Seven CAD-like Genes from Brachypodium

Cinnamyl alcohol dehydrogenase catalyzes the final step of the monolignol biosynthesis, the conversion of cinnamyl aldehydes to alcohols, using NADPH as a cofactor. Seven members of the cinnamyl alcohol dehydrogenase family were identified in the genome of Brachypodium distachyon and five of these were isolated and cloned from genomic DNA. RT-PCR revealed differential expression of the cloned genes, with BdCAD5 being expressed in all tissues and highest in root and stem while BdCAD3 was only expressed in stem and spikes. A phylogenetic analysis of CAD-like proteins place BdCAD5 on the same branch as bona fide CAD proteins from maize (ZmCAD2), rice (OsCAD2), sorghum (SbCAD2) and Arabidopsis (AtCAD4, 5). The predicted 3-D structures of both BdCAD3 and BdCAD5 resembled that of AtCAD5. However, the amino acid residues in the substrate binding domains of BdCAD3 and BdCAD5 are distributed symmetrical and BdCAD3 is similar to that of poplar sinapyl alcohol dehydrogenase (PotSAD). BdCAD3 and BdCAD5 both showed temperature optimum around 50°C. The optimal pH for the reduction of coniferyl aldehyde was 5.2 and 6.2, respectively and for the oxidation of coniferyl alcohol was 8 and 9.5 for BdCAD3 and BdCAD5, respectively. Kinetic parameters for conversion of coniferyl aldehyde and coniferyl alcohol showed that BdCAD5 was clearly the most efficient enzyme of the two. These data suggest that BdCAD5 is the main CAD enzyme for lignin biosynthesis in Brachypodium. All CAD enzymes are cytosolic except for BdCAD4 which has a putative chloroplast signal peptide adding to the diversity of CAD functions