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Abscisic acid (ABA) is a phytohormone and a major determinant of seed dormancy in plants. Seed dormancy is gradually lost during dry storage, a process known as 'after-ripening', and this dormancy decay is related to a decline in ABA content and sensitivity in seeds after imbibition. In this study, we aimed at investigating the effect of after-ripening on ABA signaling in barley, our cereal model species. Phosphosignaling networks in barley grains were investigated by a large-scale analysis of phosphopeptides to examine potential changes in response pathways to after-ripening. We used freshly harvested (FH) and after-ripened (AR) barley grains which showed different ABA sensitivity. A total of 1,730 phosphopeptides were identified in barley embryos isolated from half-cut grains. A comparative analysis showed that 329 and 235 phosphopeptides were upregulated or downregulated, respectively after ABA treatment, and phosphopeptides profiles were quite different between FH and AR embryos. These results were supported by peptide motif analysis which suggested that different sets of protein kinases are active in FH and AR grains. Furthermore, in vitro phosphorylation assays confirmed that some phosphopeptides were phosphorylated by SnRK2s, which are major protein kinases involved in ABA signaling. Taken together, our results revealed very distinctive phosphosignaling networks in FH and AR embryos of barley, and suggested that the after-ripening of barley grains is associated with differential regulation of phosphosignaling pathways leading to a decay of ABA signaling., (� The Author(s) 2019. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Previous work on maize (Zea mays L.) primary root growth under water stress showed that cell elongation is maintained in the apical region of the growth zone but progressively inhibited further from the apex. These responses involve spatially differential and coordinated regulation of osmotic adjustment, modification of cell wall extensibility, and other cellular growth processes that are required for root growth under water-stressed conditions. As the interface between the cytoplasm and the apoplast (including the cell wall), the plasma membrane likely plays critical roles in these responses. Using a simplified method for enrichment of plasma membrane proteins, the developmental distribution of plasma membrane proteins was analysed in the growth zone of well-watered and water-stressed maize primary roots. The results identified 432 proteins with differential abundances in well-watered and water-stressed roots. The majority of changes involved region-specific patterns of response, and the identities of the water stress-responsive proteins suggest involvement in diverse biological processes including modification of sugar and nutrient transport, ion homeostasis, lipid metabolism, and cell wall composition. Integration of the distinct, region-specific plasma membrane protein abundance patterns with results from previous physiological, transcriptomic and cell wall proteomic studies reveals novel insights into root growth adaptation to water stress., (© 2016 John Wiley & Sons Ltd.)

Plant roots form an intracellular symbiosis with nitrogen-fixing bacteria while maintaining the capacity for defending themselves against bacterial pathogens. To investigate the molecular relationship between these opposing cellular responses, we compared changes in the root phosphoproteome of the legume Lotus japonicus occurring within minutes after perception of nodulation factor (NF), a symbiotic signaling molecule, to those elicited by flagellin peptide (flg22), a conserved pathogen-associated peptide motif present in flagellar protein of a wide range of bacteria. Phosphoproteins were visualized by autoradiography of two-dimensional polyacrylamide gels after in vivo labeling with 33P-orthophosphate. Comparisons of NF- and flg22-induced phosphoprotein patterns revealed signal-specific responses but also a surprisingly large overlap. Specificity of the responses was observed because the NF receptor kinases NFR1 and NFR5 were both required for NF- but not for flg22-mediated changes in the phosphoproteome. Moreover, NF did not stimulate an oxidative burst or activation of mitogen-activated protein kinases, two common markers for early defense responses that were induced by flg22. Inhibitor studies revealed that phosphorylation of at least some of the proteins in response to NF requires phospholipase D (PLD) whereas regulation of the flg22 phosphoproteome is PLD-independent. Although plant signal transduction during symbiosis and defense utilizes distinct components, phosphorylation of overlapping sets of proteins is achieved.

In contrast to many mammalian pathogens, potential bacterial pathogens of plants remain outside the host cell. The plant must, therefore, promote an active resistance mechanism to combat the extracellular infection. How this resistance against bacteria is manifested and whether similar processes mediate basal, gene-for-gene, and salicylate-associated defense, however, are poorly understood. Here, we identify a specific plasma membrane syntaxin, NbSYP132, as a component contributing to gene-for-gene resistance in Nicotiana benthamiana. Silencing NbSYP132 but not NbSYP121, the apparent orthologue of a syntaxin required for resistance to powdery mildew fungus, compromised AvrPto-Pto resistance. Because syntaxins may play a role in secretion of proteins to the extracellular space, we performed a limited proteomic analysis of the apoplastic fluid. We found that NbSYP132-silenced plants were impaired in the accumulation of at least a subset of pathogenesis-related (PR) proteins in the cell wall. These results were confirmed by both immunoblot analysis and imunolocalization of a PR protein, PR1a. These results implicate NbSYP132 as the cognate target soluble N-ethylmaleimide-sensitive factor attachment protein receptor for exocytosis of vesicles containing antimicrobial PR proteins. NbSYP132 also contributes to basal and salicylate-associated defense, indicating that SYP132-dependent secretion is a component of multiple forms of defense against bacterial pathogens in plants.

Substantial evidence implicates important roles for both protein phosphorylation and protein degradation in regulation of plant defense responses. Therefore, interest is growing in applying proteomics techniques to investigate these posttranscriptional changes. We have found, however, that most proteins of interest are not visible on two-dimensional (2D) gels without previous prefractionation. This chapter describes the use of preparative denaturing isoelectric focusing to enrich for proteins of specific isoelectric points before separation by 2D gels. This method significantly increases the sensitivity of 2D gel-based comparisons.

Global analyses of protein phosphorylation require specific enrichment methods because of the typically low abundance of phosphoproteins. To date, immobilized metal ion affinity chromatography (IMAC) for phosphopeptides has shown great promise for large-scale studies, but has a reputation for poor specificity. We investigated the potential of IMAC in combination with capillary liquid chromatography coupled to tandem mass spectrometry for the identification of plasma membrane phosphoproteins of Arabidopsis. Without chemical modification of peptides, over 75% pure phosphopeptides were isolated from plasma membrane digests and detected and sequenced by mass spectrometry. We present a scheme for two-dimensional peptide separation using strong anion exchange chromatography prior to IMAC that both decreases the complexity of IMAC-purified phosphopeptides and yields a far greater coverage of monophosphorylated peptides. Among the identified sequences, six originated from different isoforms of the plasma membrane H(+)-ATPase and defined two previously unknown phosphorylation sites at the regulatory C terminus. The potential for large-scale identification of phosphorylation sites on plasma membrane proteins will have wide-ranging implications for research in signal transduction, cell-cell communication, and membrane transport processes.

The perception of microbial signal molecules is part of the strategy evolved by plants to survive attacks by potential pathogens. To gain a more complete understanding of the early signaling events involved in these responses, we used radioactive orthophosphate to pulse-label suspension-cultured cells of Arabidopsis in conjunction with two-dimensional gel electrophoresis and mass spectrometry to identify proteins that are phosphorylated rapidly in response to bacterial and fungal elicitors. One of these proteins, AtPhos43, and related proteins in tomato and rice, are phosphorylated within minutes after treatment with flagellin or chitin fragments. By measuring (32)P incorporation into AtPhos43 immunoprecipitated from extracts of elicitor-treated hormone and defense-response mutants, we found that phosphorylation of AtPhos43 after flagellin treatment but not chitin treatment is dependent on FLS2, a receptor-like kinase involved in flagellin perception. Induction by both elicitors is not dependent on salicylic acid or EDS1, a putative lipase involved in defense signaling.