Ethylene is an important plant hormone that regulates numerous cellular processes and stress responses. The mode of action of ethylene is both dose-and time-dependent. Protein phosphorylation plays a key role in ethylene signaling, which is mediated by the activities of ethylene receptors, constitutive triple response 1 (CTR1) kinase, and phosphatase. To address how ethylene alters the cellular protein phosphorylation profile in a time-dependent manner, differential and quantitative phosphoproteomics based on 15 N stable isotope labeling in Arabidopsis was performed on both one-minute ethylene-treated Arabidopsis ethylene-overly-sensitive loss-of-function mutant rcn1-1, deficient in PP2A phosphatase activity, and a pair of long-term ethylene-treated wild-type and loss-of-function ethylene signaling ctr1-1 mutants, deficient in mitogen-activated kinase kinase kinase activity. In total, 1079 phosphopeptides were identified, among which 44 were novel. Several one-minute ethylene-regulated phosphoproteins were found from the rcn1-1. Bioinformatic analysis of the rcn1-1 phosphoproteome predicted nine phosphoproteins as the putative substrates for PP2A phosphatase. In addition, from CTR1 kinase-enhanced phosphosites, we also found putative CTR1 kinase substrates including plastid transcriptionally active protein and calcium-sensing receptor. These regulatory proteins are phosphorylated in the presence of ethylene. Analysis of ethyleneregulated phosphosites using the group-based prediction system with a protein-protein interaction filter revealed a total of 14 kinase-substrate relationships that may function in both CTR1 kinase-and PP2A phosphatase-mediated phosphor-relay pathways. Ethylene is a volatile plant hormone that regulates versatile molecular and physiological processes in higher plants (1). The perception of this gaseous two-carbon hormone is achieved by a group of membrane-associated dimeric ethylene receptors that resemble bacterial two-component signaling systems and are composed of hybrid histidine (or aspartic acid) kinases, a histidine-containing phosphor-transfer domain, and response regulators (2). These receptors are made of two membrane-bound protein subunits cross-linked at the N-terminal region through two disulfide bonds (3). In Arabidopsis, there are five different ethylene receptor subunits: ethylene response 1, ethylene response 2, ethylene insensitive 4 (EIN4), 1 ethylene response sensor 1, and ethylene re- 1 The abbreviations used are: ACC, aminocyclopropane-1-carboxylic acid; ACN, acetonitrile; CIPK1, CBL-interacting protein kinase 1; CTR1, constitutive triple response 1; eer1-1, enhanced ethylene response 1; EIN, ethylene insensitive; FBH3, flowering bHLH 3 protein; FDR, false discovery rate; GPS, group-based prediction system; HMG, high mobility group; IMAC, immobilized metal-ion-affinity chromatography; iTRAQ, isobaric tag for relative and absolute quantitation; LHCB, light harvetsting chlorophyll A/B binding protein;
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