Wheat (Triticum aestivum), one of the most important cereal crops, is often threatened by drought. In this study, water deficit significantly reduced the height of plants and yield of grains. To explore further the effect of drought stress on the development and yield of grains, we first performed a large scale phosphoproteome analysis of developing grains in wheat. A total of 590 unique phosphopeptides, representing 471 phosphoproteins, were identified under well-watered conditions. Motif-X analysis showed that four motifs were enriched, including [sP], [Rxxs], [sDxE], and [sxD]. Through comparative phosphoproteome analysis between well-watered and water-deficit conditions, we found that 63 unique phosphopeptides, corresponding to 61 phosphoproteins, showed significant changes in phosphorylation level (≥2-fold intensities). Functional analysis suggested that some of these proteins may be involved in signal transduction, embryo and endosperm development of grains, and drought response and defense under water-deficit conditions. Moreover, we also found that some chaperones may play important roles in protein refolding or degradation when the plant is subjected to water stress. These results provide a detailed insight into the stress response and defense mechanisms of developmental grains at the phosphoproteome level. They also suggested some potential candidates for further study of transgenosis and drought stress as well as incorporation into molecular breeding for drought resistance.Keywords: bioinformatics; drought stress; grain development; quantitative phosphoproteomics; Triticum aestivum;

•The embryo mobilises protein and lipid while the endosperm mobilises storage starch and minor protein.•The primary energy was generated mainly in the embryo by glycolysis during seed imbibition.•The embryo increased nucleotides, indicating more active transcription and translation metabolisms.In this study, we performed the first comparative metabolomic analysis of the wheat embryo and endosperm during seed germination using GC-MS/MS. In total, 82 metabolites were identified in the embryo and endosperm. Principal component analysis (PCA), metabolite-metabolite correlation and hierarchical cluster analysis (HCA) revealed distinct dynamic changes in metabolites between the embryo and endosperm during seed germination. Generally, the metabolite changes in the embryo were much greater than those in the endosperm, suggesting that the embryo is more active than the endosperm during seed germination. Most amino acids were upregulated in both embryo and endosperm, while polysaccharides and organic acids associated with sugars were mainly downregulated in the embryo. Most of the sugars showed an upregulated trend in the endosperm, but significant changes in lipids occurred only in the embryo. Our results suggest that the embryo mobilises mainly protein and lipid metabolism, while the endosperm mobilises storage starch and minor protein metabolism during seed germination. The primary energy was generated mainly in the embryo by glycolysis during seed imbibition. The embryo containing most of the genetic information showed increased nucleotides during seed germination process, indicating more active transcription and translation metabolisms.Download high-res image (280KB)Download full-size image

Drought stress during grain development causes significant yield loss in cereal production. The phosphorylated modification of starch granule-binding proteins (SGBPs) is an important mechanism regulating wheat starch biosynthesis. In this study, we performed the first proteomics and phosphoproteomics analyses of SGBPs in elite Chinese bread wheat (Triticum aestivum L.) cultivar Jingdong 17 under well-watered and water-stress conditions.Water stress treatment caused significant reductions in spike grain numbers and weight, total starch and amylopectin content, and grain yield. Two-dimensional gel electrophoresis revealed that the quantity of SGBPs was reduced significantly by water-deficit treatment. Phosphoproteome characterization of SGBPs under water-deficit treatment demonstrated a reduced level of phosphorylation of main starch synthesis enzymes, particularly for granule-bound starch synthase (GBSS I), starch synthase II-a (SS II-a), and starch synthase III (SS III). Specifically, the Ser34 site of the GBSSI protein, the Tyr358 site of SS II-a, and the Ser837 site of SS III-a exhibited significant less phosphorylation under water-deficit treatment than well-watered treatment. Furthermore, the expression levels of several key genes related with starch biosynthesis detected by qRT-PCR were decreased significantly at 15 days post-anthesis under water-deficit treatment. Immunolocalization showed a clear movement of GBSS I from the periphery to the interior of starch granules during grain development, under both water-deficit and well-watered conditions.Our results demonstrated that the reduction in gene expression or transcription level, protein expression and phosphorylation levels of starch biosynthesis related enzymes under water-deficit conditions is responsible for the significant decrease in total starch content and grain yield.

•Proteome analysis of T. monococcum leaves under different levels of salt stress and recovery.•Phosphoproteome analysis of T. monococcum leaves under salt stress and recovery.•81 DAP spots and 20 DAPP spots were identified.•cp31BHv was regulated at both protein and phosphorylation levels under salt stress.•A synergetic mechanism of leaves responsive to salt stress and recovery was proposed.Salinity is a major abiotic stress factor affecting crops production and productivity. Triticum monococcum is closely related to Triticum urartu (AUAU), which is used as a model plant of wheat A genome study. Here, salt stress induced dynamic proteome and phosphoproteome profiling was focused. The T. monococcum seedlings were initially treated with different concentrations of NaCl ranging from 80 to 320 mM for 48 h followed by a recovery process for 48 h prior to proteomic and phosphoproteomic analysis. As a result, a total of 81 spots corresponding to salt stress and recovery were identified by MALDI-TOF/TOF-MS from 2-DE gels. These proteins were mainly involved in regulatory, stress defense, protein folding/assembly/degradation, photosynthesis, carbohydrate metabolism, energy production and transportation, protein metabolism, and cell structure. Pro-Q Diamond staining was used to detect the phosphoproteins. Finally, 20 spots with different phosphorylation levels during salt treatment or recovery compared with controls were identified. A set of potential salt stress response and defense biomarkers was identified, such as cp31BHv, betaine-aldehyde dehydrogenase, leucine aminopeptidase 2, Cu/Zn superoxide dismutase, and 2-Cys peroxiredoxin BAS1, which could lead to a better understanding of the molecular basis of salt response and defense in food crops.Biological significanceSoil salinity reduces the yield of the major crops, which is one of the severest problems in irrigated agriculture worldwide. However, how crops response and defense during different levels of salt treatment and recovery processes is still unclear, especially at the post-translational modification level. T. monococcum is a useful model for common wheat. Thus, proteomic and phosphoproteomic analyses of T. monococcum leaves were performed in our study, which provided novel insights into the underlying salt response and defense mechanisms in wheat and other crops.

Farinins are one of the oldest members of the gluten family in wheat and Aegilops species, and they influence dough properties. Here, we performed the first detailed molecular genetic study on farinin genes in Brachypodium distachyon L., the model species for Triticum aestivum. A total of 51 b-type farinin genes were cloned and characterized, including 27 functional and 24 non-functional pseudogenes from 14 different B. distachyon accessions. All genes were highly similar to those previously reported from wheat and Aegilops species. The identification of deduced amino acid sequences showed that b-type farinins across Triticeae genomes could be classified as b1-, b2-, b3-, and b4-type farinins; however, B. distachyon had only b3- and b4-type farinins. Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) revealed that farinin genes are transcribed into mRNA in B. distachyon at much lower levels than in Triticeae, despite the presence of cis-acting elements in promoter regions. Phylogenetic analysis suggested that Brachypodium farinins may have closer relationships with common wheat and further confirmed four different types of b-type farinins in Triticeae and Brachypodium genomes, corresponding to b1, b2, b3 (group 1), and b4 (group 2). A putative evolutionary origin model of farinin genes in Brachypodium, Triticum, and the related species suggests that all b-type farinins diverged from their common ancestor ~3.2 million years ago (MYA). The b3 and b4 types could be considered older in the farinin family. The results explain the loss of b1- and b2-type farinin alleles in Brachypodium.

•Brachypodium roots and leaves showed distinct proteome changes under H2O2 stress.•Ultrastructural analysis showed the damage in roots was more serious than in leaves.•84 and 53 H2O2-responsive proteins in roots and leaves were identified respectively.•14–3–3 proteins might play crucial roles during response and defense of H2O2 stress.•A synergetic mechanism of roots and leaves responsive to H2O2 stress was proposed.The plant oxidative stress response is vital for defense against various abiotic and biotic stresses. In this study, ultrastructural changes and the proteomic response to H2O2 stress in roots and leaves of the model plant Brachypodium distachyon were studied. Transmission electron microscopy (TEM) showed that the ultrastructural damage in roots was more serious than in leaves. Particularly, the ultrastructures of organelles and the nucleus in root tip cells were damaged, leading to the inhibition of normal biological activities of roots, which then spread throughout the plant. Based on two-dimensional electrophoresis (2-DE) and MALDI-TOF/TOF-MS, 84 and 53 differentially accumulated protein (DAP) spots representing 75 and 45 unique proteins responsive to H2O2 stress in roots and leaves, respectively, were identified. These protein species were mainly involved in signal transduction, energy metabolism, redox homeostasis/stress defense, protein folding/degradation, and cell wall/cell structure. Interestingly, two 14–3–3 proteins (GF14-B and GF14-D) were identified as DAPs in both roots and leaves. Protein-protein interaction (PPI) analysis revealed a synergetic H2O2-responsive network.

•Methods for obtaining high purity starch granule and associated proteins.•SSI-1, Pull, Iso and SBEIIa Expression had high differences between wheat species.•Large granule is associated with activities of multiple starch synthesis enzymes.•Phosphorylation of SSI-1 is related with the formation of B-type granule.Starch is the main component in the wheat endosperm and exists in two forms including A- and B-type granules. A bread wheat line CB037A and an Aegilops line Aegilops crassa were studied for the underlying starch biosynthesis mechanism in relation to granule types. The wheat line contains both types of starch granules while the Aegilops line only has the A-type. Differential starch granule development patterns of these two species were observed at the morphological level. A total of 190 differentially expressed proteins (DEPs) were detected between the two lines based on 2-D electrophoresis, among which 119 DEPs were identified, representing 13 unique proteins. Gene ontology annotation analysis indicated that both molecular functions and biological processes of the identified proteins are highly conserved. Different phosphorylation modification levels between the A- and B-type starch granules were found. Real-time quantitative reverse transcription PCR analysis revealed that a number of key genes including starch synthase I-1, pullulanase, isoamylase and starch branching enzyme IIa were differentially expressed between the two species. Our results demonstrated that the large granule size is associated with higher activities of multiple starch biosynthesis enzymes. The phosphorylation of starch biosynthesis enzymes is related with the formation of B-type starch granules.Biological significanceAnalyzed the proteome, transcriptome and phosphorylation of core starch granule biosynthesis enzymes and provided new insights into the differential mechanisms underlying the A- and B-type starch granule biosyntheses.

Brachypodium distachyon L., a model plant for cereal crops, has become important as an alternative and potential biofuel grass. In plants, N-glycosylation is one of the most common and important protein modifications, playing important roles in signal recognition, increase in protein activity, stability of protein structure, and formation of tissues and organs. In this study, we performed the first glycoproteome analysis in the seedling leaves of B. distachyon. Using lectin affinity chromatography enrichment and mass-spectrometry-based analysis, we identified 47 glycosylation sites representing 46 N-linked glycoproteins. Motif-X analysis showed that two conserved motifs, N-X-T/S (X is any amino acid, except Pro), were significantly enriched. Further functional analysis suggested that some of these identified glycoproteins are involved in signal transduction, protein trafficking, and quality control and the modification and remodeling of cell-wall components such as receptor-like kinases, protein disulfide isomerase, and polygalacturonase. Moreover, transmembrane helices and signal peptide prediction showed that most of these glycoproteins could participate in typical protein secretory pathways in eukaryotes. The results provide a general overview of protein N-glycosylation modifications during the early growth of seedling leaves in B. distachyon and supplement the glycoproteome databases of plants.

The embryo and endosperm of wheat have different physiological functions and large differences in protein level. In this study, we performed the first integrated proteome analysis of wheat embryo and endosperm in response to the water deficit during grain development. In total, 155 and 130 differentially expressed protein (DEP) spots in the embryo and endosperm, respectively, were identified by nonlinear two-dimensional electrophoresis and tandem mass spectrometry. These DEPs in the embryo were mainly involved in stress/defense responses such as heat shock-related proteins (HSP) and peroxidase, whereas those in endosperm were mainly related to starch and storage protein synthesis such as α-amylase inhibitor and the globulin-1 S allele. In particular, some storage proteins such as avenin-like proteins and high-molecular weight glutenin subunit Dy12 displayed higher expression levels in the mature endosperm under a water deficit, which might contribute to the improvement in the quality of breadmaking.

Wheat grain high molecular weight glutenin subunits (HMW-GS) are the major determinants of dough elasticity and viscosity, and thus of breadmaking quality. Most known HMW-GS genes in bread wheat have been already cloned except for very few such as 1By18 and 1By19. In this study, HMW-GS 1By18 gene was isolated from bread wheat for the first time. In the past, this subunit has been considered to have positive effect on gluten quality. Molecular characterization of the 1By18 subunit gene showed that its complete coding nucleotide sequence was 2163 bp long, being highly identical to 1By8, with only three single nucleotide polymorphisms (SNPs) at sites 231, 233 and 1976 of the coding sequence. The deduced amino acid sequence of 1By18 exhibited two substitutions compared to 1By8. The deduced protein was further verified by expression in E. coli and LC–MS/MS analysis. Phylogenetic analysis showed that 1By18 was more closely related to 1By8, 1By9 and 1By16 than to 1By15 and 1By20. Molecular phylogenetic analysis indicated that the Glu-B1i allele emerged after the formation of modern hexaploid wheat. Two sets of SNP-based AS-PCR markers for 1By18 gene were developed and validated on 110 bread wheat varieties with different Glu-Bly alleles, and five recombinant inbred lines. Both markers could effectively distinguish 1By18 from the other genes at the Glu-B1 locus, thus making them valuable for marker-assisted selection in wheat quality improvement programs.

Here, we conducted the first large-scale leaf phosphoproteome analysis of two bread wheat cultivars by liquid chromatography-tandem mass spectrometry. Altogether, 1802 unambiguous phosphorylation sites representing 1175 phosphoproteins implicated in various molecular functions and cellular processes were identified by gene ontology enrichment analysis. Among the 1175 phosphoproteins, 141 contained 3–10 phosphorylation sites. The phosphorylation sites were located more frequently in the N- and C-terminal regions than in internal regions, and ∼70% were located outside the conserved regions. Conservation analysis showed that 90.5% of the phosphoproteins had phosphorylated orthologs in other plant species. Eighteen significantly enriched phosphorylation motifs, of which six were new wheat phosphorylation motifs, were identified. In particular, 52 phosphorylated transcription factors (TFs), 85 protein kinases (PKs), and 16 protein phosphatases (PPs) were classified and analyzed in depth. All the Tyr phosphorylation sites were in PKs such as mitogen-activated PKs (MAPKs) and SHAGGY-like kinases. A complicated cross-talk phosphorylation regulatory network based on PKs such as Snf1-related kinases (SnRKs), calcium-dependent PKs (CDPKs), and glycogen synthase kinase 3 (GSK3) and PPs including PP2C, PP2A, and BRI1 suppressor 1 (BSU1)-like protein (BSL) was constructed and was found to be potentially involved in rapid leaf growth. Our results provide a series of phosphoproteins and phosphorylation sites in addition to a potential network of phosphorylation signaling cascades in wheat seedling leaves.

•173 phosphoproteins in HX10 were significantly regulated under drought conditions.•227 phosphoproteins in NC47 were significantly regulated under drought conditions.•30 common phosphoproteins are found in the two wheat cultivars.•Some important drought tolerant candidates were identified.•New insights into the mechanisms of drought response and defenseDrought is a major form of abiotic stress that significantly affects plant growth and development. In this study, we performed the first phosphoproteome analysis of seedling leaves from two bread wheat cultivars (Hanxuan 10 and Ningchun 47) subjected to drought stress. As a result, a total of 191 and 251 unique phosphopeptides, representing 173 and 227 phosphoproteins in two cultivars, respectively, were identified as being significant changes in phosphorylation level (SCPL) under drought stress. Through the comparison of SCPL phosphoproteins between two cultivars, 31 common SCPL phosphoproteins were found in both cultivars. Function analysis showed that the SCPL phosphoproteins in the two cultivars are mainly involved in three biological processes: RNA transcription/processing, stress/detoxification/defense, and signal transduction. Further analyses revealed that some SCPL phosphoproteins may play key roles in signal transduction and the signaling cascade under drought stress. Furthermore, some phosphoproteins related to drought tolerance and osmotic regulation exhibited significant phosphorylation changes. This study used a series of bioinformatics tools to profile the phosphorylation status of wheat seedling leaves under drought stress with greater accuracy.Biological significanceDrought is of the most studied abiotic stresses, because it severely restricts the development and yield of plants. In this study, large numbers of stress-related phosphoproteins are identified from the two bread wheat cultivars. These phosphoproteins contribute to signal transduction, osmotic regulation and ROS scavenging under water stress. This work provides a detailed insight into the mechanisms of drought response and defense in bread wheat from the perspective of phosphoproteomics, and identifies some important drought-tolerant candidates for further transgenosis study and incorporation into the breeding of resistant cultivars.

Brachypodium distachyon, a small wild grass within the Pooideae family, is a new model organism for exploring the functional genomics of cereal crops. It was shown to have close relationships to wheat, barley and rice. Here, we describe the molecular characterisation and evolutionary relationships of high molecular weight glutenin subunits (HMW-GS) genes from B. distachyon. Sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE), high performance capillary electrophoresis (HPCE) and liquid chromatography–tandem mass spectrometry (LC-MS/MS) analyses demonstrated that there was no HMW-GS expression in the Brachypodium grains due to the silencing of their encoding genes. Through allele-specific polymerase chain reaction (AS-PCR) amplification and cloning, a total of 13 HMW-GS encoding genes from diploid, tetraploid and hexaploid Brachypodium species were obtained, and all of them had typical structural features of y-type HMW-GS genes from common wheat and related species, particularly more similar to the 1Dy12 gene. However, the presence of an in-frame premature stop codon (TAG) at position 1521 in the coding region resulted in the conversion of all the genes to pseudogenes. Further, quantitative real-time PCR (qRT-PCR) analysis revealed that HMW-GS genes in B. distachyon displayed a similar trend, but with a low transcriptional expression profile during grain development due to the occurrence of the stop codon. Phylogenetic analysis showed that the highly conserved Glu-1-2 loci were presented in B. distachyon, which displayed close phylogenetic evolutionary relationships with Triticum and related species.