Stable isotope metabolic labeling-based quantitative phosphoproteomic analysis of Arabidopsis mutants reveals ethylene-regulated time-dependent phosphoproteins and putative substrates of constitutive triple response 1 kinase

Mol Cell Proteomics. 2013 Dec;12(12):3559-82. doi: 10.1074/mcp.M113.031633. Epub 2013 Sep 16.


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 ethylene-regulated 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. Finally, several ethylene-regulated post-translational modification network models have been built using molecular systems biology tools. It is proposed that ethylene regulates the phosphorylation of arginine/serine-rich splicing factor 41, plasma membrane intrinsic protein 2A, light harvesting chlorophyll A/B binding protein 1.1, and flowering bHLH 3 proteins in a dual-and-opposing fashion.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Motifs
  • Arabidopsis / drug effects
  • Arabidopsis / genetics
  • Arabidopsis / metabolism*
  • Arabidopsis Proteins / genetics
  • Arabidopsis Proteins / metabolism*
  • Calcium-Binding Proteins / genetics
  • Calcium-Binding Proteins / metabolism
  • Computational Biology
  • Ethylenes / metabolism
  • Ethylenes / pharmacology*
  • Gene Expression Regulation, Plant*
  • Isotope Labeling
  • Molecular Sequence Annotation
  • Molecular Sequence Data
  • Mutation
  • Nitrogen Isotopes
  • Phosphoproteins / genetics
  • Phosphoproteins / metabolism*
  • Phosphorylation
  • Plant Growth Regulators / metabolism
  • Plant Growth Regulators / pharmacology*
  • Protein Interaction Domains and Motifs
  • Protein Interaction Mapping
  • Protein Kinases / genetics
  • Protein Kinases / metabolism*
  • Protein Phosphatase 2 / genetics
  • Protein Phosphatase 2 / metabolism*
  • Protein Processing, Post-Translational*
  • Signal Transduction
  • Substrate Specificity
  • Time Factors


  • Arabidopsis Proteins
  • CAS protein, Arabidopsis
  • Calcium-Binding Proteins
  • Ethylenes
  • Nitrogen Isotopes
  • Phosphoproteins
  • Plant Growth Regulators
  • ethylene
  • Protein Kinases
  • CTR1 protein, Arabidopsis
  • Protein Phosphatase 2
  • RCN1 protein, Arabidopsis