Functional characterization of two chalcone isomerase (CHI) revealing their responsibility for anthocyanins accumulation in mulberry.

Mulberry (Morus sp., Moraceae) is an important economic crop plant and mulberry fruits are rich in anthocyanidins. Chalcone isomerase (CHI) catalyzes the conversion of chalcones to flavanones providing precursors for biosynthesis of anthocyanidins. In this study, bona fide CHIs were cloned and characterized from different Morus species with differently colored fruits (Morus multicaulis, Mm and Morus alba variety LvShenZi, LSZ). Enzymatic assay of MmCHI1 and MmCHI2 showed that they can utilize naringenin chalcone as substrate. The catalytic efficiency of MmCHI2 and LSZCHI2 are approximately 200 and 120-fold greater than that of MmCHI1 respectively. Phylogenetic analysis showed the two mulberry CHIs belonged to different sub-clade of Type I CHI1 named type IA (CHI2) and type IB (CHI1). Type IB CHIs are mulberry specific. MmCHI1 and MmCHI2 had similar expression profiles and showed preferred expression in fruits. In addition, both mulberry CHI1 and CHI2 played roles in the response to excess zinc stress and sclerotiniose pathogen infection. Both MmCHI1 and MmCHI2 expression levels showed positive close relationship with anthocyanins content during fruit ripening process. The co-expression of MmCHI1 and MmCHI2 was observed during fruit ripening process and in transgenic mulberry. VIGS (virus induced gene silence) targeting on MmCHI1 and MmCHI2 showed significant down-regulation of MmCHI2 instead of MmCHI1 would result in significant (about 50%) decrease in anthocyanins content. MmCHI2 is the dominant CHI for anthocyanins accumulation in mulberry. The results presented in this work provided insight on bona fide CHIs in mulberry and reveal their roles in anthocyanins accumulation.