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.
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 ethylene-enhanced 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.
Li
Hao
H
Department of Biology, The Hong Kong University of Science and Technology, Hong Kong SAR, PR China.
Wong
Wai Shing
WS
Zhu
Lin
L
Guo
Hong Wei
HW
Ecker
Joseph
J
Li
Ning
N
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000187:drug effects / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D019295:Computational Biology; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D005030:Ethylenes / Q000096:biosynthesis / Q000494:pharmacology*; D013058:Mass Spectrometry / Q000295:instrumentation; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D010748:Phosphopeptides / Q000032:analysis / Q000737:chemistry / Q000145:classification; D010750:Phosphoproteins / Q000032:analysis* / Q000737:chemistry; D010766:Phosphorylation / Q000187:drug effects; D040901:Proteomics / Q000379:methods*; D011956:Receptors, Cell Surface / Q000378:metabolism*; D015203:Reproducibility of Results; D036226:Seedlings / Q000187:drug effects / Q000378:metabolism*; D016415:Sequence Alignment; D014157:Transcription Factors / Q000032:analysis* / Q000737:chemistry
D029681:Arabidopsis Proteins; C119963:EIN2 protein, Arabidopsis; C576881:ERF110 protein, Arabidopsis; D005030:Ethylenes; D010748:Phosphopeptides; D010750:Phosphoproteins; D011956:Receptors, Cell Surface; D014157:Transcription Factors; C036216:ethylene