Identification of the CAD gene family in Cunninghamia lanceolata and functional characterization of ClCAD1 involved in lignin biosynthesis

Cinnamyl alcohol dehydrogenase (CAD), which catalyzes the final step of monolignol biosynthesis, is a key enzyme in the lignin pathway. Chinese fir (Cunninghamia lanceolata) is a major fast-growing timber species in southern China, and its wood quality is fundamentally determined by lignin content and composition. However, the systematic identification and functional characterization of the CAD gene family in gymnosperms remain limited. Based on full-length transcriptome and genomic sequencing data, we identified 12 CAD genes (designated ClCAD1-ClCAD12) and performed systematic analyses of their phylogeny, structure, expression, and cis-elements. Phylogenetic analysis categorized the 12 ClCAD proteins into three major groups. Among them, ClCAD1 and ClCAD2 clustered within the bona fide clade (Group Ia). The remaining seven members (ClCAD3–9) formed a gymnosperm-specific monophyletic group (Group Ib); these genes are physically clustered in the genome and share highly conserved structures. Expression profiling revealed that ClCAD1 was predominantly expressed in the xylem and exhibited increased transcript abundance under compression wood treatment, suggesting a potential association with lignin accumulation. Enzymatic assays demonstrated that the recombinant ClCAD1 protein efficiently converts p-coumaraldehyde, coniferaldehyde, and sinapaldehyde into their corresponding alcohols, with a potential preference for coniferaldehyde. Subcellular localization analysis showed that the ClCAD1-GFP fusion protein exhibited a distribution pattern consistent with a soluble CAD enzyme. Taken together, our findings provide the first systematic insight into the complex composition and evolutionary characteristics of the CAD family in gymnosperms, offering theoretical foundations and candidate gene resources for the genetic improvement of wood quality in Chinese fir.