Major quality regulation network of flavonoid synthesis governing the bioactivity of black wolfberry

Black wolfberry (Lycium ruthenicum Murr.) contains various bioactive metabolites represented by flavonoids, which are quite different among production regions. However, the underlying regulation mechanism of flavonoid biosynthesis governing the bioactivity of black wolfberry remains unclear. Presently, we compared the bioactivity of black wolfberry from five production regions. Multi-omics were performed to construct the regulation network associated with the fruit bioactivity. The detailed regulation mechanisms were identified using genetic and molecular methods. Typically, Qinghai (QH) fruit exhibited higher antioxidant and anti-inflammatory activities. The higher medicinal activity of QH fruit was closely associated with the accumulation of eight flavonoids, especially Kaempferol-3-O-rutinoside (K3R) and Quercetin-3-O-rutinoside (rutin). Flavonoid biosynthesis was found to be more active in QH fruit, and the upregulation of LrFLS, LrCHS, LrF3H and LrCYP75B1 caused the accumulation of K3R and rutin, leading to high medicinal bioactivities of black wolfberry. Importantly, transcription factor LrMYB94 was found to regulate LrFLS, LrCHS and LrF3H, while LrWRKY32 directly triggered LrCYP75B1 expression. Moreover, LrMYB94 interacted with LrWRKY32 to promote LrWRKY32-regulated LrCYP75B1 expression and rutin synthesis in black wolfberry. Transgenic black wolfberry overexpressing LrMYB94/LrWRKY32 contained higher levels of K3R and rutin, and exhibited high medicinal bioactivities. Importantly, the LrMYB94/LrWRKY32-regulated flavonoid biosynthesis was light-responsive, showing the importance of light intensity for the medicinal quality of black wolfberry. Overall, our results elucidated the regulation mechanisms of K3R and rutin synthesis, providing the basis for the genetic breeding of high-quality black wolfberry.