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Molecular characterisation and expression of a wound-inducible cDNA encoding a novel cinnamyl-alcohol dehydrogenase enzyme in lucerne (Medicago sativa L.)

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Abstract

A lucerne (alfalfa, Medicago sativa) stem cDNA library was screened with a cinnamyl-alcohol dehydrogenase (CAD) cDNA probe from tobacco (Nicotiana tabacum cv. Samsun). Two distinctly different cDNA clones (54% identical) were isolated and identified as putative CAD-encoding cDNAs by comparison of their nucleotide sequences with those of CAD-encoding DNA sequences from other plant species. One of the cDNAs, MsaCad2, was found to be 99.4% identical at the nucleotide level to the previously isolated lucerne cad cDNA which encodes a CAD isoform involved in lignin biosynthesis. The other cDNA, MsaCad1, has not been reported previously in lucerne, and encodes a protein related to the ELI3 class of elicitor-inducible defence-related plant proteins. The MsaCad1- and MsaCad2-encoded proteins were expressed in Escherichia coli and CAD1 was shown to be active with a range of cinnamyl, benzyl and aliphatic aldehyde substrates, while CAD2 was specific for the cinnamyl aldehydes only. Each of the respective genes is present as one or two copies. The MsaCad1 gene is expressed most actively in stem and floral tissue, whereas MsaCad2 is most actively expressed in stem, hypocotyl and root tissue. In stem tissue, expression of both genes occurs predominantly in internodes 4 and 5 (from the apex). MsaCad2, in contrast to MsaCad1, is not significantly expressed in the top three internodes of the stem. Both MsaCad1 and MsaCad2 are wound-inducible, and the wound-responsiveness of each gene is modulated by salicylic acid.

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References

  • Abrahams, S., Hayes, C.M. and Watson, J.M.1995 Expression patterns of three genes in the stem of lucerne (Medicago sativa). Plant Mol. Biol. 27: 513–528.

    Google Scholar 

  • Appels, R. and Moran, L.B.1984 Molecular analysis of alien chromatin introduced into wheat. In: Gene Manipulation and Plant Improvement, 16th Stadler Genetics Symposium, pp. 529–558.

  • Baucher, M., Monties, B., Van Montagu, M. and Boerjan, W.1998 Biosynthesis and genetic engineering of lignin. Crit. Rev. Plant Sci. 17: 125–197.

    Google Scholar 

  • Boudet, A.M., Lapierre, C. and Grima-Pettenati, J.1995 Biochemistry and molecular biology of lignification. New Phytol. 129: 203–236.

    Google Scholar 

  • Bradford, M.M.1976 A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248–254.

    Google Scholar 

  • Brill, E.M., Abrahams, S.A., McAlister, F.M., Hayes, C.M. and Watson, J.M.1996 Stem abundant expression of two distinct cDNAs encoding putative cinnamyl alcohol dehydrogenases from lucerne (Medicago sativa L.). ASBMB and ASPP Combined Conference Abstracts, Canberra, Australia.

    Google Scholar 

  • Bucholtz, D.L., Cantrell, R.P., Axtell, J.D. and Lechtenberg, V.L. 1980. Lignin biochemistry of normal and brown midrib mutant sorghum. J. Agric. Food Chem. 28: 1239–1241.

    Google Scholar 

  • Campbell, M.M. and Ellis, B.E.1992 Fungal elicitor-mediated responses in pine cell cultures. I. Induction of phenylpropanoid metabolism. Planta 186: 409–417.

    Google Scholar 

  • Chiang, V.L., Puumala, R.J., Takeuchi, H. and Eckert, R.E.1988 Comparison of softwood and hardwood kraft pulping. Tappi J. 71: 173–176.

    Google Scholar 

  • Cipollini, D.F.1997 Wind-induced mechanical stimulation increases pest resistance in common bean. Oecologia 111: 84–90.

    Google Scholar 

  • Fan, F., Lorenzen, J.A. and Plapp, B.V.1991 An aspartate residue in yeast alcohol dehydrogenase I determines the specificity for coenzyme. Biochemistry 30: 6397–6401.

    Google Scholar 

  • Feuillet, C., Boudet, A.M. and Grima-Pettenati, J.1993 Nucleotide sequence of a cDNA encoding cinnamyl alcohol dehydrogenase from Eucalyptus. Plant Physiol. 103:1447

    Google Scholar 

  • Galliano, H., Cabane, M., Eckerskorn, C., Lottspeich, F., Sandermann, H. Jr. and Ernst, D.1993 Molecular cloning, sequence analysis and elicitor-/ozone-induced accumulation of cinnamyl alcohol dehydrogenase from Norway spruce (Picea abies L). Plant Mol. Biol. 23: 145–156.

    Google Scholar 

  • Goffner, D., Joffroy, I., Grima-Pettenati, J., Halpin, C., Knight, M.E., Schuch, W. and Boudet, A.M.1992 Purification and characterization of isoforms of cinnamyl alcohol dehydrogenase from Eucalyptus xylem. Planta 188: 48–53.

    Google Scholar 

  • Goffner, D., Van Doorsselaere, J., Yahiaoui, N., Samaj, J., Grima-Pettenati, J. and Boudet, A.M.1998 A novel aromatic alcohol dehydrogenase in higher plants: molecular cloning and expression. Plant Mol. Biol. 36: 755–765.

    Google Scholar 

  • Grand, C., Sarni, F. and Lamb, C.1987 Rapid induction by fungal elicitor of the synthesis of cinnamyl alcohol dehydrogenase, a specific enzyme of lignin synthesis. Eur. J. Biochem. 169: 73–77.

    Google Scholar 

  • Grima-Pettenati, J., Campargue, C., Boudet, A. and Boudet, A.M. 1994. Purification and characterization of cinnamyl alcohol dehydrogenase isoforms from Phaseolus vulgaris. Phytochemistry 37: 941–947.

    Google Scholar 

  • Halpin, C., Holt, K., Chojecki, J., Oliver, D., Chabbert, B., Monties, B., Edwards, K., Barakate, A. and Foxon, G.A.1998 Brownmidrib maize (bm1): a mutation affecting the cinnamyl alcohol dehydrogenase gene. Plant J. 14: 545–553.

    Google Scholar 

  • Hawkins S.W. and Boudet, A.M.1994 Purification and characterization of cinnamyl alcohol dehydrogenase isoforms from the periderm of Eucalyptus gunnii Hook. Plant Physiol. 104: 75–84.

    Google Scholar 

  • Hawkins, S., Goffner, D. and Boudet, A.M.,1994 Cinnamyl alcohol dehydrogenase polymorphism and its potential role in the control of lignin heterogeneity. Acta Hort. 381: 280–285.

    Google Scholar 

  • Hibino, T., Shibata, D., Chen, J-Q. and Higuchi, T.1993 Cinnamyl alcohol dehydrogenase from Aralia cordata: cloning of the cDNA and expression of the gene in lignifying tissues. Plant Cell Physiol. 34: 659–665.

    Google Scholar 

  • Higgins, T.J., Zwar, J.A. and Jacobsen, J.V.1976 Gibberellic acid enhances the level of translatable mRNA for α-amylase in barley aleurone layers. Nature 260: 166–168.

    Google Scholar 

  • Higuchi, T.1981 Biosynthesis of lignin. In: W. Tanner and F.A. Loewus (Eds.), Encyclopedia of Plant Physiology, Vol. 13B, Springer-Verlag, Berlin, pp. 194–217.

    Google Scholar 

  • Jornvall, H., Persson, B. and Jeffery, J.1987 Characteristics of alcohol/polyol dehydrogenases. The zinc-containing long-chain alcohol dehydrogenases. Eur. J. Biochem. 167: 195–201.

    Google Scholar 

  • Kiedrowski, S., Kawalleck, P., Hahlbrock, K., Somssich, I.E. and Dangl, J.L.1992 Rapid activation of a novel plant defense gene is strictly dependent on the Arabidopsis RPM1 disease resistance locus. EMBO J. 11: 4677–4684.

    Google Scholar 

  • Knight, M.E., Halpin, C. and Schuch, W.1992 Identification and characterisation of cDNA clones encoding cinnamyl alcohol dehydrogenase from tobacco. Plant Mol. Biol. 19: 793–801.

    Google Scholar 

  • Kutsuki, H., Shimada, M. and Higuchi, T.1982 Regulatory role of cinnamyl alcohol dehydrogenase in the formation of guaiacyl and syringyl lignins. Phytochemistry 21: 19–23.

    Google Scholar 

  • Logemann, E., Reinold, S., Somssich, I.E. and Hahlbrock, K. 1997. A novel type of pathogen defense-related cinnamyl alcohol dehydrogenase. J. Biol. Chem. 378: 909–913.

    Google Scholar 

  • MacKay, J.J., Liu, W., Whetten, R., Sederoff, R.R. and O'Malley, D.M.1995 Genetic analysis of cinnamyl alcohol dehydrogenase in loblolly pine: single gene inheritance, molecular characterization and evolution. Mol. Gen. Genet. 247: 537–545.

    Google Scholar 

  • MacKay, J.J., O'Malley, D.M., Presnell, T., Booker, F.L., Campbell, M.M., Whetten, R.W. and Sederoff, R.R.1997 Inheritance, gene expression, and lignin characterisation in a mutant pine deficient in cinnamyl alcohol dehydrogenase. Proc. Natl. Acad. Sci. USA 94: 8255–8260.

    Google Scholar 

  • McKie, J.H., Jaouhari, R., Douglas, K.T., Goffner, D., Feuillet, C., Grima-Pettenati, J., Boudet, A.M., Baltas, M. and Gorrichon, L. 1993. A molecular model for cinnamyl alcohol dehydrogenase, a plant aromatic alcohol dehydrogenase involved in lignification. Biochim. Biophys. Acta 1202: 61–69.

    Google Scholar 

  • Messner, B. and Boll, M.1993 Elicitor-mediated induction of enzymes of lignin biosynthesis and formation of lignin like material in a cell suspension culture of spruce (Picea abies). Plant Cell Tissue Organ Cult. 34: 261–269.

    Google Scholar 

  • Mitchell, H.J., Hall, J.L. and Barber, M.S.1994 Elicitor-induced cinnamyl alcohol dehydrogenase activity in lignifying wheat (Triticum aestivum L.) leaves. Plant Physiol. 104: 551–556.

    Google Scholar 

  • Monties, B.1994 Chemical assessment of lignin biodegradation: some qualitative and quantitative aspects. FEMS Microbiol. Rev. 13: 277–284.

    Google Scholar 

  • Mur, L.A.J., Naylor, G., Warner, S.A.J., Sugars, J.M., White, R.F. and Draper, J.1996 Salicylic acid potentiates defence gene expression in tissue exhibiting acquired resistance to pathogen attack. Plant J. 9: 559–571.

    Google Scholar 

  • Murashige, T. and Skoog, S.1962 A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15: 473–497.

    Google Scholar 

  • Nakashima, J., Awano, T., Takabe, K., Fujita, M. and Saiki, H.1997 Immunocytochemical localisation of phenylalanine ammonialyase and cinnamyl alcohol dehydrogenase in differentiating tracheary elements derived from Zinnia mesophyll cells. Plant Cell Physiol. 38: 113–123.

    Google Scholar 

  • Porter, K.S., Axtell, J.D., Lechtenberg, V.L. and Colenbrander, V.F. 1978. Phenotype, fibre composition, and in vitro dry matter disappearance of chemically induced brown midrib (bmr) mutants of sorghum. Crop Sci. 18: 205–208.

    Google Scholar 

  • Ralph, J., MacKay, J.J., Hatfield, R.D., O'Malley, DM., Whetten, R.W. and Sederoff, R.R.1997 Abnormal lignin in a loblolly pine mutant. Science 277: 235–239.

    Google Scholar 

  • Ralph, J., Hatfield, R.D., Piquemal, J., Yahiaoui, N., Pean, M., Lapierre, C. and Boudet, A.M.1998 NMR characterization of altered lignins extracted from tobacco plants down-regulated for lignification enzymes cinnamyl-alcohol dehydrogenase and cinnamyl-CoA reductase. Proc. Natl. Acad. Sci. USA 95: 12803–12808.

    Google Scholar 

  • Ride, J.P.1975Lignification in wounded wheat leaves in response to fungi and its possible role in resistance. Physiol. Plant Pathol. 5: 125–134.

    Google Scholar 

  • Sambrook, J., Fritsch, E.F. and Maniatis, T.1989 Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

    Google Scholar 

  • Samaj, J., Hawkins, S., Lauvergeat, V., Grima-Pettenati, J. and Boudet, A.1998 Immunolocalisation of cinnamyl alcohol dehydrogenase 2 (CAD2) indicates a good correlation with cellspecific activity of CAD2 promoter in transgenic poplar shoots. Planta 204: 437–443.

    Google Scholar 

  • Smit, F. and Dubery, I.A.1997 Cell wall reinforcement in cotton hypocotyls in response to a Verticillium dahliae elicitor. Phytochemistry 44: 811–815.

    Google Scholar 

  • Somers, D.A., Nourse, J.P., Manners, J.M., Abrahams, S. and Watson, J.M.1995 A gene encoding a cinnamyl alcohol dehydrogenase homolog in Arabidopsis thaliana. Plant Physiol. 108: 1309–1310.

    Google Scholar 

  • Somssich, I.E., Bollman, J., Hahlbrock, K., Kombrink, E. and Schulz, W.1989 Differential early activation of defense-related genes in elicitor-treated parsley cells. Plant Mol. Biol. 12: 227–234.

    Google Scholar 

  • Somssich, I.E., Wernert, P., Kiedrowski, S. and Hahlbrock, K.1996 Arabidopsis thaliana defense-related protein ELI3 is an aromatic alcohol:NADP+ oxidoreductase. Proc. Natl. Acad. Sci. USA 93: 14199–14203.

    Google Scholar 

  • Vallee, B.L. and Auld, D.S.1990 Zinc coordination, function, and structure of zinc enzymes and other proteins. Biochemistry 29: 5647–5659.

    Google Scholar 

  • Van Doorsselaere, J., Baucher, M., Feuillet, C., Boudet, A.M., Van Montagu, M. and Inzé, D.1995 Isolation of cinnamyl alcohol dehydrogenase cDNAs from two important economic species: alfalfa and poplar. Demonstration of a high homology of the gene within angiosperms. Plant Physiol. Biochem. 33: 105–109.

    Google Scholar 

  • Walter, M.H.1992 Regulation of lignification in defense. In: T. Boller and F. Meins (Eds.), Plant Gene Research: Genes Involved in Plant Defense, Springer-Verlag, Vienna, pp. 327–352.

    Google Scholar 

  • Williamson, J.D., Stoop, J.M.H., Massel, M.O., Conkling, M.A. and Pharr, D.M.1995 Sequence analysis of a mannitol dehydrogenase cDNA from plants reveals a function for the pathogenesisrelated protein ELI3. Proc. Natl. Acad. Sci. USA 92: 7148–7152.

    Google Scholar 

  • Wyrambik, D. and Grisebach, H.1975 Purification and properties of isoenzymes of cinnamyl-alcohol dehydrogenase from soybean-cell-suspension cultures. Eur. J. Biochem. 59: 9–15.

    Google Scholar 

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Author notes

  1. Christine M. Hayes

    Present address: Department of Botany &, Zoology, Australian National University, PO Box 475, Canberra, ACT , 2601, Australia

Authors and Affiliations

  1. CSIRO Plant Industry, GPO Box 1600, Canberra, s[ACT , 2601, Australia

    Elizabeth M. Brill, Sharon Abrahams, Colin L.D. Jenkins & John M. Watson

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  1. Elizabeth M. Brill
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Brill, E.M., Abrahams, S., Hayes, C.M. et al. Molecular characterisation and expression of a wound-inducible cDNA encoding a novel cinnamyl-alcohol dehydrogenase enzyme in lucerne (Medicago sativa L.). Plant Mol Biol 41, 279–291 (1999). https://doi.org/10.1023/A:1006381630494

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