A Bowman-Birk type protease inhibitor is involved in the tolerance to salt stress in wheat

A salt-responsive gene WRSI5 was characterized from salt-tolerant cultivar Shanrong No. 3 (SR3), an introgression line via asymmetric somatic hybrid between Triticum aestivum L. cv. Jinan177 (JN177) and Thinopyrum ponticum Podp. The peptide encoded by WRSI5 contains a Bowman-Birk domain sharing a high level of sequence identity to monocotyledonous protease inhibitors. When expressed in vitro , the WRSI5 gene product exhibited trypsin, but not chymotrypsin inhibition. The expression level of WRSI5 was increased in SR3 roots exposed to salt, drought or oxidative stress. In situ hybridization showed that it is induced in the endodermal cells of the mature region of the SR3 root tip, with no signal detectable in the corresponding region of the salt-susceptible cultivar JN177. SR3 has a higher selectivity for K⁺ over Na⁺, and therefore limits the transport of Na⁺ from the root to the shoot. When overexpressed in Arabidopsis thaliana , WRSI5 improves the ability of seedlings to grow on a medium containing 150 m m NaCl. We suggest that WRSI5 plays an important role in regulating the plant growth rate or long-distance Na⁺ transport in SR3 plants exposed to salt stress.     

A transcriptomic analysis reveals the nature of salinity tolerance of a wheat introgression line

The bread wheat cultivar Shanrong No.3 (SR3) is a salinity tolerant derivative of an asymmetric somatic hybrid between cultivar Jinan 177 (JN177) and tall wheatgrass (Thinopyrum ponticum). To reveal some of the mechanisms underlying its elevated abiotic stress tolerance, both SR3 and JN177 were exposed to iso-osmotic NaCl and PEG stress, and the resulting gene expression was analysed using a customized microarray. Some genes associated with stress response proved to be more highly expressed in SR3 than in JN177 in non-stressed conditions. Its unsaturated fatty acid and flavonoid synthesis ability was also enhanced, and its pentose phosphate metabolism was more active than in JN177. These alterations in part accounted for the observed shift in the homeostasis related to reactive oxygen species (ROS). The specific down-regulation of certain ion transporters after a 0.5 h exposure to 340 mM NaCl demonstrated that Na(+) uptake occurred rapidly, so that the early phase of salinity stress imposes more than simply an osmotic stress. We discussed the possible effect of the introgression of new genetic materials in wheat genome on stress tolerance.     

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.     

Proteomic analysis on salicylic acid-induced salt tolerance in common wheat seedlings (Triticum aestivum L.)

The influence of salicylic acid (SA) on the salt tolerance mechanism in seedlings of common wheat (Triticum aestivum L.) was investigated using physiological measurements combined with global expression profiling (proteomics). In the present study, 0.5mM SA significantly reduced NaCl-induced growth inhibition in wheat seedlings, manifesting as increased fresh weights, dry weights, and photosynthetic pigments, but decreased lipid peroxidation. Two-week-old wheat seedlings treated with 0.5mM SA, 250mM NaCl and 250mM NaCl+0.5mM SA for 3days were used for the proteomic analyses. In total, 39 proteins differentially regulated by both salt and SA were revealed by 2D PAGE, and 38 proteins were identified by MALDI-TOF/TOF MS. The identified proteins were involved in various cellular responses and metabolic processes including signal transduction, stress defense, energy, metabolism, photosynthesis, and others of unknown function. All protein spots involved in signal transduction and the defense response were significantly upregulated by SA under salt stress, suggesting that these proteins could play a role in the SA-induced salt resistance in wheat seedlings.     

Comparative proteomic analysis of the Arabidopsis cbl1 mutant in response to salt stress

Many environmental stimuli, including light, biotic and abiotic stress factors, induce changes in cellular Ca(2+) concentrations in plants. Such Ca(2+) signatures are perceived by sensor molecules such as calcineurin B-like (CBL) proteins. AtCBL1, a member of the CBL family which is highly inducible by multiple stress signals, is known to function in the salt stress signal transduction pathway and to positively regulate the plant tolerance to salt. To shed light into the molecular mechanisms of the salt stress response mediated by AtCBL1, a two-dimensional DIGE proteomic approach was applied to identify the differentially expressed proteins in Arabidopsis wild-type and cbl1 null mutant plants in response to salt stress. Seventy-three spots were found altered in expression by least 1.2-fold and 50 proteins were identified by MALDI-TOF/TOF-MS, including some well-known and novel salt-responsive proteins. These proteins function in various processes, such as signal transduction, ROS scavenging, energy production, carbon fixation, metabolism, mRNA processing, protein processing and structural stability. Receptor for activated C kinase 1C (RACK1C, spot 715), a WD40 repeat protein, was up-regulated in the cbl1 null mutant, and two rack1c mutant lines showed decreased tolerance to salt stress, suggesting that RACK1C plays a role in salt stress resistance. In conclusion, our work demonstrated the advantages of the proteomic approach in studies of plant biology and identified candidate proteins in CBL1-mediated salt stress signaling network.     

Salt-stress induced changes in the leaf proteome of diploid and tetraploid mandarins with contrasting Na and Cl accumulation behaviour

To understand the genotypic variation of citrus to mild salt stress, a proteomic approach has been carried out in parallel on two citrus genotypes (‘Cleopatra’ and ‘Willow leaf’ mandarins), which differ for Na⁺ and Cl⁻ accumulation, and their cognate autotetraploids (4×). Using two-dimensional electrophoresis approximately 910 protein spots were reproducibly detected in control and salt-stressed leaves of all genotypes. Among them, 44 protein spots showing significant variations at least in one genotype were subjected to mass spectrometry analysis for identification. Salt-responsive proteins were involved in several functions, including photosynthetic processes, ROS scavenging, stress defence, and signalling. Genotype factors affect the salt-responsive pattern, especially that of carbon metabolism. The no ion accumulator ‘Cleopatra’ mandarin genotype showed the highest number of salt-responsive proteins, and up-regulation of Calvin cycle-related proteins. Conversely the ion accumulator ‘Willow leaf’ mandarin showed high levels of several photorespiration-related enzymes. A common set of proteins (twelve spots) displayed higher levels in salt-stressed leaves of 2× and 4× ‘Cleopatra’ and 4× ‘Willow leaf’ mandarin. Interestingly, antioxidant enzymes and heat shock proteins showed higher constitutive levels in 4× ‘Cleopatra’ mandarin and 4× ‘Willow leaf’ mandarin compared with the cognate 2× genotype. This work provides for the first time information on the effect of 8 weeks of salt stress on citrus genotypes contrasting for ion accumulation and their cognate autotetraploids. Results underline that genetic factors have a predominant effect on the salt response, although a common stress response independent from genotype was also found.     

Comparative proteomic analysis of Arabidopsis thaliana roots between wild type and its salt-tolerant mutant

Two-dimensional electrophoresis (2-DE) showed the variation expression of Arabidopsis thaliana root proteins between wild type and its salt-tolerant mutant obtained from cobalt-60 γ ray radiation. Forty-six differential root protein spots were reproducibly presented on 2-DE maps, and 29 spots were identified by matrix assisted laser desorption ionization-time of flight/time of flight mass spectrometry (MS). Fifteen protein spots corresponding to 10 proteins, and 14 protein spots corresponding to 9 proteins were constitutively up-regulated and down-regulated in the salt-tolerant mutant root. Bioinformatic analysis indicated that those differential proteins might be involved in the regulation of redox homeostasis, nucleotide metabolism, signal transduction, stress response and defense, carbohydrate metabolism, and cell wall metabolism. Peroxidase 22 might be a versatile enzyme and might play dual roles in both cell wall metabolism and regulation of redox homeostasis. Our work provides not only new insights into salt-responsive proteins in root, but also the potential salt-tolerant targets for further dissection of molecular mechanism adapted by plants during salt stress.     

小麦一个新BBI型蛋白酶抑制剂基因TaUES的克隆及初步分析

根据小麦基因表达芯片结果,以山融3号小麦叶片为试验材料,克隆获得在盐胁迫处理24 h时表达显著提高的基因TaUES(up-regulated expression under saline-stress in wheat,ID:KC408382)。序列分析显示,TaUES编码一个富含半胱氨酸的97个氨基酸的多肽,该多肽含有1个BowB结构域和10个保守的半胱氨酸残基;与小麦或其他物种BBI型蛋白酶抑制剂氨基酸序列具有较高的同源性。推测TaUES是小麦中一种新的BBI型蛋白酶抑制剂基因。基因表达分析表明,TaUES基因参与盐和干旱胁迫应答。异源过表达转基因功能初步分析表明,过表达TaUES转基因烟草后代株系耐盐性提高。     

Effects of salinity levels on proteome of Suaeda aegyptiaca leaves

Saline soils are the major problem of cultivated lands of Iran. Suaeda aegyptiaca is a salt-tolerant plant (halophytes) that grow naturally in salt-affected areas of Iran. We have employed proteomics to identify the mechanisms of salt responsiveness in leaves of S. aegyptiaca grown under different salt concentrations. Ten-day-old plants were treated with 0, 150, 300, 450, and 600 mM NaCl. After 30 days of treatment, leaf samples were collected and analyzed using 2-D-PAGE. Out of 700 protein spots reproducible detected within replications, 102 spots showed significant response to salt treatment compared to 0 mM NaCl. We analyzed expression pattern of salt-responsive proteins using a hierarchical and two nonhierarchical (Fuzzy ART and SOM) statistical methods and concluded that Fuzzy ART is the superior method. Forty proteins of 12 different expression groups were analyzed using LC/MS/MS. Of these, 27 protein spots were identified including proteins involved in oxidative stress tolerance, glycinebetain synthesis, cytoskeleton remodeling, photosynthesis, ATP production, protein degradation, cyanide detoxification, and chaperone activities. The expression pattern of these proteins and their possible roles in the adaptation of S. aegyptiaca to salinity is discussed.     

Characterization of Mitochondrial Proteomic Changes in Resistant Wheat Near‐Isogenic Line After Inoculation with Powdery Mildew

Wheat powdery mildew resistance mechanisms have been studied extensively at genomic level, however, infection induced mitochondrial proteomic changes in resistant line have not been fully characterized. Being critical organelles of chemical energy metabolism, mitochondria have also been suggested to be involved in the environmental stress response. Using proteomic approaches, we did comparative analysis of mitochondrial proteome in resistant wheat near‐isogenic line (NIL) (Brock × Jing411⁷) and its recurrent parent Jing 411 after infection of Blumeria graminis f.sp. tritici (Bgt). More than 50 down‐regulated mitochondrial protein spots were identified in NIL after 24‐h pathogen inoculation, and their abundance recovered to the levels prior to infection after extended inoculation (72‐h). We further analyzed a subgroup of down‐regulated proteins using mass spectrometry. MS/MS data analysis revealed the identities of nine protein spots and assigned them into three functional classes: synthesis of protein, disease resistance response and energy metabolism. For the first time we demonstrated pathogen stress induced mitochondrial proteomic changes and provided evidences that wheat powdery mildew resistance involves multiple biochemical events. Moreover, our results indicate that wheat mitochondrial proteome analysis can serve as a powerful tool to identify potential regulators of fungal invasion resistance.     

N+注入引起向日葵蛋白质组变异研究初报

低能离子注入可以引起生物DNA分子结构的变异,但用蛋白质组学方法对低能离子注入后所引起的蛋白质诱变效应的研究尚缺乏文献报道。以向日葵种子为实验材料,对N 注入后种子的蛋白质组进行了初步分析,共获得369个蛋白质点,其中包括对照种子中的8个特异点和处理种子中的4个特异点。这些特异点的分子量在15～34ku之间。通过基质辅助激光解吸电离飞行时间质谱鉴定发现对照种子蛋白质中的No.29特异蛋白与MADS盒转录因子HAM59有23．48％的匹配率。处理种子中的No．279特异蛋白与亮氨酸拉链蛋白的同源蛋白HAHB-4有23.20％的匹配率。     

Proteome changes induced by expression of tumor suppressor PTEN

The tumor suppressor, PTEN, located at 10q23, is one of the most frequently mutated tumor suppressors in a number of sporadic cancers and in two autosomal dominant harmatomas. It is considered one of the most important tumor suppressors in the post p53 era. To identify the molecules involved in the signal network regulated by PTEN using proteomic tools, a PTEN-inducible expression system was established in NIH 3T3 mouse embryonic fibroblast cells. We compared proteome images of PTEN-induced and non-induced cells by 2-dimensional electrophoresis. Twenty-nine differentially expressed protein spots were identified by MALDI-TOF MS and NSI MS/MS. We conclude that expression of PTEN by itself leads to protein profile changes, and those proteins affected are likely to be directly and/or indirectly involved in the function and physiology of the tumor suppressor.     

Comparative proteomic profiles of Pinus monticola needles during early compatible and incompatible interactions with Cronartium ribicola

The proteomic profiles of primary needles from Cr2-resistant and cr2-susceptible Pinus monticola seedlings were analysed post Cronartium ribicola inoculation by 2-DE. One hundred-and-five protein spots exhibiting significant differential expression were identified using LC–MS/MS. Functional classification showed that the most numerous proteins are involved in defence signalling, oxidative burst, metabolic pathways, and other physiological processes. Our results revealed that differential expression of proteins in response to C. ribicola inoculation was genotype- and infection-stage dependent. Responsive proteins in resistant seedlings with incompatible white pine blister rust (WPBR) interaction included such well-characterized proteins as heat shock proteins (HSPs), reactive oxygen species (ROS) scavenging enzymes, and intermediate factors functioning in the signal transduction pathways triggered by well-known plant R genes, as well as new candidates in plant defence like sugar epimerase, GTP-binding proteins, and chloroplastic ribonucleoproteins. Fewer proteins were regulated in susceptible seedlings; most of them were in common with resistant seedlings and related to photosynthesis among others. Quantitative RT-PCR analysis confirmed HSP- and ROS-related genes played an important role in host defence in response to C. ribicola infection. To the best of our knowledge, this is the first comparative proteomics study on WPBR interactions at the early stages of host defence, which provides a reference proteomic profile for other five-needle pines as well as resistance candidates for further understanding of host resistance in the WPBR pathosystem.     

家蚕抗BmNPV品系与感性品系血淋巴液蛋白质组的差异分析

利用遗传学的原理, 通过杂交和回交的方法, 建立家蚕抗BmNPV、感BmNPV以及近等基因系模型, 利用2-D电泳和MALDI TOF/TOF MS质谱技术, 从蛋白质组水平上研究家蚕对BmNPV抵抗性。其结果是获得家蚕高抗NB, 高感306, 近等基因系BC8五龄起蚕血淋巴液蛋白质差异表达谱, 分别获得180、190、187个蛋白点, 其中80%的蛋白点集中在等电点5~9范围之内。从三块凝胶上共获得明显差异蛋白点12个, 由质谱鉴定出5种蛋白, 其中氨基酰化酶(Aminoacylase)仅出现在抗性品系NB、近等基因系图谱中, 感性品系没有出现, 初步推测是家蚕抗BmNPV特有蛋白, 这是首次报道结果。