Stress-specific adaptation of a non-betalain DODA homolog from pansy (Viola × wittrockiana)

The mutual exclusivity of anthocyanin and betalain pigmentation in plants is a classic example of metabolic lineage-specific adaptation. Unlike L-3,4-dihydroxyphenylalanine ( L -DOPA) 4,5-dioxygenase (DODA) in betalain-producing plants, which is indispensable for betalain biosynthesis and has been extensively characterized, its LigB-type homologs in anthocyanin-producing plants lack betalain-synthetic capability, and their non-pigment physiological functions remain largely uncharacterized. In this study, we used phylogenetic reconstruction, enzymatic assays, heterologous expression and transcriptional regulation analyses to investigate VwDODA, a LigB-type DODA homolog from the anthocyanin-accumulating pansy ( Viola × wittrockiana ), a species that lacks betalain biosynthesis. Phylogenetic and enzymatic assays demonstrated that VwDODA clustered with AtLigB and catalyzes 2,3-extradiol cleavage of caffeic acid to produce arabidopyrone-related products, with negligible DODA activity. Heterologous expression in Arabidopsis demonstrated that VwDODA enhanced salt tolerance by modulating abscisic acid (ABA) signaling and enhancing the antioxidant system, while conferring resistance to Botrytis cinerea and Sclerotium rolfsii through attenuated oxidative burst. Transcriptional profiling and promoter analysis have identified VwMYB20 as a direct promoter-interacting candidate, which binds the VwDODA promoter and is correlated with its stress-responsive expression. This study unveils a non-pigmentary, stress-specific function for a non-betalain DODA homolog, highlighting its dual role in abiotic and biotic stress adaptation. While the precise in planta role of VwDODA-derived metabolites requires further investigation, our findings provide novel genetic insights and a promising candidate gene for the precision breeding of stress-resilient crops. • VwDODA is a LigB-type dioxygenase lacking betalain biosynthetic activity. • VwDODA catalyzes caffeic acid cleavage to arabidopyl alcohol. • Heterologous VwDODA in Arabidopsis enhances salt tolerance via ABA-ROS activation. • VwDODA confers pathogen resistance by attenuating oxidative burst in Arabidopsis . • VwMYB20 directly activates VwDODA to coordinate targeted stress adaptation.