The isolation and functional characterization of three liverwort genes encoding cinnamate 4-hydroxylase

The plant phenylpropanoid pathway is responsible for the synthesis of a wide variety of secondary metabolites. The second step in phenylpropanoid synthesis is carried out by the cytochrome P450 monooxygenase enzyme cinnamate 4-hydroxylase (C4H), which catalyzes the p-hydroxylation of trans-cinnamic acid to p-coumarate. Genes encoding C4H have been characterized in many vascular plant species, but as yet not in any bryophyte species. Here, a survey of the transcriptome sequences of four liverwort species was able to identify eight putative C4Hs. The three liverwort C4H genes taken forward for isolation and functional characterization were harbored by Plagiochasma appendiculatum (PaC4H) and Marchantia paleacea (MpC4H1 and MpC4H2). When the genes were heterologously expressed in yeast culture, an assay of enzyme activity indicated that PaC4H and MpC4H1 had a higher level of activity than MpC4H2. The favored substrate (trans-cinnamic acid) of all three liverwort C4Hs was the same as that of higher plant C4Hs. The co-expression of PaC4H in yeast cells harboring PaPAL (a P. appendiculatum ene encoding phenylalanine ammonia lyase) allowed the conversion of L-phenylalanine to p-coumaric acid. Furthermore, the expression level of PaC4H was enhanced after treatment with abiotic stress inducers UV irradiation or salicylic acid in the thallus of P. appendiculatum. The likelihood is that high activity C4Hs evolved in the liverworts and have remained highly conserved across the plant kingdom.