Characterization of the Lonicera japonica R2R3-MYB transcription factor gene LjaMYB305 that promotes the flavonoid biosynthesis

Lonicera japonica Thunb., commonly known as honeysuckle, is a significant medicinal plant in Asia, with its flowers, stems, and leaves widely utilized in traditional Chinese medicine due to their rich bioactive constituents. Among these, flavonoids are of particular importance for their pharmacological properties. While previous studies have shown that light significantly induces flavonoid accumulation, the molecular mechanisms remain poorly understood. In this study, we isolated and characterized a novel R2R3-MYB transcription factor, LjaMYB305, from honeysuckle. Expression analysis revealed a strong correlation between LjaMYB305 transcription and flavonoid accumulation in flowers following light treatment. Functional characterization showed that the LjaMYB305 promoter is transcriptionally activated by light. Transient overexpression in honeysuckle leaves and stable ectopic expression in transgenic tobacco both significantly upregulated key flavonoid biosynthesis genes and increased flavonoid content. Conversely, virus-induced gene silencing (VIGS) of LjaMYB305 in honeysuckle led to significantly reduced flavonoid levels, accompanied by downregulation of flavonoid pathway genes. Mechanistic studies demonstrated that LjaMYB305 directly binds to and activates the promoters of Lja4CL2 and LjaCHS3, as validated by yeast one-hybrid (Y1H) and dual-luciferase (Dual-LUC) assays. Additionally, yeast two-hybrid (Y2H), bimolecular fluorescence complementation (BiFC), Dual-LUC assays and transient co-overexpression assays revealed that LjaMYB305 interacts with LjaHY5 to form a transcriptional module, synergistically enhancing the expression of Lja4CL2 and LjaCHS3. Overall, our findings establish that LjaMYB305 acts as a key regulator of flavonoid biosynthesis, functioning both independently and in conjunction with LjaHY5 to activate the expression of Lja4CL2 and LjaCHS3. This study provides new insights into the light-responsive regulatory network governing flavonoid production in honeysuckle.