BackgroundThorough 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.ResultsOnly 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.ConclusionsBd21 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-014-0198-2) contains supplementary material, which is available to authorized users.

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.

Protein disulfide isomerases (PDI) are involved in catalyzing protein disulfide bonding and isomerization in the endoplasmic reticulum and functions as a chaperone to inhibit the aggregation of misfolded proteins. Brachypodium distachyon is a widely used model plant for temperate grass species such as wheat and barley. In this work, we report the first molecular characterization, phylogenies, and expression profiles of PDI and PDI-like (PDIL) genes in B. distachyon in different tissues under various abiotic stresses. Eleven PDI and PDIL genes in the B. distachyon genome by in silico identification were evenly distributed across all five chromosomes. The plant PDI family has three conserved motifs that are involved in catalyzing protein disulfide bonding and isomerization, but a different exon/intron structural organization showed a high degree of structural differentiation. Two pairs of genes (BdPDIL4-1 and BdPDIL4-2; BdPDIL7-1 and BdPDIL7-2) contained segmental duplications, indicating each pair originated from one progenitor. Promoter analysis showed that Brachypodium PDI family members contained important cis-acting regulatory elements involved in seed storage protein synthesis and diverse stress response. All Brachypodium PDI genes investigated were ubiquitously expressed in different organs, but differentiation in expression levels among different genes and organs was clear. BdPDIL1-1 and BdPDIL5-1 were expressed abundantly in developing grains, suggesting that they have important roles in synthesis and accumulation of seed storage proteins. Diverse treatments (drought, salt, ABA, and H2O2) induced up- and down-regulated expression of Brachypodium PDI genes in seedling leaves. Interestingly, BdPDIL1-1 displayed significantly up-regulated expression following all abiotic stress treatments, indicating that it could be involved in multiple stress responses. Our results provide new insights into the structural and functional characteristics of the plant PDI gene family.

NAC (NAM, ATAF1/2, CUC2) transcription factors are involved in regulating plant developmental processes and response to environmental stresses. Brachypodium distachyon is an emerging model system for cereals, temperate grasses and biofuel crops. In this study, a comprehensive investigation of the molecular characterizations, phylogenetics and expression profiles under various abiotic stresses of the NAC gene family in Brachypodium distachyon was performed. In total, 118 BNAC genes in B. distachyon were identified, of which 22 (18.64%) were tandemly duplicated and segmentally duplicated, respectively. The Bayesian phylogenetic inference using Markov Chain Monte Carlo (MCMC) algorithms showed that they were divided into two clades and fourteen subfamilies, supported by similar motif compositions within one subfamily. Some critical amino acids detected using DIVERGE v3.0 might contribute to functional divergence among subfamilies. The different exon-intron organizations among subfamilies revealed structural differentiation. Promoter sequence predictions showed that the BNAC genes were involved in various developmental processes and diverse stress responses. Three NAC domain-encoding genes (BNAC012, BNAC078 and BNAC108), orthologous of NAC1, were targeted by five miRNA164 (Bdi-miR164a-c, e, f), suggesting that they might function in lateral organ enlargement, floral development and the responses to abiotic stress. Eleven (~9.32%) BNAC proteins containing α-helical transmembrane motifs were identified. 23 representative BNAC genes were analyzed by quantitative real-time PCR, showing different expression patterns under various abiotic stresses, of which 18, 17 and 11 genes were up-regulated significantly under drought, H2O2 and salt stresses, respectively. Only four and two genes were up-regulated under cold and cadmium stresses, respectively. Dynamic transcriptional expression analysis revealed that six genes showed constitutive expression and period-specific expression. The current results provide novel insights into the structure and function of the plant NAC gene family.

BackgroundThe endoplasmic reticulum chaperone binding protein (BiP) is an important functional protein, which is involved in protein synthesis, folding assembly, and secretion. In order to study the role of BiP in the process of wheat seed development, we cloned three BiP homologous cDNA sequences in bread wheat (Triticum aestivum), completed by rapid amplification of cDNA ends (RACE), and examined the expression of wheat BiP in wheat tissues, particularly the relationship between BiP expression and the subunit types of HMW-GS using near-isogenic lines (NILs) of HMW-GS silencing, and under abiotic stress.ResultsSequence analysis demonstrated that all BiPs contained three highly conserved domains present in plants, animals, and microorganisms, indicating their evolutionary conservation among different biological species. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) revealed that TaBiP (Triticum aestivum BiP) expression was not organ-specific, but was predominantly localized to seed endosperm. Furthermore, immunolocalization confirmed that TaBiP was primarily located within the protein bodies (PBs) in wheat endosperm. Three TaBiP genes exhibited significantly down-regulated expression following high molecular weight-glutenin subunit (HMW-GS) silencing. Drought stress induced significantly up-regulated expression of TaBiPs in wheat roots, leaves, and developing grains.ConclusionsThe high conservation of BiP sequences suggests that BiP plays the same role, or has common mechanisms, in the folding and assembly of nascent polypeptides and protein synthesis across species. The expression of TaBiPs in different wheat tissue and under abiotic stress indicated that TaBiP is most abundant in tissues with high secretory activity and with high proportions of cells undergoing division, and that the expression level of BiP is associated with the subunit types of HMW-GS and synthesis. The expression of TaBiPs is developmentally regulated during seed development and early seedling growth, and under various abiotic stresses.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-014-0260-0) contains supplementary material, which is available to authorized users.

5-azaC treatment and TaPBF - D over-expression decrease C-methylation status of three Glu - 1 gene promoters, and aid in enhancing the expression of the Glu - 1 genes. The wheat glutenins exert a strong influence over dough elasticity, but the regulation of their encoding genes has not been firmly established. Following treatment with 5-azacytidine (5-azaC), both the weight and glutenin content of the developing and mature grains were significantly increased. The abundance of transcript produced by the Glu-1 genes (encoding high-molecular-weight glutenin subunits), as well as those encoding demethylases and transcriptional factors associated with prolamin synthesis was higher than in grain of non-treated plants. These grains also contained an enhanced content of the prolamin box binding factor (PBF) protein. Bisulfite sequencing indicated that the Glu-1 promoters were less strongly C-methylated in the developing grain than in the flag leaf, while in the developing grain of 5-azaC treated plants, the C-methylation level was lower than in equivalent grains of non-treated plants. Both Glu-1 transcript abundance and glutenin content were higher in the grain set by three independent over-expressors of the D genome homoeolog of TaPBF than in the grain set by wild type plants. When assessed 10 days after flowering, the Glu-1 promoters' methylation level was lower in the developing grains set by the TaPBF-D over-expressor than in the wild type control. An electrophoretic mobility shift assay showed that PBF-D was able to bind in vitro to the P-box of Glu-1By8 and -1Dx2, while a ChIP-qPCR analysis revealed that a lower level of C-methylation in the Glu-1By8 and -1Dx2 promoters improved the TaPBF binding. We suggest that promoter DNA C-methylation is a key determinant of Glu-1 transcription.

Background Acyl-CoA-binding proteins (ACBPs) possess a conserved acyl-CoA-binding (ACB) domain that facilitates binding to acyl-CoA esters and trafficking in eukaryotic cells. Although the various functions of ACBP have been characterized in several plant species, their structure, molecular evolution, expression profile, and function have not been fully elucidated in Zea mays L. Results Genome-wide analysis identified nine ZmACBP genes in Z. mays, which could be divided into four distinct classes (class I, class II, class III, and class IV) via construction of a phylogenetic tree that included 48 ACBP genes from six different plant species. Transient expression of a ZmACBP-GFP fusion protein in tobacco (Nicotiana tabacum) epidermal cells revealed that ZmACBPs localized to multiple different locations. Analyses of expression profiles revealed that ZmACBPs exhibited temporal and spatial expression changes during abiotic and biotic stresses. Eight of the nine ZmACBP genes were also found to have significant association with agronomic traits in a panel of 500 maize inbred lines. The heterologous constitutive expression of ZmACBP1 and ZmACBP3 in Arabidopsis enhanced the resistance of these plants to salinity and drought stress, possibly through alterations in the level of lipid metabolic and stress-responsive genes. Conclusion The ACBP gene family was highly conserved across different plant species. ZmACBP genes had clear tissue and organ expression specificity and were responsive to both biotic and abiotic stresses, suggesting their roles in plant growth and stress resistance.

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