Tandemly duplicated MYB genes are functionally diverged in the regulation of anthocyanin biosynthesis in soybean

Gene duplications have long been recognized as a driving force in the evolution of genes, giving rise to novel functions. The soybean (Glycine max) genome is characterized by a large number of duplicated genes. However, the extent and mechanisms of functional divergence among these duplicated genes in soybean remain poorly understood. In this study, we revealed that 4 MYB genes (GmMYBA5, GmMYBA2, GmMYBA1, and Glyma.09g235000)-presumably generated by tandem duplication specifically in the Phaseoleae lineage-exhibited a stronger purifying selection in soybean compared to common bean (Phaseolus vulgaris). To gain insights into the diverse functions of these tandemly duplicated MYB genes in anthocyanin biosynthesis, we examined the expression, transcriptional activity, induced metabolites, and evolutionary history of these 4 MYB genes. Our data revealed that Glyma.09g235000 is a pseudogene, while the remaining 3 MYB genes exhibit strong transcriptional activation activity, promoting anthocyanin biosynthesis in different soybean tissues. GmMYBA5, GmMYBA2, and GmMYBA1 induced anthocyanin accumulation by upregulating the expression of anthocyanin pathway-related genes. Notably, GmMYBA5 showed a lower capacity for gene induction compared to GmMYBA2 and GmMYBA1. Metabolomics analysis further demonstrated that GmMYBA5 induced distinct anthocyanin accumulation in Nicotiana benthamiana leaves and soybean hairy roots compared to GmMYBA2 and GmMYBA1, suggesting their functional divergence leading to the accumulation of different metabolites accumulation following gene duplication. Together, our data provide evidence of functional divergence within the MYB gene cluster following tandem duplication, which sheds light on the potential evolutionary directions of gene duplications during legume evolution.