An integrative proteome analysis of different seedling organs in tolerant and sensitive wheat cultivars under drought stress and recovery

Roots, leaves, and intermediate sections between roots and leaves (ISRL) of wheat seedlings show different physiological functions at the protein level. We performed the first integrative proteomic analysis of different tissues of the drought‐tolerant wheat cultivar Hanxuan 10 (HX‐10) and drought‐sensitive cultivar Chinese Spring (CS) during a simulated drought and recovery. Differentially expressed proteins (DEPs) in the roots (122), ISRLs (146), and leaves (163) showed significant changes in expression in response to drought stress and recovery. Numerous DEPs associated with cell defense and detoxifications were significantly regulated in roots and ISRLs, while in leaves, DEPs related to photosynthesis showed significant changes in expression. A significantly larger number of DEPs related to stress defense were upregulated in HX‐10 than in CS. Expression of six HSPs potentially related to drought tolerance was significantly upregulated under drought conditions, and these proteins were involved in a complex protein–protein interaction network. Further phosphorylation analysis showed that the phosphorylation levels of HSP60, HSP90, and HOP were upregulated in HX‐10 under drought stress. We present an overview of metabolic pathways in wheat seedlings based on abscisic acid signaling and important protein expression patterns.     

Comparative proteomic analysis of salt response proteins in seedling roots of two wheat varieties

A comparative proteomic analysis was made of salt response in seedling roots of wheat cultivars Jing-411 (salt tolerant) and Chinese Spring (salt sensitive) subjected to a range of salt stress concentrations (0.5%, 1.5% and 2.5%) for 2 days. One hundred and ninety eight differentially expressed protein spots (DEPs) were located with at least two-fold differences in abundance on 2-DE maps, of which 144 were identified by MALDI-TOF-TOF MS. These proteins were involved primarily in carbon metabolism (31.9%), detoxification and defense (12.5%), chaperones (5.6%) and signal transduction (4.9%). Comparative analysis showed that 41 DEPs were salt responsive with significant expression changes in both varieties under salt stress, and 99 (52 in Jing-411 and 47 in Chinese Spring) were variety specific. Only 15 and 9 DEPs in Jing-411 and Chinese Spring, respectively, were up-regulated in abundance under all three salt concentrations. All dynamics of the DEPs were analyzed across all treatments. Some salt responsive DEPs, such as guanine nucleotide-binding protein subunit beta-like protein, RuBisCO large subunit-binding protein subunit alpha and pathogenesis related protein 10, were up-regulated significantly in Jing-411 under all salt concentrations, whereas they were down-regulated in salinity-stressed Chinese Spring.     

Differences in physiological and biochemical characteristics in response to single and combined drought and salinity stresses between wheat genotypes differing in salt tolerance

The combined drought and salinity stresses pose a serious challenge for crop production, but the physiological mechanisms behind the stresses responses in wheat remains poorly understood. Greenhouse pot experiment was performed to study differences in genotype response to the single and combined (D + S) stresses of drought (4% soil moisture, D) and salinity (100 mM NaCl, S) using two wheat genotypes: Jimai22 (salt tolerant) and Yangmai20 (salt‐sensitive). Results showed that salinity, drought and/or D + S severely reduces plant growth, biomass and net photosynthetic rate, with a greater effect observed in Yangmai20 than Jimai22. A notable improvement in water use efficiency (WUE) by 239, 77 and 103% under drought, salinity and D + S, respectively, was observed in Jimai22. Moreover, Jimai22 recorded higher root K⁺ concentration in drought and salinity stressed condition and shoot K⁺ under salinity alone than that of Yangmai20. Jimai22 showed lower increase in malondialdehyde (MDA) accumulation, but higher activities of superoxide dismutase (SOD, EC 1.15.1.1) and guaicol peroxidase (POD, EC 1.11.1.7), under single and combined stresses, and catalase (CAT, EC 1.11.1.6) and ascorbate peroxidase (APX, EC 1.11.1.11) under single stress. Our results suggest that high tolerance of Jimai22 in both drought and D + S stresses is closely associated with larger root length, higher Fv/Fm and less MDA contents and improved capacity of SOD and POD. Moreover, under drought Jimai22 tolerance is firmly related to higher root K⁺ concentration level and low level of Na⁺, high‐net photosynthetic rate and WUE as well as increased CAT and APX activities to scavenge reactive oxygen species.     

Salt‐Treated Roots of Oryza australiensis Seedlings are Enriched with Proteins Involved in Energetics and Transport

Salinity is a major constraint on rice productivity worldwide. However, mechanisms of salt tolerance in wild rice relatives are unknown. Root microsomal proteins are extracted from two Oryza australiensis accessions contrasting in salt tolerance. Whole roots of 2‐week‐old seedlings are treated with 80 mM NaCl for 30 days to induce salt stress. Proteins are quantified by tandem mass tags (TMT) and triple‐stage Mass Spectrometry. More than 200 differentially expressed proteins between the salt‐treated and control samples in the two accessions (p‐value <0.05) are found. Gene Ontology (GO) analysis shows that proteins categorized as “metabolic process,” “transport,” and “transmembrane transporter” are highly responsive to salt treatment. In particular, mitochondrial ATPases and SNARE proteins are more abundant in roots of the salt‐tolerant accession and responded strongly when roots are exposed to salinity. mRNA quantification validated the elevated protein abundances of a monosaccharide transporter and an antiporter observed in the salt‐tolerant genotype. The importance of the upregulated monosaccharide transporter and a VAMP‐like protein by measuring salinity responses of two yeast knockout mutants for genes homologous to those encoding these proteins in rice are confirmed. Potential new mechanisms of salt tolerance in rice, with implications for breeding of elite cultivars are also discussed.     

Quantitative Proteomics Analysis Identifies the Potential Mechanism Underlying Yellow-Green Leave Mutant in Wheat

Enhancing photosynthesis efficiency is considered as one of the most crucial targets during wheat breeding. However, the molecular basis underlying high photosynthesis efficiency is not well understood up to now. In this study, we investigated the protein expression profile of wheat Jimai5265yg mutant, which is a yellow-green mutant with chlorophylls b deficiency but high photosynthesis efficiency. Though TMT-labeling quantitative proteomics analysis, a total of 72 differential expressed proteins (DEPs) were obtained between the mutant and wild type (WT). GO analysis found that they significantly enriched in thylakoid membrane, pigment binding, magnesium chelatase activity and response to light intensity. KEGG analysis showed that they involved in photosynthesis-antenna protein as well as porphyrin and chlorophyll metabolism. Finally, 118 RNA editing events were found between mutant and WT genotype. The A to C editing in the 3-UTR of TraesCS6D02G401500 lead to its high expression in mutant through removing the inhibition of tae-miR9781, which might have vital role in regulating the yellow-green mutant. This study provided some useful clues about the molecular basis of Jimai5265yg mutant as well as chlorophylls metabolism in wheat.     

Regulation by ABA of osmotic-stress-induced changes in protein synthesis in tomato roots

Polypeptide synthesis and accumulation were examined in the roots of tomato seedlings exposed to a polyethylene glycol‐imposed water deficit stress. In these roots, the synthesis of a number of polypeptides was induced, while that of several others was enhanced or repressed. To examine the role played by abscisic acid (ABA) in co‐ordinating the accumulation of these proteins, water‐deficit‐stress‐responsive polypeptide synthesis was investigated in the roots of the ABA‐deficient mutant flacca. In the roots of this mutant, the ability to accumulate a complete set of water‐deficit‐stress‐responsive polypeptides was impaired, indicating that ABA is required for their synthesis. The role of ABA was further examined by exposing the roots of both genotypes to exogenous ABA, which, with one exception, elicited the accumulation of all water‐deficit‐stress‐responsive proteins. Polyethylene glycol‐induced polypeptide accumulation was accompanied by a 1·6‐fold increase in the level of endogenous ABA in the roots of wild‐type plants and a 5‐fold increase in the roots of flc. Thus, although the absolute level was lower than that of the wild‐type, flc has the capacity to accumulate ABA in its roots. When fluridone was used to prevent the biosynthesis of ABA, the accumulation of several water‐deficit‐stress‐responsive polypeptides was reduced further. The synthesis of polypeptides was also examined in the roots of salt‐treated seedlings. Salt altered the accumulation of several polypeptides, all of which were previously observed in water‐deficit‐stressed roots, indicating that their synthesis was the result of the osmotic component of the salt stress. However, the accumulation of these polypeptides was not impaired in flc roots, indicating that the role played by ABA in regulating their accumulation in salt‐and polyethylene glycol‐treated roots differs. As such, salt‐ and water‐deficit‐stress‐induced changes in gene expression may be effected by different mechanisms, at least at the level of polypeptide accumulation.     

iTRAQ‐based quantitative proteomic analysis reveals new metabolic pathways of wheat seedling growth under hydrogen peroxide stress

As an abundant ROS, hydrogen peroxide (H₂O₂) plays pivotal roles in plant growth and development. In this work, we conducted for the first time an iTRAQ‐based quantitative proteomic analysis of wheat seedling growth under different exogenous H₂O₂ treatments. The growth of seedlings and roots was significantly restrained by increased H₂O₂ concentration stress. Malondialdehyde, soluble sugar, and proline contents as well as peroxidase activity increased with increasing H₂O₂ levels. A total of 3 425 proteins were identified by iTRAQ, of which 157 showed differential expression and 44 were newly identified H₂O₂‐responsive proteins. H₂O₂‐responsive proteins were mainly involved in stress/defense/detoxification, signal transduction, and carbohydrate metabolism. It is clear that up‐regulated expression of signal transduction and stress/defence/detoxification‐related proteins under H₂O₂ stress, such as plasma membrane intrinsic protein 1, fasciclin‐like arabinogalactan protein, and superoxide dismutase, could contribute to H₂O₂ tolerance of wheat seedlings. Increased gluconeogenesis (phosphoenol‐pyruvate carboxykinase) and decreased pyruvate kinase proteins are potentially related to the higher H₂O₂ tolerance of wheat seedlings. A metabolic pathway of wheat seedling growth under H₂O₂ stress is presented.     

Salinity tolerance is related to cyanide‐resistant alternative respiration in Medicago truncatula under sudden severe stress

Salt respiration is defined as the increase of respiration under early salt stress. However, the response of respiration varies depending on the degree of salt tolerance and salt stress. It has been hypothesized that the activity of the alternative pathway may increase preventing over‐reduction of the ubiquinone pool in response to salinity, which in turn can increase respiration. Three genotypes of Medicago truncatula are reputed as differently responsive to salinity: TN1.11, A17 and TN6.18. We used the oxygen‐isotope fractionation technique to study the in vivo respiratory activities of the cytochrome oxidase pathway (COP) and the alternative oxidase pathway (AOP) in leaves and roots of these genotypes treated with severe salt stress (300 mM) during 1 and 3 days. In parallel, AOX capacity, gas exchange measurements, relative water content and metabolomics were determined in control and treated plants. Our study shows for first time that salt respiration is induced by the triggered AOP in response to salinity. Moreover, this phenomenon coincides with increased levels of metabolites such as amino and organic acids, and is shown to be related with higher photosynthetic rate and water content in TN6.18.     

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.     

Global Analysis of Differentially Expressed Genes and Proteins in the Wheat Callus Infected by Agrobacterium tumefaciens

Agrobacterium-mediated plant transformation is an extremely complex and evolved process involving genetic determinants of both the bacteria and the host plant cells. However, the mechanism of the determinants remains obscure, especially in some cereal crops such as wheat, which is recalcitrant for Agrobacterium-mediated transformation. In this study, differentially expressed genes (DEGs) and differentially expressed proteins (DEPs) were analyzed in wheat callus cells co-cultured with Agrobacterium by using RNA sequencing (RNA-seq) and two-dimensional electrophoresis (2-DE) in conjunction with mass spectrometry (MS). A set of 4,889 DEGs and 90 DEPs were identified, respectively. Most of them are related to metabolism, chromatin assembly or disassembly and immune defense. After comparative analysis, 24 of the 90 DEPs were detected in RNA-seq and proteomics datasets simultaneously. In addition, real-time RT-PCR experiments were performed to check the differential expression of the 24 genes, and the results were consistent with the RNA-seq data. According to gene ontology (GO) analysis, we found that a big part of these differentially expressed genes were related to the process of stress or immunity response. Several putative determinants and candidate effectors responsive to Agrobacterium mediated transformation of wheat cells were discussed. We speculate that some of these genes are possibly related to Agrobacterium infection. Our results will help to understand the interaction between Agrobacterium and host cells, and may facilitate developing efficient transformation strategies in cereal crops.     

一氧化氮调节盐胁迫下小麦幼苗根部质膜H+-ATPase和焦磷酸酶活性提高耐盐性

采用外源一氧化氮(NO)供体硝普钠(SNP)研究了NO对盐胁迫下小麦(Triticum aestivum L.)幼苗耐盐性的影响.结果表明,0.1 mmol/L SNP处理显著缓解了1 50 mmol/L NaCl胁迫对小麦幼苗生长的抑制效应,包括水分丧失以及叶绿素降解,从而提高了小麦幼苗的耐盐性.进一步结合1 mg/mL血红蛋白处理则显著逆转了SNP诱导的上述效应;利用亚硝酸钠和铁氰化钾作为对照也证实了NO对小麦幼苗耐盐性的专一性调节作用,并可能与NO对小麦幼苗根部质膜H -ATPase和焦磷酸酶活性诱导有关.此外,尽管NO显著提高了盐胁迫下小麦幼苗根部细胞质膜H -ATPase和焦磷酸酶的ATP水解活性,但是对跨膜H 转运则没有明显影响.应用外源CaSO4和EGTA处理也证实,Ca2 可能在NO诱导的质膜H -ATPase和焦磷酸酶活性的提高过程中起信号作用.另外,分析盐胁迫下小麦幼苗根部Na 和K 含量的变化也发现,NO对Na 含量没有明显影响,但是却显著提高了K 水平和K /Na 比,这可能也是NO提高小麦幼苗耐盐性的原因之一.     

一氧化氮调节盐胁迫下小麦幼苗根部质膜H^＋-ATPase和焦磷酸酶活性提高耐盐性

采用外源一氧化氮(NO)供体硝普钠(SNP)研究了NO对盐胁迫下小麦(Triticum aestivum L.)幼苗耐盐性的影响。结果表明,0.1 mmol/L SNP处理显著缓解了150 mmol/L NaCl 胁迫对小麦幼苗生长的抑制效应,包括水分丧失以及叶绿素降解,从而提高了小麦幼苗的耐盐性。进一步结合1 mg/mL血红蛋白处理则显著逆转了SNP诱导的上述效应;利用亚硝酸钠和铁氰化钾作为对照也证实了NO对小麦幼苗耐盐性的专一性调节作用,并可能与NO对小麦幼苗根部质膜 H -ATPase和焦磷酸酶活性诱导有关。此外,尽管NO显著提高了盐胁迫下小麦幼苗根部细胞质膜H -ATPase和焦磷酸酶的ATP水解活性,但是对跨膜H 转运则没有明显影响。应用外源CaSO4 和 EGTA 处理也证实,Ca2 可能在NO诱导的质膜 H -ATPase和焦磷酸酶活性的提高过程中起信号作用。另外,分析盐胁迫下小麦幼苗根部 Na 和K 含量的变化也发现,NO对Na 含量没有明显影响,但是却显著提高了K 水平和K /Na 比,这可能也是NO提高小麦幼苗耐盐性的原因之一。     

Shotgun proteomic analysis of soybean embryonic axes during germination under salt stress

Seed imbibition and radicle emergence are generally less affected by salinity in soybean than in other crop plants. In order to unveil the mechanisms underlying this remarkable salt tolerance of soybean at seed germination, a comparative label‐free shotgun proteomic analysis of embryonic axes exposed to salinity during germination sensu stricto (GSS) was conducted. The results revealed that the application of 100 and 200 mmol/L NaCl stress was accompanied by significant changes (>2‐fold, P<0.05) of 97 and 75 proteins, respectively. Most of these salt‐responsive proteins (70%) were classified into three major functional categories: disease/defense response, protein destination and storage and primary metabolism. The involvement of these proteins in salt tolerance of soybean was discussed, and some of them were suggested to be potential salt‐tolerant proteins. Furthermore, our results suggest that the cross‐protection against aldehydes, oxidative as well as osmotic stress, is the major adaptive response to salinity in soybean.