Isolation and Functional Analysis of Chalcone Isomerase Gene from Purple-Fleshed Sweet Potato

Abstract

Chalcone isomerase (CHI, EC 5.5.1.6) is one of the essential enzymes in the anthocyanin biosynthetic pathway catalyzing the stereospecific isomerization of chalcones into their corresponding (2S)-flavanones. A full-length cDNA of CHI (844 bp in length) was isolated from purple-fleshed sweet potato [Ipomoea batatas (L.) Lam] cv. Yamakawamurasaki, designated IbCHI, containing a 732-bp open reading frame encoding a 243 amino acid polypeptide. Sequence alignment showed that the coding region of IbCHI gene is highly homologous with CHIs from other plant species and has four conserved amino acid residues (Thr50, Tyr108, Asn115, and Ser192) essential for CHI active sites. Phylogenetic analysis revealed that IbCHI was clustered into type I CHI group. Southern blotting showed that there were multicopies of IbCHI gene in I. batatas genome and IbCHI gene showed significant restriction fragment length polymorphism among different I. batatas cultivars. Overexpression of the IbCHI gene in Arabidopsis tt5 mutant fully complemented the pigmentation phenotype of the seed coat, cotyledon, and hypocotyl, indicating that the IbCHI gene encoded a fully functional CHI protein for anthocyanin and proanthocyanidin biosynthesis. The expression patterns of IbCHI and other anthocyanin biosynthesis-related genes (IbCHS, IbF3H, IbDFR, and IbANS) matched the accumulation patterns of anthocyanin in the thick roots and mature storage roots of different cultivars of sweet potato. However, in the fibrous roots, only IbCHI expressed obviously higher in purple-fleshed cultivar than white-fleshed cultivar, while IbCHS and IbDFR exhibited the opposite expression trends, and IbF3H and IbANS expressed almost equally in purple- and white-fleshed cultivars. The findings suggest that IbCHI is a key enzyme in the anthocyanin biosynthetic pathway of sweet potato, which is responsible for the activation of anthocyanin biosynthesis in the early stage of root development.