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. 2022 May 4;8(18):eabn8281. doi: 10.1126/sciadv.abn8281

Fig. 2. Dislocation of WT and W box cis-elements in 250-bp promoter of RAP2.12 and interaction with WRKY70.

Fig. 2.

(A and B) Representative shoot images of RAP2.12-GUS reporter protein fusion lines in pSic-RAP2.12:RAP2.12-GUS and pTib-RAP2.12:RAP2.12-GUS Col-0 transgenics (A) and RAP2.12-GUS reporter lines in pSic-RAP2.12:GUS (blue), pTib-RAP2.12:GUS (orange), and piecewise substitutions of RAP2.12 promoter from Sichuan to Tibet accession (B), in 4-day-old seedlings after treatment with air (untreated) or 5% (v/v) oxygen for the indicated time. Differences of the promoters are shown in fig. S7. Scale bars, 1 mm. (C) EMSA to test for interaction of WT box and W box, with WRKY70-Sic and WRKY70-Tib. Gene structure and 30-bp-length sequences used for biotin labeling and unlabeled competitive binding are shown on the top. Core cis-element sequences of WT box and W box are in blue and orange, respectively. Residue difference between GST-WRKY70-Sic and GST-WRKY70-Tib protein is shown in fig. S11A. (D and E) Repression by WRKY70 on the promoter of pTib-RAP2.12 depends on the W box cis-element. pTib-RAP2.12(WM), W box cis-element mutant to Sichuan accession form (D). Transactivation of pSic-RAP2.12 by WRKY70 depends on the WT box cis-element. pSic-RAP2.12(WTM), WT box cis-element mutant to Tibet accession form; pSic-RAP2.12(WTM + W), adding pSic-RAP2.12(WTM) promoter to the W box cis-element corresponding to the Tibet accession (E). Data are means ± SE (n = 3 repeats). **P < 0.01; NS, not significant (Student’s t test).