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Drought stress, a major abiotic stress, commonly occurs in metal-contaminated environments and affects crop growth and yield. In this study, we performed the first integrated phenotypic, physiological, and proteomic analysis of Brachypodium distachyon L. seedling leaves under polyethylene glycol (PEG) mock osmotic stress, cadmium (Cd 2+ ), and their combined stresses. Combined osmotic and Cd 2+ stress had more significant effects than each individual stress on seedling growth, and the physiological traits and ultrastructures of leaves. Totally 117 differentially accumulated protein (DAP) spots detected by two-dimensional difference gel electrophoresis (2D-DIGE) were identified, and representing 89 unique proteins under individual and combined stresses. These DAPs were involved in photosynthesis/respiration (34%), energy and carbon metabolism (21%), stress/defense/detoxification (13%), protein folding and degradation (12%), and amino acid metabolism (7%). Principal component analysis (PCA) revealed that DAPs from the Cd 2+ and combined stresses grouped much closer than those from osmotic stress, indicating Cd 2+ and combined stresses resulted in more changes to the leaf proteome than osmotic stress alone. Protein-protein interaction analyses showed that a 14-3-3 centered sub-network could play important roles in responses to abiotic stresses. An overview pathway of proteome metabolic changes in Bd21 seedling leaves under combined stresses is proposed, representing a synergistic responsive network and underlying response and defense mechanisms., Significance: Drought stress is one of the major abiotic stresses, which commonly occurs in metal-contaminated environments, and affects crop growth and yield performance. We performed the first integrated phenotypic, physiological and proteomic analysis of Brachypodium distachyon L. seedling leaves under drought (PEG), cadmium (Cd 2+ ) and their combined stresses., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Drought stress is a major abiotic stress affecting plant growth and development. In this study, we performed the first dynamic phosphoproteome analysis of Brachypodium distachyon L. seedling leaves under drought stress for different times. A total of 4924 phosphopeptides, contained 6362 phosphosites belonging to 2748 phosphoproteins. Rigorous standards were imposed to screen 484 phosphorylation sites, representing 442 unique phosphoproteins. Comparative analyses revealed significant changes in phosphorylation levels at 0, 6, and 24 h under drought stress. The most phosphorylated proteins and the highest phosphorylation level occurred at 6 h. Venn analysis showed that the up-regulated phosphopeptides at 6 h were almost two-fold those at 24 h. Motif-X analysis identified the six motifs: [sP], [Rxxs], [LxRxxs], [sxD], [sF], and [TP], among which [LxRxxs] was also previously identified in B. distachyon. Results from molecular function and protein-protein interaction analyses suggested that phosphoproteins mainly participate in signal transduction, gene expression, drought response and defense, photosynthesis and energy metabolism, and material transmembrane transport. These phosphoproteins, which showed significant changes in phosphorylation levels, play important roles in signal transduction and material transmembrane transport in response to drought conditions. Our results provide new insights into the molecular mechanism of this plant's abiotic stress response through phosphorylation modification.

The plant oxidative stress response is vital for defense against various abiotic and biotic stresses. In this study, ultrastructural changes and the proteomic response to H2O2 stress in roots and leaves of the model plant Brachypodium distachyon were studied. Transmission electron microscopy (TEM) showed that the ultrastructural damage in roots was more serious than in leaves. Particularly, the ultrastructures of organelles and the nucleus in root tip cells were damaged, leading to the inhibition of normal biological activities of roots, which then spread throughout the plant. Based on two-dimensional electrophoresis (2-DE) and MALDI-TOF/TOF-MS, 84 and 53 differentially accumulated protein (DAP) spots representing 75 and 45 unique proteins responsive to H2O2 stress in roots and leaves, respectively, were identified. These protein species were mainly involved in signal transduction, energy metabolism, redox homeostasis/stress defense, protein folding/degradation, and cell wall/cell structure. Interestingly, two 14-3-3 proteins (GF14-B and GF14-D) were identified as DAPs in both roots and leaves. Protein-protein interaction (PPI) analysis revealed a synergetic H2O2-responsive network., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Brachypodium distachyon 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 B. distachyon. 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 B. distachyon and supplement the glycoproteome databases of plants.

Background: Protein 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., Results: In 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., Conclusions: Through 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.

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.

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.

Thirteen compounds were isolated from the leaves of Rhododendron rubiginosum var. rubiginosum by various chromatographic techniques. On the basis of spectroscopic data, their structures were elucidated as 3,9-dihydroxy-megastigma-5-ene (1), 3 beta-hydroxy-5alpha ,6 alpha-epoxy-7-megastigmen-9-one (2), loliolide (3), ursolic acid(4), 2 alpha, 3 beta-dihydroxy-urs-12-en-28-oic acid (5), 2 alpha, 3 beta,23-trihydroxy-urs-12-en-28-oic acid (6), 7,9-dimethoxyrhododendrol (7), 7-methoxyrhododendrol (8), zingerone (9), isofraxidin (10), scopoletin (11), (+)-pinoresinol (12) and 3'-O-demethylepipinorisenol (13). All compounds were isolated from this plant for the first time, and compounds 1-3, 7-9, and 11-13 were isolated from the genus Rhododendron for the first time.

Objective: To study the chemical constituents from the leaf of Hydnocarpus hainanensis., Methods: Compounds were isolated and purified by silica gel and Sephadex LH-20 column chromatography, their structures were identified by spectroscopic analysis., Results: Nine compounds were isolated and identified as glutinol (I), fernenol (II), lupeol (III), a-armyrin (IV), 2, 9-dimethyldeca-2, 8-diene (V), phytenal (VI), phytol (VII), 3, 7, 11,15-tetramethylhexadecane-1,2-diol ( VI), 3, 5-dimethoxy-4-hydroxybenzaldehyde (IX)., Conclusion: All the compounds are isolated from this plant for the first time.