LvbHLH13 Regulates Anthocyanin Biosynthesis by Activating the LvMYB5 Promoter in Lily (Lilium ‘Viviana’)

Anthocyanins, constituents of flavonoid compounds prevalent in plants, possess significant value in both plant development and human nutrition. The regulation of anthocyanin biosynthesis primarily involves the orchestration of MYB, bHLH, and WD40 transcription factors. Consequently, the bHLH family assumes a pivotal role in modulating plant developmental processes. In the present investigation, a transcription factor, denoted as LvbHLH13, was identified as a positive regulator of anthocyanin pigmentation in lily petals. LvbHLH13 is classified within the IIId subgroup of Arabidopsis bHLH proteins. Functional analyses involving the transient expression and gene silencing of LvbHLH13 revealed its capacity to enhance and diminish anthocyanin accumulation, respectively, by modulating the LvMYB5 expression, thereby influencing the downstream structural gene expression. The overexpression of LvbHLH13 resulted in an increase in the expression of the downstream structural genes related to anthocyanin synthesis, whereas silencing of LvbHLH13 correspondingly decreased the expression. Yeast one-hybrid and EMSA assays demonstrated the interaction between LvbHLH13 and the LvMYB5 promoter, leading to the activation of anthocyanin biosynthesis. A further luciferase (LUC) analysis corroborated the stimulatory effect of LvbHLH13 on the LvMYB5 promoter sequence. Consequently, LvbHLH13 assumed a crucial role in lily-petal pigmentation. A yeast two-hybrid analysis revealed that LvbHLH13 diverged from typical bHLH transcription factor behavior as it did not form a complex with MYB to regulate anthocyanin biosynthesis. This discrepancy could be attributed to the deletion of the N-terminal conserved sequence of LvbHLH13. This study provides a new bHLH candidate and bHLH-MYB partner to explore the anthocyanin regulatory network in further research and provides new opportunities for breeding lilies with various anthocyanin contents. These findings lay a theoretical foundation for subsequent investigations into lily flower coloring mechanisms.