Comparative proteomic analysis of canola leaves under salinity stress

Although canola is a moderately salt‐tolerant species, its growth, seed yield, and oil production are markedly reduced under salt stress, particularly during the early vegetative growth stage. To identify the mechanisms of salt responsiveness in canola, the proteins expressed in the second and third newly developed leaves of salt‐tolerant, Hyola 308, and salt‐sensitive, Sarigol, cultivars were analyzed. Plants were exposed to 0, 175, and 350 mM NaCl during the vegetative stage. An increase in the Na content and a reduction in growth were observed in the third leaves compared to the second leaves. The accumulation of Na was more pronounced in the salt‐sensitive compared with the salt‐tolerant genotype. Out of 900 protein spots detected on 2‐DE gels, 44 and 31 proteins were differentially expressed in the tolerant and susceptible genotypes, respectively. Cluster analysis based on the expression level of total and responsive proteins indicated that the second leaves had a discriminator role between the two genotypes at both salinity levels. Using MS analysis, 46 proteins could be identified including proteins involved in responses to oxidative stress, energy production, electron transport, translation, and photosynthesis. Our results suggest that these proteins might play roles in canola adaptation to salt stress.     

Proteomic Analysis of Seedling Roots of Two Maize Inbred Lines That Differ Significantly in the Salt Stress Response

Salinity is a major abiotic stress that limits plant productivity and quality throughout the world. Roots are the sites of salt uptake. To better understand salt stress responses in maize, we performed a comparative proteomic analysis of seedling roots from the salt-tolerant genotype F63 and the salt-sensitive genotype F35 under 160 mM NaCl treatment for 2 days. Under salinity conditions, the shoot fresh weight and relative water content were significantly higher in F63 than in F35, while the osmotic potential was significantly lower and the reduction of the K⁺/Na⁺ ratio was significantly less pronounced in F63 than in F35. Using an iTRAQ approach, twenty-eight proteins showed more than 2.0- fold changes in abundance and were regarded as salt-responsive proteins. Among them, twenty-two were specifically regulated in F63 but remained constant in F35. These proteins were mainly involved in signal processing, water conservation, protein synthesis and biotic cross-tolerance, and could be the major contributors to the tolerant genotype of F63. Functional analysis of a salt-responsive protein was performed in yeast as a case study to confirm the salt-related functions of detected proteins. Taken together, the results of this study may be helpful for further elucidating salt tolerance mechanisms in maize.     

The proteome response of salt-resistant and salt-sensitive barley genotypes to long-term salinity stress

Responses of plants to salinity stress and the development of salt tolerance are extremely complex. Proteomics is a powerful technique to identify proteins associated with a particular environmental or developmental signal. We employed a proteomic approach to further understand the mechanism of plant responses to salinity in a salt-tolerant (Afzal) and a salt-sensitive (Line 527) genotype of barley. At the 4-leaf stage, plants were exposed to 0 (control) or 300 mM NaCl. Salt treatment was maintained for 3 weeks. Total proteins of leaf 4 were extracted and separated by two-dimensional gel electrophoresis. More than 500 protein spots were reproducibly detected. Of these, 44 spots showed significant changes to salt treatment compared to the control: 43 spots were upregulated and 1 spot was downregulated. Using MALDI-TOF-TOF MS, we identified 44 cellular proteins have been identified, which represented 18 different proteins and were classified into seven categories and a group with unknown biological function. These proteins were involved in various many cellular functions. Up regulation of proteins which involved in reactive oxygen species scavenging, signal transduction, protein processing and cell wall may increase plant adaptation to salt stress. The upregulation of the three of four antioxidant proteins (thioredoxin, methionine sulfoxide reductase and dehydroascorbate reductase) in susceptible genotype Line 527 suggesting a different tolerance mechanism (such as tissue tolerance) to tolerate a salinity condition in comparison with the salt sensitive genotype.     

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.     

Regulation of protein synthesis in root, shoot and embryonic tissues of germinating barley during salinity stress

Abstract Protein synthesis during seed germination, a stage vulnerable to salinity stress, was investigated. The responses of barley genotypes, CM72 (California Mariout 72) and Prato, toward salinity were different during seed germination. Germination of CM72 was unaffected up to 0.34 kmol m^?3 (2%) NaCl, but that of Prato was reduced 30% by 0.17 kmol m ³ NaCl and 75% by 0.34 kmol m^?3 NaCl. Therefore, the former genotype is relatively more salt-tolerant than the latter. Protein synthesis in roots, shoots, and embryos was investigated in these two genotypes before and after salinity stress. The uptake of S-methionine and its incorporation into protein were significantly reduced by salinity in both genotypes. The inhibition of global protein synthesis was significant in roots and shoots. Proteins from different tissues were resolved by single and two dimensional gels. The steady-state protein levels were maintained remarkably well during salinity stress in roots and shoots. Likewise, proteins in germinating embryos were stable except for a 42-kilodalton protein unique to the salt tolerant genotype which was apparently degraded during salinity stress. Salinity, around 0.34 kmol m^?3 NaCl, induced both quantitative and qualitative changes in the expression of some proteins labelled in vivo. The quantitative changes included repression or enhancement of synthesis of selected groups of proteins. Around 8% of the nearly 400 resolved proteins in a tissue was affected this way. Some of the proteins in this category were specific to each genotype. About 1 % of the total showed qualitative changes; these proteins were expressed only during salinity stress. In roots, two proteins (28, 41.7 kilodaltons) were detected in CM72 and five (28, 45, 60.5, 76.5, 82.5 kilodaltons) in Prato; only the 28-kilodalton protein was common to both genotypes. In shoots, four proteins (45, 60.5, 76.5, 82.5 kilodaltons) were found only in Prato and these were similar to those induced in roots. The four new proteins (32, 37.5, 89, 92 kilodaltons) in germinating embryos were apparently induced only in CM72; these were distinctly different from those detected in developed roots and shoots. The unique protein changes induced by salinity stress during germination (this study) and seedling growth studies reported earlier (Ramagopal, 1987b) are apparently different. The findings demonstrate that ontogeny plays an important role in the expression of tissue-specific proteins during salinity stress in the salt tolerant and sensitive barley genotypes.     

Improving salinity tolerance in crop plants: a biotechnological view

Salinity limits the production capabilities of agricultural soils in large areas of the world. Both breeding and screening germplasm for salt tolerance encounter the following limitations: (a) different phenotypic responses of plants at different growth stages, (b) different physiological mechanisms, (c) complicated genotype × environment interactions, and (d) variability of the salt-affected field in its chemical and physical soil composition. Plant molecular and physiological traits provide the bases for efficient germplasm screening procedures through traditional breeding, molecular breeding, and transgenic approaches. However, the quantitative nature of salinity stress tolerance and the problems associated with developing appropriate and replicable testing environments make it difficult to distinguish salt-tolerant lines from sensitive lines. In order to develop more efficient screening procedures for germplasm evaluation and improvement of salt tolerance, implementation of a rapid and reliable screening procedure is essential. Field selection for salinity tolerance is a laborious task; therefore, plant breeders are seeking reliable ways to assess the salt tolerance of plant germplasm. Salt tolerance in several plant species may operate at the cellular level, and glycophytes are believed to have special cellular mechanisms for salt tolerance. Ion exclusion, ion sequestration, osmotic adjustment, macromolecule protection, and membrane transport system adaptation to saline environments are important strategies that may confer salt tolerance to plants. Cell and tissue culture techniques have been used to obtain salt tolerant plants employing two in vitro culture approaches. The first approach is selection of mutant cell lines from cultured cells and plant regeneration from such cells (somaclones). In vitro screening of plant germplasm for salt tolerance is the second approach, and a successful employment of this method in durum wheat is presented here. Doubled haploid lines derived from pollen culture of F₁ hybrids of salt-tolerant parents are promising tools to further improve salt tolerance of plant cultivars. Enhancement of resistance against both hyper-osmotic stress and ion toxicity may also be achieved via molecular breeding of salt-tolerant plants using either molecular markers or genetic engineering.     

Antioxidant gene-enzyme responses in Medicago truncatula genotypes with different degree of sensitivity to salinity

Antioxidant responses and nodule function of Medicago truncatula genotypes differing in salt tolerance were studied. Salinity effects on nodules were analysed on key nitrogen fixation proteins such as nitrogenase and leghaemoglobin as well as estimating lipid peroxidation levels, and were found more dramatic in the salt-sensitive genotype. Antioxidant enzyme assays for catalase (CAT, EC 1.11.1.6), superoxide dismutase (EC 1.15.1.1), ascorbate peroxidase (EC 1.11.1.11) and guaiacol peroxidase (EC 1.11.1.7) were analysed in nodules, roots and leaves treated with increasing concentrations of NaCl for 24 and 48 h. Symbiosis tolerance level, depending essentially on plant genotype, was closely correlated with differences of enzyme activities, which increased in response to salt stress in nodules (except CAT) and roots, whereas a complex pattern was observed in leaves. Gene expression responses were generally correlated with enzymatic activities in 24-h treated roots in all genotypes. This correlation was lost after 48 h of treatment for the sensitive and the reference genotypes, but it remained positively significant for the tolerant one that manifested a high induction for all tested genes after 48 h of treatment. Indeed, tolerance behaviour could be related to the induction of antioxidant genes in plant roots, leading to more efficient enzyme stimulation and protection. High induction of CAT gene was also distinct in roots of the tolerant genotype and merits further consideration. Thus, part of the salinity tolerance in M. truncatula is related to induction and sustained expression of highly regulated antioxidant mechanisms.     

Alternative oxidase 1 (Aox1) gene expression in roots of Medicago truncatula is a genotype-specific component of salt stress tolerance

Alternative oxidase (AOX) is the central component of the non-phosphorylating alternative respiratory pathway in plants and may be important for mitochondrial function during environmental stresses. Recently it has been proposed that Aox can be used as a functional marker for breeding stress tolerant plant varieties. This requires characterization of Aox alleles in plants with different degree of tolerance in a certain stress, affecting plant phenotype in a recognizable way. In this study we examined Aox1 gene expression levels in Medicago truncatula genotypes differing in salt stress tolerance, in order to uncover any correlation between Aox expression and tolerance to salt stress. Results demonstrated a specific induction of Aox1 gene expression in roots of the tolerant genotype that presented the lowest modulation in phenotypic and biochemical stress indices such as morphologic changes, protein level, lipid peroxidation and ROS generation. Similarly, in a previous study we reported that induction of antioxidant gene expression in the tolerant genotype contributed to the support of the antioxidant cellular machinery and stress tolerance. Correlation between expression patterns of the two groups of genes was revealed mainly in 48 h treated roots. Taken together, results from both experiments suggest that M. truncatula tolerance to salt stress may in part due to an efficient control of oxidative balance thanks to (i) induction of antioxidant systems and (ii) involvement of the AOX pathway. This reinforces the conclusion that differences in antioxidant mechanisms can be essential for salt stress tolerance in M. truncatula and possibly the corresponding genes, especially Aox, could be utilized as functional marker.     

Salt tolerance of barley: Relations between expression of isoforms of vacuolar Na<Superscript>+</Superscript>/H<Superscript>+</Superscript>-antiporter and <Superscript>22</Superscript>Na<Superscript>+</Superscript> accumulation

Two barley cultivars (Hordeum vulgare L., cvs. Elo and Belogorskii) differing in salt tolerance were used to study ²²Na⁺ uptake, expression of three isoforms of the Na⁺/H⁺ antiporter HvNHX1-3, and the cellular localization of these isoforms in the elongation zone of seedling roots. During short (1 h) incubation, seedling roots of both cultivars accumulated approximately equal quantities of ²²Na⁺. However, after 24-h incubation the content of ²²Na⁺ in roots of a salt-tolerant variety Elo was 40% lower than in roots of the susceptible variety Belogorskii. The content of ²²Na⁺ accumulated in shoots of cv. Elo after 24-h incubation was 6.5 times lower than in shoots of cv. Belogorskii and it was 4 times lower after the salt stress treatment. The cytochemical examination revealed that three proteins HvNHX1-3 are co-localized in the same cells of almost all root tissues; these proteins were present in the tonoplast and prevacuolar vesicles. Western blot analysis of HvNHX1-3 has shown that the content of isoforms in vacuolar membranes increased in response to salt stress in seedling roots and shoots of both cultivars, although the increase was more pronounced in the tolerant cultivar. The content of HvNHX1 in the seedlings increased in parallel with the enhanced expression of HvNHX1, whereas the increase in HvNHX2 and HvNHX3 protein content was accompanied by only slight changes in expression of respective genes. The results provide evidence that salt tolerance of barley depends on plant ability to restrict Na⁺ transport from the root to the shoot and relies on regulatory pathways of HvNHX1-3 expression in roots and shoots during salt stress.     

Comparative proteomic analysis of seedling leaves of different salt tolerant soybean genotypes

Salinity is one of the major environmental constraints limiting yield of crop plants in many semi-arid and arid regions around the world. To understand responses in soybean seedling to salt stress at proteomic level, the extracted proteins from seedling leaves of salt-sensitive genotype Jackson and salt-tolerant genotype Lee 68 under 150 mM NaCl stress for 1, 12, 72 and 144 h, respectively, were analyzed by 2-DE. Approximately 800 protein spots were detected on 2-DE gels. Among them, 91 were found to be differently expressed, with 78 being successfully identified by MALDI-TOF-TOF. The identified proteins were involved in 14 metabolic pathways and cellular processes. Based on most of the 78 salt-responsive proteins, a salt stress-responsive protein network was proposed. This network consisted of several functional components, including balancing between ROS production and scavenging, accelerated proteolysis and reduced biosynthesis of proteins, impaired photosynthesis, abundant energy supply and enhanced biosynthesis of ethylene. Salt-tolerant genotype Lee 68 possessed the ability of higher ROS scavenging, more abundant energy supply and ethylene production, and stronger photosynthesis than salt-sensitive genotype Jackson under salt stress, which may be the major reasons why it is more salt-tolerant than Jackson.     

Phenolic content and antioxidant activity in two contrasting Medicago ciliaris lines cultivated under salt stress

The objective of this study was to determine more indepth physiological and antioxidant responses in two Medicago ciliaris lines (a salt-tolerant line TNC 1.8 and a salt-sensitive line TNC 11.9) with contrasting responses to 100 mM NaCl. Under salt stress, both lines showed a decrease in total biomass and in the growth rate for roots, but TNC 1.8 was less affected by salt than TNC 11.9 in that it maintained leaf growth even in the presence of added salt. In both lines, salt stress mainly affected micronutrient status (Fe, Mn, Cu and Zn) rather than K nutrition, but the tolerant line TNC 1.8 accumulated more Na in leaves and less in roots compared with TNC 11.9. Salt stress decreased total soluble sugars (TSS) in all organs of the sensitive line TNC 11.9, whereas TSS was only reduced in roots of the tolerant line. The salt-induced drop in growth was linked to an increase in lipid peroxidation in roots of both lines and in leaves of the sensitive line. The salt-tolerant line TNC 1.8 was more efficient at managing salt-induced oxidative damage in leaves and to a lesser extent in roots than the salt-sensitive line TNC 11.9, by preserving higher phenolic compound and superoxide dismutase levels in both organs.     

Response of Contrasting Rice Genotypes to Zinc Sources under Saline Conditions

Abiotic stresses are among the major limiting factors for plant growth and crop productivity. Among these, salinity is one of the major risk factors for plant growth and development in arid to semi-arid regions. Cultivation of salt tolerant crop genotypes is one of the imperative approaches to meet the food demand for increasing population. The current experiment was carried out to access the performance of different rice genotypes under salinity stress and Zinc (Zn) sources. Four rice genotypes were grown in a pot experiment and were exposed to salinity stress (7 dS m⁻¹), and Zn (15 mg kg⁻¹ soil) was applied from two sources, ZnSO₄ and Zn-EDTA. A control of both salinity and Zn was kept for comparison. Results showed that based on the biomass accumulation and K⁺/Na⁺ ratio, KSK-133 and BAS-198 emerged as salt tolerant and salt sensitive, respectively. Similarly, based on the Zn concentration, BAS-2000 was reported as Zn-in-efficient while IR-6 was a Zn-efficient genotype. Our results also revealed that plant growth, relative water content (RWC), physiological attributes including chlorophyll contents, ionic concentrations in straw and grains of all rice genotypes were decreased under salinity stress. However, salt tolerant and Zn-in-efficient rice genotypes showed significantly higher shoot K⁺ and Zn concentrations under saline conditions. Zinc application significantly alleviates the harmful effects of salinity by improving morpho-physiological attributes and enhancing antioxidant enzyme activities, and the uptake of K and Zn. The beneficial effect of Zn was more pronounced in salt-tolerant and Zn in-efficient rice genotypes as compared with salt-sensitive and Zn-efficient genotypes. In sum, our results confirmed that Zn application increased overall plant’s performance under saline conditions, particularly in Zn in-efficient and tolerant genotypes as compared with salt-sensitive and Zn efficient rice genotypes.     

Proteomic analysis of salt stress and recovery in leaves of Vigna unguiculata cultivars differing in salt tolerance

Key message

Cowpea cultivars differing in salt tolerance reveal differences in protein profiles and adopt different strategies to overcome salt stress. Salt-tolerant cultivar shows induction of proteins related to photosynthesis and energy metabolism.

Abstract

Salinity is a major abiotic stress affecting plant cultivation and productivity. The objective of this study was to examine differential proteomic responses to salt stress in leaves of the cowpea cultivars Pitiúba (salt tolerant) and TVu 2331 (salt sensitive). Plants of both cultivars were subjected to salt stress (75 mM NaCl) followed by a recovery period of 5 days. Proteins extracted from leaves of both cultivars were analyzed by two-dimensional electrophoresis (2-DE) under salt stress and after recovery. In total, 22 proteins differentially regulated by both salt and recovery were identified by LC–ESI–MS/MS. Our current proteome data revealed that cowpea cultivars adopted different strategies to overcome salt stress. For the salt-tolerant cultivar (Pitiúba), increase in abundance of proteins involved in photosynthesis and energy metabolism, such as rubisco activase, ribulose-5-phosphate kinase (Ru5PK) (EC 2.7.1.19), glycine decarboxylase (EC 1.4.4.2) and oxygen-evolving enhancer (OEE) protein 2, was observed. However, these vital metabolic processes were more profoundly affected in salt-sensitive cultivar (TVu), as indicated by the down-regulation of OEE protein 1, Mn-stabilizing protein-II, carbonic anhydrase (EC 4.2.1.1) and Rubisco (EC 4.1.1.39), leading to energy reduction and a decline in plant growth. Other proteins differentially regulated in both cultivars corresponded to different physiological responses. Overall, our results provide information that could lead to a better understanding of the molecular basis of salt tolerance and sensitivity in cowpea plants.     

Salt and genotype impact on plant physiology and root proteome variations in tomato

To evaluate the genotypic variation of salt stress response in tomato, physiological analyses and a proteomic approach have been conducted in parallel on four contrasting tomato genotypes. After a 14 d period of salt stress in hydroponic conditions, the genotypes exhibited different responses in terms of plant growth, particularly root growth, foliar accumulation of Na(+), and foliar K/Na ratio. As a whole, Levovil appeared to be the most tolerant genotype while Cervil was the most sensitive one. Roma and Supermarmande exhibited intermediary behaviours. Among the 1300 protein spots reproducibly detected by two-dimensional electrophoresis, 90 exhibited significant abundance variations between samples and were submitted to mass spectrometry for identification. A common set of proteins (nine spots), up- or down-regulated by salt-stress whatever the genotype, was detected. But the impact of the tomato genotype on the proteome variations was much higher than the salt effect: 33 spots that were not variable with salt stress varied with the genotype. The remaining number of variable spots (48) exhibited combined effects of the genotype and the salt factors, putatively linked to the degrees of genotype tolerance. The carbon metabolism and energy-related proteins were mainly up-regulated by salt stress and exhibited most-tolerant versus most-sensitive abundance variations. Unexpectedly, some antioxidant and defence proteins were also down-regulated, while some proteins putatively involved in osmoprotectant synthesis and cell wall reinforcement were up-regulated by salt stress mainly in tolerant genotypes. The results showed the effect of 14 d stress on the tomato root proteome and underlined significant genotype differences, suggesting the importance of making use of genetic variability.     

Cloning and functional analysis of wheat V-H+-ATPase subunit genes

The root microsomal proteomes of salt-tolerant and salt-sensitive wheat lines under salt stress were analyzed by two-dimensional electrophoresis and mass spectrum. A wheat V-H(+)-ATPase E subunit protein was obtained whose expression was enhanced by salt stress. In silicon cloning identified the full-length cDNA sequences of nine subunits and partial cDNA sequences of two subunits of wheat V-H(+)-ATPase. The expression profiles of these V-H(+)-ATPase subunits in roots and leaves of both salt-tolerant and salt-sensitive wheat lines under salt and abscisic acid (ABA) stress were analyzed. The results indicate that the coordinated enhancement of the expression of V-H(+)-ATPase subunits under salt and ABA stress is an important factor determining improved salt tolerance in wheat. The expression of these subunits was tissue-specific. Overexpression of the E subunit by transgenic Arabidopsis thaliana was able to enhance seed germination, root growth and adult seedling growth under salt stress.     

Remodeling of chloroplast proteome under salinity affects salt tolerance of <Emphasis Type="Italic">Festuca arundinacea</Emphasis>

Acclimation of photosynthetic apparatus to variable environmental conditions is an important component of tolerance to dehydration stresses, including salinity. The present study deals with the research on alterations in chloroplast proteome of the forage grasses. Based on chlorophyll fluorescence parameters, two genotypes of a model grass species—Festuca arundinacea with distinct levels of salinity tolerance: low salt tolerant (LST) and high salt tolerant (HST), were selected. Next, two-dimensional electrophoresis and mass spectrometry were applied under both control and salt stress conditions to identify proteins accumulated differentially between these two genotypes. The physiological analysis revealed that under NaCl treatment the studied plants differed in photosystem II activity, water content, and ion accumulation. The differentially accumulated proteins included ATPase B, ATP synthase, ribulose-1,5-bisphosphate carboxylase large and small subunits, cytochrome b6-f complex iron-sulfur subunit, oxygen-evolving enhancer proteins (OEE), OEE1 and OEE2, plastidic fructose-bisphosphate aldolase (pFBA), and lipocalin. A higher level of lipocalin, potentially involved in prevention of lipid peroxidation under stress, was also observed in the HST genotype. Our physiological and proteomic results performed for the first time on the species of forage grasses clearly showed that chloroplast metabolism adjustment could be a crucial factor in developing salinity tolerance.