Comprehensive metabolomic and transcriptomic analyses of the anthocyanin accumulation mechanism in the leaf veins of two Broussonetia papyrifera varieties (ZJ and CL) under cold stress.

Broussonetia papyrifera is an important native tree species in China. However, its relatively weak cold tolerance not only significantly compromises its survival and reproductive abilities in cold regions but also limits the realization of its economic potential. Notably, previous studies have demonstrated that anthocyanin accumulation enhances plant cold resistance. Despite this, the precise mechanisms underlying anthocyanin accumulation in B. papyrifera remain unclear. Therefore, this study aims to elucidate the pathways and regulatory mechanisms of anthocyanin accumulation in B. papyrifera under cold stress. Two varieties of B. papyrifera (ZJ and CL), were subjected to cold stress at 4 °C. After 21 days, a marked accumulation of anthocyanins was observed in the veins of CL, whereas severe yellowing occurred in the veins of ZJ. Quantitative analysis revealed that the anthocyanin content in CL-V-DW was significantly higher than in other groups. Integrated transcriptomic and metabolomic analyses demonstrated that differentially expressed genes were predominantly enriched in flavonoid metabolic pathways associated with anthocyanin biosynthesis. Cold stress induced upregulation of most anthocyanin synthesis-related genes in both ZJ and CL; however, the expression of glucosyltransferases (BpGST) genes in ZJ-V-DW was markedly reduced, leading to impaired transport of synthesized anthocyanins. Furthermore, genes involved in photosynthesis and chlorophyll/carotenoid metabolism were suppressed, accelerating senescence in ZJ. Metabolomic profiling identified 60 anthocyanin-related metabolites, with Cyanidin-3-O-rutinoside being the predominant pigment in CL-V-DW. The integrated analysis of the transcriptome and metabolome identified 12 differentially expressed genes (DEGs), 15 structural genes, and 20 transcription factors, revealing significant correlations between these genes and anthocyanin-3-O-rutinoside, particularly BpABR1 and chalcone isomerase (BpCHI). Notably, the accumulation of anthocyanins in CL partially attenuated photosynthetic activity. This study elucidates that CL sustains anthocyanin synthesis through the activation of BpCHI, Ethylene-responsive transcription factor ABR1 (BpABR1), and WRKY transcription factor 40 (BpWRKY40), while enhancing anthocyanin transport efficiency via BpGST activation. Collectively, these findings provide insights into the molecular mechanisms underlying vein color regulation in B. papyrifera under cold stress and offer a theoretical basis for breeding cold-tolerant varieties.