Abscisic acid (ABA) regulates various developmental processes and stress responses in plants. Protein phosphorylation/dephosphorylation is a central post-translational modification (PTM) in ABA signaling. However, the phosphoproteins regulated by ABA under osmotic stress remain unknown in maize. In this study, maize mutant vp5 (deficient in ABA biosynthesis) and wild-type Vp5 were used to identify leaf phosphoproteins regulated by ABA under osmotic stress. Up to 4052 phosphopeptides, corresponding to 3017 phosphoproteins, were identified by Multiplex run iTRAQ-based quantitative proteomic and LC-MS/MS methods. The 4052 phosphopeptides contained 5723 non-redundant phosphosites; 512 phosphopeptides (379 in Vp5, 133 in vp5) displayed at least a 1.5-fold change of phosphorylation level under osmotic stress, of which 40 shared common in both genotypes and were differentially regulated by ABA. Comparing the signaling pathways involved in vp5 response to osmotic stress and those that in Vp5, indicated that ABA played a vital role in regulating these pathways related to mRNA synthesis, protein synthesis and photosynthesis. Our results provide a comprehensive dataset of phosphopeptides and phosphorylation sites regulated by ABA in maize adaptation to osmotic stress. This will be helpful to elucidate the ABA-mediate mechanism of maize endurance to drought by triggering phosphorylation or dephosphorylation cascades.

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Roots are highly sensitive organ in plant response to drought, which commonly inhibits root growth. However, less is known about the effect of ABA on root protein expression induced by drought. To help clarify the role of ABA in protein expression of root response to drought, root protein patterns were monitored using a proteomic approach in maize ABA-deficient mutant vp5 and its wild-type Vp5 exposed to drought. Two-dimensional electrophoresis was used to identify droughtresponsive protein spots in maize roots. After coomassie brilliant blue staining, approximately 450 protein spots were reproducibly detected on each gel, wherein 22 protein spots related to ABA or drought were identified using MALDI-TOF MS. Results showed that the 22 proteins are involved in such several cellular processes as energy and metabolism, redox homeostasis and regulatory. An anionic peroxidase and two putative uncharacterized proteins were up-regulated by drought in ABA-dependent way; A glycine-rich RNA binding protein 2, pathogenesis-related protein 10, an enolase, a serine/threonine-protein kinase receptor and a cytosolic ascorbate peroxidase were up-regulated by drought in both ABA-dependent and ABAindependent way; a nuclear transport factor 2, a nucleoside diphosphate kinase, a putative uncharacterized protein and a peroxiredoxin-5 were up-regulated by drought in ABAindependent way; a superoxide dismutase 4A, a VAP27-2, a transcription factor BTF3, a glutathione S-transferase GSTF2 and a putative uncharacterized protein were up-regulated by drought in ABA-dependent way, but not exogenous ABA treatment in the absence of drought; a O-methyltransferase and a putative uncharacterized proteins were down-regulated by ABA and drought. The identification of some novel proteins in the drought response provides new insights that can lead to a better Communicated by Z

To better understand abscisic acid (ABA) regulation of the synthesis of chloroplast proteins in maize (Zea mays L.) in response to drought and light, we compared leaf proteome differences between maize ABA-deficient mutant vp5 and corresponding wild-type Vp5 green and etiolated seedlings exposed to drought stress. Proteins extracted from the leaves of Vp5 and vp5 seedlings were used for two-dimensional electrophoresis (2-DE) and subsequent matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS). After Coomassie brilliant blue staining, approximately 450 protein spots were reproducibly detected on 2-DE gels. A total of 36 differentially expressed protein spots in response to drought and light were identified using MALDI-TOF MS and their subcellular localization was determined based on the annotation of reviewed accession in UniProt Knowledgebase and the software prediction. As a result, corresponding 13 proteins of the 24 differentially expressed protein spots were definitely localized in chloroplasts and their expression was in an ABA-dependent way, including 6 up-regulated by both drought and light, 5 up-regulated by drought but down-regulated by light, 5 up-regulated by light but down-regulated by drought; 5 proteins down-regulated by drought were mainly those involved in photosynthesis and ATP synthesis. Thus, the results in the present study supported the vital role of ABA in regulating the synthesis of drought- and/or light-induced proteins in maize chloroplasts and would facilitate the functional characterization of ABA-induced chloroplast proteins in C4 plants.

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