Plant Physiol. (1999) 119: 153-164

Structural Alterations of Lignins in Transgenic Poplars with Depressed Cinnamyl Alcohol Dehydrogenase or Caffeic Acid O-Methyltransferase Activity Have an Opposite Impact on the Efficiency of Industrial Kraft Pulping¹

Catherine Lapierre^*, Brigitte Pollet, Michel Petit-Conil, Gabriel Toval, Javier Romero, Gilles Pilate, Jean-Charles Leplé, Wout Boerjan, Valérie Ferret, Véronique De Nadai, and Lise Jouanin

Laboratoire de Chimie Biologique, Institut National Agronomique, F-78850, Thiverval-Grignon, France (C.L., B.P.); Centre Technique du Papier, BP 251, F-38044, Grenoble, cedex 09, France (M.P.-C.); Centro de Investigacion de Empresa Nacional Celulosas, Carretera Campano, S/N, 36157 Pontevedra, Spain (G.T., J.R.); Station d'Amélioration des Arbres Forestiers, Institut National de la Recherche Agronomique (INRA), F-45160, Ardon, France (G.P., J-C.L.); Laboratorium voor Genetika, Vlaams Interuniversitair Instituut voor Biotechnologie VIB, Universiteit Gent, Ledeganckstraat 35, 9000 Gent Belgium (W.B.); and Laboratoire de Biologie Cellulaire, INRA, F-78026 Versailles cedex, France (V.F., V.D.N., L.J.)

	ABSTRACT

We evaluated lignin profiles and pulping performances of 2-year-old transgenic poplar (Populus tremula × Populus alba) lines severely altered in the expression of caffeic acid/5-hydroxyferulic acid O-methyltransferase (COMT) or cinnamyl alcohol dehydrogenase (CAD). Transgenic poplars with CAD or COMT antisense constructs showed growth similar to control trees. CAD down-regulated poplars displayed a red coloration mainly in the outer xylem. A 90% lower COMT activity did not change lignin content but dramatically increased the frequency of guaiacyl units and resistant biphenyl linkages in lignin. This alteration severely lowered the efficiency of kraft pulping. The Klason lignin level of CAD-transformed poplars was slightly lower than that of the control. Whereas CAD down-regulation did not change the frequency of labile ether bonds or guaiacyl units in lignin, it increased the proportion of syringaldehyde and diarylpropane structures and, more importantly with regard to kraft pulping, of free phenolic groups in lignin. In the most depressed line, ASCAD21, a substantially higher content in free phenolic units facilitated lignin solubilization and fragmentation during kraft pulping. These results point the way to genetic modification of lignin structure to improve wood quality for the pulp industry.

	INTRODUCTION

Lignins are wall polymers that are essential for mechanical support, water transport, and disease resistance in higher terrestrial plants. Their removal from wood is the basis of kraft pulping, the major process used for the production of pulp and paper. Because of the insoluble and cross-linked nature of lignins, delignification of cellulosic fibers requires harsh pulping conditions and large amounts of polluting chemicals. Currently, there is considerable interest in lignin genetic engineering as a means of improving the efficiency of the pulping process and lowering its economic and environmental costs.

Wood lignins are composed of S and/or G units linked by a series of ether and carbon-carbon bonds (Adler, 1977). The ether -O-4 linkages (Higuchi, 1990) are both frequent and labile, which makes them the target of the delignification process. In contrast, the carbon-carbon linkages are resistant, especially the biphenyl 5-5 bonds involving the aromatic C-5 position, which is available for interunit linkages only in G units (Adler, 1977). From this rationale, it can be understood why conifer wood lignins essentially made of G units are less susceptible to kraft delignification than deciduous wood lignins comprising both G and S units (Chiang et al., 1988). Tailoring plants more amenable to industrial delignification may be achieved by lowering lignin concentration and/or altering lignin structure to an extent that does not affect plant development and defense.

We recently produced transgenic poplar (Populus tremula × Populus alba) trees with down-regulated enzymes of the lignin biosynthetic pathway by introducing antisense and sense gene constructs in the plant (Van Doorsselaere et al., 1995; Baucher et al., 1996). The selected target enzymes were the bispecific COMT and CAD. The first analyses carried out on juvenile (3-month-old) poplars revealed a severe reduction of COMT and CAD activities of some of the transgenic lines. Both COMT- and CAD-transformed lines showed lignin content similar to the control. We failed to detect a substantial alteration of lignin structure in CAD-down-regulated poplar trees, which displayed a striking red coloration of the xylem. However, a noticeable fraction (approximately 5% by weight) of the lignin of the CAD-depressed samples was found to be solubilized upon mild alkaline hydrolysis. More important, these samples showed improved performances toward alkaline kraft delignification relative to the control (Baucher et al., 1996). However, the molecular basis of their altered reactivity toward alkaline treatment was not addressed. Similar to the bm3 maize mutant with depressed COMT activity (Lapierre et al., 1988), COMT-down-regulated poplars displayed a severe alteration of lignin structure, mainly revealed by a decrease in S units (Van Doorsselaere et al., 1995).

In this paper we report the in-depth evaluation of both lignin structure and pulping characteristics of 2-year-old poplars with down-regulated CAD and COMT activity. This evaluation is of fundamental importance to assess the stability of transgene expression with age and to comprehensively evaluate the pulping characteristics of the transgenic lines in relation to specific structural traits of the lignin. The lignin amount of 2-year-old COMT-transformed poplars was found to be similar to that of the control, whereas it was slightly reduced for some of the CAD-transformed lines. We show that depressed CAD activity does not change the proportions of labile -O-4 bonds and S units. However, we provide the first evidence, to our knowledge, that this transformation substantially increases the frequency of free phenolic units in lignin, an essential trait that could account for the specific reactivity of CAD-transformed samples toward alkaline treatment. COMT down-regulation dramatically affects lignin structure: relative to the control, the content in labile -O-4 bonds was lower, whereas the proportion of G units and resistant biphenyl structures was dramatically enhanced. In view of these alterations, the efficiency of kraft pulping toward CAD-transformed poplars was improved, especially for the ASCAD21 line showing the highest level in lignin-free phenolic groups. In contrast, COMT-transformed poplar lines displayed a higher resistance to kraft delignification, consistent with the reported structural changes of the lignin.

	MATERIALS AND METHODS

Plant Material

In addition, a large series of younger plants (greenhouse-grown) were individually sampled to monitor the lignin content and structure as the plant aged and to evaluate the variability of lignin profiles within the same line. A few experiments were performed on plantlets regenerated from shoot nodal explants of control and ASOMT2B poplars.

To obtain a transgenic line underexpressing both CAD and COMT, line ASCAD21 was retransformed with a construct containing the antisense COMT gene associated with a gene (hpt [hygromycin phosphotransferase]; L. Jouanin, unpublished data) conferring resistance to hygromycin. The transformation protocol reported by Leplé et al. (1992) was used. Hygromycin (20 mg L¹) was added to the culture medium to select the transformed cells. Line ASCAD21 × ASOMT7 showed a reduction of COMT activity in xylem tissues of about 90%, as observed for ASOMT2B and ASOMT10B. Lines ASCAD21 and ASCAD21 × ASOMT7 displayed a similarly reduced CAD activity. Measurements of the COMT activity toward caffeic acid and of the CAD activity in the xylem were as previously reported (Van Doorsselaere et al., 1995; Baucher et al., 1996).

Determination of Lignin Content

Analysis by Thioacidolysis

Thioacidolysis reagent was prepared by introducing 2.5 mL of BF₃ etherate (Aldrich) and 10 mL of ethane thiol EtSH (Aldrich) into a 100-mL flask and adjusting the final volume to 100 mL with dioxane (pestipur grade). The extract-free wood (10 mg) was added to 10 mL of reagent and 1 mL of a solution of GC internal standard (docosane, 0.25 mg mL¹ in CH₂Cl₂) in a glass tube closed with a Teflon-lined screwcap. Thioacidolysis was performed at 100°C (oil bath) for 4 h. The cooled reaction mixture was diluted with 30 mL of water and the pH was adjusted to 3.0 to 4.0 (aqueous 0.4 M NaHCO₃). The reaction mixture was extracted with CH₂Cl₂ (three times with 30 mL). Combined organic extracts were dried over Na₂SO₄ and then evaporated under reduced pressure at 40°C. The final residue was redissolved in approximately 1 mL of CH₂Cl₂ before silylation and GC or GC-MS analyses according to the method of Lapierre et al. (1995).

The determination of lignin-derived dimers was done after desulfurization according to the method of Lapierre et al. (1995). The analyses of lignin-derived monomers released from exhaustively CH₂N₂-methylated samples were run as previously described (Lapierre and Rolando, 1988).

Analysis of Low-M_r Phenolics Released by Mild Alkaline Hydrolysis

Wiesner Reaction Performed on Isolated Lignin Fractions

The Wiesner reaction was applied to these lignin fractions according to the method of Nakano and Meshitsuka (1992). The molar absorptivity (; liters per mole per centimeter) of the coniferaldehyde reaction product was determined with pure coniferaldehyde ( at 552 nm = 63,000). This absorptivity was used to calculate, from the A₅₅₂ of the pink solution obtained when the Wiesner reaction was applied to isolated lignins, the equivalent amount of coniferaldehyde per gram of lignin sample. The Wiesner reaction applied to sinapaldehyde led to similar results ( at 563 nm = 89,000), whereas benzaldehyde-condensation products showed weak absorptivity at a lower wavelength (503 nm for the syringaldehyde reaction product).

Pulping Experiments

	RESULTS

COMT or CAD Activity and Phenotype of Transgenic Poplar Trees

No phenotypic differences in development or growth were observed between control and transgenic poplars with antisense CAD or COMT constructs whether grown in the greenhouse or in the field. When the bark was removed during the growing season, wood of COMT-transformed poplars displayed a pale-rose coloration, whereas that of the CAD-transformed lines appeared red, particularly intense for line ASCAD21. This red coloration was essentially located in the outer part of the xylem. The red, fresh xylem turned brown after drying. This brown resisted solvent extraction. One-year-old ASCAD21 × ASOMT7 double transformants displayed the same coloration pattern as the ASCAD21 trees.

Lignin Content in Transgenic Poplars

To evaluate the variations in the lignin level within the same line, a series of 1-year-old poplars grown together in the greenhouse were individually analyzed. Whereas some variations in lignin content could be observed between trees from the same line (up to 10%), this experiment confirmed that ASCAD21 poplars have less Klason lignin than the control. This trend was observed not only in ASCAD21 (Klason lignin = 15.60 ± 0.5, mean value and SD for eight stems individually analyzed) but also in ASCAD21 × ASOMT7 (Klason lignin = 14.5 ± 0.5, nine stems individually analyzed), relative to the control (Klason lignin = 17.2 ± 0.7, five stems individually analyzed).

Analysis of Lignin-Derived Monomers Released by Thioacidolysis

The total yield in thioacidolysis main monomers Ar-CHSEt-CHSEt-CH₂SEt was monitored as a function of poplar age and line (Table II). For the sake of clarity, the data for 4- to 7-month-old poplars are not reported but are strictly consistent with the following comments. Within each line, the lowest yields calculated on the basis of the lignin content were observed in younger trees, which confirms that lignins deposited at the early stage of lignification have fewer -O-4 bonds (Terashima et al., 1993). Whatever the age, lower yields were obtained for ASOMT2B and ASOMT10B lignins, which points to a lower frequency of -O-4 bonds relative to control lignins. In contrast, lignins in CAD-transformed poplars showed a thioacidolysis yield similar to control lignins.

The relative importance (percent molar) of S and G monomers was measured to evaluate the distribution of S and G units involved only in -O-4 bonds. The proportion of S units in the lignin of 2-year-old poplars was found to be 2-fold reduced by COMT down-regulation (Table II). In contrast, this proportion remained at a high and stable level (approximately in the 50%-60% range) in the control and CAD- transformed lines, the lower level corresponding to younger plants, which is in agreement with previously published data (Terashima et al., 1993).

The first stage of lignification was studied in ASOMT2B plantlets obtained from poplar nodal explants. The decrease in the S units induced by the antisense effect could be observed very early in 4-week-old stems. In contrast, the relative frequencies of the S units in root lignin were similar in ASOMT2B and control plantlets. It is interesting that, when sections from a few-week-old ASOMT2B stems were subjected to the histochemical Wiesner stain specific for lignin, the primary xylem appeared not to be lignified, in contrast to the secondary xylem (data not shown). Taken together, these results suggest that the transformation does not affect lignification in the various plant tissues to the same extent.

Similar to maize bm3 mutants with depressed COMT activity (Lapierre et al., 1988), a substantial amount of 5-OH G units occurred in the lignin from the ASOMT lines, whereas trace amounts were found in wood of control poplars (Table II).

The increased incorporation of cinnamaldehyde units into lignin has been repeatedly reported in plants with depressed CAD activity, in sorghum (Bucholtz et al., 1980; Pillonel et al., 1991) and pine (MacKay et al., 1997) mutants, and in transgenic tobacco (Halpin et al., 1994; Higuchi et al., 1994). With this in mind, the dithioketal compounds G-CHSEt-CH₂-CH(SEt)₂ and S-CHSEt-CH₂-CH(SEt)₂, respectively, formed upon thioacidolysis of coniferaldehyde and sinapaldehyde, were quantified. Relative to the main monomers Ar-CHSEt-CHSEt-CH₂SEt (with Ar = G or S ring), these dithioketal derivatives were recovered in similar low amounts from the control and CAD-transformed lines (i.e. in the range of 5-10 µmol g¹ lignin, which is less than 0.5% of the main monomers).

To ascertain further that lignins from CAD-down-regulated poplars did not contain increased levels of cinnamaldehyde units, soluble lignin fractions isolated by mild procedures (see ``Materials and Methods'') were subjected to the Wiesner reaction according to the standard protocol (Bucholtz et al., 1980; Nakano and Meshitsuka, 1992). We found that the intensity of the pink color developed by cinnamaldehyde units was similar in samples from CAD-transformed and control lignin samples (Table III). When this coloration was expressed as equivalent to coniferaldehyde units per gram of lignin fraction, the coniferaldehyde level was found to be lower than 0.5% in the control and in line ASCAD21 (Table III). In contrast, in lignins isolated from lines ASOMT2B and ASCAD21 × ASOMT7, this level was higher, about 1% by weight. In agreement with the literature (Lai and Sarkanen, 1973), these values are lower than those obtained for conifer G lignins, which are reported to be in the 2% to 3% range (Chen, 1992).

View this table: [in this window] [in a new window]   Table III. Determination of coniferaldehyde units in soluble lignin fractions by the Wiesner reaction These lignin fractions were isolated by solvent extraction (dioxane:water, 9:1, v/v) of 1-year-old extract-free stems after ball milling and cellulase hydrolysis, which were then purified by precipitation in water. The isolation yield of the recovered lignin fractions is expressed as a weight percentage of total lignin. KL, Klason lignin. The percentage (molar) of G units in -O-4 lignin structures was determined by thioacidolysis.

In contrast to a recent study (Tsai et al., 1998), the level of coniferaldehyde units in these poplar lignin samples was not related to the wood coloration but rather seemed to be associated with the enrichment in G units induced by the antisense COMT construct (Table III). Upon thioacidolysis, ASCAD21 xylem-released S-CH(SEt)₂, which represents the dithioketal derivative of syringaldehyde, was recovered in a 2% yield relative to the major monomer S-CHSEt-CHSEt-CH₂SEt, versus 0.2% in the case of control sample. From the outer part of the xylem displaying the red coloration, the relative recovery of syringaldehyde dithioketal was still more pronounced (6% relative to the major S monomers). This result suggests that the heterogeneous pattern of the red coloration of ASCAD21 xylem might coincide with an increased incorporation of syringaldehyde units in the lignin.

Lignin characterization in a series of poplars individually analyzed revealed a fairly stable level of -O-4 bonds in control and ASCAD21 trees, as evidenced by the similar thioacidolysis yield (Fig. 1). In these lines, the relative proportion of S units involved in -O-4 bonds was found to be remarkably constant (Fig. 2). In contrast, lignins of the double transformants ASCAD21 × ASOMT7 displayed pronounced structural variations, both in -O-4 content (Fig. 1) and in S level (Fig. 2). In spite of this variability between individual plants, the antisense COMT construct introduced either in the ASCAD21 or in the wild-type background caused a similar alteration of the lignin profile, namely a reduction in -O-4 bonds, a decrease in S units, and the appearance of substantial amounts of 5-OH G units.

View larger version (44K): [in this window] [in a new window]   Figure 1. Yield in main lignin-derived thioacidolysis monomers recovered from extract-free wood of 1-year-old transgenic and control poplars grown together in the greenhouse and individually analyzed. For each tree, the main stem was collected, debarked, ground, and solvent extracted. Data are means of duplicate experiments (SE 3% to 5%) and expressed in micromoles of G, S, and 5-OH G monomers recovered per gram of lignin (evaluated as Klason lignin). This yield is a close reflection of the frequency of units involved only in labile -O-4 bonds in the lignin.

View larger version (42K): [in this window] [in a new window]   Figure 2. Relative proportion (percent molar) of S thioacidolysis monomers recovered from 1-year-old transgenic and control poplars grown together in the greenhouse and individually analyzed. For each tree, the main stem was collected, debarked, ground, and solvent extracted. Data are means of duplicate experiments (SE 3% to 5%) and expressed as a molar percentage of the main lignin-derived thioacidolysis monomers.

Evaluation of Free Phenolic Groups in -O-4-Linked Lignin Units

View this table: [in this window] [in a new window]   Table IV. Frequency of free phenolic groups in G or S units involved in -O-4 bonds, as shown from thioacidolyses of methylated extract-free poplars For each line, measurements were done from one (6-month-old poplars) or three to five (older poplars gathered for analyses) plants.

Relative Frequencies of Condensed Bonds in Lignins

View larger version (19K): [in this window] [in a new window]   Figure 3. Main dimers recovered from thioacidolysis followed by Raney nickel desulfurization of poplar lignins (R = H or O methyl; R = H or CH2OH). The S-S - dimers originate from syringaresinol structures. These dimers are representatives of resistant interunit linkages occurring in angiosperm lignins.

The determination of thioacidolysis dimers provided unique information concerning the relative frequencies of resistant interunit bonds in control and transgenic poplar trees (Table V). The main effect of depressed CAD activity in poplar lignins was the enrichment in -1 diarylpropane structures (+15%). The most remarkable effect of depressed COMT activity was the increase in 5-5 biphenyl structures, together with the severe decrease in - syringaresinol structures. In the double transformant ASCAD21 × ASOMT7, the profile for interunit bonds was similar to that of ASOMT10B single transformants.

Analysis of Low-M_r Phenolics Released by Mild Alkaline Hydrolysis

In addition to p-hydroxybenzoic acid, vanillin and syringaldehyde were released upon alkaline hydrolysis of the samples (Table VI). With the exception of line ASOMT10B, which is particularly enriched in G units, syringaldehyde was obtained in substantially higher amounts than vanillin. Past studies have shown that small amounts of benzaldehydes are released by alkaline hydrolysis of lignified walls (Hartley and Keene, 1972). In agreement with a previous study (Baucher et al., 1996), higher levels of vanillin and, more markedly, of syringaldehyde were obtained from CAD-transformed poplars, especially from line ASCAD21. From the data of Table VI, we could estimate that 1.2% of the lignin units in line ASCAD21 gave rise to syringaldehyde upon alkaline hydrolysis, whereas the figure obtained for the control lignin was 10-fold lower. Taken together with the increased recovery of syringaldehyde dithioketal from thioacidolysis of line ASCAD21, this result suggests that CAD down-regulation increased the incorporation of syringaldehyde into lignin, although the overall level remained low.

Pulp Production from Transgenic and Control Poplars

The greenhouse-grown series was subjected to cooking experiments with various alkali charges (Table VII). At a given charge, no difference in cellulose degree of polymerization (data not shown) could be observed between transgenic and control samples, which means that cellulose degradation was similar. COMT-down-regulated poplars displayed a markedly higher resistance toward kraft delignification, as evidenced by higher  numbers relative to other lines. In contrast and at a low charge of active alkali (15%), ASCAD21 poplars were more extensively delignified, whereas ASCAD52 did not display improved delignification. The measurements of mechanical properties of sheets made from the pulps did not reveal differences between transgenic and control samples. When pulps with similar  numbers from the various lines were subjected to a totally chlorine free bleaching sequence, no difference in bleachability could be evidenced (data not shown).

Evaluation of the field-grown series essentially confirmed the previous results. ASOMT2B and ASOMT10B lines proved to be substantially more resistant toward kraft delignification, as shown by their higher  number (+60% relative to the control). By contrast, the ASCAD21 line was more extensively delignified than the control. The other CAD-transformed lines, ASCAD52 and SCAD1, were not better delignified than the control. Only slight variations in bleachability and brightness of the pulps prepared from the various lines could be observed after an elementary chlorine-free or a totally free chlorine-bleaching sequence. The mechanical properties of the pulps from the various field-grown lines were similar, which means that the genetic transformation did not detrimentally affect the bonding potential of cellulosic fibers.

	DISCUSSION

Lignin Levels in Poplar Trees Are Not Altered by COMT Down-Regulation but Are Slightly Reduced by CAD Down-Regulation

Contrary to the above-mentioned studies, a moderate decrease in lignin content has been evidenced in transgenic tobacco lines (Ni et al., 1994; Sewalt et al., 1997b) and in the maize bm3 mutant (Barrière and Argillier, 1994) with depressed COMT activity. The occurrence of several O-methyltransferase activities with various substrate specificities and spatial or temporal expression patterns makes it difficult to predict the effect of selectively targeting bifunctional COMT. That primary xylem of COMT-down-regulated poplar plantlets did not display any Wiesner staining in contrast to secondary xylem suggests that the lignin level is dramatically reduced in the early stages of lignification but is restored in secondary xylem because of O-methyltransferase-compensating activities and/or of spatiotemporal variations in the effect of the antisense COMT construct.

In CAD-down-regulated poplars, our findings suggest a slightly lower Klason lignin level in the more depressed lines (ASCAD21 and SCAD1) relative to the control. This trend should be confirmed with older trees. Even though the maintenance of normal growth and lignin level in transgenic poplars with antisense CAD or COMT construct does not seem remarkable in itself, it is positive for agronomical use of the transgenic plants.

In contrast to our present findings, recent studies have shown that modifying the expression of enzymes located at an earlier stage of the lignin biosynthetic pathway may induce substantial decreases in lignin content. For instance, down-regulating cinnamate-4-hydroxylase in tobacco (Sewalt et al., 1997a) and 4-coumarate:CoA ligase in tobacco (Kajita et al., 1997) or in poplar (Hu et al., 1998) have been reported to noticeably reduce the lignin content. In cinnamoyl-CoA reductase down-regulated tobacco, an approximately 2-fold decrease in lignin content has been evidenced in the most severely depressed line, which displayed altered growth and development together with collapsed vessels (Piquemal et al., 1998).

Even though a change in lignin distribution at the tissue or cell level cannot be ruled out, the present study suggests that changes in lignin structure may be primarily responsible for the higher and lower pulping characteristics of CAD- and COMT-transformed poplar trees, respectively.

COMT-Down-Regulated Poplar Trees Display Severe Alteration in Lignin Structure and the Pulping Characteristics Are Lower

The dramatic alterations induced by COMT down-regulation gave poplar lignins some similarities with softwood lignins. Because the overall lignin level is maintained in COMT-transformed poplars, the reduction in S units, -O-4 linkages, and free phenolic units, together with the increase in resistant interunit linkages, would result in a lignin less amenable to kraft degradation. This proposition was repeatedly confirmed by our kraft cooking assays. Wood of the line ASOMT was found to be more resistant to kraft delignification, as shown by higher  numbers relative to the control. In other words, the 90% decrease in COMT activity seems detrimental for kraft pulping.

The Subtle Alteration of Lignin Structure Induced by CAD Down-Regulation Makes Poplar Lignin More Amenable to Kraft Delignification

The alteration of lignin structure induced by CAD down-regulation in poplar could be evidenced only by using the most recent developments of thioacidolysis. Whereas the proportion of G and S units and the frequency of -O-4 linkages did not appear to be affected by the transformation, we provide here the first evidence to our knowledge that CAD down-regulation induced a substantial enrichment of the lignin in free phenolic units, as well as in diarylpropane structures. That both diarylpropane and free phenolic groups increase is consistent with our current knowledge of lignin, because the former structures have been shown to be distributed mainly in lignin as end groups with free phenolic functions (Gellerstedt and Zhang, 1991). Some physical and/or chemical parameters that influence the polymerization mechanisms may be affected by the transformation. Whatever the biochemical basis of the phenomenon, the effect that can be expected from an enrichment in free phenolic units is an easier kraft delignification. Two-year-old poplar trees of the ASCAD21 line, which displayed the most severely depressed CAD activity, the brightest red coloration of the xylem, and the highest lignin content in free phenolic units, proved to be more easily delignified in two independent pulping assays. This moderate yet significant improvement in delignification efficiency can be of very high value because of the large scale of the pulping industries. In contrast, line ASCAD52, which displayed less-pronounced enzymic, phenotypic, and structural changes relative to the control, did not show any improvement in kraft-pulping characteristics. These results suggest a threshold level in free phenolic groups to be reached to beneficially affect the kraft-pulping efficiency.

Our present data were obtained from 2-year-old poplar trees regenerated from CAD- and COMT-down-regulated primary transformants. The stability of the transgene expression has therefore been demonstrated for a large series of mature plants grown in the greenhouse or in the field. It still needs to be shown that this effect is maintained for 10 years, the age at which poplars are harvested for pulping. From the present results, down-regulation of CAD activity in poplars appears to be an efficient route for improving the kraft-pulping characteristics of the trees, whereas the 90% down-regulation of COMT activity has a dramatically adverse effect on the efficiency of the kraft process. These effects may be accounted for by the changes in lignin structure induced by the transformation. In addition to the industrial interest of the transformants, the down-regulation of the CAD and COMT enzymes allowed us to obtain new insights in the complex biosynthetic pathway leading to lignin precursors and its regulatory mechanisms.

	FOOTNOTES

   Received July 31, 1998; accepted September 22, 1998.

	ABBREVIATIONS

Abbreviations: CAD, cinnamyl alcohol dehydrogenase. CCoAOMT, caffeoyl-CoA-O-methyltransferase. COMT, caffeic acid/5-hydroxyferulic acid-O-methyltransferase. G, guaiacyl. 5-OH G, 5-hydroxyguaiacyl. S, syringyl.

	ACKNOWLEDGMENT

We sincerely thank F. Legée for running the Klason lignin analyses.

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