An ancient role for the CYP73 gene family in t-cinnamic acid 4-hydroxylation, phenylpropanoid biosynthesis and embryophyte development

The phenylpropanoid pathway is a plant metabolism intimately linked to the transition to terrestrial life. It produces phenolic compounds that play essential roles in stress mitigation and ecological interactions. The pathway also provides the building blocks for hydrophobic polymers that form apoplastic diffusion barriers and make up a significant fraction of the land plant biomass. Despite its significance in embryophytes (i.e., land plants), the origin and evolutionary history of the phenylpropanoid pathway remain poorly understood. In particular, little is known about the organization and function of the pathway in bryophytes, the non-vascular embryophytes. In this study, we conducted a multidisciplinary analysis of the CYP73 gene family that encodes t-cinnamic acid 4-hydroxylase (C4H), the first plant-specific enzyme in the pathway. Our results indicate that C4H activity originated with the emergence of the CYP73 gene family in an ancestor of land plants and was supported by an arginine residue that stabilizes its substrate in the active site. C4H deficiency in the moss Physcomitrium patens, the liverwort Marchantia polymorpha and the hornwort Anthoceros agrestis resulted in a shortage of phenylpropanoids and abnormal plant development. The latter could be rescued in the moss by the exogenous supply of p-coumaric acid, the product of C4H. Our findings establish the emergence of the CYP73 gene family as a foundational event for the development of the canonical plant phenylpropanoid pathway and underscores the deep-rooted conservation of the C4H enzyme function in embryophyte biology.