Plant Molecular Biology 39: 437–447, 1999.
© 1999 Kluwer Academic Publishers. Printed in the Netherlands.
Down-regulation of cinnamyl alcohol dehydrogenase in transgenic alfalfa
(Medicago sativa L.) and the effect on lignin composition and digestibility
, Marie Andr
, Brigitte Chabbert
, Jean-Michel Besle
, Marc Van Montagu
and Johan Botterman
Laboratorium voor Genetica, Departement Genetica, Vlaams Interuniversitair Instituut voor Biotechnologie,
Universiteit Gent, K.L. Ledeganckstraat 35, 9000 Gent, Belgium (
author for correspondence);
Digestion Microbienne, SRNH INRA Theix, 63122 St. Gen`es Champanelle, France;
mol´ecules v´eg´etales, Esplanade R. Garros 2, 51686 Reims, France;
Plant Genetic Systems N.V., 9000 Gent,
Present address: Laboratoire de BiotechnologieV´eg´etale,Universit´e Libre de Bruxelles, 1850 Chauss´ee
de Wavre, 1160 Brussels, Belgium
Received 7 October 1997; accepted in revised form 2 September 1998
Key words: antisense strategy, cinnamyl alcohol dehydrogenase, digestibility, lignin, Medicago sativa
To improve the digestibility of the forage crop alfalfa (Medicago sativa L.), cinnamyl alcohol dehydrogenase
(CAD), which catalyses the last step in the biosynthesis of the lignin monomers, was down-regulated by using an
antisense approach. A subset of six transgenic lines with reduced CAD activity and control lines were analysed
when grown in the greenhouse and in the ﬁeld. The down-regulation of the CAD enzyme was associated with a
red coloration of the stem. The lignin quantity remained unchanged, but the lignin composition, as determined by
thioacidolysis, was altered. The highest reduction of CAD activity was associated with a lower syringyl/guaiacyl
(S/G) ratio and a lower S+G yield, mainly because of a decreased amount of S units. An increase in in situ
disappearance of dry matter and of cell wall residue was detected in one of the transgenic lines grown in the
greenhouse, and for two of the lines grown in the ﬁeld the rate of disappearance of dry matter slightly improved.
Furthermore, these two lines had a higher solubility in alkali as shown by the lower yield of saponiﬁed residue.
This study opens perspectives for improving forage crop digestibility by the modulation of enzymes involved in
Digestibility and intake of forage are key factors in
the growth of ruminant animals . Improvement of
digestibility would beneﬁt the recoverableyield of for-
age cropsand thereby reducethe overall cost of animal
production. One of the limiting factors for determin-
ing the digestibility of forage crops is the degree of
ligniﬁcation of cell walls [1, 10, 31].
Lignin is a major constituent of the secondary cell
walls of all vascular plants. This complex aromatic
polymer plays essential roles in strengthening and im-
permeabilization of the cell walls and constitutes me-
chanical and chemical barriers against pathogens .
Lignin is known to be synthesized de novo in response
to several sources of stress . It derives from the
oxidative polymerization of cinnamyl alcohols. These
monomers (or monolignols)are p-coumaryl, coniferyl
and sinapyl alcohols, differing only by their degree
of methoxylation. Lignin heterogeneity (quantity and
composition) among differenttaxa, differenttissues or
even different layers of individual cell walls is well
established [16, 40, 53].
Several mutants of maize, sorghum and pearl mil-
let have been characterized, with reduced lignin con-
tent or modiﬁed lignin composition and an improved
digestibility [4, 18, 30, 32, 45]. These mutants are
called brown-midrib (bm or bmr) mutants because of
the presence of a red-brown coloration of the midrib