Molecular cloning, characterization and expression analysis of cinnamate 4-hydroxylase (C4H) from Ferula pseudalliacea

Abstract

Cinnamate 4-hydroxylase (C4H) is a key enzyme in the phenylpropanoid pathway, catalyzing the hydroxylation of trans-cinnamic acid to p-coumaric acid. However, there is very limited information on the function of C4H in medicinal plants. In the present study, the C4H gene from the Ferula pseudalliacea genome (named FpC4H) was cloned and characterized. Besides, the expression levels of FpC4H in the roots, stems, leaves, flowers, and immature fruits were investigated via real-time PCR (polymerase chain reaction). FpC4H showed the most similarity with C4H proteins from Daucus carota. The results predicted that FpC4H, as well as its orthologs, is an unstable and hydrophilic protein. A high genetic distance was observed between FpC4H and C4H from monocots based on evolutionary analysis. A variable region related to the variable function of C4H was detected in the conserved domain region of C4H proteins. Protein structure analysis revealed that isoleucine and leucine residues are frequently present in the binding region of FpC4H, and two ion binding regions, cysteine258 and asparagine217, were identified in the 3D structure of FpC4H. Expression analysis revealed that the FpC4H gene is expressed in all organs of F. pseudallicea. A low expression level of FpC4H was recorded in leaves, and a high expression level was observed in flower. Based on the expression profile, FpC4H seems to be linked to a high content of phenolic compounds in flowers. In future studies related to metabolic engineering, FpC4H with a promoter linked to specific tissues (flowers and roots) could be investigated for improving the content of the phenolic compounds.