Condensed Lignins Are Synthesized in Poplar Leaves Exposed to Ozone

Abstract

Poplar (Populus tremula × alba) trees (clone INRA 717-1-B4) were cultivated for 1 month in phytotronic chambers with two different levels of ozone (60 and 120 nL L^–1). Foliar activities of shikimate dehydrogenase (EC 1.1.1.25), phenylalanine ammonia lyase (EC 4.3.1.5), and cinnamyl alcohol dehydrogenase (CAD, EC 1.1.1.195) were compared with control levels. In addition, we examined lignin content and structure in control and ozone-fumigated leaves. Under ozone exposure, CAD activity and CAD RNA levels were found to be rapidly and strongly increased whatever the foliar developmental stage. In contrast, shikimate dehydrogenase and phenylalanine ammonia lyase activities were increased in old and midaged leaves but not in the youngest ones. The increased activities of these enzymes involved in the late or early steps of the metabolic pathway leading to lignins were associated with a higher Klason lignin content in extract-free leaves. In addition, stress lignins synthesized in response to ozone displayed a distinct structure, relative to constitutive lignins. They were found substantially enriched in carbon-carbon interunit bonds and in p-hydroxyphenylpropane units, which is reminiscent of lignins formed at early developmental stages, in compression wood, or in response to fungal elicitor. The highest changes in lignification and in enzyme activities were obtained with the highest ozone dose (120 nL L^–1). These results suggest that ozone-induced lignins might contribute to the poplar tolerance to ozone because of their barrier or antioxidant effect toward reactive oxygen species.

Plants submitted to ozone generally respond with a stimulation of enzymes involved in the phenylpropanoid pathway. Both the activity and transcript level of Phe ammonia lyase (PAL), the first enzyme of the phenylpropanoid pathway, have been reported to rapidly increase under ozone exposure in various herbaceous plants and forest species (Tingey et al., 1976; Heller et al., 1990; Rosemann et al., 1991; Eckey-Kaltenbach et al., 1994; Sharma and Davis, 1994; Booker et al., 1996; Tuomainen et al., 1996; Pääkkönen et al., 1998). In poplar (Populus maximorwizzii × Populus trichocarpa), higher levels of PAL activity were found to be associated with a greater ozone tolerance (Koch et al., 1998). Other enzymes of the phenolic secondary metabolism, such as 4-coumarate CoA ligase (Eckey-Kaltenbach et al., 1994; Booker and Miller, 1998), caffeic acid O-methyl transferase (Koch et al., 1998), and cinnamyl alcohol dehydrogenase (CAD), the enzyme involved in the synthesis of the monolignols (Galliano et al., 1993a, 1993b; Eckey-Kaltenbach et al., 1994; Booker and Miller, 1998; Zinser et al., 1998), have been shown to be ozone stimulated at the activity and/or transcript level (for review, see Sandermann et al., 1998).

The activation of the phenylpropanoid metabolism also has been reported for other biotic or abiotic stresses (Dixon and Paiva, 1995) such as wounding, pathogen attack, UV irradiation, heavy metals, or drought. Stress-induced modifications of the metabolism seem to be associated with a rapid oxidative burst (Dat et al., 2000; Mittler, 2002) similar to ozone stress (Langebartels et al., 2002). The stress response could be initiated by the accumulation of reactive oxygen species exacerbating damage or signaling the activation of defense responses (Mehdy et al., 1996; Bolwell, 1999; Neill et al., 2002). In the case of wounding (Hawkins and Boudet, 1996; Brill et al., 1999), heavy metals (Diaz et al., 2001), and pathogen attack (Walter, 1992; Bennett and Wallsgrove, 1994; Bucciarelli et al., 1998), stimulation of enzyme activities involved in the more specific lignin pathway was correlated with increased lignin synthesis. Lignin deposition near the site of infection or wounding (Rittinger et al., 1987; Hawkins and Boudet, 1996) suggested a role of lignin in disease resistance. Stress lignins formed in response to pathogen elicitation or to mechanical stress have been shown to display structural features quite distinct from constitutive lignins (Boudet et al., 1995; Lange et al., 1995).

The similar response of the phenylpropanoid pathway to ozone and pathogen or wounding stress suggested a possible role of lignin in ozone resistance. A better resistance of the foliar mesophyll cells toward oxidant species might be conferred by an increased lignification of their cell walls (Pell et al., 1997). However, the ozone-induced lignins have not been characterized clearly until now (Sandermann et al., 1998), and lignin determinations of ozone-treated leaves led to conflicting results according to the species and/or the employed analytical method. Although no increase in lignin content could be detected in ozone-exposed leaves from pine (Pinus ponderosa, Pinus sylvestris, Pinus taeda), soybean (Glycine max), or cotton (Gossypium hirsutum; Tingey et al., 1976; Bonello et al., 1993; Boerner and Rebbeck, 1995; Booker et al., 1996; Booker and Miller, 1998; Booker, 2000), ozone-fumigated sugar maple (Acer saccharinum) leaves displayed a substantially higher lignin content (Boerner and Rebbeck, 1995).

The purpose of the present work was to determine to what extent metabolic pathways leading to lignin could be stimulated in the leaves of young poplar trees submitted to prolonged exposure to ozone. In addition, this work aimed at evaluating the structural peculiarities of ozone-induced lignins, if any. We tried to limit the interference between lignins and non-lignin phenolics by selecting appropriate analytical tools. Young poplar trees were cultivated in growth chambers for 1 month at two different ozone concentrations (60 and 120 nL L^–1). The foliar activities of various enzymes involved in the early or late metabolic steps leading to lignins were measured in control and ozone-treated leaves collected at three different developmental stages. Foliar lignins were localized by the specific Wiesner histochemical staining (Nakano and Meshitsuka, 1992) and then quantified by the conventional Klason procedure applied to extract-free samples (Dence, 1992). Lignin structure was evaluated by thioacidolysis (Lapierre et al., 1999).

RESULTS

Plant Growth and Visual Injuries

Young poplars (2.5 months old) were grown in charcoal-filtered (CF) air (control conditions) or fumigated for 4 weeks with CF air plus ozone (60 or 120 nL L^–1) in phytotronic chambers. Trees exposed to ozone showed a noticeably reduced growth (Fig. 1A) that was visible after 23 d. The reduction was more pronounced for the strongest concentration of ozone (120 nL L^–1). Whatever the ozone treatment, leaf injuries (red-brown spots, Fig. 1B) could only be observed in the midaged and old leaves. Necrotic areas appeared soon after the beginning of the treatment with 120 nL L^–1 ozone and later (20 d) with 60 nL L^–1 ozone.

Figure 1.

Young poplars after ozone fumigation for 23 d (A) and leaf injuries in ozone-fumigated plants (B).

Stimulation of CAD Activity and Transcripts

CAD activity and CAD transcript levels were monitored in the control and in the ozone-fumigated leaves. Three foliar levels (Fig. 2) corresponding to different developmental stages were analyzed. Leaves L1 and LL were the oldest and the youngest fully expanded leaves, respectively. L4 was an intermediate leaf that was young at the beginning of the experiment and aged during the ozone exposure time.

Figure 2.

Relative position of leaves collected during the experiment numbered from the bottom. L1, First leaf; L4, fourth leaf; LL, last fully expanded leaf.

Under ozone exposure, CAD activity was rapidly and strongly stimulated at the three foliar levels (Fig. 3, A–C). This stimulation increased up to 15-fold and 23-fold the control level for the 60 and 120 nL L^–1 exposure, respectively. This increase was found to be more rapid and stronger for the highest ozone concentration. It was maintained for at least 24 d at the three foliar levels and for both treatments. CAD transcripts were analyzed by hybridization with pGemPOPCAD1 (Baucher et al., 1996) encoding CAD2 gene from poplar. CAD RNA levels were strongly stimulated by ozone whatever the foliar stage (Fig. 3, D–F).

Figure 3.

Time course of CAD (A–C) activity and CAD RNA levels (D–F) in different leaves: L1 (A and D), L4 (B and E), and LL (C and F) of poplar trees under ozone exposure. Plants were exposed to pollutant-free air (▪), 60 nL L^–1 ozone (○), or 120 nL L^–1 ozone (•). Means ± sd (n = 3). Error bars are given when greater than symbol size.

Coordinated Stimulation of Shikimate Dehydrogenase (SHDH) and PAL

In parallel to CAD activity, we monitored the activity of two enzymes involved in earlier steps of phenolic metabolism, namely SHDH, an enzyme of the shikimate pathway involved in the synthesis of Phe, and PAL, the first enzyme of the phenylpropanoid pathway.

In contrast to CAD activity, distinct effects of ozone on SHDH and PAL could be evidenced according to the foliar stage. In control young leaves (LL), substantial constitutive SHDH and PAL activities were observed (Fig. 4, C and F), and a clear-cut effect of ozone exposure could not be observed in these physiologically active leaves. In the oldest leaves (L1), we could see that ozone stimulated both the SHDH and PAL activities (Fig. 4, A and D). The maximum activity was reached between 7 and 13 d after the onset of 60 nL L^–1 ozone exposure. A similar induction was found in leaves exposed to 120 nL L^–1 ozone, but the increase was more pronounced. The results obtained with leaves L4, which were young at the beginning of the experiment and aged during it, confirms that ozone exposure does not affect SHDH and PAL activities in young leaves: There was no effect at the beginning of the experiment, but ozone increased these activities after about 7 d of exposure once the leaves were midaged (Fig. 4, B and E).

Figure 4.

Time course of SHDH (A–C) and PAL (D–F) activities in different leaves: L1 (A and D), L4 (B and E), and LL (C and F) of poplar trees under ozone exposure. Plants were exposed to pollutant-free air (▪), 60 nL L^–1 ozone (○), or 120 nL L^–1 ozone (•). Means ± sd (n = 3). Error bars are given when greater than symbol size.

Increase of Lignin Content

All the leaves from each plant were collected at the end of the experiment for lignin quantification. The stems were also individually collected and analyzed. Each sample was ground and thoroughly extracted by various solvents to eliminate the soluble components that could interfere with the gravimetric Klason lignin (KL) determination. This solvent extraction step yielded an extract-free residue that essentially corresponds to the cell walls and will be referred to as cell wall residue (CWR). Lignin analyses of the stems did not reveal any effect of the ozone treatment on poplar wood lignins (percentage KL as weight percentage of CWR: 17.8 ± 0.4 in control and 18.2 ± 0.2 at 120 nL L^–1 ozone concentration). Consistently, PAL, SHDH, and CAD activities were not modified by ozone treatment in stems throughout the experiment (data not shown). In contrast, substantial effects of the ozone treatment were found by the analyses of lignins in leaves. The foliar CWR was recovered with similar yields ranging between 45% and 50% of the foliar dry matter (DM). When the foliar KL content was expressed as weight percentage of the CWR or of the initial DM, we observed that lignin content was substantially increased under ozone exposure (Table I), the highest increase corresponding to the highest ozone level.

Table I.

Lignin content of poplar leaves cultivated for 34 d with CF air or 60 or 120 nL L^-1 ozone

Lignin content was estimated using the Klason method. Values are expressed as percentage (w/w) of the CWR or as percentage (w/w) of DM. Four trees were grown for each culture condition, and the data represent the mean value and sd for these four replicates. The normalized values relative to the control are shown in parentheses.

Culture Condition	KL
	% CWR	% DM
Control	11.83 ± 0.68 (100)	5.93 ± 0.31 (100)
Ozone, 60 nL L-1	16.09 ± 0.66 (136)	7.08 ± 0.65 (120)
Ozone, 120 nL L-1	18.46 ± 1.31 (156)	8.58 ± 0.86 (145)

To evaluate whether the increase in lignin content under ozone was correlated with a modification of lignin distribution, leaf fragments were submitted to the Wiesner (phloroglucinol) staining reagent. This reagent colors lignins mauve, primarily from reactions of hydroxycinnamyl aldehyde end-groups in lignins (Nakano and Meshitsuka, 1992). In control leaves, the mauve coloration of lignified areas could be observed only in veins (Fig. 5A). In ozone-treated plants (120 nL L^–1), cells from the lamina reacted with the Wiesner reagent, indicating lignin in mesophyll or in epidermal cells (Fig. 5B). Colored cells appeared to be mainly localized near necrotic zones in the leaf (Fig. 5B).

Figure 5.

Phloroglucinol staining of leaves L4 from poplars cultivated in control conditions (A) or ozone fumigated (B) collected 20 d after the onset of the experiment.

Structure of Ozone-Induced Lignin

Lignin structure was evaluated by thioacidolysis. The key reaction of thioacidolysis is the cleavage of the labile β-O-4 bonds that are the major interunit bonds in native lignins (Lapierre et al., 1999). The p-hydroxyphenyl (H), guaiacyl (G), and syringyl (S) lignin units only involved in β-O-4 bonds specifically and give rise to thioethylated H, G, and S monomers, respectively (Fig. 6). Therefore, when calculated on the basis of the KL content, the recovery yield of these diagnostic monomers is a reflection of the frequency of β-O-4 bonds in lignins. Whatever the sample, we could see that yields of thioacidolysis monomers released from foliar lignins ranged between 360 and 555 μmol g^–1 KL, which is approximately 4-fold lower than yields obtained for the corresponding stem lignins (2,000 ± 90 μmol g^–1 KL in control and 1,920 ± 40 μmol g^–1 KL at 120 nL L^–1 ozone concentration). Foliar lignins have less labile β-O-4 bonds and, therefore, more carbon-carbon interunit linkages (referred to condensed linkages in the following) than stem lignins.

Figure 6.

Principle of the thioacidolysis analytical degradation. Thioethylated p-hydroxyphenyl (R¹ = R² = H) guaiacyl (R¹ = OCH₃, R² = H) and syringyl (R¹ = R² = OCH₃) monomers are specifically released from the typical β-O-4 lignin structures (R′ = H or side chain of lignin unit).

In addition, lignins from ozone-exposed leaves yielded less thioacidolysis monomers than lignins from control leaves (Table II). Lignins formed in leaves exposed to ozone are more condensed than leaf constitutive lignins.

Table II.

Lignin-derived monomers (H, G, and S) after thioacidolysis of leaves from poplars cultivated under control conditions or fumigated for 34 d with 60 or 120 nL L^-1 ozone

Four trees were grown for each culture condition, and the data represent the mean value and sd for these four replicates. The normalized values relative to the control are shown in parentheses.

Culture Condition	Yield in H + G + S Monomers	Monomer
		H	G	S
	μmol g-1 lignin		% (w/w)
Control	551 ± 22 (100)	1.4 ± 0.2 (100)	60.8 ± 0.7	37.8 ± 0.7
Ozone, 60 nL L-1	418 ± 72 (76)	3.5 ± 0.2 (250)	60.4 ± 0.9	36.1 ± 2.1
Ozone, 120 nL L-1	368 ± 41 (67)	4.9 ± 1 (350)	61.5 ± 2.2	33.6 ± 3.2

Foliar lignins in control plants predominantly released G monomers upon thioacidolysis, together with an approximately 2-fold lower recovery of S monomers and with minor amount of non-methoxylated H compounds (Table II), whereas poplar stem lignins provided S and G monomers with a 65:35 weight ratio and H monomers as trace components. The higher condensation degree of foliar lignins relative to stem lignins could be accounted for by these distinct S/G proportions as S units are essentially involved in β-O-4 interunit bonds.

A close examination of the frequency of lignin-derived H, G, and S monomers in leaves revealed that the proportion of the H compounds was noticeably increased (2.5–3.5-fold) after ozone exposure (Table II). The analyses of the thioacidolysis lignin-derived dimers that are representatives of the various condensed bonding modes in lignins (Lange et al., 1995) confirmed the ozone-induced increased frequency of H units. Besides this enrichment in condensed bonds and in H units, thioacidolysis of permethylated samples (Lapierre et al., 1999) revealed that the proportion of terminal units with free phenolic groups relative to internal units etherified at their phenolic hydroxyl was substantially higher in ozone-treated foliar lignins, relative to control lignins (data not shown). The data of Table II further show that changes in lignin structure were dose dependent, i.e. the highest ozone level induced the most severe change in lignin structure.

DISCUSSION

Coordinated Stimulation of Metabolism toward Lignin Synthesis

Phenylpropanoid metabolism and the lignin biosynthesis pathway are usually stimulated by ozone (Sandermann et al., 1998). We analyzed for the first time, to our knowledge, different enzymes from shikimate pathway to lignin biosynthesis in the same experiment. The enzyme catalyzing the final step in lignin monomer synthesis (CAD) was rapidly and strongly increased by moderate concentrations of ozone (60–120 nL L^–1), both at activity and RNA levels, in young poplar leaves. The induction of CAD activity by ozone has been demonstrated previously in conifers, Picea abies (Heller et al., 1990; Galliano et al., 1993b) and Pinus sylvestris (Zinser et al., 2000), and in two herbaceous species, soybean and parsley (Petroselinum crispum; Eckey-Kaltenbach et al., 1994; Booker and Miller, 1998). This stimulation was also shown at the RNA levels in conifers (Galliano et al., 1993a; Zinser et al., 1998, 2000). The stimulation levels of CAD activity were in good accordance with the stimulation of RNA level, 2- to 3-fold in conifers and 10-fold in poplar (this paper). These results support the hypothesis of a transcriptional control of foliar CAD activity in response to ozone treatment.

In our experiment on poplar, the stimulation of CAD was correlated with an increase of the first enzyme of the general phenylpropanoid pathway, PAL. Such a correlation was also observed in soybean and parsley (Eckey-Kaltenbach et al., 1994; Booker and Miller, 1998). Independent of CAD stimulation, studies showed the increase of PAL activity and RNA levels in response to ozone and other enzymes of the phenylpropanoid pathway such as 4-coumarate CoA ligase and caffeic acid O-methyl transferase (Booker and Miller, 1998; Koch et al., 1998). These results suggested an involvement of the general phenylpropanoid pathway. In addition, we provided the first evidence, to our knowledge, that ozone exposure stimulates not only the enzymes of the phenylpropanoid pathway but also SHDH, an enzyme of the shikimate pathway that yields Phe. Such a correlation of the shikimate pathway and secondary metabolism also was shown in response to UV (Logemann et al., 2000). Nevertheless, the ozone response is not selectively confined to the synthesis of protective agents as observed for UV light (Logemann et al., 2000). The metabolic modifications induced by ozone look more like the pathogen defense response (Somssich and Hahlbrock, 1998) because an extensive reprogramming of metabolism seems to occur. Many pathways from the primary metabolism were shown to respond: CO₂ fixation, respiration, and glycolysis (Dizengremel, 2001). These results suggested that the supply pathways providing the substrates for the production of lignin were induced by ozone in a correlated manner involving a reprogramming of cellular metabolism.

One of the major effects of ozone on primary metabolism is the decrease of photosynthesis rates (Dizengremel, 2001) probably involved in the reduced growth generally observed under ozone treatment (Bortier et al., 2000a). According to our results, the reprogramming of metabolism toward lignin synthesis could represent a substantial rerouting of carbon skeletons that may also explain the growth reduction.

Increase of Lignin Content

Similar to other authors who studied the ozone impact on plant physiology, the increased activities of the early and late enzymes involved in lignin biosynthesis led us to the hypothesis that poplar leaves might synthesize more lignins when exposed to ozone. However, in many past studies, this hypothesis was not confirmed by gravimetric (Klason assay) and/or spectrophotometric (lignothioglycolic acid assay) determinations of lignin content. Nevertheless, a few studies reported such an increase in lignin level, in the case of maple (Boerner and Rebbeck, 1995) and soybean (Booker and Miller, 1998) leaves exposed to ozone. There is no ideal method of lignin determination because both the gravimetric and the spectrophotometric methods may suffer from interference from non-lignin components. In the present study, we found that an up to 10-fold stimulation of enzyme activities was concomitant with an increase of KL in poplar leaves. Klason determination was carried out on thoroughly solvent-extracted leaves according to a standard procedure (Dence, 1992). This extraction step allows the elimination of soluble components that could condense with lignins during the Klason acidic treatment, thereby leading to overestimation of lignin content. Proanthocyanidins (condensed tannins) could resist this solvent extraction step, and their putative association with lignins during the Klason assay could also increase the apparent KL value (Booker et al., 1996; Booker and Miller, 1998). However, a recent study on the foliar chemical composition of deciduous trees (trembling aspen [Populus tremuloides] and birch [Betula papyrifera]) revealed that, in contrast to coniferous trees, the foliar level of condensed tannins was negligibly affected by a long-term exposure to 50 to 100 nL L^–1 ozone concentration (Lindroth et al., 2001). Therefore, the higher KL content observed in the ozone-fumigated leaves is not an artifact but actually corresponds to an increased lignin biosynthesis. This conclusion is further supported by the phloroglucinol lignin staining assays.

Ozone-Induced Lignin Is More Condensed

In addition to an increased lignin level in ozone-exposed leaves, we showed that the newly synthesized lignins that appear in response to ozone structurally differ from control lignins. Such a conclusion was provided by an in-depth structural investigation carried out by thioacidolysis, a degradative method that essentially does not suffer interference from non-lignin phenolics. However, there is one exception to this: Thioacidolysis cannot discriminate between native lignins and the polyphenolic domain of suberin because both contain β-O-4-linked G and S units that are the targets of the thioacidolysis procedure (Lapierre et al., 1996). We can reasonably assume that the phenolic polymers formed in response to ozone and characterized herein are lignins because we could not detect any increase of hydroxycinnamic acid derivatives, typical of the suberin polyphenolic domain (Bernards and Razem, 2001). The present method of analysis characterizes both the constitutive lignins normally formed in foliar veins (Fig. 5A) and the stress lignins more specific to necrotic areas (Fig. 5B). Despite this, we could detect a substantial structural difference between foliar lignins of control and fumigated leaves. This result supports the hypothesis that stress lignins are substantially enriched in carbon-carbon interunit linkages, in terminal units with free phenolic groups, and in p-hydroxyphenyl (H) units. In other words and relative to constitutive lignins, ozone-induced lignins are more highly branched polymers with a large proportion of interunit carbon-carbon bonds. This structural trait may be favored at high concentration of monolignols during the peroxidasic polymerization process (Sarkanen, 1971), a situation that favors the formation of β-5 and β-β condensed bonds by dimerization between two monolignols (Boerjan et al., 2003). The rapid and dramatic induction of the lignin biosynthetic pathway could therefore account for a local and high availability of lignin precursors polymerized to heavily condensed lignins. In addition, this high condensation degree is favored by the higher proportion of H units (Terashima et al., 1993). Ozone-induced lignins displayed similar structural trends as early developmental lignins (Terashima et al., 1993; Lange et al., 1995), compression wood lignins (Lange et al., 1995; for review, see Bailleres et al., 1997), or as elicitor-induced stress lignins (Lange et al., 1995). All these results suggest that a common response to mechanical, chemical (ozone), or biotic (pathogens) stress is the coordinate induction of the enzymes and mechanisms involved in the formation of heavily condensed lignins. The ozone-induced lignification localized near the foliar necrotic areas could be initiated by reactive oxygen species and could be part of the multifaceted process leading to plant programmed cell death (Rao and Davis, 2001).

Lignin Biosynthesis Is Closely Related to Stress Intensity

In agreement with other results on woody plants (Pääkkönen et al., 1995; Koch et al., 1998; Bortier et al., 2000b), chronic foliar injuries could be observed only in midaged and older leaves. The differential response according to leaf age may be because of a more active physiology of young leaves but could also be related to acclimation processes. In our experiment, enzyme activity increases were correlated with foliar injuries demonstrating the relations between stress impact and lignin metabolism. Moreover, all the parameters we analyzed, enzyme activities, lignin content, and structure (carbon-carbon linkage and H unit proportion), were remarkably dependent to ozone dose showing, once again, a tight relation between stress intensity and lignin biosynthesis. Together with the localization of ozone-induced lignin near necrosis, all these results strongly suggested a defensive role of this highly condensed lignin. Specific pathways involved in ozone-induced lignin synthesis and its regulation remain to be elucidated.

MATERIALS AND METHODS

Plant Material and Growth Conditions

Young poplars (Populus tremula × alba, clone INRA 717-1-B4) propagated from rooted cuttings was obtained from Daniel Cornu (INRA, Orléans, France) and transplanted into plastic pots filled with compost:perlite (1:1 [v/v]). The containers were covered with transparent acrylic hoods and transferred into phytotronic chambers at 75%/85% relative humidity (day/night) with a 14-h light period (Sun T Agro, Philips, Eindhoven, The Netherlands; intensity: 250–300 μmol m^–2 s^–1) and 22°C/18°C day/night temperatures. The hoods were removed after 2 weeks, and plants were transplanted into 5-L pots containing compost. Plants were initially fertilized with 20 g of slow release 13:13:13 N:P:K (Nutricot T 100, Fertil, Boulogne-Billancourt, France) and maintained in chambers for 1 month before ozone fumigation.

Ozone Treatment

Ozone treatment was performed in the phytotronic chambers used for plant acclimation. The young trees were exposed to CF air or 60 ± 5 or 120 ± 10 nL L^–1 ozone mixed with CF air. Ozone generated from pure O₂ with an CMG3-3 ozone generator (Innovatec II, Rheinbach, Germany) was distributed in the fumigation chambers during the 14-h light period. Ambient air in the different chambers was continuously analyzed by an ozone analyzer (O341M, Environment SA, Paris). Leaves were sampled over a period of 31 d during ozone exposure. Different levels of fully expanded leaves were studied as described Figure 1. Three individuals of control and exposed plants were collected at every time point. Samples were harvested in the middle of the photoperiod, frozen in liquid nitrogen, and then stored at –80°C until analysis.

Preparation of Enzyme Extracts

Frozen leaves or stems (about 300 mg) were ground in a mortar chilled with liquid nitrogen in the presence of 150 mg of polyvinylpolypyrrolidone. The resulting powder was mixed to 3.5 mL of 100 mm HEPES-KOH buffer (pH 7.5) containing 2 mm dithiothreitol, 5 mm MgCl₂, 5 mm EGTA, 20 μm 4-amidinophenylmethanesulfonyl fluoride, 1 μm pepstatin, 1 μm leupeptin, 0.5% (w/v) soluble polyvinylpyrrolidone-25, 0.5% (w/v) polyethylene glycol-20, and 10% (v/v) glycerol. The homogenate was centrifuged for 20 min at 18,500g (4°C). The supernatant was passed through a PD-10 Sephadex G-25 column (PD-10, Amersham Biosciences, Orsay, France) equilibrated with 100 mm HEPES-KOH buffer (pH 7.5) containing 2 mm dithiothreitol, 5 mm MgCl₂ and 10% (v/v) glycerol. The resulting desalted extract was used for enzyme assays.

Enzyme Activities

All analyses were performed at 30°C with a Beckman DU 640 spectrophotometer (Beckman Coulter, Roissy, France) in a final volume of 200 μL. SHDH (EC 1.1.1.25) activity was determined by following the reduction of NADP at 340 nm in 100 mm Tris-HCl (pH 8.5) containing 1 mm NADP and 10 mm shikimate (Fiedler and Schultz, 1985). CAD (EC 1.1.1.195) activity was monitored at 400 nm as described by O'Malley et al. (1992), the reaction buffer consisting of 100 mm Tris-HCl (pH 9), 0.4 mm NADP, and 70 μm coniferyl alcohol. PAL (EC 4.3.1.5) activity was assayed by measuring the release of cinnamate at 290 nm (Havir, 1987). The protein content of enzyme source was determined with the Bio-Rad Bradford protein reagent dye (Bio-Rad, Ivry sur Seine, France) using bovine serum albumin as standard (Bradford, 1976).

RNA Isolation

Total RNA were extracted from leaves according to Chang et al. (1993) with modifications (Fontaine et al., 2003). Plant material (0.5 g) was resuspended in 10 mL of extraction buffer.

Slot-Blot Analysis

Total RNA was loaded under vacuum on a nylon membrane (Roche Diagnostics, Indianapolis) using a slot-blot apparatus (Minifold II, Schleicher & Schull, Dassel, Germany). The RNA was hybridized with DIG-labeled cDNA probes according to the DIG System User's Guide for Filter Hybridization (Roche Diagnostics). The CAD RNA were detected using PGEMpopCAD1 (Van Doorsselaere et al., 1995; Baucher et al., 1996) as a probe. Membranes were prehybridized for 1 h in buffer at 50°C and hybridized overnight at 50°C with denatured probes. Membranes were washed under high-stringency conditions, and hybridization signals were visualized by chemiluminescent detection using CDP-star (Roche Diagnostics). After exposure on autoradiographic films (Lumi-Film Chemiluminescent Detection Film, Roche Diagnostics), the signal was quantified using a Bio-Rad Imaging densitometer (model GS-690), and the bands were analyzed using Multi Analyst Bio-Rad software (version 1.0.2). The linearity of the quantification was verified, and the band intensities were indicated as percentage of the maximal intensity on the film.

Lignin Characterization

Lignin analyses were carried out on dry extractive-free samples (CWR), ground to pass a 0.5-mm sieve before exhaustive solvent extraction (2:1 [v/v] toluene:ethanol, ethanol, and then water). The lignin content of the leaves or stem CWR was determined by the Klason method from 300 mg of sample according to the standard procedure (Dence, 1992). Thioacidolysis was performed as previously reported (Lapierre et al., 1999). The lignin-derived monomers were determined by gas chromatography-mass spectrometry of their silylated derivatives.

The results of lignin analyzes were reported as the mean value and se obtained for each series of four trees individually analyzed after exposure to 0 (control) of 60 or 120 nL L^–1 ozone fumigation.

Histochemical Staining of Lignin

Leaf pieces were collected and fixed in FAA (10% [v/v] formalin, 5% [v/v] acetic acid, and 60% [v/v] ethanol) for at least 24 h. Samples were then dehydrated in a graded ethanol series before coloration with phoroglucinol-HCl according to Nakano and Meshitsuka (1992). Colored samples were observed under a magnifying lens.