Identification and In Vitro Functional Characterization of the CCR Gene Family Reveals Their Regulatory Roles in Lignin Biosynthesis of Pinus yunnanensis

Cinnamoyl coenzyme A reductase (CCR) is the first rate-limiting enzyme in the monolignol-specific pathway and plays a pivotal role in lignin biosynthesis. However, CCR genes in Pinus yunnanensis remain uncharacterized, and their undefined substrate specificity further impedes mechanistic insights into lignin regulation while restricting strategies for wood property optimization. Using conserved domain and homology analysis, with a focus on the characteristic NAD(P)-binding motif (KNWYCYGK), we identified 12 CCR family members from the transcriptome data of P. yunnanensis. Phylogenetic analysis clustered the PyCCRs into two distinct clades: PyCCR1~6 fall into the CCR clade, while the remaining members form a CCR-like clade. In this study, twelve ORF regions of the P. yunnanensis CCR genes were cloned, and nine purified recombinant PyCCR proteins were obtained through prokaryotic expression. In vitro enzymatic assays demonstrated that PyCCR1, PyCCR2, PyCCR5, and PyCCR6 catalyzed the conversion of p-coumaroyl-CoA, feruloyl-CoA, and sinapoyl-CoA to p-coumaraldehyde, coniferaldehyde, and sinapaldehyde, respectively. Molecular docking of PyCCR1 to 6 with three substrates identified substrate-binding pocket domains. Within these domains, hydrogen bonds formed between ligands and residues in the R(X)5K motif of PyCCR1 to 6, whereas PyCCRL7 to 12 lacked the complete motif. RT-qPCR analysis showed tissue-specific expression patterns of the 12 genes across buds, stems, leaves, roots, and fruits. Collectively, these findings suggest that the presence of a complete R(X)5K motif may play a crucial role in maintaining the catalytic activity of PyCCRs. Our present study established a mechanistic foundation for elucidating lignin biosynthesis regulation in P. yunnanensis and offer genetic resources for improvement programs.