Cloning and functional characterization of a cinnamate 4-hydroxylase (SmC4H) from the lycophyte Selaginella moellendorffii.

Cinnamate 4-hydroxylase (C4H) is a key cytochrome P450 enzyme in the phenylpropanoid pathway, providing essential precursors for lignin and flavonoid biosynthesis. While well characterized in seed plants, the functional and regulatory features of C4H in lycophytes remain less well understood. In this study, a C4H gene (SmC4H) from the lycophyte Selaginella moellendorffii was cloned and functionally characterized. Sequence and comparative analyses showed that SmC4H retains the characteristic CYP73 framework, including a non-canonical PSRF sequence, and falls within the supported land-plant C4H/CYP73 cluster in a maximum-likelihood phylogeny. SmC4H also displayed the complete 10-motif architecture observed in the sampled land-plant C4Hs and a 3-exon/2-intron organization shared by many of these genes. Heterologous expression in yeast confirmed its cinnamate 4-hydroxylase activity, and kinetic analysis showed that SmC4H exhibited its highest apparent catalytic efficiency toward trans-cinnamic acid. In addition, SmC4H was capable of accepting the bulky cinnamate analog 2-naphthoic acid as a substrate, indicating a degree of substrate permissiveness. Subcellular localization analysis demonstrated that SmC4H is localized to the endoplasmic reticulum. Expression profiling revealed that SmC4H transcripts were most abundant in stems, which also exhibited the highest mean relative lignin content among the tissues examined. In contrast, flavonoid content was highly enriched in leaves, suggesting distinct tissue-level accumulation patterns of phenylpropanoid metabolites. Finally, promoter activity assays demonstrated that the SmC4H promoter (pSmC4H) is capable of driving gene expression in both vascular (Nicotiana benthamiana) and non-vascular (Marchantia polymorpha) plants. These findings provide a comparative reference for plant C4H biochemistry and promoter activity.