N‑Linked Glycoproteome Profiling of Seedling Leaf in <i>Brachypodium distachyon</i> L.

<i>Brachypodium distachyon</i> 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 <i>B. distachyon</i>. 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 <i>B. distachyon</i> and supplement the glycoproteome databases of plants

Integrative Network Analysis of the Signaling Cascades in Seedling Leaves of Bread Wheat by Large-Scale Phosphoproteomic Profiling

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

Comparative Phosphoproteome Analysis of the Developing Grains in Bread Wheat (<i>Triticum aestivum</i> 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

Integrative Network Analysis of the Signaling Cascades in Seedling Leaves of Bread Wheat by Large-Scale Phosphoproteomic Profiling

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

Integrative Network Analysis of the Signaling Cascades in Seedling Leaves of Bread Wheat by Large-Scale Phosphoproteomic Profiling

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

Integrative Network Analysis of the Signaling Cascades in Seedling Leaves of Bread Wheat by Large-Scale Phosphoproteomic Profiling

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

Integrative Network Analysis of the Signaling Cascades in Seedling Leaves of Bread Wheat by Large-Scale Phosphoproteomic Profiling

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

Comparative Phosphoproteome Analysis of the Developing Grains in Bread Wheat (<i>Triticum aestivum</i> L.) under Well-Watered and Water-Deficit Conditions

Wheat (<i>Triticum aestivum</i>), 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

BGLF4 binds to and phosphorylates key proteins of the spindle assembly checkpoint.

<p>(A) BGLF4 induced phosphorylation of proteins involved in SAC (spindle assembly checkpoint) activation and APC/C (anaphase promoting complex /cyclosome) inhibition. P: phosphorylation; Ac: acetylation. (B) Western blot analysis showing co-precipitation of BGLF4 with MPS1, CDC20 and p31^Comet but not BUB1. BGLF4 was co-transfected with MPS1, BUB1, CDC20 or p31^Comet in 293T cells as indicated. Cell lysates were immunoprecipitated with anti-HA antibody, anti-Myc or IgG control followed by immunoblotting with anti-BGLF4, anti-HA, anti-Flag or anti-Myc antibodies as indicated. (C) BGLF4 phosphorylates TIP60, MPS1, PP1α and CDC20. (Upper) Purified wild-type (WT) and kinase-dead (KD) BGLF4 were mixed with GST-TIP60 (aa 1–290), GST-MPS1 (a.a. 410–517), GST-PP1α, GST-CDC20 and GST. Autoradiography showing only WT BGLF4 phosphorylates TIP60, MPS1, PP1α and CDC20. GST-TIP60 (a.a. 1–290) and GST were included as positive and negative controls respectively. Arrows indicate the positions of the phosphorylated substrates. (Lower) Immunoblot showing purified WT and KD BGLF4 proteins (Elute) used in the assay. Arrows indicate the positions of purified untagged BGLF4 (Elute) and Halo-BGLF4 proteins in the total cell lysate (Lysate). See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005346#ppat.1005346.s005" target="_blank">S5 Fig</a>.</p

BGLF4-induced phosphorylation of nuclear pore associated proteins.

<p>Localization of BGLF4 up-regulated phosphoproteins associated with the nuclear pore as identified using the DAVID bioinformatics resource and manual literature curation. See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005346#ppat.1005346.s011" target="_blank">S6 Table</a>.</p