Proteome analysis of wheat leaf under salt stress by two-dimensional difference gel electrophoresis (2D-DIGE)

Salt stress is a major abiotic stress that limits agricultural productivity in many regions of the world. To understand the molecular basis of the salt stress response in wheat (Triticum aestivum L.), a proteomic approach was used to identify the salt stress-responsive proteins in an elite Chinese wheat cultivar, Zhengmai 9023, which exhibits a high yield, superior gluten quality and better biotic resistance. Three-week-old seedlings were treated with NaCl of four different concentrations (1.0%, 1.5%, 2.0%, and 2.5%). The total proteins from the leaves of untreated and NaCl-treated plants were extracted and separated by two-dimensional difference gel electrophoresis (2D-DIGE). A total of 2358 protein spots were detected on the gels, among which 125 spots showed a significant change in protein abundance, and 83 differentially expressed spots were localised on preparative gels. Using Q-TOF mass spectrometry, 52 salt-responsive spots were identified, which were classified into six functional categories that included transport-associated proteins, detoxifying enzymes, ATP synthase, carbon metabolism, protein folding, and proteins with unknown biological functions. Of the 52 differentially expressed proteins, 26 were up-regulated, 21 were down-regulated, and five spots showed multi-expression patterns. In particular, some important proteins for salt tolerance were found to be up-regulated in Zhengmai 9023 under salt stress, such as H⁺-ATPases, glutathione S-transferase, ferritin and triosephosphate isomerase.     

Proteome analysis of soybean leaves,hypocotyls and roots under salt stress

Background  

Salinity is one of the most widespread agricultural problems in arid and semi-arid regions that makes fields unproductive, and soil salinization is a serious problem in the entire world. To determine the effects of salt stress on soybean seedlings, a proteomic technique was used.     

Proteome analysis of potato under salt stress

Because salt stress is a major abiotic source of stress on potato crops, the molecular mechanism of the response of potato plants to salt stress was examined. On exposure to salt, the salt-sensitive cultivar Concord showed a greater reduction in shoot and root length than did the salt-tolerant cultivar Kennebec. For both cultivars, the reduction in the length of shoots was more severe than that of the roots. Salt exposure increased the content of free proline and total soluble sugars in shoots of Kennebec; these remained unchanged in Concord. Proteins extracted from shoots of both cultivars exposed to 90 mM NaCl were separated by two-dimensional polyacrylamide gel electrophoresis: 322 and 305 proteins were detected in shoots of Kennebec and Concord, respectively. Of these, 47 proteins were differentially expressed under NaCl treatment in shoot of both cultivars. Among the differentially expressed proteins, photosynthesis- and protein-synthesis-related proteins were drastically down-regulated, whereas osmotine-like proteins, TSI-1 protein, heat-shock proteins, protein inhibitors, calreticulin, and five novel proteins were markedly up-regulated. These results suggest that up-regulation of defense-associated proteins may confer relative salt tolerance to potato plants.     

Proteomic analysis of Tunisian grapevine cultivar Razegui under salt stress

Salt stress is one of the major abiotic stresses in agriculture worldwide, especially in the Mediterranean area. We report here a proteomic approach to investigate the salt stress-responsive proteins in grapevine (Vitis vinifera). Two-dimensional electrophoresis (2-DE) was used to analyze the proteome of the salt-tolerant Tunisian grapevine cultivar Razegui, subjected to a supply of 100mm NaCl over 15d. Analysis of 2-DE gels derived from stressed plants revealed more than 800 reproducibly detected protein spots, with 48 proteins displaying a differential expression pattern, including 32 up-regulated, 9 down-regulated and 7 new protein spots induced after salt treatment. The presence of stress-responsive proteins in the different plant organs suggests that salt spreads systemically. Edman degradation analysis and database searching aided us in identifying a major protein GP. Database analysis revealed that this peptide has a 98% sequence similarity with a pathogenesis-related (PR) protein 10 (V. vinifera). A full-length cDNA encoding the GP protein was isolated from grapevine salt-stressed leaves and sequenced. The predicted protein contained 158 amino acids and showed 98% identity with PR10 protein of of V. vinifera (accession no. Cac16165).     

Jasmonate mediates salt induced nicotine biosynthesis in tobacco (Nicotiana tabacum L.)

Jasmonate (JA), as an important signal, plays a key role in multiple processes of plant growth, deve lopment and stress response. Nicotine and related pyridine alkaloids in tobacco (Nicotiana tabacum L.) are essential secondary metabolites. Whether environmental factors control nicotine biosynthesis and the underlying mechanism remains previously unreported. Here, we applied physiological and biochemical approaches to investigate how salt stress affects nicotine biosynthesis in tobacco. We found that salt stress induced the biosynthesis of JA, which subsequently triggered the activation of JA responsive gene expression and, ultimately, nicotine synthesis. Bioinformatics analysis revealed the existence of many NtMYC2a recognized G box motifs in the promoter regions of NtLOX, NtAOS, NtAOC and NtOPR genes. Applying exogenous JA increased nicotine content, while suppressing JA biosynthesis reduced nicotine biosynthesis. Salt treatment could not efficiently induce nicotine biosynthesis in transgenic anti COI1 tobacco plants. These results demonstrate that JA acts as the essential signal which triggers nicotine biosynthesis in tobacco after salt stress.     

The physiological response of Artemisia annua L. to salt stress and salicylic acid treatment

Salinity has a great influence on plant growth and distribution. A few existing reports on Artemisia annua L. response to salinity are concentrated on plant growth and artemisinin content; the physiological response and salt damage mitigation are yet to be understood. In this study, the physiological response of varying salt stresses (50, 100, 200, 300, or 400 mM NaCl) on A. annua L. and the effect of exogenous salicylic acid (0.05 or 0.1 mM) at 300-mM salt stress were investigated. Plant growth, antioxidant enzyme activity, proline, and mineral element level were determined. In general, increasing salt concentration significantly reduced plant growth. Superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) were stimulated by salt treatment to a higher enzyme activity in treated plants than those in untreated plants. Content of proline had a visible range of increment in the salt-treated plants. Distribution of mineral elements was in inconformity: Na⁺ and Ca²⁺ were mainly accumulated in the roots; K⁺ and Mg²⁺ were concentrated in leaves and stems, respectively. Alleviation of growth arrest was observed with exogenous applications of salicylic acid (SA) under salt stress conditions. The activity of SOD and POD was notably enhanced by SA, but the CAT action was suppressed. While exogenous SA had no discernible effect on proline content, it effectively inhibited excessive Na⁺ absorption and promoted Mg²⁺ absorption. Ca²⁺ and K⁺ contents showed a slight reduction when supplemented with SA. Overall, the positive effect of SA towards resistance to the salinity of A. annua will provide some practical basis for A. annua cultivation.     

Proteome analysis of sugar beet leaves under drought stress

Drought is one of the major factors limiting the yield of sugar beet (Beta vulgaris L.). The identification of candidate genes for marker-assisted selection (MAS) could greatly improve the efficiency of breeding for increased drought tolerance. Drought-induced changes in the proteome could highlight important genes. Two genotypes of sugar beet (7112 and 7219-P.69) differing in genetic background were cultivated in the field. A line-source sprinkler irrigation system was used to apply irrigated and water deficit treatments beginning at the four-leaf stage. At 157 days after sowing, leaf samples were collected from well-watered and drought-stressed plants for protein extraction and to measure shoot biomass and leaf relative water content. Changes induced in leaf proteins were studied by two-dimensional gel electrophoresis and quantitatively analyzed using image analysis software. Out of more than 500 protein spots reproducibly detected and analyzed, 79 spots showed significant changes under drought. Some proteins showed genotype-specific patterns of up- or downregulation in response to drought. Twenty protein spots were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS), leading to identification of Rubisco and 11 other proteins involved in redox regulation, oxidative stress, signal transduction, and chaperone activities. Some of these proteins could contribute a physiological advantage under drought, making them potential targets for MAS.     

干旱胁迫条件下马铃薯耐旱品种宁蒗182叶片蛋白质组学分析

开展马铃薯抗旱分子机理的研究对培育马铃薯抗旱品种, 减少干旱造成的损失至关重要。文章利用双向电泳技术对云南地方耐旱马铃薯品种宁蒗182在干旱与正常处理条件下叶片表达差异蛋白质组进行对比研究。经电泳图谱分析和MALDI-TOF-TOF/MS质谱鉴定获得12个表达差异蛋白点, 并进行了功能分类。结果发现, 在差异蛋白中具有保护马铃薯光和系统以及线粒体正常运转的酶类; 调节该植株对环境胁迫响应的信号传导以及调控其组织内N、C运输系统的功能蛋白, 这些蛋白在受到干旱胁迫时表达量均升高。这一结果揭示出该类蛋白是马铃薯在干旱条件下产生的耐受相关蛋白。文章为阐释马铃薯抗旱品种通过多种路径和水平的调控提高其抗性的分子机理提供了理论依据。     

Effects of putrescine and ethephon on some oxidative stress enzyme activities and proline content in salt stressed spinach leaves

The effects of putrescine and ethephon on peroxidase (POD; EC 1.11.1.7), polyphenol oxidase (PPO; EC 1.14.18.1), catalase (CAT; EC 1.11.1.6) activities and proline content in spinach leaves under saline stress were investigated. In control conditions, putrescine increased PPO and CAT activities and proline content, but decreased POD activity. Ethephon increased these three enzyme activities but did not affect proline content. In saline conditions, putrescine increased POD and CAT activities and proline content, while it decreased PPO activity. Ethephon increased both PPO and CAT activities and proline content, but decreased POD activity. Putrescine and ethephon have opposite effects on the enzyme activities and proline accumulation because they acts as antagonists.     

Proteome analysis of soybean hypocotyl and root under salt stress

To evaluate the response of soybean to salt stress, the related changes in protein expression were investigated using the proteomic approach. Soybean plants were exposed to 0, 50, 100, and 200 mM NaCl. Especially at 200 mM, the length and fresh weight of the hypocotyl and root reduced under salt stress, while the proline content increased. Proteins from the hypocotyl and root treated with 100 mM NaCl were extracted and separated by two-dimensional polyacrylamide gel electrophoresis; 321 protein spots were detected. In response to salt stress, seven proteins were reproducibly found to be up- or down-regulated by two to sevenfold: late embryogenesis-abundant protein, beta-conglycinin, elicitor peptide three precursor, and basic/helix-loop-helix protein were up-regulated, while protease inhibitor, lectin, and stem 31-kDa glycoprotein precursor were down-regulated. These results indicate that salinity can change the expression level of some special proteins in the hypocotyl and root of soybean that may in turn play a role in the adaptation to saline conditions.     

Responses of Nipa palm (Nypa fruticans) seedlings,a mangrove species,to salt stress in pot culture

Nipa palm (Nypa fruticans) is the only palm that grows in mangrove vegetation. We investigated the effect of salt stress on the growth and physiology of 6-month-old seedlings of Nipa palm exposed to different degrees of salt stress (as NaCl) in pot culture. The overall growth performance of Nipa palm was unaffected by mild salt stress (8.9−16.6 dS m⁻¹), whereas seedlings grown under severe salt stress (EC = 57.2 dS m⁻¹) had lower chlorophyll content and fluorescence, reduced net photosynthesis and transpiration, which resulted in reduced growth of the plants. Na⁺ contents in leaf, petiole, and root tissues increased considerably under salt stress, depending upon the NaCl levels in the soil solution. Under salt-stress K⁺ content declined, whereas Ca²⁺ content increased somewhat, in parallel to Na⁺. Free proline accumulated in plants growing under high salt stress (EC = 57.2 dS m⁻¹). In contrast, soluble sugars were enriched under intermediate levels of salt stress (EC = 16.6 dS m⁻¹). The results obtained in the present study suggest that, based on ecophysiological data, N. fruticans is a species best adapted to grow in mangrove coastal areas with moderate only salt load, and circumscribing quite well the actual areas of occurrence of this palm in the gradient from seawater habitats to inland sites. © 2014 Elsevier GmbH     

Hypermethylation of tobacco heterochromatic loci in response to osmotic stress

 Plants have to cope with a number of envi-ronmental stresses which may potentially induce genetic and epigenetic changes and thus contribute to genome variability. In the present study we inspected the DNA methylation status of two heterochromatic loci (defined with repetitive DNA sequences HRS60 and GRS) in a tobacco cell culture exposed to osmotic stress. Investigations were performed on a TBY-2 cell suspension culture, and the stress was elicited with NaCl or D-mannitol. Using the restriction enzymes MspI/HpaII and MboI/Sau3AI in combination with Southern hydridization we observed a reversible hypermethylation of the external cytosine at the CpCpG trinucleotides in cells grown under mild osmotic stress equal to a NaCl concentration of 10 g/l. There were no changes in the methylation of the internal cytosine as the CpG dinucleotides within the CCGG motifs (HpaII sites) appeared to be fully methylated in tobacco DNA repetitive sequences under normal physiological conditions. The data suggest epigenetic changes in the plant genome based on de novo methylation of DNA in response to environmental stress. Received: 26 November 1996/Accepted: 20 December 1996     

Exogenous proline mitigates the detrimental effects of salt stress more than exogenous betaine by increasing antioxidant enzyme activities

Proline and betaine accumulate in plant cells under environmental stresses including salt stress. Here, we investigated effects of proline and betaine on the growth and activities of antioxidant enzymes in tobacco Bright Yellow-2 (BY-2) culture cells in suspension under salt stress. Both proline and betaine mitigated the inhibition of growth of BY-2 cells under salt stress and the mitigating effect of proline was more than that of betaine. Salt stress significantly decreased the activities of superoxide dismutase (SOD), catalase and peroxidase in BY-2 cells. Exogenous application of proline or betaine alleviated the reduction in catalase and peroxidase activities but not SOD activity under salt stress. In addition, proline was found to be effective in alleviating the inhibition of salt stress-induced catalase and peroxidase activities in BY-2 cells. Neither proline nor betaine directly scavenged superoxide (O(2)(-)) or hydrogen peroxide (H(2)O(2)). It is concluded that exogenous proline mitigates the detrimental effects of salt stress more than exogenous betaine because of its superior ability to increase the activities of antioxidant enzymes.     

Comparative proteomic analysis of early salt stress responsive proteins in roots and leaves of rice

Growth and productivity of rice (Oryza sativa L.) are severely affected by salinity. Understanding the mechanisms that protect rice and other important cereal crops from salt stress will help in the development of salt‐stress‐tolerant strains. In this study, rice seedlings of the same genetic species with various salt tolerances were studied. We first used 2DE to resolve the expressed proteome in rice roots and leaves and then used nanospray liquid chromatography/tandem mass spectrometry to identify the differentially expressed proteins in rice seedlings after salt treatment. The 2DE assays revealed that there were 104 differentially expressed protein spots in rice roots and 59 in leaves. Then, we identified 83 proteins in rice roots and 61 proteins in rice leaves by MS analysis. Functional classification analysis revealed that the differentially expressed proteins from roots could be classified into 18 functional categories while those from leaves could be classified into 11 functional categories. The proteins from rice seedlings that most significantly contributed to a protective effect against increased salinity were cysteine synthase, adenosine triphosphate synthase, quercetin 3‐O‐methyltransferase 1, and lipoxygenase 2. Further analysis demonstrated that the primary mechanisms underlying the ability of rice seedlings to tolerate salt stress were glycolysis, purine metabolism, and photosynthesis. Thus, we suggest that differentially expressed proteins may serve as marker group for the salt tolerance of rice.     

Genome-wide analysis of alternative splicing of pre-mRNA under salt stress in Arabidopsis

Background

Alternative splicing (AS) of precursor mRNA (pre-mRNA) is an important gene regulation process that potentially regulates many physiological processes in plants, including the response to abiotic stresses such as salt stress.

Results

To analyze global changes in AS under salt stress, we obtained high-coverage (~200 times) RNA sequencing data from Arabidopsis thaliana seedlings that were treated with different concentrations of NaCl. We detected that ~49% of all intron-containing genes were alternatively spliced under salt stress, 10% of which experienced significant differential alternative splicing (DAS). Furthermore, AS increased significantly under salt stress compared with under unstressed conditions. We demonstrated that most DAS genes were not differentially regulated by salt stress, suggesting that AS may represent an independent layer of gene regulation in response to stress. Our analysis of functional categories suggested that DAS genes were associated with specific functional pathways, such as the pathways for the responses to stresses and RNA splicing. We revealed that serine/arginine-rich (SR) splicing factors were frequently and specifically regulated in AS under salt stresses, suggesting a complex loop in AS regulation for stress adaptation. We also showed that alternative splicing site selection (SS) occurred most frequently at 4 nucleotides upstream or downstream of the dominant sites and that exon skipping tended to link with alternative SS.

Conclusions

Our study provided a comprehensive view of AS under salt stress and revealed novel insights into the potential roles of AS in plant response to salt stress.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-431) contains supplementary material, which is available to authorized users.     

Abscisic acid pretreatment enhances salt tolerance of rice seedlings: Proteomic evidence

Enhanced salt tolerance of rice seedlings by abscisic acid (ABA) pretreatment was observed from phenotypic and physiological analyses. Total proteins from rice roots treated with ABA plus subsequent salt stress were analyzed by using proteomics method. Results showed that, 40 protein spots were uniquely upregulated in the seedlings under the condition of ABA pretreatment plus subsequent salt stress, whereas only 16 under the condition of salt treatment. About 78% (31 spots) of the 40 protein spots were only upregulated in the presence of the subsequent salt stress, indicating that plants might have an economical strategy to prevent energy loss under a false alarm. The results also showed that more enzymes involved in energy metabolism, defense, primary metabolism, etc. were upregulated uniquely in ABA-pretreated rice seedlings, suggesting more abundant energy supply, more active anabolism (nitrogen, nucleotide acid, carbohydrate, etc), and more comprehensive defense systems in ABA-pretreated seedlings than in salt stressed ones.     

盐逆境下转基因耐盐小麦与其受体呼吸途径的动态变化

以转基因耐盐品种８９１２２和其受体陇春１３号两种小麦为实验材料,研究科幼苗在不同盐浓度胁迫下呼吸途径动态变化。结果表明,８９１２２出现盐呼吸明显迟于其受体;两听Ｖａｌｔ与ρＶａｌｔ变化并不同步,且Ｖａｌｔ均受高盐浓度的抑制,但低盐浓度能诱导其受体的Ｖａｌｔ;两品种的ρＶａｌｔ与ρ′Ｖｃｙｔ彼此协同调节适应盐境,且ρ′Ｖｃｙｔ仍是盐胁迫过程中线粒体电子传递的主要途径。同时讨论了盐逆境下抗氰呼吸的一些     

Genetic engineering of the biosynthesis of glycinebetaine leads to increased tolerance of photosynthesis to salt stress in transgenic tobacco plants

Genetically engineered tobacco (Nicotiana tabacum L.) with the ability to synthesis glycinebetaine (GB) in chloroplasts was established by introducing the BADH gene for betaine aldehyde dehydrogenase from spinach (Spinacia oleracea L.). The genetic engineering resulted in enhanced tolerance of growth of young seedlings to salt stress. This increased tolerance was not due to improved water status, since there were no significant differences in accumulation of sodium and chloride, leaf water potential, and relative water content between wild type and transgenic plants under salt stress. Salt stress resulted in a decrease in CO₂ assimilation and such a decrease was much greater in wild type plants than in transgenic plants. Though salt stress showed no damage to PSII, there were a decrease in the maximal PSII electron transport rate in vivo and an increase in non-photochemical quenching (NPQ) and these changes were greater in wild type plants than in transgenic plants. In addition, salt stress inhibited the activities of ribulose 1,5-bisphosphate carboxylase/oxygenase, chloroplastic fructose-1,6-bisphosphatase, fructose-1,6-bisphosphate aldolase, and phosphoribulokinase and such a decrease was also greater in wild type plants than in transgenic plants, suggesting that GB protects these enzymes against salt stress. However, there were no significant changes in the activities of phosphoglycerate kinase, triose phosphate isomerase, ribulose-5-phosphate isomerase, transketolase, and sedoheptulose-1,7-bisphosphatase in both wild type and transgenic plants. The results in this study suggest that enhanced tolerance of CO₂ assimilation to salt stress may be one of physiological bases for increased tolerance of growth of transgenic plants to salt stress.