Apr 29, 1993 - Nucleotide Sequence of a cDNA Encoding Cinnamyl Alcohol. Dehydrogenase f rom Eucalyptus'. Catherine Feuillet, Alain Michel Boudet*, and ...
Plant Physiol. (1993) 103: 1447
Plant Gene Register
Nucleotide Sequence of a cDNA Encoding Cinnamyl Alcohol Dehydrogenase f rom Eucalyptus’ Catherine Feuillet, Alain Michel Boudet*, and JacquelineCrima-Pettenati
Centre de Biologie et Physiologie Végétale, Unité de Recherche Associée au Centre National de Ia Recherche Scientifique 1457, Université Paul Sabatier, 118 Route de Narbonne, 31062 Toulouse Cédex, France CAD2 (EC 1.1.1.195) catalyzes the reduction of hydroxycinnamyl aldehydes (paracoumaryl, coniferyl, sinapyl aldehydes) to their corresponding alcohols, which are the direct monomeric precursors of lignins (Luderitz a n d Griesbach, 1981). Recently, we purified two isoforms of CAD (CAD1 and CAD2) from Eucalyptus gunnii. These isoforms are distinct in their biochemical a n d functional properties (Goffner
et al., 1992). The cDNA encoding one of these isoforms, CAD2 (pEuCAD2), was isolated by screening a Xgtll library generated from a cell-suspension culture of E. gunnii, using a heterologous probe from tobacco (Knight et al., 1992). In this paper we report the nucleotide sequence of the fulllength cDNA clone pEuCAD2 (1392 bp) obtained for the first time from a woody angiosperm. Additionally, the Eucalyptus genus is particularly important for the pulp industry. The cDNA contains one continuous open reading frame corresponding to a protein of 356 amino acid residues (calculated molecular mass 38.8 kD).The polypeptide is 78% homologous with the CAD from tobacco a n d 81% with the partia1 published sequence from a gymnosperm, Loblolly pine (O’Malley et al., 1992; Table I). The identity of the Eucdyptus clone has been unambiguously demonstrated by amino acid comparison with purified CAD2 protein from Eucalyptus and by functional analysis of the recombinant enzyme expressed in Escherichia coli (Grima-Pettenati et al., 1993).
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This work was supported by the Commission of European Communities (ECLAIR-OPLIGE No. OOZI), the Centre National de la Recherche Scientifique, and the University Paul Sabatier. * Corresponding author; fax 33-61-55-62-10. Abbreviation: CAD, cinnamyl alcohol dehydrogenase. Table 1. Characteristics of cDNA pEUCAD2 from E. gunnii
Organism: Eucalyptus gunnii, cell-suspension culture obtained from clone 832 (Teulières et al., 1989) (origin of the clone: Association Forêt Cellulose, France). Cocation on Chromosome: Unknown. Gene, Function, Pathway: pEuCAD2, CADZ (EC 1.1.1.195), lignin biosynthesis, last step in the monolignol biosynthetic pathway (Luderitz and Griesbach,
ACKNOWLEDGMENTS
We thank W. Schuch (Imperial Chemical Industries, United Kingdom) for providing tobacco CAD cDNA. Received April29, 1993; accepted June 14,1993. Copyright Clearance Center: 0032-0889/93/l03/1447/01. The EMBL accession number for the sequence reported in this article is X65631.
1981).
Techniques: cDNA library screening, plasmid sequencing, northern and Southern analysis. Method of Identification: Sequence comparison with tobacco cDNA sequence (Knight et al., 19921, amino acid comparison with purified CADZ protein, functional analysis of the recombinant protein expressed in E. coli (Crima-Pettenati et al., 1993). Expression Characteristics: CADZ transcript of about 1500 nucleotides is expressed in xylem and virtually undetectable in phloem. Features of cDNA Structure: Translational start site at nucleotide 112 and stop site at nucleotide 1180. Two putative polyadenylation sites were observed at positions 1338 and 1353 (AATGAT). Structural Features of Protein Product: Coding region codes for a polypeptide of 356 amino acids with a cakulated molecular mass of 38.8 kD and a predicted isoelectric point of 5.55. CAD2 protein is functionally expressed in E. coli, showing the same biochemical properties as the natural enzyme. Availability of Antibodies: Monoclonal antibodies.
LITERATURE CITED
Goffner D, JoffroyI, Grima-Pettenati J, Halpin C, Knigh E, Schuch W, Boudet AM (1992) Purification and characterization of isoforms of cinnamyl alcohol dehydrogenase (CAD) from Eucalyptus xylem. Planta 188: 48-53 Grima-PettenatiJ, Feuillet C, Goffner D, Borderies G, Boudet AM (1993) Molecular cloning and expression of a Eucalyptus Runnii cDNA clone encoding cinnamyl akohol dehydrogenase. Plant Mo1 Biol21: 1085-1095 Knight ME, Halpin C, Schuch W (1992) Identification and characterisation of cDNA clones encoding cinnamyl alcohol dehydrogenase from tobacco. Plant Mo1 Biol 19 793-801 Luderitz T, Griesbach H (1981) Enzymic synthesis of lignin precursors. Comparison of cinnamyl alcohols: NADP+ dehydrogenase from spruce (Picea abies L.) and soybean (Glycine max L.). Eur J Biochem 119 115-124 O’Malley DM, Porter S, Sederoff RR (1992) Purification, characterization and cloning of cinnamyl alcohol dehydrogenase in Loblolly pine (Pinus taeda L.), Plant Physiol 9 8 1364-1371 Teulières C, Feuillet C, Boudet AM (1989) Differential characteristics of cell suspension cultures initiated from Eucalyptus gunnii clones differing by their frost tolerance. Plant Cell Rep 8: 407-410 1447
