Shoumin Zhen

Comparative phosphoproteome analysis of the developing grains in bread wheat (Triticum aestivum L.) under well-watered and water-deficit conditions.

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.

First Comprehensive Proteome Analyses of Lysine Acetylation and Succinylation in Seedling Leaves of Brachypodium distachyon L.

Protein acetylation and succinylation are the most crucial protein post-translational modifications (PTMs) involved in the regulation of plant growth and development. In this study, we present the first lysine-acetylation and lysine-succinylation proteome analysis of seedling leaves in Brachypodium distachyon L (Bd). Using high accuracy nano LC-MS/MS combined with affinity purification, we identified a total of 636 lysine-acetylated sites in 353 proteins and 605 lysine-succinylated sites in 262 proteins. These proteins participated in many biology processes, with various molecular functions. In particular, 119 proteins and 115 sites were found to be both acetylated and succinylated, simultaneously. Among the 353 acetylated proteins, 148 had acetylation orthologs in Oryza sativa L., Arabidopsis thaliana, Synechocystis sp. PCC 6803, and Glycine max L. Among the 262 succinylated proteins, 170 of them were found to have homologous proteins in Oryza sativa L., Escherichia coli, Sacchayromyces cerevisiae, or Homo sapiens. Motif-X analysis of the acetylated and succinylated sites identified two new acetylated motifs (K---K and K-I-K) and twelve significantly enriched succinylated motifs for the first time, which could serve as possible binding loci for future studies in plants. Our comprehensive dataset provides a promising starting point for further functional analysis of acetylation and succinylation in Bd and other plant species.

Proteomic Analysis Reveals Key Proteins and Phosphoproteins upon Seed Germination of Wheat (Triticum aestivum L.)

Wheat (Triticum aestivum L.) is one of the oldest cultivated crops and the second most important food crop in the world. Seed germination is the key developmental process in plant growth and development, and poor germination directly affects plant growth and subsequent grain yield. In this study, we performed the first dynamic proteome analysis of wheat seed germination using a two-dimensional differential gel electrophoresis (2D-DIGE)-based proteomic approach. A total of 166 differentially expressed protein (DEP) spots representing 73 unique proteins were identified, which are mainly involved in storage, stress/defense/detoxification, carbohydrate metabolism, photosynthesis, cell metabolism, and transcription/translation/transposition. The identified DEPs and their dynamic expression profiles generally correspond to three distinct seed germination phases after imbibition: storage degradation, physiological processes/morphogenesis, and photosynthesis. Some key DEPs involved in storage substance degradation and plant defense mechanisms, such as globulin 3, sucrose synthase type I, serpin, beta-amylase, and plastid ADP-glucose pyrophosphorylase (AGPase) small subunit, were found to be phosphorylated during seed germination. Particularly, the phosphorylation site Ser355 was found to be located in the enzyme active region of beta-amylase, which promotes substrate binding. Phosphorylated modification of several proteins could promote storage substance degradation and environmental stress defense during seed germination. The central metabolic pathways involved in wheat seed germination are proposed herein, providing new insights into the molecular mechanisms of cereal seed germination.

Molecular Characterization of the 14-3-3 Gene Family in Brachypodium distachyon L. Reveals High Evolutionary Conservation and Diverse Responses to Abiotic Stresses

The 14-3-3 gene family identified in all eukaryotic organisms is involved in a wide range of biological processes, particularly in resistance to various abiotic stresses. Here, we performed the first comprehensive study on the molecular characterization, phylogenetics, and responses to various abiotic stresses of the 14-3-3 gene family in Brachypodium distachyon L. A total of seven 14-3-3 genes from B. distachyon and 120 from five main lineages among 12 species were identified, which were divided into five well-conserved subfamilies. The molecular structure analysis showed that the plant 14-3-3 gene family is highly evolutionarily conserved, although certain divergence had occurred in different subfamilies. The duplication event investigation revealed that segmental duplication seemed to be the predominant form by which the 14-3-3 gene family had expanded. Moreover, seven critical amino acids were detected, which may contribute to functional divergence. Expression profiling analysis showed that BdGF14 genes were abundantly expressed in the roots, but showed low expression in the meristems. All seven BdGF14 genes showed significant expression changes under various abiotic stresses, including heavy metal, phytohormone, osmotic, and temperature stresses, which might play important roles in responses to multiple abiotic stresses mainly through participating in ABA-dependent signaling and reactive oxygen species-mediated MAPK cascade signaling pathways. In particular, BdGF14 genes generally showed upregulated expression in response to multiple stresses of high temperature, heavy metal, abscisic acid (ABA), and salicylic acid (SA), but downregulated expression under H2O2, NaCl, and polyethylene glycol (PEG) stresses. Meanwhile, dynamic transcriptional expression analysis of BdGF14 genes under longer treatments with heavy metals (Cd2+, Cr3+, Cu2+, and Zn2+) and phytohormone (ABA) and recovery revealed two main expression trends in both roots and leaves: up-down and up-down-up expression from stress treatments to recovery. This study provides new insights into the structures and functions of plant 14-3-3 genes.

Large-scale phosphoproteome analysis in seedling leaves of Brachypodium distachyon L.

BackgroundProtein phosphorylation is one of the most important post-translational modifications involved in the regulation of plant growth and development as well as diverse stress response. As a member of the Poaceae, Brachypodium distachyon L. is a new model plant for wheat and barley as well as several potential biofuel grasses such as switchgrass. Vegetative growth is vital for biomass accumulation of plants, but knowledge regarding the role of protein phosphorylation modification during vegetative growth, especially in biofuel plants, is far from comprehensive.ResultsIn this study, we carried out the first large-scale phosphoproteome analysis of seedling leaves in Brachypodium accession Bd21 using TiO2 microcolumns combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS) and MaxQuant software. A total of 1470 phosphorylation sites in 950 phosphoproteins were identified, and these phosphoproteins were implicated in various molecular functions and basic cellular processes by gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses. Among the 950 phosphoproteins identified, 127 contained 3 to 8 phosphorylation sites. Conservation analysis showed that 93.4% of the 950 phosphoproteins had phosphorylation orthologs in other plant species. Motif-X analysis of the phosphorylation sites identified 13 significantly enriched phosphorylation motifs, of which 3 were novel phosphorylation motifs. Meanwhile, there were 91 phosphoproteins with both multiple phosphorylation sites and multiple phosphorylation motifs. In addition, we identified 58 phosphorylated transcription factors across 21 families and found out 6 significantly over-represented transcription factor families (C3H, Trihelix, CAMTA, TALE, MYB_related and CPP). Eighty-four protein kinases (PKs), 8 protein phosphatases (PPs) and 6 CESAs were recognized as phosphoproteins.ConclusionsThrough a large-scale bioinformatics analysis of the phosphorylation data in seedling leaves, a complicated PKs- and PPs- centered network related to rapid vegetative growth was deciphered in B. distachyon. We revealed a MAPK cascade network that might play the crucial roles during the phosphorylation signal transduction in leaf growth and development. The phosphoproteins and phosphosites identified from our study expanded our knowledge of protein phosphorylation modification in plants, especially in monocots.

Comparative transcriptome analysis of wheat embryo and endosperm responses to ABA and H2O2 stresses during seed germination

BackgroundWheat embryo and endosperm play important roles in seed germination, seedling survival, and subsequent vegetative growth. ABA can positively regulate dormancy induction and negatively regulates seed germination at low concentrations, while low H2O2 concentrations promote seed germination of cereal plants. In this report, we performed the first integrative transcriptome analysis of wheat embryo and endosperm responses to ABA and H2O2 stresses.ResultsWe used the GeneChip® Wheat Genome Array to conduct a comparative transcriptome microarray analysis of the embryo and endosperm of elite Chinese bread wheat cultivar Zhengmai 9023 in response to ABA and H2O2 treatments during seed germination. Transcriptome profiling showed that after H2O2 and ABA treatments, the 64 differentially expressed genes in the embryo were closely related to DNA synthesis, CHO metabolism, hormone metabolism, and protein degradation, while 121 in the endosperm were involved mainly in storage reserves, transport, biotic and abiotic stresses, hormone metabolism, cell wall metabolism, signaling, and development. Scatter plot analysis showed that ABA treatment increased the similarity of regulated patterns between the two tissues, whereas H2O2 treatment decreased the global expression similarity. MapMan analysis provided a global view of changes in several important metabolism pathways (e.g., energy reserves mobilization, cell wall metabolism, and photosynthesis), as well as related functional groups (e.g., cellular processes, hormones, and signaling and transport) in the embryo and endosperm following exposure of seeds to ABA and H2O2 treatments during germination. Quantitative RT-PCR analysis was used to validate the expression patterns of nine differentially expressed genes.ConclusionsWheat seed germination involves regulation of a large number of genes involved in many functional groups. ABA/H2O2 can repress/promote seed germination by coordinately regulating related gene expression. Our results provide novel insights into the transcriptional regulation mechanisms of embryo and endosperm in response to ABA and H2O2 treatments during seed germination.

Comparative Phosphoproteomic Analysis under High-Nitrogen Fertilizer Reveals Central Phosphoproteins Promoting Wheat Grain Starch and Protein Synthesis.

Nitrogen (N) is a macronutrient important for plant growth and development. It also strongly influences starch and protein synthesis, closely related to grain yield and quality. We performed the first comparative phosphoproteomic analysis of developing wheat grains in response to high-N fertilizer. Physiological and biochemical analyses showed that application of high-N fertilizer resulted in significant increases in leaf length and area, chlorophyll content, the activity of key enzymes in leaves such as nitrate reductase (NR), and in grains such as sucrose phosphate synthase (SPS), sucrose synthase (SuSy), and ADP glucose pyrophosphorylase (AGPase). This enhanced enzyme activity led to significant improvements in starch content, grain yield, and ultimately, bread making quality. Comparative phosphoproteomic analysis of developing grains under the application of high-N fertilizer performed 15 and 25 days post-anthesis identified 2470 phosphosites among 1372 phosphoproteins, of which 411 unique proteins displayed significant changes in phosphorylation level (>2-fold or <0.5-fold). These phosphoproteins are involved mainly in signaling transduction, starch synthesis, energy metabolism. Pro-Q diamond staining and Western blotting confirmed our phosphoproteomic results. We propose a putative pathway to elucidate the important roles of the central phosphoproteins regulating grain starch and protein synthesis. Our results provide new insights into the molecular mechanisms of protein phosphorylation modifications involved in grain development, yield and quality formation.

Deletion of the low-molecular-weight glutenin subunit allele Glu-A3a of wheat (Triticum aestivum L.) significantly reduces dough strength and breadmaking quality

BackgroundLow-molecular-weight glutenin subunits (LMW-GS), encoded by Glu-3 complex loci in hexaploid wheat, play important roles in the processing quality of wheat flour. To date, the molecular characteristics and effects on dough quality of individual Glu-3 alleles and their encoding proteins have been poorly studied. We used a Glu-A3 deletion line of the Chinese Spring (CS-n) wheat variety to conduct the first comprehensive study on the molecular characteristics and functional properties of the LMW-GS allele Glu-A3a.ResultsThe Glu-A3a allele at the Glu-A3 locus in CS and its deletion in CS-n were identified and characterized by proteome and molecular marker methods. The deletion of Glu-A3a had no significant influence on plant morphological and yield traits, but significantly reduced the dough strength and breadmaking quality compared to CS. The complete sequence of the Glu-A3a allele was cloned and characterized, which was found to encode a B-subunit with longer repetitive domains and an increased number of α-helices. The Glu-A3a-encoded B-subunit showed a higher expression level and accumulation rate during grain development. These characteristics of the Glu-A3a allele could contribute to achieving superior gluten quality and demonstrate its potential application to wheat quality improvement. Furthermore, an allele-specific polymerase chain reaction (AS-PCR) marker for the Glu-A3a allele was developed and validated using different bread wheat cultivars, including near-isogenic lines (NILs) and recombinant inbred lines (RILs), which could be used as an effective molecular marker for gluten quality improvement through marker-assisted selection.ConclusionsThis work demonstrated that the LMW-GS allele Glu-A3a encodes a specific LMW-i type B-subunit that significantly affects wheat dough strength and breadmaking quality. The Glu-A3a-encoded B-subunit has a long repetitive domain and more α-helix structures as well as a higher expression level and accumulation rate during grain development, which could facilitate the formation of wheat with a stronger dough structure and superior breadmaking quality.

Dynamic metabolome profiling reveals significant metabolic changes during grain development of bread wheat (Triticum aestivum L.)

BACKGROUND Metabolites in wheat grains greatly influence nutritional values. Wheat provides proteins, minerals, B-group vitamins and dietary fiber to humans. These metabolites are important to human health. However, the metabolome of the grain during the development of bread wheat has not been studied so far. In this work the first dynamic metabolome of the developing grain of the elite Chinese bread wheat cultivar Zhongmai 175 was analyzed, using non-targeted gas chromatography/mass spectrometry (GC/MS) for metabolite profiling. RESULTS In total, 74 metabolites were identified over the grain developmental stages. Metabolite-metabolite correlation analysis revealed that the metabolism of amino acids, carbohydrates, organic acids, amines and lipids was interrelated. An integrated metabolic map revealed a distinct regulatory profile. The results provide information that can be used by metabolic engineers and molecular breeders to improve wheat grain quality. CONCLUSION The present metabolome approach identified dynamic changes in metabolite levels, and correlations among such levels, in developing seeds. The comprehensive metabolic map may be useful when breeding programs seek to improve grain quality. The work highlights the utility of GC/MS-based metabolomics, in conjunction with univariate and multivariate data analysis, when it is sought to understand metabolic changes in developing seeds.

Integrated Proteome Analysis of the Wheat Embryo and Endosperm Reveals Central Metabolic Changes Involved in the Water Deficit Response during Grain Development.

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.