A hallmark of adaptive evolution is innovation in gene function, which is associated with the development of distinct roles for genes during plant evolution; however, assessing functional innovation over long periods of time is not trivial. Tartary buckwheat (Fagopyrum tataricum) originated in the Himalayan region and has been exposed to intense UV-B radiation for a long time, making it an ideal species for studying novel UV-B response mechanisms in plants. Here, we developed a workflow to obtain a co-functional network of UV-B responses using data from more than 10,000 samples in more than 80 projects with multi-species and multi-omics data. Dissecting the entire network revealed that flavonoid biosynthesis was most significantly related to the UV-B response. Importantly, we found that the regulatory factor MYB4R1, which resides at the core of the network, has undergone neofunctionalization. In vitro and in vivo experiments demonstrated that MYB4R1 regulates flavonoid and anthocyanin accumulation in response to UV-B in buckwheat by binding to L-box motifs in the FtCHS, FtFLS, and FtUFGT promoters. We used deep learning to develop a visual discrimination model of buckwheat flavonoid content based on natural populations exposed to global UV-B radiation. Our study highlights the critical role of gene neofunctionalization in UV-B adaptation.
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