Plant Molecular Biology

, Volume 23, Issue 1, pp 145–156 | Cite as

Molecular cloning, sequence analysis and elicitor-/ozone-induced accumulation of cinnamyl alcohol dehydrogenase from Norway spruce (Picea abies L.)

  • Hélène Galliano
  • Mireille Cabané
  • Christoph Eckerskorn
  • Friedrich Lottspeich
  • Heinrich SandermannJr.
  • Dieter Ernst
Research Article


Cinnamyl alcohol dehydrogenase (CAD) is an enzyme involved in lignin biosynthesis. We have previously isolated pure CAD enzyme from Norway spruce (Picea abies L.) cell culture. Here we report on partial protein sequences of the 42 kDa CAD polypeptide. A cDNA encoding CAD was isolated from the spruce cell culture. The open reading frame of a full-length cDNA coded for a 357 amino acid polypeptide with a calculated Mr of 38 777 Da. The identity of the deduced polypeptide was verified by comparison with amino acid sequences of tryptic peptides from the purified enzyme. Southern blot analysis showed the presence of only one gene for CAD. Sequence comparison with CAD from tobacco and with a N-terminal protein sequence from loblolly pine CAD showed an identity of 69.7% and 91.5%, respectively. Treatment of spruce cell cultures with elicitor, as well as of seedlings with ozone both markedly increased the CAD mRNA level.

Key words

CAD DNA sequence elicitor lignin ozone spruce 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Apostol I, Heinstein PF, Low PS: Rapid stimulation of an oxidative burst during elicitation of cultured plant cells. Plant Physiol 90: 109–116 (1989).Google Scholar
  2. 2.
    Bowles DJ: Defense-related proteins in higher plants. Annu Rev Biochem 59: 873–907 (1990).Google Scholar
  3. 3.
    Campbell MM, Ellis BE: Fungal elicitor-mediated response in pine cell cultures. I. Induction of phenylpropanoid metabolism. Planta 186: 409–417 (1992).Google Scholar
  4. 4.
    Chirgwin JM, Przybyla AE, MacDonald RJ, Rutter WJ: Isolation of biologically active ribonucleic acid from different sources enriched in ribonuclease. Biochemistry 18: 5294–5299 (1979).Google Scholar
  5. 5.
    Eckerskorn Ch, Lottspeich F: Internal amino acid sequence analysis of proteins separated by gel electrophoresis after tryptic digestion in polyacrylamide matrix. Chromatographia 28: 92–94 (1989).Google Scholar
  6. 6.
    Eisenberg D, Schwarz E, Komaromy M, Wall R: Analysis of membrane and surface protein sequences with the hydrophobic moment plot. J Mol Biol 179: 125–142 (1984).Google Scholar
  7. 7.
    Ernst D, Schraudner C, Langebartels C, Sandermann H: Ozone-induced changes of mRNA levels of β-1,3-glucanase, chitinase and ‘pathogenesis-related’ protein 1b in tobacco plants. Plant Mol Biol 20: 673–682 (1992).Google Scholar
  8. 8.
    Fan F, Lorenzen JA, Plapp BV: An aspartate residue in yeast alcohol dehydrogenase I determines the specificity for coenzyme. Biochemistry 30: 6397–6401 (1991).Google Scholar
  9. 9.
    Galliano H, Heller W, Sandermann H: Ozone induction and purification of spruce cinnamyl alcohol dehydrogenase. Phytochemistry 32: 557–563 (1993).Google Scholar
  10. 10.
    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).Google Scholar
  11. 11.
    Grand C, Sarni F, Lamb CJ: Rapid induction by fungal elicitor of the synthesis of cinnamyl-alcohol dehydrogenase, a specific enzyme of lignin synthesis. Eur J Biochem 169: 73–77 (1987).Google Scholar
  12. 12.
    Grisebach H: Lignins. In: Stumpf PK, Conn EE (eds) The Biochemistry of Plants, vol 7, pp. 457–478. Academic Press, New York (1981).Google Scholar
  13. 13.
    Halpin C, Knight ME, Grima-Pettenati J, Goffner D, Boudet A, Schuch W: Purification and characterization of cinnamyl alcohol dehydrogenase from tobacco stems. Plant Physiol 98: 12–16 (1992).Google Scholar
  14. 14.
    Heath RL: Biochemical mechanisms of pollutant stress. In: Heck WW, Taylor OC, Tingey DT (eds) Assessment of Crop Loss from Air Pollutants, pp 259–286. Elsevier. London (1988).Google Scholar
  15. 15.
    Heath RL, Castillo FJ: Membrane disturbances in response to air pollutants. In Schulte-Hostede S, Darrall NM, Blank LW, Wellburn AR (eds) Air Pollution and Plant Metabolism, pp. 55–75, Elsevier, London (1988).Google Scholar
  16. 16.
    Helfman DM, Feramisco JR, Fiddes JC, Thomas GP, Hughes SH: Immunological screening of cDNA expression libraries. Focus 6: 1–5 (1984).Google Scholar
  17. 17.
    Henikoff S: Unidirectional digestion with exonuclease III creates targeted breakpoints for DNA sequencing. Gene 28: 351–359 (1984).Google Scholar
  18. 18.
    Inoue H, Nojima H, Okayama H: High efficiency transformation of Escherichia coli with plasmids. Gene 96: 23–28 (1990).Google Scholar
  19. 19.
    Jansson S, Gustafsson P: Type I and type II genes for the chlorophyll a/b-binding protein in the gymnosperm Pinus sylvestris (Scots pine): cDNA cloning and sequence analysis. Plant Mol Biol 14: 287–296 (1990).Google Scholar
  20. 20.
    Jörnvall H, Persson B, Jeffery J: Characteristics of alcohol/polyol dehydrogenases. The zinc-containing longchain alcohol dehydrogenase. Eur J Biochem 167: 195–201 (1987).Google Scholar
  21. 21.
    Knight ME, Halpin C, Schuch W: Identification and characterization of cDNA clones encoding cinnamyl alcohol dehydrogenase from tobacco. Plant Mol Biol 19: 793–801 (1992).Google Scholar
  22. 22.
    Lewis NG, Yamamoto E: Lignin: occurrence, biogenesis and biodegradation. Annu Rev Plant Physiol Plant Mol Biol 41: 455–496 (1990).Google Scholar
  23. 23.
    Lüderitz T, Grisebach H: Enzymic synthesis of lignin precursors comparison of cinnamoyl-CoA reductase and cinnamyl alcohol: NADP + dehydrogenase from spruce (Picea abies L.) and soybean (Glycine max L.) Eur J Biochem 119: 115–124 (1981).Google Scholar
  24. 24.
    Messner B, Boll M, Berndt J: L-Phenylalanine ammonialyase in suspension culture cells of spruce (Picea abies). Plant Cell Tiss Organ Cult 27: 267–274 (1991).Google Scholar
  25. 25.
    Möller EM, Bahnweg G, Sandermann H, Geiger HH: A simple and efficient protocol for isolation of high molecular weight DNA from filamentous fungi, fruit bodies, and infected plant tissue. Nucl Acids Res 20: 6115–6116 (1992).Google Scholar
  26. 26.
    O'Malley DM, Porter S, Sederoff RR: Purification, characterization, and cloning of cinnamyl alcohol dehydrogenase in loblolly pine (Pinus toeda L.). Plant Physiol. 98: 1364–1371 (1992).Google Scholar
  27. 27.
    Pillonel C, Hunziker P, Binder A: Multiple forms of the constitutive wheat cinnamyl alcohol dehydrogenase. J Exp Bot 43: 299–305 (1992).Google Scholar
  28. 28.
    Rao M, Argos P: A conformational preference to predict helices in integral membrane proteins. Biochim Biophys Acta 869: 197–214 (1986).Google Scholar
  29. 29.
    Rosemann D, Heller W, Sandermann J: Biochemical plants responses to ozone. II. Induction of stilbene biosynthesis in Scots pine (Pinus sylvestris L.) seedlings. Plant Physiol 97: 1280–1286 (1991).Google Scholar
  30. 30.
    Sambrook J, Fritsch EF, Maniatis T: Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989).Google Scholar
  31. 31.
    Sandermann H, Schmitt R, Heller W, Rosemann D, Langebartels C: Ozone-induced early biochemical reactions in conifers. In Longhurst JWS (ed) Acidic Deposition. Sources, Effects and Controls, pp. 243–254. British Library, London (1989).Google Scholar
  32. 32.
    Sanger F, Nicklen S, Coulson AR: DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74: 5463–5467 (1977).Google Scholar
  33. 33.
    Sarni F, Grand C, Boudct AM: Purification and properties of cinnamoyl-Co reductase and cinnamyl alcohol dehydrogenase from poplar stems (Populus X euramericana). Eur J Biochem 139: 259–265 (1984).Google Scholar
  34. 34.
    Schraudner M, Ernst D, Langebartels C, Sandermann H: Biochemical plant responses to ozone. III. Activation of the defense-related proteins β-1,3-glucanase and chitinase in tobacco leaves. Plant Physiol 99: 1321–1328 (1992).Google Scholar
  35. 35.
    Schwacke R, Hager A: Fungal elicitors induce a transient release of active oxygen species from cultured spruce cells that is dependent on Ca2+ and protein-kinase activity. Planta 187: 136–141 (1992).Google Scholar
  36. 36.
    Vallee BL, Auld DS: Zinc coordination, function, and structure of zinc enzymes and other proteins. Biochemistry 29: 5647–5659 (1990).Google Scholar
  37. 37.
    Walter MH, Grima-Pattenati, 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).Google Scholar
  38. 38.
    Walter MH, Grima-Pattenati J, Grand C, Boudet AM, Lamb CJ: Extensive sequence similarity of the bean CAD4 (cinnamyl-alcohol dehydrogenase) to a maize malic enzyme. Plant Mol Biol 15: 525–526 (1990).Google Scholar
  39. 39.
    Wardrop AB: Occurrence and formation in plants. In: Sarkanen KV, Ludwig CH (eds), Lignins: Occurrence, Formation Structure and Reactions, pp. 29–41. Wiley-Interscience, New York (1971).Google Scholar
  40. 40.
    Wyrambik D, Grisebach H: Purification and properties of isoenzymes of cinnamyl-alcohol dehydrogenase from soybean-cell-supension cultures. Eur J Biochem 59: 9–15 (1975).Google Scholar

Copyright information

© Kluwer Academic Publishers 1993

Authors and Affiliations

  • Hélène Galliano
    • 1
  • Mireille Cabané
    • 1
  • Christoph Eckerskorn
    • 2
  • Friedrich Lottspeich
    • 2
  • Heinrich SandermannJr.
    • 1
  • Dieter Ernst
    • 1
  1. 1.GSF-Institut für Biochemische PflanzenpathologieOberschlei\heimGermany
  2. 2.MPI für BiochemieMartinsriedGermany

Personalised recommendations