Plant Molecular Biology 41: 279–291, 1999.
© 1999 Kluwer Academic Publishers. Printed in the Netherlands.
Molecular characterisation and expression of a wound-inducible cDNA
encoding a novel cinnamyl-alcohol dehydrogenase enzyme in lucerne
(Medicago sativa L.)
Elizabeth M. Brill, Sharon Abrahams, Christine M. Hayes
, Colin L.D. Jenkins and John M.
CSIRO Plant Industry, GPO Box 1600, Canberra, ACT 2601, Australia (
author for correspondence);
address: Department of Botany & Zoology, Australian National University, PO Box 475, Canberra, ACT 2601,
Received 19 October 1998; accepted in revised form 18 August 1999
Key words: alfalfa, cinnamyl alcohol dehydrogenase, ELI3, ligniﬁcation, lucerne, stem abundant cDNA, substrate
speciﬁcity, wound induction
A lucerne (alfalfa, Medicago sativa) stem cDNA library was screened with a cinnamyl-alcohol dehydrogenase
(CAD) cDNA probe from tobacco (Nicotiana tabacum cv. Samsun). Two distinctly different cDNA clones (54%
identical) were isolated and identiﬁed as putative CAD-encoding cDNAs by comparison of their nucleotide se-
quences with those of CAD-encoding DNA sequences from other 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, MsaCad1, has not been reported previously in
lucerne, and encodes a protein related to the ELI3 class of elicitor-inducible defence-related plant proteins. The
MsaCad1-andMsaCad2-encoded proteins were expressed in Escherichia coli and CAD1 was shown to be active
with a range of cinnamyl, benzyl and aliphatic aldehyde substrates, while CAD2 was speciﬁc for the cinnamyl
aldehydes only. Each of the respective genes is present as one or two copies. The MsaCad1 gene is expressed most
actively in stem and ﬂoral tissue, whereas 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 signiﬁcantly expressed in the top three internodes of the stem. Both MsaCad1 and
MsaCad2 are wound-inducible, and the wound-responsiveness of each gene is modulated by salicylic acid.
Lignin is a complex aromatic polymer formed from
phenolic precursors which are products of the plant
phenylpropanoid pathway. It imparts rigidity and
structural support to the plant cell wall and assists in
the transport of solutes within xylem tissue by decreas-
ing the permeability of the cell wall (Higuchi et al.,
1981). Ligniﬁcation of plant tissue also occurs in re-
The nucleotide sequence data reported will appear in the
EMBL, GenBank and DDBJ Nucleotide Sequence Databases under
the accession numbers AF083333 and AF083332.
sponse to wounding and fungal infection as part of the
general defence mechanism of plants (Walter, 1992).
The monomeric composition of lignin differs be-
tween angiosperms and gymnosperms, and also be-
tween healthy and wounded or pathogen-challenged
tissue (Ride, 1975; Kutsuki et al., 1982; Walter, 1992).
Wood from angiosperms has been shown to have better
pulping characteristics than wood from gymnosperms
(Chiang et al., 1988), and this may relate to the
differences in lignin monomeric composition. These
different lignin compositions may result from differ-
ing speciﬁcities or activities of enzymes involved in
lignin biosynthesis. Two enzymes, cinnamoyl-CoA