Systematic functional characterization of cinnamyl alcohol dehydrogenase family members revealed their functional divergence in lignin biosynthesis and stress responses in mulberry.

Mulberry (Morus) is used as a feed additive and biofuel materials. Cinnamyl alcohol dehydrogenase (CAD; EC 1.1.1.95) catalyzes the final step of monolignol biosynthesis and is responsible for various monolignols. Five MaCADs from Morus alba were cloned and functionally characterized in the present study. These MaCADs encoded proteins with 357-364 amino acids, and the putative protein sequences conservatively possessed two Zn2+ binding motifs and an NADP(H) cofactor binding motif. However, MaCAD1, 2, and 5 shared similar amino acids at substrate binding positions that differed from those possessed by bona fide CADs. MaCAD3 and 4 had conservative substrate binding sites, and both phylogenetic and expression profile analysis indicated they were bona fide CADs involved in lignin biosynthesis. The enzymatic assay showed that MaCAD1 and 5 had a high affinity to p-coumaryl aldehyde. MaCAD4 preferentially used coniferyl aldehyde and sinapyl aldehyde as substrates. His-72 and Tyr-124 in MaCAD1 stabilized p-coumaryl aldehyde, and may have resulted in the substrate preference for p-coumaryl aldehyde. Down-regulation of MaCADs in mulberry showed that MaCAD3/4 were dominant CADs that functioned in monolignol biosynthesis, and decreased MaCAD3/4 resulted in significant decreases of lignin content in both stems and leaves. MaCADs exhibited different expression patterns in response to various stresses, indicating their possible diverse roles. MaCAD2 and MaCAD5 may play positive roles in response to drought and cold stresses, respectively. These results provide a systematic functional analysis of MaCADs in mulberry and an important foundation for the genetic modification of the monolignol pathway in mulberry.