Cloning and Functional Characterization of Chalcone Isomerase Genes Involved in Anthocyanin Biosynthesis in Clivia miniata

Abstract

Chalcone isomerase (CHI), catalyzing isomerization of chalcones, is a crucial enzyme in flavonoid biosynthesis. Three CHI genes were isolated from Clivia miniata and designated as CmCHI1, CmCHI2 and CmCHI3, respectively. Multiple sequence alignments and phylogenetic analysis showed that CmCHI1 and CmCHI2 were members of type I CHI proteins, whereas CmCHI3 belonged to type IV CHI proteins. Subcellular localization analysis found that all three CmCHIs had diffused distribution in the cytoplasm similar to green fluorescent protein (GFP). Anthocyanin biosynthesis and gene expression analysis demonstrated that CmCHIs were highly expressed in anthocyanin accumulated tissues. To further functionally characterize the role of CmCHIs, an in vitro enzymatic activity assay was carried out using the purified recombinant proteins. Results showed that CmCHI1 and CmCHI2 could completely convert the substrate naringenin chalcone (NC) into the product naringenin (NA), whereas CmCHI3 seemed nonfunctional as no increment of NA was detected. Further genetic transformation of Arabidopsis tt5-1 mutant validated that CmCHI1 and CmCHI2 rather than CmCHI3 could complement the chi deficient phenotypes. In summary, CmCHI1 and CmCHI2 are the real active CHI genes in Clivia miniata. The results not only broaden our knowledge on flavonoid biosynthesis in C. miniata but also lay a new foundation for further flavonoid modification in C. miniata.