Systematic characterization of cinnamyl alcohol dehydrogenase members revealed classification and function divergence in Haplomitrium mnioides.

Cinnamyl alcohol dehydrogenase (CAD; EC 1.1.1.195) is considered to be a key enzyme in lignin biosynthesis, which can catalyze cinnamyl aldehyde to produce cinnamyl alcohol. In this study, three putative CADs were characterized from the liverwort Haplomitrium mnioides. The sequence alignment and phylogenetic analysis revealed that HmCADs belonged to a multigene family, with three HmCADs belonging to class II, class III, and class IV, respectively. In vitro enzymatic studies demonstrated that HmCAD2 exhibited high affinity and catalytic activity towards five cinnamyl aldehydes, followed by HmCAD3 with poor catalytic activity, and HmCAD1 catalyzed only the reaction of p-coumaryl aldehyde and coniferyl aldehyde with extremely low catalytic capacity. Protein-substrate binding simulations were performed to investigate the differences in catalytic activity exhibited when proteins catalyzed different substrates. Furthermore, distinct expression patterns of three HmCADs were identified in different plant tissues. Subcellular localization tests confirmed that HmCAD1/2/3 was located in the cytoplasm. The simulated responses of HmCADs to different stresses showed that HmCAD1 played a positive role in coping with each stress, while HmCAD2/3 was weak. These findings demonstrate the diversity of CADs in liverwort, highlight the divergent role of HmCAD1/2/3 in substrate catalysis, and also suggest their possible involvement in stress response, thereby providing new insights into CAD evolution while emphasizing their potential distinctive and collaborative contributions to the normal growth of primitive liverworts.