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.

In order to explore the changes in the content of phenolic compounds and the activity of antioxidant enzymes during the growth and metabolism of red raspberry leaves, the leaves of ‘Heritage’ and ‘Autumn Bliss’ red raspberry were analyzed and compared for the contents of total flavonoids and seven phenolic substances and the activities of three antioxidant enzymes. The results showed that from May to December, the content of ellagic acid and peroxidase (POD) activity in the upper, middle and lower leaves of the two varieties showed a double peak curve during the process of growth and metabolism, the content of salicylic acid showed a downward trend, the contents of ferulic acid, catechin, epicatechin, rutin and total flavones showed a single peak curve, the superoxide dismutase (SOD) activity remained at a high level, and the catalase (CAT) activity decreased, then increased and finally decreased again. The content of caffeic acid in the upper leaves of the two varieties showed a downward trend, while those in the middle and lower leaves showed a single peak curve. The highest contents of salicylic acid, ferulic acid, ellagic acid and epicatechin were determined in the upper leaves of ‘Autumn Bliss’ in May, upper leaves in June, middle leaves in July and upper leaves in July, which were 59.33 mg/100 g, 158.18 mg/100 g, 250.84 mg/g and 350.04 mg/100 g, respectively. The highest contents of rutin, caffeic acid, catechin and total flavonoids were determined in the lower leaves of ‘Heritage’ in June, middle leaves in July, middle leaves in July and upper leaves in July, which were 8.00 mg/100 g, 100.48 mg/100 g, 18.71 mg/g and 34.18 mg/g, respectively. The highest activities of POD, SOD and CAT were determined in the upper leaves of ‘Autumn Bliss’ in July, the lower leaves of ‘Heritage’ in July and the lower leaves of ‘Heritage’ in July, which were 13.05 U/(g·min), 37.00 U/g and 41.12 U/(g·min), respectively. In conclusion, the contents of phenolic substances and antioxidant enzyme activities in the leaves of ‘Heritage’ and ‘Autumn Bliss’ red raspberry are highest from May to July. This study provides an important theoretical basis for the timely harvest and better development and utilization of red raspberry leaves.