Up-regulation of PsbHLH3 assists the induction of anthocyanin biosynthesis in the amber-flesh of black plums trigged by cold storage

Low temperature storage induced flesh reddening of the amber-fleshed plum cultivar. 10 candidate PsbHLH s differentially expressed in reddened flesh were screened out. PsbHLH3 was validated to be crucial in cold-induced plum anthocyanin biosynthesis. PsbHLH3 interacted with PsMYB10.1 to enhance activation on the promoter of PsUFGT . Cold storage of amber-fleshed black plums induced the biosynthesis and accumulation of anthocyanin in plum fruit, which caused the color change of the flesh tissue when stored at low temperature. bHLH transcription factors had 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. cv. Friar and cv. Angeleno) were selected as fruit material to conduct transcriptomic and bioinformatics analysis. The differentially expressed bHLH s genes were identified via RNA sequencing and verified by qRT-PCR. Ten bHLH s genes, containing 5 up-regulated and 5 down-regulated, were identified as the most significantly altered during cold-induced flesh reddening. The structure of these 10 bHLH proteins was characterized, and a phylogenetic tree was constructed. Among them, the gene evm.TU.Chr8.2504, identified as PsbHLH3 , exhibits low temperature responsiveness and contains MYB-binding sites. Further experiments involving transient overexpression were performed to validate the function of PsbHLH3 in facilitating anthocyanin biosynthesis. The yeast two-hybrid and bimolecular fluorescence complementation 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 augmenting the transcription of structural genes related to anthocyanin biosynthesis. The study indicated that low temperatures induced the up-regulation of PsbHLH3 , facilitating the opening of ON/OFF molecular switch responsible for the flesh-reddening of plum fruit. This elucidates the role of PsbHLH3 in postharvest regulation of anthocyanin biosynthesis in plums trigged by cold storage.