.; Kleczkowski, K.

    1979-01-01

    Tobacco leaf mesophyll protoplasts exhibit low incorporation of (3H)uridine and 32P into RNA, up to 12 h of cultivation, irrespective of the presence of phytohormones. After 24 h of cultivation a dramatic increase in RNA synthesis is observed. The protoplasts cultivated in the absence of phytohormones show lower incorporation of precursors.

  18. Isolation of protoplast from soybean, cowpea, and tobacco and their fusion

    International Nuclear Information System (INIS)

    Protoplast were isolated from leaf and callus. Young leaf of 3-4 weeks old plant of soybean T219 and A24, A27, C4, E1, and H6 of cowpeas strains (strains named by Prof. S. Sakamoto, University of Kyoto) were suspended in digestive medium containing cellulase 'Onuzuka' R-10, macerozyme R-10, mannitol, CaCl, and 2 (N-morpholilno) echane sulfonic acid (MES). For soybean leaf, the medium was enriched with driselase and pectolyase Y-23. They were incibated in full darkness at 27 Celcius centigrade by constant shaking at 50 rpm orbitor shaker. Callus wich has been two times resubcultured was suspended in the digestive medium without driselase, CaCl2, and MES and incubated in lowlight intensity by constant shaking at 100 rpm in reciprocal water shaker at 30 celcius centigrade. Leaf protoplast were releasaed in 10-14 h, soybean and tobacco callus protoplast in 3-4 h, and cowpeas callus protoplast in 4-6 h of incubation. Protoplast were collected by centrifugation of 400 g and a thin layer of the suspension was irradiated with ultraviolet light. Fusion was induced with PEG 6000 solution according to Uchimia and fused protoplasts were collected by centrifugation of 200 g. Protoplast were cultured on the medium of Ikeda and Uchimia. On both medium leaf protoplast, irradiated protoplasts and their fused do not regenerate cell wall and all cultured died out within four weeks incubation. Cell wall generation was observed. Regeneration of cell wall observed progessively in mother protoplast from tobacco, cowpea (A27, E1, and H6) and fused protoplast of soybean with tobacco, tobacco with cowpea (C4, E1, and H6), soybean with cowpea (C4) and between cowpea (C4) and cowpea (E1). (author). 25 refs, 4 tabs

  19. Protocols for Studying Protein Stability in an Arabidopsis Protoplast Transient Expression System.

    Science.gov (United States)

    Planchais, Séverine; Camborde, Laurent; Jupin, Isabelle

    2016-01-01

    Protein stability influences many aspects of biology, and measuring their stability in vivo can provide important insights into biological systems.This chapter describes in details two methods to assess the stability of a specific protein based on its transient expression in Arabidopsis protoplasts. First, a pulse-chase assay based on radioactive metabolic labeling of cellular proteins, followed by immunoprecipitation of the protein of interest. The decrease in radioactive signal is monitored over time and can be used to determine the protein's half-life.Alternatively, we also present a nonradioactive assay based on the use of reporter proteins, whose ratio can be quantified. This assay can be used to determine the relative stability of a protein of interest under specific conditions. PMID:27424754

  20. 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.

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

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    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.

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

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    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.

  3. A Study on the isolation of protoplasts from the callus of lilium longiflorum overig

    International Nuclear Information System (INIS)

    Lilium longiflorum Overig is a Lilaceous plant grown for ornamental as well as certain other general purposes. The research presented here focuses on the method of protoplast isolation from the In vitro grown callus of Lilium longiflorum Overig. Series of experiments were conducted in order to optimize the conditions. Four different amounts of callus and a range of incubation time were used to achieve maximum number of viable protoplasts. However, the calli used were treated with Enzyme solution containing 0.5 percentage (w/v) Macerozyme, 2 percentage (w/v) Cellulase and 0.1 percentage (w/v) Pectinase in combination, to obtain maximum number of viable protoplasts through the process of cell-wall digestion. Furthermore, an osmoticum of 20 percentage (w/v) sucrose and a washing solution consisting of 0.1 percentage (w/v) MES and 0.5M Mannitol were added to suspend a ring of protoplast at the interphase of both the solutions. This ring of protoplast was then isolated and tested for viability and yield. Consequently, amongst the various sets of experiment, callus of 1.5 grams and incubation time of 4 hours was found an ideal approach to obtain maximum number of viable protoplasts. (author)

  4. Studies on the isolation and culture of protoplasts from Kappaphycus alvarezii

    Institute of Scientific and Technical Information of China (English)

    ZHANG Si; LIU Cui; JIN Yuemei; CHI Shan; TANG Xianming; CHEN Fuxiao; FANG Xu; LIU Tao

    2014-01-01

    In this study, protoplasts were successfully isolated from Kappaphycus alvarezii using snail enzymes, aba-lone enzymes and cellulase. The optimum enzymic ratio was fixed to be 20%of abalone enzyme, 12%of cellulase and the osmotic stabilizer was 2.0 mol/L glucose. The optimum enzymic hydrolysis conditions were found to be dark enzymolysis at 30°C continuing for 4.0 h. The resultant density and yield of protoplasts achieved 32.60×104 mL-1, 65.20×104 g-1 tissue for Kappaphycus alvarezii. Finally, under the temperature of 20°C, light intensity of 1 500-2 000 lx and photoperiod of 12 h/d, two developmental pathways were investi-gated:(1) callus-like cell mass and regenerated plantlet occurred on protoplast;(2) young shoots and callus-like cell mass occurred in tissue blocks after enzymolysis.

  5. 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.

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

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    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.

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

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    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

  8. 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.

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

    OpenAIRE

    Esmaeil CHAMANI; Seyyed Karim TAHAMI; ZARE, Nasser; Rasool Asghari-ZAKARIA; Mehdi MOHEBODINI; Joyce, Daryl

    2012-01-01

    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...

  10. Photorespiratory properties of total leaf protoplasts isolated from C3-C4 intermediate species of MORICANDIA

    International Nuclear Information System (INIS)

    In comparison to C3 species, intact leaf tissue of M. arvensis (C3-C4) exhibits a reduced sensitivity of photosynthesis to inhibition by O2 and an enhanced capacity for the refixation of 14CO2 evolved during decarboxylation of exogenous [1-14C]glycine. In contrast, purified protoplast preparations from leaves of M. arvensis and M. spinosa (C3-C4), containing 8.4 +/- 0.6% (S.E) bundle-sheath protoplasts and approx. 90% mesophyll protoplasts, showed no significant differences from protoplasts of the C3 species, M. foetida and Triticum aestivum with respect to the following parameters: (a) percentage O2-inhibition of photosynthetic 14CO2 fixation; (b) apparent Km(CO2) of photosynthesis; and (c) dark/light ratios of 14CO2 evolution during metabolism of exogenous [1-14C]glycine. It was concluded that the structural arrangement of mesophyll and bundle-sheath cells and organelles in situ in leaf tissue of M. arvensis and M. spinosa is of importance in facilitating reduced apparent photorespiration in these intermediate species by enhanced recycling of CO2

  11. Isolation and culture of protoplasts of Ma-phut (Garcinia dulcis derived from cell suspension culture

    Directory of Open Access Journals (Sweden)

    Sompong Te-chato

    2008-09-01

    Full Text Available Friable callus induced from young leaves of Ma-phut on Murashige and Skoog (MS medium containing 3% sucrose,1 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D, 0.5 mg/l benzyladenine (BA and 500 mg/l polyvinylpyrrolidone (PVP, was cultured in liquid medium with the same components. Various ages of cell suspension at weekly intervals were then incubated in various kinds and concentrations of cell wall digestion enzymes combined with 1% macerozyme R-10 on a rotary shaker at 100 rpm under 1500 lux illumination at 26±4oC. Purified protoplasts were cultured at various densities in MS medium (adjusted osmoticum to 0.4 M by mannitol supplemented with 3% sucrose and two types of auxin, 2,4-D and NAA at four concentrations (1, 2, 3 and 4 mg/l together with 1 mg/l BA. The results revealed that a four-day old cell suspension culture incubated in 2% cellulase Onozuka R-10 (CR10 in combination with 1% macerozyme R-10 gave an optimum result in both yield and viability of protoplasts at 5.7x106/1 ml PCV and 80%, respectively. Embedding protoplasts at a density of 2.5x105/ml in 0.2% phytagel containing MS medium supplemented with 3 mg/l NAA and 1 mg/l BA promoted the most effective division of the protoplasts (20%. The first division of the protoplasts was obtained after 2 days of culture and further divisions to form micro- and macro-colonies could be observed after 7-10 days of culture. However, callusformation and plantlet regeneration was not obtained.

  12. Isolation and Characterization of Protoplasts and their Utilization for Model Membrane

    Czech Academy of Sciences Publication Activity Database

    Nováková, Kateřina; Navrátil, Tomáš; Šestáková, Ivana; Langmaier, Jan; Heyrovský, Michael; Zámečníková, B.; Vodičková, H.

    Ústí nad Labem : Best Servis, 2014 - (Navrátil, T.; Fojta, M.; Pecková, K.), s. 114-117 ISBN 978-80-905221-2-1. [Moderní Elektrochemické Metody /34./. Jetřichovice (CZ), 19.05.2014-23.05.2014] R&D Projects: GA ČR(CZ) GAP208/12/1645 Institutional support: RVO:61388955 Keywords : Protoplasts * Cells * Model membrane Subject RIV: CG - Electrochemistry

  13. 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. PMID:27574524

  14. 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.

  15. 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.

  16. Protoplast isolation and plant regeneration of guava (Psidium guajava L.) using experiments in mixture-amount design

    Science.gov (United States)

    A protocol was established for plant regeneration from leaf protoplasts of guava (Psidium guajava L.) using mixture-amount (concentration) experiments. A protoplast yield of 3.7 × 106 (viability > 90 percent) was obtained when 1 g leaf strips were digested in a solution of approximately 0.75 M osmot...

  17. Effect of X-Rays on Growth Rate of Rose Shoot Cultures and the Ability of Isolated Protoplasts to Form Cell Colonies

    International Nuclear Information System (INIS)

    The popularity of rose as a garden plant, allied with its use in the production of cut flowers and also as a source of aromatic rose oils, make it one of the most important ornamental crops. Roses, however, have suffered from a narrow genetic base to which only few species have contributed significantly. In vitro culture of plants might facilitate the improvement of rose via the exploitation of somaclonal variation to generate new genetic variability and selection within the variation for desirable traits. The application of mutagens for in vitro cultures, in addition to the induced mutations, may lead to increase the somaclonal variation, thus providing additional variation for selection. On the other hand, plant protoplasts offer exciting possibilities to establish in vitro selection programs based on single cells. Induced variation in isolated protoplasts using mutagen agents may be one mean to select useful mutants. Thus the present experiments were conducted to determine the effect of X-rays on shoot cultures and the isolated protoplasts of rose (Rosa sp.). The materials consisted of the three rose varieties Rosa wichuriana, Paricer charm and Heckenzauber.The applied doses were 0, 10, 20, 30, 40, 50 and 60 Gy. Obtained results indicated that the genotypes differed in their sensitivity to X-rays. Rosa wichuriana seemed to be the most sensitive variety to radiation, where a dose of 20 Gy caused approximately 50% reduction in growth rate of shoot cultures, while the same dose decreased the growth rate of Paricer charm only by 25% and did not affect the growth of Heckenzauber. Results also revealed that the ability of irradiated protoplasts to form cell colonies increased when a dose of 10 Gy was performed. Doses higher than that level caused gradual decreasing in the forming of cell colonies, but however, the protoplasts could form colonies even when a dose of 60 Gy was applied. (Author)

  18. Embryogenic Calls Protoplast Isolation of Rubber Tree and Transient Expression of Red Fluorescent Protein%橡胶树原生质体的分离及红色荧光蛋白的瞬时表达研究

    Institute of Scientific and Technical Information of China (English)

    于晓玲; 阮孟斌; 王树昌

    2013-01-01

    The aim was to isolate protoplasts of callus in rubber tree and to build the system of transient expression in protoplasts. The friable callus of rubber tree was used as materials to isolate protoplast by enzyme digestion;many high purity protoplasts were isolated. The transient expression vector with reported gene coded red fluorescent protein (RFP) was transferred into protoplasts by electric shock method, and the expression of RFP in protoplast was examined by laser scanning confocal microscopy. It showed that the system of transient expression of target gene in protoplasts of rubber has been established.%分离巴西橡胶树松散愈伤组织原生质体,建立目标基因在橡胶原生质体的瞬时表达系统。以巴西橡胶树松散愈伤组织为材料,酶解分离得到高纯度原生质体,通过电击介导的转化方法,将编码红色荧光蛋白(RFP)的植物表达载体转入原生质体中,用激光扫描共聚焦显微镜检测原生质体中RFP的表达情况。初步建立了橡胶原生质体瞬时表达系统。

  19. 小青杨叶肉原生质体分离条件的研究%Isolation of Mesophyll Protoplasts from Populus pseudo-simonii Kitag.

    Institute of Scientific and Technical Information of China (English)

    蔡肖; 康向阳

    2011-01-01

    To obtain high yield and high viability protoplasts, an efficiency protocol was presented here using leaves of in vitro shoot culture of Populus pseudo-simonii Kitag.. The yield of protoplasts depended on factors such as enzyme concentration, osmoticum concentration, incubation time and the age of leaves. The results showed that the optimal protocol for isolating protoplasts was treating 35 days aged leaves with CPW salts + 3.0% Cellulase R-10 + 0.5% Macerozyme R-10 + 0.1% Pectolase Y-23 + 0.6 mol/L mannitol + 0.6 g/L MES+ 1 g/L BSA for 8 h. Protoplast yields were up to 2.44× 107 protoplasts per gram and the viability was 78.7%. This protocol that resulted in high yield and high viability of protoplasts could meet the demands of protoplast culture-based techniques.%为获得大量、有活力的原生质体,建立起高效的小青杨(Populuspseudo-simonii Kitag.)原生体分离体系,本研究以小青杨无菌苗叶片为材料进行原生质体分离条件的研究.结果表明:酶的种类和浓度、渗透压稳定剂浓度、酶解时间、叶龄是影响原生质体分离的重要因素;将叶龄35天的无菌苗第2~3片叶片置于酶液组成为CPW+3.0%纤维素酶R-10+0.5%离析酶R-10+0.1%果胶酶Y-23+0.6 mol/L甘露醇+0.6 g/LMES+1 g/L BSA中酶解8 h,原生质体产量和活力分别为2.44×107个/g和78.7%.通过该分离体系稳定的获得了高产量、高活力的原生质体,可以满足进一步的原生质体培养等技术的要求.

  20. 忍冬原生质体分离条件研究%Researches on Isolation Conditions of Protoplasts of Lonicera japonica

    Institute of Scientific and Technical Information of China (English)

    刘颖; 郭明晔; 王朝阳; 白根本

    2012-01-01

    目的:探讨忍冬原生质体分离的最佳条件,为忍冬原生质体培养、细胞融合以及遗传转化提供大量优质的原生质体.方法:分别对忍冬原生质体分离过程中的若干影响因子如:酶液组成、酶解方式、酶解时间、酶解温度、渗透压进行比较分析,以确定最佳的分离条件.结果:确定了最优化的忍冬原生质体分离条件,即以生长旺盛的疏松的忍冬愈伤组织作为分离原生质体所需材料,以1.5%纤维素酶+0.25%果胶酶作为分离原生质体所用酶液,以含有0.7 mol·L-1甘露醇作为渗透保护剂的细胞-原生质体清洗液(CPW)溶液进行酶液的配制,于25℃静置条件下酶解12 h.结论:利用该实验所确定的忍冬原生质体最佳分离条件可获得大量优质的原生质体,为后续忍冬细胞融合以及遗传转化等实验奠定良好的实验基础.%Objective: To find out the best isolation conditions of protoplasts of Lonicera japonica. Method: Some influence factors on isolation of protoplasts, such as composition of enzyme solution, methods, hours and temperature of enzyme dissolving, osmotic pressure, and so on, were compared and analyzed to find out the best conditions of protoplasts isolation of L. japonica. Result: The callus flourishing well of L. japonica was used as materials for protoplasts isolation. 0.7 mol·L-1 mannitol + 1.5% cellulose Onozuka R-10 +0.25% pectolase Yakult Y-23 was the best composition of enzyme solution, which was dissolved in cell protoplast wash medium (CPW) solution. 12-hour enzyme dissolving at 25℃ was the best methods of protoplasts isolation. And standing was better than shaking. Conclusion: Abundant and superior protoplasts of L. japonica can be obtained under the best isolation conditions that this study has found out which will give the necessary help for protoplasts culture, cell fusion and genetic transformation of L. japonica.

  1. Isolation of mutants with altered pigments after irradiating haploid protoplasts from Datura innoxia Mill. with X-rays

    International Nuclear Information System (INIS)

    Ten different mutants with altered pigment patterns were isolated following X-irradiation of approximately 105 haploid protoplasts of Datura innoxia Mill. Seven of the selected strains gave rise to shoots and 3 to leaves only. The mutants were selected from light green or white calli, which had developed 4 weeks after transfer of developing cell clusters onto B5 agar medium containing 0.5 mg/1 BAP. Of the 10 mutant strains 5 were light green, two were yellow, one was pale yellow and one was white. One additional strain does not posses anthocyanin in its stems; a feature characteristic of the wildtype is the possession of anthocyanin. This strain is able to grow in soil and has now flowered. None of the mutants obtained is haploid. Nine are diploid and the other is tetraploid. The chlorophyll deficient strains can be propagated on B5 agar medium supplemented with higher concentration of sucrose than normally required for the growth of the wild-type. (orig.)

  2. 新疆杨愈伤组织原生质体的游离与纯化%Isolation and Purification of Callus-Derived Protoplasts of Populus alba L.var.pyramidalis

    Institute of Scientific and Technical Information of China (English)

    白姗姗; 尹敏娟; 张磊; 康向阳

    2011-01-01

    目的:以愈伤组织为材料,研究新疆杨原生质体的游离、纯化.方法:以新疆杨愈伤组织为材料,采用简单试验设计和方差分析方法,对新疆杨原生质体游离的影响因素进行研究,并利用二乙酸荧光素染色法观察原生质体活力.结果:适宜新疆杨愈伤组织原生质体游离的较适宜条件是:CPW+2.0%纤维素酶R-10+1.0%离析酶R-10+1.0%果胶酶Y-23+0.6 mol/L甘露醇,酶解温度27℃,酶解时间8 h.在此条件下,原生质体产量达8.5×106个/(g·FW),活力达83.6%.原生质体纯化可采用蔗糖等密度离心法,较适蔗糖浓度为30%.结论:研究筛选出的酶解因素组合与等密度离心条件较适宜新疆杨愈伤组织原生质体的游离和纯化.%Objective: Protoplasts of Populus alba L.var.pyramidalis were isolated from calli, isolation and purification of protoplasts were studied.Methods: Using simple experimental design and variance analysis method, according to the influence factors of protoplasts isolation, isolated time and mannitol concentration of protoplasts were studied, by using FDA staining method observing viability of calli protoplasts.Results: The more appropriate yield of protoplasts was produced by using CPW salts solution containing 2.0% cellulase R-10, 1.0% macerozyme R-10,1.0% pectolase Y-23, 0.6 mol/L mannitol which incubated at 27℃ for 8 hours.The protoplasts yield was 8.5×106/ (g·FW) and the viability was 83.6% under the condition.Moreover, the more suitable protoplasts purification method was sucrose gradient centrifugation at concentration of 30%.Conclusion: The combination of different enzyme concentrations and factors is appropriate conducted on investigation into isolation and purification of calli-derived protoplasts.

  3. 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

  4. 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

  5. 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

  6. Electrofusion of Penicillium protoplasts after dielectrophoresis

    International Nuclear Information System (INIS)

    The protoplast fusion of auxotrophic Penicillium mutants after dielectrophoretic cell contact and rectangular field pulses (4.2 kV/cm, 25 μs) was microscopically observed in a microchamber consisting of two vapor-plated chromium electrodes on a microslide (gap between the electrodes of about 150 μm). The conditions for protoplasting of Penicillium and for electrofusion are described. Isolation of auxotrophic mutants was realized after treatment of spores with UV light and X-rays. (author)

  7. 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.

  8. 酿酒葡萄‘桂葡1号’幼叶原生质体分离研究%Research on Protoplast Isolation from Young Leaves of Wine Grape ‘ GuiPu No.1 '

    Institute of Scientific and Technical Information of China (English)

    唐文忠; 廖芬; 黄茂康; 卢塔山; 黄伟雄

    2011-01-01

    The optimal isolation conditions of protoplasts from young leaves of wine grape 'GuiPu No.l' was studied in this paper. The influence of some factors, such as enzyme composition, time of enzymatic hydrolysis, concentration of stabilizer, as well as speed and time of centrifugation, on isolation of protoplasts was comparatively analyzed, using young leaves of ' GuiPu No. 1' aseptic seedling with 20-30 day as materials. The appropriate enzyme composition was 2%cellulase + 0.5%pectinase. The protoplast yield gradually increased with extension of enzymatic hydrolysis time. The appropriate enzymatic hydrolysis time was 8 hours for protoplast isolation from young leaves of 'GuiPu No.l'. The protoplast yield significantly increased with rise of mannitol concentration in the range from 0.45-0.55 mol/L and the yield reached highest at mannitol concentration 0.55 mol/L 2.48 × 106 protoplast/(g ? FW) and activity 79.78%. The centrifugation speed of 1000 r/min and the centrifugation time of 6-8 min was appropriate for suspension purification of protoplast. The optimal enzyme solution for protoplast isolation was as follows: 2% Celluasw Onozuka R-10 + 0.5% PectolyaseY-23+25 mmol/L MES+0.55 mol/L Mannitol. Protoplasts with high yield and viability were obtained when incubation for 8 h. The centrifugation at 1000 r/min for 6-8 min in 30% sucrose was good for suspension purification of protoplast.%研究酿酒葡萄‘桂葡1号’幼叶原生质体的最佳分离条件.以‘桂葡1号’20~30日龄无菌苗幼叶为分离原生质体试材,对分离过程中酶液组合、酶解时间、稳定剂浓度、纯化时离心速度及离心时间进行比较分析.酶组合以2%纤维素酶+0.5%果胶酶为宜.随着酶解时间的延长,原生质体的产量逐渐增高,适合‘桂葡1号’幼叶原生质体的酶解时间以8h为宜.在0.45~0.55 mol/L的甘露醇范围内,随浓度提高,原生质的产率明显上升,0.55 mol/L时达到最大2.48×106

  9. 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.

  10. Optimized condition for protoplast isolation from maize,wheat and rice leaves%玉米、小麦、水稻原生质体制备条件优化

    Institute of Scientific and Technical Information of China (English)

    孙鹤; 郎志宏; 朱莉; 黄大昉

    2013-01-01

    Maize (Zea mays L.), wheat (Triticum aestivum L.) and rice (Oryza sativa L.) are three staple crops and accordingly it is very meaningful to optimize the condition of their protoplasts isolation. The concentration of the enzyme, the time of isolation and centrifugal force in protoplast isolation were investigated to find their effects on protoplast yield and viability using leaves of maize (Zong 3), wheat (Chinese Spring) and rice (Nipponbare). The results show that the concentration of the enzyme and the time of isolation affected the protoplast yield significantly. Although the yield of protoplast was increased with high concentration of enzyme and long incubated time, it led to too much cells breakdown. The orthogonal experimental design results show that the best condition of maize protoplast isolation was Cellulase R-10 1.5%, Macerozyme R-10 0.5%, 50 r/min 7 h, 100×g 2 min and the protoplasts yield was 7×106 cells/g fresh weight (FW); the best condition of wheat protoplast isolation was Cellulase R-10 1.5%, Macerozyme R-10 0.5%, 50 r/min 5 h, l00×g 2 min and the protoplasts yield was 6×106 cells/g FW; the best condition of rice protoplast isolation was Cellulase R-10 2.0%, Macerozyme R-10 0.7%, 50 r/min 7 h, 1 000×g 2 min and the protoplasts yield was 6×106 cells/g FW. The vitalities were more than 90% using fluorescein diacetate staining method. 50%-80% transformation efficiency was obtained when protoplasts were transformed by green fluorescent protein using PEG-Ca2+ method.%玉米Zea mays L.、小麦Triticum aestivum L.、水稻Oryza sativaL.是三大重要粮食作物,对其原生质体制备条件的优化具有重要意义.以玉米(综3)、小麦(中国春)、水稻(日本晴)10日龄幼苗为材料,研究了叶肉细胞原生质体分离过程中的酶浓度、酶解时间和离心力大小等因素对产量和活力的影响.结果表明:酶浓度和酶解时间对原生质体产量影响显著,随着酶解液浓度和酶解时间的提高,原

  11. Formation of interspecies fusants of Agaricus bisporus and Agaricus bitorquis mushroom by protoplast fusion

    OpenAIRE

    Singh, Rana Inder; Aarti, Kanojiya; Singh, Sandhu Sardul

    2007-01-01

    Interspecies fusants are formed between Agaricus bisporus and Agaricus bitorquis by protoplast fusion technique. Protoplasts were isolated and regenerated by using Novozyme 234 lytic enzyme. Twenty slow growing isolates were separated from the protoplast regenerated colonies, which were assumed as homokaryons (putative homokaryons). These twenty isolates were subjected to growth rate, colony morphology and spawn run studies for screening of true homokaryons. Antifungal markers were developed ...

  12. Interaction of E. coli DNA with tobacco mesophyll protoplasts

    International Nuclear Information System (INIS)

    This chapter is part of a dissertation dealing with the interaction of DNA with protoplasts. Having established the length of time during which tobacco mesophyll protoplasts do not synthesize DNA following their isolation, it is important to know the extent of DNA uptake just before the onset of DNA synthesis (and possible integration) and to find optimal conditions for this uptake. Therefore, the association of E. coli DNA with tobacco protoplasts was studied. Care should be taken with the interpretation of ''uptake'' results: adsorption phenomena play a very important role and may do so at the plasmalemma of naked protoplasts. To solve the problems involved, the use of radiation-damaged DNA was attempted. With E. coli DNA possessing a large number of thymine containing pyrimidine dimers, the loss of dimers from DNA recovered from treated protoplasts was tested in order to obtain an indication of ''real'' uptake. The results are reported

  13. Protoplasts and plant viruses

    International Nuclear Information System (INIS)

    The use of protoplasts in the study of plant viruses has attracted considerable attention since its inception in the late 1960s. This article is an attempt to assess the current status of protoplasts (primarily) and all cell cultures (in some instances) in studies of virus infection, virus replication, cytopathology, cross-protection, virus resistance, and the use of in vitro methods and genetic engineering to recover virus-resistant plants. These areas of study proved difficult to do entirely with whole plants or plant parts. However, because protoplasts could be synchronously infected with virus, they provided a valuable alternative means of following biochemical and cytological events in relation to the virus growth cycle in a more precise manner than previously possible

  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. Valine-Resistance, a Potential Marker in Plant Cell Genetics. I. Distinction between Two Types of Valine-Resistant Tobacco Mutants Isolated from Protoplast-Derived Cells

    OpenAIRE

    Bourgin, J. P.; Goujaud, J.; Missonier, C.; Pethe, C.

    1985-01-01

    In previous experiments, seven lines of valine-resistant plants were regenerated from protoplast-derived haploid tobacco mesophyll cells which had been UV mutagenized and submitted to selection by toxic concentrations of valine. In this study we described the transmission of valine-resistance to progeny and a preliminary phenotypical and biochemical characterization of the resistant plants.—Two types were thus distinguished among the seven mutant lines. Valine-resistance of the mutants of the...

  17. Protoplast preparation from monokaryotic mycelium of Pleurotus sajor-caju using lysing enzyme

    International Nuclear Information System (INIS)

    The objective of this study was to determine the optimum parameters of the factors influencing protoplast isolation from monokaryotic mycelium of Pleurotus sajor-caju using lysing enzyme from Trichoderma harzianurm. The study was conducted by manipulating the variables of the factors affecting protoplast isolation, such as age of mycelium culture, period for lysing of mycelium, concentration of lysing enzyme and concentration of osmotic stabilizer. The highest protoplast yield of 8.3 x 104 protoplast/ml was achieved when a 3-day P. sajor-caju mycelium, cultured statically, was incubated for 3 hours in a lytic mixture containing 7.5 mg/ml lysing enzyme and 1.2 M ammonium sulfate as osmotic stabilizer. This protoplast yield, however, is insufficient for regeneration and protoplast fusion works. (Author)

  18. Protoplast formation and regeneration in Lactobacillus delbrueckii

    OpenAIRE

    Singhvi, Mamta; Joshi, Dipti; Gaikaiwari, Shalaka; Digambar V. Gokhale

    2010-01-01

    Method for production and regeneration of Lactobacillus delbrueckii protoplasts are described. The protoplasts were obtained by treatment with a mixture of lysozyme and mutanolysin in protoplast buffer at pH 6.5 with different osmotic stabilizers. The protoplasts were regenerated on deMan, Rogosa and Sharpe (MRS) with various osmotic stabilizers. Maximum protoplast formation was obtained in protoplast buffer with sucrose as an osmotic stabilizer using a combination of lysozyme (1 mg/ml) and m...

  19. Research on protoplast preparation and culture in Gladiolus hybridus Hort.

    Institute of Scientific and Technical Information of China (English)

    ZHANG Ying; GONG Sufang; WANG Jingang; CHE Daidi

    2007-01-01

    Protoplasts prepared from the aseptic leaves of Rose Supreme in Gladiolus hybridus Hort. were cultured. The yield of protoplasts with vigour was the highest under the following conditions: 1.5% cellulase Onzuka RS, 0.5% pectinase Y-23, 0.6 mol· L-1 mannitol, pH 5.8, digestion time 2 h, and temperature 27 ℃. After isolation and purification, protoplasts were cultured in solid and liquid combined medium, which included KM8P, 0.6 mol· L-1 mannitol, 1.0 mg· L-1 2, 4-D, 0.2 mg· L-1 NAA and 0.2 mg· L-1 KT. The division of the protoplasts first emerged in medium after cultured 4 days, and emerged only a few times.

  20. 3D fluorescent in situ hybridization using Arabidopsis leaf cryosections and isolated nuclei

    Directory of Open Access Journals (Sweden)

    Biot Eric

    2009-08-01

    Full Text Available Abstract Background Fluorescent hybridization techniques are widely used to study the functional organization of different compartments within the mammalian nucleus. However, few examples of such studies are known in the plant kingdom. Indeed, preservation of nuclei 3D structure, which is required for nuclear organization studies, is difficult to fulfill. Results We report a rapid protocol for fluorescent in situ hybridization (FISH performed on 3D isolated nuclei and thin cryosectioned leaves of Arabidopsis thaliana. The use of direct labeling minimized treatment steps, shortening the overall procedure. Using image analysis, we measured different parameters related to nucleus morphology and overall 3D structure. Conclusion Our work describes a 3D-FISH protocol that preserves the 3D structure of Arabidopsis interphase nuclei. Moreover, we report for the first time FISH using cryosections of Arabidopsis leaves. This protocol is a valuable tool to investigate nuclear architecture and chromatin organization.

  1. Polyamine metabolism and osmotic stress. I. Relation to protoplast viability

    Science.gov (United States)

    Tiburcio, A. F.; Masdeu, M. A.; Dumortier, F. M.; Galston, A. W.

    1986-01-01

    Cereal leaves subjected to the osmotica routinely used for protoplast isolation show a rapid increase in arginine decarboxylase activity, a massive accumulation of putrescine, and slow conversion of putrescine to the higher polyamines, spermidine and spermine (HE Flores, AW Galston 1984 Plant Physiol 75: 102). Mesophyll protoplasts from these leaves, which have a high putrescine:polyamine ratio, do not undergo sustained division. By contrast, in Nicotiana, Capsicum, Datura, Trigonella, and Vigna, dicot genera that readily regenerate plants from mesophyll protoplasts, the response of leaves to osmotic stress is opposite to that in cereals. Putrescine titer as well as arginine and ornithine decarboxylase activities decline in these osmotically stressed dicot leaves, while spermidine and spermine titers increase. Thus, the putrescine:polyamine ratio in Vigna protoplasts, which divide readily, is 4-fold lower than in oat protoplasts, which divide poorly. We suggest that this differing response of polyamine metabolism to osmotic stress may account in part for the failure of cereal mesophyll protoplasts to develop readily in vitro.

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

    NARCIS (Netherlands)

    Vliegenthart, J.F.G.; 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 retaine

  3. Lentil root protoplasts: a transient expression system suitable for coelectroporation of monoclonal antibodies and plasmid molecules

    NARCIS (Netherlands)

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

    1995-01-01

    Protoplasts were isolated from lentil (Lens culinaris) roots and their suitability as a transient expression system was investigated. After transfecting the protoplasts with the -glucuronidase (GUS) gene by either electroporation or polyethylene glycol (PEG), the specific activity of the reporter en

  4. 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)

  5. Cell fusion hybrids. [Nicotiana protoplasts

    Energy Technology Data Exchange (ETDEWEB)

    Smith, H H

    1976-01-01

    Cell fusion hybrids were obtained by fusing protoplasts of Nicotiana glauca and N. langsdorffii in the presence of polyethylene glycol. The hybrid protoplasts were selected out of a mixed population by growing on a culture medium that does not support the growth of parental protoplasts. The cell fusion hybrids had chromosome numbers that were higher (56 to 64) than in the amphiploid (2n = 42). Most of these hyper-aneuploids were fertile and their progeny retained the characteristic morphology and approximate chromosome number of their hybrid parent.

  6. 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.

  7. Protoplast culture of some economically important plants: Studies on plant regeneration

    International Nuclear Information System (INIS)

    Protoplast culture was attempted for three economically important plants - Santalum album (Indian sandalwood tree), Tylophora indica (a medicinal plant) and Arachis hypogaea (peanut). In sandalwood, protoplasts were successfully isolated from various explants, such as leaf mesophylls, stem and hypocotyl calli and cell suspensions derived from a callus. In the cases of Tylophora callus tissues and Arachis, leaf mesophylls were used as source materials for isolating protoplasts. Different enzyme mixtures (pH 5.5-5.8), containing combinations of cellulase, hemicellulase, pectinase, macerozyme and driselase, were employed in various concentrations. Protoplasts of all three plants divided and yielded rapidly proliferating callus tissues. In sandalwood and Tylophora, plant regeneration was achieved through somatic embryogenesis/organogenesis. Protoplast-derived calli of peanut lacked the ability for plant regeneration. (author)

  8. 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.

  9. 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.

  10. Improving the culture of cucumber protoplasts by using an agarose-disc procedure

    International Nuclear Information System (INIS)

    A method is described for the isolation of protoplasts from cotyledons of cucumber (Cucumis sativus L.). After isolation, protoplasts were embedded in a mixture of agarose and Murashige and Skoog medium supplemented with 250 mg/L trypton, 2% sucrose, 5 μM naphthaleneacetic acid and 15 μM 2iP. The culture of the protoplasts was improved by the application of an agarose-disc culture procedure. The embedded protoplasts were plated in small (100 μL) droplets in Petri dishes to which, after gelling of the agarose, liquid medium was added. For comparison, protoplasts were also cultured according to the agarose-bead procedure. The plating efficiency ranged from 50 to 80% if the protoplasts were plated at high density (105 protoplasts per mL). In agarose-bead culture, divisions were induced at a lower rate. At lower densities the plating efficiency was dramatically decreased. Growth of microcalli was determined by homogenizing culture samples and measuring their density spectrophotometrically. The growth rate of the developing cell clusters was much higher in agarose-disc cultures as compared with bead-type cultures. It is concluded that for cucumber protoplasts the agarose-disc culture procedure provided optimal conditions for both the initiation of cell division and the growth of microcalli. (author)

  11. 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%.

  12. Genotypic variability for protoplast regeneration in Saintpaulia ionantha (H. Wendl.).

    Science.gov (United States)

    Winkelmann, T; Grunewaldt, J

    1995-08-01

    The behaviour of eleven Saintpaulia ionantha (H. Wendl.) genotypes in protoplast culture was compared. Isolation of protoplasts from young shootlets regenerated in vitro on leaf explants, yielded 0.7 to 1.8 × 10(6) protoplasts per gram fresh weight. In all cultivars and breeding lines tested, cell divisions were observed. The mean division frequencies varied between 1.0 and 5.0% after 14 days, and between 6.4 and 13.8% after 24 days of culture. In ten genotypes callussing and shoot regeneration were achieved. The difference between the genotypes in shoot regeneration rate, between 2 and 68%, was more pronounced. The comparison of four cytokinins indicated hat thidiazuron was most effective for shoot regeneration, but often resulted in poorer shoot quality than benzylaminopurine. PMID:24186626

  13. 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.

  14. Isolation and Functional Analysis of ZmLTP3, a Homologue to Arabidopsis LTP3

    Directory of Open Access Journals (Sweden)

    Guo-Hui Ma

    2013-03-01

    Full Text Available Plant lipid transfer proteins (LTPs are encoded by multigene families and play important roles in plant physiology. One full-length cDNA encoding an Arabidopsis LTP3 homologue was isolated from maize by RT-PCR and named as ZmLTP3. RT-PCR analysis indicated that the ZmLTP3 expression is induced by salicylic acid (SA, mannitol and salt. Furthermore, in different tissues the ZmLTP3 displayed different expression patterns, indicating that ZmLTP3 may play multiple roles in stress resistance. Over-expression of ZmLTP3 in wild-type Arabidopsis resulted in the increased salt tolerance. Under salt stress condition, compared to wild-type (WT plants, transgenic Arabidopsis grew better, had higher seedling fresh (FW, dry weight (DW, seed yields, proline content and lower MDA content and relative electric conductivity level. Our results suggest that maize ZmLTP3 might encode a member of LTPs family and play roles in salt resistance.

  15. Isolation and Functional Analysis of ZmLTP3, a Homologue to Arabidopsis LTP3.

    Science.gov (United States)

    Zou, Hua-Wen; Tian, Xiao-Hai; Ma, Guo-Hui; Li, Zhi-Xin

    2013-01-01

    Plant lipid transfer proteins (LTPs) are encoded by multigene families and play important roles in plant physiology. One full-length cDNA encoding an Arabidopsis LTP3 homologue was isolated from maize by RT-PCR and named as ZmLTP3. RT-PCR analysis indicated that the ZmLTP3 expression is induced by salicylic acid (SA), mannitol and salt. Furthermore, in different tissues the ZmLTP3 displayed different expression patterns, indicating that ZmLTP3 may play multiple roles in stress resistance. Over-expression of ZmLTP3 in wild-type Arabidopsis resulted in the increased salt tolerance. Under salt stress condition, compared to wild-type (WT) plants, transgenic Arabidopsis grew better, had higher seedling fresh (FW), dry weight (DW), seed yields, proline content and lower MDA content and relative electric conductivity level. Our results suggest that maize ZmLTP3 might encode a member of LTPs family and play roles in salt resistance. PMID:23455470

  16. Isolation and Functional Analysis of ZmLTP3, a Homologue to Arabidopsis LTP3

    OpenAIRE

    Guo-Hui Ma; Xiao-Hai Tian; Hua-Wen Zou; Zhi-Xin Li

    2013-01-01

    Plant lipid transfer proteins (LTPs) are encoded by multigene families and play important roles in plant physiology. One full-length cDNA encoding an Arabidopsis LTP3 homologue was isolated from maize by RT-PCR and named as ZmLTP3. RT-PCR analysis indicated that the ZmLTP3 expression is induced by salicylic acid (SA), mannitol and salt. Furthermore, in different tissues the ZmLTP3 displayed different expression patterns, indicating that ZmLTP3 may play multiple roles in stress resistance. Ove...

  17. 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.

  18. Calogênese, embriogênese somática e isolamento de protoplastos em variedades de laranja doce Callus induction, somatic embryogenesis and protoplast isolation from sweet orange varieties

    Directory of Open Access Journals (Sweden)

    Vagner Augusto Benedito

    2000-03-01

    Full Text Available Com o objetivo de produzir calos embriogênicos de citros, óvulos abortados de frutos maduros de 6 variedades de laranja doce (Citrus sinensis L. Osbeck, ‘Bahia Cabula’, ‘Baianinha’, ‘Hamlin’, ‘Orvalho de Mel’, ‘Rubi’ e ‘Valência’ foram introduzidos em meio de cultivo MT modificado com adição de extrato de malte, com e sem adição de 5 mg L-1 benziladenina (BA. Os calos obtidos das variedades ‘Bahia Cabula’, ‘Orvalho de Mel’, ‘Rubi’ e ‘Valência’ foram cultivados em meio basal MT, contendo os carboidratos maltose, galactose, lactose, glicose ou sacarose nas concentrações de 18; 37; 75; 110 e 150 mM para a indução da embriogênese somática. Os calos destas mesmas variedades foram submetidos ao isolamento de protoplastos, com o uso de 3 soluções enzimáticas. Os resultados revelaram que há influência do genótipo sobre a calogênese. A embriogênese somática foi estimulada na presença de galactose, para a maioria das variedades. A solução enzimática composta de 1% de celulase, 1% de macerase e 0,2% de pectoliase foi a mais adequada para o isolamento de protoplastos a partir de calos das variedades de laranja doce estudadas.Intending to produce embryogenic citrus calli (Citrus sinensis L. Osbeck, aborted ovules of ripe fruits from six sweet orange varieties (‘Bahia Cabula’, ‘Baianinha’, ‘Hamlin’, ‘Orvalho de Mel’, ‘Rubi’ and ‘Valencia’ were introduced onto modified MT medium with and without benzyladenine (BA. Calli obtained from ‘Bahia Cabula’, ‘Orvalho de Mel’, ‘Rubi’ and ‘Valencia’ varieties were grown on MT basal medium modified with the carbohydrates maltose, galactose, lactose, glucose and sucrose at 18; 37; 75; 110 and 150 mM. The effect on somatic embryogenesis was studied. Calli from the same genotypes were submitted to protoplast isolation, using three different enzymatic solutions. Results revealed the genotype effect in the callus

  19. A Sulfonylurea Herbicide Resistance Gene from Arabidopsis thaliana as a New Selectable Marker for Production of Fertile Transgenic Rice Plants.

    Science.gov (United States)

    Li, Z; Hayashimoto, A; Murai, N

    1992-10-01

    A mutant acetolactate synthase (ALS) gene, csr1-1, isolated from sulfonylurea herbicide-resistant Arabidopsis thaliana, was placed under control of a cauliflower mosaic virus 35S promoter (35S). Rice protoplasts were transformed with the 35S/ALS chimeric gene and regenerated into fertile transgenic rice (Oryza sativa) plants. The 35S/ALS gene was expressed effectively as demonstrated by northern blot hybridization analysis, and conferred to transformed calli at least 200-fold greater chlorsulfuron resistance than nontransformed control calli. Effective selection of 35S/ALS-transformed protoplasts was achieved at extremely low chlorsulfuron concentrations of 10 nm. The results demonstrated that the 35S/ALS gene is an alternative selectable marker for rice protoplast transformation and fertile transgenic rice production. The results also suggest that the mutant form of Arabidopsis ALS enzyme operates normally in rice cells. Thus, the mechanism of protein transport to chloroplast and ALS inhibition by chlorsulfuron is apparently conserved among plant species as diverse as Arabidopsis (dicotyledon) and rice (monocotyledon). PMID:16653044

  20. Progress towards sugar beet improvement through somatic hybridization. Pt. 1. Inactivation of nuclei and cytoplasm in donor and recipient protoplasts

    International Nuclear Information System (INIS)

    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. (author)

  1. 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.

  2. 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.

  3. The location of coat protein and viral RNAs of alfalfa mosaic virus in infected tobacco leaves and protoplasts

    NARCIS (Netherlands)

    Pelt-Heerschap, H. van; Verbeek, H.; Slot, J.W.; Vloten-Doting, L. van

    1987-01-01

    The location of coat protein of alfalfa mosaic virus (AIMV) strain 425 was determined in protoplasts isolated from infected tobacco leaves and in in vitro inoculated tobacco protoplasts, using immunocytochemistry on ultrathin frozen sections labeled with colloidal gold. In infected tobacco leaves 5

  4. Variability of enzymatic activities in ligninolytic fungi Pleurotus ostreatus and Lentinus tigrinus after protoplasting and UV-mutagenization

    International Nuclear Information System (INIS)

    Protoplast preparation and UV-irradiation of mycelial fragments were used to study the variability of production of laccase, peroxidase and manganese-dependent peroxidase (MnP) involved in lignin degradation in Pleurotus ostreatus and Lentinus tigrinus. After protoplasting, the variability of production of all enzymes increased substantially and was comparable to that of isolates after mutagenesis. (author)

  5. Microcolony formation from embryogenic callus-derived protoplasts of oil palm

    Directory of Open Access Journals (Sweden)

    Sompong Te-chato

    2005-07-01

    Full Text Available Embryogenic callus of oil palm induced from young leaves of seedlings DxP was used as initial material for protoplast isolation. Various combinations of cellulase Onozuka RS and macerozyme R-10 were tested. Isolated protoplasts were cultured by various methods in MS medium supplemented with different phytohormones. The results revealed that 2% cellulase RS in combination with 2% macerozyme R-10 (adjusted osmoticum to 0.4 M by manitol yielded the highest number of viable protoplasts (1x107 per gram fresh weight. Dicamba at concentration 2 mg/l with 1 mg/l 6-benzyladenin (BA containing in phytagel semisolidified MS medium promoted the highest division of 2.3-4.0%. First division of the protoplasts was observed at 4 days after culture. Microcolony formation (8-10 cells was seen after three weeks of culture. Unfortunately, neither callus formation nor plantlet regeneration were obtained.

  6. 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.

  7. Protoplast fusion between Pleurotus ostreatus and P. djamor

    Directory of Open Access Journals (Sweden)

    Chaninan Pornsuriya

    2005-09-01

    Full Text Available Protoplast fusion between Pleurotus ostreatus and P. djamor was carried out by isolating protoplasts from 4-day-old monokaryotic mycelia cultured on malt extract broth. The mycelia were then agitated at 100 rpm for 2 h with 9 mg Lysing Enzyme (Sigma L-1412 in 1 ml osmotic stabilizer (0.6 M MgSO4·7H2O in 0.05 M sodium maleate buffer,pH 5. The freshly prepared protoplasts were then mixed and incubated in 40% PEG (polyethylene glycol 6,000/0.05 M CaCl2·2H2O for 20 min at room temperature. All protoplasts were regenerated on Regeneration Medium for 7-12 days. There were 412 regenerated colonies detected but only two of them were selected as fusants by posessing clamp connections on their mycelia. The fusants were proved to be "hybrids" of P. ostreatus and P. djamor. Their mycelia were significantly faster in growth and larger in size than the parental strains. They showed bands common to their parents when esterase was used for isozyme studies. The fruiting bodies of the fusants also showed recombined characteristics of the parental strains.

  8. Dimethomorph and metalaxyl sensitivity in somatic hybrids of Phytophthora parasitica obtained by protoplast fusion

    OpenAIRE

    Chabanne, Kamel; Leroux, P; Bompeix, G.; Maia, N.

    1996-01-01

    Protoplasts were successfully isolated from wild-type and mutant strains of #Phytophthora nicotianae$ var. #parasitica$ using Novozym 234. Putative somatic hybrids were recovered following protoplast fusions from the first time to dimethomorph resistant strain P 310 (Dim r) or metalaxyl P 26 (Met r) by selection on agar amended with dimethomorph and metalaxyl. Fusion products from this cross were resistant to dimethomorph and metalaxyl. Zoospore progeny from the fusion products retained this ...

  9. 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. PMID:26058149

  10. Assessment of resistance pathways induced in Arabidopsis thaliana by hypovirulent Rhizoctonia spp. isolates.

    Science.gov (United States)

    Sharon, Michal; Freeman, Stanley; Sneh, Baruch

    2011-07-01

    Certain hypovirulent Rhizoctonia isolates effectively protect plants against well-known important pathogens among Rhizoctonia isolates as well as against other pathogens. The modes of action involved in this protection include resistance induced in plants by colonization with hypovirulent Rhizoctonia isolates. The qualifications of hypovirulent isolates (efficient protection, rapid growth, effective colonization of the plants, and easy application in the field) provide a significant potential for the development of a commercial microbial preparation for application as biological control agents. Understanding of the modes of action involved in protection is important for improving the various aspects of development and application of such preparations. The hypothesis of the present study is that resistance pathways such as systemic acquired resistance (SAR), induced systemic resistance (ISR), and phytoalexins are induced in plants colonized by the protective hypovirulent Rhizoctonia isolates and are involved in the protection of these plants against pathogenic Rhizoctonia. Changes in protection levels of Arabidopsis thaliana mutants defective in defense-related genes (npr1-1, npr1-2, ndr1-1, npr1-2/ndr1-1, cim6, wrky70.1, snc1, and pbs3-1) and colonized with the hypovirulent Rhizoctonia isolates compared with that of the wild type (wt) plants colonized with the same isolates confirmed the involvement of induced resistance in the protection of the plants against pathogenic Rhizoctonia spp., although protection levels of mutants constantly expressing SAR genes (snc1 and cim6) were lower than that of wt plants. Plant colonization by hypovirulent Rhizoctonia isolates induced elevated expression levels of the following genes: PR5 (SAR), PDF1.2, LOX2, LOX1, CORI3 (ISR), and PAD3 (phytoalexin production), which indicated that all of these pathways were induced in the hypovirulent-colonized plants. When SAR or ISR were induced separately in plants after application of the

  11. 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.

  12. 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. PMID:26473358

  13. 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.

  14. Isolation of uvh1, an Arabidopsis mutant hypersensitive to ultraviolet light and ionizing radiation

    International Nuclear Information System (INIS)

    A genetic screen for mutants of Arabidopsis that are hypersensitive to UV light was developed and used to isolate a new mutant designated uvh1. UV hypersensitivity in uvh1 was due to a single recessive trait that is probably located on chromosome 3. Although isolated as hypersensitive to an acute exposure to UV-C light, uvh1 was also hypersensitive to UV-B wavelengths, which are present in sunlight that reaches the earth's surface. UV-B damage to both wild-type and uvh1 plants could be significantly reduced by subsequent exposure of UV-irradiated plants to photoreactivating light, showing that photoreactivation of UV-B damage is important for plant viability and that uvh1 plants are not defective in photoreactivation. A new assay for DNA damage, the Dral assay, was developed and used to show that exposure of wild-type and uvh1 plants to a given dose of UV light induces the same amount of damage in chloroplast and nuclear DNA. Thus, uvh1 is not defective in a UV protective mechanism. uvh1 plants were also found to be hypersensitive to ionizing radiation. These results suggest that uvh1 is defective in a repair or tolerance mechanism that normally provides plants with resistance to several types of DNA damage

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

    International Nuclear Information System (INIS)

    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 host DNA excision repair system. Previous results (R.E. Yasbin, J.D. Fernwalt, and P.I. Fields, J. Bacteriol.; 137: 391-396) showed that ultraviolet-irradiated bacteriophage DNA could not be repaired via the excision repair system of competent cells. Therefore, the processing of bacteriophage DNA by protoplasts and by competent cells must be different. This sensitive protoplast assay can be used to identify and to isolate various types of DNA repair enzymes

  16. 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

  17. The Saharan isolate Saccharothrix algeriensis NRRL B-24137 induces systemic resistance in Arabidopsis thaliana seedlings against Botrytis cinerea

    OpenAIRE

    Muzammil, Saima; Graillon, Clotilde; Saria, Rayenne; Mathieu, Florence; Lebrihi, Ahmed; Compant, Stéphane

    2013-01-01

    Background and aim Saccharothrix algeriensis NRRL B-24137, isolated from a Saharan soil, has been described as a potential biocontrol agent against Botrytis cinerea and other phytopathogens. However, the plant protection mechanisms involved still need to be described. The aim of this study was to determine this protection phenomenon as well as parts of the mechanisms involved, using Arabidopsis thaliana seedlings and B. cinerea. Methods The bacterial colonization process was evaluated on A. t...

  18. Isolation and characterization of Arabidopsis mutants defective in the induction of ethylene biosynthesis by cytokinin

    Science.gov (United States)

    Vogel, J. P.; Schuerman, P.; Woeste, K.; Brandstatter, I.; Kieber, J. J.; Evans, M. L. (Principal Investigator)

    1998-01-01

    Cytokinins elevate ethylene biosynthesis in etiolated Arabidopsis seedlings via a post-transcriptional modification of one isoform of the key biosynthetic enzyme ACC synthase. In order to begin to dissect the signaling events leading from cytokinin perception to this modification, we have isolated a series of mutants that lack the ethylene-mediated triple response in the presence of cytokinin due to their failure to increase ethylene biosynthesis. Analysis of genetic complementation and mapping revealed that these Cin mutants (cytokinin-insensitive) represent four distinct complementation groups, one of which, cin4, is allelic to the constitutive photomorphogenic mutant fus9/cop10. The Cin mutants have subtle effects on the morphology of adult plants. We further characterized the Cin mutants by analyzing ethylene biosynthesis in response to various other inducers and in adult tissues, as well as by assaying additional cytokinin responses. The cin3 mutant did not disrupt ethylene biosynthesis under any other conditions, nor did it disrupt any other cytokinin responses. Only cin2 disrupted ethylene biosynthesis in multiple circumstances. cin1 and cin2 made less anthocyanin in response to cytokinin. cin1 also displayed reduced shoot initiation in tissue culture in response to cytokinin, suggesting that it affects a cytokinin signaling element.

  19. Isolation and characterization of hormone-autonomous tumours of Arabidopsis thaliana

    International Nuclear Information System (INIS)

    In order to study the molecular genetics of factors controlling plant cell growth, we have isolated and begun to characterize a set of tumours on the small crucifer Arabidopsis thaliana. Seeds or seedlings were exposed to 60Co gamma radiation and, 30–60 d after germination, tumours developed either on the hypocotyl or in the region of the apical meristem of about 1% of the plants. When excised and placed in culture, some of these tumours were found to be capable of hormone-independent growth. The tumours exhibit a number of different phenotypes, varying in colour, texture, and degree of differentiation. Some tumours appear to be completely undifferentiated, one consistently produces roots, and others show the sporadic appearance of shoots or leaflets. Doubling times of the tumours on hormone-free medium range from approximately 2 d to 9 d. We propose that these tumours arose due to heritable changes in the genome which result in altered expression of important growth-regulatory genes. Preliminary investigations of gene expression in the tumours have led to the identification of an mRNA that is abundant in all of the tumours, differentially expressed in plant organs and hormone-dependent callus grown on different auxins, and which encodes a putative glycine-rich protein. (author)

  20. Isolation of a strong Arabidopsis guard cell promoter and its potential as a research tool

    Directory of Open Access Journals (Sweden)

    Siegel Robert S

    2008-02-01

    Full Text Available Abstract Background A common limitation in guard cell signaling research is that it is difficult to obtain consistent high expression of transgenes of interest in Arabidopsis guard cells using known guard cell promoters or the constitutive 35S cauliflower mosaic virus promoter. An additional drawback of the 35S promoter is that ectopically expressing a gene throughout the organism could cause pleiotropic effects. To improve available methods for targeted gene expression in guard cells, we isolated strong guard cell promoter candidates based on new guard cell-specific microarray analyses of 23,000 genes that are made available together with this report. Results A promoter, pGC1(At1g22690, drove strong and relatively specific reporter gene expression in guard cells including GUS (beta-glucuronidase and yellow cameleon YC3.60 (GFP-based calcium FRET reporter. Reporter gene expression was weaker in immature guard cells. The expression of YC3.60 was sufficiently strong to image intracellular Ca2+ dynamics in guard cells of intact plants and resolved spontaneous calcium transients in guard cells. The GC1 promoter also mediated strong reporter expression in clustered stomata in the stomatal development mutant too-many-mouths (tmm. Furthermore, the same promoter::reporter constructs also drove guard cell specific reporter expression in tobacco, illustrating the potential of this promoter as a method for high level expression in guard cells. A serial deletion of the promoter defined a guard cell expression promoter region. In addition, anti-sense repression using pGC1 was powerful for reducing specific GFP gene expression in guard cells while expression in leaf epidermal cells was not repressed, demonstrating strong cell-type preferential gene repression. Conclusion The pGC1 promoter described here drives strong reporter expression in guard cells of Arabidopsis and tobacco plants. It provides a potent research tool for targeted guard cell expression or

  1. 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.

  2. Transformation of Schizosaccharomyces pombe: Protoplast Procedure.

    Science.gov (United States)

    Murray, Johanne M; Watson, Adam T; Carr, Antony M

    2016-04-01

    Transformation of Schizosaccharomyces pombe with DNA requires the conditioning of cells to promote DNA uptake followed by cell growth under conditions that select and maintain the plasmid or integration event. The three main methodologies are electroporation, treatment with lithium cations, and transformation of protoplasts. The protocol for protoplast transformation, which is described here, is more complicated than those for electroporation or lithium acetate and thus less often used. However, for some strains, it remains the only reliable protocol. PMID:27037076

  3. Uptake of sodium in quince, sugar beet, and wheat protoplasts determined by the fluorescent sodium-binding dye benzofuran isophthalate.

    Science.gov (United States)

    D'Onofrio, Claudio; Kader, Abdul; Lindberg, Sylvia

    2005-04-01

    The uptake of sodium into protoplasts of quince (Cydonia oblonga Mill, clone BA29), sugar beet (Beta vulgaris L. cv. Monohill), and wheat (Triticum aestivum L. cv. Kadett) was determined by use of the acetoxy methyl ester of the fluorescent sodium-binding benzofuran isopthalate (SBFI-AM). In the presence of 1 mM CaCl2, little sodium was taken up in the cytosol of quince mesophyll cells compared to cytosols of sugar beet and wheat. Upon addition of 40 mM NaCl, approximately the same amount of sodium was taken up in leaf and root protoplasts of wheat, but no sodium was taken up in quince. However, in calcium-free medium, obtained by addition of ethylene glycol tetra acetic acid (EGTA), quince protoplasts transiently took up sodium in the cytosol when 200-400 mM NaCl was added to the protoplast medium. Moreover, after cultivation of quince in the presence of 200 mM sodium for 4 weeks, the cytosol of isolated protoplasts did not take up any sodium at all from a calcium-free medium. The results show that protoplasts from salt tolerant quince only temporarily take up sodium in the cytosol and that they have a mechanism for fast extrusion of sodium from that compartment. These mechanisms are probably important for the high salt tolerance of quince. Calcium blocks the sodium uptake into the cytosol of both quince and wheat protoplasts. PMID:15900884

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

    Directory of Open Access Journals (Sweden)

    Boyu Cui

    Full Text Available 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.

  5. 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.

  6. Cloning of a Nicotiana plumbaginifolia protoplast-specific enhancer-like sequence

    OpenAIRE

    Horth, Marie; Negrutiu, Ioan; Burny, Arséne; Van Montagu, Marc; Herrera-Estrella, Luis

    1987-01-01

    We have isolated a 1.5-kb plant DNA fragment (called insert 7) from Nicotiana plumbaginifolia DNA that contains a protoplast-specific enhancer-like sequence. The presence of this sequence on a plasmid carrying a chimeric nos-npt-II gene conferring kanamycin resistance to plant cells, produces an overexpression of the npt-II gene during at least eight days after protoplast transformation. This effect on the expression of the nos promoter was independent of the orientation and was observed both...

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

    Directory of Open Access Journals (Sweden)

    Wacław Orczyk

    2014-02-01

    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.

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

    OpenAIRE

    Wacław Orczyk; Anna Nadolska-Orczyk

    2014-01-01

    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...

  9. Transformation of Bacillus thuringiensis subsp. galleria protoplasts by plasmid pBC16.

    OpenAIRE

    Alikhanian, S. I.; Ryabchenko, N F; Bukanov, N O; Sakanyan, V A

    1981-01-01

    Protoplasts of the entomopathogenic bacterium Bacillus thuringiensis subsp. galleria were transformed by plasmid pBC16. The frequency of transformation was much lower than that of Bacillus subtilis. All isolated B. thuringiensis transformants were characterized by increased sensitivity to lysozyme as compared with the original strain.

  10. Photorespiratory properties of protoplasts from C3-C4intermediate species of moricandia

    International Nuclear Information System (INIS)

    Protoplasts were isolated from leaves of the C3-C4 intermediate species, Moricandia arvensis (L.) DC. and Moricandia spinosa Pomel. Analysis by light and transmission electron microscopy indicated that these purified preparations contained both mesophyll protoplasts (MP) and bundle-sheath protoplasts (BSP). Conventional density gradient centrifugation procedures failed to yield separations of pure protoplasts from each cell-type. With these heterogeneous suspensions of MP and BSP, values measured for (i) the percentage inhibition of photosynthetic CO2 fixation by O2, (ii) the apparent K (CO2) of photosynthesis, and (iii) dark/light ratios of the rate of 14CO2 evolution during decarboxylation of exogenous [1-14C]glycine were not significantly different from those determined for protoplasts preparations from related or representative C3 plants, including M. foetida, Nicotiana tavacum, and Triticum aestivum. In contrast, previous comparisons with C3 species, using intact leaf tissue from M, arvensis, have shown a reduced sensitivity of new photosynethic to inhibition by O2 [Holaday et al., Plant Sci. Lett., 27 (1982) 181] and an enhanced capacity for the photosynthetic refixation of CO2 evolved during decarboxylation of exogenous photorespiratory substrates [Holbrook et al., Plant Physiol., 77 (1985) 578]. We conclude that these photosynthetic properties, associated with reduced photorespiration by M. arvensis and M. spinosa, are dependent upon the integrity of the anatomical and ultrastructural arrangement of bundle-sheath and mesophyll cells in these C3-C4intermediate species. (author)

  11. Cytoplasmic calcium levels in protoplasts from the cap and elongation zone of maize roots

    Science.gov (United States)

    Kiss, H. G.; Evans, M. L.; Johnson, J. D.

    1991-01-01

    Calcium has been implicated as a key component in the signal transduction process of root gravitropism. We measured cytoplasmic free calcium in protoplasts isolated from the elongation zone and cap of primary roots of light-grown, vertically oriented seedlings of Zea mays L. Protoplasts were loaded with the penta-potassium salts of fura-2 and indo-1 by incubation in acidic solutions of these calcium indicators. Loading increased with decreasing pH but the pH dependence was stronger for indo-1 than for fura-2. In the case of fura-2, loading was enhanced only at the lowest pH (4.5) tested. Dyes loaded in this manner were distributed predominantly in the cytoplasm as indicated by fluorescence patterns. As an alternative method of loading, protoplasts were incubated with the acetoxymethylesters of fura-2 and indo-1. Protoplasts loaded by this method exhibited fluorescence both in the cytoplasm and in association with various organelles. Cytoplasmic calcium levels measured using spectrofluorometry, were found to be 160 +/- 40 nM and 257 +/- 27 nM, respectively, in populations of protoplasts from the root cap and elongation zone. Cytoplasmic free calcium did not increase upon addition of calcium to the incubation medium, indicating that the passive permeability to calcium was low.

  12. 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.

  13. A rapid method for isolation of low-molecular-weight RNA from Arabidopsis using low salt concentration buffer

    Directory of Open Access Journals (Sweden)

    Han Cheng

    2010-08-01

    Full Text Available Normal 0 7.8 pt 0 2 false false false EN-US ZH-CN X-NONE MicrosoftInternetExplorer4 We have developed a rapid extraction method using low salt concentration buffer for the isolation of low-molecular-weight RNA from Arabidopsis tissues. The method was quick and efficient, and the small scale extraction process took no more than 1 hour, while yield and RNA quality were comparable with those of previously reported. The LMW RNA isolated using this method was high quality, abundant in small RNA and free of high molecular weight RNA. This method can be used to extract low-molecular-weight RNA for the purpose of small RNA cloning and detection, and library construction.

  14. An improved, simple, inexpensive and highly flexible hydroponic setup for root mitochondria isolation from arabidopsis and nicotiana pants

    International Nuclear Information System (INIS)

    Hydroponic setups are frequently developed and improved as they are convenient platforms for studying whole plant physiology. Mostly, the available systems produce small amounts of plant material and are therefore, unsuitable for studies requiring large quantities of plant material like isolation of mitochondria. To address this issue, we have modified a hydroponic setup that can sustain hundreds of Arabidopsis and tobacco plants until adult plants are established. The setup is very flexible and easy to construct. It is based on the use of recyclable and sterilizable plastic-net-pots and media containers, which are easily available from the local suppliers. The modified seed-pots and styrofoam sheets facilitate the transfer and harvesting of seedlings. We have used the Percoll based two-step density gradient centrifugation method for the isolation of root mitochondria from the hydroponically grown plants. (author)

  15. 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

  16. Patterns of indole alkaloids synthesis in response to heat shock, 5-azacytidine and Na-butyrate treatment of cultured catharanthus roseus mesophyll protoplasts

    International Nuclear Information System (INIS)

    Alkaloids of C. roseus are in high demand for therapeutic and other reasons. Cultured Catharanthus cells can produce limited quantities of these alkaloids. The authors have found that cultured mesophyll protoplasts in the presence of 14C-Tryptamine are capable of synthesizing alkaloids. The pattern of alkaloids synthesis changes when protoplasts are subjected to a heat shock at 370C. The heat shocked protoplasts incorporated 33% more 14C-Tryptamine and produced 3 new types of alkaloids. Treatment of protoplasts with 5-azacytidine, a DNA hypomethylating agent and Na-butyrate which induces hyperacetylation of histones produced qualitative and quantitative changes in the alkaloid pattern. Four new alkaloids following the above treatments were detected by TLC and HPLC of the extracts. It is suggested that the alkaloid pattern of the cultured protoplasts can be altered by treatment with compounds known as regulators of gene expression. Work is in progress to isolate and identify these new alkaloids

  17. Patterns of indole alkaloids synthesis in response to heat shock, 5-azacytidine and Na-butyrate treatment of cultured catharanthus roseus mesophyll protoplasts

    Energy Technology Data Exchange (ETDEWEB)

    Saleem, M.; Cutler, A.J.

    1986-04-01

    Alkaloids of C. roseus are in high demand for therapeutic and other reasons. Cultured Catharanthus cells can produce limited quantities of these alkaloids. The authors have found that cultured mesophyll protoplasts in the presence of /sup 14/C-Tryptamine are capable of synthesizing alkaloids. The pattern of alkaloids synthesis changes when protoplasts are subjected to a heat shock at 37/sup 0/C. The heat shocked protoplasts incorporated 33% more /sup 14/C-Tryptamine and produced 3 new types of alkaloids. Treatment of protoplasts with 5-azacytidine, a DNA hypomethylating agent and Na-butyrate which induces hyperacetylation of histones produced qualitative and quantitative changes in the alkaloid pattern. Four new alkaloids following the above treatments were detected by TLC and HPLC of the extracts. It is suggested that the alkaloid pattern of the cultured protoplasts can be altered by treatment with compounds known as regulators of gene expression. Work is in progress to isolate and identify these new alkaloids.

  18. 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.

  19. 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.

  20. An Effective Strategy for Reliably Isolating Heritable and Cas9-Free Arabidopsis Mutants Generated by CRISPR/Cas9-Mediated Genome Editing1[OPEN

    Science.gov (United States)

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

    2016-01-01

    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. PMID:27208253

  1. 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.

  2. 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.

  3. Polyamine binding to proteins in oat and Petunia protoplasts

    Science.gov (United States)

    Mizrahi, Y.; Applewhite, P. B.; Galston, A. W.

    1989-01-01

    Previous work (A Apelbaum et al. [1988] Plant Physiol 88: 996-998) has demonstrated binding of labeled spermidine (Spd) to a developmentally regulated 18 kilodalton protein in tobacco tissue cultures derived from thin surface layer explants. To assess the general importance of such Spd-protein complexes, we attempted bulk isolation from protoplasts of Petunia and oat (Avena sativa). In Petunia, as in tobacco, fed radioactive Spd is bound to protein, but in oat, Spd is first converted to 1,3,-diaminopropane (DAP), probably by polyamine oxidase action. In oat, binding of DAP to protein depends on age of donor leaf and conditions of illumination and temperature, and the extraction of the DAP-protein complex depends upon buffer and pH. The yield of the DAP-protein complex was maximized by extraction of frozen-thawed protoplasts with a pH 8.8 carbonate buffer containing SDS. Its molecular size, based on Sephacryl column fractionation of ammonium sulfate precipitated material, exceeded 45 kilodaltons. Bound Spd or DAP can be released from their complexes by the action of Pronase, but not DNAse, RNAse, or strong salt solutions, indicating covalent attachment to protein.

  4. 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.

  5. 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.

  6. 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. PMID:26577784

  7. Guard Cell Chloroplasts Are Essential for Blue Light-Dependent Stomatal Opening in Arabidopsis

    Science.gov (United States)

    Suetsugu, Noriyuki; Takami, Tsuneaki; Ebisu, Yuuta; Watanabe, Harutaka; Iiboshi, Chihoko; Doi, Michio; Shimazaki, Ken-ichiro

    2014-01-01

    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. PMID:25250952

  8. Genetic engineering with tobacco protoplasts. [Hybridization by fusion of leaf protoplasts

    Energy Technology Data Exchange (ETDEWEB)

    Smith, H H

    1976-01-01

    Interspecific hybridization by fusion of leaf protoplasts of Nicotiana glauca (GG) and N. langsdorffii (LL) was confirmed and extended. Enzymatic digestion of leaf tissues to obtain protoplats was followed by fusion with the aid of polyethylene glycol. The hybrid calli were selected by their better growth on defined culture media. Mature hybrid plants were identified by their morphology and tumor formation. Cytological examination revealed a range in chromosome numbers from 56 to 64 rather than the amphiploid GGLL number of 42. About 75 percent of the hybrids were fertile. The potential range in combining widely disparate genotypes by somatic cell fusion was demonstrated by fusing tobacco GGLL protoplasts with human HeLa cells. The HeLa nucleus was observed inside the plant protoplasts, thus forming an interkingdom heterokaryon.

  9. Transformation of Bacillus thuringiensis protoplasts by plasmid deoxyribonucleic acid.

    OpenAIRE

    Martin, P A; Lohr, J. R.; Dean, D H

    1981-01-01

    A method has been developed to transform plasmid deoxyribonucleic acid into protoplasts of the insect pathogen Bacillus thuringiensis. Protoplasts were formed by treatment of cells with lysozyme. The efficiency of formation of protoplasts was affected by the strain, the media, and the cell density. Deoxyribonucleic acid uptake was induced by polyethylene glycol. Deoxyribonucleic acid from the Staphylococcus aureus plasmid pC194 was used for transformation. Although this plasmid could not be i...

  10. Polyethylene glycol-assisted transfection of Streptomyces protoplasts.

    OpenAIRE

    Suarez, J E; Chater, K F

    1980-01-01

    In the presence of polyethylene glycol (concentration optimum 20%), protoplasts of appropriate Streptomyces strains could be transfected by deoxyribonucleic acid (DNA) of five temperate phages (phi C31, VP5, R4, phi 448, and S14) belonging to four different immunity groups. Quantitation of transfection was made possible by plating the transfection mixture with excess uninfected protoplasts in soft agar overlays on protoplast regeneration medium so that plaques were easily detected. Optimum fr...

  11. 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

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

    OpenAIRE

    Pitzschke Andrea; Persak Helene

    2012-01-01

    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 protoplast...

  13. Purification and functional characterization of protoplasts and intact vacuoles from grape cells

    OpenAIRE

    Gerós Hernâni; Lecourieux Fatma; Vignault Céline; Silva Rui; Fontes Natacha; Delrot Serge

    2010-01-01

    Abstract Background During grape berry ripening, the vacuoles accumulate water, sugars and secondary metabolites, causing great impact in plant productivity and wine quality. However, the molecular basis of these compartmentation processes is still poorly understood. As in many species, the major bottleneck to study these aspects in grapevine is to obtain highly purified vacuoles with a good yield. The present paper describes an isolation method of protoplasts and intact vacuoles from grape b...

  14. Protoplasting impact on polyketide activity and characterization of the interspecific fusants from Streptomyces spp

    International Nuclear Information System (INIS)

    Streptomycetes are gram-positive, soil-inhabiting bacteria of the order Actinomycetales. These organisms exhibit an unusual, developmentally complex life cycle and produce many economically important secondary metabolites, such as antibiotics, immunosuppressants, insecticides, and antitumor agents. Streptomyces species have been the subject of genetic investigation for over 50 years, with many studies focusing on the production of bioactives compounds. The protoplast formation and regeneration are important processes, and they are a major step following genetic manipulations such as fusion and DNA-mediated transformation, which can improve antibiotic production. The protoplast fusion, transformation and improved fermentation features can be used to regenerate strains with increased antibiotic activity. Local Streptomyces spp. CN207 produce a broad range of secondary metabolites which is active against bacteria and fungi. This strain was used as a donor and S. coelicolor strain M145 was used as a recipient host for protoplast fusion. The protoplast fusion resulted in increased isolation of variants with higher antibiotic activity. Recombinant Streptomyces coelicolor PF04 was increased 10 times more than the wild strain. The antimicrobial activity from PF04 strain was studied using the disc method agar. TLC analysis confirmed that the Rf of cell extract for PF04 strain is identical to antimicrobial compound of Streptomyces CN207. Our results confirm the possibility of transferring antibiotics cluster genes by fusion. In fact, many of the selective markers such as Ticarcillin, Cefalotin, Oxacillin and Cefotaxim were transferred during the protoplast fusion. PFGE analysis and DNA-hybridization confirmed the presence of homologous fragments between a wild-type Streptomyces CN207 and a recombinant S. coelicolor PF04

  15. Fusion of protoplasts with irradiated micro protoplasts as a tool for radiation hybrid panel in citrus

    International Nuclear Information System (INIS)

    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)

  16. 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...

  17. Studies on color type variants from mutagenized protoplasts of Porphyra haitanensis Chang et Zheng & P. Yezoensis ueda (rhodophycease)

    Science.gov (United States)

    Yan, Xinghong

    1993-09-01

    Isolated protoplasts from thalli of Porphyra haitanensis and Porphyra yezoensis were treated with colchicine or irradiated by ultraviolet (UV). Several types of color variants were observed among the protoplast offspring. After treatment with colchicine: (1) 0.04 0.09% of red type variants in P. haitanensis were obtained; (2) The rate of red type variants and the variegated chimeral thalli composed of red type and wild type of sectors were 0.31 1.11% in P. yezoensis. After irradiation with UV: (1) 3.5 10.5% of red type variants in P. yezoensis were obtained: (2) 0.5 2.0% of red type variants and the variegated chimeral thalli composed of red type and wild type of sectors were obtained in P. haitanensis. Colchicine and UV’s mutangenic effects on P. yezoensis protoplasts were stronger than those on P. haitanensis protoplasts. The most efficient concentration of colchicine was 0.05%. The optimal length of UV-radiation was 1/2 min (radiation distance 5 cm). The red type variants induced by colchicine treatment grew faster than the wild type thalli. The clones of vegetative propagation from protoplasts of red type variants were still red type thalli. The red type variants will be good materials for genetic studies and improvement of Porphyra strains.

  18. Effet de la pectolyase Y-23 et de la cellulase Onozuka RS sur le rendement en protoplastes viables de Prunus cerasus L.

    Directory of Open Access Journals (Sweden)

    Mehri-Kamoun R.

    2001-01-01

    Full Text Available Effect of pectolyase Y-23 and cellulase Onozuka RS on the yield of viable protoplasts of Prunus cerasus L. ""Montmorency"". To isolate leaf mesophyll, leaf and root callus protoplasts of Prunus cerasus L. ""Montmorency"", we have determined the optimum enzymatic mixtures to be used, and characterized the specific activity of these enzymes. The analysis of the specific activities of enzymes allows to compare the different cellulases and pectinases used to obtain protoplasts in relation with the tissue sources. This analysis concerned the FPase (degradation of filter paper and CMCase activities for cellulases Onozuka RS and R-10, and the PME (pectinmethylesterase, PL (pectate lyase and PG (polygalacturonase activities for the pectinases Macerozyme R-10 and Pectolyase Y-23. The results show that the digestion of leaf mesophyll tissues need cellulase Onozuka RS and Pectolyase Y-23 while callus protoplasts of the same material, can be isolated with cellulase Onozuka R-10 and Macerozyme R-10. The enzymes cellulase Onozuka RS and Pectolyase Y-23 (as pectinase improved significantly the yield and the viability of leaf mesophyll protoplasts compared to cellulase Onozuka R-10 and Macerozyme R-10. These results were correlated to the specific activities of the enzymes. Significant differences between the 2 pectinases are observed for PME, PL and PG activities and between the 2 cellulases for CMCase activity. From callus, the maximum amount of viable protoplasts was obtained with cellulase Onozuka R-10 (low CMCase activity and Macerozyme R-10 (low PG activity.

  19. Isolation of AtNUDT5 gene promoter and characterization of its activity in transgenic Arabidopsis thaliana.

    Science.gov (United States)

    Zhang, Xiu-Chun; Li, Mei-Ying; Ruan, Meng-Bin; Xia, Yi-Ji; Wu, Kun-Xin; Peng, Ming

    2013-03-01

    AtNUDT5 is a cytosol Nudix that catalyzes the hydrolysis of a variety of substrates. In this report, a 1,387-bp 5'-flanking region of the AtNUDT5 gene was isolated from Arabidopsis thaliana. The tissue-specific activity of the 5'-flanking region was investigated by using the GUS gene as a reporter in transgenic A. thaliana plants. Weak GUS activity appeared in vascular tissues of young plants, strong GUS activity appeared in the axial roots, but no GUS activity was observed in the root cap, lateral roots, rosette leaf, mature silique and reproductive tissues such as stamen, pistil, and petal. Furthermore, by using these transgenic A. thaliana plants, results of the histochemical staining and fluorometric assays of GUS activity showed that the AtNUDT5 promoter can be activated by both avirulent Pst avrRpm1 and virulent Pst strains at 5 h post-infiltration and that the activity of AtNUDT5 promoter increased significantly at 24 h post-infiltration. Taken together, our results demonstrated that the AtNUDT5 promoter is pathogen-responsive. The promoter may be used to develop transgenic plants with an increased tolerance to pathogenic stresses. PMID:23322251

  20. A new approach for cytokinin isolation from Arabidopsis tissues using miniaturized purification: pipette tip solid-phase extraction

    Directory of Open Access Journals (Sweden)

    Svačinová Jana

    2012-05-01

    Full Text Available Abstract Background We have developed a new analytical approach for isolation and quantification of cytokinins (CK in minute amounts of fresh plant material, which combines a simple one-step purification with ultra-high performance liquid chromatography–fast scanning tandem mass spectrometry. Results Plant tissue samples (1–5 mg FW were purified by stop-and-go-microextraction (StageTip purification, which previously has only been applied for clean-up and pre-concentration of peptides. We found that a combination of two reverse phases and one cation-exchange phase, was the best tool, giving a total extraction recovery higher than 80%. The process was completed by a single chromatographic analysis of a wide range of naturally occurring cytokinins (bases, ribosides, O- and N-glucosides, and nucleotides in 24.5 minutes using an analytical column packed with sub-2-microne particles. In multiple reaction monitoring mode, the detection limits ranged from 0.05 to 5 fmol and the linear ranges for most cytokinins were at least five orders of magnitude. The StageTip purification was validated and optimized using samples of Arabidopsis thaliana seedlings, roots and shoots where eighteen cytokinins were successfully determined. Conclusions The combination of microextraction with one-step high-throughput purification provides fast, effective and cheap sample preparation prior to qualitative and quantitative measurements. Our procedure can be used after modification also for other phytohormones, depending on selectivity, affinity and capacity of the selected sorbents.

  1. Growth of Streptococcus mutans protoplasts is not inhibited by penicillin.

    Science.gov (United States)

    Parks, L C; Shockman, G D; Higgins, M L

    1980-01-01

    A method is described in which cells of Streptococcus mutans BHT can be converted to spherical, osmotically fragile protoplasts. Exponential-phase cells were suspended in a solution containing 0.5 M melezitose, and their cell walls were hydrolyzed with mutanolysin (M-1 enzyme). When the resultant protoplasts were incubated in a chemically defined growth medium containing 0.5 M NH4Cl, the protoplast suspensions increased in turbidity, protein, ribonucleic acid, and deoxyribonucleic acid in a balanced fashion. In the presence of benzylpenicillin (5 microgram/ml), balanced growth of protoplasts was indistinguishable from untreated controls. This absence of inhibition of protoplast growth in the presence of benzylpenicillin was apparently not due to inactivation of the antibiotic. When exponential-phase cells of S. mutans BHT were first exposed to 5 microgram of benzyl-penicillin per ml for 1 h and then converted to protoplasts, these protoplasts were also able to grow in chemically defined, osmotically stabilized medium. The ability of wall-free protoplasts to grow and to synthesize ribonucleic acid and protein in the presence of a relatively high concentration of benzylpenicillin contrasts with the previously reported rapid inhibition of ribonucleic acid and protein synthesis in intact streptococci. These data suggest that this secondary inhibition of ribonucleic acid and protein synthesis in whole cells is due to factors involved with the continued assembly of an intact, insoluble cell wall rather than with earlier stages of peptidoglycan synthesis. Images PMID:6997274

  2. Genetic recombination in Actinoplanes brasiliensis by protoplast fusion.

    OpenAIRE

    Palleroni, N. J.

    1983-01-01

    Protoplast formation, fusion, and cell regeneration have been achieved with mutant strains of Actinoplanes brasiliensis. Three-, four-, and five-factor crosses have shown genetic recombination among the markers, and a five-factor cross is analyzed and discussed. Possibilities of using protoplast fusion for gene mapping and strain improvement are suggested.

  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. 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.

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

    OpenAIRE

    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...

  6. 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

  7. Transfection of Bacillus subtilis protoplasts by bacteriophage phi do7 DNA.

    OpenAIRE

    Perkins, J B; Dean, D H

    1983-01-01

    DNA from the Bacillus subtilis temperate bacteriophage phi do7 was found to efficiently transfect B. subtilis protoplasts; protoplast transfection was more efficient than competent cell transfection by a magnitude of 10(3). Unlike competent cell transfection, protoplast transfection did not require primary recombination, suggesting that phi do7 DNA enters the protoplast as double-stranded molecules.

  8. RcLEA, a late embryogenesis abundant protein gene isolated from Rosa chinensis, confers tolerance to Escherichia coli and Arabidopsis thaliana and stabilizes enzyme activity under diverse stresses.

    Science.gov (United States)

    Zhang, Xuan; Lu, Songchong; Jiang, Changhua; Wang, Yaofeng; Lv, Bo; Shen, Jiabin; Ming, Feng

    2014-07-01

    The late embryogenesis abundant (LEA) protein family is a large protein family that is closely associated with resistance to abiotic stresses in many organisms, such as plants, bacteria and animals. In this study, we isolated a LEA gene, RcLEA, which was cytoplasm-localized, from Rosa chinensis. RcLEA was found to be induced by high temperature through RT-PCR. Overexpression of RcLEA in Escherichia coli improved its growth performance compared with the control under high temperature, low temperature, NaCl and oxidative stress conditions. RcLEA was also overexpressed in Arabidopsis thaliana. The transgenic Arabidopsis showed better growth after high and low temperature treatment and exhibited less peroxide according to 3, 3-diaminobenzidine staining. However, RcLEA did not improve the tolerance to NaCl or osmotic stress in Arabidopsis. In vitro analysis showed that RcLEA was able to prevent the freeze-thaw-induced inactivation or heat-induced aggregation of various substrates, such as lactate dehydrogenase and citrate synthase. It also protected the proteome of E. coli from denaturation when the proteins were heat-shocked or subjected to acidic conditions. Furthermore, bimolecular fluorescence complementation assays suggested that RcLEA proteins function in a complex manner by making the form of homodimers. PMID:24760474

  9. 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.

  10. 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.

  11. Plant regeneration from cell suspension-derived protoplasts of Saintpaulia ionantha Wendl.

    Science.gov (United States)

    Hoshino, Y; Nakano, M; Mii, M

    1995-03-01

    Friable calli were induced on leaf segments of Saintpaulia ionantha Wendl. on B5 medium containing 1 mg l(-1) 2,4-D and 2 g l(-1) casein hydrolysate. Cell suspension cultures were readily established from these friable calli and protoplasts could be isolated from the cells with yields of 1-3×10(7)/g f. wt.. By culturing in 0.1 % gellan gum-solidified B5 medium supplemented with 1 mg l(-1) 2,4-D and 0.1 M each of sucrose and mannitol at a density of 1×10(5)/ml, the protoplasts divided within 6 days and formed macro-colonies after 2 months of culture. Shoot regeneration from protoplast-derived calli was obtained by sequential treatment of the calli with plant growth regulators: initially with 1 mg l(-1) each of NAA and BA for 2 months followed by 0.01 mg l(-1) NAA and 5 mg l(-1) BA for 4 months. Regenerated plants were established after rooting of the shoots on half-strength MS medium, and successfully transferred to the greenhouse. The regenerated plants grew into flowering stage and showed the same phenotype as the parent plant. PMID:24185329

  12. 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.

  13. 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.

  14. ZmSOC1, a MADS-Box Transcription Factor from Zea mays, Promotes Flowering in Arabidopsis

    Directory of Open Access Journals (Sweden)

    Suzhou Zhao

    2014-11-01

    Full Text Available 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.

  15. 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.

  16. Viral protein synthesis in cowpea mosaic virus infected protoplasts

    International Nuclear Information System (INIS)

    Some aspects of cowpea mosaic virus (CPMV) multiplication in cowpea mesophyll protoplasts were studied. The detection and characterization of proteins whose synthesis is induced or is stimulated upon virus infection was performed with the aid of radioactive labelling. (Auth.)

  17. Viral protein synthesis in cowpea mosaic virus-infected protoplasts

    OpenAIRE

    Rottier, P. J. M.

    1980-01-01

    In contrast to the situation concerning bacterial and, to a lesser extent, animal RNA viruses, little is known about the biochemical processes occurring in plant cells due to plant RNA virus infection. Such processes are difficult to study using intact plants or leaves. Great effort has therefore been spent in developing in vitro cultures of plant protoplasts, but the use of these protoplasts has been seriously hampered by various technical problems.It is clear that plant RNA virus infections...

  18. Proteins synthesized in tobacco mosaic virus infected protoplasts

    International Nuclear Information System (INIS)

    The author deals with research on the multiplication of tobacco mosaic virus (TMV) in leaf cell protoplasts. An attempt is made to answer three questions: (1) Which proteins are synthesized in TMV infected protoplasts as a result of TMV multiplication. (2) Which of the synthesized proteins are made under the direction of the TMV genome and, if any, which of the proteins are host specific. (3) In which functions are these proteins involved. (Auth.)

  19. [Conditions for protoplast preparation of spinosyn-producing strain and the physiological properties of protoplast-regenerated strains].

    Science.gov (United States)

    Luo, Yushuang; Ding, Xuezhi; Xia, Liqiu; Wang, Hailong; Huang, Fan; Tang, Ying

    2009-03-01

    To improve spinosyn-producing strain and enhance spinosyns yield, we studied the effects of glycin concentration and the operational time, temperature and lysozyme concentration on protoplast preparation of Saccharopolyspora spinosa SP06081. We also studied different regeneration media and osmotic stabilizing agents. In addition, we compared the change of morphology and spinosyns yield of the regenerated strains. The results showed that the Saccharopolyspora spinosa SP06081 protoplast yield was the highest under these conditions: the collected mycelium from SP06081 grown in Tryptic Soy Broth (TSB) medium with 0.2% glycin for 48 h was treated by 0.1 mg/mL lysozyme at 28 degrees C for 20 min, then plated on the R2YE medium with sucrose as osmotic stabilizer, the number of regeneration protoplast was up to 10(8)/mL. The protoplast-regenerated strains exhibited changes in morphology and antibiotic production, 29.3% protoplast-regenerated strains was characterized by loose mycelium and abundant broken branches as did their parent. Among them, 58.2% strains presented the trend to positive variation in spinosad yield, with the highest spinosad yield of up to 582.0 mg/L, 85.6% higher than that of their parent. There is significant correlation between the morphological differentiation and antibiotic yield of the protoplast-regenerated strains from spinosyn-producing strain. PMID:19621575

  20. Expression of photosynthesis-related gene fusions is restricted by cell type in transgenic plants and in transfected protoplasts.

    OpenAIRE

    Harkins, K R; Jefferson, R A; Kavanagh, T A; Bevan, M W; Galbraith, D W

    1990-01-01

    We have analyzed the expression of chimeric genes in populations of protoplasts isolated from the photosynthetic and nonphotosynthetic tissues within leaves of transgenic tobacco plants and separated by fluorescence-activated cell sorting. Expression of transcriptional gene fusions controlled by promoters from photosynthesis-associated genes showed a striking dependence on cell type. These patterns of expression were preserved when the gene fusions were transfected into normal (nontransgenic)...

  1. Comparative cytological investigations on protoplasts, tissue cultures and seedlings from Beta vulgaris (sugar-beet)

    International Nuclear Information System (INIS)

    Investigations were carried out with the aim of determining ploidy status, at short and long intervals, using suspension and protoplast cultures and seedlings of Beta vulgaris L. var. altissima cv. Hymona (sugar-beet). Two rapid-growing strains of sugar-beet were used, strain B.14.1, with a ploidy level of 8c to 64c (with maxima between 8c and 16c) and strain B.1.9, varying in DNA content from 16c to 128c (with a maximum frequency between 32c and 64c). Long-term studies of about two years resulted in constant ploidy spectrum, whereas short-term analyses under turbidostatic conditions showed more or less regular oscillations in the frequency distribution, with an amplitude of 20-40% of the medium ploidy level and with an oscillation period of 1-2 days. The isolation of protoplasts from the two strains and the measurement of their ploidy levels before and after isolation, and at longer periods thereafter, showed a shift in ploidy level immediately after isolation. Studies on the ploidy levels in seeds and seedlings of sugar-beet could yield evidence that heterogeneity in the ploidy patterns of cell cultures is not a feature of cultivated cells or tissue alone, but also occurs naturally during plant development. (author)

  2. Interspecific transfer of only part of genome by fusion between non-irradiated protoplasts of Nicotiana glauca and X-ray irradiated protoplasts of N. Langsdorffii

    International Nuclear Information System (INIS)

    To transfer only part of genome, X-ray irradiated suspension cell protoplasts of N. langsdorffii were fused with suspension cell protoplasts of N. glauca by polyethylene glycol. Somatic hybrid calli were selected by the growth in the hormone-free medium. Some of somatic hybrid calli from fusion with irradiated protoplasts indicated the loss of small subunit polypeptide of fraction 1 protein which was coded by N. langsdorffii nuclear DNA. Cytological analysis provided an information on significant decrease of chromosomes in somatic hybrid calli from fusion with irradiated protoplasts, compared with the somatic hybrid calli from fusion with non-irradiated protoplasts. In addition, isozyme analysis revealed that somatic hybrid calli from fusion with irradiated protoplasts lost particular bands of N. langsdorffli. These results demonstrate the tranfer of only part of genome from N, langsdorffii to N, glauca by fusion with X-ray irradiated protoplasts

  3. 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

  4. A new approach for cytokinin isolation from Arabidopsis tissues using miniaturized purification: pipette tip solid-phase extraction

    Czech Academy of Sciences Publication Activity Database

    Svačinová, Jana; Novák, Ondřej; Plačková, Lenka; Lenobel, René; Holík, Josef; Strnad, Miroslav; Doležal, Karel

    2012-01-01

    Roč. 8, _ (2012), s. 17. ISSN 1746-4811 R&D Projects: GA TA ČR TA01010861; GA AV ČR KAN200380801 Grant ostatní: GA MŠk(CZ) ED0007/01/01 Institutional research plan: CEZ:AV0Z50380511 Keywords : Pipette tip solid-phase extraction (PT-SPE) * Arabidopsis thaliana * Cytokinins Subject RIV: EC - Immunology Impact factor: 2.667, year: 2012

  5. 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.

  6. 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.

  7. Factors affecting callus and protoplast production and regeneration of plants from garlic tissue cultures

    International Nuclear Information System (INIS)

    Five cultivars of garlic, two explants, six callusing media, six regeneration media, two kinds of light and several doses of gamma irradiation were used to determine the best conditions for callus induction and plant regeneration from garlic tissue cultures. Also, some experiments were conducted to study the possibility to isolate protoplast and regenerate plants. The experiment showed that medium MS9 was good for regenerating plant directly from basal plate without going through callus phase. ANOVA exhibited significant differences among used cultivars in their ability to form callus. No significant difference was observed between 16 hr light and complete darkness in callus growth. However, appearance of callus was generally better on darkness. Cultivar varied in their ability to regenerate and interaction between cultivars and media was observed. Cultivar kisswany was the best in regeneration (38%) and medium MS47 was the best among used media (35%). Light type played a significant role in regeneration of plants where red light was much better than white light in inducing regeneration (68% vs 36%). ANOVA revealed significant effect of low doses of gamma irradiation on stimulation regeneration of plant whereas high doses prevented regeneration. Many experiments were conducted to isolate protoplast and regenerate plants. The best method for culturing was the droplet and the best conditions for incubation were complete darkness at 25 Degreed centigrade. This lead to formation of cell wall but no cell division was observed (author)

  8. Methanogenesis and ATP synthesis in a protoplast system of Methanobacterium thermoautotrophicum.

    OpenAIRE

    Mountfort, D O; Mörschel, E; Beimborn, D B; Schönheit, P

    1986-01-01

    When Methanobacterium thermoautotrophicum cells were incubated in 50 mM potassium phosphate buffer (pH 7.0) containing 1 M sucrose and autolysate from Methanobacterium wolfei, they were transformed into protoplasts. The protoplasts, which possessed no cell wall, lysed in buffer without sucrose. Unlike whole cells, the protoplasts did not show convoluted internal membrane structures. The protoplasts produced methane from H2-CO2 (approximately 1 mumol min-1 mg of protein-1) at about 50% the rat...

  9. Mutanolysin-induced spheroplasts of Streptococcus mutants are true protoplasts.

    Science.gov (United States)

    Siegel, J L; Hurst, S F; Liberman, E S; Coleman, S E; Bleiweis, A S

    1981-01-01

    A method is described for the preparation of protoplasts of Streptococcus mutans BHT. The muralytic enzyme mutanolysin was prepared free of contaminating proteinases and shown to completely dissolve cell walls of this strain. Whole cells were converted to stabilizable protoplasts by using the enzyme in an isotonic medium containing 40% raffinose. Experiments using [3H]thymidine and [14C]leucine as cytoplasmic pool markers revealed only minimal (10%) leakage during a 1-h incubation. Examination by electron microscopy revealed the apparent absence of structural cell wall on the enlarged spherical bodies. Quantitative chemical analyses of membranes prepared by lysing protoplasts demonstrated only very small amounts of rhamnose and trace amounts of galactose. These sugars are the principal components of the BHT cell wall polysaccharide. Also, there were only small amounts of peptidoglycan components (e.g., N-acetylglucosamine) in the purified membranes obtained by this method. Images PMID:7012022

  10. 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.

  11. 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.

  12. Ethylene Antagonizes Salt-Induced Growth Retardation and Cell Death Process via Transcriptional Controlling of Ethylene-, BAG- and Senescence-Associated Genes in Arabidopsis

    Science.gov (United States)

    Pan, Ya-Jie; Liu, Ling; Lin, Ying-Chao; Zu, Yuan-Gang; Li, Lei-Peng; Tang, Zhong-Hua

    2016-01-01

    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. Ethylene Antagonizes Salt-Induced Growth Retardation and Cell Death Process via Transcriptional Controlling of Ethylene-, BAG- and Senescence-Associated Genes in Arabidopsis.

    Science.gov (United States)

    Pan, Ya-Jie; Liu, Ling; Lin, Ying-Chao; Zu, Yuan-Gang; Li, Lei-Peng; Tang, Zhong-Hua

    2016-01-01

    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. PMID:27242886

  14. Two distinct signaling pathways participate in auxin-induced swelling of pea epidermal protoplasts.

    Science.gov (United States)

    Yamagami, Mutsumi; Haga, Ken; Napier, Richard M; Iino, Moritoshi

    2004-02-01

    Protoplast swelling was used to investigate auxin signaling in the growth-limiting stem epidermis. The protoplasts of epidermal cells were isolated from elongating internodes of pea (Pisum sativum). These protoplasts swelled in response to auxin, providing the clearest evidence that the epidermis can directly perceive auxin. The swelling response to the natural auxin IAA showed a biphasic dose response curve but that to the synthetic auxin 1-naphthalene acetic acid (NAA) showed a simple bell-shaped dose response curve. The responses to IAA and NAA were further analyzed using antibodies raised against ABP1 (auxin-binding protein 1), and their dependency on extracellular ions was investigated. Two signaling pathways were resolved for IAA, an ABP1-dependent pathway and an ABP1-independent pathway that is much more sensitive to IAA than the former. The response by the ABP1 pathway was eliminated by anti-ABP1 antibodies, had a higher sensitivity to NAA, and did not depend on extracellular Ca(2+). In contrast, the response by the non-ABP1 pathway was not affected by anti-ABP1 antibodies, had no sensitivity to NAA, and depended on extracellular Ca(2+). The swelling by either pathway required extracellular K(+) and Cl(-). The auxin-induced growth of pea internode segments showed similar response patterns, including the occurrence of two peaks in the dose response curve for IAA and the difference in Ca(2+) requirements. It is suggested that two signaling pathways participate in auxin-induced internode growth and that the non-ABP1 pathway is more likely to be involved in the control of growth by constitutive concentrations of endogenous auxin. PMID:14764902

  15. 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.

  16. Protoplast water content of bacterial spores determined by buoyant density sedimentation.

    OpenAIRE

    Lindsay, J A; Beaman, T C; Gerhardt, P

    1985-01-01

    Protoplast wet densities (1.315 to 1.400 g/ml), determined by buoyant density sedimentation in Metrizamide gradients, were correlated inversely with the protoplast water contents (26.4 to 55.0 g of water/100 g of wet protoplast) of nine diverse types of pure lysozyme-sensitive dormant bacterial spores. The correlation equation provided a precise method for obtaining the protoplast water contents of other spore types with small impure samples and indicated that the average protoplast dry densi...

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

    OpenAIRE

    John Graham

    2002-01-01

    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.

  18. 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.

  19. Characterization of Overexpressed cDNAs Isolated from a Hormone-Autonomous, Radiation-Induced Tumor Tissue Line of Arabidopsis thaliana.

    Science.gov (United States)

    Campell, B R; Town, C D

    1992-12-01

    To investigate the molecular mechanisms of hormonal control of growth, we constructed a subtracted cDNA library enriched for sequences expressed more in a hormone-autonomous, radiation-induced tumor tissue line of Arabidopsis thaliana than in normal, hormone-dependent callus. Ten cDNA clones, which are expressed 1.3- to 10-fold more in the tumor line, were isolated and partially characterized. The clones differ greatly in their level of expression in tumor tissue and in their pattern of expression in plant organs. Southern blot hybridization and sequence analysis showed that this group contains three pairs of closely related clones. Northern blot analysis indicates that one pair of clones represents two members of a gene family that are expressed in different plant organs. One of the isolated sequences shows strong sequence similarity to a cDNA encoding a lipid transfer protein. Two sequences are highly similar to those of previously described membrane channel proteins but have different organ specificities. Two other cDNAs have significant sequence similarity to glycine-rich proteins and hydroxy-proline-rich glycoproteins. When used to probe Southern blots, none of the cDNAs identified polymorphisms between tumor and callus DNA, which might be expected if their overexpression were due to local genome rearrangements induced by radiation. The diversity observed among these 10 clones suggests that some are likely to be involved in tumorous growth and not simply specific to a certain cell or tissue type present in the tumor. PMID:16653233

  20. Effect of microgravity environment on cell wall regeneration, cell divisions, growth, and differentiation of plants from protoplasts (7-IML-1)

    Science.gov (United States)

    Rasmussen, Ole

    1992-01-01

    The primary goal of this project is to investigate if microgravity has any influence on growth and differentiation of protoplasts. Formation of new cell walls on rapeseed protoplasts takes place within the first 24 hours after isolation. Cell division can be observed after 2-4 days and formation of cell aggregates after 5-7 days. Therefore, it is possible during the 7 day IML-1 Mission to investigate if cell wall formation, cell division, and cell differentiation are influenced by microgravity. Protoplasts of rapeseeds and carrot will be prepared shortly before launch and injected into 0.6 ml polyethylene bags. Eight bags are placed in an aluminum block inside the ESA Type 1 container. The containers are placed at 4 C in PTCU's and transferred to orbiter mid-deck. At 4 C all cell processes are slowed down, including cell wall formation. Latest access to the shuttle will be 12 hours before launch. In orbit the containers will be transferred from the PTC box to the 22 C Biorack incubator. The installation of a 1 g centrifuge in Biorack will make it possible to distinguish between effects of near weightlessness and effects caused by cosmic radiation and other space flight factors including vibrations. Parallel control experiments will be carried out on the ground. Other aspects of the experiment are discussed.

  1. 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.

  2. 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.

  3. Plasmalemma- and tonoplast-ATPase activity in mesophyll protoplasts, vacuoles and microsomes of the Crassulacean-acid-metabolism plant Kalanchoe daigremontiana.

    Science.gov (United States)

    Balsamo, R A; Uribe, E G

    1988-02-01

    Adenosine-triphosphatase activity on the plasmalemma and tonoplast of isolated mesophyll protoplasts, isolated vacuoles and tonoplast-derived microsomes of the Crassulacean-acid-metabolism plant Kalanchoe daigremontiana Hamet et Perr., was localized by a cytochemical procedure using lead citrate. Enzyme activity was detected on the cytoplasmic surfaces of the plasmalemma and tonoplast. The identity of the enzymes was confirmed by various treatments differentiating the enzymes by their sensitivity to inhibitors of plasmalemma and tonoplast H(+)-ATPase. Isolated vacuoles and microsomes prepared from isolated vacuoles clearly exhibited single-sided deposition on membrane surfaces. PMID:24226399

  4. PpCBF3 from Cold-Tolerant Kentucky Bluegrass Involved in Freezing Tolerance Associated with Up-Regulation of Cold-Related Genes in Transgenic Arabidopsis thaliana.

    Directory of Open Access Journals (Sweden)

    Lili Zhuang

    Full Text Available Dehydration-Responsive Element Binding proteins (DREB/C-repeat (CRT Binding Factors (CBF have been identified as transcriptional activators during plant responses to cold stress. The objective of this study was to determine the physiological roles of a CBF gene isolated from a cold-tolerant perennial grass species, Kentucky bluegrass (Poa pratensis L., which designated as PpCBF3, in regulating plant tolerance to freezing stress. Transient transformation of Arabidopsis thaliana mesophyll protoplast with PpCBF3-eGFP fused protein showed that PpCBF3 was localized to the nucleus. RT-PCR analysis showed that PpCBF3 was specifically induced by cold stress (4°C but not by drought stress [induced by 20% polyethylene glycol 6000 solution (PEG-6000] or salt stress (150 mM NaCl. Transgenic Arabidopsis overexpressing PpCBF3 showed significant improvement in freezing (-20°C tolerance demonstrated by a lower percentage of chlorotic leaves, lower cellular electrolyte leakage (EL and H2O2 and O2.- content, and higher chlorophyll content and photochemical efficiency compared to the wild type. Relative mRNA expression level analysis by qRT-PCR indicated that the improved freezing tolerance of transgenic Arabidopsis plants overexpressing PpCBF3 was conferred by sustained activation of downstream cold responsive (COR genes. Other interesting phenotypic changes in the PpCBF3-transgenic Arabidopsis plants included late flowering and slow growth or 'dwarfism', both of which are desirable phenotypic traits for perennial turfgrasses. Therefore, PpCBF3 has potential to be used in genetic engineering for improvement of turfgrass freezing tolerance and other desirable traits.

  5. 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.

  6. 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.

  7. Characterization of Overexpressed cDNAs Isolated from a Hormone-Autonomous, Radiation-Induced Tumor Tissue Line of Arabidopsis thaliana1

    Science.gov (United States)

    Campell, Bruce R.; Town, Christopher D.

    1992-01-01

    To investigate the molecular mechanisms of hormonal control of growth, we constructed a subtracted cDNA library enriched for sequences expressed more in a hormone-autonomous, radiation-induced tumor tissue line of Arabidopsis thaliana than in normal, hormone-dependent callus. Ten cDNA clones, which are expressed 1.3- to 10-fold more in the tumor line, were isolated and partially characterized. The clones differ greatly in their level of expression in tumor tissue and in their pattern of expression in plant organs. Southern blot hybridization and sequence analysis showed that this group contains three pairs of closely related clones. Northern blot analysis indicates that one pair of clones represents two members of a gene family that are expressed in different plant organs. One of the isolated sequences shows strong sequence similarity to a cDNA encoding a lipid transfer protein. Two sequences are highly similar to those of previously described membrane channel proteins but have different organ specificities. Two other cDNAs have significant sequence similarity to glycine-rich proteins and hydroxy-proline-rich glycoproteins. When used to probe Southern blots, none of the cDNAs identified polymorphisms between tumor and callus DNA, which might be expected if their overexpression were due to local genome rearrangements induced by radiation. The diversity observed among these 10 clones suggests that some are likely to be involved in tumorous growth and not simply specific to a certain cell or tissue type present in the tumor. Images Figure 1 Figure 2 PMID:16653233

  8. IP3 stimulates CA++ efflux from fusogenic carrot protoplasts

    International Nuclear Information System (INIS)

    Polyphosphoinositide breakdown plays an important role in signal transduction in animal cells (Berridge and Irvine, 1984, Nature, 312:315). Upon stimulation, phospholipase C hydrolyzes phosphatidylinositol 4,5-bisphosphate to inositol 1,4,5-trisphosphate (IP3) and diacylglycerol both of which act as cellular second messengers. IP3 mobilizes Ca++ from internal stores, hence the cytosolic free Ca++ concentration increases and those physiological activities regulated by Ca++ are stimulated. To test if plant cells also responded to IP3, Ca++ efflux studies were done with fusogenic carrot protoplasts released in EGTA. The protoplasts were preloaded with 45Ca++ placed in a Ca++-free medium, and efflux determined as 45Ca++ loss from the protoplasts. IP3 (10-20μM) caused enhanced 45Ca++ efflux and the response was sustained for at least 15 min. In plants, as in animals, the observed IP3-enhanced 45Ca++ efflux suggested that IP3 released Ca++ from internal stores, and the increased free cytosolic Ca++ activated Ca++ pumping mechanisms which restored the Ca++ concentration in the cytosol to the normal level

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

    OpenAIRE

    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 Ca2+ signals induced by a single stimulus vary, depending on the physiological state of the cell and experimental conditions. We compared Ca2+ homeostasis and stimulus-induced Ca2+ signals in guard cells of intact plants, epidermal strips, and isolated protoplasts. Single-cell ratiometric imaging with the Ca2+-s...

  10. Selecting the Electrofusion Condition of Alfalfa Protoplast%苜蓿原生质体电融合适宜条件的筛选

    Institute of Scientific and Technical Information of China (English)

    张凌云; 师尚礼

    2013-01-01

    为改良培育苜蓿(Medicago)新品种,拟建立苜蓿原生质体融合体系.通过电融合方法,使俄罗斯杂花苜蓿(M.varia)原生质体和甘农4号紫花苜蓿(M.sativa‘Gannong No.4’)原生质体进行非对称融合,获得了杂交细胞,研究不同失活处理条件、电场条件、原生质体密度对苜蓿原生质体融合的影响.结果表明:经过紫外灯辐射5min的俄罗斯杂花苜蓿原生质体和6 mmol· L-1 IOA处理的甘农4号紫花苜蓿原生质体,密度调至3×105~5×105个·mL-1,以交流电场强度15~20 V·cm-1、交流频率2000~2500 kHz,直流脉冲场强200~250 V·cm-1、脉冲宽幅40 μs、脉冲个数为3的条件为最适电融合条件,此时一对一有效融合率最高可达13.8%.%The somatic hybridization of asymmetric fusion between protoplasts isolated from callus of Russian variegated alfalfa and ‘Gannong No.4' alfalfa was obtained through electrofusion method.The effects of different inactivation pretreatments,electric field conditions and protoplast densities on alfalfa protoplast fusion were studied.Test results showed that the optimum inactivation pretreatments were that Russian variegated alfalfa protoplast was treated with ultraviolet radiation by 5 min and ‘Gannong No.4'alfalfa protoplast was treated with 6 mmol · L-1 iodoacetamide.The optimum electrofusion parameterswere AC electric field intensity for 15~20 V · cm-1,frequency for 2000~2500 kHz,DC electric field intensity for 200~250 V · cm-1,pulse width for 40 μs and pulse 3 times.The optimum protoplast density is 3 × 105 ~5 ×105 mL-1.The highest protoplast binary fusion frequency reached 13.8% with above conditions.

  11. Isolation and proteomic analysis of the SYP61 compartment reveal its role in exocytic trafficking in Arabidopsis

    Institute of Scientific and Technical Information of China (English)

    Georgia Drakakaki; Glenn Hicks; Natasha Raikhel; Wilhelmina van de Ven; Songqin Pan; Yansong Miao; Junqi Wang; Nana F Keinath; Brent Weatherly; Liwen Jiang; Karin Schumacher

    2012-01-01

    The endomembrane system is a complex and dynamic intracellular trafficking network.It is very challenging to track individual vesicles and their cargos in real time; however,affinity purification allows vesicles to be isolated in their natural state so that their constituent proteins can be identified.Pioneering this approach in plants,we isolated the SYP61 trans-Golgi network compartment and carried out a comprehensive proteomic analysis of its contents with only minimal interference from other organelles.The proteome of SYP61 revealed the association of proteins of unknown function that have previously not been ascribed to this compartment.We identified a complete SYP61 SNARE complex,including regulatory proteins and validated the proteome data by showing that several of these proteins associated with SYP61 in planta.We further identified the SYP121-complex and cellulose synthases,suggesting that SYP61 plays a role in the exocytic trafficking and the transport of cell wall components to the plasma membrane.The presence of proteins of unknown function in the SYP61 proteome including ECHIDNA offers the opportunity to identify novel trafficking components and cargos.The affinity purification of plant vesicles in their natural state provides a basis for further analysis and dissection of complex endomembrane networks.The approach is widely applicable and can afford the study of several vesicle populations in plants,which can be compared with the SYP61 vesicle proteome.

  12. High-frequency transfer of cloned herpes simplex virus type 1 sequences to mammalian cells by protoplast fusion.

    OpenAIRE

    Sandri-Goldin, R M; Goldin, A L; Levine, M.; Glorioso, J C

    1981-01-01

    The protoplast fusion technique of Schaffner (W. Schaffner, Proc. Natl. Acad. Sci. U.S.A. 77:2163-2167, 1980) has been adapted to introduce cloned herpes simplex virus genes into cultured mammalian cells. The technique involves digesting bacterial cell walls with lysozyme to produce protoplasts and then fusing the protoplasts to mammalian cells by treatment with polyethylene glycol. For monitoring transfer, protoplasts were labeled with the fluorescent dye fluorescein isothiocyanate before fu...

  13. 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.

  14. The use of morphogenic suspension cultures for the development of a protoplast regeneration system in lily

    NARCIS (Netherlands)

    Famelaer, L.; Bordas, M.; Baliu', E.; Ennik, E.; Meijer, H.; Tuyl, van J.M.; Creemers-Molenaar, J.

    1997-01-01

    The present study reports data on the development of a protoplast regeneration procedure in lily. Established morphogenic suspension cultures were obtained from callus cultures induced on mature embryos from crosses between cultivars of L. longiflorum. The effect on the frequency of protoplast divis

  15. Cytoplasmic transfer of oligomycin resistance during protoplast fusion of Saccharomycopsis lipolytica.

    OpenAIRE

    Matsuoka, M; Uchida, K.(Physikalisches Institut, University of Bonn, Bonn, Germany); Aiba, S

    1982-01-01

    Mitotic segregation of oligomycin resistance and oligomycin sensitivity was observed among the prototrophic progeny of protoplast fusion between drug-resistant and drug-sensitive complementary auxotrophs of Saccharomycopsis lipolytica. The transfer of oligomycin resistance by protoplast fusion without karyogamy suggests a cytoplasmic inheritance of this drug resistance determinant.

  16. 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. PMID:27192440

  17. PROTOPLAST FORMATION AND DNA-MEDIATED TRANSFORMATION OF FUSARIUM-CULMORUM TO HYGROMYCIN-B RESISTANCE

    NARCIS (Netherlands)

    CURRAGH, HJ; MOOIBROEK, H; WESSELS, JGH; MARCHANT, R; MULLAN, E

    1993-01-01

    This work involved firstly optimizing the protoplast yields for F. culmorum 159026, then setting up a system for DNA-mediated transformation. Higher protoplast yields and more rapid regeneration were obtained when the organic stabilizers sucrose and sorbitol were used rather than NH4Cl. Successful t

  18. 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.

  19. Rapid and simple isolation of vascular, epidermal and mesophyll cells from plant leaf tissue.

    Science.gov (United States)

    Endo, Motomu; Shimizu, Hanako; Araki, Takashi

    2016-08-01

    To understand physiological phenomena at the tissue level, elucidation of tissue-specific molecular functions in vivo is required. As an example of the current state of affairs, many genes in plants have been reported to have discordant levels of expression between bulk tissues and the specific tissues in which the respective gene product is principally functional. The principal challenge in deciphering such tissue-specific functions lies in separating tissues with high spatiotemporal resolution to evaluate accurate gene expression profiles. Here, we provide a simple and rapid tissue isolation protocol to isolate all three major leaf tissues (mesophyll, vasculature and epidermis) from Arabidopsis within 30 min with high purity. On the basis of the different cell-to-cell connectivities of tissues, the mesophyll isolation is achieved by making protoplasts, and the vasculature and epidermis isolation is achieved through sonication and enzymatic digestion of leaves. We have successfully tested the protocol on several other plant species, including crop plants such as soybean, tomato and wheat. Furthermore, isolated tissues can be used not only for tissue-specific transcriptome assays but also potentially for tissue-specific proteome and methylome assays. PMID:27388555

  20. Liposome-enhanced transformation of Streptococcus lactis and plasmid transfer by intergeneric protoplast fusion of Streptococcus lactis and Bacillus subtilis

    NARCIS (Netherlands)

    Vossen, Jos M.B.M. van der; Kok, Jan; Lelie, Daniel van der; Venema, Gerhardus

    1988-01-01

    An efficient protoplast transformation system and a procedure of plasmid transfer by means of protoplast fusion is described for Streptococcus lactis. Protoplasts of S. lactis IL1403 and S. lactis MG1363 were transformed by pGK12 [2.9 MDa erythromycin resistance (Emr)] with an efficiency of 3 × 10^5

  1. 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.

  2. Myo-Inositol trisphosphate mobilizes calcium from fusogenic carrot (Daucus carota L.) protoplasts

    International Nuclear Information System (INIS)

    To determine whether or not inositol trisphosphate (IP3) mobilizes calcium in higher plant cells; they investigated the effect of IP3 on Ca2+ fluxes in fusogenic carrot (Daucus carota L.) protoplasts. The protoplasts were incubated in 45Ca2+-containing medium and the 45Ca2+ associated with the protoplasts was monitored with time. Addition of IP3 (20 micromolar) caused a 17% net loss of the accumulated 45Ca2+ within 4 minutes. There was a reuptake of 45Ca2+ and the protoplasts recovered to their initial value by 10 minutes. Phytic acid (IP6), also stimulated 45Ca2+ efflux from the protoplasts. Both the IP3- and the IP6-induced 45Ca2+ efflux were inhibited by the calmodulin antagonist, trifluoperazine

  3. 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.

  4. 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.

  5. Interspecific hybridization by protoplast fusion in Nicotiana. Confirmation and extension

    Energy Technology Data Exchange (ETDEWEB)

    Smith, H.H. (Brookhaven National Lab., Upton, NY); Kao, K.N.; Combatti, N.C.

    1976-01-01

    Protoplasts of Nicotiana glauca and N. langsdorffi were prepared from leaf tissue by enzymatic digestion and were fused with the aid of polyethylene glycol. The mixed population of protoplasts was grown first on an enriched medium (M3 of Kao, et al.) and was then transferred to a medium lacking phytohormones, which selects against the parental types. A total of 174 calli that grew on the hormoneless medium were obtained. Mature flowering plants were differentiated from 19 different calli, and more than one from three of these, making 23 regenerated plants in all. Each of the plants was shown to be a parasexual hybrid in that it formed tumors; and the corolla, leaf, and plant habit were similar to, but somewhat different from, the amphiploid produced by cross pollination. The parasexual hybrids were examined cytologically and, instead of the amphiploid number 42, they were found to have a range of from 56 to 64 chromosomes, which accounted for the different characteristics observed. At meiosis mostly bivalents were formed and pollen fertility was high, averaging 84 percent. Seeds or progeny have been obtained from 16 of the hybrids. The unusual chromosome numbers found in the parasexual hybrids may have been due primarily to triple fusions (giving 60-66 chromosomes) followed by losses during callus growth, accompanied by selection for a particular range of aneuploidy (2n = 56 to 64) favorable for development of plantlets from calli.

  6. Molecular screening tools to study Arabidopsis transcription factors

    Directory of Open Access Journals (Sweden)

    Nora eWehner

    2011-11-01

    Full Text Available In the model plant Arabidopsis thaliana, more than 2000 genes are estimated to encode transcription factors (TFs, which clearly emphasizes the importance of transcriptional control. Although genomic approaches have generated large TF Open Reading Frame (ORF collections, only a limited number of these genes is functionally characterized, yet. This review evaluates strategies and methods to identify TF functions. In particular, we focus on two recently developed TF screening platforms, which make use of publi-cally available GATEWAY® compatible ORF collections. (1 The Arabidopsis thaliana TF ORF over-Expression (AtTORF-Ex library provides pooled collections of transgenic lines over-expressing HA-tagged TF genes, which are suited for screening approaches to define TF functions in stress defense and development. (2 A high-throughput microtiter plate based Protoplast Trans Activation (PTA system has been established to screen for TFs which are regulating a given promoter:Luciferase construct in planta.

  7. The effect of exposure to microgravity on the development and structural organisation of plant protoplasts flown on Biokosmos 9.

    Science.gov (United States)

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

    1992-01-01

    Preparatory experiments for the IML-1 (International Microgravity Laboratory) mission to be flown on the Space Shuttle in January, 1992, were performed on a 14 day flight on Biokosmos 9 (Kosmos 2044) in September 1989. The purpose of the experiment was to study the effect of weightlessness on protoplast regeneration. Problems with late access to the space vehicle meant that the newly isolated protoplasts from hypocotyl cells of rapeseed (Brassica napus L. cv Niklas) and suspension cultures of carrot (Daucus carota L, cv Nobo) had to be stored at 4 degrees C for 36 h prior to the launch of the biosatellite, in order to delay cell wall regeneration until the samples were in orbit. In the flight samples and the ground controls, a portion of the total number of protoplasts regenerated cell walls. The growth of flight rapeseed cells was only 56% compared to the ground control; the respective growth of carrot cells in orbit was 82% of the ground control. Analysis demonstrated that the peroxidase activity and the amount of protein was lower in the flight samples than in the ground controls. The number of different isoenzymes was also decreased in the flight samples. A 54% decrease in the production of cellulose was found in rapeseed, and a 71% decrease in carrot. Hemicellulose production was also decreased in the flight samples compared to the ground controls. Ultrastructural analysis of the cell aggregates from the protoplasts cultured in orbit, demonstrated that hydrolysis and disappearance of reserve starch occurred in the flight cell plastids. The mitochondria were more varied in appearance in the flight samples than in the ground control cells. An increased frequency of the occurrence of folds formed by the plasmalemma together with an increase in the degree of complexity of these folds was also observed. Fluorescence analysis showed a decrease of the calcium content in cell cultures under space flight compared to the ground controls. One general effect of the stay

  8. 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

  9. Rapid isolation and sequencing of purified plasmid DNA from Bacillus subtilis.

    OpenAIRE

    Voskuil, M. I.; Chambliss, G H

    1993-01-01

    We report two methods for isolation of plasmid DNA from the gram-positive bacterium Bacillus subtilis. The protoplast alkaline lysis procedure was developed for general use, and the protoplast alkaline lysis magic procedure was developed for isolation of DNA for sequencing. Both procedures yielded large amounts of high-quality DNA in less than 1 h, while current protocols require 4 to 7 h to perform and give lower yields and quality. Plasmid DNA was obtained from strains containing either hig...

  10. The Elucidation of the Interactome of 16 Arabidopsis bZIP Factors Reveals Three Independent Functional Networks

    Science.gov (United States)

    Llorca, Carles Marco; Berendzen, Kenneth Wayne; Malik, Waqas Ahmed; Mahn, Stefan; Piepho, Hans-Peter; Zentgraf, Ulrike

    2015-01-01

    The function of the bZIP transcription factors is strictly dependent on their ability to dimerize. Heterodimerization has proven to be highly specific and is postulated to operate as a combinatorial mechanism allowing the generation of a large variety of dimers with unique qualities by specifically combining a small set of monomers; an assumption that has not yet been tested systematically. Here, the interaction pattern and the transactivation properties of 16 Arabidopsis thaliana bZIPs are examined in transiently transformed Arabidopsis protoplasts to deliver a perspective on the relationship between bZIP dimerization and function. An interaction matrix of bZIPs belonging to the C, G, H, and S1 bZIP groups was resolved by Bimolecular Fluorescent Complementation (BiFC) coupled to quantitative flow cytometric analysis, while an extensive GUS reporter gene assay was carried out to determine the effect of different bZIP pairs on the expression of four different known bZIP-targeted promoters. Statistical data treatment and complementary bioinformatic analysis were performed to substantiate the biological findings. According to these results, the 16 bZIPs interact in three isolated networks, within which their members dimerize non-specifically and exhibit a significant level of functional redundancy. A coherent explanation for these results is supported by in silico analysis of differences in the length, structure and composition of their leucine zippers and appears to explain their dimerization specificity and dynamics observed in vivo quite well. A model in which the bZIP networks act as functional units is proposed. PMID:26452049

  11. 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.

  12. 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.

  13. Redox modulation of the expression of bacterial genes encoding cysteine-rich proteins in plant protoplasts.

    OpenAIRE

    Piñeiro Galvin, Manuel; García Olmedo, Francisco; Diaz Rodriguez, Isabel

    1994-01-01

    Activity of neomycin phosphotransferase II (NPTII; gene, neo; five cysteines) in tobacco protoplasts transfected with fusions of the octopine TR2' or cauliflower mosaic virus 35S promoter and the neo gene, with or without a signal peptide, increased up to 8-fold in response to externally added dithiothreitol at concentrations that did not affect protoplast viability (up to 2.5 mM). Activity of phosphinothricin acetyltransferase (PAT; gene, bar; one cysteine) expressed under control of the TR1...

  14. Overexpression of SpCBL6, a calcineurin B-like protein of Stipa purpurea, enhanced cold tolerance and reduced drought tolerance in transgenic Arabidopsis.

    Science.gov (United States)

    Zhou, Yanli; Cheng, Ying; Yang, Yunqiang; Li, Xiong; Supriyo, Basak; Sun, Xudong; Yang, Yongping

    2016-09-01

    The purpose of the present study was to characterize SpCBL6 (GenBank accession number: KT780442) from Stipa purpurea and elucidate the function of this protein in abiotic stress. The full-length cDNA of SpCBL6 was isolated from S. purpurea by rapid amplification of cDNA ends methods. Laser confocal microscopy was used to analyze the subcellular localization of SpCBL6. The constructs of 35S:GFP-SpCBL6 was used to transform wild-type (WT) Arabidopsis plants (ecotype Columbia-0) with the floral dip method. Quantitative reverse-transcription PCR (qRT-PCR), water potential, photosynthetic efficiency (F v/F m), and ion leakage was performed to investigate the role of SpCBL6 in abiotic stress. The open reading frame of SpCBL6 contains 681 bp nucleotides and encodes a 227-amino acid polypeptide. Phylogenetic analysis indicated that SpCBL6 showed the highest similarity with rice OsCBL6. SpCBL6 transcripts were induced by freezing and drought treatments. Subcellular localization analysis showed that SpCBL6 was located in membrane of protoplast. Overexpression of SpCBL6 in Arabidopsis thaliana demonstrated that the transgenic plants were more tolerant to cold treatment, but less tolerant to drought, compared with the plants. qRT-PCR analysis showed that the drought stress marker genes were inhibited in transgenic plants, whereas the cold stress marker genes were enhanced. Further analysis showed that SpCBL6-overexpressing plants showed enhanced water potential, photosynthetic efficiency (F v/F m), and reduced ion leakage compared with the wild-type after cold treatment. Collectively, these results indicate that SpCBL6, a new member of the CBL gene family isolated from S. purpurea, enhances cold tolerance and reduces drought tolerance in plants. PMID:27393148

  15. Activation of Tag1 transposable elements in Arabidopsis dedifferentiating cells and their regulation by CHROMOMETHYLASE 3-mediated CHG methylation.

    Science.gov (United States)

    Khan, Asif; Yadav, Narendra Singh; Morgenstern, Yaakov; Zemach, Assaf; Grafi, Gideon

    2016-10-01

    Dedifferentiation, that is, the acquisition of stem cell-like state, commonly induced by stress (e.g., protoplasting), is characterized by open chromatin conformation, a chromatin state that could lead to activation of transposable elements (TEs). Here, we studied the activation of the Arabidopsis class II TE Tag1, in which two copies, situated close to each other (near genes) on chromosome 1 are found in Landsberg erecta (Ler) but not in Columbia (Col). We first transformed protoplasts with a construct in which a truncated Tag1 (ΔTag1 non-autonomous) blocks the expression of a reporter gene AtMBD5-GFP and found a relatively high ectopic excision of ΔTag1 accompanied by expression of AtMBD5-GFP in protoplasts derived from Ler compared to Col; further increase was observed in ddm1 (decrease in DNA methylation1) protoplasts (Ler background). Ectopic excision was associated with transcription of the endogenous Tag1 and changes in histone H3 methylation at the promoter region. Focusing on the endogenous Tag1 elements we found low level of excision in Ler protoplasts, which was slightly and strongly enhanced in ddm1 and cmt3 (chromomethylase3) protoplasts, respectively, concomitantly with reduction in Tag1 gene body (GB) CHG methylation and increased Tag1 transcription; strong activation of Tag1 was also observed in cmt3 leaves. Notably, in cmt3, but not in ddm1, Tag1 elements were excised out from their original sites and transposed elsewhere in the genome. Our results suggest that dedifferentiation is associated with Tag1 activation and that CMT3 rather than DDM1 plays a central role in restraining Tag1 activation via inducing GB CHG methylation. PMID:27475038

  16. 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.

  17. 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. PMID:25988244

  18. Photorepair mutants of Arabidopsis

    International Nuclear Information System (INIS)

    UV radiation induces two major DNA damage products, the cyclobutane pyrimidine dimer (CPD) and, at a lower frequency, the pyrimidine (6-4) pyrimidinone dimer (6-4 product). Although Escherichia coli and Saccharomyces cerevisiae produce a CPD-specific photolyase that eliminates only this class of dimer, Arabidopsis thaliana, Drosophila melanogaster, Crotalus atrox, and Xenopus laevis have recently been shown to photoreactivate both CPDs and 6-4 products. We describe the isolation and characterization of two new classes of mutants of Arabidopsis, termed uvr2 and uvr3, that are defective in the photoreactivation of CPDs and 6-4 products, respectively. We demonstrate that the CPD photolyase mutation is genetically linked to a DNA sequence encoding a type II (metazoan) CPD photolyase. In addition, we are able to generate plants in which only CPDs or 6-4 products are photoreactivated in the nuclear genome by exposing these mutants to UV light and then allowing them to repair one or the other class of dimers. This provides us with a unique opportunity to study the biological consequences of each of these two major UV-induced photoproducts in an intact living system

  19. Responses of corn root protoplasts to exogenous reduced nicotinamide adenine dinucleotide: Oxygen consumption, ion uptake, and membrane potential

    OpenAIRE

    Lin, Willy

    1982-01-01

    Addition of 1.5 mM NADH tripled the O2 consumption in corn root protoplasts. The stimulation was temperature and pH dependent, specific to NADH, and accompanied by a 2- to 3-fold increase in K+ and Pi uptake into protoplasts. The increase in ion uptake was not due to the accumulation of NADH into protoplasts. The effect of exogenous NADH on O2 consumption and ion uptake was also evident in corn root segments but to a lesser extent. A 20-mV hyperpolarization of protoplast membrane potential oc...

  20. Hydrogen peroxide affects ion channels in lily pollen grain protoplasts.

    Science.gov (United States)

    Breygina, M A; Abramochkin, D V; Maksimov, N M; Yermakov, I P

    2016-09-01

    Ion homeostasis plays a central role in polarisation and polar growth. In several cell types ion channels are controlled by reactive oxygen species (ROS). One of the most important cells in the plant life cycle is the male gametophyte, which grows under the tight control of both ion fluxes and ROS balance. The precise relationship between these two factors in pollen tubes has not been completely elucidated, and in pollen grains it has never been studied to date. In the present study we used a simple model - protoplasts obtained from lily pollen grains at the early germination stage - to reveal the effect of H2 O2 on cation fluxes crucial for pollen germination. Here we present direct evidence for two ROS-sensitive currents on the pollen grain plasma membrane: the hyperpolarisation-activated calcium current, which is strongly enhanced by H2 O2 , and the outward potassium current, which is modestly enhanced by H2 O2 . We used low concentrations of H2 O2 that do not cause an intracellular oxidative burst and do not damage cells, as demonstrated with fluorescent staining. PMID:27115728

  1. Protoplast fusion technology for somatic hybridisation in Phaseolus

    Directory of Open Access Journals (Sweden)

    Ochatt S.

    2008-01-01

    Full Text Available The success of interspecific breeding between Phaseolus vulgaris L. (PV and the two donor species Phaseolus coccineus L. (PC or Phaseolus polyanthus Greenm. (PP requires the utilization of the donor species as female parents. Although incompatibility barriers are post-zygotic, success in such F1 crosses is very limited due to early hybrid embryo abortion. Rescue techniques for globular or early heart-shaped embryos have been improved but hybrid plant regeneration remains very difficult. In this study we describe the use of protoplast fusion techniques within the genus Phaseolus, as an alternative to succeed crosses between PP or PC and PV. Large numbers of heterokaryons have been produced using different genotypes and procedures for fusion, based either on electro-fusion (750 or 1500 V.cm-1, or on the use of a chemical micro-method with polyethylene glycol (PEG 6000 as the fusing agent. Both divisions of heterokaryons and the formation of heterokaryon-derived microcalli were observed.

  2. 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.

  3. Analysis of DNA polymerase activity in Petunia protoplasts treated with clastogenic agents

    International Nuclear Information System (INIS)

    Clastogenic agents, i.e. agents that can induce chromosome or DNA breakage, have been shown to enhance the role of direct gene transfer to protoplasts. The effect was analysed at the enzymatic level using protoplast homogenates as well as intact protoplasts. For that purpose existing procedures were modified to enable measurement of DNA polymerase in vivo. In the system used, external DNA was able to enter the cells without the addition of membrane-permeabilizing compounds. When comparing total DNA polymerase activity of protoplasts irradiated with X-rays or UV-light with that of untreated cells we did not observe significant differences. Incubation of protoplasts with high doses of bleomycin affected total DNA polymerase activity negatively. but dideoxythymidine triphosphate-sensitive activity was not influenced. We conclude that the DNA strand-breaks induced by low doses of X-rays. UV-light or bleomycin do not increase the total or the repair-DNA polymerase activity and. therefore. that the increase in the transformation rates after DNA strand-breaking is not preceded by enhanced DNA polymerase activity. (author)

  4. 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.

  5. 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'

  6. Analysis of mutant plants resistant to salt or water stress and to proline analogues obtained from the protoplasts of Nicotiana plumbaginifolia viviani

    International Nuclear Information System (INIS)

    Salt resistant, proline analogue resistant and osmotolerant cell lines were isolated by submitting to selection the mutagenized protoplasts from the haploid of Nicotiana plumbaginifolia viviani. One day after isolation, the protoplasts were exposed to UV light (2.5 x 10-6J.mm-2.s-1 for 20 s). The frequency of mutation was about 10-5. NaCl and KCl (200 mM) were used for salt stress and 25% polyethyleneglycol (6000) for osmotic stress. Two analogues of proline, azetidine-2-carboxylate (A2C) and 4-hydroxyproline (Hyp) (0.2 mM), were used to inhibit more specifically enzymes of the pathway and cell division. All the resistant lines were maintained in a stable condition after three passages in the absence of selection pressure. Resistant calli growing on the non-selective media accumulated free proline at levels that were five to ten times higher than those in the wild type. The levels of proline overproduction in salt resistant and osmotolerant lines were higher than those observed in the proline analogue resistant line. All the selected traits were transmitted to the progeny as a single dominant gene mutation. (author). 3 refs, 2 figs, 1 tab

  7. 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.

  8. Aluminum ions induce oat protoplasts to produce an extracellular (1 yields 3). beta. -D-glucan

    Energy Technology Data Exchange (ETDEWEB)

    Schaeffer, H.J.; Walton, J.D. (Michigan State Univ., East Lansing (USA))

    1990-09-01

    Aluminum chloride induced mesophyll protoplasts of oat (Avena sativa) to produce an extracellular polysaccharide (EPS). EPS induced by AlCl{sub 3} appeared identical to that produced in response to the phytotoxin victorin. Al ions at 1 millimolar were toxic to protoplasts, but maximum EPS production occurred at a sublethal concentration of 200 micromolar, assayed at pH 6.0. As measured by incorporation of ({sup 14}C)glucose, AlCl{sub 3} stimulated EPS production 10- to 15-fold. Pretreatment of protoplasts with cycloheximide prevented EPS production but not cell death in response to AlCl{sub 3}, indicating that protein synthesis was necessary for EPS production but not for the phytotoxicity of Al ions. The trivalent salts of Y, Yb, Gd, and In also induced EPS production but those of Sc, Fe, Ga, Cr, and La did not. Mesophyll protoplasts from an acid-soil tolerant oat cultivar produced less EPS in response to AlCl{sub 3} than the acid-soil sensitive cultivar Fla 501. EPS was also produced by wheat (Triticum aestivum) and barley (Hordeum vulgare) protoplasts in response to AlCl{sub 3}. An Al-tolerant cultivar of wheat, Atlas, produced less EPS than an Al-sensitive cultivar, Scout, but an Al-tolerant cultivar of barley, Dayton, produced more than the Al-sensitive cultivar Kearney. Therefore, production of EPS by protoplasts in response to Al ions did not appear to be related to Al ion tolerance at the level of whole plants. EPS fluoresced in the presence of Calcofluor and Sirofluor and was degraded by purified laminarinase ((1{yields}3){beta}-D-glucanase) but did not pectinase (polygalacturonase). EPS was composed solely of glucose in 1{yields}3 linkages; hence it is a (1{yields}3){beta}-D-glucan (callose).

  9. Aluminum ions induce oat protoplasts to produce an extracellular (1→3)β-D-glucan

    International Nuclear Information System (INIS)

    Aluminum chloride induced mesophyll protoplasts of oat (Avena sativa) to produce an extracellular polysaccharide (EPS). EPS induced by AlCl3 appeared identical to that produced in response to the phytotoxin victorin. Al ions at 1 millimolar were toxic to protoplasts, but maximum EPS production occurred at a sublethal concentration of 200 micromolar, assayed at pH 6.0. As measured by incorporation of [14C]glucose, AlCl3 stimulated EPS production 10- to 15-fold. Pretreatment of protoplasts with cycloheximide prevented EPS production but not cell death in response to AlCl3, indicating that protein synthesis was necessary for EPS production but not for the phytotoxicity of Al ions. The trivalent salts of Y, Yb, Gd, and In also induced EPS production but those of Sc, Fe, Ga, Cr, and La did not. Mesophyll protoplasts from an acid-soil tolerant oat cultivar produced less EPS in response to AlCl3 than the acid-soil sensitive cultivar Fla 501. EPS was also produced by wheat (Triticum aestivum) and barley (Hordeum vulgare) protoplasts in response to AlCl3. An Al-tolerant cultivar of wheat, Atlas, produced less EPS than an Al-sensitive cultivar, Scout, but an Al-tolerant cultivar of barley, Dayton, produced more than the Al-sensitive cultivar Kearney. Therefore, production of EPS by protoplasts in response to Al ions did not appear to be related to Al ion tolerance at the level of whole plants. EPS fluoresced in the presence of Calcofluor and Sirofluor and was degraded by purified laminarinase [(1→3)β-D-glucanase] but did not pectinase (polygalacturonase). EPS was composed solely of glucose in 1→3 linkages; hence it is a (1→3)β-D-glucan (callose)

  10. 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

    inhibitor tolerance of this yeast, a combination of UV mutagenesis and protoplast fusion was used to construct strains with improved performance. Firstly, UVinduced mutants were screened and selected for improved tolerance towards furfural. The most promised mutant, S. passalidarum M7, produced 50% more...... 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...

  11. Arabidopsis thaliana is an asymptomatic host of Alfalfa mosaic virus.

    Science.gov (United States)

    Balasubramaniam, Muthukumar; Ibrahim, Amr; Kim, Bong-Suk; Loesch-Fries, L Sue

    2006-11-01

    The susceptibility of Arabidopsis thaliana ecotypes to infection by Alfalfa mosaic virus (AMV) was evaluated. Thirty-nine ecotypes supported both local and systemic infection, 26 ecotypes supported only local infection, and three ecotypes could not be infected. No obvious symptoms characteristic of virus infection developed on the susceptible ecotypes under standard conditions of culture. Parameters of AMV infection were characterized in ecotype Col-0, which supported systemic infection and accumulated higher levels of AMV than the symptomatic host Nicotiana tabacum. The formation of infectious AMV particles in infected Col-0 was confirmed by infectivity assays on a hypersensitive host and by electron microscopy of purified virions. Replication and transcription of AMV was confirmed by de novo synthesis of AMV subgenomic RNA in Col-0 protoplasts transfected with AMV RNA or plasmids harboring AMV cDNAs. PMID:16875753

  12. Fluorescence-Activated Nucleolus Sorting in Arabidopsis.

    Science.gov (United States)

    Pontvianne, Frédéric; Boyer-Clavel, Myriam; Sáez-Vásquez, Julio

    2016-01-01

    Nucleolar isolation allows exhaustive characterization of the nucleolar content. Centrifugation-based protocols are not adapted to isolation of nucleoli directly from a plant tissue because of copurification of cellular debris. We describe here a method that allows the purification of nucleoli using fluorescent-activated cell sorting from Arabidopsis thaliana leaves. This approach requires the expression of a specific nucleolar protein such as fibrillarin fused to green fluorescent protein in planta. PMID:27576720

  13. 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.

  14. 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

  15. Inhibition of lipoxygenase in lentil protoplasts by expression of antisense RNA

    NARCIS (Netherlands)

    Vliegenthart, J.F.G.; Maccarrone, M.; Hilbers, M.P.; Finazzi Agrò, A.

    1995-01-01

    A number of plasmids were constructed containing chimeric genes consisting of fragments of antisense-oriented lentil lipoxygenase cDNA. The different constructs were tested for their ability to lower lipoxygenase activity in lentil protoplasts. Plasmids containing a full length lentil lipoxygenase c

  16. [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. PMID:25985526

  17. 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.

  18. Regulation of WRKY46 Transcription Factor Function by Mitogen-Activated Protein Kinases in Arabidopsis thaliana.

    Science.gov (United States)

    Sheikh, Arsheed H; Eschen-Lippold, Lennart; Pecher, Pascal; Hoehenwarter, Wolfgang; Sinha, Alok K; Scheel, Dierk; Lee, Justin

    2016-01-01

    Mitogen-activated protein kinase (MAPK) cascades are central signaling 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 defense 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 defense. PMID:26870073

  19. 3D gel map of Arabidopsis complex I

    OpenAIRE

    Katrin ePeters; Katharina eBelt; Hans-Peter eBraun

    2013-01-01

    Complex I has a unique structure in plants and includes extra subunits. Here, we present a novel study to define its protein constituents. Mitochondria were isolated from Arabidopsis thaliana cell cultures, leaves and roots. Subunits of complex I were resolved by 3D blue native (BN)/SDS/SDS-PAGE and identified by mass spectrometry. Overall, 55 distinct proteins were found, 7 of which occur in pairs of isoforms. We present evidence that Arabidopsis complex I consists of 49 distinct types of su...

  20. Evidence for five divergent thioredoxin h sequences in Arabidopsis thaliana.

    OpenAIRE

    Rivera-Madrid, R.; Mestres, D; Marinho, P.; Jacquot, J P; Decottignies, P; Miginiac-Maslow, M; Meyer, Y.

    1995-01-01

    Five different clones encoding thioredoxin homologues were isolated from Arabidopsis thaliana cDNA libraries. On the basis of the sequences they encode divergent proteins, but all belong to the cytoplasmic thioredoxins h previously described in higher plants. The five proteins obtained by overexpressing the coding sequences in Escherichia coli present typical thioredoxin activities (NADP(+)-malate dehydrogenase activation and reduction by Arabidopsis thioredoxin reductase) despite the presenc...

  1. 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.

  2. Use of a temperature-sensitive, protoplast-forming Neurospora crassa strain for the detection of antifungal antibiotics.

    OpenAIRE

    Selitrennikoff, C P

    1983-01-01

    Protoplasts of the temperature-sensitive osmotic-1 mutant of Neurospora crassa grew and divided as cell wall-less cells when incubated under certain conditions at 37 degrees C. Each protoplast regenerated cell wall and formed a mycelium when the temperature was shifted to 22 degrees C. Cell wall regeneration, but not cell growth, was prevented by the inhibition of cell wall assembly functions. Thus, the inhibition of cell wall regeneration could serve as an indicator of the mode of action of ...

  3. Red light stimulates an electrogenic proton pump in Vicia guard cell protoplasts.

    OpenAIRE

    Serrano, E E; Zeiger, E; Hagiwara, S.

    1988-01-01

    Stomatal opening in response to light has a component that matches the absorption spectrum of chlorophyll; however, the intervening sensory transduction steps are not well understood. To study this process, we illuminated Vicia faba guard cell protoplasts with red light and simultaneously recorded current flow across the plasma membrane, utilizing the patch clamp technique in the whole cell configuration. We report evidence that under voltage clamp conditions, red light (1 mmol of photons.m-2...

  4. Glutathione and fungal elicitor regulation of a plant defense gene promoter in electroporated protoplasts

    OpenAIRE

    Dron, Michel; Clouse, Steven D.; Dixon, Richard A.; Lawton, Michael A; Lamb, Christopher J.

    1988-01-01

    To investigate the mechanisms underlying activation of plant defenses against microbial attack we have studied elicitor regulation of a chimeric gene comprising the 5′ flanking region of a defense gene encoding the phytoalexin biosynthetic enzyme chalcone synthase fused to a bacterial chloramphenicol acetyltransferase gene. Glutathione or fungal elicitor caused a rapid, marked but transient expression of the chimeric gene electroporated into soybean protoplasts. The response closely resembled...

  5. Expression of Bottom Component RNA of Cowpea Mosaic Virus in Cowpea Protoplasts

    OpenAIRE

    Rezelman, Geertje; Goldbach, Rob; van Kammen, Albert

    1980-01-01

    Upon inoculation of cowpea protoplasts with the bottom component of cowpea mosaic virus, at least six virus-induced proteins (with sizes of 170, 110, 87, 84, 60, and 32 kilodaltons) are synthesized, but not the capsid proteins (37 and 23 kilodaltons). These bottom-component-induced proteins were studied with respect to their genetic origin and mode of synthesis. The analyses were based on their electrophoretic peptide patterns resulting from partial digestion with Staphylococcus aureus protea...

  6. Heat resistance of bacterial spores correlated with protoplast dehydration, mineralization, and thermal adaptation.

    OpenAIRE

    Beaman, T C; Gerhardt, P

    1986-01-01

    Twenty-eight types of lysozyme-sensitive spores among seven Bacillus species representative of thermophiles, mesophiles, and psychrophiles were obtained spanning a 3,000-fold range in moist-heat resistance. The resistance within species was altered by demineralization of the native spores to protonated spores and remineralization of the protonated spores to calcified spores and by thermal adaptation at maximum, optimum, and minimum sporulation temperatures. Protoplast wet densities, and there...

  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

    1992-01-01

    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. PMID:11536947

  8. 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.

  9. Improving Protoplast Dissociation Yield and Division Frequency of Glycyrrhizia in flata Batal.%提高甘草原生质体游离产量及分裂频率的研究

    Institute of Scientific and Technical Information of China (English)

    刘昕; 余斌; 陈晓燕; 王清

    2011-01-01

    This study reports influencing factors of protoplast isolation, protoplast yield and division frequency of Glycyrrhizia inflata Batal.. Five enzyme combinations with different concentrations of cellulase, pectolase, macerozyme and three enzymolysis methods were used to dissociate the protoplast of leaves, hypocotyl and calli from Glycyrrhizia inflata Batal.. Results show that the best materials for protoplast isolation are leaves and hypocotyls from 11-day seedlings as well as calli subcultured five times. The highest frequency of protoplast isolation is obtained by using either 2.0% cellulase+0.5% pectolase, or 2% cellulase+0.25 % macerozyme+0.5% pectolase. Protoplast yield and viability for each of three methods are 1.25×105~1.26×105 individual · g-1 and 65.7%~70.5%; 1.43 × 105~1.44× 105 individual ·g-land 69.5%~72.8%; 1.10×105 ~1.16×105 individual · g-1 and 61.9%~69.4%, respectively. The average yield and viability of protoplast from hypocotyls cut longitudinally (1.19×105 individual · g-1 and 65.2%) was higher than from leaves and calli. Furthermore, the highest protoplast yield was obtained by shaking (40 r · min-1) 7h after enzymolysis. However, both optimizing protoplast yield and viability were reached after standing a while then shaking (40 r · min-1 ). The highest rate of protoplast division (2.34 %) was obtained by adjusting the protoplast density to 1 × 105 individual · g-1 then cultured in KM8P medium.%为探讨影响胀果甘草(Glycyrrhiza inflata Batal.)原生质体游离的因素,提高原生质体游离产量和分裂频率,试验采用纤维素酶、果胶酶、离析酶不同浓度组合的酶解液,在3种酶解方式下对甘草叶片、下胚轴、愈伤组织进行了原生质体游离.结果表明:苗龄11 d的甘草叶片、下胚轴及继代5次的愈伤组织是原生质体游离的良好材料,三者均在2.0%纤维素酶+0.5%果胶酶和2.O%纤维素酶+0.25%离析酶+0.5%果

  10. 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; Welinder, K G

    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 ...

  11. 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.

  12. A Superfamily of Arabidopsis Thaliana Retrotransposons

    OpenAIRE

    Konieczny, A; Voytas, D. F.; Cummings, M. P.; Ausubel, F M

    1991-01-01

    We describe a superfamily of Arabidopsis thaliana retrotransposable elements that consists of at least ten related families designated Ta1-Ta10. The Ta1 family has been described previously. Two genomic clones representing the Ta2 and Ta3 elements were isolated from an A. thaliana (race Landsberg erecta) λ library using sequences derived from the reverse transcriptase region of Ta1 as hybridization probes. Nucleotide sequence analysis showed that the Ta1, Ta2 and Ta3 families share >75% amino...

  13. 不同质膜稳定剂对烟草原生质体细胞壁再生的影响%The Effect of Different Cell Membrane Stabilizer on Reformation of Cell Wall from Tobacco Protoplasts

    Institute of Scientific and Technical Information of China (English)

    朱俊; 聂琼; 杨川龙; 陈茜

    2012-01-01

    The cell membrane stabilizer can increase the number of intact protoplasts, so as to prevent the disruption of the cell membrane, and promote the cell wall reformation from protoplast and formation of mitotic cell clusters. To investigate the effect of membrane stabilizers on the reformation of tobacco cell wall from protoplasts, three kinds of cell membrane stabilizers including either CaCl2 · 2H2O, or KH2PO4 or MES was added to the enzyme solution of protoplast, or their combination was supplemented in the solution, and protoplast was isolated, purified and cultrued. The results showed that three kinds of membrane stabilizers had certain effects on the protoplast quality, cell wall reformation and protoplast division. The optimum concentration was CaCl2 · 2H2O 10 mM, KH2PO4 0. 7 mM, and MES 5 mM. Among the tested membrane stabilizer combinations, the best one was CaCl2 · 2H2O 5 mM, KH2P04 0.35 mM and MES 2.5 mM, and the cell wall reformation rate and cell division rate were as high as 88.5% and 77.5% , respectively.%质膜稳定剂能增加完整原生质体数量,防止质膜破坏,促进原生质体细胞壁再生和细胞分裂形成细胞团.本研究拟在原生质体酶解液中分别加入CaCl2 ·2H2O、KH2 PO4、MES 3种质膜稳定剂,或3种质膜稳定剂组合分离、提纯、培养原生质体,探讨3种质膜稳定剂对烟草原生质体细胞壁再生的影响.结果表明:3种质膜稳定剂对于解离出的原生质体的质量及原生质体细胞壁再生和原生质体细胞分裂都有一定影响,最佳浓度分别为CaCl2·2H2O10mM、KH2PO4 0.7 mM、MES 5 mM;3种质膜稳定剂组合以CaCl2·2H2O 5 mM、KH2PO4 0.35 mM与MES 2.5 mM的组合最佳,原生质体壁再生率达88.5%,细胞分裂率达77.5%,表明能最快促进烟草原生质体细胞壁再生及细胞分裂生长.

  14. 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.

  15. 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.

  16. 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%.

  17. 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.

  18. 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)

  19. Protoplast transformation of recalcitrant alkaliphilic Bacillus sp. with methylated plasmid DNA and a developed hard agar regeneration medium.

    Directory of Open Access Journals (Sweden)

    Chenghua Gao

    Full Text Available Among the diverse alkaliphilic Bacillus strains, only a little have been reported to be genetically transformed. In this study, an efficient protoplast transformation procedure was developed for recalcitrant alkaliphilic Bacillus sp. N16-5. The procedure involved polyethylene glycol-induced DNA uptake by the protoplasts and subsequent protoplast regeneration with a developed hard agar regeneration medium. An in vivo methylation strategy was introduced to methylate the exogenous plasmid DNA for improving the transformation efficiency. The transformation efficiency reached to 1.1×10(5 transformants per µg plasmid DNA with methylated plasmid pHCMC04 and the developed hard agar regeneration medium. This procedure might also be applicable to the genetic transformation of other Bacillus strains.

  20. Production of enkephalin in tobacco protoplasts using tobacco mosaic virus RNA vector.

    Science.gov (United States)

    Takamatsu, N; Watanabe, Y; Yanagi, H; Meshi, T; Shiba, T; Okada, Y

    1990-08-20

    To examine the validity of the strategy to express a foreign gene as a fusion protein with the coat protein (CP) of tobacco mosaic virus (TMV), we have constructed ENK RNA by using an in vitro transcription system of TMV RNA. ENK RNA differs from TMV RNA only in that ENK RNA carries an additional sequence coding for Leu-enkephalin (Tyr-Gly-Gly-Phe-Leu) (Enk) with a preceding in-frame methionine just before the termination codon of CP gene. In protoplasts inoculated with ENK RNA, CP + Enk fusion protein accumulated as the major protein. PMID:2387417

  1. High-frequency transformation of Brevibacterium lactofermentum protoplasts by plasmid DNA.

    OpenAIRE

    Santamaria, R I; Gil, J.A.; Martin, J. F.

    1985-01-01

    An efficient polyethylene glycol-assisted method for transformation of Brevibacterium lactofermentum protoplasts that uses plasmid vectors has been developed. Two small plasmids, pUL330 (5.2 kilobases) and pUL340 (5.8 kilobases), both containing the kanamycin resistance gene from transposon Tn5 and the replication origin of the natural plasmid pBL1 of B. lactofermentum, were selected as vectors. Supercoiled forms of the plasmids yielded a 100-fold higher transformation frequency than did line...

  2. UV-killed protoplast fusion as a method for breeding killer yeasts

    International Nuclear Information System (INIS)

    A simple method for breeding killer yeasts without changing the nuclear genotype was described. Killer plasmids were introduced into recipient cells just after killing the protoplasts carrying killer plasmids with UV-rays. For the donors, UV-sensitive strains harboring killer plasmids having 100 base pair deletion and drug resistant mitochondria were constructed. Almost all fusants obtained showed the same nuclear phenotype as the recipient but had killer activity and drug resistance. Killer sake yeast were bred using a commercial strain, Kyokai 7,as the recipient, and they were confirmend to produce sake of the same quality as that produced with strain Kyokai 7 with no contamination by wild yeast

  3. Plastid sedimentation kinetics in roots of wild-type and starch-deficient mutants of Arabidopsis

    Science.gov (United States)

    MacCleery, S. A.; Kiss, J. Z.

    1999-01-01

    Sedimentation and movement of plastids in columella cells of the root cap were measured in seedlings of wild-type, a reduced starch mutant, and a starchless mutant of Arabidopsis. To assay for sedimentation, we used both linear measurements and the change of angle from the cell center as indices in vertical and reoriented plants with the aid of computer-assisted image analysis. Seedlings were fixed at short periods after reorientation, and plastid sedimentation correlated with starch content in the three strains of Arabidopsis. Amyloplasts of wild-type seedlings showed the greatest sedimentation, whereas plastids of the starchless mutant showed no significant sedimentation in the vertically grown and reoriented seedlings. Because previous research has shown that a full complement of starch is needed for full gravitropic sensitivity, this study correlates increased sensitivity with plastid sedimentation. However, although plastid sedimentation contributed to gravisensitivity, it was not required, because the gravitropic starchless mutant had plastids that did not sediment. This is the first study, to our knowledge, to measure plastid sedimentation in Arabidopsis roots after reorientation of seedlings. Taken together, the results of this study are consistent with the classic plastid-based and protoplast-based models of graviperception and suggest that multiple systems of perception exist in plant cells.

  4. Regulation of Arabidopsis thaliana Em genes : role of AB15

    NARCIS (Netherlands)

    Carles, C.; Bies-Etheve, N.; Aspart, L.; Léon-Kloosterziel, K.M.; Koornneef, M.; Echeverria, M.; Delseny, M.

    2002-01-01

    In order to identify new factors involved in Em (a class I Late Embryogenesis Abundant protein) gene expression, Arabidopsis mutants with an altered expression of an Em promoter GUS fusion construct and a modified accumulation of Em transcripts and proteins were isolated. Germination tests on ABA sh

  5. 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.

  6. Analysis of Microbe-Associated Molecular Pattern-Responsive Synthetic Promoters with the Parsley Protoplast System.

    Science.gov (United States)

    Kanofsky, Konstantin; Lehmeyer, Mona; Schulze, Jutta; Hehl, Reinhard

    2016-01-01

    Plants recognize pathogens by microbe-associated molecular patterns (MAMPs) and subsequently induce an immune response. The regulation of gene expression during the immune response depends largely on cis-sequences conserved in promoters of MAMP-responsive genes. These cis-sequences can be analyzed by constructing synthetic promoters linked to a reporter gene and by testing these constructs in transient expression systems. Here, the use of the parsley (Petroselinum crispum) protoplast system for analyzing MAMP-responsive synthetic promoters is described. The synthetic promoter consists of four copies of a potential MAMP-responsive cis-sequence cloned upstream of a minimal promoter and the uidA reporter gene. The reporter plasmid contains a second reporter gene, which is constitutively expressed and hence eliminates the requirement of a second plasmid used as a transformation control. The reporter plasmid is transformed into parsley protoplasts that are elicited by the MAMP Pep25. The MAMP responsiveness is validated by comparing the reporter gene activity from MAMP-treated and untreated cells and by normalizing reporter gene activity using the constitutively expressed reporter gene. PMID:27557767

  7. 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.

  8. Gravitropism and gravimorphism during regeneration from protoplasts of the moss Ceratodon purpureus (Hedw.) Brid

    Science.gov (United States)

    Wagner, T. A.; Sack, F. D.

    1998-01-01

    Wild-type (WT) protonemata of the moss Ceratodon purpureus grow upwards in darkness (negative gravitropism), whereas protonemata of the mutant, wrong-way response (wwr-1) grow down. Since Ceratodon protoplasts regenerate to form new protonemata, we analyzed whether the direction of filament emergence was influenced by gravity (gravimorphism) and determined the cytological events that correlated with the onset of gravitropism in WT and wwr-1 filaments formed de novo. In the WT the direction of filament emergence appeared to be gravimorphic as more than 66% of the new filaments emerged above the horizontal. In contrast, the direction of filament emergence was random in wwr-1. Tip-growing cells of both genotypes became gravitropic within a total of one to two cell divisions. Gravitropic curvature in wwr-1 was opposite in direction to that of WT, and the timing of curvature was comparable, indicating that the wwr-1 mutation acts during the onset of gravitropic competence. In time-lapse studies of both genotypes, neither a plastid-free zone nor obvious and extensive plastid sedimentation characteristic of mature dark-grown protonemata was observed in the new filaments prior to gravitropic curvature. Thus, it appears that these latter two features are not required for gravitropism in new protonemal filaments from protoplasts.

  9. Extracellular Nucleotides and Apyrases Regulate Stomatal Aperture in Arabidopsis1[W][OA

    Science.gov (United States)

    Clark, Greg; Fraley, Devin; Steinebrunner, Iris; Cervantes, Andrew; Onyirimba, James; Liu, Angela; Torres, Jonathan; Tang, Wenqiang; Kim, Joshua; Roux, Stanley J.

    2011-01-01

    This study investigates the role of extracellular nucleotides and apyrase enzymes in regulating stomatal aperture. Prior data indicate that the expression of two apyrases in Arabidopsis (Arabidopsis thaliana), APY1 and APY2, is strongly correlated with cell growth and secretory activity. Both are expressed strongly in guard cell protoplasts, as determined by reverse transcription-polymerase chain reaction and immunoblot analyses. Promoter activity assays for APY1 and APY2 show that expression of both apyrases correlates with conditions that favor stomatal opening. Correspondingly, immunoblot data indicate that APY expression in guard cell protoplasts rises quickly when these cells are moved from darkness into light. Both short-term inhibition of ectoapyrase activity by polyclonal antibodies and long-term suppression of APY1 and APY2 transcript levels significantly disrupt normal stomatal behavior in light. Stomatal aperture shows a biphasic response to applied adenosine 5′-[γ-thio]triphosphate (ATPγS) or adenosine 5′-[β-thio] diphosphate, with lower concentrations inducing stomatal opening and higher concentrations inducing closure. Equivalent concentrations of adenosine 5′-O-thiomonophosphate have no effect on aperture. Two mammalian purinoceptor inhibitors block ATPγS- and adenosine 5′-[β-thio] diphosphate-induced opening and closing and also partially block the ability of abscisic acid to induce stomatal closure and of light to induce stomatal opening. Treatment of epidermal peels with ATPγS induces increased levels of nitric oxide and reactive oxygen species, and genetically suppressing the synthesis of these agents blocks the effects of nucleotides on stomatal aperture. A luciferase assay indicates that treatments that induce either the closing or opening of stomates also induce the release of ATP from guard cells. These data favor the novel conclusion that ectoapyrases and extracellular nucleotides play key roles in regulating stomatal functions

  10. 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. PMID:27208266

  11. Characterization of a calmodulin binding protein kinase from Arabidopsis thalian

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    A full-length calmodulin binding protein kinase cDNA, AtCBK1, from Arabidopsis has been isolated by screening of an Arabidopsis cDNA library and by 5′-RACE. Northern blot and in situ hybridization indicated that the expression of AtCBK1 was more abundant in the vascular bundles and the meristems than in other tissues. The phylogenetic analyses reveal that AtCBK1 is different from animal CaMKs and it falls into CRK subgroup, indicating that they may come from different ancestors. The result suggests that AtCBK1 encodes a CaM-binding serine/threonine protein kinase.

  12. 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 (

  13. 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.

  14. Efficient gusA transient expression in Porphyra yezoensis protoplasts mediated by endogenous beta-tubulin flanking sequences

    Science.gov (United States)

    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 unknown. 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®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 35S 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.

  15. Expression of wild-type PtrIAA14.1, a poplar Aux/IAA gene causes morphological changes in Arabidopsis

    Directory of Open Access Journals (Sweden)

    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.

  16. 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.

  17. [14C]-Sucrose uptake by guard cell protoplasts of pisum sativum, argenteum mutant

    International Nuclear Information System (INIS)

    Guard cells rely on import for their supply with reduced carbon. The authors tested by silicone oil centrifugation the ability of guard cell protoplasts to accumulated [14C]-sucrose. Uptake rates were corrected after measurement of 14C-sorbitol and 3H2O spaces. Sucrose uptake followed biphasic kinetics, with a high-affinity component below 1 mM external sucrose (apparent Km 0.8 mM at 25C) and a low-affinity nonsaturable component above. Uptake depended on pH (optimum at pH 5.0). Variations in the concentrations of external KCl, CCCP, and valinomycin indicated that about one-half of the sucrose uptake rate could be related to an electrochemical gradient across the plasmalemma. Total uptake rates measured at 5 mM external sucrose seem to be sufficient to replenish emptied plastids with starch within a few hours

  18. 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.

  19. Break of symmetry in regenerating tobacco protoplasts is independent of nuclear positioning.

    Science.gov (United States)

    Brochhausen, Linda; Maisch, Jan; Nick, Peter

    2016-09-01

    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 (perinuclear 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 prerequisite 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. PMID:26898230

  20. Characterization of Alkaloid Uptake by Catharanthus roseus (L.) G. Don Protoplasts 1

    Science.gov (United States)

    McCaskill, David G.; Martin, DeAndra L.; Scott, A. Ian

    1988-01-01

    The accumulation of alkaloids by protoplasts of Catharanthus roseus (L.) G. Don var. Little Bright Eye was studied to determine the specificity of uptake and the role of ion trapping in the storage of alkaloids. Accumulation of the indole alkaloids vindoline, ajmalicine, tabersonine, and vinblastine was found to be biphasic, with an initial burst of uptake followed by a slow, prolonged phase of accumulation. The concentration and pH dependence of the initial burst of uptake for vindoline suggested that uptake occurred by simple diffusion. Uptake of nicotine was monophasic, with a half life of 5.2 minutes. The accumulation ratio (Ci/Ce) for nicotine at steady state and for the initial burst of uptake for vindoline and ajmalicine suggested that accumulation was driven by the pH gradient between the vacuole and the external assay medium. The second, sustained phase of uptake of vindoline was sensitive to inhibition by either 20 millimolar NaN3 or 0.5 millimolar Cu2+. In azide-treated protoplasts, the uptake for vindoline conformed to the kinetics of simple diffusion, with a half life of 4 minutes. The second phase of uptake for ajmalicine, although sensitive to inhibition by Cu2+, was insensitive to inhibition by NaN3. The biphasic uptake of the indole alkaloids was not due to any significant metabolism. It is concluded that accumulation and storage of the indole alkaloids is due only partly to ion trapping of the alkaloids by the low pH of the vacuole lumen. In the case of vindoline, there appears to be a specific energy-requiring uptake that is not seen with nicotine (which is not endogenous to Catharanthus). Accumulation of ajmalicine appears to involve both ion trapping and an azide-insensitive component, which may be due to complexation with organic counterions and phenolics. PMID:16666154

  1. Arabidopsis in Wageningen

    OpenAIRE

    Koornneef, M

    2013-01-01

    Arabidopsis thaliana is the plant species that in the past 25 years has developed into the major model species in plant biology research. This was due to its properties such as short generation time, its small genome and its easiness to be transformed. Wageningen University has played an important role in the development of this model, based on interdisciplinary collaborations using genetics as a major tool to investigate aspects of physiology, development, plant-microbe interactions and evol...

  2. 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 signaling...... Arabidopsis family of 34 genes. The predicted peptides are characterized by a conserved C-terminal sequence motif and additional primary structure conservation in a core region. The majority of these genes had not previously been annotated. A subset of the predicted peptides show high overall sequence...

  3. Use of Arabidopsis thaliana and Pseudomonas syringae in the Study of Plant Disease Resistance and Tolerance

    OpenAIRE

    Bent, Andrew F.; Kunkel, Barbara N.; Innes, Roger W.; Staskawicz, Brian J.

    1993-01-01

    The interaction between Arabidopsis thaliana and the bacterium Pseudomonas syringae is being developed as a model experimental system for plant pathology research. Race-specific ("gene-for-gene") resistance has been demonstrated for this interaction, and pathogen genes that determine avirulence have been isolated and characterized. Because certain lines of both Arabidopsis and soybean are resistant to bacteria carrying the avirulence genes avrRpt2 and avrB, extremely similar pathogen recognit...

  4. Plant-microbes interactions : Implication of Phyllobacterium brassicacearum in Arabidopsis responses to water deficit

    OpenAIRE

    Bresson, Justine

    2013-01-01

    Plant growth promoting rhizobacteria (PGPR) can enhance plant performance and plant tolerance to environmental stresses. Arabidopsis thaliana is a useful organism to study the mechanisms involved in plant-PGPR interactions. We analyzed multiple plant traits related to growth dynamics, development and physiology in order to assess the effects of Phyllobacterium brassicacearum STM196 strain, isolated from the rhizosphere of oilseed rape, on Arabidopsis responses to well-defined soil water avail...

  5. 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.

  6. Dithiothreitol increases f3-glucuronidase accumulation in transformed tobacco (Nicotiana tabacum) protoplasts without altering their viability or the synthesis and export of cellular proteins

    OpenAIRE

    Piñeiro Galvin, Manuel; Alamillo, Josefa M; García Olmedo, Francisco

    1999-01-01

    The effect of dithiothreitol (DTT) on the expression of the β-glucuronidase (GUS) reporter gene under the control of the CaMV-35 S promoter has been investigated by radioactive labelling and immunoprecipitation of the enzyme in protoplasts from stably transformed tobacco plants and compared with that observed in protoplasts transiently expressing the same gene construct. An increase in net accumulation of GUS during the culture period in response to externally added DTT (2 mm) was observed bo...

  7. Ectopic expression of R3 MYB transcription factor gene OsTCL1 in Arabidopsis, but not rice, affects trichome and root hair formation

    Science.gov (United States)

    Zheng, Kaijie; Tian, Hainan; Hu, Qingnan; Guo, Hongyan; Yang, Li; Cai, Ling; Wang, Xutong; Liu, Bao; Wang, Shucai

    2016-01-01

    In Arabidopsis, a MYB-bHLH-WD40 (MBW) transcriptional activator complex activates the homeodomain protein gene GLABRA2 (GL2), leading to the promotion of trichome formation and inhibition of root hair formation. The same MBW complex also activates single-repeat R3 MYB genes. R3 MYBs in turn, play a negative feedback role by competing with R2R3 MYB proteins for binding bHLH proteins, thus blocking the formation of the MBW complex. By BLASTing the rice (Oryza sativa) protein database using the entire amino acid sequence of Arabidopsis R3 MYB transcription factor TRICHOMELESS1 (TCL1), we found that there are two genes in rice genome encoding R3 MYB transcription factors, namely Oryza sativa TRICHOMELESS1 (OsTCL1) and OsTCL2. Expressing OsTCL1 in Arabidopsis inhibited trichome formation and promoted root hair formation, and OsTCL1 interacted with GL3 when tested in Arabidopsis protoplasts. Consistent with these observations, expression levels of GL2, R2R3 MYB transcription factor gene GLABRA1 (GL1) and several R3 MYB genes were greatly reduced, indicating that OsTCL1 is functional R3 MYB. However, trichome and root hair formation in transgenic rice plants overexpressing OsTCL1 remained largely unchanged, and elevated expression of OsGL2 was observed in the transgenic rice plants, indicating that rice may use different mechanisms to regulate trichome formation. PMID:26758286

  8. Constitutively expressed ERF-VII transcription factors redundantly activate the core anaerobic response in Arabidopsis thaliana.

    Science.gov (United States)

    Bui, Liem T; Giuntoli, Beatrice; Kosmacz, Monika; Parlanti, Sandro; Licausi, Francesco

    2015-07-01

    Plant adaptation to hypoxic conditions is mediated by the transcriptional activation of genes involved in the metabolic reprogramming of plant cells to cope with reduced oxygen availability. Recent studies indicated that members of the group VII of the Ethylene Responsive Transcription Factor (ERFs) family act as positive regulators of this molecular response. In the current study, the five ERF-VII transcription factors of Arabidopsis thaliana were compared to infer a hierarchy in their role with respect to the anaerobic response. When the activity of each transcription factor was tested on a set of hypoxia-responsive promoters, RAP2.2, RAP2.3 and RAP2.12 appeared to be the most powerful activators. RAP2.12 was further dissected in transactivation assays in Arabidopsis protoplasts to identify responsible regions for transcriptional activation. An ultimate C-terminal motif was identified as sufficient to drive gene transcription. Finally, using realtime RT-PCR in single and double mutants for the corresponding genes, we confirmed that RAP2.2 and RAP2.12 exert major control upon the anaerobic response. PMID:26025519

  9. Effects of dicyclohexylamine on polyamine biosynthesis and incorporation into turnip yellow mosaic virus in Chinese cabbage protoplasts infected in vitro

    International Nuclear Information System (INIS)

    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

  10. Somaclonal variation in potato cv. Bintje: Mosaic composition of protoplast calli and segregation of various phenotypes after vegetation propagation

    International Nuclear Information System (INIS)

    The occurrence of spontaneous variation among plants regenerated from in vitro culture of cells or protoplasts ('somaclonal variation') in a number of crops, including potato, has stimulated great interest in recent years from the point of view of using it in plant-improvement programmes. The plant regeneration process from shoot-culture-derived protoplasts of the Dutch potato cultivar Bintje makes it possible to recover a wide array of variants, including undesirable gross-aberrant types. After tuber propagation it was found that some of the normal and variant protoclones (regenerated plants) segregated or showed new types, probably due to chimerism. For plant-breeding purposes, it is highly desirable to know whether or not small groups of genetically different cells present in heterogeneous calli actually express in regenerated plants, or appear in later generations of vegetatively propagated material. The analysis carried out in this regard on the origin of 'segregating protoclones', through characterization of protoplast calli, protoclones and tuber progeny, suggests that a high frequency of protoplast calli are mosaic in composition, consisting of groups of variant (presumably mutated) cells. Two types of mosaicism can be generally distinguished. (1) Fine-grade mosaicism, occurring because small groups of variant cells do not affect the phenotype of regenerated shoots (plants), but form new phenotypes among tuber progeny. (2) Coarse mosaicism, showing the occurrence of large groups of variant cells in a given callus regenerating variant/chimeric shoots. A majority of the mosaic calli gave rise to variant protoclones and mixtures of normal and variant protoclones. The analysis of second-generation tuber progeny showed that part stabilized in phenotype and part segregated again into various phenotypes, indicating the persistence of at least some chimerism. (author)

  11. Novel symbiotic protoplasts formed by endophytic fungi explain their hidden existence, lifestyle switching, and diversity within the plant kingdom

    OpenAIRE

    Atsatt, PR; Whiteside, MD

    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 an...

  12. Cadmium uptake and sequestration kinetics in individual leaf cell protoplasts of the Cd/Zn hyperaccumulator Thlaspi caerulescens

    OpenAIRE

    Leitenmaier, Barbara; Küpper, Hendrik

    2011-01-01

    Hyperaccumulators store accumulated metals in the vacuoles of large leaf epidermal cells (storage cells). For investigating cadmium uptake, we incubated protoplasts obtained from leaves of Thlaspi caerulescens (Ganges ecotype) with a Cd-specific fluorescent dye. A fluorescence kinetic microscope was used for selectively measuring Cd-uptake and photosynthesis in different cell types, so that physical separation of cell types was not necessary. Few minutes after its addition, cadmium accumulate...

  13. Sodium induces simultaneous changes in cytosolic calcium and pH in salt-tolerant quince protoplasts.

    Science.gov (United States)

    D'Onofrio, Cladio; Lindberg, Sylvia

    2009-11-01

    Previous experiments with salt-resistant quince BA29 (Cydonia oblonga cv. Mill.) have shown that this cultivar takes up sodium transiently into the cytosol of shoot protoplasts only in the absence of calcium chloride, or at or =100mM to single protoplasts from in vitro-cultivated quince in the presence of 1.0mM calcium induced instant changes in the cytosolic concentrations of calcium and protons. These changes were investigated by use of tetra [acetoxymethyl] esters of the fluorescent stilbene chromophores Fura 2 and bis-carboxyethyl-carboxyfluorescein (BCECF), respectively. The cytosolic Ca(2+) dynamics in the protoplasts were dependent on the concentration of NaCl added. The changes in calcium differed in amplitude and final concentration and were correlated in time mainly with changes in pH. Addition of 100-400mM NaCl to the protoplasts caused an oscillating increase in the cytosolic level of calcium, and then a decrease. Addition of mannitol, of equiosmolar concentration to NaCl, did not increase the cytosolic calcium concentration. Moreover, there was no increase in cytosolic calcium when NaCl was added in the presence of calcium binding ethylene glycol-bis(beta-aminoethylether)-N,N,N',N'-tetra acetic acid (EGTA), or lantan or verapamil, two inhibitors of plasma membrane calcium channels. Therefore, we conclude that, in salt-resistant quince, sodium induces an influx of calcium into the cytosol by plasma membrane calcium channels, and a simultaneous increase in cytosolic pH. Because these changes were obtained in the presence of 1mM calcium in the medium, they were not due to sodium uptake into the cytosol. PMID:19556023

  14. Occurrence and reduction of acytokinesis in leaf protoplast cultures of potato and tobacco. Implications for chromosome number variation.

    OpenAIRE

    Everdink, Willem Jacobus van

    1994-01-01

    The studies described in this thesis were carried out to investigate and find means to lower early chromosome number variation during leaf protoplast culture of Solanum tuberosum (potato). In a number of plant species, maldistribution of chromosomes had been related to the early occurence of polynucleate cells. In potato, however, neither the causes of polynucleation nor its impact on poly- and aneuploidization had been investigated. Some general and relevant aspects concerning the encountere...

  15. Dissecting RNA silencing in protoplasts uncovers novel effects of viral suppressors on the silencing pathway at the cellular level

    OpenAIRE

    Qi, Yijun; Zhong, Xuehua; Itaya, Asuka; Ding, Biao

    2004-01-01

    Short interfering RNA (siRNA)-mediated RNA silencing plays an important role in cellular defence against viral infection and abnormal gene expression in multiple organisms. Many viruses have evolved silencing suppressors for counter-defence. We have developed an RNA silencing system in the protoplasts of Nicotiana benthamiana to investigate the functions of viral suppressors at the cellular level. We showed that RNA silencing against a green fluorescent protein (GFP) reporter gene in the prot...

  16. Probing plasma membrane microdomains in cowpea protoplasts using lipidated GFP-fusion proteins and multimode FRET microscopy.

    OpenAIRE

    Vermeer, J.E.M.; Munster, van, B.C.; Vischer, N O; Gadella, Th.W.J.

    2004-01-01

    Multimode fluorescence resonance energy transfer (FRET) microscopy was applied to study the plasma membrane organization using different lipidated green fluorescent protein (GFP)-fusion proteins co-expressed in cowpea protoplasts. Cyan fluorescent protein (CFP) was fused to the hyper variable region of a small maize GTPase (ROP7) and yellow fluorescent protein (YFP) was fused to the N-myristoylation motif of the calcium-dependent protein kinase 1 (LeCPK1) of tomato. Upon co-expressing in cowp...

  17. An Arabidopsis callose synthase

    DEFF Research Database (Denmark)

    Ostergaard, Lars; Petersen, Morten; Mattsson, Ole;

    2002-01-01

    unclear whether callose synthases can also produce cellulose and whether plant cellulose synthases may also produce beta-1,3-glucans. We describe here an Arabidopsis gene, AtGsl5, encoding a plasma membrane-localized protein homologous to yeast beta-1,3-glucan synthase whose expression partially......Beta-1,3-glucan polymers are major structural components of fungal cell walls, while cellulosic beta-1,4-glucan is the predominant polysaccharide in plant cell walls. Plant beta-1,3-glucan, called callose, is produced in pollen and in response to pathogen attack and wounding, but it has been...

  18. Identification of an Arabidopsis thaliana protein that binds to tomato mosaic virus genomic RNA and inhibits its multiplication

    International Nuclear Information System (INIS)

    The genomic RNAs of positive-strand RNA viruses carry RNA elements that play positive, or in some cases, negative roles in virus multiplication by interacting with viral and cellular proteins. In this study, we purified Arabidopsis thaliana proteins that specifically bind to 5' or 3' terminal regions of tomato mosaic virus (ToMV) genomic RNA, which contain important regulatory elements for translation and RNA replication, and identified these proteins by mass spectrometry analyses. One of these host proteins, named BTR1, harbored three heterogeneous nuclear ribonucleoprotein K-homology RNA-binding domains and preferentially bound to RNA fragments that contained a sequence around the initiation codon of the 130K and 180K replication protein genes. The knockout and overexpression of BTR1 specifically enhanced and inhibited, respectively, ToMV multiplication in inoculated A. thaliana leaves, while such effect was hardly detectable in protoplasts. These results suggest that BTR1 negatively regulates the local spread of ToMV

  19. CRISPR/Cas-Mediated Site-Specific Mutagenesis in Arabidopsis thaliana Using Cas9 Nucleases and Paired Nickases.

    Science.gov (United States)

    Schiml, Simon; Fauser, Friedrich; Puchta, Holger

    2016-01-01

    The CRISPR/Cas system has recently become the most important tool for genome engineering due to its simple architecture that allows for rapidly changing the target sequence and its applicability to organisms throughout all kingdoms of life. The need for an easy-to-use and reliable nuclease is especially high in plant research, as precise genome modifications are almost impossible to achieve by Agrobacterium-mediated transformation and the regeneration of plants from protoplast cultures is very labor intensive. Here, we describe the application of the Cas9 nuclease to Arabidopsis thaliana for the induction of heritable targeted mutations, which may also be used for other plant species. To cover the concern for off-target activity, we also describe the generation of stable mutants using paired Cas9 nickases. PMID:27557689

  20. Is the LIM-domain protein HaWLIM1 associated with cortical microtubules in sunflower protoplasts?

    Science.gov (United States)

    Brière, Christian; Bordel, Anne-Claire; Barthou, Henri; Jauneau, Alain; Steinmetz, André; Alibert, Gilbert; Petitprez, Michel

    2003-10-01

    Flowering plants express several LIM-domain proteins related to the animal cystein-rich proteins. The expression of sunflower LIM genes was followed by RT-PCR in cultured sunflower protoplasts. A transcript was detected only for HaWLIM1, but not for the other two genes HaPLIM1 and HaPLIM2. Polyclonal antibodies raised against either full length recombinant HaWLIM1 protein or peptides recognized a 27 kDa polypeptide on Western blots. Immunocytolocalization studies showed that HaWLIM1 is located in the cytoplasm and in the nucleus. In the cytoplasm, HaWLIM1 is localized in punctate structures, distributed along microtubule bundles. Depolymerizing microtubules with oryzalin resulted in a strong modification of the HaWLIM1 cortical pattern. In contrast, treatment of protoplasts with latrunculin B, which disrupts actin filaments, had no effect on HaWLIM1 localization. HaWLIM1 was also located within the nucleus of interphase protoplasts. During mitosis, nuclear labelling was observed in prophase, which decreased in metaphase, disappeared in anaphase, and recovered in telophase. These results suggest a dual role for HaWLIM1: in the cytoplasm, as a component of molecular complexes which may interact with microtubules, and in the nucleus, as a partner of transcription factors during interphase. PMID:14581630

  1. Transformation efficiencies and progeny analysis after varying different parameters of direct gene transfer of Nicotiana tabacum protoplasts

    International Nuclear Information System (INIS)

    Nicotiana tabacum protoplasts were transformed by polyethylene glycol (PEG)-mediated uptake and electroporation, with circular and linear DNA, and with or without X-ray irradiation. We investigated the influence on the transient expression by these parameters as well as on the frequencies for stable transformation. Plants were regenerated and selfed, and the progenies of the transformed plants were analysed and used to compare the pattern of gene integration by these different variations in transformation methods. The results from the transient expression as judged by glucuronidase (GUS) activity, showed electroporation to give higher and more reproducible results than PEG-mediated uptake. Using linear instead of circular DNA increased the rate of stable transformation about 3 times. Including a mild X-ray treatment gave an increase in the same range. When the inheritance of the transferred trait was investigated, it was found that protoplasts transformed with linear DNA resulted in the highest number of plants with single-copy insertions. Protoplasts transformed with circular DNA showed the highest incidence of losing the trait, while plants in which the transformation included an X-ray treatment, had the highest frequency of multicopy insertion events

  2. 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.

  3. 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.

  4. 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.

  5. 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.

  6. 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. PMID:26442049

  7. Characterization of a new lectin involved in the protoplast regeneration of Bryopsis hypnoides

    Science.gov (United States)

    Niu, Jianfeng; Wang, Guangce; Lü, Fang; Zhou, Baicheng; Peng, Guang

    2009-09-01

    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.

  8. 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.

  9. 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. PMID:25451863

  10. Water consumption and biomass production of protoplast fusion lines of poplar hybrids under drought stress.

    Science.gov (United States)

    Hennig, Anne; Kleinschmit, Jörg R G; Schoneberg, Sebastian; Löffler, Sonja; Janßen, Alwin; Polle, Andrea

    2015-01-01

    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 9-month-old plants of four tetraploid Populus tremula (L.) × 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. PMID:26042130

  11. Studies on Protoplasts Culture of Zygophyllum xanthoxylum%霸王的原生质体培养的研究

    Institute of Scientific and Technical Information of China (English)

    张改娜; 施江

    2009-01-01

    目的:为利用原生质体融合技术转移霸王抗旱基因.方法:采用酶解法分离霸王原生质体,比较了霸王子叶和愈伤组织游离原生质体的产量和活力,不同渗透压和起始密度对原生质体分裂频率的影响.结果:愈伤组织游离的原生质体产量和活力均高于子叶,原生质体产率可达2.4×10~6个/g·FW,活力达89%.采用液体浅层培养,在附加2,4-D(2mg/L)、6-BA(1.0mg/L)、2%蔗糖和甘露醇(0.4mol/L)的DPD培养基中,原生质体分裂频率最高,达68.6%.转移到附加2-iP(3mg/L)、KT(1.0mg/L)、6-BA(1.0mg/L)的分化培养基上,获得2个再生苗.结论:采用酶解法游离霸王愈伤组织,可获得高活力和高分裂频率的霸王原生质体.%Objective:To transfer resistent gene of Zygophyllum xanthoxylum by protoplast fusion.Method: Z.xanthoxylum protoplasts were obtained through enzyme digestion method,they were compared that the yield and viability of protoplasts of cotyledon and calli from Z.xanthoxylum and protoplast division frequecy in different osmotic potentia and densities.Result: The protoplast yield and viability from calli of Zygophyllum xanthoxylum were higher than those from the cotyledons,respectively 2.4×10~6/g·FW and 89%.The protoplast cell division frequency was up to 68.6% in DPD medium containing 2,4-D(2mg/L),6-BA(1.0mg/L), 2% sucrose and mannitol(0.4mol/L) by liquid thin layer culture.Two regenerated plantlets were obtained when these calli were transferred to MS medium containing 2-iP(3mg/L),KT(1.0mg/L),6-BA(1.0mg/L).Conclusion: the high protoplast iability and cell division frequency of were obtained through enzyme digesting calli of Z.xanthoxylum.

  12. 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.

  13. Multidimensional fluorescence microscopy of multiple organelles in Arabidopsis seedlings

    Directory of Open Access Journals (Sweden)

    Morales Andrea

    2008-05-01

    Full Text Available Abstract Background The isolation of green fluorescent protein (GFP and the development of spectral variants over the past decade have begun to reveal the dynamic nature of protein trafficking and organelle motility. In planta analyses of this dynamic process have typically been limited to only two organelles or proteins at a time in only a few cell types. Results We generated a transgenic Arabidopsis plant that contains four spectrally different fluorescent proteins. Nuclei, plastids, mitochondria and plasma membranes were genetically tagged with cyan, red, yellow and green fluorescent proteins, respectively. In addition, methods to track nuclei, mitochondria and chloroplasts and quantify the interaction between these organelles at a submicron resolution were developed. These analyzes revealed that N-ethylmaleimide disrupts nuclear-mitochondrial but not nuclear-plastids interactions in root epidermal cells of live Arabidopsis seedlings. Conclusion We developed a tool and associated methods for analyzing the complex dynamic of organelle-organelle interactions in real time in planta. Homozygous transgenic Arabidopsis (Kaleidocell is available through Arabidopsis Biological Resource Center.

  14. Transgenic Arabidopsis Gene Expression System

    Science.gov (United States)

    Ferl, Robert; Paul, Anna-Lisa

    2009-01-01

    The Transgenic Arabidopsis Gene Expression System (TAGES) investigation is one in a pair of investigations that use the Advanced Biological Research System (ABRS) facility. TAGES uses Arabidopsis thaliana, thale cress, with sensor promoter-reporter gene constructs that render the plants as biomonitors (an organism used to determine the quality of the surrounding environment) of their environment using real-time nondestructive Green Fluorescent Protein (GFP) imagery and traditional postflight analyses.

  15. Phenotypic analysis of Arabidopsis mutants: oomycete pathogens.

    Science.gov (United States)

    Clarke, Joseph D

    2009-10-01

    Various fungal pathogens are used in Arabidopsis pathogen studies, including Fusarium oxysporum, Alternaria brassicicola, Botrytis cinerea, and others. The oomycete pathogen Peronospora parasitica has been used by several groups and is described in this protocol. Working with Peronospora is complicated by the fact that it is an obligate biotroph, and consequently cultures must be maintained on living plants. There is no central repository for Peronospora stocks, but most investigators who work with them are willing to provide samples of infected tissue. These can be used to initiate new stock cultures, or they can be maintained as live cultures on seedlings. One of the most important factors in maintaining Peronospora is the humidity of the growth chamber, which must be kept at a minimum of 80%. Various Peronospora isolates are available. These vary with respect to which Arabidopsis ecotypes they can infect, because some combinations trigger gene-for-gene resistance. Thus, it is important that the appropriate ecotype is inoculated with the appropriate strain of pathogen. The extent of infections can be rated or quantitatively measured as the number of spores produced per plant, and frozen tissue stocks can be prepared from heavily infected tissue. PMID:20147042

  16. Characterization of minisatellites in Arabidopsis thaliana with sequence similarity to the human minisatellite core sequence.

    OpenAIRE

    Tourmente, S; Deragon, J M; Lafleuriel, J; Tutois, S; Pélissier, T; Cuvillier, C.; Espagnol, M C; G. Picard

    1994-01-01

    A strategy based on random PCR amplification was used to isolate new repetitive elements of Arabidopsis thaliana. One of the random PCR product analyzed by this approach contained a tandem repetitive minisatellite sequence composed of 33 bp repeated units. The genomic locus corresponding to this PCR product was isolated by screening a lambda genomic library. New related loci were also isolated from the genomic library by screening with a 14 mer oligonucleotide representing a region conserved ...

  17. 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

  18. Covisualization by computational optical-sectioning microscopy of integrin and associated proteins at the cell membrane of living onion protoplasts

    Science.gov (United States)

    Gens, J. S.; Reuzeau, C.; Doolittle, K. W.; McNally, J. G.; Pickard, B. G.; Evans, M. L. (Principal Investigator)

    1996-01-01

    Using higher-resolution wide-field computational optical-sectioning fluorescence microscopy, the distribution of antigens recognized by antibodies against animal beta 1 integrin, fibronectin, and vitronectin has been visualized at the outer surface of enzymatically protoplasted onion epidermis cells and in depectinated cell wall fragments. On the protoplast all three antigens are colocalized in an array of small spots, as seen in raw images, in Gaussian filtered images, and in images restored by two different algorithms. Fibronectin and vitronectin but not beta 1 integrin antigenicities colocalize as puncta in comparably prepared and processed images of the wall fragments. Several control visualizations suggest considerable specifity of antibody recognition. Affinity purification of onion cell extract with the same anti-integrin used for visualization has yielded protein that separates in SDS-PAGE into two bands of about 105-110 and 115-125 kDa. These bands are again recognized by the visualization antibody, which was raised against the extracellular domain of chicken beta 1 integrin, and are also recognized by an antibody against the intracellular domain of chicken beta 1 integrin. Because beta 1 integrin is a key protein in numerous animal adhesion sites, it appears that the punctate distribution of this protein in the cell membranes of onion epidermis represents the adhesion sites long known to occur in cells of this tissue. Because vitronectin and fibronection are matrix proteins that bind to integrin in animals, the punctate occurrence of antigenically similar proteins both in the wall (matrix) and on enzymatically prepared protoplasts reinforces the concept that onion cells have adhesion sites with some similarity to certain kinds of adhesion sites in animals.

  19. Is the LIM-domain Protein HaWLIM1 Associated with Cortical Microtubules in Sunflower Protoplasts?

    OpenAIRE

    Brière, Christian; Bordel, Anne-Claire; Barthou, Henri; Jauneau, Alain; Steinmetz, André; Alibert, Gilbert; Petitprez, Michel

    2003-01-01

    Flowering plants express several LIM-domain proteins related to the animal cystein-rich proteins. The expression of sunflower LIM genes was followed by RTPCR in cultured sunflower protoplasts. A transcript was detected only for HaWLIM1, but not for the other two genes HaPLIM1 and HaPLIM2. Polyclonal antibodies raised against either full length recombinant HaWLIM1 protein or peptides recognized a 27 kDa polypeptide on Western blots. Immunocytolocalization studies showed that HaWLIM1 is located...

  20. 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.

  1. Arabidopsis Pol II-Dependent in Vitro Transcription System Reveals Role of Chromatin for Light-Inducible rbcS Gene Transcription.

    Science.gov (United States)

    Ido, Ayaka; Iwata, Shinya; Iwata, Yuka; Igarashi, Hisako; Hamada, Takahiro; Sonobe, Seiji; Sugiura, Masahiro; Yukawa, Yasushi

    2016-02-01

    In vitro transcription is an essential tool to study the molecular mechanisms of transcription. For over a decade, we have developed an in vitro transcription system from tobacco (Nicotiana tabacum)-cultured cells (BY-2), and this system supported the basic activities of the three RNA polymerases (Pol I, Pol II, and Pol III). However, it was not suitable to study photosynthetic genes, because BY-2 cells have lost their photosynthetic activity. Therefore, Arabidopsis (Arabidopsis thaliana) in vitro transcription systems were developed from green and etiolated suspension cells. Sufficient in vitro Pol II activity was detected after the minor modification of the nuclear soluble extracts preparation method; removal of vacuoles from protoplasts and L-ascorbic acid supplementation in the extraction buffer were particularly effective. Surprisingly, all four Arabidopsis Rubisco small subunit (rbcS-1A, rbcS-1B, rbcS-2B, and rbcS-3B) gene members were in vitro transcribed from the naked DNA templates without any light-dependent manner. However, clear light-inducible transcriptions were observed using chromatin template of rbcS-1A gene, which was prepared with a human nucleosome assembly protein 1 (hNAP1) and HeLa histones. This suggested that a key determinant of light-dependency through the rbcS gene transcription was a higher order of DNA structure (i.e. chromatin). PMID:26662274

  2. Study on Electrofusion of Barley Protoplasts using Fluorescent Dextra%用荧光葡聚糖研究大麦细胞电融合

    Institute of Scientific and Technical Information of China (English)

    王明艳; 龚叶芳; 江志裕

    2001-01-01

    利用阴离子表面活性物质荧光葡聚糖(F-DX)研究了表面活性物质对大麦细胞电融合的影响.结果表明,F-DX可抑制电融合过程.对放置过大麦细胞原生质体的F-DX溶液,在荧光显微镜下可观察到其膜表面的荧光圈,证明F-DX在膜上的吸附.添加F-DX可增加原生质体的电泳速度,说明吸附后原生质体表面负电荷增多.由于相互间静电斥力的增强,使细胞的电融合率下降. 此外,还利用荧光显微技术研究了细胞电生孔现象.观察到经电脉冲后溶液中的F-DX可进入原生质体内部,间接证明了细胞电生孔的存在.%The influence of interfacial active substance on the electrofusion of barley protoplasts was investigated using fluorescent dextra (F-DX) as an anion additive. It was found that fluorescent dextra could inhibit the electrofusion process. The adsorption of fluorescent dextra on the membrane of protoplasts was detected by fluorescent microscopy technique. It was observed that after being stored in a solution containing fluorescent dextra a fluorescent ring appeared on the membrane of the protoplast, reflecting the adsorption of fluorescent dextra. The results of electrophoresis experiment also proved the adsorption of F-DX on the surface of protoplasts, because the relative mobility of protoplasts increased in the solution containing F-DX. The increase of negative charge on the membrane of protoplasts could increase the repulsion between protoplasts, and therefore inhibit the electrofusion process. The electroporation phenomena were also investigated using fluorescent microscopy technique. It was found that F-DX could pass into the inside of protoplasts under an electronic pulse. It proves the presence of pores formed in the elecroporation process.

  3. Co-localisation studies of Arabidopsis SR splicing factors reveal different types of speckles in plant cell nuclei

    International Nuclear Information System (INIS)

    SR proteins are multidomain splicing factors which are important for spliceosome assembly and for regulation of alternative splicing. In mammalian nuclei these proteins localise to speckles from where they are recruited to transcription sites. By using fluorescent protein fusion technology and different experimental approaches it has been shown that Arabidopsis SR proteins, in addition to diffuse nucleoplasmic staining, localise into an irregular nucleoplasmic network resembling speckles in mammalian cells. As Arabidopsis SR proteins fall into seven conserved sub-families we investigated co-localisation of members of the different sub-families in transiently transformed tobacco protoplast. Here we demonstrate the new finding that members of different SR protein sub-families localise into distinct populations of nuclear speckles with no, partial or complete co-localisation. This is particularly interesting as we also show that these proteins do interact in a yeast two-hybrid assay as well as in pull-down and in co-immunopreciptiation assays. Our data raise the interesting possibility that SR proteins are partitioned into distinct populations of nuclear speckles to allow a more specific recruitment to the transcription/pre-mRNA processing sites of particular genes depending on cell type and developmental stage

  4. Supermolecular organization of photosystem II and its associated light-harvesting antenna in Arabidopsis thaliana

    NARCIS (Netherlands)

    Yakushevska, AE; Jensen, PE; Keegstra, W; van Roon, H; Scheller, HV; Boekema, EJ; Dekker, JP; Yakushevska, Alevtyna E.; Jensen, Poul E.; Scheller, Henrik V.; Dekker, Jan P.

    2001-01-01

    The organization of Arabidopsis thaliana photosystem II (PSII) and its associated light-harvesting antenna (LHCII) was studied in isolated PSII-LHCII supercomplexes and native membrane-bound crystals by transmission electron microscopy and image analysis. Over 4000 single-particle projections of PSI

  5. A multiple-method approach reveals a declining amount of chloroplast DNA during development in Arabidopsis

    Directory of Open Access Journals (Sweden)

    Oldenburg Delene J

    2009-01-01

    Full Text Available Abstract Background A decline in chloroplast DNA (cpDNA during leaf maturity has been reported previously for eight plant species, including Arabidopsis thaliana. Recent studies, however, concluded that the amount of cpDNA during leaf development in Arabidopsis remained constant. Results To evaluate alternative hypotheses for these two contradictory observations, we examined cpDNA in Arabidopsis shoot tissues at different times during development using several methods: staining leaf sections as well as individual isolated chloroplasts with 4',6-diamidino-2-phenylindole (DAPI, real-time quantitative PCR with DNA prepared from total tissue as well as from isolated chloroplasts, fluorescence microscopy of ethidium-stained DNA molecules prepared in gel from isolated plastids, and blot-hybridization of restriction-digested total tissue DNA. We observed a developmental decline of about two- to three-fold in mean DNA per chloroplast and two- to five-fold in the fraction of cellular DNA represented by chloroplast DNA. Conclusion Since the two- to five-fold reduction in cpDNA content could not be attributed to an artifact of chloroplast isolation, we conclude that DNA within Arabidopsis chloroplasts is degraded in vivo as leaves mature.

  6. 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

  7. 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

  8. Cadmium uptake and sequestration kinetics in individual leaf cell protoplasts of the Cd/Zn hyperaccumulator Thlaspi caerulescens.

    Science.gov (United States)

    Leitenmaier, Barbara; Küpper, Hendrik

    2011-02-01

    Hyperaccumulators store accumulated metals in the vacuoles of large leaf epidermal cells (storage cells). For investigating cadmium uptake, we incubated protoplasts obtained from leaves of Thlaspi caerulescens (Ganges ecotype) with a Cd-specific fluorescent dye. A fluorescence kinetic microscope was used for selectively measuring Cd-uptake and photosynthesis in different cell types, so that physical separation of cell types was not necessary. Few minutes after its addition, cadmium accumulated in the cytoplasm before its transport into the vacuole. This demonstrated that vacuolar sequestration is the rate-limiting step in cadmium uptake into protoplasts of all leaf cell types. During accumulation in the cytoplasm, Cd-rich vesicle-like structures were observed. Cd uptake rates into epidermal storage cells were higher than into standard-sized epidermal cells and mesophyll cells. This shows that the preferential heavy metal accumulation in epidermal storage cells, previously observed for several metals in intact leaves of various hyperaccumulator species, is due to differences in active metal transport and not differences in passive mechanisms like transpiration stream transport or cell wall adhesion. Combining this with previous studies, it seems likely that the transport steps over the plasma and tonoplast membranes of leaf epidermal storage cells are driving forces behind the hyperaccumulation phenotype. PMID:20880204

  9. Reinvestigation of intracellular localization of the 30K protein in tobacco protoplasts infected with tobacco mosaic virus RNA.

    Science.gov (United States)

    Meshi, T; Hosokawa, D; Kawagishi, M; Watanabe, Y; Okada, Y

    1992-04-01

    It has been shown that the 30K protein of tobacco mosaic virus (TMV) is responsible for the cell-to-cell movement function of the virus. It is still obscure how the protein is involved in this function at the molecular level. We formerly found that the 30K protein is localized to the plasmodesmata of TMV-infected plants. We also reported that the 30K protein was detected in a nuclei-rich fraction of TMV-infected protoplasts after biochemical fractionation. To clarify the inconsistency, the 30K protein was immunocytologically localized in TMV-infected protoplasts using a newly prepared antibody against the 30K protein. On some sections, the 30K protein was found near the nucleus but not in or on the nucleus. At later stages of infection a novel electron-transparent structure was detected in the cytoplasm where the 30K proteins were localized. This structure might reflect an intermediate form between its synthesis in the cytoplasm and its targeting to the plasmodesmata in whole plants. PMID:1546469

  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. Construction of Porphyra yezoensis Pure Line from Protoplasts and Its 18S rDNA Sequence Determination

    Institute of Scientific and Technical Information of China (English)

    LIU Hongquan; YU Wengong; DAI Jixun; GONG Qianhong; SHI Xiaochong; YANG Kunfeng

    2004-01-01

    The wild Porphyra yezoensis collected from the Qingdao coast was used to prepare protoplasts by enzyme digestion. The pure line was constructed by cultivating the protoplasts. The 18S rDNA of the P. yezoensis pure line was cloned and sequenced. Sequence analysis was executed for this sequence and other 22 sequences retrieved from GenBank. A phylogenetic tree was constructed using the neighbor-joining method. The results revealed a high diversity of 18S rDNA sequences in genus Porphyra and the considerable variation of 18S rDNA sequences in different strains of the same species P. yezoensis and P. tenera. Significant difference of 18S rDNA sequence was observed between P. yezoensis from Qingdao, China, and the two strains of P. yezoensis from Japan, but the three strains of P. yezoensis formed a stable clade in the phylogenetic tree. These results indicate the possibility of interspecies and intraspecies discrimination of Porphyra using the 18S rDNA sequences.

  12. 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.

  13. 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.

  14. In Vitro Morphogenesis of Arabidopsis to Search for Novel Endophytic Fungi Modulating Plant Growth

    OpenAIRE

    Francesco Dovana; Marco Mucciarelli; Maurizio Mascarello; Anna Fusconi

    2015-01-01

    Fungal endophytes have shown to affect plant growth and to confer stress tolerance to the host; however, effects of endophytes isolated from water plants have been poorly investigated. In this study, fungi isolated from stems (stem-E) and roots (root-E) of Mentha aquatica L. (water mint) were identified, and their morphogenetic properties analysed on in vitro cultured Arabidopsis (L.) Heynh., 14 and 21 days after inoculation (DAI). Nineteen fungi were analysed and, based on ITS analysis, 17 i...

  15. Biosynthesis of acid phosphatase of baker's yeast . Characterization of a protoplast-bound fraction containing precursors of the exo-enzyme

    NARCIS (Netherlands)

    Boer, Pieter; Rijn, Herman J.M. van; Reinking, A.; Steyn-Parvé, Elizabeth P.

    1975-01-01

    1. 1.|Yest protoplasts, secreting acid phosphatase (orthophosphoric-monoester phosphohydrolase (acid optimum) EC 3.1.3.2) contain a small amount of firmly bound enzyme, even after lysis (Van Rijn, H.J.M.; Boer, P. and Steyn-Parvé, E.P. (1972) Biochim. Biophys. Acta 268, 431–441). The major part (70%

  16. 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.

  17. Defects in Peroxisomal 6-Phosphogluconate Dehydrogenase Isoform PGD2 Prevent Gametophytic Interaction in Arabidopsis thaliana.

    Science.gov (United States)

    Hölscher, Christian; Lutterbey, Marie-Christin; Lansing, Hannes; Meyer, Tanja; Fischer, Kerstin; von Schaewen, Antje

    2016-05-01

    We studied the localization of 6-phosphogluconate dehydrogenase (PGD) isoforms of Arabidopsis (Arabidopsis thaliana). Similar polypeptide lengths of PGD1, PGD2, and PGD3 obscured which isoform may represent the cytosolic and/or plastidic enzyme plus whether PGD2 with a peroxisomal targeting motif also might target plastids. Reporter-fusion analyses in protoplasts revealed that, with a free N terminus, PGD1 and PGD3 accumulate in the cytosol and chloroplasts, whereas PGD2 remains in the cytosol. Mutagenesis of a conserved second ATG enhanced the plastidic localization of PGD1 and PGD3 but not PGD2. Amino-terminal deletions of PGD2 fusions with a free C terminus resulted in peroxisomal import after dimerization, and PGD2 could be immunodetected in purified peroxisomes. Repeated selfing of pgd2 transfer (T-)DNA alleles yielded no homozygous mutants, although siliques and seeds of heterozygous plants developed normally. Detailed analyses of the C-terminally truncated PGD2-1 protein showed that peroxisomal import and catalytic activity are abolished. Reciprocal backcrosses of pgd2-1 suggested that missing PGD activity in peroxisomes primarily affects the male gametophyte. Tetrad analyses in the quartet1-2 background revealed that pgd2-1 pollen is vital and in vitro germination normal, but pollen tube growth inside stylar tissues appeared less directed. Mutual gametophytic sterility was overcome by complementation with a genomic construct but not with a version lacking the first ATG. These analyses showed that peroxisomal PGD2 activity is required for guided growth of the male gametophytes and pollen tube-ovule interaction. Our report finally demonstrates an essential role of oxidative pentose-phosphate pathway reactions in peroxisomes, likely needed to sustain critical levels of nitric oxide and/or jasmonic acid, whose biosynthesis both depend on NADPH provision. PMID:26941195

  18. Overexpression of Heat Shock Factor Gene HsfA3 Increases Galactinol Levels and Oxidative Stress Tolerance in Arabidopsis.

    Science.gov (United States)

    Song, Chieun; Chung, Woo Sik; Lim, Chae Oh

    2016-06-30

    Heat shock factors (Hsfs) are central regulators of abiotic stress responses, especially heat stress responses, in plants. In the current study, we characterized the activity of the Hsf gene HsfA3 in Arabidopsis under oxidative stress conditions. HsfA3 transcription in seedlings was induced by reactive oxygen species (ROS), exogenous hydrogen peroxide (H2O2), and an endogenous H2O2 propagator, 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB). HsfA3-overexpressing transgenic plants exhibited increased oxidative stress tolerance compared to untransformed wild-type plants (WT), as revealed by changes in fresh weight, chlorophyll fluorescence, and ion leakage under light conditions. The expression of several genes encoding galactinol synthase (GolS), a key enzyme in the biosynthesis of raffinose family oligosaccharides (RFOs), which function as antioxidants in plant cells, was induced in HsfA3 overexpressors. In addition, galactinol levels were higher in HsfA3 overexpressors than in WT under unstressed conditions. In transient transactivation assays using Arabidopsis leaf protoplasts, HsfA3 activated the transcription of a reporter gene driven by the GolS1 or GolS2 promoter. Electrophoretic mobility shift assays showed that GolS1 and GolS2 are directly regulated by HsfA3. Taken together, these findings provide evidence that GolS1 and GolS2 are directly regulated by HsfA3 and that GolS enzymes play an important role in improving oxidative stress tolerance by increasing galactinol biosynthesis in Arabidopsis. PMID:27109422

  19. Somatic hybrids between Arabidopsis thaliana and cytoplasmic male-sterile radish (Raphanus sativus).

    Science.gov (United States)

    Yamagishi, H; Glimelius, K

    2003-08-01

    Somatic hybrids were produced by protoplast fusion between Arabidopsis thaliana ecotype Columbia and a male-sterile radish line MS-Gensuke ( Raphanus sativus) with the Ogura cytoplasm. Forty-one shoots were differentiated from the regenerated calli and established as shoot cultures in vitro. About 20 of these shoots were judged to be hybrids based on growth characteristics and morphology. Molecular analyses of 11 shoots were performed, confirming the hybrid features. Of these 11 shoots, eight were established as rooted plants in the greenhouse. Polymerase chain reaction and randomly amplified polymorphic DNA analyses of the nuclear genomes of all analyzed shoots and plants confirmed that they contained hybrid DNA patterns. Their chromosome numbers also supported the hybrid nature of the plants. Investigations of the organelles in the hybrids revealed that the chloroplast (cp) genome was exclusively represented by radish cpDNA, while the mitochondrial DNA configuration showed a combination of both parental genomes as well as fragments unique to the hybrids. Hybrid plants that flowered were male-sterile independent of the presence of the Ogura CMS-gene orf138. PMID:12827437

  20. 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.

  1. An early nodulin-like protein accumulates in the sieve element plasma membrane of Arabidopsis

    DEFF Research Database (Denmark)

    Khan, Junaid A.; Wang, Qi; Sjölund, Richard D.;

    2007-01-01

    ) tissue cultures, recognizes an antigen in the Arabidopsis (Arabidopsis thaliana) ecotype Columbia that is associated specifically with the plasma membrane of sieve elements, but not companion cells, and accumulates at the earliest stages of sieve element differentiation. The identity of the RS6 antigen...... cleaved from the precursor protein, resulting in a mature peptide of approximately 15 kD that is attached to the sieve element plasma membrane via a carboxy-terminal glycosylphosphatidylinositol membrane anchor. Many of the Arabidopsis ENOD-like proteins accumulate in gametophytic tissues, whereas in both......Membrane proteins within the sieve element-companion cell complex have essential roles in the physiological functioning of the phloem. The monoclonal antibody line RS6, selected from hybridomas raised against sieve elements isolated from California shield leaf (Streptanthus tortuosus; Brassicaceae...

  2. In vitro induction, isolation and transfer of chloroplast mutations in Nicotiana

    International Nuclear Information System (INIS)

    Protoplast cultures of Nicotiana plumbaginifolia have been used for the isolation of mutants resistant to antibiotics and to photosynthesis-inhibiting herbicides. The effectiveness of N-ethyl-N-nitrosourea in inducing different chloroplast mutations, establishing proper selective conditions in cell cultures, plant regeneration from resistant cell lines and inheritance of these markers are presented. Bacterial protein-synthesis inhibitors (streptomycin and lincomycin) were used in the selection of a large number of chloroplast mutants under standard culture conditions, while special 'photomixotrophic' culture conditions, for selecting mutants resistant to photosynthesis-inhibiting herbicides, had to be established. Under these culture conditions, the primary symptom of photosynthetic electron-transport inhibition by herbicides (bleaching) can be observed and selection for resistance can be carried out. Protoplast fusion has been shown to be suitable for the 'rescue' of mutant plastids from an undesirable nuclear background, from transfer into a different background, or even transfer into a completely different species. (author)

  3. Construction and evaluation of an exopolysaccharide-producing engineered bacterial strain by protoplast fusion for microbial enhanced oil recovery.

    Science.gov (United States)

    Sun, Shanshan; Luo, Yijing; Cao, Siyuan; Li, Wenhong; Zhang, Zhongzhi; Jiang, Lingxi; Dong, Hanping; Yu, Li; Wu, Wei-Min

    2013-09-01

    Enterobacter cloacae strain JD, which produces water-insoluble biopolymers at optimal temperature of 30°C, and a thermophilic Geobacillus strain were used to construct an engineered strain for exopolysaccharide production at high temperatures by protoplast fusion. The obtained fusant strain ZR3 produced exopolysaccharides at up to 45°C with optimal growth temperature at 35°C. The fusant produced exopolysaccharides of approximately 7.5 g/L or more at pH between 7.0 and 9.0. The feasibility of the enhancement of crude oil recovery with the fusant was tested in a sand-packed column at 40°C. The results demonstrated that bioaugmentation of the fusant was promising approach for MEOR. Mass growth of the fusant was confirmed in fermentor tests. PMID:23856587

  4. The transfer of 'Polima' cytoplasmic male sterility from oilseed rape (Brassica napus) to broccoli (B. oleracea) by protoplast fusion.

    Science.gov (United States)

    Yarrow, S A; Burnett, L A; Wildeman, R P; Kemble, R J

    1990-08-01

    Protoplast fusion was utilised to transfer Polima type cytoplasmic male sterility (CMS) from Brassica napus, canola cv. Polima Karat (Pol-Karat) to B. oleracea, broccoli, var. "Green Comet". Southern and RFLP analysis confirmed that four cybrids possessed nuclear genomes of broccoli with Polima mitochondria and chloroplasts. A fifth cybrid was a nuclear hybrid between broccoli and Pol-Karat, with Polima mitochondria and chloroplasts of broccoli. The broccoli type cybrids were morphologically similar to "Green Comet", while the hybrid type was an intermediate of the two fusion parents. Flowers on the cybrids were distinctive in that although they possessed a morphology typical of Polima, they had very reduced petals. The broccoli type cybrids exhibited some female fertility, albeit low, establishing potential for F1 hybrid production. PMID:24226699

  5. 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.

  6. An ABA down-regulated bHLH transcription repressor gene, bHLH129 regulates root elongation and ABA response when overexpressed in Arabidopsis

    Science.gov (United States)

    Tian, Hainan; Guo, Hongyan; Dai, Xuemei; Cheng, Yuxin; Zheng, Kaijie; Wang, Xiaoping; Wang, Shucai

    2015-01-01

    Plant hormone abscisic acid (ABA) plays a crucial role in modulating plant responses to environmental stresses. Basic helix-loop-helix (bHLH) transcription factors are one of the largest transcription factor families that regulate multiple aspects of plant growth and development, as well as of plant metabolism in Arabidopsis. Several bHLH transcription factors have been shown to be involved in the regulation of ABA signaling. We report here the characterization of bHLH129, a bHLH transcription factor in Arabidopsis. We found that the expression level of bHLH129 was reduced in response to exogenously applied ABA, and elevated in the ABA biosynthesis mutant aba1-5. Florescence observation of transgenic plants expressing bHLH129-GFP showed that bHLH129 was localized in the nucleus, and transient expression of bHLH129 in protoplasts inhibited reporter gene expression. When expressed in Arabidopsis under the control of the 35S promoter, bHLH129 promoted root elongation, and the transgenic plants were less sensitivity to ABA in root elongation assays. Quantitative RT-PCR results showed that ABA response of several genes involved in ABA signaling, including ABI1, SnRK2.2, SnRK2.3 and SnRK2.6 were altered in the transgenic plants overexpressing bHLH129. Taken together, our study suggests that bHLH129 is a transcription repressor that negatively regulates ABA response in Arabidopsis. PMID:26625868

  7. Characterization of minisatellites in Arabidopsis thaliana with sequence similarity to the human minisatellite core sequence.

    Science.gov (United States)

    Tourmente, S; Deragon, J M; Lafleuriel, J; Tutois, S; Pélissier, T; Cuvillier, C; Espagnol, M C; Picard, G

    1994-08-25

    A strategy based on random PCR amplification was used to isolate new repetitive elements of Arabidopsis thaliana. One of the random PCR product analyzed by this approach contained a tandem repetitive minisatellite sequence composed of 33 bp repeated units. The genomic locus corresponding to this PCR product was isolated by screening a lambda genomic library. New related loci were also isolated from the genomic library by screening with a 14 mer oligonucleotide representing a region conserved among the different repeated units. Alignment of the consensus sequence for each minisatellite locus allowed the definition of an Arabidopsis thaliana core sequence that shows strong sequence similarities with the human core sequence and with the generalized recombination signal Chi of Escherichia coli. The minisatellites were tested for their ability to detect polymorphism, and their chromosomal position was established. PMID:8078766

  8. Arabidopsis thaliana—Aphid Interaction

    OpenAIRE

    Louis, Joe; Singh, Vijay,; Shah, Jyoti

    2012-01-01

    Aphids are important pests of plants that use their stylets to tap into the sieve elements to consume phloem sap. Besides the removal of photosynthates, aphid infestation also alters source-sink patterns. Most aphids also vector viral diseases. In this chapter, we will summarize on recent significant findings in plant-aphid interaction, and how studies involving Arabidopsis thaliana and Myzus persicae (Sülzer), more commonly known as the green peach aphid (GPA), are beginning to provide impor...

  9. Selenium Speciation in Arabidopsis Thaliana

    OpenAIRE

    Wang, Xiaoou

    2011-01-01

    Selenium has been proved as an essential micronutrient and is beneficial to animals and humans. It is a structural component of the important antioxidant enzyme, glutathione peroxidase, which catalyzes reactions to detoxify reactive oxygen species. However, the essentiality of Se in plants remains controversial and the protective role of Se in plants has rarely been investigated. In this study, Arabidopsis thaliana was grown in controlled environments having selenate or selenite enriched medi...

  10. Stem cell organization in Arabidopsis

    OpenAIRE

    Wendrich, J.R.

    2016-01-01

    Growth of plant tissues and organs depends on continuous production of new cells, by niches of stem cells. Stem cells typically divide to give rise to one differentiating daughter and one non-differentiating daughter. This constant process of self-renewal ensures that the niches of stem cells or meristems stay active throughout plant-life. Specification of stem cells occurs very early during development of the emrbyo and they are maintained during later stages. The Arabidopsis embryo is a hig...

  11. Comparison of quantitative and qualitative antibody-producing cell responses to lipopolysaccharide in cell walls of the bacterial form and in membranes of the protoplast L-form of Proteus mirabilis.

    OpenAIRE

    Karch, H; Nixdorff, K

    1980-01-01

    Membranes of the stable protoplast L-form of Proteus mirabilis strain VI were highly immunogenic carriers of lipopolysaccharide when compared with the immune responses to lipopolysaccharide contained in cell walls of the bacterial form of this organism.

  12. Formation Conditions of Aspergiilus oryzae Protoplast%米曲霉原生质体生成条件的研究

    Institute of Scientific and Technical Information of China (English)

    屈二军; 李辰曦; 李文建; 冯振; 陈兰英

    2010-01-01

    [目的]探讨米曲霉原生质体制备的高效方法.[方法]统计米曲霉菌丝体检测不同生长时间(72、84、96、108、120 h)的菌丝、不同渗透压(分别使用0.8 mol/L蔗糖、氯化钠和葡萄糖溶液作为渗透压稳定剂)和不同组合酶系(溶菌酶、纤维素酶和蜗牛酶、混合酶系的配比按照L9(34)正交设计)下的原生质体产量.[结果]经检测,培养108 h菌丝体、0.8 mol/L NaCl渗透压稳定剂原生质体产量较高.同时溶菌酶(2 mg/ml)+纤维素酶(6 mg/ml)+蜗牛酶(6 mg/ml)组合酶系原生质体产量较高,达9×105个/ml.[结论]原生质体的产量受菌体自身状况和外界条件影响.该研究为大量制备高活力的原生质体及进行细胞融合试验奠定了基础.%[Objective] To explore the efficient way for the preparation of Aspergillus oryzae protoplast.[Method] The statistics of protoplast yield by the enzymolysis of mycelia with different culture time,different osmotic stabilizers and different concentrations of enzyme system was carried out in this study.[Result] The protoplast yield was higher when the mycelium was cultivated for 108 h,and the osmotic stabilizer was 0.8 mol/L of NaCl.With the enzymes system of 2 mg/ml of lysozyme,6 mg/ml of cellulose and 6 mg/ml of glusulase,the protoplast yield achieved its highest,which was up to 9.0×105 per ml.[Conclusion] The yield of protoplast was affected by the situation of mycelia themselves and external conditions.This study had provided a basis for the preparation of a large number of active protoplasts and the further researches on cell fusion.

  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: AK106750 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK106750 002-115-C09 At4g15560.1 1-deoxy-D-xylulose 5-phosphate synthase, putative / 1-deoxyxylu ... phate synthase, putative / DXP-synthase, putative (DEF ) (CLA1) identical to SP|Q38854 Probable 1-deoxy-D- ... (DXPS). [Mouse-ear cress] {Arabidopsis thaliana}, DEF ... (def icient in photosynthesis) protein [Arabidopsis ...

  15. Arabidopsis CDS blastp result: AK104851 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK104851 001-043-A10 At4g15560.1 1-deoxy-D-xylulose 5-phosphate synthase, putative / 1-deoxyxylu ... phate synthase, putative / DXP-synthase, putative (DEF ) (CLA1) identical to SP|Q38854 Probable 1-deoxy-D- ... (DXPS). [Mouse-ear cress] {Arabidopsis thaliana}, DEF ... (def icient in photosynthesis) protein [Arabidopsis ...

  16. Arabidopsis CDS blastp result: AK100909 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK100909 J023132G24 At4g15560.1 1-deoxy-D-xylulose 5-phosphate synthase, putative / 1-deoxyxylul ... phate synthase, putative / DXP-synthase, putative (DEF ) (CLA1) identical to SP|Q38854 Probable 1-deoxy-D- ... (DXPS). [Mouse-ear cress] {Arabidopsis thaliana}, DEF ... (def icient in photosynthesis) protein [Arabidopsis ...

  17. Arabidopsis CDS blastp result: AK058950 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK058950 001-020-A07 At4g15560.1 1-deoxy-D-xylulose 5-phosphate synthase, putative / 1-deoxyxylu ... phate synthase, putative / DXP-synthase, putative (DEF ) (CLA1) identical to SP|Q38854 Probable 1-deoxy-D- ... (DXPS). [Mouse-ear cress] {Arabidopsis thaliana}, DEF ... (def icient in photosynthesis) protein [Arabidopsis ...

  18. Arabidopsis CDS blastp result: AK059821 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK059821 006-205-D11 At4g15560.1 1-deoxy-D-xylulose 5-phosphate synthase, putative / 1-deoxyxylu ... phate synthase, putative / DXP-synthase, putative (DEF ) (CLA1) identical to SP|Q38854 Probable 1-deoxy-D- ... (DXPS). [Mouse-ear cress] {Arabidopsis thaliana}, DEF ... (def icient in photosynthesis) protein [Arabidopsis ...

  19. Arabidopsis CDS blastp result: AK064944 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK064944 J013000P14 At4g15560.1 1-deoxy-D-xylulose 5-phosphate synthase, putative / 1-deoxyxylul ... phate synthase, putative / DXP-synthase, putative (DEF ) (CLA1) identical to SP|Q38854 Probable 1-deoxy-D- ... (DXPS). [Mouse-ear cress] {Arabidopsis thaliana}, DEF ... (def icient in photosynthesis) protein [Arabidopsis ...

  20. Arabidopsis CDS blastp result: AK068400 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK068400 J013151M04 At3g45810.1 ferric reductase-like transmembrane component family protein sim ... ilar to respiratory burst ... oxidase protein D RbohD from Arabidopsis thaliana, ... EMBL:AF055357 [gi:3242789], similar to respiratory burst ... oxidase protein D RbohD from Arabidopsis thaliana, ...

  1. Arabidopsis CDS blastp result: AK066013 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK066013 J013047I12 At3g45810.1 ferric reductase-like transmembrane component family protein sim ... ilar to respiratory burst ... oxidase protein D RbohD from Arabidopsis thaliana, ... EMBL:AF055357 [gi:3242789], similar to respiratory burst ... oxidase protein D RbohD from Arabidopsis thaliana, ...

  2. Arabidopsis CDS blastp result: AK100241 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK100241 J023054P13 At3g45810.1 ferric reductase-like transmembrane component family protein sim ... ilar to respiratory burst ... oxidase protein D RbohD from Arabidopsis thaliana, ... EMBL:AF055357 [gi:3242789], similar to respiratory burst ... oxidase protein D RbohD from Arabidopsis thaliana, ...

  3. Arabidopsis CDS blastp result: AK318553 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK318553 J075145A22 At3g45810.1 68416.m04958 ferric reductase-like transmembrane component famil ... y protein similar to respiratory burst ... oxidase protein D RbohD from Arabidopsis thaliana, ... EMBL:AF055357 [gi:3242789], similar to respiratory burst ... oxidase protein D RbohD from Arabidopsis thaliana, ...

  4. 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 RLK) genetic…

  5. 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.

  6. 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.

  7. Protoplast formation and regeneration for Saccharopolyspora spinosa%刺糖多孢菌原生质体制备与再生条件优化

    Institute of Scientific and Technical Information of China (English)

    甘邱锋; 张晓琳; 王洁颖; 汪洋; 黄必旺; 关雄

    2011-01-01

    目的 研究多杀菌素产生菌刺糖多孢菌(Saccharopolyspora spinosa)原生质体制备与再生的最佳条件.方法 利用数理统计的方法研究了不同制备培养基、菌龄、甘氨酸浓度、溶菌酶处理条件以及再生培养基对原生质体制备和再生的影响,并考察了原生质体的适宜保藏温度.结果 菌体在添加0.3%甘氨酸的EHC培养基中培养72h,用2mg/mL溶菌酶32℃酶解40min后,涂布在再生培养基R6上再生,原生质体制备率超过99%,再生数町达到10cfu/mL.刺糖多孢菌原生质体可置于4℃短期保存72h,长期保存需要放置于-80℃条件下.结论 优化的结果为刺糖多孢菌原生质体融合育种和遗传转化体系建立奠定了基础.%Objective The optimal conditions for the formation, regeneration and preservation of protoplasts were established in Saccharopolyspora spinosa N-15-50-4B, a spinosad-producing strain. Methods Several crucial factors were tested on protoplast formation and regeneration including the preparation medium, spawn age, glycine concentration, regeneration medium and the treatment conditions of lysozyme. Results The results presented that the protoplast could be efficiently formed and regenerated under a certain condition. When the collected mycelia from S. spinosa N-15-50-4B grown in EHC medium with 0.3% glycine for 72h and were treated by 2mg/mL lysozyme at 32℃ for 40min, then plated on the R6 medium, the rate of protoplasts formation could reach more than 99% and the number of regeneration protoplast was up to 107cfu/mL. We also studied the effects of temperature on protoplast preservation. Short-term storage for protoplasts was 4℃ for 72h, and long-term storage needed -80℃. Conclusion These studies enable to improve spinosad-producing strains though protoplast fusion and to establish genetic transformation system orS. spinosa.

  8. Integration of Bioinformatics and Synthetic Promoters Leads to the Discovery of Novel Elicitor-Responsive cis-Regulatory Sequences in Arabidopsis1[C][W][OA

    Science.gov (United States)

    Koschmann, Jeannette; Machens, Fabian; Becker, Marlies; Niemeyer, Julia; Schulze, Jutta; Bülow, Lorenz; Stahl, Dietmar J.; Hehl, Reinhard

    2012-01-01

    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. PMID:22744985

  9. 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. PMID:22744985

  10. GpDSR7, a Novel E3 Ubiquitin Ligase Gene in Grimmia pilifera Is Involved in Tolerance to Drought Stress in Arabidopsis.

    Science.gov (United States)

    Li, Mengmeng; Li, Yihao; Zhao, Junyi; Liu, Hai; Jia, Shenghua; Li, Jie; Zhao, Heping; Han, Shengcheng; Wang, Yingdian

    2016-01-01

    The growth and development of plants under drought stress depends mainly on the expression levels of various genes and modification of proteins. To clarify the molecular mechanism of drought-tolerance of plants, suppression subtractive hybridisation cDNA libraries were screened to identify drought-stress-responsive unigenes in Grimmia pilifera, and a novel E3 ubiquitin ligase gene, GpDSR7, was identified among the 240 responsive unigenes. GpDSR7 expression was induced by various abiotic stresses, particularly by drought. GpDSR7 displayed E3 ubiquitin ligase activity in vitro and was exclusively localised on the ER membrane in Arabidopsis mesophyll protoplasts. GpDSR7-overexpressing transgenic Arabidopsis plants showed a high water content and survival ratio under drought stress. Moreover, the expression levels of some marker genes involved in drought stress were higher in the transgenic plants than in wild-type plants. These results suggest that GpDSR7, an E3 ubiquitin ligase, is involved in tolerance to drought stress at the protein modification level. PMID:27228205

  11. Auxin-induced regulation of protein synthesis in tobacco mesophyll protoplasts cultivated in vitro: I. Characteristics of auxin-sensitive proteins.

    Science.gov (United States)

    Meyer, Y; Aspart, L; Chartier, Y

    1984-08-01

    The presence of auxin (2,4-D), in the culture medium of tobacco (Nicotiana tabacum var Maryland) mesophyll protoplasts is necessary both for cell wall regeneration and for passage of the cells from phase G(0) to phase G(1) of the cell cycle. Among about 250 proteins synthesized by protoplasts and characterized by their migration in a two-dimensional electrophoresis gel, 2,4-dichlorophenoxyacetic acid affects the synthesis of 11.Nine proteins are synthesized at a reduced level in the presence of the hormone, of which three are rapidly labeled and short-lived, while the others, which are long-lived, become detectable only after 2 hours of radioactive labeling, suggesting that they undergo slow posttranslational maturation. These nine proteins are proline-rich but the proline radicals are not strongly hydroxylated. The synthesis of these proteins is no longer inhibited by auxin if dichlorobenzonitril, a weed-killer which inhibits cell wall reformation of tobacco protoplasts, is added to the culture medium.Two proteins are only synthesized if protoplasts are cultivated in an auxin-containing medium. These polypeptides are rapidly labeled, and are long-lived. The inhibition of cell wall reformation by dichlorobenzonitril does not modify their synthesis.These results suggest that proteins whose synthesis is reduced by auxin are related to cell wall reformation and that they do not play a role in the induction of the cell cycle. In contrast, proteins whose synthesis is stimulated in the presence of auxin are good candidates for a role in the induction of the cell cycle. PMID:16663728

  12. Valine-Resistance, a Potential Marker in Plant Cell Genetics. II. Optimization of Uv Mutagenesis and Selection of Valine-Resistant Colonies Derived from Tobacco Mesophyll Protoplasts

    OpenAIRE

    Grandbastien, M. A.; Bourgin, J. P.; Caboche, M.

    1985-01-01

    The induction and selection of valine-resistant mutants from haploid tobacco (Nicotiana tabacum L.) mesophyll protoplast-derived cells have been studied. Using cells from an original mutant plant obtained previously, we performed reconstruction experiments in order to determine the best conditions for the recovery of resistant cells among a population of sensitive cells. Optimal selective conditions were shown to depend on various factors including cell density, time of addition of valine and...

  13. Expression profile analysis of genes involved in cell wall regeneration during protoplast culture in cotton by suppression subtractive hybridization and macroarray

    OpenAIRE

    Yang, Xiyan; Tu, Lili; Zhu, Longfu; Fu, Lili; Min, Ling; Zhang, Xianlong

    2008-01-01

    The molecular mechanisms underlying cell wall biosynthesis are poorly understood. In this study, microscopic analysis showed that protoplasts generated a new cell wall within 48 h after transfer to a wall-regeneration medium. To identify genes related to cell wall biosynthesis in cotton, suppression subtractive hybridization was used to visualize differential gene expression at seven time points within the first 48 h. In total, 412 differentially expressed sequence tags (ESTs; >3-fold) were i...

  14. 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

  15. Arabidopsis Pumilio protein APUM5 suppresses Cucumber mosaic virus infection via direct binding of viral RNAs

    OpenAIRE

    Huh, Sung Un; Kim, Min Jung; Paek, Kyung-Hee

    2012-01-01

    Posttranscriptional/translational regulation of gene expression is mediated by diverse RNA binding proteins and plays an important role in development and defense processes. Among the RNA-binding proteins, the mammalian Pumilio RNA-binding family (Puf) acts as posttranscriptional and translational repressors. An Arabidopsis Puf mutant, apum5-D, was isolated during a T-DNA insertional mutant screen for mutants with reduced susceptibility to Cucumber mosaic virus (CMV) infection. Interestingly,...

  16. Multiple receptor complexes assembled for transmitting CLV3 signaling in Arabidopsis

    OpenAIRE

    Zhu, Yingfang; Wan, Yinglang; Lin, Jinxing

    2010-01-01

    In Arabidopsis, the feedback regulatory loop between CLAVATA3 (CLV3) signaling pathway and transcription factor, WUSCHEL (WUS) plays a significant role in shoot apical meristems (SAM) maintenance. Previously, CLV1/CLV2 heterodimers were supposed to perceive and transmit CLV3 signaling. Recent genetic analysis isolated a novel receptor kinase, CORYNE (CRN), which was found to be involved in the CLV3 pathway. Therefore, new hypothesis was put forward that CRN probably acts with CLV2 to transmit...

  17. Photorespiration mutants of Arabidopsis thaliana deficient in serine-glyoxylate aminotransferase activity

    OpenAIRE

    Somerville, C. R.; Ogren, W L

    1980-01-01

    Three mutants of the crucifer Arabidopsis thaliana (Linnaeus) Heynhold were isolated that are completely lacking in activity catalyzed by serine-glyoxylate aminotransferase (EC 2.6.1.45), a peroxisomal enzyme involved in photorespiratory carbon metabolism. These mutants were viable and exhibited normal photosynthesis under conditions that suppressed photorespiration, but they were inviable and photosynthesized at greatly reduced rates under conditions that promoted photorespiration. Serine an...

  18. Microscopic Evaluation of Interactions between Varieties of Arabidopsis thaliana Challenged by Peronospora parasitica

    OpenAIRE

    TÜRK*, Figen MERT

    2002-01-01

    Peronospora parasitica (Pers ex Fr.) Pers. is an obligate biotrophic pathogen that causes downy mildew in Arabidopsis thaliana (L.) Heynh. In this study, cotyledons of four A. thaliana varieties were inoculated with the Cala2 isolate of P. parasitica and the degree of susceptibility was observed under the microscope 1, 2, 3 and 7 days after inoculation (DAI). Microscopic examination of infected tissues revealed that early restriction of the pathogen was accompanied by a hypersensitive respons...

  19. SPL8, an SBP-box gene that affects pollen sac development in Arabidopsis

    OpenAIRE

    Unte, Ulrike S.; Sorensen, Anna-Marie; Pesaresi, Paolo; Gandikota, Madhuri; Leister, Dario; Saedler, Heinz; Huijser, Peter

    2003-01-01

    SQUAMOSA PROMOTER BINDING PROTEIN-box genes (SBP-box genes) encode plant-specific proteins that share a highly conserved DNA binding domain, the SBP domain. Although likely to represent transcription factors, little is known about their role in development. In Arabidopsis, SBP-box genes constitute a structurally heterogeneous family of 16 members known as SPL genes. For one of these genes, SPL8, we isolated three independent transposon-tagged mutants, all of which exhibited a strong reduction...

  20. Increased Ac excision (iae): Arabidopsis thaliana mutations affecting Ac transposition

    International Nuclear Information System (INIS)

    The maize transposable element Ac is highly active in the heterologous hosts tobacco and tomato, but shows very much reduced levels of activity in Arabidopsis. A mutagenesis experiment was undertaken with the aim of identifying Arabidopsis host factors responsible for the observed low levels of Ac activity. Seed from a line carrying a single copy of the Ac element inserted into the streptomycin phosphotransferase (SPT) reporter fusion, and which displayed typically low levels of Ac activity, were mutagenized using gamma rays. Nineteen mutants displaying high levels of somatic Ac activity, as judged by their highly variegated phenotypes, were isolated after screening the M2 generation on streptomycin-containing medium. The mutations fall into two complementation groups, iae1 and iae2, are unlinked to the SPT::Ac locus and segregate in a Mendelian fashion. The iae1 mutation is recessive and the iae2 mutation is semi-dominant. The iae1 and iae2 mutants show 550- and 70-fold increases, respectively, in the average number of Ac excision sectors per cotyledon. The IAE1 locus maps to chromosome 2, whereas the SPT::Ac reporter maps to chromosome 3. A molecular study of Ac activity in the iae1 mutant confirmed the very high levels of Ac excision predicted using the phenotypic assay, but revealed only low levels of Ac re-insertion. Analyses of germinal transposition in the iae1 mutant demonstrated an average germinal excision frequency of 3% and a frequency of independent Ac re-insertions following germinal excision of 22%. The iae mutants represents a possible means of improving the efficiency of Ac/Ds transposon tagging systems in Arabidopsis, and will enable the dissection of host involvement in Ac transposition and the mechanisms employed for controlling transposable element activity

  1. Radiosensitivity of Nicotiana protoplasts. Action on cell; cycle effects of low dose and fractionated irradiations; biological repair

    International Nuclear Information System (INIS)

    Leaf protoplasts of Nicotiana plumbaginifolia and Nicotiana sylvestris demonstrate five main qualities: they can be maintained as haploid lines; they constitute starting populations with a remarkable cytological homogeneity; they show a transient initial lag-phase; they yield very high plating efficiencies and retain permanently a complete differentiation capacity; being derived of a cell wall, they appear well adapted for fusion experiments or enzymatic dosages. The resumption of mitotic activity was followed by cytophotometric measurements, labelling experiments, nuclear sizing and enzymatic assays. The action of 5 Gy gamma-ray irradiations delayed entrance in the S-phase, provoked an otherwise not verified dependency between transcription, translation and protein synthesis, increased nuclear volumes in the G2-phase, and slightly stimulated the activity of a repair enzyme. The plating efficiency was a sensitive end-point which allowed the evaluation of the biological effectiveness of low to medium radiation-doses after gamma-ray and fast neutron irradiations. The neutron dose-RBE relationship increased from 3 to 25 when the dose decreased from 5 Gy to 5 mGy. When fractionated into low single doses only, a neutron dose of 300 mGy markedly increased its biological effectiveness: this phenomenon could not be explained by cell progression, and necessitated additional hypotheses involving other mechanisms in the specific action of low radiation doses. Radiation-induced UDS was measured in presence of aphidicolin. A beta-like DNA-polymerase was shown to be definitely involved in nuclear repair synthesis

  2. Bioethanol production by a flocculent hybrid, CHFY0321 obtained by protoplast fusion between Saccharomyces cerevisiae and Saccharomyces bayanus

    International Nuclear Information System (INIS)

    Fusion hybrid yeast, CHFY0321, was obtained by protoplast fusion between non-flocculent-high ethanol fermentative Saccharomyces cerevisiae CHY1011 and flocculent-low ethanol fermentative Saccharomyces bayanus KCCM12633. The hybrid yeast was used together with the parental strains to examine ethanol production in batch fermentation. Under the conditions tested, the fusion hybrid CHFY0321 flocculated to the highest degree and had the capacity to ferment well at pH 4.5 and 32 oC. Simultaneous saccharification and fermentation for ethanol production was carried out using a cassava (Manihot esculenta) powder hydrolysate medium containing 19.5% (w v-1) total sugar in a 5 l lab scale jar fermenter at 32 oC for 65 h with an agitation speed of 2 Hz. Under these conditions, CHFY0321 showed the highest flocculating ability and the best fermentation efficiency for ethanol production compared with those of the wild-type parent strains. CHFY0321 gave a final ethanol concentration of 89.8 ± 0.13 g l-1, a volumetric ethanol productivity of 1.38 ± 0.13 g l-1 h-1, and a theoretical yield of 94.2 ± 1.58%. These results suggest that CHFY0321 exhibited the fermentation characteristics of S. cerevisiae CHY1011 and the flocculent ability of S. bayanus KCCM12633. Therefore, the strong highly flocculent ethanol fermentative CHFY0321 has potential for improving biotechnological ethanol fermentation processes.

  3. Characterization of IRE1 ribonuclease-mediated mRNA decay in plants using transient expression analyses in rice protoplasts.

    Science.gov (United States)

    Hayashi, Shimpei; Wakasa, Yuhya; Ozawa, Kenjirou; Takaiwa, Fumio

    2016-06-01

    In some eukaryotes, endoplasmic reticulum (ER) stress induces regulated inositol-requiring enzyme 1 (IRE1)-dependent decay (RIDD) of mRNAs. Recently, the expression levels of the mRNAs encoding some secretory proteins were reported to be downregulated by RIDD in the vegetative tissues of plants. However, the characteristics of plant RIDD have been insufficiently investigated due to difficulty of in planta analyses. Here, the RIDD susceptibilities of various mRNAs that are difficult to analyze in planta were examined using transient expression analyses of rice protoplasts. In this system, the mRNAs encoding three rice seed storage proteins (SSPs) - namely α-globulin, 16-kDa prolamin and 10-kDa prolamin - were downregulated in response to ER stress. The rapid ER stress-induced degradation of these mRNAs was repressed in cells in which the ribonuclease activity of IRE1 was specifically abolished by genome editing, suggesting that the mRNAs encoding certain SSPs are strong targets of RIDD. Furthermore, we investigated whether these RIDD targets are substrates of the IRE1 ribonuclease using a recombinant IRE1 protein, and identified candidate IRE1-mediated cleavage sites. Overall, the results demonstrate the existence of a post-transcriptional mechanism of regulation of SSPs, and illustrate the basic and multifaceted characteristics of RIDD in higher plants. PMID:26831622

  4. Replication of TMV-L and Lta1 RNAs and their recombinants in TMV-resistant Tm-1 tomato protoplasts.

    Science.gov (United States)

    Yamafuji, R; Watanabe, Y; Meshi, T; Okada, Y

    1991-07-01

    Tm-1 is a gene that provides resistance to tomato plants against tobacco mosaic virus (TMV) infection. In tomato cells carrying the Tm-1 gene, multiplication of TMV is inhibited. From previous analysis of resistance-breaking mutants, the involvement of the 130- and 180-kDa proteins, putative viral replicases, in the resistance conferred by the Tm-1 gene was suggested. When wild-type TMV RNA was co-inoculated with a resistance-breaking mutant RNA, replication of the wild-type TMV genomic RNA could not be rescued by the 130- and 180-kDa proteins of a resistance-breaking strain, Lta1. To investigate how the putative resistance factor interacts with the 130- and 180-kDa proteins, we expressed the wild-type TMV protein sequence that is associated with the resistance-breaking phenomenon as part of a recombinant virus derived from Lta1 in Tm-1/Tm-1 protoplasts. No specific degradation of wild-type TMV protein sequences was observed, suggesting that the mechanism of the resistance does not involve the instability of a viral protein. PMID:2053299

  5. Bioethanol production by a flocculent hybrid, CHFY0321 obtained by protoplast fusion between Saccharomyces cerevisiae and Saccharomyces bayanus

    Energy Technology Data Exchange (ETDEWEB)

    Choi, Gi-Wook; Kang, Hyun-Woo; Kim, Yule [Changhae Institute of Cassava and Ethanol Research, Changhae Ethanol Co., LTD, Palbok-Dong 829, Dukjin-Gu, Jeonju 561-203 (Korea); Um, Hyun-Ju; Kim, Mina; Kim, Yang-Hoon [Department of Microbiology, Chungbuk National University, 410 Sungbong-Ro, Heungduk-Gu, Cheongju 361-763 (Korea)

    2010-08-15

    Fusion hybrid yeast, CHFY0321, was obtained by protoplast fusion between non-flocculent-high ethanol fermentative Saccharomyces cerevisiae CHY1011 and flocculent-low ethanol fermentative Saccharomyces bayanus KCCM12633. The hybrid yeast was used together with the parental strains to examine ethanol production in batch fermentation. Under the conditions tested, the fusion hybrid CHFY0321 flocculated to the highest degree and had the capacity to ferment well at pH 4.5 and 32 C. Simultaneous saccharification and fermentation for ethanol production was carried out using a cassava (Manihot esculenta) powder hydrolysate medium containing 19.5% (w v{sup -1}) total sugar in a 5 l lab scale jar fermenter at 32 C for 65 h with an agitation speed of 2 Hz. Under these conditions, CHFY0321 showed the highest flocculating ability and the best fermentation efficiency for ethanol production compared with those of the wild-type parent strains. CHFY0321 gave a final ethanol concentration of 89.8 {+-} 0.13 g l{sup -1}, a volumetric ethanol productivity of 1.38 {+-} 0.13 g l{sup -1} h{sup -1}, and a theoretical yield of 94.2 {+-} 1.58%. These results suggest that CHFY0321 exhibited the fermentation characteristics of S. cerevisiae CHY1011 and the flocculent ability of S. bayanus KCCM12633. Therefore, the strong highly flocculent ethanol fermentative CHFY0321 has potential for improving biotechnological ethanol fermentation processes. (author)

  6. 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.

  7. The Arabidopsis transcriptional regulator DPB3-1 enhances heat stress tolerance without growth retardation in rice.

    Science.gov (United States)

    Sato, Hikaru; Todaka, Daisuke; Kudo, Madoka; Mizoi, Junya; Kidokoro, Satoshi; Zhao, Yu; Shinozaki, Kazuo; Yamaguchi-Shinozaki, Kazuko

    2016-08-01

    The enhancement of heat stress tolerance in crops is an important challenge for food security to facilitate adaptation to global warming. In Arabidopsis thaliana, the transcriptional regulator DNA polymerase II subunit B3-1 (DPB3-1)/nuclear factor Y subunit C10 (NF-YC10) has been reported as a positive regulator of Dehydration-responsive element binding protein 2A (DREB2A), and the overexpression of DPB3-1 enhances heat stress tolerance without growth retardation. Here, we show that DPB3-1 interacts with DREB2A homologues in rice and soya bean. Transactivation analyses with Arabidopsis and rice mesophyll protoplasts indicate that DPB3-1 and its rice homologue OsDPB3-2 function as positive regulators of DREB2A homologues. Overexpression of DPB3-1 did not affect plant growth or yield in rice under nonstress conditions. Moreover, DPB3-1-overexpressing rice showed enhanced heat stress tolerance. Microarray analysis revealed that many heat stress-inducible genes were up-regulated in DPB3-1-overexpressing rice under heat stress conditions. However, the overexpression of DPB3-1 using a constitutive promoter had almost no effect on the expression of these genes under nonstress conditions. This may be because DPB3-1 is a coactivator and thus lacks inherent transcriptional activity. We conclude that DPB3-1, a coactivator that functions specifically under abiotic stress conditions, could be utilized to increase heat stress tolerance in crops without negative effects on vegetative and reproductive growth. PMID:26841113

  8. 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.

  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. The FAST technique: a simplified Agrobacterium-based transformation method for transient gene expression analysis in seedlings of Arabidopsis and other plant species

    Directory of Open Access Journals (Sweden)

    von Arnim Albrecht G

    2009-05-01

    Full Text Available Abstract Background Plant genome sequencing has resulted in the identification of a large number of uncharacterized genes. To investigate these unknown gene functions, several transient transformation systems have been developed as quick and convenient alternatives to the lengthy transgenic assay. These transient assays include biolistic bombardment, protoplast transfection and Agrobacterium-mediated transient transformation, each having advantages and disadvantages depending on the research purposes. Results We present a novel transient assay based on cocultivation of young Arabidopsis (Arabidopsis thaliana seedlings with Agrobacterium tumefaciens in the presence of a surfactant which does not require any dedicated equipment and can be carried out within one week from sowing seeds to protein analysis. This Fast Agro-mediated Seedling Transformation (FAST was used successfully to express a wide variety of constructs driven by different promoters in Arabidopsis seedling cotyledons (but not roots in diverse genetic backgrounds. Localizations of three previously uncharacterized proteins were identified by cotransformation with fluorescent organelle markers. The FAST procedure requires minimal handling of seedlings and was also adaptable for use in 96-well plates. The high transformation efficiency of the FAST procedure enabled protein detection from eight transformed seedlings by immunoblotting. Protein-protein interaction, in this case HY5 homodimerization, was readily detected in FAST-treated seedlings with Förster resonance energy transfer and bimolecular fluorescence complementation techniques. Initial tests demonstrated that the FAST procedure can also be applied to other dicot and monocot species, including tobacco, tomato, rice and switchgrass. Conclusion The FAST system provides a rapid, efficient and economical assay of gene function in intact plants with minimal manual handling and without dedicated device. This method is potentially

  11. Arabidopsis CDS blastp result: AK119708 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK119708 002-157-E08 At1g28330.1 dormancy-associated protein, putative (DRM1) identical to dormancy...-associated protein [Arabidopsis thaliana] GI:2995990; similar to dormancy-associated protei

  12. Arabidopsis CDS blastp result: AK060981 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK060981 006-202-H08 At1g28330.1 dormancy-associated protein, putative (DRM1) identical to dormancy...-associated protein [Arabidopsis thaliana] GI:2995990; similar to dormancy-associated protei

  13. Arabidopsis CDS blastp result: AK111736 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK111736 J023047L09 At1g68370.1 gravity -responsive protein / altered response to gravity ... protein ... (ARG1) identical to Altered Response to Gravity ... [Arabidopsis thaliana] GI:4249662; contains Pfam p ...

  14. Arabidopsis CDS blastp result: AK070093 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK070093 J023041M10 At2g39290.1 phosphatidylglycerolphosphate synthase (PGS1) identical to phosphati...dylglycerolphosphate synthase GI:13365519 from [Arabidopsis thaliana] 7e-78 ...

  15. Arabidopsis CDS blastp result: AK060009 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK060009 006-302-D03 At2g39290.1 phosphatidylglycerolphosphate synthase (PGS1) identical to phosphati...dylglycerolphosphate synthase GI:13365519 from [Arabidopsis thaliana] 8e-71 ...

  16. Arabidopsis CDS blastp result: AK058419 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK058419 001-015-D06 At4g16280.3 flowering time ... control protein / FCA gamma (FCA) identical to S ... P|O04425 Flowering time ... control protein FCA {Arabidopsis thaliana}; four a ...

  17. Arabidopsis CDS blastp result: AK073225 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK073225 J033023C04 At4g16280.3 flowering time ... control protein / FCA gamma (FCA) identical to SP ... |O04425 Flowering time ... control protein FCA {Arabidopsis thaliana}; four a ...

  18. 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 ...

  19. 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 ...

  20. 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 ...

  1. 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 ...

  2. 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 ...

  3. Arabidopsis CDS blastp result: AK288065 [KOME

    Lifescience Database Archive (English)

    Full Text Available al to sulfate tansporter Sultr1;3 [Arabidopsis thaliana] GI:10716805; contains Pfam profile PF00916: Sulfate... transporter family; contains Pfam profile PF01740: STAS domain; contains TIGRfam profile TIGR00815: sulfate permease 1e-145 ...

  4. Arabidopsis CDS blastp result: AK288002 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK288002 J075110B01 At1g68510.1 68414.m07826 LOB domain protein 42 ... / lateral organ boundaries do ... main protein 42 ... (LBD42 ) identical to LOB DOMAIN 42 ... [Arabidopsis th ...

  5. Arabidopsis CDS blastp result: AK241043 [KOME

    Lifescience Database Archive (English)

    Full Text Available upted by a stop codon, creating non-consensus donor and acceptor splice sites. 2e-41 ... ...tical to SP|P92997 Germin-like protein subfamily 1 member 13 precursor {Arabidopsis thaliana}; exon 2 interr

  6. Arabidopsis CDS blastp result: AK243135 [KOME

    Lifescience Database Archive (English)

    Full Text Available upted by a stop codon, creating non-consensus donor and acceptor splice sites. 7e-43 ... ...tical to SP|P92997 Germin-like protein subfamily 1 member 13 precursor {Arabidopsis thaliana}; exon 2 interr

  7. Arabidopsis CDS blastp result: AK111785 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK111785 J023089N11 At5g62310.1 incomplete root hair ... elongation (IRE) / protein kinase, putative ... nearly identical to IRE (incomplete root hair ... elongation) [Arabidopsis thaliana] gi|6729346|dbj| ...

  8. Arabidopsis CDS blastp result: AK243050 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK243050 J100011E04 At5g62310.1 68418.m07822 incomplete root hair ... elongation (IRE) / protein kin ... putative nearly identical to IRE (incomplete root hair ... elongation) [Arabidopsis thaliana] gi|6729346|dbj| ...

  9. Arabidopsis CDS blastp result: AK242758 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK242758 J090051H03 At5g62310.1 68418.m07822 incomplete root hair ... elongation (IRE) / protein kin ... putative nearly identical to IRE (incomplete root hair ... elongation) [Arabidopsis thaliana] gi|6729346|dbj| ...

  10. Arabidopsis CDS blastp result: AK242717 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK242717 J090043H19 At5g62310.1 68418.m07822 incomplete root hair ... elongation (IRE) / protein kin ... putative nearly identical to IRE (incomplete root hair ... elongation) [Arabidopsis thaliana] gi|6729346|dbj| ...

  11. Arabidopsis CDS blastp result: AK288095 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK288095 J075191E21 At5g62310.1 68418.m07822 incomplete root hair ... elongation (IRE) / protein kin ... putative nearly identical to IRE (incomplete root hair ... elongation) [Arabidopsis thaliana] gi|6729346|dbj| ...

  12. Arabidopsis CDS blastp result: AK242638 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK242638 J090023J02 At5g62310.1 68418.m07822 incomplete root hair ... elongation (IRE) / protein kin ... putative nearly identical to IRE (incomplete root hair ... elongation) [Arabidopsis thaliana] gi|6729346|dbj| ...

  13. Arabidopsis CDS blastp result: AK242651 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK242651 J090026B08 At5g62310.1 68418.m07822 incomplete root hair ... elongation (IRE) / protein kin ... putative nearly identical to IRE (incomplete root hair ... elongation) [Arabidopsis thaliana] gi|6729346|dbj| ...

  14. Arabidopsis CDS blastp result: AK287631 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK287631 J065073J24 At5g62310.1 68418.m07822 incomplete root hair ... elongation (IRE) / protein kin ... putative nearly identical to IRE (incomplete root hair ... elongation) [Arabidopsis thaliana] gi|6729346|dbj| ...

  15. Arabidopsis CDS blastp result: AK288923 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK288923 J090081P06 At5g62310.1 68418.m07822 incomplete root hair ... elongation (IRE) / protein kin ... putative nearly identical to IRE (incomplete root hair ... elongation) [Arabidopsis thaliana] gi|6729346|dbj| ...

  16. Arabidopsis CDS blastp result: AK242271 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK242271 J075187A19 At5g62310.1 68418.m07822 incomplete root hair ... elongation (IRE) / protein kin ... putative nearly identical to IRE (incomplete root hair ... elongation) [Arabidopsis thaliana] gi|6729346|dbj| ...

  17. Arabidopsis CDS blastp result: AK242681 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK242681 J090032N04 At5g62310.1 68418.m07822 incomplete root hair ... elongation (IRE) / protein kin ... putative nearly identical to IRE (incomplete root hair ... elongation) [Arabidopsis thaliana] gi|6729346|dbj| ...

  18. Arabidopsis CDS blastp result: AK243656 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK243656 J100088L22 At5g62310.1 68418.m07822 incomplete root hair ... elongation (IRE) / protein kin ... putative nearly identical to IRE (incomplete root hair ... elongation) [Arabidopsis thaliana] gi|6729346|dbj| ...

  19. Arabidopsis CDS blastp result: AK241519 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK241519 J065170E12 At5g62310.1 68418.m07822 incomplete root hair ... elongation (IRE) / protein kin ... putative nearly identical to IRE (incomplete root hair ... elongation) [Arabidopsis thaliana] gi|6729346|dbj| ...

  20. Arabidopsis CDS blastp result: AK240655 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK240655 J023135E11 At5g62310.1 68418.m07822 incomplete root hair ... elongation (IRE) / protein kin ... putative nearly identical to IRE (incomplete root hair ... elongation) [Arabidopsis thaliana] gi|6729346|dbj| ...

  1. Arabidopsis CDS blastp result: AK242733 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK242733 J090047O22 At5g62310.1 68418.m07822 incomplete root hair ... elongation (IRE) / protein kin ... putative nearly identical to IRE (incomplete root hair ... elongation) [Arabidopsis thaliana] gi|6729346|dbj| ...

  2. Arabidopsis CDS blastp result: AK242859 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK242859 J090073L24 At5g62310.1 68418.m07822 incomplete root hair ... elongation (IRE) / protein kin ... putative nearly identical to IRE (incomplete root hair ... elongation) [Arabidopsis thaliana] gi|6729346|dbj| ...

  3. Arabidopsis CDS blastp result: AK243187 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK243187 J100039E11 At5g62310.1 68418.m07822 incomplete root hair ... elongation (IRE) / protein kin ... putative nearly identical to IRE (incomplete root hair ... elongation) [Arabidopsis thaliana] gi|6729346|dbj| ...

  4. Arabidopsis CDS blastp result: AK242550 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK242550 J080319D10 At2g35630.1 68415.m04369 microtubule organization 1 protein (MO...R1) identical to microtubule organization 1 protein GI:14317953 from [Arabidopsis thaliana] 5e-44 ...

  5. Arabidopsis CDS blastp result: AK101368 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK101368 J033035L13 At5g24270.1 calcineurin B-like protein, putative / calcium sensor ... homolog (S ... OS3) identical to calcium sensor ... homolog [Arabidopsis thaliana] GI:3309575; similar ...

  6. Arabidopsis CDS blastp result: AK111570 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK111570 J013071C24 At5g24270.1 calcineurin B-like protein, putative / calcium sensor ... homolog (S ... OS3) identical to calcium sensor ... homolog [Arabidopsis thaliana] GI:3309575; similar ...

  7. Arabidopsis CDS blastp result: AK243065 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK243065 J100015N03 At5g24270.1 68418.m02855 calcineurin B-like protein, putative / calcium sensor ... or homolog (SOS3) identical to calcium sensor ... homolog [Arabidopsis thaliana] GI:3309575; similar ...

  8. The fifth international conference on Arabidopsis research

    Energy Technology Data Exchange (ETDEWEB)

    Hangarter, R.; Scholl, R.; Davis, K.; Feldmann, K.

    1993-12-31

    This volume contains abstracts of oral and poster presentations made in conjunction with the Fifth International Conference on Arabidopsis Research held August 19--22, 1993 at the Ohio State University, Columbus, Ohio.

  9. Arabidopsis CDS blastp result: AK070528 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK070528 J023060D13 At3g10920.1 superoxide dismutase [Mn], mitochondrial (SODA) / manganese ... supe ... roxide dismutase (MSD1) identical to manganese ... superoxide dismutase [Arabidopsis thaliana] gi|327 ...

  10. Arabidopsis CDS blastp result: AK119904 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK119904 002-182-A05 At3g10920.1 superoxide dismutase [Mn], mitochondrial (SODA) / manganese ... sup ... eroxide dismutase (MSD1) identical to manganese ... superoxide dismutase [Arabidopsis thaliana] gi|327 ...

  11. Arabidopsis CDS blastp result: AK104030 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK104030 001-020-C01 At3g10920.1 superoxide dismutase [Mn], mitochondrial (SODA) / manganese ... sup ... eroxide dismutase (MSD1) identical to manganese ... superoxide dismutase [Arabidopsis thaliana] gi|327 ...

  12. Arabidopsis CDS blastp result: AK104160 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK104160 006-211-E09 At3g10920.1 superoxide dismutase [Mn], mitochondrial (SODA) / manganese ... sup ... eroxide dismutase (MSD1) identical to manganese ... superoxide dismutase [Arabidopsis thaliana] gi|327 ...

  13. Arabidopsis CDS blastp result: AK287459 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK287459 J043019O07 At4g37000.1 68417.m05242 accelerated cell death ... 2 (ACD2) identical to accele ... rated cell death ... 2 (ACD2) GI:12484129 from [Arabidopsis thaliana] 4 ...

  14. Arabidopsis CDS blastp result: AK288034 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK288034 J075140H07 At4g37000.1 68417.m05242 accelerated cell death ... 2 (ACD2) identical to accele ... rated cell death ... 2 (ACD2) GI:12484129 from [Arabidopsis thaliana] 5 ...

  15. Arabidopsis CDS blastp result: AK111576 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK111576 J013075J23 At1g01510.1 C-terminal binding protein (ANGUSTIFOLIA) nearly id...entical to C-terminal binding protein ANGUSTIFOLIA [Arabidopsis thaliana] GI:15408535; contains Pfam profile

  16. Arabidopsis CDS blastp result: AK120838 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK120838 J023022B11 At1g01510.1 C-terminal binding protein (ANGUSTIFOLIA) nearly id...entical to C-terminal binding protein ANGUSTIFOLIA [Arabidopsis thaliana] GI:15408535; contains Pfam profile

  17. Arabidopsis CDS blastp result: AK111921 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK111921 001-013-A10 At1g01510.1 C-terminal binding protein (ANGUSTIFOLIA) nearly i...dentical to C-terminal binding protein ANGUSTIFOLIA [Arabidopsis thaliana] GI:15408535; contains Pfam profil

  18. Functional analysis of the theobroma cacao NPR1 gene in arabidopsis

    Directory of Open Access Journals (Sweden)

    Verica Joseph

    2010-11-01

    Full Text Available Abstract Background The Arabidopsis thaliana NPR1 gene encodes a transcription coactivator (NPR1 that plays a major role in the mechanisms regulating plant defense response. After pathogen infection and in response to salicylic acid (SA accumulation, NPR1 translocates from the cytoplasm into the nucleus where it interacts with other transcription factors resulting in increased expression of over 2000 plant defense genes contributing to a pathogen resistance response. Results A putative Theobroma cacao NPR1 cDNA was isolated by RT-PCR using degenerate primers based on homologous sequences from Brassica, Arabidopsis and Carica papaya. The cDNA was used to isolate a genomic clone from Theobroma cacao containing a putative TcNPR1 gene. DNA sequencing revealed the presence of a 4.5 kb coding region containing three introns and encoding a polypeptide of 591 amino acids. The predicted TcNPR1 protein shares 55% identity and 78% similarity to Arabidopsis NPR1, and contains each of the highly conserved functional domains indicative of this class of transcription factors (BTB/POZ and ankyrin repeat protein-protein interaction domains and a nuclear localization sequence (NLS. To functionally define the TcNPR1 gene, we transferred TcNPR1 into an Arabidopsis npr1 mutant that is highly susceptible to infection by the plant pathogen Pseudomonas syringae pv. tomato DC3000. Driven by the constitutive CaMV35S promoter, the cacao TcNPR1 gene partially complemented the npr1 mutation in transgenic Arabidopsis plants, resulting in 100 fold less bacterial growth in a leaf infection assay. Upon induction with SA, TcNPR1 was shown to translocate into the nucleus of leaf and root cells in a manner identical to Arabidopsis NPR1. Cacao NPR1 was also capable of participating in SA-JA signaling crosstalk, as evidenced by the suppression of JA responsive gene expression in TcNPR1 overexpressing transgenic plants. Conclusion Our data indicate that the TcNPR1 is a functional

  19. 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.

  20. CHAPERONIN 20 mediates iron superoxide dismutase (FeSOD) activity independent of its co-chaperonin role in Arabidopsis chloroplasts.

    Science.gov (United States)

    Kuo, W Y; Huang, C H; Liu, A C; Cheng, C P; Li, S H; Chang, W C; Weiss, C; Azem, A; Jinn, T L

    2013-01-01

    Iron superoxide dismutases (FeSODs; FSDs) are primary antioxidant enzymes in Arabidopsis thaliana chloroplasts. The stromal FSD1 conferred the only detectable FeSOD activity, whereas the thylakoid membrane- and nucleoid-co-localized FSD2 and FSD3 double mutant showed arrested chloroplast development. FeSOD requires cofactor Fe for its activity, but its mechanism of activation is unclear. We used reversed-phase high-performance liquid chromatography (HPLC), gel filtration chromatography, LC-MS/MS, protoplast transient expression and virus-induced gene silencing (VIGS) analyses to identify and characterize a factor involved in FeSOD activation. We identified the chloroplast-localized co-chaperonin CHAPERONIN 20 (CPN20) as a mediator of FeSOD activation by direct interaction. The relationship between CPN20 and FeSOD was confirmed by in vitro experiments showing that CPN20 alone could enhance FSD1, FSD2 and FSD3 activity. The in vivo results showed that CPN20-overexpressing mutants and mutants with defective co-chaperonin activity increased FSD1 activity, without changing the chaperonin CPN60 protein level, and VIGS-induced downregulation of CPN20 also led to decreased FeSOD activity. Our findings reveal that CPN20 can mediate FeSOD activation in chloroplasts, a role independent of its known function in the chaperonin system. PMID:23057508

  1. Arabidopsis CDS blastp result: AK073140 [KOME

    Lifescience Database Archive (English)

    Full Text Available me 4 (EC 3.1.3.16) {Arabidopsis thaliana}, phosphoprotein phosphatase 1 GI:166801 (Arabidopsis thaliana); contains a Ser/Thr protein...AK073140 J033022I01 At2g39840.1 serine/threonine protein phosphatase PP1 isozyme 4 (TOPP4) / phosphoprotein... phosphatase 1 identical to SP|P48484 Serine/threonine protein phosphatase PP1 isozy... phosphatase signature (PDOC00115); contains a metallo-phosphoesterase motif (QDOC50185) 1e-168 ...

  2. Arabidopsis CDS blastp result: AK120439 [KOME

    Lifescience Database Archive (English)

    Full Text Available me 4 (EC 3.1.3.16) {Arabidopsis thaliana}, phosphoprotein phosphatase 1 GI:166801 (Arabidopsis thaliana); contains a Ser/Thr protein...AK120439 J013098H20 At2g39840.1 serine/threonine protein phosphatase PP1 isozyme 4 (TOPP4) / phosphoprotein... phosphatase 1 identical to SP|P48484 Serine/threonine protein phosphatase PP1 isozy... phosphatase signature (PDOC00115); contains a metallo-phosphoesterase motif (QDOC50185) 1e-154 ...

  3. Arabidopsis CDS blastp result: AK121378 [KOME

    Lifescience Database Archive (English)

    Full Text Available me 4 (EC 3.1.3.16) {Arabidopsis thaliana}, phosphoprotein phosphatase 1 GI:166801 (Arabidopsis thaliana); contains a Ser/Thr protein...AK121378 J023127F14 At2g39840.1 serine/threonine protein phosphatase PP1 isozyme 4 (TOPP4) / phosphoprotein... phosphatase 1 identical to SP|P48484 Serine/threonine protein phosphatase PP1 isozy... phosphatase signature (PDOC00115); contains a metallo-phosphoesterase motif (QDOC50185) 1e-142 ...

  4. Arabidopsis CDS blastp result: AK063856 [KOME

    Lifescience Database Archive (English)

    Full Text Available yme 4 (EC 3.1.3.16) {Arabidopsis thaliana}, phosphoprotein phosphatase 1 GI:166801 (Arabidopsis thaliana); contains a Ser/Thr protein...AK063856 001-122-D05 At2g39840.1 serine/threonine protein phosphatase PP1 isozyme 4 (TOPP4) / phosphoprotein... phosphatase 1 identical to SP|P48484 Serine/threonine protein phosphatase PP1 isoz... phosphatase signature (PDOC00115); contains a metallo-phosphoesterase motif (QDOC50185) 6e-46 ...

  5. Terpene Specialized Metabolism in Arabidopsis thaliana

    OpenAIRE

    Tholl, Dorothea; Lee, Sungbeom

    2011-01-01

    Terpenes constitute the largest class of plant secondary (or specialized) metabolites, which are compounds of ecological function in plant defense or the attraction of beneficial organisms. Using biochemical and genetic approaches, nearly all Arabidopsis thaliana (Arabidopsis) enzymes of the core biosynthetic pathways producing the 5-carbon building blocks of terpenes have been characterized and closer insight has been gained into the transcriptional and posttranscriptional/translational mech...

  6. BODYGUARD is required for the biosynthesis of cutin in Arabidopsis.

    Science.gov (United States)

    Jakobson, Liina; Lindgren, Leif Ove; Verdier, Gaëtan; Laanemets, Kristiina; Brosché, Mikael; Beisson, Fred; Kollist, Hannes

    2016-07-01

    The cuticle plays a critical role in plant survival during extreme drought conditions. There are, however, surprisingly, many gaps in our understanding of cuticle biosynthesis. An Arabidopsis thaliana T-DNA mutant library was screened for mutants with enhanced transpiration using a simple condensation spot method. Five mutants, named cool breath (cb), were isolated. The cb5 mutant was found to be allelic to bodyguard (bdg), which is affected in an α/β-hydrolase fold protein important for cuticle structure. The analysis of cuticle components in cb5 (renamed as bdg-6) and another T-DNA mutant allele (bdg-7) revealed no impairment in wax synthesis, but a strong decrease in total cutin monomer load in young leaves and flowers. Root suberin content was also reduced. Overexpression of BDG increased total leaf cutin monomer content nearly four times by affecting preferentially C18 polyunsaturated ω-OH fatty acids and dicarboxylic acids. Whole-plant gas exchange analysis showed that bdg-6 had higher cuticular conductance and rate of transpiration; however, plant lines overexpressing BDG resembled the wild-type with regard to these characteristics. This study identifies BDG as an important component of the cutin biosynthesis machinery in Arabidopsis. We also show that, using BDG, cutin can be greatly modified without altering the cuticular water barrier properties and transpiration. PMID:26990896

  7. Amyloplast movement and gravityperception in Arabidopsis endoderm

    Science.gov (United States)

    Tasaka, M.; Saito, T.; Morita, M. T.

    Gravitropism of higher plant is a growth response regulating the orientation of organs elongation, which includes four sequential steps, the perception of gravistimulus, transduction of the physical stimulus to chemical signal, transmission of the signal, and differential cell elongation depending on the signal. To elucidate the molecular mechanism of these steps, we have isolated a number of Arabidopsis mutants with abnormal shoot gravitropic response. zig (zigzag)/sgr4(shoot gravitropism 4) shows little gravitropism in their shoots. Besides, their inflorescence stems elongate in a zigzag-fashion to bend at each node. ZIG encodes a SNARE, AtVTI11. sgr3 with reduced gravitropic response in inflorescence stems had a missense mutation in other SNARE, AtVAM3. These two SNAREs make a complex in the shoot endoderm cells that are gravity-sensing cells, suggesting that the vesicle transport from trans-Golgi network (TGN) to prevacuolar compartment (PVC) and/or vacuole is involved in gravitropism. Abnormal vesicular/vacuolar structures were observed in several tissues of both mutants. Moreover, SGR2 encodes phospholipase A1-like protein that resides in the vacuolar membrane. Endodermis-specific expression of these genes could complement gravitropism in each mutant. In addition, amyloplasts thought to be statoliths localized abnormally in their endoderm cells. These results strongly suggest that formation and function of vacuole in the endoderm cells are important for amyloplasts sedimentation, which is involved in the early process of shoot gravitropism. To reveal this, we constructed vertical stage microscope system to visualize the behavior of amyloplasts and vacuolar membrane in living endodermal cells. We hope to discuss the mechanism of gravity perception after showing their movements.

  8. 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

    2015-01-01

    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. PMID:25900618

  9. 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. PMID:25900618

  10. In Vitro Morphogenesis of Arabidopsis to Search for Novel Endophytic Fungi Modulating Plant Growth.

    Directory of Open Access Journals (Sweden)

    Francesco Dovana

    Full Text Available Fungal endophytes have shown to affect plant growth and to confer stress tolerance to the host; however, effects of endophytes isolated from water plants have been poorly investigated. In this study, fungi isolated from stems (stem-E and roots (root-E of Mentha aquatica L. (water mint were identified, and their morphogenetic properties analysed on in vitro cultured Arabidopsis (L. Heynh., 14 and 21 days after inoculation (DAI. Nineteen fungi were analysed and, based on ITS analysis, 17 isolates showed to be genetically distinct. The overall effect of water mint endophytes on Arabidopsis fresh (FW and dry weight (DW was neutral and positive, respectively, and the increased DW, mainly occurring 14 DAI, was possibly related to plant defence mechanism. Only three fungi increased both FW and DW of Arabidopsis at 14 and 21 DAI, thus behaving as plant growth promoting (PGP fungi. E-treatment caused a reduction of root depth and primary root length in most cases and inhibition-to-promotion of root area and lateral root length, from 14 DAI. Only Phoma macrostoma, among the water mint PGP fungi, increased both root area and depth, 21 DAI. Root depth and area 14 DAI were shown to influence DWs, indicating that the extension of the root system, and thus nutrient uptake, was an important determinant of plant dry biomass. Reduction of Arabidopsis root depth occurred to a great extent when plants where treated with stem-E while root area decreased or increased under the effects of stem-E and root-E, respectively, pointing to an influence of the endophyte origin on root extension. M. aquatica and many other perennial hydrophytes have growing worldwide application in water pollution remediation. The present study provided a model for directed screening of endophytes able to modulate plant growth in the perspective of future field applications of these fungi.

  11. Male sterility in Arabidopsis induced by overexpression of a MYC5-SRDX chimeric repressor.

    Science.gov (United States)

    Figueroa, Pablo; Browse, John

    2015-03-01

    Jasmonate hormone (JA) plays critical roles in both plant defense and reproductive development. Arabidopsis thaliana plants deficient in JA-biosynthesis or -signaling are male-sterile, with defects in stamen and pollen development. MYC2, MYC3 and MYC4 are JAZ-interacting bHLH transcription factors that play a major role in controlling JA responses in vegetative tissue, but are not likely to play a role in reproductive tissue. We found that a closely related transcription factor, MYC5 (bHLH28), was able to induce JAZ promoters that control some of the early JA-responsive genes in a Daucus carota (carrot) protoplast expression system. A G-box sequence in the JAZ2 promoter was necessary and sufficient for induction by MYC5 (as it is for MYC2, MYC3 and MYC4), and induction of JAZ genes was repressed by co-expression of a stabilized, JAZ1ΔJas repressor. Two allelic myc5 mutants exhibited no overt phenotype; however, transgenic lines expressing MYC5 fused to an SRDX (SUPERMAN repressive domain X) motif phenocopied mutants defective in JA signaling. In particular, MYC5-SRDX plants were male-sterile, with defects in stamen filament elongation, anther dehiscence and pollen viability. Importantly, expression of MYB21 and other transcription factors required for stamen and pollen maturation was strongly reduced in stamens of MYC5-SRDX plants relative to the wild type. Taken together, these results indicate that MYC5, probably together with other, redundant transcription factors, may be activated by JA signaling to induce the expression of MYB21 and components required for male fertility. PMID:25627909

  12. Functional analysis of U1-70K interacting SR proteins in pre-mRNA splicing in Arabidopsis

    International Nuclear Information System (INIS)

    Proteins of a serine/arginine-rich (SR) family are part of the spliceosome and are implicated in both constitutive and alternative splicing of pre-mRNAs. With the funding from DOE we have been studying alternative of splicing of genes encoding serine/arginine-rich (SR) proteins and the roles of SR proteins that interact with U1-70K in regulating basic and alternative splicing. Alternative splicing of pre-mRNAs of Arabidopsis serine/arginine-rich proteins and its regulation by hormones and stresses: We analyzed the splicing of all 19 Arabidopsis genes in different tissues, during different seedling stages and in response to various hormonal and stress treatments. Remarkably, about 90 different transcripts are produced from 15 SR genes, thereby increasing the transcriptome complexity of SR genes by about five fold. Using the RNA isolated from polysomes we have shown that most of the splice variants are recruited for translation. Alternative splicing of some SR genes is controlled in a developmental and tissue-specific manner (Palusa et al., 2007). Interestingly, among the various hormones and abiotic stresses tested, temperature stress (cold and heat) and ultraviolet light dramatically altered alternative splicing of pre-mRNAs of several SR genes whereas hormones altered the splicing of only two SR genes (Palusa et al., 2007). Localization and dynamics of a novel serine/arginine-rich protein that interacts with U1-70K: We analyzed the intranuclear movement of SR45 fused to GFP by fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP). We demonstrate that the movement of GFP-SR45 is ATP-dependent. Interestingly, inhibition of transcription or phosphorylation slowed the mobility of GFP-SR45 (Ali et al., 2006). Our studies have revealed that the nuclear localization signals are located in arg/ser-rich domains (RS) 1 and 2, whereas the speckle targeting signals are exclusively present in RS2 (Ali et al., 2006). The regulation of

  13. Protoplast fusion technology for improved production of coenzyme Q10 using Paracoccus denitrificans ATCC 19367 mutant strains

    Directory of Open Access Journals (Sweden)

    Pradipta Tokdar

    2014-01-01

    Full Text Available Normal 0 false false false EN-US X-NONE X-NONE MicrosoftInternetExplorer4 Induced mutants generated from Paracoccus denitrificans ATCC 19367 having antibiotic resistant markers, were used as parent strains to carry out protoplast fusion. The generated fusants were screened using standardized protocol for CoQ10 production. Among the generated fusants, one fusant namely PF-P1 showed 1.73 folds enhancements in specific CoQ10 content than wild type strain. Fusant PF-P1 was characterized by biochemical and molecular approaches where it showed differences than wild type strain. The fusant was further identified by 16S rRNA gene sequence analysis that showed eight nucleotide base pair mutation on conserved region and 99% homology with Paracoccus denitrificans strains. /* Style Definitions */ table.MsoNormalTable {mso-style-name:"Table Normal"; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-qformat:yes; mso-style-parent:""; mso-padding-alt:0in 5.4pt 0in 5.4pt; mso-para-margin:0in; mso-para-margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:11.0pt; font-family:"Calibri","sans-serif"; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-fareast-font-family:"Times New Roman"; mso-fareast-theme-font:minor-fareast; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin; mso-bidi-font-family:"Times New Roman"; mso-bidi-theme-font:minor-bidi;}

  14. Lazarus1, a DUF300 Protein, Contributes to Programmed Cell Death Associated with Arabidopsis acd11 and the Hypersensitive Response

    DEFF Research Database (Denmark)

    Malinovsky, F.G.; Brodersen, P.; Fiil, B.K.;

    2010-01-01

    ) mutant exhibits HR-like accelerated cell death, and cell death execution in acd11 shares genetic requirements for HR execution triggered by one subclass of R proteins. Methodology/Principal Findings: To identify genes required for this PCD pathway, we conducted a genetic screen for suppressors of acd11......, here called lazarus (laz) mutants. In addition to known suppressors of R protein-mediated HR, we isolated 13 novel complementation groups of dominant and recessive laz mutants. Here we describe laz1, which encodes a protein with a domain of unknown function (DUF300), and demonstrate that LAZ1....... Finally, we demonstrate by transient expression of reporter fusions in protoplasts that localization of LAZ1 is distributed between the cytosol, the plasma membrane and FM4-64 stained vesicles. Conclusions/Significance: Our findings indicate that LAZ1 functions as a regulator or effector of plant PCD...

  15. Advances in Arabidopsis research in China from 2006 to 2007

    Institute of Scientific and Technical Information of China (English)

    LIANG Yan; ZUO JianRu; YANG WeiCai

    2007-01-01

    @@ Arabidopsis thaliana, a model plant species, has a number of advantages over other plant species as an experimental organism due to many of its genetic and genomic features. The Chinese Arabidopsis community has made significant contributions to plant biology research in recent years[1,2]. In 2006, studies of plant biology in China received more attention than ever before, especially those pertaining to Arabidopsis research. Here we briefly summarize recent advances in Arabidopsis research in China.

  16. Genetic transformation of marine Actinomycete sp. Isolate M048 and expression of a recombinant plasmid carrying the apc gene

    Institute of Scientific and Technical Information of China (English)

    HOU Yanhua; LI Fuchao; QIN Song; WANG Quanfu

    2006-01-01

    Optimal conditions for protoplasts formation of marine Actinomycete sp. isolate M048 were described, dense and disperse mycelia were cultured in SGGP medium, 0.5% glycine, lysozyme exposure (2 mg/cm3, 37 ℃, 40 min), and the concentration of sucrose in protoplast buffer was 0.4 mol/dm3 for keeping the balance of osmotic pressure. Using PEG-mediated protoplasts transformation, the transformation frequency was 89 transformants per microgramme of pIJ702. Meanwhile, an effective transformation procedure was established based on intergeneric conjugation from E. coli ET12567 (pUZ8002) using shuttle vectors pPM801, pPM803 and a(ψ)C31-derived integration vector pIJ8600 containing oriT and attP fragments. Transformation frequencies were 5.30×10-4±0.26×10-4, 8.92×10-4±0.19×10-4 and 6.38×10-5±0.41×10-5, respectively. Further, the heterologous expression of the allophycocyanin gene (apc) in the strain M048 was used to demonstrate this transformation system. SDS-PAGE and Western blot analysis confirmed the expression of recombinant APC (rAPC).

  17. Isolation of dimorphic chloroplasts from the single-cell C4 species Bienertia sinuspersici

    Directory of Open Access Journals (Sweden)

    Lung Shiu-Cheung

    2012-03-01

    Full Text Available Abstract Three terrestrial plants are known to perform C4 photosynthesis without the dual-cell system by partitioning two distinct types of chloroplasts in separate cytoplasmic compartments. We report herein a protocol for isolating the dimorphic chloroplasts from Bienertia sinuspersici. Hypo-osmotically lysed protoplasts under our defined conditions released intact compartments containing the central chloroplasts and intact vacuoles with adhering peripheral chloroplasts. Following Percoll step gradient purification both chloroplast preparations demonstrated high homogeneities as evaluated from the relative abundance of respective protein markers. This protocol will open novel research directions toward understanding the mechanism of single-cell C4 photosynthesis.

  18. Expression of aberrant forms of AUXIN RESPONSE FACTOR8 stimulates parthenocarpy in Arabidopsis and tomato.

    Science.gov (United States)

    Goetz, Marc; Hooper, Lauren C; Johnson, Susan D; Rodrigues, Julio Carlyle Macedo; Vivian-Smith, Adam; Koltunow, Anna M

    2007-10-01

    Fruit initiation in Arabidopsis (Arabidopsis thaliana) is generally repressed until fertilization occurs. However, mutations in AUXIN RESPONSE FACTOR8 (ARF8) uncouple fruit initiation from fertilization, resulting in the formation of seedless, parthenocarpic fruit. Here we induced parthenocarpy in wild-type Arabidopsis by introducing either the mutant genomic (g) Atarf8-4 sequence or gAtARF8:beta-glucuronidase translational fusion constructs by plant transformation. Silencing of endogenous AtARF8 transcription was not observed, indicating that the introduced, aberrant ARF8 transcripts were compromising the function of endogenous ARF8 and/or associated factors involved in suppressing fruit initiation. To analyze the role of ARF8 in tomato (Solanum lycopersicum) we initially emasculated 23 tomato cultivars to test for background parthenocarpy. Surprisingly, all had a predisposition to initiate fertilization-independent fruit growth. Expression of gAtarf8-4 in transgenic tomato ('Monalbo') resulted in a significant increase in the number and size of parthenocarpic fruit. Isolation of tomato ARF8 cDNA indicated significant sequence conservation with AtARF8. SlARF8 may therefore control tomato fruit initiation in a similar manner as AtARF8 does in Arabidopsis. Two SlARF8 cDNAs differing in size by 5 bp were found, both arising from the same gene. The smaller cDNA is a splice variant and is also present in Arabidopsis. We propose that low endogenous levels of the splice variant products might interfere with efficient formation/function of a complex repressing fruit initiation, thereby providing an explanation for the observed ovary expansion in tomato and also Arabidopsis after emasculation. Increasing the levels of aberrant Atarf8-4 transcripts may further destabilize formation/function of the complex in a dosage-dependent manner enhancing tomato parthenocarpic fruit initiation frequency and size and mimicking the parthenocarpic dehiscent silique phenotype found in

  19. Mining the active proteome of Arabidopsis thaliana

    Directory of Open Access Journals (Sweden)

    Renier A. L. Van Der Hoorn

    2011-11-01

    Full Text Available Assigning functions to the >30.000 proteins encoded by the Arabidopsis genome is a challenging task of the Arabidopsis Functional Genomics Network. Although genome-wide technologies like proteomics and transcriptomics have generated a wealth of information that significantly accelerated gene annotation, protein activities are poorly predicted by transcript or protein levels as protein activities are post-translationally regulated. To directly display protein activities in Arabidopsis proteomes, we developed and applied Activity-based Protein Profiling (ABPP. ABPP is based on the use of small molecule probes that react with the catalytic residues of distinct protein classes in an activity-dependent manner. Labeled proteins are separated and detected from proteins gels and purified and identified by mass spectrometry. Using probes of six different chemotypes we have displayed of activities of 76 Arabidopsis proteins. These proteins represent over ten different protein classes that contain over 250 Arabidopsis proteins, including cysteine- serine- and metallo-proteases, lipases, acyltransferases, and the proteasome. We have developed methods for identification of in vivo labeled proteins using click-chemistry and for in vivo imaging with fluorescent probes. In vivo labeling has revealed novel protein activities and unexpected subcellular activities of the proteasome. Labeling of extracts displayed several differential activities e.g. of the proteasome during immune response and methylesterases during infection. These studies illustrate the power of ABPP to display the functional proteome and testify to a successful interdisciplinary collaboration involving chemical biology, organic chemistry and proteomics.

  20. Bioavailability of nanoparticulate hematite to Arabidopsis thaliana

    International Nuclear Information System (INIS)

    The environmental effects and bioavailability of nanoparticulate iron (Fe) to plants are currently unknown. Here, plant bioavailability of synthesized hematite Fe nanoparticles was evaluated using Arabidopsis thaliana (A. thaliana) as a model. Over 56-days of growing wild-type A. thaliana, the nanoparticle-Fe and no-Fe treatments had lower plant biomass, lower chlorophyll concentrations, and lower internal Fe concentrations than the Fe-treatment. Results for the no-Fe and nanoparticle-Fe treatments were consistently similar throughout the experiment. These results suggest that nanoparticles (mean diameter 40.9 nm, range 22.3–67.0 nm) were not taken up and therefore not bioavailable to A. thaliana. Over 14-days growing wild-type and transgenic (Type I/II proton pump overexpression) A. thaliana, the Type I plant grew more than the wild-type in the nanoparticle-Fe treatment, suggesting Type I plants cope better with Fe limitation; however, the nanoparticle-Fe and no-Fe treatments had similar growth for all plant types. -- Highlights: ► Iron nanoparticles were synthesized and assessed for bioavailability to Arabidopsis. ► Arabidopsis grew better in the presence of EDTA-bound iron than nanoparticulate iron. ► Arabidopsis grew the same in the presence of nanoparticulate iron compared to no iron. -- Synthesized iron nanoparticles were not bioavailable to Arabidopsis thaliana in agar nutrient media

  1. Sulfonamides identified as plant immune-priming compounds in high-throughput chemical screening increase disease resistance in Arabidopsis thaliana

    Directory of Open Access Journals (Sweden)

    Yoshiteru eNoutoshi

    2012-10-01

    Full Text Available Plant activators are agrochemicals that protect crops from diseases by activating the plant immune system. To isolate lead compounds for use as practical plant activators, we screened 2 different chemical libraries composed of various bioactive substances by using an established screening procedure that can selectively identify immune-priming compounds. We identified and characterized a group of sulfonamide compounds—sulfameter, sulfamethoxypyridazine, sulfabenzamide, and sulfachloropyridazine—among the various isolated candidate molecules. These sulfonamide compounds enhanced the avirulent Pseudomonas-induced cell death of Arabidopsis suspension cell cultures and increased disease resistance in Arabidopsis plants against both avirulent and virulent strains of the bacterium. These compounds did not prevent the growth of pathogenic bacteria in minimal liquid media at 200 µM. They also did not induce the expression of defense-related genes in Arabidopsis seedlings, at least not at 24 and 48 h after treatment, suggesting that they do not act as salicylic acid analogs. In addition, although sulfonamides are known to be folate biosynthesis inhibitors, the application of folate did not restore the potentiation effects of the sulfonamides on pathogen-induced cell death. Our data suggest that sulfonamides potentiate Arabidopsis disease resistance by their novel chemical properties.

  2. The small ethylene response factor ERF96 is involved in the regulation of the abscisic acid response in Arabidopsis

    Directory of Open Access Journals (Sweden)

    Xiaoping eWang

    2015-11-01

    Full Text Available Ethylene regulates many aspects of plant growth and development including seed germination, leaf senescence, and fruit ripening, and of plant responses to environmental stimuli including both biotic and abiotic stresses. Ethylene Response Factors (ERFs are plant-specific transcription factors and are a subfamily of the AP2 (APETALA2/ERF transcription factor family. The function of many members in this large gene family remains largely unknown. ERF96, a member of the Group IX ERF family transcription factors, has recently been shown to be a transcriptional activator that is involved in plant defense response in Arabidopsis. Here we provide evidence that ERF96 is a positive regulator of abscisic acid (ABA responses. Bioinformatics analysis indicated that there are a total four small ERFs in Arabidopsis including ERF95, ERF96, ERF97 and ERF98, and that ERF96 forms a cluster with ERF95 and ERF97. By using quantitative RT-PCR, we found that ERF96 is expressed in all tissues and organs examined except roots, with relatively high expression in flowers and seeds. Results from the protoplast transfection assay results indicated that the EDLL motif-containing C-terminal domain is responsible for ERF96’s transcriptional activity. Although loss-of-function mutant of ERF96 was morphologically similar to wild type plants, transgenic plants overexpressing ERF96 had smaller rosette size and were delayed in flowering time. In ABA sensitivity assays, we found that ERF96 overexpression plants were hypersensitive to ABA in terms of ABA inhibition of seed germination, early seedling development and root elongation. Consistent with these observations, elevated transcript levels of some ABA-responsive genes including RD29A, ABI5, ABF3, ABF4, P5CS and COR15A were observed in the transgenic plants in the presence of ABA. However, in the absence of ABA treatment, the transcript levels of these ABA-responsive genes remained largely unchanged. Our experiments also showed

  3. PEG介导的玉米弯孢叶斑病菌遗传转化%PEG-mediated protoplast transformation of Curvularia lunata, the causal agent of Curvularia leaf spot in maize

    Institute of Scientific and Technical Information of China (English)

    刘铜; 侯巨梅; 陈捷; 荆晶; 王玉莹; 左豫虎

    2012-01-01

    为了探索不同酶系组成对玉米弯孢叶斑病菌原生质体制备的影响,建立该病菌原生质体遗传转化系统,采用酶系混合物裂解菌丝体制备原生质体,采用PEG介导方法进行原生质遗传转化,通过PCR和Southern blotting技术对转化子进行验证.结果表明:1%溶壁酶+1%蜗牛酶+1%纤维素酶混合酶系为玉米弯孢叶斑病菌原生质体制备的最佳酶系,可以产生原生质体6.78×106 du/mL.用PEG介导方法转化共获得16个稳定的转化子,从中随机挑取5个转化子发现质粒pV2已被成功整合到基因组中.本研究获得了制备玉米弯孢叶斑病菌原生质体的最佳酶系,建立了PEG介导的原生质体遗传转化体系,为开展该菌致病相关基因克隆和基因功能研究提供了一种手段.%The effects of the composition of various enzymes on protoplast of Curvularia lunata preparation were explored, and its protoplast-mediated genetic transformation system was established. Protoplast was obtained through cleavage of mycelium using enzyme mixture. Protoplast genetic transformation was mediated by polyethylene glycol (PEG), and transformants were analyzed by PCR and Southern blotting. The best composition of enzymes for protoplast preparation was 1% snailase, 1% cellulase and 1% wallzyme, which could produce 6.78× 106 protoplasts/mL. Sixteen stable transformants were obtained by PEG, and the plasmid pV2 was successfully integrated into the genome in 5 randomly selected transformants. The best composition of enzymes for protoplasts preparation was obtained, and the protoplast-mediated genetic transformation system of C. lunata was established, which provided a tool for cloning pathogenicity genes and studying the gene function.

  4. Breeding a Strain Produced Oils with Cellulose by Protoplast Fusion%原生质体融合技术选育纤维素发酵产油菌株

    Institute of Scientific and Technical Information of China (English)

    徐新丽

    2011-01-01

    Through inactivated protoplast fusion technology to filtrate the direct use of cellulose raw materials,oil-producing strains.Experiments were from parents strains protoplast inactivated way and the selection of fusion conditions.%利用灭活原生质体进行融合的方法来选育能直接利用纤维素原料产油脂菌株。实验分别从双亲株原生质体灭活方式以及融合条件的选择进行了研究。

  5. Arabidopsis Pumilio protein APUM5 suppresses Cucumber mosaic virus infection via direct binding of viral RNAs.

    Science.gov (United States)

    Huh, Sung Un; Kim, Min Jung; Paek, Kyung-Hee

    2013-01-01

    Posttranscriptional/translational regulation of gene expression is mediated by diverse RNA binding proteins and plays an important role in development and defense processes. Among the RNA-binding proteins, the mammalian Pumilio RNA-binding family (Puf) acts as posttranscriptional and translational repressors. An Arabidopsis Puf mutant, apum5-D, was isolated during a T-DNA insertional mutant screen for mutants with reduced susceptibility to Cucumber mosaic virus (CMV) infection. Interestingly, CMV RNA contained putative Pumilio-homology domain binding motifs in its 3' untranslated region (UTR) and internal places in its genome. APUM5 directly bound to the 3' UTR motifs and some internal binding motifs in CMV RNAs in vitro and in vivo. We showed that APUM5 acts as a translational repressor that regulates the 3' UTR of CMV and affects CMV replication. This study uncovered a unique defense system that Arabidopsis APUM5 specifically regulates CMV infection by the direct binding of CMV RNAs. PMID:23269841

  6. Recent Progress in Arabidopsis Research in China: A Preface

    Institute of Scientific and Technical Information of China (English)

    Zhi-Hong Xu

    2006-01-01

    @@ In 2002, a workshop on Arabidopsis research in China was held in Shanghai, when a small group of Chinese plant scientists was working on this model species. Since then, we have witnessed the rapid growth of Arabidopsis research in China. This special issue of Journal of Integrative Plant Biology is dedicated exclusively to the Fourth Workshop on Arabidopsis Research in China, scheduled on November 30, 2005, in Beijing. In addition to reports collected in this special issue, the Chinese Arabidopsis community has been able to make significant contributions to many research fields. Here, I briefly summarize recent advances in Arabidopsis research in China.

  7. The arabidopsis cyclic nucleotide interactome

    KAUST Repository

    Donaldson, Lara

    2016-05-11

    Background Cyclic nucleotides have been shown to play important signaling roles in many physiological processes in plants including photosynthesis and defence. Despite this, little is known about cyclic nucleotide-dependent signaling mechanisms in plants since the downstream target proteins remain unknown. This is largely due to the fact that bioinformatics searches fail to identify plant homologs of protein kinases and phosphodiesterases that are the main targets of cyclic nucleotides in animals. Methods An affinity purification technique was used to identify cyclic nucleotide binding proteins in Arabidopsis thaliana. The identified proteins were subjected to a computational analysis that included a sequence, transcriptional co-expression and functional annotation analysis in order to assess their potential role in plant cyclic nucleotide signaling. Results A total of twelve cyclic nucleotide binding proteins were identified experimentally including key enzymes in the Calvin cycle and photorespiration pathway. Importantly, eight of the twelve proteins were shown to contain putative cyclic nucleotide binding domains. Moreover, the identified proteins are post-translationally modified by nitric oxide, transcriptionally co-expressed and annotated to function in hydrogen peroxide signaling and the defence response. The activity of one of these proteins, GLYGOLATE OXIDASE 1, a photorespiratory enzyme that produces hydrogen peroxide in response to Pseudomonas, was shown to be repressed by a combination of cGMP and nitric oxide treatment. Conclusions We propose that the identified proteins function together as points of cross-talk between cyclic nucleotide, nitric oxide and reactive oxygen species signaling during the defence response.

  8. Arabidopsis CDS blastp result: AK243152 [KOME

    Lifescience Database Archive (English)

    Full Text Available ase PP1 isozyme 4 (EC 3.1.3.16) {Arabidopsis thaliana}, phosphoprotein phosphatase 1 GI:166801 (Arabidopsis thaliana); contains...P1 isozyme 4 (TOPP4) / phosphoprotein phosphatase 1 identical to SP|P48484 Serine/threonine protein phosphat... a Ser/Thr protein phosphatase signature (PDOC00115); contains a metallo-phosphoesterase motif (QDOC50185) 1e-154 ... ...AK243152 J100032N02 At2g39840.1 68415.m04893 serine/threonine protein phosphatase P

  9. Arabidopsis CDS blastp result: AK288069 [KOME

    Lifescience Database Archive (English)

    Full Text Available ase PP1 isozyme 4 (EC 3.1.3.16) {Arabidopsis thaliana}, phosphoprotein phosphatase 1 GI:166801 (Arabidopsis thaliana); contains...P1 isozyme 4 (TOPP4) / phosphoprotein phosphatase 1 identical to SP|P48484 Serine/threonine protein phosphat... a Ser/Thr protein phosphatase signature (PDOC00115); contains a metallo-phosphoesterase motif (QDOC50185) 6e-70 ... ...AK288069 J075158N05 At2g39840.1 68415.m04893 serine/threonine protein phosphatase P

  10. Gibberellins control fruit patterning in Arabidopsis thaliana.

    Science.gov (United States)

    Arnaud, Nicolas; Girin, Thomas; Sorefan, Karim; Fuentes, Sara; Wood, Thomas A; Lawrenson, Tom; Sablowski, Robert; Østergaard, Lars

    2010-10-01

    The Arabidopsis basic helix-loop-helix (bHLH) proteins INDEHISCENT (IND) and ALCATRAZ (ALC) specify tissues required for fruit opening that have major roles in seed dispersal and plant domestication. Here, we show that synthesis of the phytohormone gibberellin is a direct and necessary target of IND, and that ALC interacts directly with DELLA repressors, which antagonize ALC function but are destabilized by gibberellin. Thus, the gibberellin/DELLA pathway has a key role in patterning the Arabidopsis fruit, and the interaction between DELLA and bHLH proteins, previously shown to connect gibberellin and light responses, is a versatile regulatory module also used in tissue patterning. PMID:20889713

  11. Impacts of banning protoplast fusion on the range of varieties available for organic arable cropping and vegetable production - Auswirkungen des Verbots der Protoplastenfusion auf das Sortenspektrum im ökologischen Acker- und Gemüsebau

    OpenAIRE

    Billmann, Bettina

    2008-01-01

    The use of protoplast fusion (PF) in plant breeding was often discussed between stakeholders of organic agriculture. The main criticism was the combination of genetic material under circumstances, which naturally do not exist. The results of a literature research and expert interviews summarize the scientific and legal state of affairs in Europe and provide a basis for decision-making of standard setting bodies.

  12. Arabidopsis PIZZA has the capacity to acylate brassinosteroids.

    Directory of Open Access Journals (Sweden)

    Katja Schneider

    Full Text Available Brassinosteroids (BRs affect a wide range of developmental processes in plants and compromised production or signalling of BRs causes severe growth defects. To identify new regulators of plant organ growth, we searched the Arabidopsis FOX (Full-length cDNA Over-eXpressor gene collection for mutants with altered organ size and isolated two overexpression lines that display typical BR deficient dwarf phenotypes. The phenotype of these lines, caused by an overexpression of a putative acyltransferase gene PIZZA (PIZ, was partly rescued by supplying exogenous brassinolide (BL and castasterone (CS, indicating that endogenous BR levels are rate-limiting for the growth of PIZ overexpression lines. Our transcript analysis further showed that PIZ overexpression leads to an elevated expression of genes involved in BR biosynthesis and a reduced expression of BR inactivating hydroxylases, a transcriptional response typical to low BR levels. Taking the advantage of relatively high endogenous BR accumulation in a mild bri1-301 background, we found that overexpression of PIZ results in moderately reduced levels of BL and CS and a strong reduction of typhasterol (TY and 6-deoxocastasterone (6-deoxoCS, suggesting a role of PIZ in BR metabolism. We tested a set of potential substrates in vitro for heterologously expressed PIZ and confirmed its acyltransferase activity with BL, CS and TY. The PIZ gene is expressed in various tissues but as reported for other genes involved in BR metabolism, the loss-of-function mutants did not display obvious growth phenotypes under standard growth conditions. Together, our data suggest that PIZ can modify BRs by acylation and that these properties might help modulating endogenous BR levels in Arabidopsis.

  13. Characterization of Sugar Insensitive (sis) Mutants of Arabidopsis

    Energy Technology Data Exchange (ETDEWEB)

    Gibson, Susan I.

    2009-06-08

    Despite the fact that soluble sugar levels have been postulated to play an important role in the control of a wide variety of plant metabolic and developmental pathways, the mechanisms by which plants respond to soluble sugar levels remain poorly understood. Plant responses to soluble sugar levels are also important in bioenergy production, as plant sugar responses are believed to help regulate both carbon fixation and carbon partitioning. For example, accumulation of soluble sugars, such as sucrose and glucose, in source tissues leads to feedback inhibition of photosynthesis, thereby decreasing rates of carbon fixation. Soluble sugar levels can also affect sink strengths, affecting the rates of accumulation of carbon-based compounds into both particular molecular forms (e.g. carbohydrates versus lipids versus proteins) and particular plant organs and tissues. Mutants of Arabidopsis that are defective in the ability to respond to soluble sugar levels were isolated and used as tools to identify some of the factors involved in plant sugar response. These sugar insensitive (sis) mutants were isolated by screening mutagenized seeds for those that were able to germinate and develop relatively normal shoot systems on media containing 0.3 M glucose or 0.3 M sucrose. At these sugar concentrations, wild-type Arabidopsis germinate and produce substantial root systems, but show little to no shoot development. Twenty-eight sis mutants were isolated during the course of four independent mutant screens. Based on a preliminary characterization of all of these mutants, sis3 and sis6 were chosen for further study. Both of these mutations appear to lie in previously uncharacterized loci. Unlike many other sugar-response mutants, sis3 mutants exhibit a wild-type or near wild-type response in all phytohormone-response assays conducted to date. The sis6-1 mutation is unusual in that it appears to be due to overexpression of a gene, rather than representing a loss of function mutation

  14. Arabidopsis CDS blastp result: AK066771 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK066771 J013083K07 At1g01170.1 ozone-responsive stress-related protein, putative s...imilar to stress-related ozone-induced protein AtOZI1 (GI:790583) [Arabidopsis thaliana]; contains 1 predicted transmembrane domain; 2e-29 ...

  15. Arabidopsis CDS blastp result: AK059353 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK059353 001-026-D01 At1g01170.1 ozone-responsive stress-related protein, putative ...similar to stress-related ozone-induced protein AtOZI1 (GI:790583) [Arabidopsis thaliana]; contains 1 predicted transmembrane domain; 2e-29 ...

  16. Arabidopsis CDS blastp result: AK059160 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK059160 001-023-D05 At1g01170.1 ozone-responsive stress-related protein, putative ...similar to stress-related ozone-induced protein AtOZI1 (GI:790583) [Arabidopsis thaliana]; contains 1 predicted transmembrane domain; 3e-28 ...

  17. Arabidopsis CDS blastp result: AK242849 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK242849 J090072M15 At1g68370.1 68414.m07809 gravity -responsive protein / altered response to gravity ... ty protein (ARG1) identical to Altered Response to Gravity ... [Arabidopsis thaliana] GI:4249662; contains Pfam p ...

  18. Arabidopsis CDS blastp result: AK288959 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK288959 J090084E19 At1g68370.1 68414.m07809 gravity -responsive protein / altered response to gravity ... ty protein (ARG1) identical to Altered Response to Gravity ... [Arabidopsis thaliana] GI:4249662; contains Pfam p ...

  19. Arabidopsis CDS blastp result: AK243008 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK243008 J090097H12 At1g68370.1 68414.m07809 gravity -responsive protein / altered response to gravity ... ty protein (ARG1) identical to Altered Response to Gravity ... [Arabidopsis thaliana] GI:4249662; contains Pfam p ...

  20. Arabidopsis CDS blastp result: AK288072 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK288072 J075161I05 At1g68370.1 68414.m07809 gravity -responsive protein / altered response to gravity ... ty protein (ARG1) identical to Altered Response to Gravity ... [Arabidopsis thaliana] GI:4249662; contains Pfam p ...

  1. Arabidopsis CDS blastp result: AK243178 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK243178 J100036P15 At1g68370.1 68414.m07809 gravity -responsive protein / altered response to gravity ... ty protein (ARG1) identical to Altered Response to Gravity ... [Arabidopsis thaliana] GI:4249662; contains Pfam p ...

  2. Arabidopsis CDS blastp result: AK243505 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK243505 J100074N19 At1g68370.1 68414.m07809 gravity -responsive protein / altered response to gravity ... ty protein (ARG1) identical to Altered Response to Gravity ... [Arabidopsis thaliana] GI:4249662; contains Pfam p ...

  3. Arabidopsis CDS blastp result: AK287577 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK287577 J065037N08 At1g68370.1 68414.m07809 gravity -responsive protein / altered response to gravity ... ty protein (ARG1) identical to Altered Response to Gravity ... [Arabidopsis thaliana] GI:4249662; contains Pfam p ...

  4. Protease gene families in Populus and Arabidopsis

    Directory of Open Access Journals (Sweden)

    Jansson Stefan

    2006-12-01

    Full Text Available Abstract Background Proteases play key roles in plants, maintaining strict protein quality control and degrading specific sets of proteins in response to diverse environmental and developmental stimuli. Similarities and differences between the proteases expressed in different species may give valuable insights into their physiological roles and evolution. Results We have performed a comparative analysis of protease genes in the two sequenced dicot genomes, Arabidopsis thaliana and Populus trichocarpa by using genes coding for proteases in the MEROPS database 1 for Arabidopsis to identify homologous sequences in Populus. A multigene-based phylogenetic analysis was performed. Most protease families were found to be larger in Populus than in Arabidopsis, reflecting recent genome duplication. Detailed studies on e.g. the DegP, Clp, FtsH, Lon, rhomboid and papain-Like protease families showed the pattern of gene family expansion and gene loss was complex. We finally show that different Populus tissues express unique suites of protease genes and that the mRNA levels of different classes of proteases change along a developmental gradient. Conclusion Recent gene family expansion and contractions have made the Arabidopsis and Populus complements of proteases different and this, together with expression patterns, gives indications about the roles of the individual gene products or groups of proteases.

  5. Arabidopsis CDS blastp result: AK241402 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK241402 J065159A02 At4g19070.1 68417.m02810 cadmium-responsive protein / cadmium i...nduced protein (AS8) identical to cadmium induced protein AS8 SP:P42735 from [Arabidopsis thaliana] 3e-11 ...

  6. Arabidopsis CDS blastp result: AK242143 [KOME

    Lifescience Database Archive (English)

    Full Text Available ar to GI:6573119 from [Lycopersicon esculentum] (Plant Physiol. 122 (1), 292 (2000)) 3e-12 ... ... identical to SP|Q9C888 Phospholipase D epsilon (EC 3.1.4.4) (AtPLDepsilon) (PLD epsilon) (PLDalpha3) {Arabidopsis thaliana}; simil

  7. Arabidopsis CDS blastp result: AK242143 [KOME

    Lifescience Database Archive (English)

    Full Text Available ar to GI:6573119 from [Lycopersicon esculentum] (Plant Physiol. 122 (1), 292 (2000)) 6e-22 ... ... identical to SP|Q9C888 Phospholipase D epsilon (EC 3.1.4.4) (AtPLDepsilon) (PLD epsilon) (PLDalpha3) {Arabidopsis thaliana}; simil

  8. Arabidopsis CDS blastp result: AK240654 [KOME

    Lifescience Database Archive (English)

    Full Text Available ar to GI:6573119 from [Lycopersicon esculentum] (Plant Physiol. 122 (1), 292 (2000)) 1e-160 ... ... identical to SP|Q9C888 Phospholipase D epsilon (EC 3.1.4.4) (AtPLDepsilon) (PLD epsilon) (PLDalpha3) {Arabidopsis thaliana}; simil

  9. Arabidopsis CDS blastp result: AK242290 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK242290 J075191E07 At4g13870.1 68417.m02148 Werner Syndrome-like exonuclease (WEX)... contains Pfam profile PF01612: 3'-5' exonuclease; identical to Werner Syndrome-like exonuclease [Arabidopsis thaliana] GP:28195109 gb:AAO33765 1e-20 ...

  10. Arabidopsis CDS blastp result: AK063585 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK063585 001-118-A04 At4g13870.2 Werner Syndrome-like exonuclease (WEX) contains Pf...am profile PF01612: 3'-5' exonuclease; identical to Werner Syndrome-like exonuclease [Arabidopsis thaliana] GP:28195109 gb:AAO33765 6e-16 ...

  11. Arabidopsis CDS blastp result: AK242290 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK242290 J075191E07 At4g13870.2 68417.m02149 Werner Syndrome-like exonuclease (WEX)... contains Pfam profile PF01612: 3'-5' exonuclease; identical to Werner Syndrome-like exonuclease [Arabidopsis thaliana] GP:28195109 gb:AAO33765 1e-20 ...

  12. Arabidopsis CDS blastp result: AK243230 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK243230 J100044L04 At1g19850.1 68414.m02490 transcription factor MONOPTEROS (MP) /... auxin-responsive protein (IAA24) / auxin response factor 5 (ARF5) identical to transcription factor MONOPTEROS (MP/IAA24/ARF5) SP:P93024 from [Arabidopsis thaliana] 2e-65 ...

  13. Arabidopsis CDS blastp result: AK103452 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK103452 J033129I11 At1g19850.1 transcription factor MONOPTEROS (MP) / auxin-respon...sive protein (IAA24) / auxin response factor 5 (ARF5) identical to transcription factor MONOPTEROS (MP/IAA24/ARF5) SP:P93024 from [Arabidopsis thaliana] 1e-166 ...

  14. Arabidopsis CDS blastp result: AK318617 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK318617 J100090H20 At1g19850.1 68414.m02490 transcription factor MONOPTEROS (MP) /... auxin-responsive protein (IAA24) / auxin response factor 5 (ARF5) identical to transcription factor MONOPTEROS (MP/IAA24/ARF5) SP:P93024 from [Arabidopsis thaliana] 2e-63 ...

  15. Arabidopsis CDS blastp result: AK287832 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK287832 J065187F20 At1g30950.1 68414.m03790 unusual floral organ (UFO ) / F-box family protein ( ... ubunit; almost identical to unusual floral organs (UFO )GI:4376159 from [Arabidopsis thaliana] Landsberg-e ...

  16. Arabidopsis CDS blastp result: AK241547 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK241547 J065176G22 At1g30950.1 68414.m03790 unusual floral organ (UFO ) / F-box family protein ( ... ubunit; almost identical to unusual floral organs (UFO )GI:4376159 from [Arabidopsis thaliana] Landsberg-e ...

  17. Arabidopsis CDS blastp result: AK242616 [KOME

    Lifescience Database Archive (English)

    Full Text Available ve contains PF00481: Protein phosphatase 2C domain; identical to protein phosphatase 2C (GI:4587992) [Arabidopsis thaliana] 2e-34 ... ...AK242616 J090017C19 At2g40180.1 68415.m04941 protein phosphatase 2C, putative / PP2C, putati

  18. Arabidopsis CDS blastp result: AK242846 [KOME

    Lifescience Database Archive (English)

    Full Text Available ve contains PF00481: Protein phosphatase 2C domain; identical to protein phosphatase 2C (GI:4587992) [Arabidopsis thaliana] 9e-12 ... ...AK242846 J090071I10 At2g40180.1 68415.m04941 protein phosphatase 2C, putative / PP2C, putati

  19. Arabidopsis CDS blastp result: AK241162 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK241162 J065116A05 At5g54800.1 68418.m06826 glucose-6-phosphate/phosphate translocator, putative identic...al to glucose 6 phosphate/phosphate translocator [Arabidopsis thaliana] gi|7229675|gb|AAF42936 2e-11 ...

  20. Arabidopsis CDS blastp result: AK242098 [KOME

    Lifescience Database Archive (English)

    Full Text Available ve contains PF00481: Protein phosphatase 2C domain; identical to protein phosphatase 2C (GI:4587992) [Arabidopsis thaliana] 3e-22 ... ...AK242098 J075143H11 At2g40180.1 68415.m04941 protein phosphatase 2C, putative / PP2C, putati