LvWRKY75 enhances the transcription of LvMYB5 and promotes anthocyanin biosynthesis in lily petals during the blooming phase

Anthocyanin accumulation plays a crucial role in enhancing Lilium petal colouration; however, breeding efforts are hindered by our lack of understanding of the complex molecular mechanism behind the pigment's synthesis. This study explores the potential role of the WRKY family gene WRKY75 in anthocyanin synthesis in lilies. Contrary to the inhibitory effect observed in Arabidopsis thaliana, both transient silencing and overexpression analyses of LvWRKY75 indicate that the gene positively regulates anthocyanin synthesis in lilies. The overexpression of LvWRKY75 was found to cause a significant upregulation of structural genes pivotal for anthocyanin biosynthesis in lilies, including Lv3GT, LvDFR and LvANS, as well as the anthocyanin synthesis regulatory gene LvMYB5. Further in-depth analyses, including yeast one-hybrid, electrophoretic mobility shift assay, and dual-luciferase assays, demonstrated that LvWRKY75 binds to the promoter of LvMYB5, enhancing its transcriptional activity. In turn, the increased expression of LvMYB5 upregulates the transcription of downstream genes such as LvDFR and LvANS. In summary, this study provides a deeper understanding of the mechanisms behind anthocyanin synthesis in lilies, contributing to improving molecular breeding strategies for enhancing the flowers' ornamental value and commercial appeal.