Iftekhar Alam

Comparative proteomic study of arsenic-induced differentially expressed proteins in rice roots reveals glutathione plays a central role during As stress

While the phytotoxic responses of arsenic (As) on plants have been studied extensively, based on physiological and biochemical aspects, very little is known about As stress‐elicited changes in plants at the proteome level. Hydroponically grown 2‐wk‐old rice seedlings were exposed to different doses of arsenate, and roots were collected after 4 days of treatment, as well as after a recovery period. To gain a comprehensive understanding of the precise mechanisms underlying As toxicity, metabolism, and the defense reactions in plants, a comparative proteomic analysis of rice roots has been conducted in combination with physiological and biochemical analyses. Arsenic treatment resulted in increases of As accumulation, lipid peroxidation, and in vivo H2O2 contents in roots. A total of 23 As‐regulated proteins including predicted and novel ones were identified using 2‐DE coupled with MS analyses. The expression levels of S‐adenosylmethionine synthetase (SAMS), GSTs, cysteine synthase (CS), GST‐tau, and tyrosine‐specific protein phosphatase proteins (TSPP) were markedly up‐regulated in response to arsenate, whereas treatment by H2O2 also regulated the levels of CS suggesting that its expression was certainly regulated by As or As‐induced oxidative stress. In addition, an omega domain containing GST was induced only by arsenate. However, it was not altered by treatment of arsenite, copper, or aluminum, suggesting that it may play a particular role in arsenate stress. Analysis of the total glutathione (GSH) content and enzymatic activity of glutathione reductase (GR) in rice roots during As stress revealed that their activities respond in a dose‐dependent manner of As. These results suggest that SAMS, CS, GSTs, and GR presumably work synchronously wherein GSH plays a central role in protecting cells against As stress.

Glyphosate-induced oxidative stress in rice leaves revealed by proteomic approach

Glyphosate is one of the most widely used herbicides in cereal-growing regions worldwide. In the present work, the protein expression profile of rice leaves exposed to glyphosate was analyzed in order to investigate the alternative effects of glyphosate on plants. Two-week-old rice leaves were subjected to glyphosate or a reactive oxygen species (ROS) inducing herbicide paraquat, and total soluble proteins were extracted and analyzed by two-dimensional gel electrophoresis (2-DE) coupled with matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry (MS) analysis. A total of 25 differentially expressed proteins were identified from the glyphosate treated sample, wherein 18 proteins were up-regulated and 7 proteins were down-regulated. These proteins had shown a parallel expression pattern in response to paraquat. Results from the 2-DE analysis, combined with immunoblotting, clearly revealed that ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subunit was significantly decreased by the treatment of both herbicides. An increased accumulation of antioxidant enzymes including ascorbate peroxidase, glutathione S-transferase, thioredoxin h-type, nucleoside diphosphate kinase 1, peroxiredoxin and a superoxide dismutase [Cu-Zn] chloroplast precursor in the glyphosate-treated sample suggests that a glyphosate treatment possibly generates oxidative stress in plants. Moreover, a gene expression analysis of five antioxidant enzymes by Northern blot confirmed their mRNA levels in the rice leaves. A histo-cytochemical investigation with DAB (3,3-diaminobenzidine) to localize H(2)O(2) and increases of the thiobarbituric acid reactive substances (TBARS) concentration revealed that the glyphosate application generates ROS, which resulted in the peroxidation and destruction of lipids in the rice leaves.

Analysis of arsenic stress-induced differentially expressed proteins in rice leaves by two-dimensional gel electrophoresis coupled with mass spectrometry.

In the present study, we have investigated the protein expression profile of rice leaves under arsenic (As) stress. Two-week-old rice seedlings were exposed to two concentrations of arsenate (50 or 100 microM), and leaf samples were collected 4d after treatment. To elucidate the As stress-induced differentially expressed proteins in rice leaves, proteins were extracted from the control and treated samples, separated by two-dimensional gel electrophoresis (2-DE), and visualized by staining with Coomassie Brilliant Blue (CBB). A total of 14 protein spots showed reproducible changes in expression of at least 1.5-fold when compared to the control and showed a similar expression pattern in both treatments. Of these 14 spots, 8 were up-regulated and 6 were down-regulated following exposure to As. These proteins were identified using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS). The increased expression of several proteins associated with energy production and metabolism suggests that higher energy is required for activation of the metabolic processes in leaves exposed to As. On the other hand, results from the 2-DE analysis, combined with immunoblotting, clearly revealed that the ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) large subunit was significantly decreased under As stress. Thus, the down-regulation of RuBisCO and chloroplast 29 kDa ribonucleoproteins might be the possible causes of the decreased photosynthesis rate under As stress.

Proteome analysis of soybean roots under waterlogging stress at an early vegetative stage

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.

Chromium-induced physiological and proteomic alterations in roots of Miscanthus sinensis.

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.

Improved drought and salt tolerance of Arabidopsis thaliana by transgenic expression of a novel DREB gene from Leymus chinensis.

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, Leymuschinensis, 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.

Overexpression of a chloroplast-localized small heat shock protein OsHSP26 confers enhanced tolerance against oxidative and heat stresses in tall fescue

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.

Mapping the leaf proteome of Miscanthus sinensis and its application to the identification of heat-responsive proteins.

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.

Agrobacterium‐mediated transformation of reed (Phragmites communis Trinius) using mature seed‐derived calli

Reed (Phragmites communis) is a potential bioenergy plant. We report on its first Agrobacterium‐mediated transformation using mature seed‐derived calli. The Agrobacterium strains LBA4404, EHA105, and GV3101, each harboring the binary vector pIG121Hm, were used to optimize T‐DNA delivery into the reed genome. Bacterial strain, cocultivation period and acetosyringone concentration significantly influenced the T‐DNA transfer. About 48% transient expression and 3.5% stable transformation were achieved when calli were infected with strain EHA105 for 10 min under 800 mbar negative pressure and cocultivated for 3 days in 200 μm acetosyringone containing medium. Putative transformants were selected in 25 mg l−1 hygromycin B. PCR, and Southern blot analysis confirmed the presence of the transgenes and their stable integration. Independent transgenic lines contained one to three copies of the transgene. Transgene expression was validated by RT‐PCR and GUS staining of stems and leaves.

Screening for salt-responsive proteins in two contrasting alfalfa cultivars using a comparative proteome approach

A comparative proteomic approach was carried out between two contrasting alfalfa cultivars, nonomu (NM-801; salt tolerant) and vernal (VN; salt intolerant) in terms of salt tolerance. Seedlings were subjected to salt stress (50 and 100 mM NaCl) for three days. Several physiological parameters (leaf water, chlorophyll, root Na(+), K(+), and Ca(2+)) and root proteome profile were analyzed. Comparison of physiological status revealed that NM-801 is more tolerant to salt than VN. Eighty three differentially expressed proteins were found on 2-DE maps, of which 50 were identified by MALDI-TOF or MALDI-TOF/TOF mass spectrometry. These proteins were involved in ion homeostasis, protein turnover and signaling, protein folding, cell wall components, carbohydrate and energy metabolism, reactive oxygen species regulation and detoxification, and purine and fatty acid metabolism. The comparative proteome analysis showed that 33 salt-responsive proteins were significantly changed in both cultivars, while 17 (14 in VN and 3 in NM-801) were cultivar-specific. Peroxidase, protein disulfide-isomerase, NAD synthetase, and isoflavone reductase were up-regulated significantly only in NM-801 in all salt concentrations. In addition, we identified novel proteins including NAD synthetase and biotin carboxylase-3 that were not reported previously as salt-responsive. Taken together, these results provide new insights of salt stress tolerance in alfalfa.