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. 2021 Feb 24;12:634040. doi: 10.3389/fpls.2021.634040

FIGURE 4.

FIGURE 4

CAT2 is a direct target of NAC075. (A) Relative expression level of CAT2 in the third and fourth rosette leaves of 25-day-old Col-0 and nac075 mutant plants. Data are means ± SD, n = 3. (B) NAC075 directly binds to the promoter of CAT2. Schematic diagram indicates the locations of two putative NAC075 binding sites (P1 and P2) in the CAT2 promoter (top). ChIP-qPCR was performed to examine the relative NAC075 binding to the CAT2 promoter (bottom). An anti-GFP monoclonal antibody was used for DNA immunoprecipitation from 28-day-old Pro35S:NAC075-GFP/Col-0 transgenic plants. The relative enrichment of NAC075 binding to CAT2 promoter was normalized to TUBULIN2 (TUB2). Student’s t-test, ∗∗P < 0 .01, ∗∗∗P < 0.001, ns, not significant. Data are means ± SD of three independent biological replicates. (C) EMSA assay of the binding of NAC075 to the CAT2 promoter in vitro. Biotin-labeled probe was used to EMSA experiment and non-labeled fragments were used as competitors. The + and − symbols represent the presence and absence of components. Three biological replicates were performed with similar results. (D) qRT-PCR analysis of NAC075 and CAT2 expressions in the pER8-FLAG-NAC075 plants treated with 50 mM β-estradiol for the indicated time points. Data are represented as means ± SD, n = 3.