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 H(2)O(2) 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 H(2)O(2) 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.     

铝胁迫下柱花草SSH文库构建及表达序列标签分析

柱花草栽培种热研2号(Stylosanthes guianensis ‘Reyan 2’)对铝毒有较强的耐受性。为了鉴定其在铝胁迫下的诱导 基因, 利用抑制消减杂交(SSH)技术构建在300 μmol·L^–1铝胁迫下正向cDNA文库。挑选插入片段大于300 bp的600个克隆进行测序, 共获得504条表达序列标签(EST)。序列重复性分析表明, 其中12.1%的EST只有1次重复, 61.4%的EST有2–16次重复, 重复出现次数较高的EST是细胞色素P450(53次, 占10.5%)、病原诱导型胰蛋白酶抑制剂(44次, 占8.7%)和衰老相关蛋白(37次, 占7.3%)。BLASTX分析显示, 504条EST中有97种非冗余基因, 其中包括46条功能已知基因和51条功能未知序列。46条功能已知EST中有30个为已报道铝胁迫相关基因, 16个是新发现的铝胁迫相关基因。SSH cDNA文库提供的信息为阐明柱花草耐铝毒的分子机制提供了重要线索。     

Proteomic analysis of the aging-related proteins in human normal colon epithelial tissue

In order to screen the aging related proteins in human normal colon epithelia, the comparative proteomics analysis was applied to get the two-dimensional electrophoresis (2-DE) profiles with high resolution and reproducibility from normal colon epithelial tissues of young and aged people. Differential proteins between the colon epithelia of two age groups were found with PDQuest software. The thirty five differential protein-spots were identified by peptide mass fingerprint (PMF) based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) and database searching. Among them there are sixteen proteins which are significantly up-regulated in the colonic mucosal epithelia of young people group, which include ATP synthase beta chain, electron transfer flavoprotein alpha-subunit, catalase, glutathione peroxidase 1, annexin A2 and heat shock cognate 71 kDa protein, etc.; There are nineteen proteins which are significantly up-regulated in the colonic mucosal epithelia of aged people group, which include far upstream element-binding protein 1, nucleoside diphosphate kinase B, protein disulfide-isomerase precursor and VDAC-2, etc.. The identified differential proteins appear to be involved in metabolism, energy generation, chaperone, antioxidation, signal transduction, protein folding and apoptosis. The data will help to understand the molecular mechanisms of human colon epithelial aging.     

Possible involvement of protein phosphorylation in aluminum-responsive malate efflux from wheat root apex

In many plants, efflux of organic anions from roots has been proposed as one of the major Al resistance mechanisms. However it remains unknown how plants regulate efflux of organic anions in response to Al. In this study, the regulatory mechanisms of Al-responsive malate efflux in wheat (Triticum aestivum) were characterized focusing on the role of protein phosphorylation. Al-resistant wheat (cv Atlas) initiated malate efflux at 5 min after addition of Al, and this response was sensitive to temperature. K-252a, a broad range inhibitor of protein kinases, effectively blocked the Al-induced malate efflux accompanied with an increased accumulation of Al and intensified Al-induced root growth inhibition. A transient activation of a 48-kD protein kinase and an irreversible repression of a 42-kD protein kinase were observed preceding the initiation of malate efflux, and these changes were canceled by K-252a. Malate efflux was accompanied with a rapid decrease in the contents of organic anions in the root apex, such as citrate, succinate, and malate but with no change in the contents of inorganic anions such as chloride, nitrate, and phosphate. These results suggest that protein phosphorylation is involved in the Al-responsive malate efflux in the wheat root apex and that the organic anion-specific channel might be a terminal target that responds to Al signaling mediated by phosphorylation.     

UV radiation-responsive proteins in rice leaves: a proteomic analysis

Depletion of stratospheric ozone has led to increased UV radiation reaching the surface of the Earth. This may damage plants. Using physiological, proteomic and quantitative real-time PCR (qPCR) methods, we systematically studied the response of 16-day-old rice seedlings to UV [0.67 W m(-2) biologically effective UVB (UVB(BE)) and 0.28 W m(-2) UVA] exposure for 6, 12 and 24 h. UV exposure resulted in the appearance of light brown patches on leaves, a decrease in the net photosynthetic rate (Pn), lipid peroxidation, accumulation of UV-absorbing compounds (including flavonoids and other phenolic pigments) and differential expression of 22 proteins. Both physiological and molecular responses became stronger with increasing UV exposure time, indicating the effects of UV accumulation on plants. UV-induced responses included (i) phytohormone-regulative responses (up-regulation of proteins related to phytohormone synthesis such as IAA and ethylene); (ii) injurious responses (photosynthesis suppression, lipid peroxidation and visible injury); and (iii) protective responses (accumulation of UV-absorbing compounds and differential expression of proteins involved in detoxification/antioxidation, defense, protein processing, RNA processing, carbohydrate metabolism and secondary metabolism). The identification of UV-responsive proteins provided a better understanding of the molecular mechanism of plant responses to UV stress. Proteomic and qPCR analysis identified one up-regulated and two induced proteins with important functions: tryptophan synthase α chain (production of radical oxygen species), glyoxalase I (detoxification/antioxidation) and a Bet v I family protein (defense). These results will contribute to future research into their roles in UV stress responses in plants.     

Proteomic analysis of rice seedlings during cold stress

Low temperature is one of the important environmental changes that affect plant growth and agricultural production. To investigate the responses of rice to cold stress, changes in protein expression were analyzed using a proteomic approach. Two-week-old rice seedlings were exposed to 5 degrees C for 48 h, then total crude proteins were extracted from leaf blades, leaf sheaths and roots, separated by 2-DE and stained with CBB. Of the 250-400 protein spots from each organ, 39 proteins changed in abundance after cold stress, with 19 proteins increasing, and 20 proteins decreasing. In leaf blades, it was difficult to detect the changes in stress-responsive proteins due to the presence of an abundant protein, ribulose bisphosphate carboxylase/oxygenase large subunit (RuBisCO LSU), which accounted for about 50% of the total proteins. To overcome this problem, an antibody-affinity column was prepared to trap RuBisCO LSU, and the remaining proteins in the flow through from the column were subsequently separated using 2-DE. As a result, slight changes in stress responsive proteins were clearly displayed, and four proteins were newly detected after cold stress. From identified proteins, it was concluded that proteins related to energy metabolism were up-regulated, and defense-related proteins were down-regulated in leaf blades, by cold stress. These results suggest that energy production is activated in the chilling environment; furthermore, stress-related proteins are rapidly up-regulated, while defense-related proteins disappear, under long-term cold stress.     

Expressed sequence tag-based gene expression analysis under aluminum stress in rye

To understand the mechanisms responsible for aluminum (Al) toxicity and tolerance in plants, an expressed sequence tag (EST) approach was used to analyze changes in gene expression in roots of rye (Secale cereale L. cv Blanco) under Al stress. Two cDNA libraries were constructed (Al stressed and unstressed), and a total of 1,194 and 774 ESTs were generated, respectively. The putative proteins encoded by these cDNAs were uncovered by Basic Local Alignment Search Tool searches, and those ESTs showing similarity to proteins of known function were classified according to 13 different functional categories. A total of 671 known function genes were used to analyze the gene expression patterns in rye cv Blanco root tips under Al stress. Many of the previously identified Al-responsive genes showed expression differences between the libraries within 6 h of Al stress. Certain genes were selected, and their expression profiles were studied during a 48-h period using northern analysis. A total of 13 novel genes involved in cell elongation and division (tonoplast aquaporin and ubiquitin-like protein SMT3), oxidative stress (glutathione peroxidase, glucose-6-phosphate-dehydrogenase, and ascorbate peroxidase), iron metabolism (iron deficiency-specific proteins IDS3a, IDS3b, and IDS1; S-adenosyl methionine synthase; and methionine synthase), and other cellular mechanisms (pathogenesis-related protein 1.2, heme oxygenase, and epoxide hydrolase) were demonstrated to be regulated by Al stress. These genes provide new insights about the response of Al-tolerant plants to toxic levels of Al.     

Local regulation of auxin transport in root-apex transition zone mediates aluminium-induced Arabidopsis root-growth inhibition

Aluminium (Al) stress is a major limiting factor for worldwide crop production in acid soils. In Arabidopsis thaliana, the TAA1-dependent local auxin biosynthesis in the root-apex transition zone (TZ), the major perception site for Al toxicity, is crucial for the Al-induced root-growth inhibition, while the mechanism underlying Al-regulated auxin accumulation in the TZ is not fully understood. In the present study, the role of auxin transport in Al-induced local auxin accumulation in the TZ and root-growth inhibition was investigated. Our results showed that PIN-FORMED (PIN) proteins such as PIN1, PIN3, PIN4 and PIN7 and AUX1/LAX proteins such as AUX1, LAX1 and LAX2 were all ectopically up-regulated in the root-apex TZ in response to Al stress and coordinately regulated local auxin accumulation in the TZ and root-growth inhibition. The ectopic up-regulation of PIN1 in the TZ under Al stress was regulated by both ethylene and auxin, with auxin signalling acting downstream of ethylene. Al-induced PIN1 up-regulation and auxin accumulation in the root-apex TZ was also regulated by the calossin-like protein BIG. Together, our results provide insight into how Al stress induces local auxin accumulation in the TZ and root-growth inhibition through the local regulation of auxin transport.     

Effect of early cold stress on the maturation of rice anthers

Male reproductive development in rice (Oryza sativa Linnaeus is very sensitive to various forms of environmental stresses including low temperature. Here, we present our findings on the proteomic analysis of the later developmental consequences of low temperature treatment on rice anthers. Anther proteins at the trinucleate stage, with or without cold treatment for four days at 12 degrees C at the young microspore stage, were extracted, separated by two-dimensional gel electrophoresis (2-DE) and compared. More than 3000 rice anther proteins of cold-sensitive cultivar Doongara plants at the trinucleate stage were resolved on 2-DE gels over a pH range of 4-7 and detected by silver-staining. Seventy protein spots were differentially displayed after four days of cold treatment at the young microspore stage. Of these, 12 protein spots were newly-induced, 47 were up-regulated, and 11 were down-regulated by cold treatment at the early microspore stage. We identified 18 by matrix-assisted laser desorption/ionization mass spectrometry time of flight (MALDI-TOF) analysis. Of the identified proteins, seven were observed as breakdown (cleavage) products by a combination of 2-DE and MALDI-TOF analysis, thus demonstrating for the first time that cold temperature stress at the young microspore stage enhances and induces partial degradation of proteins in the rice anthers at the trinucleate stage.     

Rice proteome analysis: a step toward functional analysis of the rice genome

The technique of proteome analysis using 2-DE has the power to monitor global changes that occur in the protein complement of tissues and subcellular compartments. In this review, we describe construction of the rice proteome database, the cataloging of rice proteins, and the functional characterization of some of the proteins identified. Initially, proteins extracted from various tissues and organelles were separated by 2-DE and an image analyzer was used to construct a display or reference map of the proteins. The rice proteome database currently contains 23 reference maps based on 2-DE of proteins from different rice tissues and subcellular compartments. These reference maps comprise 13 129 rice proteins, and the amino acid sequences of 5092 of these proteins are entered in the database. Major proteins involved in growth or stress responses have been identified by using a proteomics approach and some of these proteins have unique functions. Furthermore, initial work has also begun on analyzing the phosphoproteome and protein-protein interactions in rice. The information obtained from the rice proteome database will aid in the molecular cloning of rice genes and in predicting the function of unknown proteins.     

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 H₂O₂ 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.     

Recent progress in the research of external Al detoxification in higher plants: a minireview

Aluminum (Al) is highly toxic to plant growth. The toxicity is characterized by rapid inhibition of root elongation. However, some plant species and cultivars have evolved some mechanisms for detoxifying Al both internally and externally. In this review, the recent progress made in the research of external detoxification of Al is described. Accumulating evidence has shown that organic acids play an important role in the detoxification of Al. Some plant species and cultivars respond to Al by secreting citrate, malate or oxalate from the roots. Recently, the anion channel of malate and citrate in the plasma membrane has been characterized and a gene encoding the malate channel has been cloned. The metabolism of organic acids seems to be poorly correlated with the Al-induced secretion of organic acid anions. A number of QTLs (quantitative trait loci) for Al resistance have been identified in rice, Arabidopsis, and other species. Transgenic plants with enhanced resistance to Al have also been reported, but introduction of multiple genes may be required to gain high Al resistance in future.     

Al-induced cell wall hydroxyproline-rich glycoprotein accumulation is involved in alleviating Al toxicity in rice

Cell wall components such as pectin and hemicelluloses have been proposed to be involved in aluminum resistance mechanisms in plants. However, whether hydroxyproline-rich glycoproteins (HRGPs), one of the most abundant proteins of the cell walls, are involved in Al resistance mechanisms remains elusive. In this study, two rice cultivars Xiushui 03 (Al resistant) and Xiushui 128 (Al sensitive) significantly differing in Al resistance were identified. In the absence of Al, no significant difference was observed in contents of glycoproteins and hydroxyproline in cell wall fractions of these two cultivars. At the early stage of Al toxicity, glycoproteins and hydroxyproline were significantly induced in these two cultivars, but levels of their accumulation in cell walls were much higher in cv. Xiushui 03 than in cv. Xiushui 128. At the late stage of Al toxicity, their accumulation in cell walls dramatically decreased in cv. Xiushui 128 and, however, still kept a high level in cv. Xiushui 03. The finding that Al-induced changes of glycoproteins and hydroxyproline were completely consistent indicates that Al-induced glycoproteins are HRGPs. Further observation utilizing transmission electron microscope showed that HRGPs were greatly accumulated in cell walls leading to thickening of cell walls in cv. Xiushui 03, however, HRGPs and cell walls greatly decreased in cv. Xiushui 128. These data suggest that Al-induced HRGP accumulation in cell walls is involved in alleviating Al toxicity in rice.