Phosphoproteomics-identified ERF110 affects ... - Plant Physiology

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32 and interaction network. Molecular Plant 4: 626-634
Figure legends 33 Figure 1. Bioinformatics prediction and validation of a phosphosite on ERF110 A: The alignment of a phosphosite motif PRVDSS between the authentic MS-derived phosphopeptide with those of Arabidopsis gene accessions. The top amino acid sequence motif derives from a phosphopeptide from a segment of aluminum-induced protein (At5g43830). The remaining peptide sequences were retrieved from the Arabidopsis gene database according to the alignment conducted by the BLAST program using the authentic MS-derived phosphosite motif PRVDSS. The phosphorylation site (S) is marked with a black asterisk. The synthetic peptides marked with red asterisks on the left (Figure 1A) were successfully phosphorylated by the in-vitro kinase assay. The conserved motif sequence (PRVDSS) among these members is shown at the bottom of the alignment. The conservation score (%) and the sequence logo are also shown at the bottom of alignment, representing a frequency of the conserved amino acid present in the motif. B: Quantitation of the Ser62 phosphosite of ERF110 using the in-vitro kinase assay coupled with iTRAQ. The bars indicate the relative intensities of reporter ions for the phosphorylated ERF110 peptide catalyzed by the in-vitro kinase assay (*** p < 0.001 by the Student’s t-test, ethylene versus air treatment). The ion intensity of the air-treated sample was set as 1. For each pair of comparisons, the experiments were repeated with at least two independent biological samples using reciprocal labeling (Supplementary Figures 2 and 3). Representative MS/MS spectra are presented in Supplementary Figures 2 and 3. The in-vitro kinase activity of Ser62 was ethylene-dependent but EIN2-independent. White and black bars represent Arabidopsis kinase extracts prepared from air- and ethylene-treated plants, respectively (see Materials and Methods for the detailed kinase assay protocol). C: MS/MS spectra of the in vivo phosphorylation of ERF110. The precursor ion (VDpSSHNPIEESMSK) was isolated and fragmented by collision-induced dissociation. Both b-type and y-type ions, including the H3PO4 neutral loss ions (indicated as –H3PO4 and # in spectra), were labeled to determine the peptide sequence of the precursor ion and to locate the sites of phosphorylation. An enlarged portion of the spectrum (100-500 m/z) that showed the b3 neutral loss ion (284.1158) was put at the bottom of the full MS/MS spectrum obtained from LC-MS/MS (Q-TOF, Waters). Figure 2. Molecular characterization of ERF110 RNAi lines A: Delayed bolting phenotype of two ERF110-RNAi lines (erf110-1, erf110-2) at 23 days of growth; the wild-type (Col-0) was the control. B: Western blot analysis of ERF110 protein in both wild-type and mutants was performed using rabbit anti-ERF110 and -actin polyclonal antibodies, respectively. Actin signal was used as the sample protein loading control.
Page 1 and 2: Plant Physiology Preview. Published
Page 3 and 4: 3 Acknowledgement: This research wo
Page 5 and 6: 5 Introduction Ethylene is known to
Page 7 and 8: 7 whereas ethylene up-regulated ERF
Page 9 and 10: 9 matrix-assisted laser desorption/
Page 11 and 12: 11 To minimize the effect of endoge
Page 13 and 14: 13 Materials and Methods). Western
Page 15 and 16: 15 transcript level in Pro35S-ERF11
Page 17 and 18: 17 Table 2) in the presence of AOA,
Page 19 and 20: 19 role in the regulation of boltin
Page 21 and 22: 21 ethylene-response mutant that ac
Page 23 and 24: 23 sample was set as 1, whereas the
Page 25 and 26: 25 C18 Zip-tip column and resuspend
Page 27 and 28: 27 phosphopeptide ARVDpSSHNPIEESM w
Page 29 and 30: 29 acid-enhanced 1 gene plays a rol
Page 31: 31 events in plants. Plant Cell 5:
Page 35 and 36: 35 Figure 5. Ethylene represses in-
Page 37 and 38: Table 1. Bolting time of ERF110 WT
Page 39 and 40: Figure 2 A B Anti-ERF110 Anti-Actin
Page 41 and 42: Figure 4 A B C a b Col-0 ctr1-1 erf
Page 43 and 44: Figure 6 A B WT-OX WT-OXA WT-OXD O
Page 45: Figure 8

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