Lilies (Lilium spp.) are globally important ornamental crops which are constrained by their narrow thermal tolerance range. However, tiger lily (Lilium lancifolium), a wild lily species, exhibits remarkable cold tolerance. Based on our previous findings, we proposed that LlR3MYB, an R3-MYB transcription factor (TF), confers cold tolerance via transcriptional regulation of flavonoid metabolism in tiger lily. Here, we revealed that LlR3MYB represents a unique CPC-type R3-MYB TF exhibiting a bifunctional role in flavonoid metabolism. Specifically, LlR3MYB suppresses anthocyanin biosynthesis while promoting non-anthocyanin flavonoid accumulation (i.e. flavonols, flavones, and chalcones) responding to cold stress. Overexpression of LlR3MYB in tobacco and tiger lily increased total flavonoid content but reduced anthocyanin levels, consistent with the upregulation of early biosynthesis genes (e.g. CHS and FLS) and repression of late biosynthesis genes (e.g. DFR and ANS) in the pathway. In contrast, silencing LlR3MYB in tiger lily reduced total flavonoid production, enhanced anthocyanin accumulation, and compromised cold resistance. Mechanistically, LlR3MYB can directly bind to the AC-I element (ACCTACC) and MBSI motif (CAACGGTT) in the LlCHS2 promoter and activating its transcription, with enhanced activation under low temperature conditions. Mutations of critical residues within the C1/C2 repressor motifs may endow LlR3MYB with this transcriptional activation function. Furthermore, LlDREB can directly bind to the DRE motif (ACCGAC) in the LlR3MYB promoter and activating its transcription in a low-temperature-dependent manner. Our findings uncover a branch-specific regulatory mechanism by which MYB TFs fine-tune flavonoid biosynthesis, highlighting their essential role in plant cold stress responses.

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