Coumarin is an important secondary metabolite that affects plant physiology. It is a lactone of cis- o -hydroxycinnamic acid and widely exists in medicinal plants . Clematis terniflora DC. is a plant belonging to Ranunculaceae and is rich in variety of coumarins. Feruloyl-CoA 6′-hydroxylase has been reported as a key enzyme in the formation of coumarin basic skeleton only in some common plants, however, its evidence in other species is still lacking especially for the biosynthesis of coumarins in C . terniflora . In the present study, we identified a feruloyl-CoA 6′-hydroxylase CtF6′H in C . terniflora , and functional characterization indicated that CtF6′H could hydroxylate feruloyl-CoA to 6-hydroxyferuloyl-CoA. Furthermore, the expression level of CtF6′H was differed among different tissues in C. terniflora , while under UV-B radiation, the level of CtF6′H was increased in the leaves. Biochemical characteristics and subcellular location showed that CtF6′H was mainly present in the cytosol. The crystal structure of CtF6′H was simulated by homology modeling to predict the potential residues affecting enzyme activity . This study provides the additional evidence of feruloyl-CoA 6′-hydroxylase in different plant species and enriches our understanding of biosynthetic mechanism of coumarin in C . terniflora .
Coumarin synthase (COSY) is the key enzyme involved in the coumarin biosynthesis. Although it has been identified firstly in Arabidopisis thaliana , there is little known about the COSY gene function in Clematis terniflora DC. Our previous study indicated that the contents of coumarins in C. terniflora leaf were increased under ultraviolet-B radiation and dark treatments (UV-D). To reveal the function of COSY in C. terniflora and its role in response to UV-D stress, the functional characterization of CtCOSY which was identified as a BADH acyltransferase was performed. The results showed that coupled reaction of CtCOSY with 4-coumarinyl CoA-ligase in vitro to produce umbelliferone . Moreover, the functions of CtCOSY in response to UV-D stress was studied using proteomic and metabolomic techniques. Multi-omics analyses of CtCOSY -silenced C. terniflora indicated that as well as a limitation of coumarin synthesis, aspects of the energy metabolism, such as the TCA cycle, were disturbed in these plants. Under UV-D stress, CtCOSY affected the biosynthesis of phenylpropanoids in C. terniflora , resulting in the improvement of coumarin biosynthesis and affecting purine metabolism. This study provides the evidences of CtCOSY responsible for the biosynthesis of coumarin in C. terniflora and the resistance to UV-D stress.
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