Cloning and functional characterization of a cinnamate 4-hydroxylase gene from the hornwort Anthoceros angustus

Hornworts, as the sister group to liverworts and mosses, comprise bryophytes, which are critical in understanding the evolution of key land plant traits. Cinnamate 4-hydroxylase (C4H) catalyzes the second step of the phenylpropanoid pathway to synthesize the precursor of numerous phenolic compounds, such as lignin and flavonoids. However, C4H in the hornwort Anthoceros angustus has not yet been cloned and functionally characterized. In this work, we screened the transcriptome database of A. angustus and identified one C4H gene, AnanC4H. AnanC4H maintained conserved cytochrome P450 domains with other typical plant C4Hs. Ultraviolet B irradiation and exogenous application of methyl jasmonate (MeJA) induced the expression of AnanC4H to varying degrees. The coding sequence of AnanC4H was expressed in yeast, and the recombinant proteins were isolated. The recombinant proteins of AnanC4H catalyzed the conversion of trans-cinnamic acid to p-coumaric acid and catalyzed the conversion of 3-hydroxycinnamic acid to caffeic acid. AnanC4H showed higher affinity for trans-cinnamic acid than for 3-hydroxycinnamic acid, but there was no significant difference in the catalytic efficiency of AnanC4H for the two substrates in vitro. Moreover, the expression of AnanC4H in Arabidopsis thaliana led to an increase in both the lignin content and the number of lignified cells in stems. However, there was no significant change in flavonoid content in transgenic Arabidopsis plants.