Nagib Ahsan

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.

Recent developments in the application of proteomics to the analysis of plant responses to heavy metals

Pollution of soils by heavy metals is an ever‐growing problem throughout the world, and is the result of human activities as well as geochemical weathering of rocks and other environmental causes such as volcanic eruptions, acid rain and continental dusts. Plants everywhere are continuously exposed to metal‐contaminated soils. The uptake of heavy metals not only constrains crop yields, but can also be a major hazard to the health of humans and to the entire ecosystem. Although analysis of gene expression at the mRNA level has enhanced our understanding of the response of plants to heavy metals, many questions regarding the functional translated portions of plant genomes under metal stress remain unanswered. Proteomics offers a new platform for studying complex biological functions involving large numbers and networks of proteins, and can serve as a key tool for revealing the molecular mechanisms that are involved in interactions between toxic metals and plant species. This review focuses on recent developments in the applications of proteomics to the analysis of the responses of plants to heavy metals; such studies provide a deeper understanding of protein responses and the interactions among the possible pathways that are involved in detoxification of toxic metals in plant cells. In addition, the challenges faced by proteomics in understanding the responses of plants to toxic metal are discussed, and some possible future strategies for meeting these challenges are proposed.

Chilling stress-induced proteomic changes in rice roots.

Roots are highly sensitive organs in plants. To gain a better knowledge of the chilling stress responses of plants, it is imperative to analyze the tissue-specific proteome patterns under chilling stress. In the present study, two-dimensional gel electrophoresis (2-DE) coupled with mass spectrometry, has been adopted to investigate the protein expression patterns of rice roots in response to chilling stress. Rice seedlings were subjected to 10 degrees C and samples were collected 24 and 72h after treatment. To identify the low-abundant proteins in root tissues, samples were fractionated by 15% polyethylene glycol (PEG), separated by 2-DE, and visualized by silver or CBB staining. A total of 27 up-regulated proteins 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. Together with the previously identified cold-stress-responsive proteins, a group of novel proteins were identified including acetyl transferase, phosphogluconate dehydrogenase, NADP-specific isocitrate dehydrogenase, fructokinase, PrMC3, putative alpha-soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein, and glyoxalase 1. These proteins are involved in several cellular processes, including energy production and metabolism, vesicular trafficking, and detoxification. Gene expression at the mRNA level of some selected proteins revealed that transcription levels are not always concomitant to the translational level. Thus, investigation of root proteome expression and identification of some novel proteins could be useful in better understanding the molecular basis of chilling stress responses in plants.

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.

Soybean proteomics and its application to functional analysis.

Complete genome sequences, which are available for rice and Arabidopsis, provide insights into many fundamental aspects of plant biology; they do not, however, address some important aspects of legume biology. Legumes are important for maintenance of human health and as crops for sustainable agriculture. Two model species of legume, Lotus japonicus and Medicago truncatula, have been the focus of projects on genome sequencing and functional genomics. A project aimed at sequencing the genome of the agricultural legume soybean recently began, but functional genomics studies of this plant are in their infancy, and therefore proteomics approaches could be a powerful tool for functional analysis. In this review, we discuss the strengths and weaknesses of proteomics technologies in soybean biology and we examine the limitations of current techniques.

Tissue-Specific Defense and Thermo-Adaptive Mechanisms of Soybean Seedlings under Heat Stress Revealed by Proteomic Approach

A comparative proteomic approach was employed to explore tissue-specific protein expression patterns in soybean seedlings under heat stress. The changes in the protein expression profiles of soybean seedling leaves, stems, and roots were analyzed after exposure to high temperatures. A total of 54, 35, and 61 differentially expressed proteins were identified from heat-treated leaves, stems, and roots, respectively. Differentially expressed heat shock proteins (HSPs) and proteins involved in antioxidant defense were mostly up-regulated, whereas proteins associated with photosynthesis, secondary metabolism, and amino acid and protein biosynthesis were down-regulated in response to heat stress. A group of proteins, specifically low molecular weight HSPs and HSP70, were up-regulated and expressed in a similar manner in all tissues. Proteomic analysis indicated that the responses of HSP70, CPN-60 beta, and ChsHSP were tissue specific, and this observation was validated by immunoblot analysis. The heat-responsive sHSPs were not induced by other stresses such as cold and hydrogen peroxide. Taken together, these results suggest that to cope with heat stress soybean seedlings operate tissue-specific defenses and adaptive mechanisms, whereas a common defense mechanism associated with the induction of several HSPs was employed in all three tissues. In addition, tissue-specific proteins may play a crucial role in defending each type of tissues against thermal stress.

Ozone stress-induced proteomic changes in leaf total soluble and chloroplast proteins of soybean reveal that carbon allocation is involved in adaptation in the early developmental stage

Considerable soybean yield losses caused by ozone (O3) stress have been demonstrated by large‐scale meta‐analyses of free‐gas concentration enrichment systems. In this study, comparative proteomic approach was employed to explore the differential changes of proteins in O3 target structures such as leaf and chloroplasts of soybean seedlings. Acute O3 exposure (120 parts‐per‐billion) for 3 days did not cause any visible symptoms in developing leaves. However, higher amounts of ROS and lipid peroxidation indicated that severe oxidative burst occurred. Immunoblot analysis of O3‐induced known proteins revealed that proteins were modulated before symptoms became visible. Proteomic analysis identified a total of 20 and 32 differentially expressed proteins from O3‐treated leaf and chloroplast, respectively. Proteins associated with photosynthesis, including photosystem I/II and carbon assimilation decreased following exposure to O3. In contrast, proteins involved in antioxidant defense and carbon metabolism increased. The activity of enzymes involved in carbohydrate metabolism increased following exposure to O3, which is consistent with the decrease in starch and increase in sucrose concentrations. Taken together, these results suggest that carbon allocation is tightly programmed, and starch degradation probably feeds the tricarboxylic acid cycle while the photosynthesis pathway is severely affected during O3 stress.

The impact of atmospheric composition on plants: a case study of ozone and poplar.

Tropospheric ozone is the main atmospheric pollutant that causes damages to trees. The estimation of the threshold for ozone risk assessment depends on the evaluation of the means that this pollutant impacts the plant and, especially, the foliar organs. The available results show that, before any visible symptom appears, carbon assimilation and the underlying metabolic processes are decreased under chronic ozone exposure. By contrast, the catabolic pathways are enhanced, and contribute to the supply of sufficient reducing power necessary to feed the detoxification processes. Reactive oxygen species delivered during ozone exposure serve as toxic compounds and messengers for the signaling system. In this review, we show that the contribution of genomic tools (transcriptomics, proteomics, and metabolomics) for a better understanding of the mechanistic cellular responses to ozone largely relies on spectrometric measurements.

Differential responses of microsomal proteins and metabolites in two contrasting cadmium (Cd)-accumulating soybean cultivars under Cd stress

While there are significant genotypic differences in cadmium (Cd) uptake and distribution in soybean cultivars, little attention has been paid to the underlying molecular mechanisms. We adopted a comparative proteomic approach coupled with metabolite analysis to examine Cd uptake and translocation in two contrasting Cd-accumulating soybean cultivars, Enrei and Harosoy, which accumulate higher amount of Cd in the roots and aerial parts, respectively. Proteins extracted from the root microsomal fraction were evaluated by immunoblot analysis using different subcellular marker proteins. Analysis of control and Cd-exposed samples by two-dimensional gel electrophoresis coupled with mass spectrometry revealed a total of 13 and 11 differentially expressed proteins in the Enrei and Harosoy cultivars, respectively. Metabolome profiling identified a total of 32 metabolites, the expression of 18 of which was significantly altered in at least in one cultivar in response to Cd stress. Analysis of the combined proteomic and metabolomic results revealed that proteins and amino acids associate with Cd-chelating pathways are highly active in the Enrei cultivar. In addition, proteins associated with lignin biosynthesis are significantly upregulated in the Enrei cultivar under Cd stress. Our results indicate that in the Enrei cultivar, Cd-chelating agents may bind excess free Cd ion and that translocation of Cd from the roots to the aerial parts might be prevented by increased xylem lignification.