Identification of PIF Gene Family and Functional Study of PbPIF3a/PbPIF4 in Anthocyanin Biosynthesis of Pear

Anthocyanins are key metabolites that determine red pigmentation in pear skin (Pyrus spp.) and their biosynthesis is controlled by multiple transcription factors. Although phytochrome-interacting factors (PIFs) of the bHLH family have been shown to regulate anthocyanin biosynthesis in Arabidopsis thaliana, their genome-wide identification and regulatory mechanisms in pear (Pyrus spp.) anthocyanin synthesis remain unclear. Here, we characterized PIFs family in pear, identifying eight PbPIF proteins. Promoter cis-elements and expression patterns analysis suggested that PbPIF3a and PbPIF4 might be involved in anthocyanin biosynthesis. Subcellular localization confirmed nuclear enrichment of PbPIF3a and PbPIF4. Functional studies demonstrated that overexpression of PbPIF3a and PbPIF4 significantly suppressed anthocyanin accumulation in fruit skins, downregulating key biosynthetic genes such as PbDFR and PbUFGT. In contrast, the silencing of related genes led to an enhancement of anthocyanin accumulation. Dual-luciferase reporter assays and yeast one-hybrid assays confirmed that PbPIF3a directly bound to the promoters of PbDFR and PbUFGT and repressed their transcriptional activation, while PbPIF4 specifically inhibited the activity of the PbDFR promoter. Taken together, we demonstrated that PbPIF3a and PbPIF4 negatively regulated pear fruit coloration by directly repressing the transcriptional activity of key anthocyanin biosynthesis genes, providing novel insights into PIF-mediated regulation of anthocyanin biosynthesis.