Leguminous plants have many paralogous genes encoding enzymes involved in the flavonoid biosynthetic pathway. Duplicate genes are predicted to contribute to the production of various flavonoid compounds and to have resulted in a diversity of legume species. We identified gene duplication in the transcription factors regulating flavonoid biosynthesis in the model legume Lotus japonicus. Three copies of a homolog of Arabidopsis thaliana TRANSPARENT TESTA2 (TT2), which is a MYB transcription factor that regulates proanthocyanidin biosynthesis, were present in the L. japonicus genome. The organ specificity and stress responsiveness differed among the three LjTT2s, and correlations between proanthocyanidin accumulation and the expression levels of LjTT2s were observed during seedling development. Moreover, three LjTT2s functionally complemented TT2 in transient expression experiments in A. thaliana leaf cells. The different reporter activity caused by LjTT2a was consistent with the affinity of physical interactions with TT8 and TTG1 in yeast two-hybrid experiments as well as the branching pattern of the phylogenetic tree. These results suggest that LjTT2 factors have diverse functions in the tissues in which they are expressed; in particular, LjTT2a is predicted to have evolved flexibility in interaction with other transcription regulators to resist environmental stresses.

In the genome of the model legume Lotus japonicus, dihydroflavonol 4-reductase (DFR), which is the first committed enzyme of the anthocyanin and proanthocyanidin (PA) pathways, is encoded as a tandemly arrayed five-gene family. Expression analysis revealed that both organ specificity and stress responsiveness differ among the DFRs. To elucidate the regulatory mechanisms underlying the expression of DFRs, we investigated the transcriptional control of each member of the DFR multigene family. Ectopic expression of a combination of the transcription factors MYB, bHLH, and WDR showed that only the DFR2 promoter was activated, indicating that each member of the DFR gene family is regulated independently.