Phosphoproteomics-identified ERF110 affects ... - Plant Physiology

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ERF110 is involved in ethylene-regulated bolting time 10 Before an extensive experiment could be performed on the post-translational modification of ERF110 protein, i.e. phosphorylation, the biological function of the ERF110 gene needed to be addressed first in Arabidopsis. To that end, we investigated the in-planta role of the ERF110 gene. ERF110 RNA-interfering (RNAi) constructs were created to suppress the endogenous ERF110 transcripts in wild-type Arabidopsis. As expected, two ERF110 RNAi lines were found to have severely reduced ERF110 transcripts and protein in T2 plants (Figures 2B and 2C). These transgenic lines are called erf110-1 and erf110-2 knockout lines. When examined in these transgenic plants, it was found that both RNAi lines showed a delayed bolting time (25.8 ± 1.19 and 25.1 ± 1.00 days (p < 0.001) for erf110-1 and erf110-2, respectively; Figure 2A and Supplementary Table 2) compared with the wild-type (Col-0; 19.8 ± 0.18 days). These erf110 mutants also had a greater number of rosette leaves (10.4 ± 0.56 and 10.6 ± 0.62) compared with the wild-type (7.1 ± 0.08 leaves per plant, p < 0.001; Supplementary Table 2), suggesting that ERF110 is involved in the control of the floral transition in Arabidopsis. This interesting phenomenon led us to extend our investigation to incorporate the role of ethylene-regulated ERF110 Ser62-phosphorylation in bolting. It has been reported that the application of the ethylene biosynthesis precursor 1-aminocyclopropane-1-carboxylic acid (ACC) delays bolting time significantly in wild-type Arabidopsis (Achard et al., 2007), whereas the phenotypic characterization of a single or multiple ACC synthase (ACS) mutants also supports an inhibitory role for ACC in bolting (Tsuchisaka et al., 2009). Moreover, the constitutive ethylene-response mutant ctr1-1 is well known to delay bolting (Hua and Meyerowitz, 1998; Achard et al., 2007). To confirm this role of ethylene, the immediate ethylene biosynthesis precursor ACC was used. As Figure 3 shows, the application of ACC delayed the bolting time significantly in a dose-dependent manner in wild-type Arabidopsis. In comparison with the untreated group, 1 µM ACC delayed the bolting time from 19.8 ± 0.18 to 21.3 ± 0.19 days (p < 0.001) in Col-0 and caused a significant increase in the number of leaves, whereas 5 µM ACC further delayed the bolting time to 22.9 ± 0.17 days (p < 0.001) in Col-0 background with 9.1 ± 0.24 leaves at the time of bolting (p < 0.01).
11 To minimize the effect of endogenous ethylene, an ACC synthase inhibitor, aminooxyacetic acid (AOA), is usually added to the growth medium to reduce the background level of ethylene. ACC is frequently used in combination with AOA to study the effect of ethylene on bolting time among the wild-type, erf110 knockout lines and ethylene-response mutants such as ctr1-1. Because it has been reported that ctr1-1 inflorescence has a faster gravicurvature response to gravistimulation following a long-term ethylene treatment (Li, 2008), we deliberately treated ctr1-1 with ethylene to address whether its bolting time could be altered in response to treatment. Interestingly, treatment with 5 µM ACC delayed the bolting time of ctr1-1 from 21.8 ± 0.24 to 24.3 ± 0.35 days (p < 0.001; Figure 3B and Supplementary Table 2), whereas, in the presence of AOA, a similar delay in bolting was observed from 22.0 ± 0.21 to 24.1 ± 0.36 days in ACC-treated ctr1-1 (p < 0.001; Figure 3B and Supplementary Table 2). Similarly, the two ERF110 RNAi lines also exhibited a delayed bolting phenotype after ACC treatment (from 23.2 ± 0.83 days to 25.6 ± 0.75 days (p = 0.044) and from 23.8 ± 0.70 days to 25.8 ± 0.69 days (p = 0.045) for erf110-1 and erf110-2, respectively; Figure 3B and Supplementary Table 2). As the delayed bolting of erf110 knockout lines was more obvious in the presence of AOA, endogenous background ethylene is believed to influence the bolting phenotype measurement and several other ethylene-regulated flowering regulatory factors may exist in addition to ERF110. Ethylene regulates bolting by enhancing ERF110 gene expression Initially, the delayed bolting phenotype of the ERF110-knockout lines erf110-1 and erf110-2 suggests that ethylene (or ACC) may down-regulate ERF110 gene expression, which delays the bolting of Arabidopsis. When the 0.5-Kb promoter region of ERF110 (–289 to + 226) was fused to a β-glucuronidase (GUS) reporter gene and transformed into Arabidopsis Col-0, the application of ethylene actually enhanced rather than inhibited ERF110 promoter activity at the 2-3-week-old stage (Supplementary Figure 4 and Supplementary Table 3). ACC-treated seedlings had a GUS activity of 454.1 ± 21.8 pmol 4-MU/h/µg, which was significantly higher than that in the untreated group (372.6 ± 33.4 pmol 4-MU/h/µg, p = 0.03). The ERF110 promoter activities (GUS activities) of AOA-treated background plants was 390.4 ± 26.1 pmol 4-MU/h/µg, whereas plants treated with both AOA and ACC had a GUS
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: 9 matrix-assisted laser desorption/
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 and 32: 31 events in plants. Plant Cell 5:
Page 33 and 34: Figure legends 33 Figure 1. Bioinfo
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
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Phosphoproteomics-identified ERF110 affects ... - Plant Physiology

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