Molecular cloning and expression of a Eucalyptus gunnii cDNA clone encoding cinnamyl alcohol dehydrogenase Authors
Received: 08 October 1992 Accepted: 10 January 1993 DOI:
Cite this article as: Grima-Pettenati, J., Feuillet, C., Goffner, D. et al. Plant Mol Biol (1993) 21: 1085. doi:10.1007/BF00023605 Abstract
Cinnamyl alcohol dehydrogenase (CAD) catalyses the reduction of hydroxycinnamyl aldehydes (sinapyl, paracoumaryl, coniferyl aldehydes) to the corresponding alcohols which are the direct monomeric precursors of lignins. Recently, we have purified from
Eucalyptus gunnii two isoforms of CAD (CAD1 and CAD2), distinct in their biochemical and functional properties. In this paper, we report the cloning of a CAD cDNA (pEuCAD2) isolated by screening a λgt11 library generated from cell suspension culture of Eucalyptus gunnii, using a tobacco CAD cDNA as a probe. This full-length clone (1392 bp) encodes a protein of 356 amino acids which corresponds to the subunit molecular weight of the CAD2 isoform. Sequence analysis revealed that CAD2 is very well conserved among species (78% homology with CAD from tobacco, a herbaceous angiosperm, and 81% with the partial sequence from a gymnosperm, loblolly pine). The identity of this clone was unambiguously demonstrated (1) by comparison with peptide sequence data from purified CAD2 and (2) by functional expression of the recombinant enzyme in Escherichia coli. Recombinant CAD showed the same properties as the natural isoform CAD2, in terms of electrophoretic mobility, polypeptide structure, substrate specificity and antigenicity. The CAD2 transcript is equally abundant in stems and leaves and at the limit of detection in roots. At the tissue level the CAD2 gene is highly expressed in xylem and virtually undetectable in phloem. Key words cinnamyl alcohol dehydrogenase cDNA sequence eucalyptus gene cloning lignins References
Dellaporta SL, Wood J, Hicks JB: A plant DNA minipreparation: version II. Plant Mol Biol Rep 1: 19–21 (1983).
Dennis ES, Gerlach WL, Pryor AJ, Bennetzen JL, Inglis A, Llewllyn D, Sachs MM, Ferl RJ and Peacock WJ: Molecular analysis of the alcohol dehydrogenase (Adh1) gene of maize. Nucl Acids Res 12: 3982–4000 (1984).
Esau K: Anatomy of Seed Plants, 2nd ed. J. Wiley, New York (1977).
Fan F, Lerentzen JA, Plapp BV: An aspartate residue in yeast alcohol dehydrogenase I determines the specificity for coenzyme. Biochemistry 30: 6397–6401 (1991).
Goffner D, Joffroy I, Grima-Pettenati J, Halpin C, Knight ME, Schuch W, Boudet AM: Purification and characterization of isoforms of cinnamyl alcohol dehydrogenase (CAD) from eucalyptus xylem. Planta 188: 48–53 (1992).
Gould SJ, Keller GA, Subramani S: Identification of peroxisomal targeting signals located at the carboxy terminus of four peroxisomal proteins. J Cell Biol 107: 897–905 (1988).
Higuchi T: Biosynthesis of lignins. In: Biosynthesis and Biodegradation of Wood Components, pp. 141–160. Academic Press, Orlando, FL (1985).
Jornvall H, Persson B, Jeffery J: Characteristics of alcohol/polyol dehydrogenases. The zinc-containing long chain alcohol dehydrogenases. Eur J Biochem 167: 195–201 (1987).
Kolattukudy PE: Structure, biosynthesis and biodegradation of cutin and suberin. Annu Rev Plant Physiol 32: 539–567 (1981).
Knight ME, Halpin C, Schuch W: Identification and characterisation of cDNA clones encoding cinnamyl alcohol dehydrogenase from tobacco. Plant Mol Biol 19: 793–801 (1992).
Jones JDG, Duinsmuir P, Bedbrook J: High level expression of introduced chimeric genes in regenerated transformed plants. EMBO J 4: 2411–2418 (1985).
Laemmli UK: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685 (1970).
Lewis NG, Yamamoto E: Lignin: occurrence, biogenesis and biodegradation. Annu Rev Plant Physiol Plant Mol Biol 41: 445–496 (1990).
Logemann J, Schell J, Willmitzer L: Improved method for the isolation of RNA from plant tissue. Anal Biochem 163: 16–20 (1987).
Luderitz T, Grisebach H: Enzymic synthesis of lignin precursors. Comparison of cinnamyl alcohol: NADP
dehydrogenase from spruce (
L.) and soybean (
L.). Eur J Biochem 119: 115–124 (1981).
Lynn DG, Chang M: Phenolic signals in cohabitation: implication for plant development. Annu Rev Plant Physiol Plant Mol Biol 41: 497–526 (1990).
Maniatis T, Fritsch EF, Sambrook J: Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1982).
Mansell RB, Babbel GR, Zenk MH: Multiple forms and specificity of coniferyl alcohol dehydrogenase from cambial regions of higher plants. Phytochemistry 15: 1849–1853 (1976).
Monties B: Recent advances in lignin inhomogeneity. In: Van Sumere CF, Lea PJ (eds) The Biochemistry of Plant Phenolics, pp. 161–181. Clarendon Press, London (1985).
O'Malley DM, Porter S, Sederoff RR: Purification, characterization and cloning of cinnamyl alcohol dehydrogenase in Loblolly Pine (
Pinus taeda L.). Plant Physiol 98: 1364–1371 (1992).
Pillonel C, Hunziker P, Binder A: multiple forms of the constitutive wheat cinnamyl alcohol dehydrogenase. J Exp Bot 43: 299–305 (1992).
Pognonec P, Kato H, Sumimoto H, Kretzschmar M, Roeder RG: A quick procedure for purification of functional recombinant proteins overexpressed in
E. coli. Nucl Acids Res 23: 6650 (1991).
Sanger F, Nicklen S, Coulson AR: DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74: 5463–5467 (1977).
Studier FW, Moffat BA: Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J Mol Biol 189: 113–130 (1986).
Tabor S, Richardson CC: A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes. Proc Natl Acad Sci USA 82: 1074–1078 (1985).
Terashima N, Fukushima K: Heterogeneity in formation of lignin. XI. An autoradiographic study of the heterogeneous formation and structure of pine lignin. Wood Sci Technol 22: 259–270 (1988).
Tuelières C, Feuillet C, Boudet AM: Differential characteristics of cell suspension cultures initiated from
clones differing by their frost tolerance. Plant Cell Rep 8: 407–410 (1989).
Umezawa T, Davin LB, Lewis NG: Formation of lignans (-)secoisolariciresinol and (-)matairesinol with
cell-free extracts. J Biol Chem 266: 10210–10217 (1991).
Vallee BL, Auld DS: Zinc coordination, function and structure of zinc enzymes and other proteins. Biochemistry 29: 5647–5659 (1990).
Walter MH, Grima-Pettenati J, Grand C, Boudet AM, Lamb CJ: Cinnamyl alcohol dehydrogenase, a molecular marker specific for lignin synthesis: cDNA cloning and mRNA induction by fungal elicitor. Proc Natl Acad Sci USA 85: 5546–5550 (1988).
Walter MH, Grima-Pettenati J, Grand C, Boudet AM, Lamb CJ: Extensive sequence similarity of the bean CAD4 ‘cinnamyl alcohol dehydrogenase’ clone to a maize malic enzyme. Plant Mol Biol 15: 525–526 (1990).
Wyrambik D, Griseback H: Purification and properties of isoenzymes of cinnamoyl alcohol dehydrogenase from soybean cell suspension cultures. Eur J Biochem 59: 9–15 (1975).
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