Plant Physiol. (1994) 104: 75-84 Purification and Characterization of Cinnamyl Alcohol Dehydrogenase lsoforms from the Periderm of Eucalyptus gunnii Hook’ Simon William Hawkins and Alain Michel Boudet* Université Paul Sabatier, Centre de Biologie et Physiologie Végétales, Unité de Recherche Associée au Centre National de Ia Recherche Scientifique 1457, 118 Route de Narbonne, 31062 Toulouse Cédex, France water conduction (Northcote, 1989), and defense (Vance et Cinnamyl alcohol dehydrogenase (CAD, EC 1.1.1.195) isoforms al., 1980). However, although they are of vital importance to were purified from the periderm (containing both suberized and the survival of a11 higher land plants, high lignin levels are lignified cell layers) of Eucalyptus gunnii Hook stems. lwoisoforms problematical in the agro-industrial exploitation of various (CAD 1P and CAD 2P) were initially characterized, and the major plant species. Therefore, numerous biotechnological pro- form, CAD 2P, was resolved into three further isoforms by ion- grams are focused on the biosynthesis of this polymer with exchange chromatography. Crude extracts contained two aliphatic the intention of reducing total lignin content in commercially alcohol dehydrogenases (ADH) and one aromatic ADH, which was important tree and forage crop species. later resolved into two further isoforms. Aliphatic ADHs did not As well as being the precursors of lignin, recent work has use hydroxycinnamyl alcohols as substrates, whereas both aromatic shown that monolignols also constitute the monomeric pre- ADH isoforms used coniferyl and sinapyl alcohol as substrates but cursors of other phenolic compounds such as lignans (Lewis with a much lower specific activity when compared with benzyl and Yamamoto, 1990) and suberin (Kolattukudy, 1987). Sub- alcohol. lhe minor form, CAD lP, was a monomer with a molecular erin is a complex polymer with a structure that has still not weight of 34,000 that did not co-elute with either aromatic or aliphatic ADH activity. Sodium dodecyl sulfate-polyacrylamide gel been fully elucidated. It consists of an aliphatic and an electrophoresis (SDS-PACE) and western blot analysis demon- aromatic domain, and analysis has shown that monolignols strated that this protein was very similar to another CAD isoform are the monomeric precursors of the latter domain. Unlike purified from Eucalyptus xylem tissue. CAD ZP had a native mo- lignin, suberin is not implicated in mechanical support and lecular weight of approximately 84,000 and was a dimer consisting water conduction but it is intimately concemed with the of two heterogenous subunits (with molecular weights of 42,000 defense of the plant (Bostock and Stermer, 1989), where, like and 44,000). These subunits were differentially combined to give lignin, it plays an important role in the response to mechan- the heterodimer and two homodimers. SDS-PACE, western blots, ical wounding or pathogen attack. In higher plants showing and nondenaturing PACE indicated that the CAD 2P heterodimer secondary growth, it is the suberized and often lignified cell was very similar to the main CAD isoform previously purified in layers (phellem) of the periderm that, after the loss of the our laboratory from differentiating xylem tissue of E. gunnii (D. cuticle, constitute the physical bamer between the environ- Coffner, 1. joffroy, 1. Crima-Pettenati, C. Halpin, M.E. Knight, W. ment and the underlying plant tissues and that are respon- Schuch, A.M. Boudet [1992] Planta 188: 48-53). Kinetic data indi- sible for preventing moisture loss and opportunistic pathogen cated that the different CAD 2P isoforms may be implicated in the attack. preferential production of different monolignols used in the syn- thesis of lignin and/or suberin. Although the down-regulation of monolignol synthesis in xylem tissue would be economically advantageous, it is clear that such down-regulation could also have potentially un- CAD is one of the two branch enzymes of general phen- desirable effects on, for example, the synthesis of surface, ylpropanoid metabolism specific to hydroxycinnamyl alcohol ‘defense lignin,” or suberin. However, it is also possible that (monolignol) synthesis catalyzing the conversion of hydrox- tissue-specific isoforms of such enzymes may exist that, in ycinnamaldehydes to the corresponding alcohols (Grisebach, conjunction with the use of tissue-specific promoters, could 1981). Monolignols are the monomeric precursors of the lead to the independent manipulation of different product complex phenolic polymer lignin that, after cellulose, consti- (e.g. lignin/suberin) levels in different tissues. Although CAD tutes the most abundant biopolymer on Earth and accounts has previously been purified and characterized from whole for up to 30% dry weight of secondary xylem in woody organs (stems [Mansell et al., 1974; Halpin et al., 19921, roots species. [Rhodes and Wooltorton, 19741, pine megagametophytes In the plant, the rigidity and hydrophobicity of the lignin [O’Malley et al., 1992]), cell-suspension cultures (Wryambik polymer is important for mechanical support (Monties, 1989), and Grisebach, 1975; Galliano et al., 1993), and differentiat- ’ This work was supported by a European Economic Community Abbreviations: ADH, alcohol dehydrogenase; CAD, cinnamyl al- grant (contract No. AGRE 913011) and by Eurosilva. cohol dehydrogenase; HMW, high molecular weight; LMW, low * Corresponding author; fax 33-61-55-62-10. molecular weight. 75 76 Hawkins and Boudet Plant Physiol. Vol. 104, 1994 ing xylem tissue (Savidge, 1989; Goffner et al., 1992; O’Mal- Agronomique, Grignon, France) essentially as described by ley et al., 1992), it has not been purified from other lignified Lapierre et al. (1986). plant tissues and so little is known conceming the nature of tissue-specific forms of this enzyme. Purification Following the observation that CAD activity could be detected in the surface layers of Eucalyptus gunnii Hook CAD isoforms were purified using a combination of ion- branches, it was hypothesized that (a) this enzyme might be exchange and affinity chromatography. A11 steps were canied implicated in the synthesis of surface lignin monolignols or out at 4OC, and CAD activity was monitored by the oxidation the monomeric precursors of the aromatic domain of suberin, of coniferyl alcohol. Samples showing activity were pooled, and (b) it might represent a tissue-specific isoform of this desalted, and buffer exchanged by centrifugation through enzyme. To test these hypotheses we have purified and Sephadex G-25 columns (Penefsky, 1977) before proceeding characterized CAD isoforms from the periderm of E. gunnii to the next chromatographic step. Hook and compared them with isoforms previously charac- terized in differentiating xylem tissue from the same clone. Preparation of Crude Extract E. gunnii Hook was chosen as the experimental system first Frozen periderm tissue (250 g) was ground up in a coffee because of the molecular tools and data already available mil1 prechilled with liquid nitrogen and extracted conceming Eucalyptus xylem CAD (Goffner et al., 1992), (buffer:tissue 4:l [v/w]) by stirring with a magnetic bar for second because it is a tree species of economic importance 45 min in cold extraction buffer (250 m Tris-HC1, 100 mM and is already the target of transformation experiments, and sodium ascorbate, 10% polyvinylpolypyrrolidone,5 % ethyl- third because a knowledge of the enzymes and genes present ene glycol, 2% PEG, 0.1% P-mercaptoethanol, and the pro- in different tissues could, potentially, lead to a strategy for tease inhibitors PMSF [l m~],leupeptin [l p~],and pepstatin the independent manipulation of lignin/suberin in different [l PM],pH 7.5). Crude extract was filtered through two layers tissues. of Miracloth, and a sample was removed, desalted, and assayed for activity. Crude extract was then clarified by MATERIALS AND METHODS centrifugation (18,00Og, 45 min) and the supematant was taken on to the next purification step. Plant Material Periderm tissue was obtained from the trunks of young (4- Amberlite XAD-7 year-old) Eucalyptus gunnii Hook trees (clone No. 800859) growing on the AFOCEL (Association FÔret Cellulose) Plan- Amberlite XAD-7 polyphenol-absorbing resin (Sigma) tation at Longages, southem France, toward the end of the equilibrated in 250 rmTris-HCI, 5% ethylene glycol, and 5 growing season (September 15-20, 1992). Material was m DTT was added to the crude, filtered extract (resin:extract saaped into a beaker with a scalpel, dropped directly into [1:10, w/v]) and stirred for 5 min before filtering through liquid nitrogen, and stored at -8OOC until needed. Previous two layers of Miracloth. detailed microscopic examination had resulted in a thorough knowledge of surface layer anatomy, which enabled the Ammonium Sulfate Precipitation and Concentration remova1 of periderm tissue without contamination from other The crude extract was brought to 20% saturation with solid lignifying tissue (i.e. secondary phloem). (NH4)*S04and then centrifuged (18,00Og, 45 min). The re- sulting supematant was then brought to 80% saturation with Microscopic and Histochemical Analysis solid (NH4)2S04and centrifuged (18,OOOg, 45 min), and the resulting pellet was resuspended in 100 m Tris-HC1, 5% For general anatomical investigations, semithin (30- to 60- ethylene glycol, 5 m DTT, pH 7.5. PM) transverse sections of E. gunnii branches (2 to 3 cm in diameter) were made on a Reichart Wood Microtome. Sec- DEAE-Sephacel tions were stained as follows: for general anatomical study, safranin O/fast green, carmine/iodine green (Johansen, The resuspended pellet was desalted into DEAE-Sephacel 1940); for lignin detection, Weisner reaction, Maule reaction, start buffer (20 m Tris-HC1, 5% ethylene glycol, 5 m DTT, Cross and Bevan reaction (Monties, 1987); for suberin detec- pH 7.5) and then loaded onto a 60-mL DEAE-Sephacel tion, sudan black, sudan III/IV, oil red O (Gahan, 1984). For (Pharmacia) column (IBF-Sepracor, 1.6 cm diameter) con- more detailed investigation of the bark (secondary phloem, nected to a Bio-Rad Econo system low-pressure liquid chro- cortex, and periderm), samples were fixed in formalin:70% matography system at a flow rate of 0.25 mL min-’.