Genome-wide identification of the gene family and functional analysis of putative genes in tobacco ( L.).

Cinnamyl alcohol dehydrogenase (CAD) plays a crucial role in lignin biosynthesis, and the gene family encoding various CAD isozymes has been cloned and characterized in numerous plant species. However, limited information regarding the gene family in tobacco is currently available. In this study, we identified 10 genes in , four in , and six in . The nucleotide and amino acid sequences of these tobacco CADs demonstrate high levels of similarity, whereas the putative protein sequences conservatively possessed two Zn binding motifs and an NADP(H) cofactor binding motif. Both NtCAD1 and NtCAD2 had conservative substrate binding sites, similar to those possessed by CADs, and evidence from phylogenetic analysis as well as expression profiling supported their role as CADs involved in lignin biosynthesis. NtCAD1 has two paralogous genes, and . Enzyme activity analysis revealed that NtCAD1-1 and NtCAD1-2 had a high affinity to coniferyl aldehyde, -coumaryl aldehyde, and sinapyl aldehyde, whereas NtCAD2 preferred coniferyl aldehyde and -coumaryl aldehyde as substrates. The kinetic parameter assay revealed that NtCAD1-2 functions as the most efficient enzyme. Downregulation of both NtCAD1-1 and NtCAD1-2 resulted in reddish-brown stems without significant changes in lignin content. Furthermore, NtCAD1-1, NtCAD1-2, and NtCAD2 showed distinct expression patterns in response to biotic and abiotic stresses, as well as different phytohormones. Our findings suggest that NtCAD1-1 and NtCAD1-2 are involved in lignin biosynthesis, with NtCAD1-2 also participating in both biological and abiotic stresses, whereas NtCAD2 plays a distinct role mainly in responding to biological and abiotic stresses in tobacco.