Phosphoproteomics-identified ERF110 affects ... - Plant Physiology

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alignment to identify a putative Ser62 phosphosite on a putative
bioinformatics-predicted transcription factor, ERF110 (Li et al., 2009), delineate a
functional role for the Ser62-phosphorylated isomer of the ERF110 transcription
factor and provide a demonstration of functional phosphoproteomics by integrating
quantitative post-translation modification (PTM) proteomics with bioinformatics
prediction and in-vitro and in-vivo validations.
Kinase- and phosphatase-mediated phosphorylation is known to be an important
molecular mechanism that regulates ethylene responses. CTR1 has been predicted to
encode a MAPK. A loss-of-function mutation (ctr1-1) in the CTR1 gene leads to a
strong constitutive triple response phenotype, which acts as if the plant had constantly
been exposed to ethylene. However, in this ctr1-1 Arabidopsis mutant, a higher level
of ERF110 protein was found (Figure 5). The enhanced accumulation of ERF110
protein in ctr1-1 may result from the suppression of Ub/26S proteasome-mediated
protein degradation by constitutive ethylene signaling. This speculation is supported
by Western blot analysis of an ethylene-insensitive mutant, etr1-1, in which ERF110
protein appears to be dramatically decreased (data not shown) compared with that of
the wild-type. An unexpected finding from Western blot analysis of ctr1-1 was that
Ser62-phosphorylation of ERF110 could not be detected (Figures 4 and 5). The
diminished Ser62-phosphorylated isoform in ctr1-1 might be attributed to two factors:
it may result from an increase in phosphatase activities or a reduction in kinase
activities, or the combined effects of both enzymes on Ser62-phosphosite that are
indirectly activated by ctr1-1 mutation. Alternatively, the Ser62 phosphosite motif of
ERF110 may serve as a direct substrate for the CTR1 kinase. The in vitro kinase
assays that include an excess of synthetic substrate, as shown in Figure 1, suggest that
a functional ethylene perception is involved in the regulation of Ser62-specifc
kinase/phosphatase activities (Figure 1).
Because ctr1-1 has an enhanced level of ERF110 protein and its bolting time is also
delayed, it was speculated that an ERF110 over-expression transgenic plant,
Pro35S-ERF110 WT ::Col-0, may confer delayed bolting. ERF110 protein was
over-expressed 100 fold or more (Supplementary Figure 5) in this transgenic plant;
however, it did not produce a significant difference in the bolting time compared with
that of the wild-type (19.8 ± 0.18 versus 19.6 ± 0.22 days; Table 1 and Supplementary