Biochemical Characterization of the Rice Cinnamyl Alcohol Dehydrogenase Gene Family.

Cinnamyl alcohol dehydrogenase (CAD) is involved in the final step of the phenylpropanod pathway, catalyzing the NADPH-dependent reduction of hydroxy-cinnamaldehydes into the corresponding alcohols. The rice genome contains twelve and genes, collectively called s. To elucidate the biochemical function of the s, , , , and , which are highly expressed in rice, were cloned from rice tissues. The cloned s were heterologously expressed in as His-tag fusion proteins. The activity assay of the recombinant OsCADs showed that OsCAD2, 6, and 7 have CAD activity toward hydroxycinnamaldehydes, but OsCAD1 has no detectable catalytic activity. The kinetic parameters of the enzyme reactions demonstrated that has the highest catalytic activity among the examined enzymes. This result agrees well with the finding that the Zn binding and NADPH binding motifs and the residues constituting the substrate binding pocket in plant CADs were fully conserved in . Although they have large variations in the residue for the substrate binding pocket, OsCAD6 and 7 catalyzed the reduction of hydroxycinnamaldehydes with a similar efficiency. Alignment of amino acid sequences showed that OsCAD1 lacks the GxxxxP motif for NADPH binding and has mismatches in residues important in the reduction process, which could be responsible for the loss of catalytic activity. OsCAD2 belongs to CAD Class I with CADs from other plant species and is constitutively expressed throughout the developmental stages of rice, with preferential expression in actively lignifying tissues such as the root, stem, and panicle, suggesting that it is mainly involved in developmental lignification in rice. The expression of was also induced by biotic and abiotic stresses such as pv. () infection and UV-irradiation, suggesting that it plays a role in the defense response of rice, in addition to a role in developmental lignification. and 7 belong in CAD Class II. Their expression is relatively lower than that of and is confined to certain tissues, such as the leaf sheath, stem, and panicle. The expression of was stimulated by infection and UV-irradiation. Thus appears to be an inducible OsCAD that is likely involved in the defense response of rice against biotic and abiotic stresses.