Up-regulation of PsbHLH3 promotes cold-triggered anthocyanin biosynthesis in the amber flesh of black plums

Cold storage of amber-fleshed black plums triggers anthocyanin biosynthesis and accumulation in fruit flesh, resulting in flesh reddening under low-temperature conditions. bHLH transcription factors have potential functions in response to cold induction and anthocyanin biosynthesis. To investigate the role of bHLH genes, two amber-fleshed and black-skinned cultivars (Prunus salicina Lindl. ‘Friar’ and ‘Angeleno’) were selected as fruit materials to conduct transcriptomic and bioinformatics analyses. Differentially expressed bHLH genes were identified by RNA-seq and validated using qRT-PCR. Ten bHLH genes (five upregulated, five downregulated) showed the most significant expression changes during cold- triggered flesh reddening. We characterized the protein structures of these 10 bHLH members and constructed a phylogenetic tree. Among them, the gene evm.TU.Chr8.2504 (the corresponding protein ID is evm.model.Chr8.2504), named PsbHLH3, was cold-responsive and contains MYB-binding sites. Further experiments involving transient overexpression were performed to validate the function of PsbHLH3 in facilitating anthocyanin biosynthesis. Yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays demonstrated that PsbHLH3 interacts with PsMYB10.1. Dual-luciferase assay showed that PsbHLH3 promoted the activation of PsMYB10.1 on the promoter of PsUFGT, and consequently augmented the transcription of structural genes related to anthocyanin biosynthesis. The study reveals that cold storage upregulates PsbHLH3, which acts as a molecular switch governing cold-triggered flesh reddening in plum fruit. These findings clarify the regulatory function of PsbHLH3 in cold-triggered anthocyanin biosynthesis in postharvest plums.