Salinity is a major abiotic stress affecting plant growth and development. Understanding the molecular mechanisms of salt response and defense in plants will help in efforts to improve the salt tolerance of crops. Brachypodium distachyon is a new model plant for wheat, barley, and several potential biofuel grasses. In the current study, proteome and phosphoproteome changes induced by salt stress were the focus. The Bd21 leaves were initially treated with salt in concentrations ranging from 80 to 320 mm and then underwent a recovery process prior to proteome analysis. A total of 80 differentially expressed protein spots corresponding to 60 unique proteins were identified. The sample treated with a median salt level of 240 mm and the control were selected for phosphopeptide purification using TiO₂ microcolumns and LC-MS/MS for phosphoproteome analysis to identify the phosphorylation sites and phosphoproteins. A total of 1509 phosphoproteins and 2839 phosphorylation sites were identified. Among them, 468 phosphoproteins containing 496 phosphorylation sites demonstrated significant changes at the phosphorylation level. Nine phosphorylation motifs were extracted from the 496 phosphorylation sites. Of the 60 unique differentially expressed proteins, 14 were also identified as phosphoproteins. Many proteins and phosphoproteins, as well as potential signal pathways associated with salt response and defense, were found, including three 14-3-3s (GF14A, GF14B, and 14-3-3A) for signal transduction and several ABA signal-associated proteins such as ABF2, TRAB1, and SAPK8. Finally, a schematic salt response and defense mechanism in B. distachyon was proposed.

Background: The analyses of protein synthesis, accumulation and regulation during grain development in wheat are more complex because of its larger genome size compared to model plants such as Arabidopsis and rice. In this study, grains from two wheat cultivars Jimai 20 and Zhoumai 16 with different gluten quality properties were harvested at five development stages, and were used to displayed variable expression patterns of grain proteins.Results: Proteome characterization during grain development in Chinese bread wheat cultivars Jimai 20 and Zhoumai 16 with different quality properties was investigated by 2-DE and tandem MALDI-TOF/TOF-MS. Identification of 117 differentially accumulated protein spots representing 82 unique proteins and five main expression patterns enabled a chronological description of wheat grain formation. Significant proteome expression differences between the two cultivars were found; these included 14 protein spots that accumulated in both cultivars but with different patterns and 27 cultivar-different spots. Among the cultivar-different protein spots, 14 accumulated in higher abundance in Jimai 20 than in Zhoumai 16, and included NAD-dependent isocitrate dehydrogenase, triticin precursor, LMW-s glutenin subunit and replication factor C-like protein. These proteins are likely to be associated with superior gluten quality. In addition, some proteins such as class II chitinase and peroxidase 1 with isoforms in developing grains were shown to be phosphorylated by Pro-Q Diamond staining and phosphorprotein site prediction. Phosphorylation could have important roles in wheat grain development. qRT-PCR analysis demonstrated that transcriptional and translational expression patterns of many genes were significantly different.Conclusions: Wheat grain proteins displayed variable expression patterns at different developmental stages and a considerable number of protein spots showed differential accumulation between two cultivars. Differences in seed storage proteins were considered to be related to different quality performance of the flour from these wheat cultivars. Some proteins with isoforms were phosphorylated, and this may reflect their importance in grain development. Our results provide new insights into proteome characterization during grain development in different wheat genotypes.

Thorough understanding of seed starch biosynthesis and accumulation mechanisms is of great importance for agriculture and crop improvement strategies. We conducted the first comprehensive study of the dynamic development of starch granules and the regulation of starch biosynthesis in Brachypodium distachyon and compared the findings with those reported for common wheat (Chinese Spring, CS) and Aegilops peregrina. Only B-granules were identified in Brachypodium Bd21, and the shape variation and development of starch granules were similar in the B-granules of CS and Bd21. Phylogenetic analysis showed that most of the Bd21 starch synthesis-related genes were more similar to those in wheat than in rice. Early expression of key genes in Bd21 starch biosynthesis mediate starch synthesis in the pericarp; intermediate-stage expression increases the number and size of starch granules. In contrast, these enzymes in CS and Ae. peregrina were mostly expressed at intermediate stages, driving production of new B-granules and increasing the granule size, respectively. Immunogold labeling showed that granule-bound starch synthase (GBSSI; related to amylose synthesis) was mainly present in starch granules: at lower levels in the B-granules of Bd21 than in CS. Furthermore, GBSSI was phosphorylated at threonine 183 and tyrosine 185 in the starch synthase catalytic domain in CS and Ae. peregrina, but neither site was phosphorylated in Bd21, suggesting GBSSI phosphorylation could improve amylose biosynthesis. Bd21 contains only B-granules, and the expression of key genes in the three studied genera is consistent with the dynamic development of starch granules. GBSSI is present in greater amounts in the B-granules of CS than in Bd21; two phosphorylation sites (Thr183 and Tyr185) were found in Triticum and Aegilops; these sites were not phosphorylated in Bd21. GBSSI phosphorylation may reflect its importance in amylose synthesis.

The rice gene seed dormancy 4 (OsSdr4) functions in seed dormancy and is a major factor associated with pre-harvest sprouting (PHS). Although previous studies of this protein family were reported for rice and other species, knowledge of the evolution of genes homologous to OsSdr4 in plants remains inadequate. Fifty four Sdr4-like (hereafter designated Sdr4L) genes were identified in nine plant lineages including 36 species. Phylogenetic analysis placed these genes in eight subfamilies (I-VIII). Genes from the same lineage clustered together, supported by analysis of conserved motifs and exon-intron patterns. Segmental duplications were present in both dicot and monocot clusters, while tandemly duplicated genes occurred only in monocot clusters indicating that both tandem and segmental duplications contributed to expansion of the grass I and II subfamilies. Estimation of the approximate ages of the duplication events indicated that ancestral Sdr4 genes evolved from a common angiosperm ancestor, about 160 million years ago (MYA). Moreover, diversification of Sdr4L genes in mono and dicot plants was mainly associated with genome-wide duplication and speciation events. Functional divergence was observed in all subfamily pairs, except IV/VIIIa. Further analysis indicated that functional constraints between subfamily pairs I/II, I/VIIIb, II/VI, II/VIIIb, II/IV, and VI/VIIIb were statistically significant. Site and branch-site model analyses of positive selection suggested that these genes were under strong adaptive selection pressure. Critical amino acids detected for both functional divergence and positive selection were mostly located in the loops, pointing to functional importance of these regions in this protein family. In addition, differential expression studies by transcriptome atlas of 11 Sdr4L genes showed that the duplicated genes may have undergone divergence in expression between plant species. Our findings showed that Sdr4L genes are functionally divergent and positively selected. These may contribute to further functional analysis and molecular evolution of Sdr4L gene families in land plants.