New Insights into the Role of Cytokinin in Regulating Anthocyanin Biosynthesis and Leaf Expansion: An Integrated Transcriptomic, Metabolomic, and Physiological Analysis of Hypericum monogynum

Hypericum monogynum is a valuable perennial species with multiple uses, one of which is its ornamental value. In this study, we found that cytokinin treatment not only efficiently induced anthocyanin biosynthesis but also promoted leaf expansion in H. monogynum, both of which could potentially enhance its ornamental qualities. To investigate the molecular regulatory network underlying these processes, time-resolved transcriptomic sequencing and widely targeted metabolomic analysis were conducted. The results revealed 752, 385, and 1009 differentially expressed genes (DEGs) at 6, 12, and 24 h after 6-BA (6-benzylaminopurine) treatment, respectively. A total of 101 DEGs were co-regulated at all three time points, including key components of cytokinin metabolism and signaling. KEGG analysis identified metabolic pathways, biosynthesis of secondary metabolites, and plant hormone signal transduction as the most significantly enriched pathways. Key DEGs associated with the MYB and bHLH families, involved in flavonoid biosynthesis and cell proliferation, were also identified. Specifically, four MYB113 genes were found to be cytokinin-responsive and upregulated by 6-BA treatment at various time points. Several genes in the anthocyanin biosynthesis pathway, such as CHS, F3H, and F3 ' H, were upregulated by 6-BA treatment. Additionally, many DEGs related to nutrient transport, sugar metabolism, cell cycle, and cell expansion were identified, most of which were upregulated by 6-BA treatment, supporting cytokinin's role in promoting leaf growth and expansion. Furthermore, metabolomic analysis revealed key differentially accumulated metabolites in the flavonoid biosynthesis pathway, with major anthocyanins being identified. In conclusion, this study suggests that cytokinin application is an effective strategy for enhancing the ornamental value of H. monogynum and provides new insights into its role in regulating anthocyanin biosynthesis and leaf expansion in tree species.