Plant Molecular Biology 36: 755–765, 1998.
1998 Kluwer Academic Publishers. Printed in Belgium.
A novel aromatic alcohol dehydrogenase in higher plants: molecular cloning
, Jan Van Doorsselaere
, Nabila Yahiaoui, Josef Samaj
Jacqueline Grima-Pettenati and Alain M. Boudet
Signaux et Messages Cellulaires chez les V
etaux-UMR CNRS-UPS 5546, Centre de Physiologie et Biologie
e Paul Sabatier, 118 route de Narbonne, 31062 Toulouse cedex, France (
correspondence); Present addresses:
Laboratorium voor Genetica, K.L. Ledeganckstraat 35, 9000 Gent,
Institute of Plant Genetics, Slovak Academy of Sciences, Akademia 2, 949 01 Nitra, Slovak Republic
Received 15 May 1997; accepted in revised form 12 November 1997
Key words: cinnamyl alcohol dehydrogenase, Eucalyptus gunnii, lignin
Cinnamyl alcohol dehydrogenase (CAD; EC 1.1.195) catalyses the conversion ofp-hydroxy-cinnamaldehydes to the
corresponding alcohols and is considered a key enzyme in lignin biosynthesis. In a previous study, an atypical form
of CAD (CAD 1) was identiﬁed in Eucalyptus gunnii. We reporthere the molecularcloningand characterization
of the corresponding cDNA, CAD 1-5, which encodes this novel aromatic alcohol dehydrogenase. The identity
of CAD 1-5 was unambiguously conﬁrmed by sequence comparison of the cDNA with peptide sequences derived
from puriﬁed CAD 1 protein and by functional expression of CAD 1 recombinant protein in Escherichia coli.
Both native and recombinant CAD 1 exhibit high afﬁnity towards lignin precursors including 4-coumaraldehyde
and coniferaldehyde, but they do not accept sinapaldehyde. Moreover, recombinant CAD 1 can also utilize a wide
range of aromatic substrates including unsubstituted and substituted benzaldehydes. The open reading frame of
CAD 1-5 encodes a protein with a calculated molecular mass of 35 790 Da and an isoelectric point of 8.1. Although
sequencecomparisonswith proteins in databases revealed signiﬁcant similarities with dihydroﬂavonol-4-reductases
(DFR; EC 188.8.131.52) from a wide range of plant species, the most striking similarity was found with cinnamoyl-
CoA reductase (CCR; EC 184.108.40.206), the enzyme which directly precedes CAD in the lignin biosynthetic pathway.
RNA blot analysis and immunolocalization experiments indicated that CAD 1 is expressed in both ligniﬁed and
unligniﬁed tissues/cells. Based on the catalytic activity of CAD 1 in vitro and its localization in planta,CAD1
may function as an ‘alternative’ enzyme in the lignin biosynthetic pathway. However, additional roles in phenolic
metabolism are not excluded.
Lignin biochemistry and molecular biology have
received widespread attention in recent years [45, 3, 4].
This emphasis is due not only to the abundance of lign-
ins in nature (ca. 25% of the earth’s biomass), but also
to the diversity of essential functions they afford, ran-
ging from physical support and conduction to defence.
It is generally accepted that the appearance of lignins
The nucleotide and protein sequences of the CAD 1 cDNA from
Eucalyptus gunnii are available in the EMBL Nucleotide Sequence
Database under the accesssion number X88797.
may be considered an evolutionaryturningpointessen-
tial for the adaptation of land plants to a non-aquatic
environment . Whether the original raison d’
of lignins was structural (i.e. conduction and support)
or defence-related remains speculative. However, the
presence of lignin-like compounds with a role in resist-
ance to microbial attack in charophycean green algae
suggests that the original role of these compounds may
well have been associated with defence . Lignins
are also an important consideration in the pulp and
paper industry in that lignins must be removed from
cellulose microﬁbrils by a costly and environmentally