FUNCTIONAL GENOMICS OF MAIZE CAD

¹ North Carolina State University, 6113 Jordan Hall, Raleigh, NC 27695

² Pioneer Hi-Bred Int. 7300 NW 62nd Ave. P.O. Box 1004 Johnston, IA 50131-1004

³ North Carolina State University, Williams 1236, Raleigh, NC 27695

Cinnamyl alcohol dehydrogenase (CAD; EC1.1.1.195) catalyzes the final step of monolignol biosynthesis. In young maize seedlings, we have biochemically characterized two CAD-like enzymes that are both present in CAD deficient bm1 mutant plants. One of these was a dimeric, and the other was a monomeric enzyme. If redundant CAD-like gene products are involved in monolignol biosynthesis, manipulation of lignin content and quality in plants could be complicated. We carried out a functional genomic approach to identify the genes corresponding to these CAD-like enzymes. An extensive maize EST database showed several candidate genes for CAD-like enzymes, cad1 homologues and eli3 homologues. CAD1 in eucalyptus has enzyme activity similar to CAD, and a defense response gene eli3 in parsley has a high sequence similarity to cad. Full-length cDNA clones for two eli3 and a cad1 gene homologues were expressed in E. coli. One of the ELI3 proteins had substrate specificity similar to the dimeric CAD-like enzyme. Anti-ELI3 serum made from this recombinant protein recognized the dimeric CAD-like enzyme, and F2 segregation patterns on native gels for the dimeric CAD-like enzyme and the corresponding western blots with anti-ELI3 serum were the same. The CAD1 recombinant protein had substrate specificity similar to the monomeric CAD-like enzyme. The genetic map location for this cDNA coincided with the location of the monomeric CAD-like enzyme. We thus concluded that the CAD-like enzyme activity in maize seedlings could be attributed to the proteins encoded by three different genes: cad, eli3 and cad1. ELI3 enzyme activity is expressed in non-stressed, disease-free plant tissues. Further studies on the tissue and cell specificities of expression of these genes will help to determine the extent to which the gene products are redundant.