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.

[Technological parameter optimization method for washing of coarse bear gall powder extracts based on NIR spectroscopy].

During the washing process of coarse bear gall powder extracts, it is necessary to adjust the amount of ethyl acetate according to the properties of raw materials, which aims to improving the yield and purity of the final product. In the research, using NIR spectra to reflect the comprehensive properties of coarse bear gall powder extracts, the process is optimized in a flexible way. Forty batches experiments are designed according to the weight ratio of ethyl acetate and coarse extracts of bear gall powder. The NIR spectra of the coarse extracts of bear gall powder are collected and processed using principal component analysis (PCA) method. The first 8 principal components combined with the amount of the ethyl acetate are used as the input variables, and calibration models are established to predict the yield and purity of the final product 30 batches are used as calibration set, which is used to establish the models, and other 10 batches are used as validation set, which is used for the performance appraisal of the established models. The correlation coefficients of the calibration, inner cross-validation and external validation for the purity model are 0.902, 0.896 and 0.883, respectively, and the RMSEC, RMSECV and RMSEP are 1.22%, 1.48% and 1.59%, respectively. The correlation coefficients of the calibration, inner cross-validation and external validation for the yield model are 0.921, 0.859 and 0.916, respectively, and the RMSEC, RMSECV and RMSEP are 1.39%, 1.65% and 1.53% respectively. This work demonstrated that NIR spectra combined with technology parameter could be used to predict the yield and purity of the final product. Using the established models, the most appropriate amount of the ethyl acetate can be determined according to the properties of the coarse bear gall powder extracts, and the yield and purity of the final product can be improved.

The protein elicitor Hrip1 enhances resistance to insects and early bolting and flowering in Arabidopsis thaliana.

The elicitor Hrip1 isolated from necrotrophic fungus Alternaria tenuissima, could induce systemic acquired resistance in tobacco to enhance resistance to tobacco mosaic virus. In the present study, we found that the transgenic lines of Hrip1-overexpression in wild type (WT) Arabidopsis thaliana were more resistant to Spodoptera exigua and were early bolting and flowering than the WT. A profiling of transcription assay using digital gene expression profiling was used for transgenic and WT Arabidopsis thaliana. Differentially expressed genes including 40 upregulated and three downregulated genes were identified. In transgenic lines of Hrip1-overexpression, three genes related to jasmonate (JA) biosynthesis were significantly upregulated, and the JA level was found to be higher than WT. Two GDSL family members (GLIP1 and GLIP4) and pathogen-related gene, which participated in pathogen defense action, were upregulated in the transgenic line of Hrip1-overexpression. Thus, Hrip1 is involved in affecting the flower bolting time and regulating endogenous JA biosynthesis and regulatory network to enhance resistance to insect.

Integrated physiological and proteomic analysis reveals underlying response and defense mechanisms of Brachypodium distachyon seedling leaves under osmotic stress, cadmium and their combined stresses.

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 (Cd2+), and their combined stresses. Combined osmotic and Cd2+ 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 Cd2+ and combined stresses grouped much closer than those from osmotic stress, indicating Cd2+ 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 (Cd2+) and their combined stresses.

Proteomic and phosphoproteomic analysis reveals the response and defense mechanism in leaves of diploid wheat T. monococcum under salt stress and recovery.

UNLABELLED: Salinity is a major abiotic stress factor affecting crops production and productivity. Triticum monococcum is closely related to Triticum urartu (A(U)A(U)), which is used as a model plant of wheat A genome study. Here, salt stress induced dynamic proteome and phosphoproteome profiling was focused. The T. monococcum seedlings were initially treated with different concentrations of NaCl ranging from 80 to 320mM for 48h followed by a recovery process for 48h prior to proteomic and phosphoproteomic analysis. As a result, a total of 81 spots corresponding to salt stress and recovery were identified by MALDI-TOF/TOF-MS from 2-DE gels. These proteins were mainly involved in regulatory, stress defense, protein folding/assembly/degradation, photosynthesis, carbohydrate metabolism, energy production and transportation, protein metabolism, and cell structure. Pro-Q Diamond staining was used to detect the phosphoproteins. Finally, 20 spots with different phosphorylation levels during salt treatment or recovery compared with controls were identified. A set of potential salt stress response and defense biomarkers was identified, such as cp31BHv, betaine-aldehyde dehydrogenase, leucine aminopeptidase 2, Cu/Zn superoxide dismutase, and 2-Cys peroxiredoxin BAS1, which could lead to a better understanding of the molecular basis of salt response and defense in food crops. BIOLOGICAL SIGNIFICANCE: Soil salinity reduces the yield of the major crops, which is one of the severest problems in irrigated agriculture worldwide. However, how crops response and defense during different levels of salt treatment and recovery processes is still unclear, especially at the post-translational modification level. T. monococcum is a useful model for common wheat. Thus, proteomic and phosphoproteomic analyses of T. monococcum leaves were performed in our study, which provided novel insights into the underlying salt response and defense mechanisms in wheat and other crops.

Advancements in the maintenance of skin barrier/skin lipid composition and the involvement of metabolic enzymes.

The human skin barrier has an important role in protection and defense, reflected not only in the ability to resist entry of harmful substances into the human body, but also in the ability to prevent loss of water and nutrients. Once the skin barrier is damaged, the skin may become dry, scaly, and wrinkled, and a series of skin problems may occur. In this article, we review the composition of lipids, such as ceramides, cholesterol, and free fatty acids, in the skin and examine the expression of enzymes related to lipid metabolism, such as kallikreins, elongase of elongation of very long-chain fatty acids, hydrolases, and lipid synthases. Additionally, we discuss the involvement of these proteins in skin barrier function and structure. The information presented in this review is expected to provide a theoretical basis for the development of skin care products facilitating the maintenance and repair of skin barrier function.

Integrative proteome analysis of Brachypodium distachyon roots and leaves reveals a synergetic responsive network under H2O2 stress.

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.

[Mutational analysis of Meq, RLORF4, RLORF12 and 132bpr genes of epidemic Marek's disease virus strains highly passaged on chicken embryo fibroblast].

Recently, much work has been devoted to study MD-induced oncogenesis and the genes involved in this process. Among many genes in the MDV genome, several genes such as Meq, RLORF4, RLORF12, and 132bpr have been considered recently associated with virulence of MDV. In this paper, primers of Meq, RLORF4, RLORF12 and 132bpr genes were designed and synthesized, based on the published whole genome sequence of MDV strain GA. The genes of Meq, RLORF4 and RLORF12 from four Chinese epidemic MDV strains highly passaged on chicken embryo fibroblast (CEF), i. e. L-SYp85C, L-MSp75C, L-CZp75C, and L-ZYp75C, as well as their corresponding parent strains, i. e. L-SY, L-MS, L-CZ, and L-ZY, the reference virulent strain J-1 and the vaccine strain 814 were amplified by PCR respectively. Then the PCR products of interest were cloned and sequenced respectively. The results of sequence comparison and analysis of Meq genes in the study indicated that Meq genes from the two strains L-ZYp75C and L-CZp75C contained single nucleotide insertion and deletion. The Meq gene from strain L-ZYp75C contained an extra cytidine (C) insertion at nucleotide position 529 and a single thymidine (T) deletion at nucleotide position 602, resulting in a frameshift mutation. And this frameshift mutation could lead to changes in deduced amino acid sequence from position 177 to 200 of Meq gene. The extra C insertion at nucleotide position 625 in Meq gene of strain L-CZp75C was also predicted to cause frameshift mutation in three overlapping genes (Meq, RLORF6 and 23KD genes). The comparison of nucleotide sequences of RLORF4 genes in the study revealed that the RLORF4 gene of strain L-SYp85C contained a fragment deletion in Open Reading Frame (ORF) from nucleotide position 215 to 265, resulting in 17 amino acids deletions, which were not found in other sequenced strains. Comparison of nucleotide sequences of RLORF12 genes in the study revealed several mutations. The RLORF12 gene of strain L-MSp75C contained a single T deletion at nucleotide position 67 and of 814 vaccine strain a large fragment deletion from nucleotide position 18 to 86, both of the deletions located in Origin of replication site (Ori) of MDV genome. But strain L-ZYp75C possessed an unique "TGTTGGG" deletion in its RLORF12 gene. When the four Chinese epidemic MDV strains were serially passaged on CEF, the number of copies of the 132bp repeats increased from 2 to more than 10 copies. All of above results indicated that deletion and/or insertion mutation occurred in Meq, RLORF4, RLORF12 and 132bpr after serial passage of these four Chinese epidemic MDV strains on CEF.