[Show abstract][Hide abstract] ABSTRACT: We have previously investigated the proteome changes of rice leaves under heat stress (Lee et al. in Proteomics 2007a, 7:3369-3383), wherein a group of antioxidant proteins and heat shock proteins (HSPs) were found to be regulated differently. The present study focuses on the biochemical changes and gene expression profiles of heat shock protein and antioxidant genes in rice leaves in response to heat stress (42°C) during a wide range of exposure times. The results show that hydrogen peroxide and proline
contents increased significantly, suggesting an oxidative burst and osmotic imbalance under heat stress. The mRNA levels of chaperone 60, HSP70, HSP100, chloroplastic HSP26, and mitochondrial small HSP responded rapidly and showed maximum expression after 0.5 or 2 h under heat stress. Transcript levels of ascorbate peroxidase (APX), dehydroascorbate reductase (DHAR) and Cu-Zn superoxide dismutase (Cu-Zn SOD) showed a rapid and marked accumulation upon heat stress. While prolonged exposure to heat stress resulted in increased transcript levels of monodehydroascorbate reductase, peroxidase, glyoxalase 1,glutathione reductase, thioredoxin peroxidase, 2-Cysteine peroxiredoxin, and nucleoside diphosphate kinase 1, while the transcription of catalase was suppressed. Consistent with their changes in gene expression, the enzyme activities of APX and DHAR also increased significantly following exposure to heat stress. These results suggest that oxidative stress is usually caused by heat stress, and plants apply complex HSP- and antioxidant-mediated defense mechanisms to cope with heat stress.
[Show abstract][Hide abstract] ABSTRACT: Miscanthus sinensis is a promising bioenergy crop; however, its genome is poorly represented in sequence databases. As an initial step in the comprehensive analysis of the M. sinensis proteome, we report a reference 2-DE protein map of the leaf. A total of 316 protein spots were excised from the gels, digested with trypsin and subjected to matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) or MALDI-TOF/TOF MS. Two hundred and thirty-two protein spots were identified, which are involved in a variety of cellular functions through distinct metabolic pathways. Functional annotation of the proteins revealed a nearly complete C3 and C4 cycle, starch and sugar synthesis pathway, glycolysis pathway, a significant portion of the pentose phosphate pathway, and many enzymes involved in secondary metabolism such as flavonoid/isoflavonoid, kaurene, chalcone, sesquiterpene and lignin biosynthesis. Other proteins belong to primary metabolism, transcription, protein synthesis, protein destination/storage, disease/defense, cell growth/division, transportation and signal transduction. To test the applicability of the constructed map, we studied the effect of heat stress on M. sinensis leaf proteome. Twenty-five protein spots were upregulated, five were newly induced and twenty-five spots were downregulated by heat treatment. The differentially accumulated proteins were involved in photosynthesis, energy metabolism, gene transcription, protein kinases and phosphatases, signal transduction, protein synthesis and heat shock responses. C4-specific pyruvate orthophosphate dikinase, Rubisco large subunit, Rubisco activase and some associated proteins were upregulated during heat stress and tend to restore upon recovery. Identification of these proteins provides some important clues regarding the way M. sinensis copes with hot climate. This work represents the first extensive proteomic description of M. sinensis and provides a reference map and heat-responsive candidates for future molecular and physiological studies of this bioenergy crop.
[Show abstract][Hide abstract] ABSTRACT: Zoysiagrass (Zoysia japonica Steud.) is an important forage and turfgrass that spreads by stolons and rhizomes. Zoysiagrass stolon can be used directly for Agrobacterium-mediated genetic transformation by exploiting the potential of direct shoot formation. However, surface sterilization of field-grown stolons is difficult and remains to be explored. We developed an effective surface sterilization and culture method using the stolon explant for infection with Agrobacterium tumefaciens. Among various treatments, sequential disinfection in 30% bleach for 15 min followed by 0.1% mercuric chloride for 25 min resulted in the highest number of clean stolons. The efficacy of mercuric chloride was increased under vacuum conditions by incubating at 800 mbar for 5 min. The inclusion of 2.5 mg/l amphotericin B further prevents fungal growth in in vitro cultures. This protocol would speed up the development of transgenic plants by utilizing field-grown stolon nodes.
Journal of The Korean Society of Grassland and Forage Science. 01/2013; 33(2).
[Show abstract][Hide abstract] ABSTRACT: This study compares the differences in proteomes expressed in tuberous roots of a light orange-fleshed sweetpotato (Ipomoea batatas (L.) Lam. cultivar Yulmi) and a purple-fleshed sweetpotato cultivar (Shinjami). More than 370 protein spots were reproducibly detected by two-dimensional gel electrophoresis, in which 35 spots were up-regulated (Yulmi vs. Shinjami) or uniquely expressed (only Yulmi or Shinjami) in either of the two cultivars. Of these 35 protein spots, 23 were expressed in Yulmi and 12 were expressed in Shinjami. These protein spots were analyzed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry and electrospray ionization tandem mass spectrometry. Fifteen proteins in Yulmi and eight proteins in Shinjami were identified from the up-regulated (Yulmi vs. Shinjami) or uniquely expressed (only Yulmi or Shinjami) proteins, respectively. In Yulmi, α-amylase and isomerase precursor-like protein were uniquely expressed or up-regulated and activities of α-amylase, monodehydroascorbate reductase, and dehydroascorbate reductase were higher than in Shinjami. In Shinjami, peroxidase precursor and aldo-keto reductase were uniquely expressed or up-regulated and peroxidase and aldo-keto reductase activities were higher than in Yulmi. PSG-RGH7 uniquely expressed only in Shinjami and the cultivar was evaluated more resistant than Yulmi against the root-knot nematode, Meloidogyne incognita (Kofold and White, 1919) Chitwood 1949 on the basis of shoot and root growth. Egg mass formation was 14.9-fold less in Shinjami than in Yulmi. These results provide important clues that can provide a foundation for sweetpotato proteomics and lead to the characterization of the physiological function of differentially expressed proteins.
[Show abstract][Hide abstract] ABSTRACT: The gene expression profiles of hybrid poplar (Populus alba × Populus tremula var. glandulosa) cells in suspension culture after exposure to salinity (NaCl) induced stress were examined by constructing two suppression subtractive hybridization (SSH) libraries. cDNA from non-treated cells was used as a driver and cDNA samples from cell suspension cultures exposed to 150 mM NaCl for 2 or 10 h were used as testers. Randomly selected clones from each SSH library were sequenced and 727 high-quality expressed sequence tags (ESTs) were obtained and analyzed. Four novel ESTs were identified. Between the two libraries, 542 unique SSH clones were selected for placement on a cDNA microarray. In total, 18 differentially expressed genes were identified with 4 and 12 genes being significantly differentially expressed 2 and 10 h after the treatment, respectively. Genes related to metabolism and protein synthesis and several genes whose protein products are implicated in salt or other abiotic stress-related responses were expressed in the salt-stressed cells.
Plant Physiology and Biochemistry 05/2012; 58:151-8. · 2.78 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Despite the widespread occurrence of chromium toxicity, its molecular mechanism is poorly documented in plants compared to other heavy metals. To investigate the molecular mechanisms that regulate the response of Miscanthus sinensis roots to elevated level of chromium, seedlings were grown for 4 weeks and exposed to potassium dichromate for 3 days. Physiological, biochemical and proteomic changes in roots were investigated. Lipid peroxidation and H₂O₂ content in roots were significantly increased. Protein profiles analyzed by two-dimensional gel electrophoresis revealed that 36 protein spots were differentially expressed in chromium-treated root samples. Of these, 13 protein spots were up-regulated, 21 protein spots were down-regulated and 2 spots were newly induced. These differentially displayed proteins were identified by MALDI-TOF and MALDI-TOF/TOF mass spectrometry. The identified proteins included known heavy metal-inducible proteins such as carbohydrate and nitrogen metabolism, molecular chaperone proteins and novel proteins such as inositol monophosphatase, nitrate reductase, adenine phosphoribosyl transferase, formate dehydrogenase and a putative dihydrolipoamide dehydrogenase that were not known previously as chromium-responsive. Taken together, these results suggest that Cr toxicity is linked to heavy metal tolerance and senescence pathways, and associated with altered vacuole sequestration, nitrogen metabolism and lipid peroxidation in Miscanthus roots.
[Show abstract][Hide abstract] ABSTRACT: Small heat shock proteins are involved in stress tolerance. We previously isolated and characterized a rice cDNA clone, Oshsp26, encoding a chloroplast-localized small heat shock protein that is expressed following oxidative or heat stress. In this study, we transferred this gene to tall fescue plants by an Agrobacterium-mediated transformation system. The integration and expression of the transgene was confirmed by PCR, Southern, northern, and immunoblot analyzes. Compared to the control plants, the transgenic plants had significantly lower electrolyte leakage and accumulation of thiobarbituric acid-reactive substances when exposed to heat or methyl viologen. The photochemical efficiency of photosystem II (PSII) (Fv/Fm) in the transgenic tall fescue plants was higher than that in the control plants during heat stress (42°C). These results suggest that the OsHSP26 protein plays an important role in the protection of PSII during heat and oxidative stress in vivo.
[Show abstract][Hide abstract] ABSTRACT: The cloning and characterization of a gene (MsHSP23) coding for a heat shock protein in alfalfa in a prokaryotic and model plant system is described. MsHSP23 contains a 633 bp ORF encoding a polypeptide of 213 amino acids and exhibits greater sequence similarity to mitochondrial sHSPs from dicotyledons than to those from monocotyledons. When expressed in bacteria, recombinant MsHSP23 conferred tolerance to salinity and arsenic stress. Furthermore, MsHSP23 was cloned in a plant expressing vector and transformed into tobacco, a eukaryotic model organism. The transgenic plants exhibited enhanced tolerance to salinity and arsenic stress under ex vitro conditions. In comparison to wild type plants, the transgenic plants exhibited significantly lower electrolyte leakage. Moreover, the transgenic plants had superior germination rates when placed on medium containing arsenic. Taken together, these overexpression results imply that MsHSP23 plays an important role in salinity and arsenic stress tolerance in transgenic tobacco. This approach could be useful to develop stress tolerant crops including forage crops.
[Show abstract][Hide abstract] ABSTRACT: Dehydration-responsive element-binding (DREB) proteins are important transcription factors in plant stress responses and signal transduction. Based on high-throughput sequencing results, a new cDNA sequence encoding an LcDREB3a transcription factor from the drought-resistant forage grass, Leymus chinensis, was isolated by RACE PCR. Sequence similarity analysis indicates that the gene product is active in the ABA-responsive pathway, and real-time PCR-based expression analysis shows the transcript accumulates in response to a variety of stress treatments. These results indicate that LcDREB3a is involved in both ABA-dependent and -independent signal transduction in the stress-responsive process of L. chinensis. The identity of the gene product as a DREB transcription factor is supported by observations of its nuclear localization when transiently expressed as a GFP fusion in onion epidermal cells. Furthermore, LcDREB3a is able to activate reporter gene expression, and the protein is shown to specifically bind to the conserved DRE element in a yeast one-hybrid assay. The transgenic expression of LcDREB3a in Arabidopsis causes no growth retardation and induces the increased expression of stress tolerance genes compared to control, resulting in improved drought and salt stress tolerance. Thus, LcDREB3a, encoding a stress-inducible DREB transcription factor, could enhance the abiotic stress tolerance of plants.
[Show abstract][Hide abstract] ABSTRACT: The present study was conducted to determine the optimum in vitro culture condition for callus induction and plant regeneration from mature seeds of bermudagrass (Cynodon dactylon cv. Common). It was revealed that mature seeds cultured on MS medium supplemented with 2 mg/L 2,4-D, 0.5 g/L proline, 0.5 g/L casamino acid and 3 g/L Gelrite under light condition produced the highest percentage of callus formation (39.2%). The most suitable medium for plant regeneration from dehydrated calli was MS agar medium supplemented with 0.5 mg/L 2,4-D, 2 mg/L BA, 0.5 g/L proline, 0.5 g/L casamino acid 3 g/L Gelrite which induced the highest percentage of calli forming shoots (57.7%). The frequency of callus induction and plant regeneration were the highest on sucrose, followed by maltose. The shoots were rooted at the highest rate (100%) when transferred onto 1/2 MS medium. Regenerated plants were morphologically uniform with normal growth pattern.
Journal of The Korean Society of Grassland and Forage Science. 01/2011; 31(3).
[Show abstract][Hide abstract] ABSTRACT: The present study was conducted to determine the optimum in vitro conditions for callus induction and plant regeneration from mature seed derived callus of four cultivars of Timothy. In order to investigate the effects of genetic variations of timothy in tissue culture, calli were induced from mature seeds of four varieties, 'Colt', 'Chair', 'Richmond' and 'Hokuo' and plant regeneration frequency was compared. Significant differences were observed among the cultivars in both callus induction and plant regeneration. Genotype 'Colt' consistently performed best in the callus subculture and plant regeneration. The complete plantlets were thereafter transplanted to grow under greenhouse condition. Regenerated timothy plants were morphologically uniform with normal leaf and growth pattern.
Journal of The Korean Society of Grassland and Forage Science. 01/2011; 31(3).
[Show abstract][Hide abstract] ABSTRACT: In order to investigate rice stem proteome in response to heat stress, rice plants were subjected to heat treatment at 42 and total soluble proteins were extracted from stem tissues, and were fractionated with 15% PEG (poly ethylene glycol) and separated by two-dimensional polyacrylamide gel electrophoresis (2-DE). After staining of 2-DE gels, 46 of differentially expressed proteins were extracted, digested by trypsin, and subjected to matrix assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis. Proteins were identified through database search by using peptide mass fingerprints. Among them, 10 proteins were successfully identified. Seven proteins were up- and 3 proteins were down-regulated, respectively. These proteins are involved in energy and metabolism, redox homeostasis, and mitochondrial small heat shock proteins. The identification of some novel proteins in the heat stress response provides new insights that can lead to a better understanding of the molecular basis of heat-sensitivity in plants, and also useful to molecular breeding of thermotolerant forage crops.
Journal of The Korean Society of Grassland and Forage Science. 01/2011; 31(2).
[Show abstract][Hide abstract] ABSTRACT: In order to improve forage characteristics of Italian ryegrass by genetic transformation, an efficient callus induction from mature seed and optimal plant regeneration system were established using a domestic cultivar 'Kospeed'. Addition of 5 mg/L of 2,4-D showed highest frequency of embryogenic callus induction from mature seeds. N6 medium showed higher frequency of both callus induction and plant regeneration as compared with MS and SH medium. The highest plant regeneration frequency 67% was obtained when embryogenic calli were transferred to N6 medium containing 1 mg/L 2,4-D and 5 mg/L BA. Supplementation of regeneration medium with sucrose at 30 g/L level maximized regeneration frequency as compared to the other concentrations. These data would be very helpful for molecular breeding of domestic Italian ryegrass cultivar through genetic transformation.
Journal of The Korean Society of Grassland and Forage Science. 01/2011; 31(3).
[Show abstract][Hide abstract] ABSTRACT: Herbicide-resistant creeping bentgrass plants (Agrostis stolonifera L.) without antibiotic-resistant markers were produced by Agrobacterium-mediated transformation. Embryogenic callus tissues were infected with Agrobacterium tumefaciens EHA105, harboring the bar and the CP4-EPSPS genes for bialaphos and glyphosate resistance. Phosphinothricin-resistant calli and plants were selected. Soil-grown plants were obtained at 14-16 weeks after transformation. Genetic transformation of the selected, regenerated plants was validated by PCR. Southern blot analysis revealed that at least one copy of the transgene was integrated into the genome of the transgenic plants. Transgene expression was confirmed by Northern blot. CP4-EPSPS protein was detected by ELISA. Transgenic plants remained green and healthy when sprayed with Basta, containing 0.5% glufosinate ammonium or glyphosate. The optimized Agrobacterium-mediated transformation method resulted in an average of 9.4% transgenic plants. The results of the present study suggest that the optimized marker-free technique could be used as an effective and reliable method for routine transformation, which may facilitate the development of varieties of new antibiotic-free grass species.
[Show abstract][Hide abstract] ABSTRACT: Salinity together with waterlogging or flooding, a condition that occurs frequently in the field, can cause severe damage
to crops. Combined flooding and salinity decreases the growth and survival of plants more than either stress alone. We report
here the first proteomic analysis to investigate the global effects of saline flooding on multiple metabolic pathways. Soybean
seedlings at the emergence (VE) stage were treated with 100mM NaCl and flooded with water or 100mM sodium chloride solution
for 2days. Proteins were extracted from hypocotyl and root samples and analyzed by two-dimensional gel electrophoresis followed
by MALDI-TOF, MALDI-TOF/TOF mass spectrometry or immunoblotting. A total of 43 reproducibly resolved, differentially expressed
protein spots visualized by Coomassie brilliant blue staining were identified by MALDI-TOF MS. Identities of several proteins
were also validated by MS/MS analysis or immunoblot analysis. Twenty-nine proteins were upregulated, eight proteins were downregulated
and six spots were newly induced. The identified proteins include well-known salt and flooding induced proteins as well as
novel proteins expressed by the salinity-flooding combined stress. The comparative analysis identified changes at the proteome
level that are both specific and part of a common or shared response. The identification of such differentially expressed
proteins provides new targets for future studies that will allow assessment of their physiological roles and significance
in the response of glycophytes to a combination of flooding and salinity.
KeywordsAbiotic stress–Combined stress–Flooding–Proteome–Salinity–Soybean
Plant and Soil 01/2011; 346(1):45-62. · 3.24 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In order to develop an efficient, reliable and reproducible tissue culture system for reed (Phragmites communis Trinius), an efficient plant regeneration system via callus induction was established using mature seeds as explants. MS medium containing 1 mg/L 2,4-D and 0.5 mg/L BA was optimal for callus induction from mature seeds. The highest frequency (88.7%) of callus formation was obtained in 1.0 mg/L 2,4-D. The highest plant regeneration frequency (59.6%) was found when the embryogenic calli were subcultured on MS medium supplemented with 100 mg/L myo-inositol, whereas, adding of plant growth regulators was not so promising in this case. Our result would be useful for development of transgenic reed plants.
Journal of The Korean Society of Grassland and Forage Science. 01/2011; 31(3).
[Show abstract][Hide abstract] ABSTRACT: To gain better insight into how soybean roots respond to waterlogging stress, we carried out proteomic profiling combined with physiological analysis at two time points for soybean seedlings in their early vegetative stage. Seedlings at the V2 stage were subjected to 3 and 7 days of waterlogging treatments. Waterlogging stress resulted in a gradual increase of lipid peroxidation and in vivo H2O2 level in roots. Total proteins were extracted from root samples and separated by two-dimensional gel electrophoresis (2-DE). A total of 24 reproducibly resolved, differentially expressed protein spots visualized by Coomassie brilliant blue (CBB) staining were identified by matrix assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry or electrospray ionization tandem mass spectrometry (ESI-MS/MS) analysis. Of these, 14 proteins were upregulated; 5 proteins were decreased; and 5 were newly induced in waterlogged roots. The identified proteins include well-known classical anaerobically induced proteins as well as novel waterlogging-responsive proteins that were not known previously as being waterlogging responsive. The novel proteins are involved in several processes, i.e. signal transduction, programmed cell death, RNA processing, redox homeostasis and metabolisms of energy. An increase in abundance of several typical anaerobically induced proteins, such as glycolysis and fermentation pathway enzymes, suggests that plants meet energy requirement via the fermentation pathway due to lack of oxygen. Additionally, the impact of waterlogging on the several programmed cell death- and signal transduction-related proteins suggest that they have a role to play during stress. RNA gel blot analysis for three programmed cell death-related genes also revealed a differential mRNA level but did not correlate well with the protein level. These results demonstrate that the soybean plant can cope with waterlogging through the management of carbohydrate consumption and by regulating programmed cell death. The identification of novel proteins such as a translation initiation factor, apyrase, auxin-amidohydrolase and coproporphyrinogen oxidase in response to waterlogging stress may provide new insight into the molecular basis of the waterlogging-stress response of soybean.
Journal of Biosciences 03/2010; 35(1):49-62. · 1.76 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Plant 2-Cys peroxiredoxins (2-Cys Prx) has both peroxidase and chaperon function. We overexpressed an Arabidopsis 2-Cys Prx in transgenic tall fescue (Festuca arundinacea) plants to confer tolerance against heat and methyl viologen (MV) stress. Transgenic plants were generated by Agrobacterium-mediated genetic transformation, and integration and expression of the transgene was confirmed by Southern, northern and
western blot analyses. Compared to control plants, transgenic plants had significantly less electrolyte leakage and thiobarbituric
acid-reactive substances (TBARS) when exposed to heat or MV. Under heat stress (42°C), transgenic plants maintained their
chlorophyll fluorescence (Fv/Fm) for 24 h while control plants lost chlorophyll fluorescence very quickly. We conclude that the high levels of 2-Cys Prx
proteins in transgenic plants protect leaves from oxidative damage probably due to chaperon activity.