[Show abstract][Hide abstract] ABSTRACT: In order to reveal the aluminum (Al) stress tolerance mechanisms in alfalfa plant at low pH soil, a proteomic approach has been conducted. Alfalfa plants were exposed to Al stress for 5 days. The plant growth and total chlorophyll content are greatly affected by Al stress. The malondialdehyde (MDA) and H 2 O 2 contents were increased in a low amount but free proline and soluble sugar contents, and the DPPH-radical scavenging activity were highly increased. These results indicate that antioxidant activity (DPPH activity) and osmoprotectants (proline and sugar) may involve in ROS (H 2 O 2) homeostasis under Al stress. In proteomic analysis, over 500 protein spots were detected by 2-dimentional gel electrophoresis analysis. Total 17 Al stress-induced proteins were identified, of which 8 protein spots were up-regulated and 9 were down-regulated. The differential expression patterns of protein spots were selected and analyzed by the peptide mass fingerprinting (PMF) using MALDI-TOF MS analysis. Three protein spots corresponding to Rubisco were significantly down-regulated whereas peroxiredoxin and glutamine synthetase were up-regulated in response to Al stress. The different regulation patterns of identified proteins were involved in energy metabolism and antioxidant / ROS detoxification during Al stress in alfalfa. Taken together, these results provide new insight to understand the molecular mechanisms of alfalfa plant in terms of Al stress tolerance. (Key words : Alfalfa, Aluminum stress, Proteome)
[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: 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.
[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: Previously, we identified more than 1,500 genes that were induced by high salt stress in sheepgrass (Leymus chinensis, Gramineae: Triticeae) when comparing the changes in their transcription levels in response to high salt stress by next-generation sequencing. Among the identified genes, a gene of unknown function (designated as Leymus chinensis salt-induced 1, LcSAIN1) showed a high sequence identity to its homologs from wheat, Hordeum vulgare and Oryza sativa, but LcSAIN1 and its homologs produce hypothetical proteins with no conserved functional domains. The LcSAIN1 gene transcription was up-regulated by various stresses. The over-expression of LcSAIN1 in Arabidopsis and rice increased the greening rate of cotyledons, the fresh weight, root elongation, plant height, and the plant survival rate when compared with control plants and conferred a tolerance against salt stress. Subcellular localization analysis indicated that LcSAIN1 is localized predominantly in the nucleus. Our results show that the LcSAIN1 gene might play an important positive modulation role in increasing the expression of transcription factors (MYB2 and DREB2A) and functional genes (P5CS and RAB18) in transgenic plants under salt stress and that it augments stress tolerance through the accumulation of compatible solutes (proline and soluble sugar) and the alleviation of ROS changes. The LcSAIN1 gene could be a potential resource for engineering salinity tolerance in important crop species.
[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.
[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.
Electronic supplementary material
The online version of this article (doi:10.1007/s10529-011-0750-1) contains supplementary material, which is available to authorized users.
[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.
[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.