Abstract
Two distinct isoforms of cinnamyl alcohol dehydrogenase, CAD 1 and CAD 2, have been purified to homogeneity from xylem-enriched fractions of Eucalyptus gunii Hook and partially characterized. They differ greatly in terms of both physical and biochemical properties, and can be separated by hydrophobic interaction chromatography on Phenyl Sepharose CL-4B. The native molecular weight of of CAD 1 is 38 kDa as determined by gel-filtration chromatography on Superose 6, and this isoform is likely to be a monomer since it yields a polypeptide of 35 kDa upon sodium dodecyl sulfatepolyacrylamide gel electrophoresis. It has a low substrate affinity for coniferyl and p-coumaryl alcohols and their corresponding aldehydes. No activity with sinapyl aldehyde and alcohol was detected. The more abundant isoform is CAD 2, which has a native molecular weight of 83 kDa and is a dinier composed of two subunits of slightly different molecular weights (42–43 kDa). These subunits show identical peptide patterns after digestion with N-chlorosuccinimide. The isoform, CAD 2, has a high substrate affinity for all the substrates tested. The two isoforms are immunologically distinct as polyclonal antibodies raised against CAD 2 do not cross-react with CAD 1. The characterization of two forms of CAD exhibiting such marked differences indicates their involvement in specific pathways of monolignol utilisation.
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Abbreviations
- CAD:
-
cinnamyl alcohol dehydrogenase
- DTT:
-
dithiothreitol
- NCS N:
-
chlorosuccinimide
- SDS-PAGE:
-
sodium dodecyl sulfate-polyacrylamide gel electrophoresis
References
Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilising the principle of protein-dye binding. Anal. Biochem. 72, 248–254
Boudet, A.M., Grand, C. (1987) Lignin synthesis inhibitors: potential tools for imporving nutritional value of plant crops. In: Plant growth regulators for agricultural and amenity Use, pp. 67–77, Hawkins, A.F., Stead, A.D., Pinfield, N.F., eds British Crop Protection Council, UK
Cordonnier, M.M., Smith, C., Greppin, H., Pratt, L.H. (1983) Production and purification of monoclonal antibodies to Pisum and Avena phytochrome. Planta 158, 369–376
Damerval, C., LeGuilloux, M., Blaisonneau, J., DeVienne, D. (1987) A simplification of Heukeneshoven and Dermick's silver staining of proteins. Electrophoresis 8, 158–159
Duran, E., Duran, H., Cazaux, L., Gorrichon, L., Tisnes, P., Sarni, F. (1987) Synthèse de parahydroxythiocinnamates de S-phényle précurseurs d'esters de S-CoA. Bulletin Societé Chimique de France No 1, 143–148
Halpin, C., Knight, M.E., Grima-Pettenati, J., Goffner, D., Boudet, A.M., Schuch, W.(1991) Purification and characterisation of cinnamyl alcohol dehydrogenase from tobacco stems. Plant Physiol., in press
Kawamura, I., Bland, D.E. (1967) The lignins of Eucalyptus wood from tropical and temperate zones. Holzforschung 21, 65–74
Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685
Lewis, N.G., Yamamoto, E. (1990) Lignin: occurrence, biogenesis and biodegradation. Annu. Rev. Plant Physiol. Plant. Mol. Biol. 41, 455–496
Lischwe, M.A., Ochs, D. (1982) A new method for partial peptide mapping using N-chlorosuccinimide/urea and peptide silver staining in sodium dodecyl sulphate polyacrylamide gels. Anal. Biochem. 127, 453–457
Luderitz, T., Grisebach, H. (1981) 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
Mansell, R.B., Babbel, G.R., Zenk, M.H. (1976) Multiple forms and specificity of coniferyl alcohol dehydrogenase from cambial regions of higher plants. Phytochemistry 15, 1849–1853
Moerschbacher, B.M., Noll, U., Gorrichon, L., Reisener, H.J. (1990) Specific inhibition of lignification breaks hypersensitive resistance of wheat to stem rust. Plant Physiol. 93, 465–470
O'Malley, D.M., Sederoff, R.R. (1990) Purification and characterization of cinnamyl alcohol dehydrogenase from developing xylem of loblolly pine, and its role in strategies to modify the lignin content in wood. J. Cellular Biochem. Suppl. 14E, 355
Sarni, F., Grand, C., Boudet, A.M. (1984) Purification and properties of cinnamyl CoA reductase and cinnamyl alcohol dehydrogenase from poplar stems (Populus x euramericana). Eur. J. Biochem. 139, 259–265
Umezawa, T., Davin, L.B., Lewis, N.G. (1990) Formation of the lignan, (-) secoisolariciresinol, by cell free extracts of Forsythia intermedia. Biochem. Biophys. Res. Comm. 171, 1008–1114
Walter, M.H., Grima-Pettenati, J., Grand, C., Boudet, A.M., Lamb, C.J. (1988) 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
Wyrambik, D., Grisebach, H. (1975) Purification and properties of isoenzymes of cinnamyl alcohol dehydrogenase from soybean cell suspension cultures. Eur. J. Biochem. 59, 9–15
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This work was supported by the European Economic Community project AGRE 0021 (OPLIGE) in the scope of the ECLAIR PROGRAMME. The authors whis to thank Drs. L. Davin and N. Lewis (Washington State University) for kindly providing synthesized substrates, Dr. Annie Boudet for excellent technical assistance, and Dr. M. Campbell for fruitful discussions (Université Paul Sabatier, Toulouse, France). We would also like to thank Dr. M. M. Cordonnier-Pratt and Dr. L. Pratt (University of Georgia, Athens, USA) for helpful advice and antibody production.
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Goffner, D., Joffroy, I., Grima-Pettenati, J. et al. Purification and characterization of isoforms of cinnamyl alcohol dehydrogenase from Eucalyptus xylem. Planta 188, 48–53 (1992). https://doi.org/10.1007/BF00198938
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DOI: https://doi.org/10.1007/BF00198938