Endoplasmic Reticulum Subproteome Analysis Reveals Underlying Defense Mechanisms of Wheat Seedling Leaves under Salt Stress.

Salt stress is the second most important abiotic stress factor in the world, which seriously affects crop growth, development and grain production. In this study, we performed the first integrated physiological and endoplasmic reticulum (ER) proteome analysis of wheat seedling leaves under salt stress using a label-free-based quantitative proteomic approach. Salt stress caused significant decrease in seedling height, root length, relative water content and chlorophyll content of wheat seedling leaves, indicating that wheat seedling growth was significantly inhibited under salt stress. The ER proteome analysis identified 233 ER-localized differentially accumulated proteins (DAPs) in response to salt stress, including 202 upregulated and 31 downregulated proteins. The upregulated proteins were mainly involved in the oxidation-reduction process, transmembrane transport, the carboxylic acid metabolic process, stress response, the arbohydrate metabolic process and proteolysis, while the downregulated proteins mainly participated in the metabolic process, biological regulation and the cellular process. In particular, salt stress induced significant upregulation of protein disulfide isomerase-like proteins and heat shock proteins and significant downregulation of ribosomal protein abundance. Further transcript expression analysis revealed that half of the detected DAP genes showed a consistent pattern with their protein levels under salt stress. A putative metabolic pathway of ER subproteome of wheat seedling leaves in response to salt stress was proposed, which reveals the potential roles of wheat ER proteome in salt stress response and defense.

Genome-wide analysis of wheat DNA-binding with one finger (Dof) transcription factor genes: evolutionary characteristics and diverse abiotic stress responses.

BACKGROUND: DNA binding with one finger (Dof) transcription factors play important roles in plant growth and abiotic stress responses. Although genome-wide identification and analysis of the DOF transcription factor family has been reported in other species, no relevant studies have emerged in wheat. The aim of this study was to investigate the evolutionary and functional characteristics associated with plant growth and abiotic stress responses by genome-wide analysis of the wheat Dof transcription factor gene family. RESULTS: Using the recently released wheat genome database (IWGSC RefSeq v1.0), we identified 96 wheat Dof gene family members, which were phylogenetically clustered into five distinct subfamilies. Gene duplication analysis revealed a broad and heterogeneous distribution of TaDofs on the chromosome groups 1 to 7, and obvious tandem duplication genes were present on chromosomes 2 and 3.Members of the same gene subfamily had similar exon-intron structures, while members of different subfamilies had obvious differences. Functional divergence analysis indicated that type-II functional divergence played a major role in the differentiation of the TaDof gene family. Positive selection analysis revealed that the Dof gene family experienced different degrees of positive selection pressure during the process of evolution, and five significant positive selection sites (30A, 31 T, 33A, 102G and 104S) were identified. Additionally, nine groups of coevolving amino acid sites, which may play a key role in maintaining the structural and functional stability of Dof proteins, were identified. The results from the RNA-seq data and qRT-PCR analysis revealed that TaDof genes exhibited obvious expression preference or specificity in different organs and developmental stages, as well as in diverse abiotic stress responses. Most TaDof genes were significantly upregulated by heat, PEG and heavy metal stresses. CONCLUSIONS: The genome-wide analysis and identification of wheat DOF transcription factor family and the discovery of important amino acid sites are expected to provide new insights into the structure, evolution and function of the plant Dof gene family.

Retraction Note to: Integrated physiological and proteomic analysis of embryo and endosperm reveals central salt stress response proteins during seed germination of winter wheat cultivar Zhengmai 366.

The editor has retracted this article [1] because parts of Figs. 1 and 4 were duplicated from a previously published paper by the same authors [2] without appropriate disclosure. None of the authors have responded to any correspondence from the editor about publication of this retraction notice.

Genome-wide identification and expression analysis of expansin gene family in common wheat (Triticum aestivum L.).

BACKGROUND: Expansin loosens plant cell walls and involves in cell enlargement and various abiotic stresses. Plant expansin superfamily contains four subfamilies: α-expansin (EXPA), ß-expansin (EXPB), expansin-like A (EXLA), and expansin-like B (EXLB). In this work, we performed a comprehensive study on the molecular characterization, phylogenetic relationship and expression profiling of common wheat (Triticum aestivum) expansin gene family using the recently released wheat genome database (IWGSC RefSeq v1.1 with a coverage rate of 94%). RESULTS: Genome-wide analysis identified 241 expansin genes in the wheat genome, which were grouped into three subfamilies (EXPA, EXPB and EXLA) by phylogenetic tree. Molecular structure analysis showed that wheat expansin gene family showed high evolutionary conservation although some differences were present in different subfamilies. Some key amino acid sites that contribute to functional divergence, positive selection, and coevolution were detected. Evolutionary analysis revealed that wheat expansin gene superfamily underwent strong positive selection. The transcriptome map and qRT-PCR analysis found that wheat expansin genes had tissue/organ expression specificity and preference, and generally highly expressed in the roots. The expression levels of some expansin genes were significantly induced by NaCl and polyethylene glycol stresses, which was consistent with the differential distribution of the cis-elements in the promoter region. CONCLUSIONS: Wheat expansin gene family showed high evolutionary conservation and wide range of functional divergence. Different selection constraints may influence the evolution of the three expansin subfamilies. The different expression patterns demonstrated that expansin genes could play important roles in plant growth and abiotic stress responses. This study provides new insights into the structures, evolution and functions of wheat expansin gene family.

Integrated physiological and proteomic analysis of embryo and endosperm reveals central salt stress response proteins during seed germination of winter wheat cultivar Zhengmai 366.

BACKGROUND: Salinity is a major abiotic stressor that affects seed germination, plant growth, and crop production. Seed germination represents the beginning of plant growth and is closely linked with subsequent crop development and ultimate yield formation. This study attempted to extend findings regarding the potential proteomic dynamics during wheat seed germination under salt stress and to explore the mechanism of crop salt response. RESULTS: Salt stress significantly affected seed physiological activities during the germination process, resulting in significant decreases in phytohormone and α-amylase activity and significant increases in soluble sugar, starch, and ADP glucose pyrophosphorylase activity. A comparative proteomics approach was applied to analyze the dynamic proteome changes of embryo and endosperm during seed germination in Chinese winter wheat cultivar Zhengmai 366 under salt stress. Two-dimensional electrophoresis identified 92 and 61 differentially accumulated proteins (DAPs) in response to salt stress in embryo and endosperm, respectively. Both organs contained a high proportion of DAPs involved in stress defense, energy metabolism, and protein/amino acid metabolism. The endosperm had more DAPs related to storage proteins and starch metabolism than the embryo, and 2% of DAPs participating in lipid and sterol metabolism were specifically detected in the embryo. CONCLUSIONS: Seed physiological activities were significantly affected during the germination process when subjected to salt stress. The DAPs involved in stress defense and energy metabolism were upregulated whereas those related to reserve substance degradation and protein/amino acid metabolism were significantly downregulated, leading to delayed seed germination under salt stress. Our proteomic results revealed synergistic regulation of the response to salt stress during seed germination.

Integrated proteome analyses of wheat glume and awn reveal central drought response proteins under water deficit conditions.

MAIN CONCLUSION: Integrated proteome analyses revealed differentially accumulated proteins in the non-leaf green organs in wheat glume and awn that play important roles in photosynthesis and drought resistance. Two non-leaf green organs in wheat, glume and awn, have photosynthetic potential, contribute to grain yield, and also play roles in resistance to adverse conditions. We performed the first integrated proteome analysis of wheat glume and awn in response to water deficit. Water deficit caused a significant decrease in important agronomic traits and grain yield. A total of 120 and 77 differentially accumulated protein (DAP) spots, representing 100 and 67 unique proteins responsive to water deficit, were identified by two-dimensional difference gel electrophoresis (2D-DIGE) in glumes and awns, respectively, of the elite Chinese bread wheat cultivar Zhongmai 175. The DAPs of both organs showed similar functional classification and proportion and were mainly involved in photosynthesis, detoxification/defense, carbon/energy metabolism, and proteometabolism. Comparative proteome analyses revealed many more drought-responsive DAP spots in glumes than in awns, which indicate that glumes underwent more proteome changes in response to water deficit. The main DAPs involved in photosynthesis and carbon metabolism were significantly downregulated, whereas those related to detoxification/defense and energy metabolism were markedly upregulated under water deficit. The potential functions of the identified DAPs revealed an intricate interaction network that responds synergistically to drought stress during grain development. Our results from the proteome perspective illustrate the potential roles of wheat non-leaf green organs glume and awn in photosynthetic and defensive responses under drought stress.

Genome-wide identification, molecular evolution, and expression analysis of auxin response factor (ARF) gene family in Brachypodium distachyon L.

BACKGROUND: The auxin response factor (ARF) gene family is involved in plant development and hormone regulation. Although the ARF gene family has been studied in some plant species, its structural features, molecular evolution, and expression profiling in Brachypodium distachyon L. are still not clear. RESULTS: Genome-wide analysis identified 19 ARF genes in B. distachyon. A phylogenetic tree constructed with 182 ARF genes from seven plant species revealed three different clades, and the ARF genes from within a clade exhibited structural conservation, although certain divergences occurred in different clades. The branch-site model identified some sites where positive selection may have occurred, and functional divergence analysis found more Type II divergence sites than Type I. In particular, both positive selection and functional divergence may have occurred in 241H, 243G, 244 L, 310 T, 340G and 355 T. Subcellular localization prediction and experimental verification indicated that BdARF proteins were present in the nucleus. Transcript expression analysis revealed that BdARFs were mainly expressed in the leaf and root tips, stems, and developing seeds. Some BdARF genes exhibited significantly upregulated expression under various abiotic stressors. Particularly, BdARF4 and BdARF8 were significantly upregulated in response to abiotic stress factors such as salicylic acid and heavy metals. CONCLUSION: The ARF gene family in B. distachyon was highly conserved. Several important amino acid sites were identified where positive selection and functional divergence occurred, and they may play important roles in functional differentiation. BdARF genes had clear tissue and organ expression preference and were involved in abiotic stress response, suggesting their roles in plant growth and stress resistance.

Integrated physiology and proteome analysis of embryo and endosperm highlights complex metabolic networks involved in seed germination in wheat (Triticum aestivum L.).

The aim of this study was to investigate the physiological and proteomic changes in the embryo and endosperm during seed germination in the elite Chinese bread wheat cultivar Zhengmai 366. Upon imbibition, seed size and water content increased rapidly, followed by a series of metabolic changes including increases in soluble sugar content and α-amylase activity, a decrease in starch content, and a rapid increase in plant hormones. In total, 57 and 45 differentially accumulated proteins (DAPs) from the embryo and endosperm, respectively, were identified at five germination stages (0, 6, 12, 18, and 24 h). Principal component analysis revealed a significant proteome difference between embryo and endosperm as well as the different germination stages. The largest proteome changes occurred 24 h after seed imbibition. Embryo DAP spots were mainly involved in energy metabolism, amino acid metabolism, stress/defense, and protein metabolism; those from the endosperm were primarily related to storage protein and carbohydrate metabolism. Protein-protein interaction analysis revealed a complicated interaction network between energy-related proteins and other proteins. Metabolic pathway analysis highlighted complex regulatory networks in the embryo and endosperm that regulate wheat seed germination. These results provide new insights into the molecular mechanisms of seed germination.

Comparative Proteome Analysis of Wheat Flag Leaves and Developing Grains Under Water Deficit.

In this study, we performed the first comparative proteomic analysis of wheat flag leaves and developing grains in response to drought stress. Drought stress caused a significant decrease in several important physiological and biochemical parameters and grain yield traits, particularly those related to photosynthesis and starch biosynthesis. In contrast, some key indicators related to drought stress were significantly increased, including malondialdehyde, soluble sugar, proline, glycine betaine, abscisic acid content, and peroxidase activity. Two-dimensional difference gel electrophoresis (2D-DIGE) identified 87 and 132 differentially accumulated protein (DAP) spots representing 66 and 105 unique proteins following exposure to drought stress in flag leaves and developing grains, respectively. The proteomes of the two organs varied markedly, and most DAPS were related to the oxidative stress response, photosynthesis and energy metabolism, and starch biosynthesis. In particular, DAPs in flag leaves mainly participated in photosynthesis while those in developing grains were primarily involved in carbon metabolism and the drought stress response. Western blotting and quantitative real-time polymerase chain reaction (qRT-PCR) further validated some key DAPs such as rubisco large subunit (RBSCL), ADP glucose pyrophosphorylase (AGPase), chaperonin 60 subunit alpha (CPN-60 alpha) and oxalate oxidase 2 (OxO 2). The potential functions of the identified DAPs revealed that a complex network synergistically regulates drought resistance during grain development. Our results from proteome perspective provide new insight into the molecular regulatory mechanisms used by different wheat organs to respond to drought stress.

Effects of water deficit on breadmaking quality and storage protein compositions in bread wheat (Triticum aestivum L.).

BACKGROUND: Water deficiency affects grain proteome dynamics and storage protein compositions, resulting in changes in gluten viscoelasticity. In this study, the effects of field water deficit on wheat breadmaking quality and grain storage proteins were investigated. RESULTS: Water deficiency produced a shorter grain-filling period, a decrease in grain number, grain weight and grain yield, a reduced starch granule size and increased protein content and glutenin macropolymer contents, resulting in superior dough properties and breadmaking quality. Reverse phase ultra-performance liquid chromatography analysis showed that the total gliadin and glutenin content and the accumulation of individual components were significantly increased by water deficiency. Two-dimensional gel electrophoresis detected 144 individual storage protein spots with significant accumulation changes in developing grains under water deficit. Comparative proteomic analysis revealed that water deficiency resulted in significant upregulation of 12 gliadins, 12 high-molecular-weight glutenin subunits and 46 low-molecular-weight glutenin subunits. Quantitative real-time polymerase chain reaction analysis revealed that the expression of storage protein biosynthesis-related transcription factors Dof and Spa was upregulated by water deficiency. CONCLUSION: The present results illustrated that water deficiency leads to increased accumulation of storage protein components and upregulated expression of Dof and Spa, resulting in an improvement in glutenin strength and breadmaking quality. © 2018 Society of Chemical Industry.