PURIFICATION AND CHARACTERIZATION OF CINNAMYL ALCOHOL-DEHYDROGENASE ISOFORMS FROM THE PERIDERM OF EUCALYPTUS GUNNII HOOK

Cinnamyl alcohol dehydrogenase (CAD, EC 1.1.1.195) isoforms were purif ied from the periderm (containing both suberized and lignified cell la yers) of Eucalyptus gunnii Hook stems. Two isoforms (CAD 1P and CAD 2P ) were initially characterized, and the major form, CAD 2P, was resolv ed into three further isoforms by ion-exchange chromatography. Crude e xtracts contained two aliphatic alcohol dehydrogenases (ADH) and one a romatic ADH, which was later resolved into two further isoforms. Aliph atic ADHs did not use hydroxycinnamyl alcohols as substrates, whereas both aromatic ADH isoforms used coniferyl and sinapyl alcohol as subst rates but with a much lower specific activity when compared with benzy l alcohol. The minor form, CAD 1P, was a monomer with a molecular weig ht of 34,000 that did not co-elute with either aromatic or aliphatic A DH activity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PACE) and western blot analysis demonstrated that this protein w as very similar to another CAD isoform purified from Eucalyptus xylem tissue. CAD 2P had a native molecular weight of approximately 84,000 a nd was a dimer consisting of two heterogenous subunits (with molecular weights of 42,000 and 44,000). These subunits were differentially com bined to give the heterodimer and two homodimers. SDS-PACE, western bl ots, and nondenaturing PACE indicated that the CAD 2P heterodimer was very similar to the main CAD isoform previously purified in our labora tory from differentiating xylem tissue of E. gunnii (D. Goffner, I. Jo ffroy, J. Grima-Pettenati, C. Halpin, M.E. Knight, W. Schuch, A.M. Bou det [1992] Planta 188: 48-53). Kinetic data indicated that the differe nt CAD 2P isoforms may be implicated in the preferential production of different monolignols used in the synthesis of lignin and/or suberin.