Low molecular weight glutenin subunits (LMW-GS), as important seed storage proteins, together with HMW-GS significantly define unique dough viscoelastic properties. In this study, a rapid ultraperformance liquid chromatography (UPLC) method for the separation and characterization of LMW-GS in wheat was optimized and established. The fast, reproducible, and high-resolution UPLC separation of LMW-GS could be obtained by gradually increasing eluting gradient from 21 to 47% in 30 min at flow rate of 0.55 mL/min and 60 °C for separation temperature. By this method, analysis of one sample could be completed in <20 min, significantly less time than the traditional reversed-phase high-performance liquid chromatography (RP-HPLC) method. Under the optimized conditions, the genetic features of LMW-GS and genotype × environmental interaction were successfully analyzed, leading to a fast identification of 17 main LMW-GS alleles that were related to different quality properties in wheat. The results demonstrated that UPLC could be a powerful and alternative tool for genetic and proteomic studies of wheat grain proteins and fast identification or screening of desirable LMW-GS alleles in wheat quality improvement.

Fifteen novel α-gliadin genes were cloned and sequenced from Triticum and related Aegilops genomes by allele-specific polymerase chain reaction (AS-PCR). Sequence comparison displayed high diversities in the α-gliadin gene family. Four toxic epitopes and glutamine residues in the two polyglutamine domains facilitated these α-gliadins to be assigned to specific chromosomes. Five representative α-gliadin genes were successfully expressed in Escherichia coli, and their amount reached a maximum after 4 h induced by isopropyl-β-D-thiogalactoside (IPTG), indicating a high level of expression under the control of T7 promoter. The transcriptional expression of α-gliadin genes during grain development detected by quantitative real-time polymerase chain reaction (qRT-PCR) showed a similar up–down regulation pattern in different genotypes. A neighbor-joining tree constructed with both full-open reading frame (ORF) α-gliadin genes and pseudogenes further revealed the origin and phylogenetic relationships among Triticum and related Aegilops genomes. The evolutionary analysis demonstrated that α-gliadin genes evolved mainly by synonymous substitutions under strong purifying selection during the evolutionary process.

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.Highlights► One highly salt tolerant wheat cultivar and one salt sensitive wheat line were studied. ► Their seedling roots were used for a comparative proteome analysis for salt response. ► 114 DEPs were identified which fall into 4 functional categories.

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.Graphical abstractThe proteomics analysis of wheat leaf under salt stress, we found out some key proteins for salt resistance and illuminated some mechanism.Research highlights► 2D-DIGE was used for the first time to identify salt response proteins in wheat. ► More salt concentrations reveal more detailed information salt response proteins. ► Some very important proteins were identified in the elite cultivar Zhengmai 9023. ► The results provide biochemical bases for the good salt tolerance of Zhengmai 9023.

Proteome Dynamics of albumins and globulins during grain development of elite Chinese wheat cultivar Xiaoyan 6 were studied. A total of 210 differentially expressed protein spots were recognized by two-dimensional differential gel electrophoresis (2D-DIGE) and 146 of these that represented 89 unique proteins were identified by MALDI-TOF mass spectrometry. Four protein expression patterns were observed across five grain developmental stages. Among the identified proteins, more than 80% were enzymes in eight functional categories, including carbohydrate metabolism (27%), protein metabolism (27%), stress/defense/detoxification (11%), cell metabolism (6%), transcription/translation (4%), nitrogen metabolism (4%), photosynthesis (4%), and signal transduction (1%). Quantitative real-time polymerase chain reaction (qRT-PCR) analysis of 18 representative genes demonstrated that about two thirds of the genes displayed different expression profiles between the transcriptional and translational stages.Highlights► The albumin/globulin proteome of the Chinese elite cultivar Xiaoyan 6 was studied. ► 144 proteins differentially expressed during grain development were identified by MS. ► More than 80% of the proteins were enzymes involved in nine functional categories. ► Transcript profiles were developed for 18 genes and compared with protein profiles.

Water-soluble proteins account for about 10% of total grain proteins in wheat (Triticum aestivum L.) and have specific functions in plant growth and development. In this study, a reversed-phase ultra performance liquid chromatography (RP-UPLC) method was trialed and the experimental conditions were optimized for rapidly separating and characterizing water-soluble proteins in wheat grains, and a comparative analysis with traditional RP-HPLC was performed. Under optimized separation conditions, fast, high resolution and reproducible RP-UPLC separation for water-soluble proteins could be obtained by gradually increasing eluting gradient from 21% to 47% in 30 min at 0.6 ml/min and 60 °C. Using this method, separation and characterization of water-soluble protein in wheat grains could be completed in less than 20 min for one sample, and the resolution and efficiency were significantly higher than those obtained with RP-HPLC. In addition, RP-UPLC consumed smaller amounts of samples and reagents as well. The optimized RP-UPLC could be used as an effective and alternative method for rapid separation and characterization of water-soluble proteins in wheat cultivar and germplasm evaluation, genetic and biochemical studies on grain proteins, and environmental influence analysis of water-soluble proteins.Highlights► RP-UPLC conditions for separating whet water-soluble proteins were optimized. ► A comparative analysis between RP-UPLC and RP-HPLC was carried out. ► Different wheat cultivars and germplasm were identified by the optimized RP-UPLC. ► Genetic controls and environmental effects of water-soluble proteins were studied.