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. 2021 Apr 27;186(4):2169–2189. doi: 10.1093/plphys/kiab190

Figure 3.

Figure 3

CaHSFA5 Is a Direct Transcriptional Target of CaNAC2c upon HTS. A, Schematics of the CaHSFA5 promoter, with the CATGTG-containing fragment and a CATGTG-free fragment highlighted for ChIP-PCR of CaHSFA5 by CaNAC2c. B, CaNAC2c directly targets the CaHSFA5 promoter, as shown by ChIP-PCR. Chromatin was isolated from pepper leaves transiently overexpressing CaNAC2c-GFP, sheared into 300–500 bp fragments. The DNA was immunoprecipitated with antibodies against GFP, and the purified DNA was used as a template with specific primer pair of CATGTG-containing promoter region. C, CaNAC2c shows enhanced binding to the CaHSFA5 promoter under HTS, as seen by ChIP-qPCR. D, MST analysis of CaNAC2c binding to the CaHSFA5 promoter, using CaNAC2c-GFP fusion protein transiently overexpressed in pepper leaves and immunoprecipitated with anti-GFP antibody and a CATGTG-containing CaHSFA5 promoter fragment. E, EMSA analysis of CaNAC2x binding to the CaHSFA5 promoter, with recombinant CaNAC2c-GST and a CaHSFA5 promoter fragment containing CATGTG or its mutant version (GGGGGG). F, CaHSFA5 is downregulated by silencing CaNAC2c under HTS. G, CaHSFA5 is upregulated in N. benthamiana plants overexpressing CaNAC2c or by the transient overexpression of CaNAC2c in pepper leaves. In C, F, and G, data presented are means ± standard error (SE) of four replicates, different capital letters indicate significant differences among means (P < 0.01), as calculated with Fisher’s protected LSD test.