Molecular characterisation and expression of a wound-inducible cDNA encoding a novel cinnamyl-alcohol dehydrogenase enzyme in lucerne (Medicago sativa L.)

A lucerne (alfalfa, Medicago sativa) stem cDNA library was screened with a cinnamyl-alcohol dehydrogenase (CAD) cDNA probe from tobacco (Nicotiana tab acum cv. Samsun). Two distinctly different cDNA clones (54% identical) were isolated and identified as putative CAD-encoding cDNAs by comparison of th eir nucleotide sequences with those of CAD-encoding DNA sequences from othe r plant species. One of the cDNAs, MsaCad2, was found to be 99.4% identical at the nucleotide level to the previously isolated lucerne cad cDNA which encodes a CAD isoform involved in lignin biosynthesis. The other cDNA, MsaC ad1, has not been reported previously in lucerne, and encodes a protein rel ated to the ELI3 class of elicitor-inducible defence-related plant proteins . The MsaCad1- and MsaCad2-encoded proteins were expressed in Escherichia c oli and CAD1 was shown to be active with a range of cinnamyl, benzyl and al iphatic aldehyde substrates, while CAD2 was specific for the cinnamyl aldeh ydes only. Each of the respective genes is present as one or two copies. Th e MsaCad1 gene is expressed most actively in stem and floral tissue, wherea s MsaCad2 is most actively expressed in stem, hypocotyl and root tissue. In stem tissue, expression of both genes occurs predominantly in internodes 4 and 5 (from the apex). MsaCad2, in contrast to MsaCad1, is not significant ly expressed in the top three internodes of the stem. Both MsaCad1 and MsaC ad2 are wound-inducible, and the wound-responsiveness of each gene is modul ated by salicylic acid.