Effects of ozone on the carbon metabolism of forest trees

Plant Physiol. Biochem. 39 (2001) 729−742 © 2001 Éditions scientifiques et médicales Elsevier SAS. All rights reserved S0981942801012918/REV

Review Effects of ozone on the carbon metabolism of forest trees Pierre Dizengremel* Écologie et écophysiologie forestières, Inra - UHP Nancy-1 (UMR 1137), Équipe écophysiologie cellulaire et moléculaire, BP 239, 54506 Vandœuvre, France

Received 1 March 2001; accepted 10 May 2001 Abstract – Chronic long-term exposure to near-ambient concentrations of ozone could contribute to forest decline in several regions of the world, in combination with other biotic and abiotic factors. It is generally admitted that, under ozone stress, biochemical events occur before any development of visible symptoms of damage. Photosynthesis is impaired whereas respiration is increased. The activity and quantity of Rubisco and Rubisco activase are diminished as well as the transcription of the proteins. Concomitantly, there is a general increase in the functioning of the catabolic pathways (glycolysis, pentose phosphate pathway). The mitochondrial respiration is also activated with an increased transcription of the alternative oxidase. The most impressive event is the huge increase in activity of phosphoenolpyruvate carboxylase linked to a stimulation of the enzyme biosynthesis. Therefore, the high ratio between the two carboxylases, which reaches about 25 in ozone-free air, falls to about 2 under ozone fumigation. There is also an increase in the detoxifying processes (chloroplastic superoxidase isoform). All these changes in cellular metabolism are directed towards repair and maintenance of the cell structure. In this respect, a general increase in the phenylpropanoid metabolism is also observed with the production of more phenolic compounds and a stimulation of the lignin biosynthetic pathway through the activation of several enzymes (phenylalanine ammonia lyase, cinnamyl alcohol dehydrogenase, etc.). The mechanism of ozone action however still remains to be elucidated. Ozone causes an oxidative stress producing reactive oxygen species, which are the probable source for signal chains with messenger molecules such as jasmonic acid, salicylic acid and ethylene. The problem remains of the existence of a specific series of events starting from ozone penetrating through the stomata to the repression/stimulation of gene transcription in foliar cells. © 2001 Éditions scientifiques et médicales Elsevier SAS carbon metabolism / enzymes / gene expression / ozone stress / photosynthesis / respiration AOT40, accumulated hourly exposure over a threshold of 40 nL·L–1 O3 / AOX, alternative oxidase / Cab, chlorophyll a/b binding protein / CAD, cinnamyl alcohol dehydrogenase / CF, charcoal-filtered / CHS, chalcone synthase / G6PDH, glucose 6-phosphate dehydrogenase / IDH, isocitrate dehydrogenase / JA, jasmonic acid / LSU, Rubisco large subunit / ME, malic enzyme (NAD and NADP) / NF, non-filtered / OTC, open-top chamber / PAL, phenylalanine ammonia lyase / PEPc, phosphoenolpyruvate carboxylase / PFK, phosphofructokinase / PFP, pyrophosphate fructose 6-phosphate phosphotransferase / PK, pyruvate kinase / PMT, pinosylvin methyl transferase / ROS, reactive oxygen species / Rubisco, ribulose 1,5-biphosphate carboxylase/oxygenase / SA, salicylic acid / SHDH, shikimate dehydrogenase / SOD, superoxide dismutase / SSU, Rubisco small subunit / STS, stilbene synthase

1. INTRODUCTION Although definite proof of a link between ozone and forest damage exists only in California, a large number of data supports a role for this tropospheric pollutant in forest decline in eastern USA and Europe [43]. During sunny days, elevated concentrations of ozone are found in remote areas, due to the transport of the pollutant *Correspondence and reprints: fax +33 3 83 91 25 64. E-mail address: [email protected] (P. Dizengremel).

from urban source areas (mainly car traffic) over large distances. This is particularly evident in the Los Angeles basin where the sea breeze causes polluted air to flow to the east and the surrounding mountains: a range of increasing ozone concentrations from the west to the east is thus found, peaking to about 200 nL·L–1 in the San Bernardino mountains [72]. Analyses of historic ozone measurements reveal largely more than a doubling of tropospheric ozone concentration in the northern hemisphere in the last century and this increase was comprised between 1 and 2 % per year in the last two decades [72]. A critical

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level of ozone for forest trees, the AOT40 value (sum of 1-h mean ozone concentrations above a threshold of 40 nL·L–1) of 10 µL·L–1-h, calculated for 24 h a day during the 6-month growing season was slightly modified later on by using only daylight hours [25]. It is obvious that this threshold is exceeded every year over the USA and Europe [72], exposing trees to a potential phytotoxicity. It must however be kept in mind that phytotoxicity will depend on the ozone flux within the leaf cells which in turn will depend on stomatal opening linked to various climatic factors. Mixed conifer forests growing on the mountains surrounding the Los Angeles basin are effectively injured by ozone exposure and the visible symptoms of damage on the needles are described as a typical chlorotic mottle [46]. Ozone-induced visible injury was also demonstrated on fruit trees and conifers in the Mediterranean basin [3]. The situation is less clear-cut in western Europe [32]. In addition, inhibitions of photosynthesis and growth can happen in the absence of visible injury. In fact, ozone appears to be phytotoxic in two ways. Short-term exposures of trees to high ozone concentrations cause severe damages such as cell death, leading to the rapid appearance of a chlorotic mottle. Chronic long-term exposures to lower ozone concentrations may affect physiological processes prior to any development of premature senescence and visible symptoms of damage. The situation is made even more complicated since the sensitivity to ozone, generally lower for coniferous trees than for deciduous ones, depends also on the species (e.g. Scots pine is more sensitive than Norway spruce), on the provenance and on the clone [32]. The age of the needles is also of importance, the older needles being more sensitive to the pollutant [13, 33]. The developmental stage affects ozone-induced visible symptoms which generally appear after the maximum rate of leaf expansion [20]. In conifers, the delayed visible symptoms which follow the early biochemical events have been termed ‘carry-over effects’ or ‘memory effects’ [32, 61]. Except for rare field cases such as the clearly demonstrated ozone toxicity for Pinus ponderosa in California, it is difficult to link physiological and biochemical responses of adult trees to identified individual stresses. Therefore, most of the knowledge on the effects of ozone has been completed on young trees growing during months or years in controlled chambers (open-top chambers, OTC, and phytotronic chambers) in which the ambient air was filtered or supplemented with ozone, the final concentration being never higher than 200 nL·L–1.

This paper will focus on the biochemical processes linked to carbon metabolism in response to ozone attack, attempting to explain these events in the light of a signal chain system.

2. PHOTOSYNTHESIS Decreased rates of photosynthesis generally result from long-term chronic exposure of trees to realistic concentrations of ozone [7, 8, 36, 56, 78]. An example of an experiment made on loblolly pine trees [62] is given in figure 1. The photosynthetic response of the first 1987 flush needles of loblolly pine trees growing in OTC for 3 years in charcoal-filtered (CF) air or two times ambient ozone (2 × O3 = 92 nL·L–1, 12 h seasonal mean) was plotted as a function of intercellular CO2 (Ci). The stomatal resistance to CO2 diffusion decreases the CO2 concentration from 350 µmol·mol–1 outside (Ca) to Ci and the A-Ci curves provide a means to calculate the possible stomatal and biochemical limitations of photosynthesis resulting from a stress [12]. The curve for loblolly pine needles exposed to ozone was lower than that for filtered trees, with a 27 % decrease in photosynthesis at normal ambient CO2 concentration (Ca). It should also be noted that the calculated values of Ci were slightly different between the two treatments, with 233 and 253 µmol·mol–1 CO2 for respectively filtered and ozone-treated trees. However, the supply function (broken line), traced from Ca to the measured A values, and which corresponds to the rate of diffusion of CO2

Figure 1. Response of photosynthesis of the first 1987 flush needles to intercellular CO2 concentration in 3-year-old loblolly pine seedlings grown in charcoal-filtered or 2 × ambient ozone. The ambient CO2 concentration of 350 µmol·mol–1 is indicated as Ca and the arrows correspond to the intercellular CO2 concentrations (Ci) respectively for the filtered (233 µmol·mol–1) and the ozone treatment (253 µmol·mol–1). The broken line represents the supply function. Redrawn after Sasek and Richardson [62].

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from the atmosphere to the intercellular spaces (i.e. the stomatal conductance), was the same in the two conditions (figure 1). This means that the decreased photosynthetic rate in ozone-fumigated needles was not due to a higher stomatal resistance. A biochemical effect can thus be suspected, which is suggested by the 20 % decrease in the initial slope of the 2 × O3 curve, which could correspond to a decrease in the Rubiscomediated carboxylation efficiency [62]. Although a direct effect of the pollutant on stomata was sometimes suspected [83] with either an increase or decrease of stomatal resistance depending on the developmental age of the plant and measurement conditions [20], it appears that the decreasing effect of ozone on the photosynthetic rate is due to a decrease in carboxylation efficiency rather than changes in stomatal conductance [21, 40, 41, 49]. A decrease in the photochemicaldependent light-use efficiency is occurring at later stages of ozone exposure. It must be noticed, however, that ozone generally has a more pronounced decreasing effect on the photosynthetic rate of older needles, the current-year needles even showing a stimulation of photosynthesis suggesting a sort of compensatory process [79]. In deciduous trees, the detrimental effect of ozone on photosynthesis is generally more marked on mature leaves [49]. A transient stimulation of photosynthesis was even observed in young leaves of ozone-treated beech trees followed by a decrease later in the season [38]. This phenomenon was also observed in ozonetreated birch trees but only under low fertilization [11]. The effect of ozone could result in an accelerated maturation of the young leaves [79] linked to an increased nitrogen mobilization from old leaves [11]. It must also be noticed that the CO2 compensation point was shifted from 80 µmol·mol–1 in filtered loblolly pine needles to 105 µmol·mol–1 in ozone-treated needles (figure 1), suggesting an increase in respiratory loss.

3. RUBISCO ACTIVITY AND QUANTITY The initial slope of the A-Ci curve, indicative of carboxylation activity, decreases upon ozone exposure (figure 1), suggesting a decrease in the activity of the CO2-fixing enzyme of C3 plants, ribulose 1,5bisphosphate carboxylase/oxygenase (Rubisco). A parallel decrease in photosynthetic rate and Rubisco activity was thus demonstrated in loblolly pine needles (figure 2; [9]). The 3-year-old trees were placed in OTCs with CF air (8 nL·L–1 O3, 12 h daily mean), non-filtered (NF) ambient air (45 nL·L–1 O3, 12 h daily

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Figure 2. Photosynthesis (A) and Rubisco activity (B) expressed relative to the charcoal-filtered treatment in relation to the cumulative ozone exposure (12 h summation) for loblolly pine needles exposed to charcoal-filtered air (CF), non-filtered air (NF), and 2 × ambient ozone in the 1989 first flush (89-1), 1989 third flush (89-3) and 1990 first flush (90-1). Values are from experiments conducted each month from May to August 1990. R2 was 0.78 and 0.85 for the photosynthesis and Rubisco curves, respectively (taken from [9]). The original photosynthetic rates and the Rubisco activities were expressed on the same basis (needle surface area).

mean) and in 2 × ambient ozone (86 nL·L–1 O3, 12 h daily mean). Data from the various sampling dates obtained in 1990 from May to August on three different flushes (first and third flushes 1989 and first flush 1990) were plotted and expressed in terms of cumulative ozone exposure in ppm-h (µL·L–1-h). By mid-August 1990, the 1989 first flush had accumulated an ozone exposure of 380 µL·L–1-h in the 2 × O3 treatment compared with 125 µL·L–1-h in the CF treatment. In contrast, the younger 1990 first flush had only accumulated 120 µL·L–1-h in the 2 × O3 treatment and less than 20 µL·L–1-h in the CF treatment. There was a similar decline, relative to the CF treatment, observed for photosynthesis and Rubisco activity, with increasing cumulative ozone exposure (figure 2). A loss of Rubisco activity was also found in experiments carried out with realistic ozone fumigation of various deciduous and evergreen species:

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hybrid poplar [50], sugar maple [17], beech [38], birch [40], Norway spruce [1, 78], Scots pine [70] and Aleppo pine [14, 18]. Generally, over a season, the young leaves from ozone-treated trees show similar or higher Rubisco activities compared to the control whereas a decrease in Rubisco activity characterizes mature leaves, accompanied by a progressive appearance of visible symptoms [4, 38, 49]. A similar situation is found with young top leaves of deciduous ozone-treated trees which present a stimulated Rubisco activity compared to the filtered trees, followed by a progressive decrease with increasing leaf age [17, 30, 31]. In conifers, current-year needles of ozone-treated trees can show no change or a slightly stimulated Rubisco activity whereas there is a clear decrease in the older needle classes [13, 50]. Ozone generally induces a decrease in both the activity and quantity of Rubisco in various tree species: hybrid poplar [49, 50], Norway spruce [1] and Aleppo pine [14, 53]. As for Rubisco activity, the loss of Rubisco protein is more marked in mature than in young leaves of ozone-treated hybrid poplar [4, 49], a higher level of the enzyme protein even being observed in young leaves at the top of ozone-treated aspen seedlings compared to control ones [50]. Rubisco is made up of two types of subunits, designated as the large subunit (LSU) encoded by chloroplastic DNA and the small subunit (SSU) encoded by nuclear DNA. The quantity of LSU was found to be diminished in the foliage of trees submitted to ozone [4, 50, 53]. Decreased levels of rbcS (small subunit) and rbcL (large subunit) mRNA transcripts were reported in leaves of ozone-treated trees [1, 4, 35, 50, 53]. Figure 3 shows such reductions for rbcS in Norway spruce (figure 3A) and for rbcL in Aleppo pine (figure 3B) in response to ozone. It was recently shown that the quantity of Rubisco activase, implicated in the regulation of Rubisco synthesis, is decreased in 1-yearold needles of Aleppo pine trees submitted to ozone [53]. This result can be linked to a parallel decrease of the level of rca mRNA transcripts (figure 3B; [53]). Although ozone clearly reduces the amount of Rubisco and Rubisco activase, it is not yet totally clear whether the loss in transcripts is the result of mRNA degradation or reduced transcription [50]. In fact, the O3-induced decrease in Rubisco quantity may involve several mechanisms such as an increased rate of protease-mediated protein degradation, an alteration of the protein through an oxidative process or an inhibition of the protein synthesis [4, 23, 50]. These processes could occur together or separately, depending on ozone concentration, exposure time and leaf age [23].

Figure 3. Effect of ozone on Rubisco SSU and LSU and Rubisco activase mRNA quantities. A, Slot blots with the rbcS probe, over the duration of the ozone treatment; total RNA was extracted from 1-year-old Norway spruce needles fumigated with 200 nL·L–1 ozone (taken from [1]). B, Slot blots with the rbcL and rca probes; total RNA was extracted from 1-year-old Aleppo pine needles after 34 d of treatment with 200 nL·L–1 ozone (taken from [53]).

4. PHOSPHOENOLPYRUVATE CARBOXYLASE In C3 plants, the low activity of PEPc, a cytosolic enzyme, is thought to contribute to an anaplerotic pathway accounting for the fixation of respiratory CO2 and the replenishment of the tricarboxylic acid cycle ([34]; figure 4). A strong stimulation of PEPc activity is generally observed in ozone-treated trees compared to filtered ones (Scots pine, [37]; hybrid poplar, [31]; birch, [30]; Norway spruce, [68]; Aleppo pine, [14]; beech, [38]). In leaves of hybrid poplar exposed to ozone for 3 months, the PEPc activity was always two-fold increased compared to the control while the activity of Rubisco decreased only in the older leaves ([31]; figure 5). The same kind of results were obtained with birch trees grown in low nutrient regimes [30]. The ratio between Rubisco and PEPc activities, very high in leaves of trees growing in controlled conditions (about 20 to 25), is strongly diminished to reach between 1 and 5 in ozone-treated trees [14, 68]. The opposite behaviour of the activities of the two carboxylases over 3 months of treatment of Aleppo pine trees with 200 nL·L–1 O3 is shown in figure 6. The Rubisco activity was half reduced while the maximum PEPc activity was increased by about 4.5-fold in ozone-treated needles (figure 6A, total cumulative dose of ozone 146 µL·L–1-h). The effect of ozone on Rubisco

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Figure 4. Scheme of the pathways of carbon metabolism. AOX, alternative oxidase; CAD, cinnamyl alcohol dehydrogenase; COX, cytochrome oxidase; G6PDH, glucose 6-phosphate dehydrogenase; GlycOX, glycolate oxidase; HPR, hydroxypyruvate reductase; IDH, isocitrate dehydrogenase; MDH, malate dehydrogenase; ME, malic enzyme (NAD and NADP); PAL, phenylalanine ammonia lyase; PEPc, phosphoenolpyruvate carboxylase; PFK, phosphofructokinase; PFP, pyrophosphate fructose 6-phosphate phosphotransferase; PK, pyruvate kinase; SHDH, shikimate dehydrogenase; SPS, sucrose phosphate synthase; SuSy, sucrose synthase.

LSU and PEPc amounts is shown at 46 d in figure 6B in the form of protein bands reacting with the corresponding antibodies. There is a clear decrease in Rubisco LSU and an increase in PEPc quantities. This opposite effect of ozone on the two carboxylase

proteins is found throughout the duration of the treatment as well as the parallels between the changes in activity and quantity (figure 6A). Moreover, the rbcL and pepc mRNA quantities also vary under ozone in the same way as the amounts of proteins (figure 6C).

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Figure 5. Effect of ozone (40 nL·L–1 from 21:00 to 7:00 hours, 90 nL·L–1 from 7:00 to 21:00 hours, 7 d a week) on the activity of Rubisco and PEPc in the leaves of hybrid poplar (Populus × euramericana, var. ‘Dorskamp’). The leaf number 14 is the oldest one. Results are given as the ratio of the enzyme activity in ozone-treated trees relative to the activity in control trees. The original activities were expressed in µmol CO2·h–1·g–1 fresh weight (redrawn after [31]).

5. RESPIRATION It is widely admitted that the respiratory processes are increased in response to ozone [7, 8]. A rise in dark respiration, more marked in young than in old leaves, was thus demonstrated in hybrid poplar exposed to a realistic ozone concentration (figure 7, [56]). Ozone also increased dark respiration of Norway spruce needles [29], Scots pine shoots [70] and birch leaves [40]. In current-year and 1-year-old needles of Norway spruce trees, this increase in dark respiration was obtained after a long-term exposure (up to three growing seasons) to a low level of ozone (no more than 50 nL·L–1) without any visible symptoms of injury [29, 78].

Figure 6. Effect of ozone on the activity, quantity and amount of transcripts for Rubisco and PEPc in 1-year-old Aleppo pine needles. The 3-year-old trees were fumigated for 3 months with 200 nL·L–1 ozone. A, Effect of ozone on the activity of the two enzymes and on LSU and PEPc quantities all over the treatment (redrawn after [14]). Results are given as the ratios of the activities and quantities in ozone-treated trees relative to control trees. The original activities were expressed on a milligram protein basis. B, Western blots of Rubisco LSU and PEPc and C, slot blots with the rbcL and pepc probes at day 24 of the ozone treatment (taken from [13] and [53]).

6. PATHWAYS OF CARBOHYDRATE BREAKDOWN 6.1. Glycolysis and pentose phosphate pathway Compared to photosynthesis and its associated Rubisco enzyme, less information is available on the impact of ozone on the enzymes of the degradative pathways, which can be considered as possible targets for the pollutant [8]. The scheme in figure 4 shows the different pathways and the most studied enzymes. It is generally admitted that, aside from the strong stimulation of PEPc activity, there is a general enhancement in both the glycolysis-TCA cycle and pentose phos-

Figure 7. Effect of ozone on dark respiration of hybrid poplar leaves (redrawn after Reich [56]).

phate pathway, this latter showing a more marked increase ([8, 9, 13, 68]; figure 8). The pentose phosphate pathway enzyme, glucose 6-phosphate dehydro

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genase (G6PDH) is thus known to show an enhanced activity in leaves of trees fumigated with ozone [8, 9, 13, 68]. The activity of G6PDH is enhanced more than that of the glycolytic enzyme, phosphofructokinase (PFK) (figure 8A; [8, 68]). No clear change was detected in the activity of pyrophosphate fructose 6-phosphate phosphotransferase (PFP) [8, 68]. An increased pyruvate kinase (PK) activity was observed in current-year and 1-year-old needles of Aleppo pine trees fumigated with 200 nL·L–1 ozone during the 14-h photoperiod [13]. Generally, the activities are more enhanced in 1-year-old than in current-year needles [13]. It nevertheless must be noticed that a number of enzymes are multicompartmented (G6PDH, PFK, maybe PK), which renders it difficult to know exactly which part of the change can be attributed to the cytosolic and chloroplast isoforms. It appears that the shunt initiated by PEPc and leading to malate (figure 4) is more stimulated than the classical glycolytic pathway delivering pyruvate. An increase in the activity of the NADP malic enzyme (NADP-ME) was also demonstrated in needles of ozone-treated Aleppo pine [13] which, in addition to the increased G6PDH activity, could help furnish NADPH in the cytosol for detoxification and repair (see below). Finally, although this process accounts for a great loss of fixed CO2 in C3 plants, the response of photorespiration to ozone has been barely studied. The decreased activity of both glycolate oxidase and hydroxypyruvate reductase observed in current-year needles of Norway spruce fumigated with 200 nL·L–1 ozone could indicate a lower photorespiratory activity (figure 8B). This result is in accordance with the decrease in mRNA for the peroxisomal catalase observed in ozone-treated tobacco [82].

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Figure 8. Effect of ozone (200 nL·L–1) on the activity of several enzymes of carbohydrate breakdown in current-year needles of Norway spruce trees (A, redrawn after Sehmer et al. [68]) and on the activity of photorespiratory enzymes in current-year needles of Aleppo pine trees (B, Martin, Dizengremel and Gérant, unpubl.). The results are given as the ratios of the activities in ozone-treated trees relative to control trees. The original activities were expressed on a milligrams protein basis. G6PDH, glucose 6-phosphate dehydrogenase; GlycOX, glycolate oxidase; HPR, hydroxypyruvate reductase; PEPc, phosphoenolpyruvate carboxylase; PFK, phosphofructokinase.

6.2. Mitochondria and oxidation of organic acids Pyruvate and malate can be transferred into the mitochondria and, whereas pyruvate enters the TCA cycle, malate can be first decarboxylated into pyruvate by NAD malic enzyme (NAD-ME; figure 4), thus providing NADH to the matrix. An increase in NAD-ME activity was demonstrated in needles of ozone-treated Norway spruce [8] and Aleppo pine [13, 18]. The activity of the TCA cycle enzyme, fumarase, was also found to be slightly enhanced in trees submitted to realistic concentrations of ozone (figure 8A; [9, 68]). An increased isocitrate dehydrogenase (IDH) activity was also shown in needles of Norway spruce trees fumigated with ozone [8, 13]. This gives an increased reducing power, mainly in the form of

NADH, which will then be reoxidized by the respiratory chain allowing the production of ATP through the oxidative phosphorylation process. It must however be noted that several IDH isoforms are located in different compartments in plant cells. In plant mitochondria, a non-phosphorylating pathway, terminating with a quinol oxidase (alternative oxidase, AOX) exists in parallel with the cytochrome phosphorylating pathway, terminated with cytochrome oxidase. A slight stimulation of cytochrome oxidase activity was observed in needles of 4-year-old Scots pine trees fumigated for 6 weeks with 100 nL·L–1 ozone [37]. An increased transcription of a mitochondrial phosphate translocator was demonstrated in leaves of birch trees

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exposed for 8 h to 150 nL·L–1 ozone [26]. More recently, a net increase in the level of mRNA transcripts for AOX was found in leaves of poplar trees treated with 100 nL·L–1 ozone for 4 weeks [19].

7. SECONDARY METABOLISM Ozone induces an accumulation of polyphenolic compounds, flavonoids and cell wall-bound polyphenols such as lignins, in various tree species [32]. The first part of the phenylpropanoid pathway is formed by the shikimic acid pathway which links the primary carbon metabolism to the secondary one (figure 4). The first step is the condensation of erythrose-4phosphate from the pentose phosphate pathway and PEP from glycolysis, leading to the amino acid phenylalanine. The activity of the shikimate dehydrogenase (SHDH) was found to be increased in Scots pine and poplar trees fumigated with ozone [35, 37]. Phenylalanine is further transformed in cinnamoylCoA, a precursor at the start of three important pathways leading to the production of stilbenes, catechin and lignins. The activity of phenylalanine ammonia lyase (PAL) is known to be increased in trees submitted to ozone [24, 32, 35]. The activities of the biosynthetic enzymes, stilbene synthase (STS), pinosylvin methyl transferase (PMT) and chalcone synthase (CHS, on the way to catechin), are stimulated by ozone in Scots pine [84]. The exposure of Norway spruce and hybrid poplar trees to sub-acute doses of ozone produced a strong increase in the activity of cinnamyl alcohol dehydrogenase (CAD; [32, 35]). For several of these enzymes (PAL, STS, PMT, CAD), the ozone-dependent increased activity could be related to an increase at the transcript level [5, 16, 32, 35, 76, 84]. The biosynthesis of polyamines, mainly putrescine, is known to be increased upon ozone exposure [24, 32]. These compounds could play an indirect role in preventing membrane lipid peroxidation, acting as scavengers of oxygen radicals [24, 32]. The production of the wounding compound ethylene is also stimulated upon ozone exposure [45] and, since ethylene and polyamines share a common precursor, S-adenosylmethionine, there could be a competition between the two biosynthetic pathways, linked to greater or lesser tolerance of the tree to the pollutant [21, 32]. In fact, ethylene and polyamines are mutually repressing enzymes of their respective biosynthetic pathways [24, 64]. Ethylene could also act as a second messenger molecule, inducing defence gene expression [24, 60].

8. CARBON METABOLISM AND OZONE: A SUMMARY It clearly appears that, before the occurrence of any visible symptoms of injury (for a cumulative dose of external ozone varying, according to the species, from around 50–70 µL·L–1-h for the sensitive poplar to 150–200 µL·L–1-h for the more tolerant Norway spruce), the carbon metabolism is deeply modified (figure 9). There is a loss of carbon fixation through Rubisco and problems later at the level of the photochemical reactions (pigments, for example, with a fall in cab mRNA level [16, 51, 84]). The functioning of the Calvin cycle is progressively reduced along with the decrease in enzyme activity (mainly Rubisco) as well as a reduction in the available NADPH coming from photochemical reactions since a large part of this NADPH is used for detoxifying processes [8]. The reducing power is thus used to trigger the ascorbate peroxidase-glutathione reductase pathway reducing hydroperoxide derived from the superoxide dismutase

Figure 9. Implication of pathways and enzymes of carbon metabolism in trees exposed to ozone. AOX, alternative oxidase; CAD, cinnamyl alcohol dehydrogenase; CHS, chalcone synthase; COX, cytochrome oxidase; G6PDH, glucose 6-phosphate dehydrogenase; IDH, isocitrate dehydrogenase; ME, malic enzyme (NAD and NADP); PAL, phenylalanine ammonia lyase; PEPc, phosphoenolpyruvate carboxylase; PFK, phosphofructokinase; PFP, pyrophosphate fructose 6-phosphate phosphotransferase; PK, pyruvate kinase; PPP, pentose phosphate pathway; PMT, pinosylvin methyl transferase; SHDH, shikimate dehydrogenase; STS, stilbene synthase. Dashed arrows: inhibition; continuous arrows: stimulation, more and more marked according to the thickness of the arrow.

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(SOD) activity. The detoxifying system, known to be present in plant cell compartments including chloroplasts, is activated upon ozone exposure [2, 24, 47, 82]. Total SOD activity is thus increased in ozonetreated birch [76] and Norway spruce trees [66]. The chloroplastic Cu,Zn SOD isoform identified in Norway spruce trees [67] was found to be the more responsive isoform in ozone-treated trees [68]. The chloroplastic SOD isoform was also found to be implicated in ozone stress response in tobacco overexpressing SOD [77]. It is however to be noted that conflicting results exist about an increased SOD activity in ozone-exposed trees [21, 57]. The synthesis of sucrose is also diminished and sucrose degradation is favoured, as demonstrated in mature leaves of ozone-treated birch trees, which showed a reduction in sucrose phosphate synthase activity linked to an increased level of fructose 2,6-bis phosphate and an increased activity of sucrose synthase and alkaline invertase [11]. A reduction in sucrose export is also known to occur in ozone-treated plants [31, 71, 80]. In addition, the resulting accumulation of carbohydrates in leaves could inhibit the photosynthetic process [28] and stimulate the carbon degradative pathways. An increased carbohydrate breakdown, mainly from sucrose, is known to result from ozone exposure, and, in connection with a decreased production of photosynthates, this could ultimately lead to a disequilibrium at the cell level [8]. The PEPc-linked anaplerotic pathway is favoured as well as the non-phosphorylating mitochondrial pathway, suggesting at least in part (PFP activity is not modified) an avoidance of adenylate control. It could also be questioned whether there is a possible compensatory role for PEPc in ozone-stressed leaves, which could participate in the fixation of atmospheric CO2 besides the refixation of respiratory CO2 (the increase in PEPc activity is higher than the enhancement in respiration). The enzymes delivering NADPH (pentose phosphate pathway and NADPmalic enzyme) are also stimulated to provide reducing power for detoxification processes and the functioning of dehydrogenases (e.g. CAD) linked to lignin synthesis. NADPH could also be used by the cell wall NADPH oxidase which delivers the superoxide ion ([52]; see below). There is a general increase in mitochondrial metabolism. The increased level in AOX could serve to limit the production of reactive oxygen species (ROS) in mitochondria [42, 54] whereas a stimulated phosphate translocator could be related to energy production needed for biosynthetic activities [26]. An enhanced flux from PEP and erythrose

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4-phosphate towards the shikimate and phenylpropanoid pathways leads to the production of protective compounds, including lignins. In brief, there is a general modification of the primary and secondary carbon metabolisms under ozone exposure for detoxification, protection and repair, at the expense of growth.

9. MECHANISM OF OZONE ACTION The exact means by which ozone exerts its oxidative stress on trees has still not been entirely elucidated [21, 51]. A putative mechanism is proposed in the scheme of figure 10. This scheme, inspired from different papers dealing with plant defence mechanisms [22, 60, 64, 69], although not differentiating

Figure 10. Possible cellular mechanisms of ozone-induced defence reactions in trees. Aox, alternative oxidase; APX, ascorbate peroxidase; Cab, chlorophyll a/b-binding protein; Cad, cinnamyl alcohol dehydrogenase; CAT, catalase; EC-POD, extracellular peroxidase; EC-SOD, extracellular superoxide dismutase; GR, glutathione reductase; JA, jasmonic acid; Pal, phenylalanine ammonia lyase; Pepc, phosphoenolpyruvate carboxylase; RbcL, RbcS, large and small subunits of Rubisco; Rca, Rubisco activase; ROS, reactive oxygen species; SA, salicylic acid; Sts, stilbene synthase.

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between ozone-tolerant and -sensitive species, underlines the biochemical events and the probable sequence of signals from the pollutant attack to the gene response. Ozone diffuses through the stomata in the intercellular spaces and reaching the cells, is decomposed, both in the apoplasm and the rest of the cell, in toxic ROS such as hydrogen peroxide (H2O2), superoxide ion (O2–) and hydroxyl radical (OH·) [21, 24]. The release of ROS can also be an active process mediated through a plasma membrane-linked NADPH oxidase (giving O2–) and extracellular/apoplastic peroxidases delivering H2O2, as shown in ozone-treated birch trees [52]. An extracellular Cu,Zn SOD, associated with the NADPH oxidase could dismutate the superoxide ion in hydroperoxide [52, 73]. The oxidative burst also comprised a diminution of the plasma membrane K+-stimulated ATPase and an alteration of the Ca2+transport system, resulting in an increased Ca2+ influx [21]. Based on the analogies recently emphasized between plant responses to ozone and pathogen defence reactions [24, 60, 69], Heath and Taylor [21] proposed that the increased flow of Ca2+ into the cytoplasm could activate a protein kinase triggering the plasma membrane NADPH oxidase activity. ROS themselves (mainly H2O2) as well as several molecules (salicylic acid, jasmonic acid and ethylene) have been proposed in signal transduction pathways for plant defence reactions and could serve as secondary messengers to change the gene expression through transcription factors [24, 48, 60, 69]. The exact mechanisms and possible interactions between these molecules and pathways are not yet clear. Salicylic acid (SA) could be implicated since it is known that this molecule, which is synthesized via the phenylpropanoid pathway, is implicated in ozone-induced plant defence [55]. An increase in the amount of SA was thus observed in leaves of poplar trees fumigated with ozone [19, 27]. ROS could stimulate the production of both SA and jasmonic acid (JA), the JA- and SA-signalling pathways contributing, sometimes in an antagonistic way, to the control of the cellular response to ozone stress [55]. In fact, ozone activates multiple interactive pathways [27, 48, 55]. Although ozone does not penetrate into the chloroplast, an oxidative stress occurs with a rather strong deleterious effect on photosynthetic functions, which is exerted (without excluding an increased degradation of mRNAs) via a decreased level of transcripts of chloroplast (cab, rbcL) and nuclear (rbcS, rca) genes. The detoxifying system is stimulated in chloroplasts as well as in the cytoplasm, involving an induced tran-

scription of antioxidative enzymes [59]. Many other transcripts for enzyme proteins are stimulated through the signal transduction pathway(s), including enzymes of primary metabolism (among them, PEPc, AOX, SHDH) and secondary metabolism (phytoalexins- and flavonoids-synthesizing enzymes, enzymes for lignin biosynthesis). There are often multiple genes coding for these proteins and the exact mode of action of transcription factors on the promoters of these genes is far from being totally known. The question of the specificity of an ozone-dependent cascade of signals has not been elucidated since other stresses (in particular, pathogen attack) may activate the same, or overlapping signal transduction pathways. There is, however, some selective induction of stress-related proteins isoforms, at the level of genes coding for enzymes of ethylene biosynthesis and peroxidases [65]. Moreover, some ozone-responsive regions of enzyme promoters could be different from other stressresponsive sequences (i.e. pathogen attack) [66]. This scheme is general and it is clear that differences in the cascade of signals can exist, allowing one to explain the differences in sensitivity of trees to ozone, for example at the level of ethylene emission [45, 64] or salicylic acid production [27, 55]. Ozone sensitivity can also be correlated with the absence of a SA- and JA-mediated signal transduction pathway [27]. In addition, there is a strong difference between deciduous trees and conifers, these latter trees showing a significant delay between the first ozone-induced biochemical events and the appearance of visible symptoms of injury [32].

10. CONCLUSION Although the model is not yet totally clear, the series of biochemical events resulting from a chronic exposure of trees to realistic concentrations of ozone are more and more correctly described. More work is necessary to gain a better understanding of the implication of the different signalling pathways including the alterations in gene expression. The problem of the specificity of ozone-response processes must also be solved in this respect as well as the cellular bases of the differences in sensitivity to the pollutant. An overall integrated biochemical and physiological view of the responses of carbon metabolism to ozone in tree leaves therefore emerges with a net increase in carbohydrate breakdown at the expense of photosynthate production. The sucrose translocation is impaired resulting in a modified partitioning of carbon above

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and below ground, with root growth more affected than shoot growth [6, 7, 18, 21, 41, 71, 79, 80]. The interaction of ozone with other abiotic and biotic stresses can also modify the response of the trees. There have been many works on the combination of ozone and water stress, which is the major recurrent stress in forest ecosystems. Generally, drought stress was shown to protect the tree against ozone injury [10, 21, 36, 75] even though some contradictory results exist [18, 58]. In fact, the closure of stomata could reduce the amount of fixed carbon and ultimately lead to decreased growth [3, 18]. Concerning the interactions between ozone and pathogens, as mentioned above, the two stresses cause similar responses and controlled experiments gave contradictory results with either an ozone-induced resistance to pathogens or an increased disease [32, 39, 59]. The other important combination, namely elevated ozone and CO2, also has to be considered with the prediction that co-exposure could restrict the expected gains from high CO2 level and increase the damages due to ozone [44]. The results obtained in controlled conditions with trees (often young trees or seedlings) submitted to ozone alone, although necessary, are not sufficient to address the question of the possible impact of this pollutant as concerns forest stands. The possibility of using a biochemical molecule as a biomarker for the presence of an ozone stress is unrealistic, even though the strong PEPc activity increase in ozone-treated trees has been suggested to serve as an indicator of air pollution in forest stands [81]. Chronic levels of ozone, present on a local or regional scale are supposed to affect trees individually and it is rather difficult to assess the impact on the health of forest ecosystems. Many parameters must be taken into account such as the time scale of the responses of trees to ozone which differ between deciduous trees (and sensitive conifers) and tolerant conifers, which can be of importance in mixed forests, the differences in age of the trees, the spatial and temporal distribution of the pollutant, the interactions with other biotic and abiotic stresses. Even though it is clear that ozone can be a causative factor of forest damages in several regions of the world [63], particularly in western and southern USA and the Mediterranean basin [15, 43, 44, 46], the points mentioned above must be carefully considered [21, 32]. The good knowledge gained on the biochemical and physiological responses of individual trees to ozone must be reinforced by deeper insights into the signal transduction/gene expression processes. This will facili-

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tate the necessary integration from the molecular to the cellular and whole plant levels. In this respect, the UN/ECE defined AOT40 yearly threshold of 10 µL·L–1-h for forest damage is not well adapted since the ozone cumulative dose is very often 2 to 4 times higher [41]. Furthermore, the exact actual dose of ozone reacting within the leaf must be considered rather than the external dose of the pollutant in the surrounding air [21, 41, 74]. The most probable risk for a forest tree will be a lower resource acquisition and a disturbed carbon allocation with a probable root growth restriction and reduction of tree growth. This could lead to a lower competitive power towards the neighbouring trees, ultimately affecting biodiversity [41]. Finally, to integrate the different levels of study of ozone effects, cellular biochemistry, leaf physiology, whole tree growth, forest ecosystem viability and the different possible interactions, it is necessary to use process-based models [21]. It would also be necessary, however to include more information on the below ground system and to extend the individual model to the stand.

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