Planta

Planta (1992)188:48-53

9 Springer-Verlag1992

Purification and characterization of isoforms of cinnamyl alcohol dehydrogenase from Eucalyptus xylem D. Goffner 1, I. Joffroy 1, J. Grima-PettenatP, C. Halpin 2, M.E. Knight 2, W. Schuch 2, and A.M. Boudet 1. 1 Universit~ Paul Sabatier, Centre de Biologie et Physiologie V6g&ale, URA CNRS 1457, 118, route de Narbonne, F 31062 Toulouse Cedex, France 2 ICI Seeds, Plant Biotechnology Section, Jealott's Hill Research Station, Bracknell RG12 6EY, UK Received 5 December 1991; accepted 13 March 1992

Abstract. Two distinct isoforms of cinnamyl alcohol dehydrogenase, CAD 1 and CAD 2, have been purified to homogeneity from xylem-enriched fractions of E u c a l y p ~ tus g u n i i 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 dimer 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. Key words: Cinnamyl alcohol dehydrogenase (isoforms) Lignin - Xylem

thesis of lignin monomers, the general phenylpropanoid pathway, have been extensively studied, few data exist concerning the enzymatic steps specifically involved in lignin formation. In addition to peroxidases, which are assumed to be involved in the polymerization of lignin monomers, there are two reductive steps catalysing the reduction of cinnamoyl CoA to cinnamyl alcohol via cinnamoyl CoA reductase (EC 1.2.1.44) and cinnamyl alcohol dehydrogenase (CAD; EC 1.1.1.195). Mainly as a consequence of technical problems such as nonavailability of natural substrates, and low enzyme activity and stability, these two enzymes are poorly characterized. No data are available on their cellular location and the molecular mechanisms involved in the regulation of their synthesis and activity. Additional questions relate to the existence of a possible polymorphism of CAD (Wyrambik and Grisebach 1975; Mansell et al. 1976). Recent data also indicate the potential involvement of coniferyl alcohol in the synthesis of compounds including lignans and surface polymers such as cutin and suberin (Lewis and Yamamoto 1990). As a prerequisite for further molecular studies of CAD in woody plants, we have purified to homogeneity and characterized this enzyme from E u c a l y p t u s , a fastgrowing species of economic interest. Our results indicate that CAD is polymorphic, with two major forms that differ greatly in terms of their molecular weight and substrate affinities.

- Eucalyptus -

Introduction Lignins may represent up to 30% of the dry matter of woody plants. Even though the early steps of the syn* To whom correspondence should be addressed; FAX (+33) 61556210 Abbreviations. CAD=cinnamyl alcohol dehydrogenase DTT= dithiothreitol; NCS = N-chlorosuccinimide; SDS-PAGE = sodium dodecyl sulfate-polyacrylamide gel electrophoresis

Material and methods Chemicals. Synthesized CAD substrates (p-coumaryl, sinapyl, and coniferyl aldehydes, and p-coumaryl and sinapyl alcohols) were generous gifts from Drs. N. Lewisand L. Davin (Washington State University, Pullman, USA). Coniferyl alcohol was purchased from Fluka (Buchs, Switzerland), leupeptin, pepstatin A, phenylmethylsulfonylfluoride (PMSF), and polyvinylpolypyrrolidone (PVPP), from Sigma (St. Louis, Mo., USA), and lubrol from Serva (Heidelberg, FRG). All chromatographic gels were purchased from Pharmacia (Uppsala, Sweden).

D. Goffner et al. : Cinnamyl alcohol dehydrogenases in Eucalyptus

49

Spectrophotometric measurement of CAD activity and K,, determination. The activity of CAD during the course of purification was

hol dehydrogenase was eluted by a gradient of 0-4 mM NADP § in equilibration buffer (total volume 40 ml).

measured spectrophotometrically at 400 nm. The reaction mixture contained 100 mM Tris-HC1 (pH 8.8), 20 mM coniferyl alcohol, 5 mM NADP +, and 5-100 ~tl of extract. The reaction was carried out at 30~ C. Paper chromatography of reaction products was performed as described by Duran et al. (1987) with slight modifications. For reduction of coniferyl, sinapyl, and p-coumaryl aldehydes, assays were performed as in Sarni et al. (1984). Determinations of Km were performed by varying the substrate concentrations. Values were determined by extrapolation from linear Lineweaver-Burke plots.

Extraction of CAD. Young branches from three-year-old trees of Eucalyptus gunnii Hook (clone 800859) grown on AFOCEL (Association F6ret Cellulose-Longages, France) plantations were used for extractions. At harvest, the outer bark was removed. For each purification, 600 g of soft xylem were scraped and immediately immersed in liquid nitrogen. The peels were ground in liquid nitrogen to a fine powder and then placed in a Waling blender in 100 mM Tris-HC1 (pH 7.5), 15 mM 13-mercaptoethanol, 2% polyethylene glycol 6000 (PEG) and 300 g of PVPP. All procedures were then carried out at 4~ C. The extraction medium contained the following protease inhibitors: PMSF (174tug'l-a), leupeptin (1 rag" 1-1), pepstatin A (1 m g ' l-i), and EDTA (370 m g - l - l ) . After filtration through cheesecloth, the crude extract was brought to 40% saturation in ammonium sulfate and stirred at 4~ C for 1 h. The extract was then centrifuged for 50 min at 19 000 9g. The supernatant containing CAD activity was used for further purification steps.

Purification of CAD. The supernatant after ammonium precipitation was loaded at a flow rate of 150 ml 9h- i onto a Phenyl Sepharose (Pharmacia) column (22 cm long, 5 cm i.d.) equilibrated in 20 mM Tris-HC1 (pH 7.5), 5 mM dithiothreitol (DTT), and 0.4 M ammonium sulfate. After rinsing the column with 300 ml of equilibration buffer, two successive linear gradients were carried out: 0-20% ethylene glycol (total volume 1 1), and 20-30% ethylene glycol containing 0.5 % lubrol (1 1). Further elution was carried out with 20 mM Tris-HC1 (pH 7.5), 5 mM DTT, 30% ethylene glycol, and 0.5% lubrol until the two peaks of enzyme activity corresponding to CAD 1 and CAD 2 were completely eluted. Elutions were performed at a flow rate of 150 ml" h -1. Each peak was then treated separately for subsequent chromatographic steps. Fractions containing CAD activity were pooled, and after adjusting the pH to 6.5, loaded onto a Blue Sepharose column (20 cm long, 4 cm i.d.) equilibrated in 20 mM Tris-HC1 (pH 6), 5 mM DTT, and 5 % ethylene glycol at a flow rate of 100 ml 9h-1. After enzyme fixation, the column was rinsed with 200 ml equilibration buffer containing 4 mM NAD +. A linear gradient of 0-4 mM NADP + in equilibration buffer (500 ml) was used to elute CAD. For elution of CAD 1, the column was then rinsed with 8 mM NADP +. Elutions were performed at a flow rate of 100 ml - h- 1. Pooled CAD 2 fractions were then applied to an FPLC (fast protein liquid chromatography) Mono-Q column equilibrated in 20 mM Tris-HC1 (pH 7.5), 5 mM DTT, and 5% ethylene glycol at a flow rate of 1 ml 9min-i. Cinnamyl alcohol dehydrogenase was eluted at the same flow rate with a linear gradient of equilibration buffer and 300 mM Tris-HC1, 5 mM DTT, 5% ethylene glycol. The two peaks of CAD activity from the Mono-Q column, A and B, were loaded separately on a Sephacryl 200 HR (Pharmacia) column (100 cm long, 2.6 cm i.d.) and eluted with 25 mM 2-[bis(2hydroxyethyl)amino]-2-(hydroxymethyl)- 1,3-propanediol (Bistris) (pH 7.5), 5 mM DTT, and 10mM NaC1 at a flow rate of 40ml 9h 1. After gel filtration, fractions containing CAD activity were loaded on a 2'5' ADP Sepharose column (5 cm long, 1.5 cm i.d.) equilibrated in 100 mM Tris (pH 7.5), 5 mM DTT, and 5% ethylene glycol at a flow rate of 12 ml - h - 1. The column was then rinsed with 15 ml equilibration buffer containing 4 mM NAD +. Cinnamyl alco-

Determination of native molecular weight. Native molecular weights for CAD 1 and CAD 2 were determined by Superose 6 (HR 10/30) gel-filtration chromatography on FPLC. Pure, active protein was loaded and eluted in 75 mM Tris-HC1 (pH 8.8), 50 mM NaC1. A calibration curve was generated using the following protein standards: ferritin (440 kDa), alcohol dehydrogenase (150 kDa), bovine serum albumin (66 kDa), and chymotrypsinogen A (25 kDa).

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Denaturing polyacrylamide electrophoresis was performed according to Laemmli (1974). Gels contained 12% acrylamide. Proteins were stained with silver nitrate (Damerval et al. 1987). Staining of CAD activity in native gels. Activity staining was done as described in Halipin et al. (1991). After electrophoresis, native gels containing 7.5% acrylamide were transferred to 10ml of 100 mM Tris-glycine (pH 8.8) containing 1.5 mg nitro blue tetrazolium, 0.1 mg phenazine methosulfate, 2.5 mg NADP +, and 2.5 mg coniferyl alcohol. Incubation was for 30 min in the dark at 37~ C.

Protein determination. Protein content was determined by the BioRad assay (Bradford, 1976).

Digestion with N-chlorosuccinimide (NCS). Digestion of polypeptides in the presence of NCS was carried out essentially as described in Lischwe and Ochs (1982). First-dimension SDS-PAGE polypeptides visualized with Coomassie Blue were excised from the gel and incubated with 0.3M NCS in urea :water :acetic acid (1 g : 1 ml : 1 ml) for 1 h under constant agitation at room temperature. After treatment, gel slices were equilibrated in 10% glycerol, 15% 13-mercaptoethanol, 3% SDS, and 0.0625 M Tris-HC1 (pH 6.8) and loaded onto an SDS-polyacrylamide gel (15% acrylamide). Peptides were stained with silver nitrate.

Generation of antibodies. Mice were immunized with 200-300 ~tg of pure CAD 2 following the schedule given in Cordonnier et al. (1983), except that the final injection was intraperitoneal rather than intravenous. Serum containing polyclonal antibodies was separated from blood that was collected by heart puncture at the time the mouse was sacrificed, preparatory to performing a fusion for the purpose of generating monoclonal antibodies.

Results T h e p u r i f i c a t i o n o f two isoforms o f C A D , C A D 1 a n d C A D 2, f r o m Eucalyptus x y l e m was achieved u s i n g the scheme i n d i c a t e d in Fig. 1. Several b i o c h e m i c a l p r o p e r ties, i n c l u d i n g p r o t e i n h y d r o p h o b i c i t y , N A D P + affinity, charge, a n d m o l e c u l a r weight, were exploited to purify C A D to h o m o g e n e i t y . A n initial p o l y m o r p h i s m was observed o n P h e n y l Sepharose C L - 4 B as i n d i c a t e d b y two peaks o f C A D activity (Fig. 2). These two peaks gave rise to C A D 1 a n d 2, respectively. E v e n t h o u g h their relative intensities varied greately d e p e n d i n g o n the s a m p l i n g date, C A D 2 was always m o r e a b u n d a n t . T a b l e 1 shows the yields at the different steps used to purify the two isoforms o f C A D . T h e i s o f o r m C A D 1 was easily purified to h o m o g e n e i t y since it was s t r o n g l y b o u n d to Blue Sepharose. I n d e e d , 8 m M N A D P + was necessary to elute this isoform. This step yielded a 439fold p u r i f i c a t i o n for C A D 1. A s for C A D 2, a greater

50

D. Goffner et al. : Cinnamyl alcohol dehydrogenases in Eucalyptus Ammonium sulfate precipitation

(40%)

CAD 5

I

4

Phenyl Sepharose CL 4B

/

Blue Sepharose CAD1

2

\

c

Blue Sepharose

! ! !

Mono Q

SephacrMl

0

.

0

200

.

.

.

.

20

40

60

80

100

120

140

160

180

200

220

Fraction number

2' 5' ADP Sepharose CAD 2

Fig. 1. Purification scheme for the two isoforms of CAD from Eucalyptus xylem. Purification to homogeneity of CAD 1 and CAD 2 was achieved after Blue Sepharose and 2'5' ADP Sepharose respectively

than 6700-fold purification factor was achieved after its ultimate purification step, 2'5' ADP Sepharose. Figure 3 shows the electrophoretic profiles of the denatured proteins for each of the purification steps. In the more crude fractions (Phenyl Sepharose: Lanes 1 and 6 for CAD 2 and CAD 1, respectively), CAD was barely detectable, indicating that from a quantitative standpoint CAD is a relatively minor enzyme in Eucalyptus xylem (calculated as approx. 0.06% of total protein). After chromatography on Blue Sepharose, CAD 1 was pure, as indicated by the presence of a single subunit on SDS-PAGE with an apparent molecular weight of 35 kDa. At the ultimate purification step, CAD 2 ap-

Fig. 2. Elution profile of CAD activity on Phenyl Sepharose CL-4B. Fraction 0 corresponds to the beginning of the 20-30% ethylene glycol gradient. The arrow indicates the beginning of the elution with 20 mM Tris-HC1 (pH 7.5), 5 mM DTT, 30% ethylene glycol, and 0.5 % lubrol. Fraction volume = 6.1 ml

peared, after SDS gel electrophoresis, as a doublet of 42-43 kDa in the fractions containing the highest activity. Individual digestion of the two CAD 2 polypeptides (42, and 43 kDa) with NCS-urea gave rise to the same electrophoretic pattern, indicating a very close relationship between these two subunits (Fig. 4). It should be noted that in some experiments, two distinct peaks of CAD 2 activity were observed on Mono Q chromatography (data not shown). However, after the ultimate purification step, 2'-5' ADP Sepharose chromatography, the pure protein derived from each peak gave rise to the same electrophoretic patterns on both SDS-PAGE (data not shown) and native gel electrophoresis (Fig. 5). They are therefore assumed to be very closely related species differing only slightly in charge.

Table 1. Purification table for CAD from Eucalyptus Purification step

Protein (mg)

Specific activity (nkat 9m g - 1)

Purification factor

Total activity (nkat)

CAD 1 and CAD 2

Crude extract Ammonium sulfate 40% supernatant

i515

12 626

0.12

4550

0.26

2.2

1183

426 0.04

0.45 52.7

3.8 439

192 2

61 12.2 0.6 0.18 0.01

3.93 23.4 91.7 226 815

CAD 1

Phenyl Sepharose CL-4B Blue Sepharose a CAD 2

Phenyl Sepharose CL-4B Blue Sepharose Mono Qb Sephacryl-200 2'5' ADP Sepharose a

33 195 768 1883 6791

240 285 55 55 11

a Values for tubes containing maximum activity only b Two peaks of C A D activity with slight charge variation. Each gave rise to very similar molecular species after chromatography on 2'5' ADP Sepharose

D. Goffner et al. : Cinnamyl alcohol dehydrogenases in Eucalyptus

51

Fig. 3. Electrophoretic protein profiles (SDS-PAGE 12%) of Eucalyptus xylem during CAD purification. All chromatographic steps are shown. Lanes 1-5, CAD 2; lanes 6, 7, CAD 1. Lane 1, Phenyl Sepharose CL 4B; lane 2, Blue Sepharose; lane 3, Mono Q; lane 4, Sephacryl 200; lane 5, 2'5' ADP Sepharose; lane 6, Phenyl Sepharose CL 4B; lane 7, Blue Sepharose; lane 8, molecularweight markers. Both CAD 1 and CAD 2 are indicated beneath the lanes corresponding to their ultimate purification steps

Fig. 4. Analysis by SDS-PAGE (15%) of CAD 2 polypeptides (42 and 43 kDa) after digestion with NCS. The 42-kDa (lane 1) and 43-kDa (lane 2) polypeptides give rise to identical digestion patterns

Fig. 5. Nondenaturing gel electrophoresis (A) and corresponding CAD-activity gels (B) of pure CAD 2 after 2'5' ADP Sepharose chromatography. Lanes 1, 3, CAD 2 originally derived from Mono Q, peak A; lanes 2, 4, CAD 2 derived from Mono Q, peak B; MW, bovine serum albumin as molecular-weight marker

52

D. Goffner et al. : Cinnamyl alcohol dehydrogenases in Eucalyptus

Table 2. Michaelisconstants (K~ values, ~tM)for CAD 1 and CAD 2

distinct since polyclonal antibodies raised against CAD 2 did not cross-react with CAD 1 in immunoblot analysis (data not shown). Previous reports indicate that in most plant species, with very few exceptions, only one form of CAD is detected (Mansell et al. t976). However, the detection of CAD activity in starch or polyacrylamide gels is limited to abundant and-or high-activity forms. For example, in crude extracts of Eucalyptus xylem, initial attempts to detect CAD 1 were unsuccessful because of the relatively low activity of this isoform, which must be concentrated in order to detect activity on gels. Therefore, it is possible that multiple isoforms are more common than previously determined. Interestingly, Wyrambik and Grisebach (1975) have reported the occurrence of two CAD isoenzymes in soybean cell-suspension cultures. As in Eucalyptus, the substrate affinities of these two isoenzymes differ greatly. In addition, the low-affinity isoform of soybean uses only coniferyl alcohol as its substrate. The differences observed between the two isoforms of CAD characterized in Eucalyptus indicate that they are involved in rather different processes. The fact that CAD 1 does not catalyze the conversion of ethanol into acetylaldehyde rules out the possibility of it being a broad-range ethanol dehydrogenase. Several hypotheses can account for this polymorphism that could be related to different locations or functions of lignins in plants. The question as to whether there are separate CAD enzymes/genes for developmental and defense lignins was recently raised (Walter et al. 1988). Furthermore, monolignols seem to be used as precursors of complex molecules such as cutin and suberin. Different CAD isoforrns could provide the cinnamyl alcohol moiety of these polymers. Other polymers, such as lignans, also composed of monolignol units, have been found in many woody plants and their synthesis could be dependent on a specific form of CAD (Umezawa et al. 1990). Immunocytolocalisation studies of these respective isoforms may provide essential information concerning the function of these two isoforms. As for CAD 2, it appears as a doublet of 42-43 kDa after SDS-PAGE. This situation is identical to that of CAD from tobacco (Halpin et al. 1991). The identical NCS digestion patterns of the two Eucalyptus polypeptides indicate, as in tobacco, a close relationship between them. In addition, monoclonal antibodies raised against CAD 2 from Eucalyptus and CAD polyclonal antibodies from tobacco recognize the members of the doublet to the same extent (data not shown). Regarding the native conformation of CAD 2, it seems evident that this protein is a dimer. This is in agreement with previous reports of CAD from poplar (Sarni et al. 1984), spruce (Luderitz and Grisebach 1981), loblolly pine (O'Malley and Sederoff 1990), and tobacco (Halpin et al. 1991). The Km values of CAD 2 are very similar to those already reported for the enzyme isolated poplar, soybean (isoenzyme 2), loblolly pine, and tobacco. However, the significance of CAD 1 specificity for coniferyl and pcoumaryl alcohol and aldehydes, remains to be elucidated. Detailed analysis of lignin composition in Eu~ calyptus indicated that it is representative of typical

CAD 1

CAD 2

Aldehydes coniferyl sinapyl p-coumaryl

25 70

1.2

Alcohols coniferyl sinapyl p-coumaryl

560 760

6 7.2 4.9

1.7 1.5

Superose 6 chromatography indicated that CAD 2 has a native molecular weight of 83 kDa. From this molecular-weight determination, it appears evident that CAD 2 is a dimer consisting of two subunits of 42-43 kDa. On the other hand, CAD 1 eluted on Superose 6 as a 38 kDa protein, indicating that, under these running conditions, CAD 1 is an active monomer. The/(ms for the two isoforms in their pure form were determined in order to compare CAD 1 and CAD 2 substrate affinities for coniferyl, sinapyl, and p-coumaryl alcohol and their corresponding aldehydes. The results are indicated in Table 2. The isoform CAD 1 used coniferyl and p-coumaryl aldehyde/alcohol exclusively as substrates, showing a slightly higher affinity for the coniferyl substrates. No activity with sinapyl aldehyde and alcohol was observed. As for CAD 2, it used all substrates indifferently whether alcohol or aldehyde, as determined by equivalent Kms for either alcohols or aldehydes. It is clear that for both CAD 1 and CAD 2 the aldehydes, rather than the corresponding alcohols, were the preferred substrates. In addition it should be noted that for both CAD 1 and CAD 2, no alcohol dehydrogenase activity was observed. Both isoforms used NADP + as a cofactor but slight activity with N A D + could be determined (less than 3% as compared to NADP+). The pH optima for CAD 1 and CAD 2 were also determined. Maximum activity in the basic pH range, with an optimum of 9.5 for CAD 1 and 10.2 for CAD 2, was observed for the oxidation of coniferyl alcohol. Discussion

Few data are currently available concerning ligninspecific enzymes in plants. Recently, we have reported the purification from tobacco stems of a CAD enzyme which is composed of two similar but different-sized polypeptides varying slightly in amino-acid composition (Halpin et al. 1991). In this paper, we describe the purification to homogeneity of CAD from Eucalyptus xylemenriched fractions. On the whole, CAD is present in very small amounts because even in a CAD-enriched tissue such as xylem, this protein accounts for only 0.06% of the total soluble protein. Two distinct isoforms of CAD were separated by hydrophobic interaction chromatography on Phenyl Sepharose CL-4B. They are considered as immunologically

D. Goffner et al. : Cinnamyl alcohol dehydrogenases in Eucalyptus

53

angiosperm lignin, containing both guaiacyl and syringyl units ( K a w a m u r a and Bland 1967). These data indicate that C A D 2 m a y be more directly involved in lignification. The characterization of several C A D s in Eucalyptus shows that the involvement of C A D in different biochemical pathways is p r o b a b l y more complex than initially expected. It is evident that the process of lignification m a y be regulated quantitatively by lignin-specific enzymes. This hypothesis is supported by the fact that specific C A D inhibitors such as N(o-hydroxyphenyl)- and N(o-aminophenyl) sulfinamoyl tertiobutyl acetate ( O H P A S and NH2PAS, respectively) m a y dramatically decrease lignin content (Boudet and G r a n d 1987; Moerschbacher et al. 1990). These arguments point to C A D as an interesting target in the manipulation o f lignin synthesis, the basic regulation of which is poorly understood. It is clear that the molecular probes including c D N A clones and antibodies corresponding to C A D 1 and C A D 2 resulting f r o m this enzymological work will be i m p o r t a n t tools in gaining a better understanding o f the control o f lignification both for basic and applied purposes.

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) Synth~se de parahydroxytbiocinnamates de S-phrnyle prrcurseurs d'esters de S-CoA. Bulletin Societ6 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 • 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

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 (Universit6 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.

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 Arena phytochrome. Planta 158, 369-376