Blumea balsamifera is a renowned medicinal plant in the Miao ethnic tradition, with flavonoids constituting its primary pharmacologically active components. However, the functional mechanisms of the key enzymes involved in their biosynthesis remain poorly understood. In this study, we identified the chalcone isomerase (CHI) gene family from the B. balsamifera transcriptome and selected a pivotal member, BbCHI3, for in-depth functional investigation. Phylogenetic and sequence analyses indicated that BbCHI3 belongs to the type I CHI subfamily and possesses conserved catalytic active sites. Expression pattern analysis revealed that BbCHI3 is predominantly expressed in flowers and leaf bud of B. balsamifera, and its transcription is strongly induced by various stress treatments, including high salinity (HS), polyethylene glycol (PEG), abscisic acid (ABA), methyl jasmonate (MeJA), and herbivory by Spodoptera litura (SL), suggesting a crucial role in plant stress and defense responses and secondary metabolite accumulation. Subcellular localization results demonstrated that the BbCHI3 protein is localized in the cytoplasm. In vitro enzyme activity assays confirmed that recombinant BbCHI3 protein participates in the biosynthesis of various flavonoid compounds in B. balsamifera, including anthocyanidins, flavones, and flavonols. Furthermore, heterologous complementation experiments revealed that overexpression of BbCHI3 successfully restored anthocyanin accumulation in the cotyledons and seed coats of the Arabidopsis thaliana chi loss-of-function mutant (tt5). Additionally, heterologous expression and functional assays in Escherichia coli BL21(DE3) demonstrated that BbCHI3 confers enhanced tolerance to extremely high salt stress. This study systematically elucidates the core function of BbCHI3 in the flavonoid biosynthesis pathway of B. balsamifera, providing a key genetic resource for deciphering the molecular basis of its Daodi (geo-authentic) quality and for the targeted breeding of superior varieties through metabolic engineering.
Sha et al. (Mon,) studied this question.