Extended Data Fig. 2: Identification and verification of BolPIF4 promoter-binding proteins in Brassica oleracea (cabbage).
(a) Diagram of the target site in the BolPIF4 promoter. (b) Schematic of cabbage leaf treatment. (c) Venn analysis of proteins captured in different treatments using LC-MS/MS. BolT0 and BolT1 represent the non-target and BolT1 target of the BolPIF4 promoter, while 0B and 200B represent the 0 μM and 200 μM biotin treatments, respectively. The red number indicates the proteins selected for further analysis. (d) Identification of nuclear-localized proteins by DeepLoc 2.1 and transcription factors (TFs) by PlantTFDB. Previously reported and newly identified TFs in this study are shown below the circle. (e) Diagrams of constructs used for the LUC/REN assay. The BolPIF4 promoter was used as the reporter and BolGTL2, BolTGA6 or BolC3H as the effectors. Meanwhile, the empty vector pGreen II 62-SK was used as mock control. (f) The results of the LUC/REN assay showed that BolGTL2, BolTGA6, and BolC3H activate the BolPIF4 expression (n = 13-15). Unpaired t-test (two tailed) was used to evaluate the statistical significance. The data are reported as the mean ± SD. Boxes represent the interquartile range of data, whiskers mean the 5-95 pecentile, and shading indicates the density profile. (g) The Y1H assay validated the regulatory abilities of BolGTL2, BolTGA6, and BolC3H to the BolPIF4 promoter. AbA: Aureobasidin A. SD-UL+ represents the SD-UL medium supplied with certain amount of AbA. (h–j) EMSA assessing the binding ability of BolGTL2, BolTGA6, and BolC3H to the BolPIF4 promoter. ‘Competitor’ represents unlabeled probes and ‘Mut’ represents the mutated probes. The experiment was repeated two times independently with similar results. Figure created with BioRender.com.
