Integrating molecular characterization and metabolites profile revealed CtCHI1's significant role in Carthamus tinctorius L.

As a traditional Chinese herb, safflower (Carthamus tinctorius L.) is valued for its florets to prevent cardiovascular and cerebrovascular diseases. Basing on previous chemical analysis, the main active compounds are flavonoids in its florets. Although flavonoid biosynthetic pathway has been well-documented in many model species, unique biosynthetic pathway remains to be explored in safflower. Of note, as an important class of transitional enzymes, chalcone isomerase (CHI) has not been characterized in safflower.According to our previous research, CHIs were identified in a safflower transcriptome library built by our lab. To characterize CHI in safflower, a CHI gene named CtCHI1 was identified. A multiple sequences alignment and phylogenetic tree demonstrate that CtCHI1 shares 92% amino acid identity and close relationship with CHI to Saussurea medusa. Additionally, subcellular localization analysis indicated CtCHI1-GFP fusion protein was mainly in the cell nucleus. Further, we purified CtCHI1 protein from E. coli which can effectively catalyze isomerization of 2',4',4,6'-tetrahydroxychalcone into naringenin in vitro. Via genetic engineer technology, we successfully obtained transgenic tobacco and safflower lines. In transgenic tobacco, overexpression of CtCHI1 significantly inhibited main secondary metabolites accumulation, including quercetin (~ 79.63% for ovx-5 line) and anthocyanins (~ 64.55% for ovx-15 line). As shown in transgenic safflower, overexpression of CtCHI1 resulted in upstream genes CtPAL3 and CtC4H1 increasing dramatically (up to ~ 3.9fold) while Ct4CL3, CtF3H and CtDFR2 were inhibited. Also, comparing the whole metabolomics database by PCA and PLS-DA between transgenic and control group, 788 potential differential metabolites were marked and most of them displayed up-regulated trends. In parallel, some isolated secondary metabolites, such as hydroxysafflor yellow A (HSYA), rutin, kaempferol-3-O-β-rutinoside and dihydrokaempferol, accumulated in transgenic safflower plants.In this study, we found that CtCHI1 is an active, functional, catalytic protein. Moreover, CtCHI1 can negatively and competitively regulate anthocyanins and quercetin pathway branches in tobacco. By contrast, CtCHI1 can positively regulate flavonol and chalcone metabolic flow in safflower. This research provides some clues to understand CHI's differential biochemical functional characterization involving in flavonoid pathway. More molecular mechanisms of CHI remain to be explored in the near future.