Alternative First Exons Drive Enzymatic Activity Variation in Chalcone Synthase 3 of Dendrobium sinense

Abstract

Dendrobium sinense, a native orchid species of Hainan Island, is cultivated for its ornamental flowers. Recently, this species has gained significant attention due to its medicinal value. This study focuses on the identification of type III polyketide synthase (PKS), which catalyzes the formation of crucial intermediates in secondary metabolites. Through analysis of previous transcriptome data, a total of ten type III DsPKS genes were identified. Phylogenetic analysis categorized the type III PKS proteins into CHS, BBS, and PKS groups. Interestingly, the DsCHS3 gene exhibited alternative first exons, resulting in two splice variants, namely DsCHS3-1 and DsCHS3-2. Full-length cDNA sequencing revealed that DsCHS3-1 was the more prevalent splice variant. Prokaryotic expression and purification of DsCHS3-1 and DsCHS3-2 proteins were successfully achieved. Enzyme activity analysis demonstrated significantly higher catalytic activity in DsCHS3-2 compared to DsCHS3-1, particularly in the conversion of p-coumaryol-CoA and malonyl-CoA to naringin chalcone. Functional complementation assays in Arabidopsis mutants confirmed the higher catalytic activity of DsCHS3-2, as it restored flavonoid biosynthesis to a greater extent compared to DsCHS3-1. Overall, these findings offer valuable insights into the alternative splicing patterns and functional divergence of DsCHS3 genes in D. sinense.