Fig. 1

A Multiple sequence alignment of homologous amino acid sequences of different C2H2-type zinc finger proteins revealed conserved C2H2 domains, which are demarcated by box. B Subcellular localization studies in tobaco leaves demonstrated that the 35S::MdZAT1-GFP fusion protein was localized to the nucleus. C Y1H assays indicated that MdZAT1 interacts with the promoter of MdMYB114. D EMSAs further confirmed the direct binding of MdZAT1-HIS to a TTGGGT motif within the MdMYB114 promoter. The biotin-labeled probes were used for detection, while unlabeled (cold) and mutant probes served as competitors. E Luciferase reporter assays showed that MdZAT1 suppresses the transcriptional activity of the MdMYB114 promoter. F ChIP-qPCR assays demonstrated the in vivo binding of MdZAT1 to the MdMYB114 promoter. G Fruit development was observed at 0, 6, 12, and 18 days. H The anthocyanin content and MdZAT1 expression levels during the fruit coloration. I Y1H assays indicated that MdZAT1 also interacts with the promoter of MdCHI. J EMSAs confirmed the direct binding of MdZAT1-HIS to two TTGGGT motifs within the MdCHI promoter. The biotin-labeled probes for detection and unlabeled (cold) and mutant probes as competitors. K Luciferase reporter assays demonstrated that MdZAT1 suppresses the transcriptional activity of the MdCHI promoter. L ChIP-qPCR assays confirmed the in vivo binding of MdZAT1 to the MdCHI promoter. M Y1H assays also indicated an interaction between MdZAT1 and the promoter of MdANS. N EMSAs confirmed the direct binding of MdZAT1-HIS to two TTGGGT motifs within the MdANS promoter. The biotin-labeled probes for detection and unlabeled (cold) and mutant probes as competitors. O Luciferase reporter assays showed that MdZAT1 suppresses the transcriptional activity of the MdANS promoter. P ChIP-qPCR assays confirmed the in vivo binding of MdZAT1 to the MdANS promoter. Q MdZAT1 directly represses anthocyanin biosynthesis by binding to the promoters of MdCHI and MdANS, and potentially regulates anthocyanin biosynthesis through MdMYB114. Data are presented as mean ± standard deviation (SD) from three biological replicates (n = 3). Asterisks indicate statistically significant differences based on Student’s t-tests (*p < 0.05, **p < 0.01)