Phosphoproteomic analysis of ethylene-regulated protein phosphorylation in etiolated seedlings of Arabidopsis mutant ein2 using two-dimensional separations coupled with a hybrid quadrupole time-of-flight mass spectrometer

Hao Li, Wai Shing Wong, Lin Zhu, Hong Wei Guo, Joseph Ecker, Ning Li*

*Corresponding author for this work

Research output: Contribution to journalJournal articlepeer-review

73 Citations (Scopus)

Abstract

Ethylene regulates a variety of stress responses and developmental adaptation in plants. In the present study, the phosphoproteomics is adopted to investigate the differential protein phosphorylation by ethylene in Arabidopsis ethylene-insensitive 2 (ein2) mutant. A total of 224 phosphopeptides were identified, of which 64 phosphopeptides were detected three or more times. Ethylene induces a general reduction in phosphorylated proteins in ein2. Totally, three ethyleneenhanced and three ethylene-repressible unique phosphopeptides were identified, respectively. Classification of the cellular functions of these phosphoproteins revealed that 55.5% of them are related to signaling and gene expression. Peptide sequence alignment reveals two highly conserved phosphorylation motifs, PRVD/GSx and SPDYxx. Alignment of these phosphopeptides with Arabidopsis proteins reveals five phosphorylation motifs. Both ethylene-enhanced and -repressible phosphopeptides present in these motifs. EIL-1, ERF110 transcription factors and Hua enhancer 4 (HEN4) are predicted to contain one of the phosphorylation motifs. The phosphorylation of the motif-containing peptides has been validated by the in vitro kinase assays coupled with MS analysis. The differential regulation of phosphorylation by ethylene is substantiated by Western dot blot analysis. Taken together, these results suggest that ethylene signals may be transduced by a phosphor-relay from receptors to transcriptional events via both ein2-dependent and -independent pathways.

Original languageEnglish
Pages (from-to)1646-1661
Number of pages16
JournalProteomics
Volume9
Issue number6
DOIs
Publication statusPublished - Mar 2009

Scopus Subject Areas

  • Biochemistry
  • Molecular Biology

User-Defined Keywords

  • Arabidopsis mutant
  • Differential treatment
  • Phosphoproteome
  • Plant hormone
  • Time-of-flight mass spectrometry
  • Two-dimensional separations

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