Plant Physiol. (1998) 117: 565-574

Overexpression of Iron Superoxide Dismutase in Transformed Poplar Modifies the Regulation of Photosynthesis at Low CO₂ Partial Pressures or Following Exposure to the Prooxidant Herbicide Methyl Viologen¹

Ana-Carolina M. Arisi, Gabriel Cornic, Lise Jouanin, and Christine H. Foyer^*

Laboratoire de Biologie Cellulaire and Laboratoire du Metabolisme, Institut National de la Recherche Agronomique, Route de Saint Cyr, 78026 Versailles cedex, France (A.-C.M.A., L.J.); Laboratoire d'Ecologie Végétale, Groupe Photosynthèse et Environnement, Unité de Recherche Associée, Centre National de la Recherche Scientifique 2154, bât 362, Université de Paris XI, F-91405 Orsay cedex, France (A.-C.M.A., G.C.); and Department of Environmental Biology, Institute of Grassland and Environmental Research, Plas Gogerddan, Ceredigion SY23 3EB, United Kingdom (C.H.F.)

	ABSTRACT

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Chloroplast-targeted overexpression of an Fe superoxide dismutase (SOD) from Arabidopsis thaliana resulted in substantially increased foliar SOD activities. Ascorbate peroxidase, glutathione reductase, and monodehydroascorbate reductase activities were similar in the leaves from all of the lines, but dehydroascorbate reductase activity was increased in the leaves of the FeSOD transformants relative to untransformed controls. Foliar H₂O₂, ascorbate, and glutathione contents were comparable in all lines of plants. Irradiance-dependent changes in net CO₂ assimilation and chlorophyll a fluorescence quenching parameters were similar in all lines both in air (21% O₂) and at low (1%) O₂. CO₂-response curves for photosynthesis showed similar net CO₂-exchange characteristics in all lines. In contrast, values of photochemical quenching declined in leaves from untransformed controls at intercellular CO₂ (Ci) values below 200 µL L¹ but remained constant with decreasing Ci in leaves of FeSOD transformants. When the O₂ concentration was decreased from 21 to 1%, the effect of FeSOD overexpression on photochemical quenching at limiting Ci was abolished. At high light (1000 µmol m² s¹) a progressive decrease in the ratio of variable (F_v) to maximal (F_m) fluorescence was observed with decreasing temperature. At 6^oC the high-light-induced decrease in the F_v/F_m ratio was partially prevented by low O₂ but values were comparable in all lines. Methyl viologen caused decreased F_v/F_m ratios, but this was less marked in the FeSOD transformants than in the untransformed controls. These observations suggest that the rate of superoxide dismutation limits flux through the Mehler-peroxidase cycle in certain conditions.

	INTRODUCTION

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During pseudocyclic electron flow O₂ is reduced to superoxide in the Mehler reaction (Mehler, 1951). Efficient elimination of this radical at the site of production on the thylakoid membrane is required to prevent interaction with other electron-transport components (Richter et al., 1990; Miyake and Asada, 1994). SODs (EC 1.15.1.1), which rapidly convert superoxide to H₂O₂, are found on the thylakoid membrane and in the chloroplast stroma (Jackson et al., 1978; Asada, 1994). Three types of SODs, defined by their component metal prosthetic groups (Mn, Fe, and CuZn), are found in leaves (Bowler et al., 1994; Scandalios, 1997). Chloroplasts contain CuZnSODs (encoded by the nuclear sodCp gene) and FeSODs, which are coded by nuclear sodB genes (Jackson et al., 1978; Bowler et al., 1994; Van Camp et al., 1994; Scandalios, 1997). An early report of a thylakoid-bound MnSOD has never been substantiated (Hayakawa et al., 1985). FeSOD is a highly hydrophillic protein indicating localization in the chloroplast stroma, but Van Camp et al. (1996) suggested that it could also associate with the thylakoid membranes. The presence of both FeSOD and CuZnSOD isoforms in the chloroplasts could be a physiological advantage in stress conditions because of the different characteristics of the enzymes (Droillard and Paulin, 1990; Scandalios, 1997). FeSOD expression appears to be tightly linked to photosynthetic activity (Kurepa et al., 1997). Whereas FeSOD transcripts were most abundant in young tobacco leaves, highest SOD activities were found in mature and senescent leaves (Kurepa et al., 1997). Application of the herbicide paraquat (MV) increased sodB mRNA abundance, whereas prolonged darkness decreased FeSOD transcripts in tobacco leaves (Tsang et al., 1991).

SOD has been used many times for overexpression studies in plants (for reviews, see Foyer et al., 1994; Allen, 1995) largely because various SOD cDNAs have been available for nearly a decade (Perl-Treves et al., 1988; Scioli and Zilinskas, 1988; Karpinski et al., 1992), whereas cDNAs for other antioxidant enzymes have only recently become available. Studies have addressed the overproduction of CuZnSOD (Tepperman and Dunsmuir, 1990; Perl et al., 1993; Sen Gupta et al., 1993a), of MnSOD (McKersie et al., 1993, 1996; Bowler et al., 1994; Foyer et al., 1994; Van Camp et al., 1994; Slooten et al., 1995; Payton et al., 1997), or of FeSOD (Van Camp et al., 1996) in the chloroplasts. With one exception (Tepperman and Dunsmuir, 1990) SOD overproduction resulted in enhanced tolerance to oxidative stress. Overproduction of FeSOD in tobacco chloroplasts was more effective in protecting against MV-induced damage than overproduction of MnSOD (Van Camp et al., 1996). The mechanism by which SOD-dependent increases in stress tolerance are achieved, however, is unknown, since SOD activity has never been considered to be a constraint on flux through the Mehler-peroxidase cycle (Schreiber and Neubauer, 1990; Miyake and Asada, 1994; Foyer, 1997).

Relationships between O₂ metabolism and photosynthesis are complex, and hypotheses on the role of O₂ reduction in the regulation of electron transport are frequently controversial (Osmond and Grace, 1995). The knowledge that H₂O₂ is a signal-transducing molecule in plant cells prompted Prasad et al. (1994) to suggest that benefits caused by SOD overexpression may be due to enhanced H₂O₂ production. To explore the relationships between SOD activity and photosynthesis, we have expressed an FeSOD cDNA from Arabidopsis thaliana in the chloroplasts of poplar (Populus tremula × Populus alba) and compared photosynthetic performance in untransformed controls and in FeSOD transformants. The results presented here suggest that FeSOD overexpression can protect PSII from overreduction in situations when intracellular CO₂ is depleted and from MV-induced photoinhibition. A clear role for O₂ photoinhibition is demonstrated, but FeSOD does not appear to contribute to protection against decreases in PSII efficiency in these circumstances.

	MATERIALS AND METHODS

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Transformation and Growth Conditions

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Detection of SOD Activity on Gels following IEF Gels

Enzyme Extraction and Analysis

H₂O₂, Ascorbate, and Glutathione Determinations

Gas-Exchange and Chlorophyll a Fluorescence Measurements

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High-Light and Low-Temperature Treatments

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MV Treatments

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	RESULTS

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Hybrid poplars overexpressing an FeSOD cDNA from A. thaliana were obtained by A. tumefaciens-mediated transformation using the binary plasmid pEXSOD10 (Van Camp et al., 1996), enabling constitutive expression in the chloroplast. After kanamycin selection, screening of the transformed lines was performed by RNA/DNA hybridization of total RNA from leaves of plants cultivated in vitro. Whereas sodB mRNA was not detectable in total RNA fractions extracted from WT, it was identified in certain kanamycin-resistant lines. The lines expressing sodB mRNA were multiplied in vitro, transferred to soil, and placed in the greenhouse. Five independent, FeSOD-overexpressing lines were obtained containing either one (lines 4SOD, 6SOD, 8SOD, and 9SOD) or two copies of inserted T-DNA (line 5SOD).

SOD isoforms were detected by SOD-activity staining of soluble leaf proteins on polyacrylamide gels following IEF (Fig. 1) in the absence or in the presence of the SOD inhibitors CN and H₂O₂. In the absence of either of these inhibitors, FeSOD transformants contained one band of SOD activity in addition to those observed in the untransformed controls (Fig. 1A). Untransformed poplar leaves did not contain detectable FeSOD activity. For effective comparison of relative activities of the SOD isoforms present in leaf extracts, the wells containing FeSOD-transformant leaf extracts had to be loaded with 10 times less protein than those from untransformed controls (Fig. 1). When gels were loaded with equal quantities of total soluble protein, the FeSOD activity band in the transformants was so intense that it masked all other SOD bands (data not shown). The additional band in the transformants was sensitive to inhibition by H₂O₂ (Fig. 1C) but was not inhibited by KCN (Fig. 1B), characteristic of an FeSOD. Furthermore, an FeSOD band with the same pI (approximately 5.20) as the additional SOD band was found in soluble leaf-protein extracts from A. thaliana (At; Fig. 1A). In untransformed poplar and in A. thaliana, closely running KCN-sensitive bands with a pI value of approximately 4.55 were observed (Fig. 1A). These may be isoforms of Cu/ZnSOD, since they were inhibited by KCN. In transformed poplar extracts containing 5 µg of total protein, these Cu/ZnSOD bands were below detection level.

View larger version (80K): [in this window] [in a new window]   Figure 1. Polyacrylamide gels stained for SOD activity following IEF in the absence (A) or presence of the inhibitors CN (B) and H2O2 (C). The top gels show a comparison between SOD activity from A. thaliana leaves (At), WT, and three lines of FeSOD-transformed poplars (4SOD, 5SOD, and 6SOD). The bottom gels show FeSOD transformants 8SOD and 9SOD in addition to At, WT, and 4SOD. Extracts were loaded on a total soluble protein basis (5 µg per well for extracts from transformed poplar leaves, 50 µg per well for WT leaves, and 35 µg per well for A. thaliana leaves).

The maximal extractable SOD activity of untransformed leaves varied with the time of year when samples were harvested (compare, for example, maximal extractable SOD activities for untransformed controls in Table I and Fig. 2). In comparison, the maximal extractable SOD activity from FeSOD transformants was more constant. As a result, extractable foliar SOD activity of FeSOD transformants from line 6 SOD was at maximum 7 times higher than that found in the leaves of untransformed controls (Table I) and from 2 to 3 times higher even when values for untransformed controls were highest (Fig. 2). The untransformed control line with the highest SOD activity was chosen for the study of SOD foliar distribution (Fig. 2). High SOD activity was maintained in the leaves of the FeSOD transformants 1 year after in vitro micropropagation (data not shown). Maximal extractable SOD activities were similar in leaves from different positions on the stem (Fig. 2). SOD activity varied slightly with leaf age in transformed poplars and WT, but was always substantially higher in the former than in the latter. SOD activities assayed by an alternative method gave comparable results (Beyer and Fridovich, 1987). Similar to SOD, seasonal variations were observed in foliar APX and MDHAR activities, but they were always similar in all lines of plants regardless of the SOD activity (Table I). Similar results were obtained in plants grown either in the greenhouse or in controlled-environment chambers (data not shown). GR activity was also similar in transformed (34.8 ± 1.5 nmol min¹ mg¹ protein) and untransformed plants (33.9 ± 1.8 nmol min¹ mg¹ protein). Of the antioxidant enzymes measured, DHAR was the only enzyme in which the activity was increased in the FeSOD transformants compared with the untransformed controls (Table I). Increases in foliar DHAR were not significant, however, at any time of the year.

View larger version (9K): [in this window] [in a new window]   Figure 2. The relationship between the maximal extractable SOD activity of leaves and their position on the stem of WT (open bars) and transformed poplars overexpressing FeSOD (line 6 SOD [closed bars]). Position 1 contained the youngest green leaf on the stem, whereas leaf 16 was the oldest mature leaf. Leaf 3 was the youngest mature leaf on the stem when leaf 16 showed no sign of senescence, such as decreased total leaf-extractable soluble protein. U, Units; prot, protein.

The maximal extractable SOD activity was similar in lines 4SOD, 6SOD, 8SOD, and 9SOD, and values for all physiological parameters were similar in all of these lines. The data presented in Figures 3 9 therefore represent the mean values of measurements of plants from all of these transformed lines. It is important to note, however, that we have data records of each line used in the following experiments.

H₂O₂, ascorbate, and total glutathione contents of leaves were similar in FeSOD transformants and in untransformed controls both at the end of the dark period and in the middle of the light period (Table II). The foliar H₂O₂ pool was similar in the dark and in the light. Total foliar glutathione increased slightly during the photoperiod in both control and transformed plants. The percentage reduction of the glutathione pool was similar in all conditions in leaves of all lines. Total foliar ascorbate was similar during the light and dark periods, but the percentage reduction of the ascorbate pool decreased during the photoperiod in all lines (Table II).

Photosynthesis

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View larger version (25K): [in this window] [in a new window]   Figure 3. Light (A)- and CO2 (B)-response curves for chlorophyll a fluorescence quenching parameters and net CO2 uptake by leaves from WT () and poplar overexpressing FeSOD (). A, CO2 concentration was 350 µL L1; B, PPFD was 590 µmol m2 s1. Values are means ± SE of three (A) or four (B) individual plant measurements.

The CO₂-response curves for photosynthesis show that net CO₂ uptake was similar in all lines of plants (Fig. 3B). However, q_P values were always highest in the FeSOD transformants (Fig. 3B; third panel from top). Whereas q_P values declined in leaves of untransformed controls at below 200 µL L¹, they remained relatively constant in the leaves of untransformed controls under these conditions (Figs. 3B and 4A). The decline in q_P values at low Ci measured in WT (P < 0.001 for Ci 90 µL L¹) are related to differences in F/F_m and F_v/F_m (Genty et al., 1989). A slight but consistent difference between leaves of FeSOD transformants and untransformed controls was found for both of these parameters under low Ci (Fig. 3B; top two panels). F/F_m values were higher and F_v/F_m values were lower in the FeSOD plants than in the untransformed controls under comparable conditions (Fig. 3B; top two panels). When the O₂ concentration of the air was decreased from 21 to 1% to decrease photorespiration (Fig. 4), the effect of Fe-SOD overexpression on q_P at limiting CO₂ was abolished, with q_P decreasing with decreasing Ci in all lines (Fig. 4).

View larger version (12K): [in this window] [in a new window]   Figure 4. The relationship between Ci and qP in leaves of WT () and transformed poplars overexpressing SOD () in air containing 1% O2. The mean values ± SE for three individual untransformed poplar leaves and three FeSOD transformants are given. PPFD was 590 µmol m2 s1.

The relationships between quantum yield of electron transport and quantum yield of CO₂ assimilation determined by the sum of CO₂ assimilation and CO₂ evolution due to respiration explored under nonphotorespiratory conditions (1% O₂) were similar in all lines of plants (Fig. 5). This shows that F/F_m can be used as a relative measure of whole-chain electron transport to compare untransformed and transformed lines.

View larger version (17K): [in this window] [in a new window]   Figure 5. The relationship between PSII and CO2 in WT (, ) and poplar overexpressing FeSOD (, ) measured under nonphotorespiratory conditions, 1% O2. Measurements were obtained with either varying irradiance (, ) or varying CO2 partial pressures (, ). Each point represents an individual measurement.

High-Light Treatments

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View larger version (17K): [in this window] [in a new window]   Figure 6. Net CO2 uptake by WT () and transformed poplar () leaves after a 2-h high-light (1100 µmol m2 s1) treatment at different temperatures in 350 µL L1 CO2, 21% O2. Each point is an independent measurement on a different plant.

View larger version (17K): [in this window] [in a new window]   Figure 7. The effect of temperature during a high-light treatment on Fv/Fm ratios measured in leaves of WT ( and white bars) and poplar overexpressing FeSOD ( and black bars). In A, the O2 concentration of the surrounding air was maintained at 21%, whereas in B, the O2 concentration was maintained at either 21 or 1% throughout the duration of the high-light treatment (2 h). Measurements of Fv/Fm were made on leaves maintained in the dark at 24°C for 1 h (, ) or on leaves subjected to 2 h of high light and temperatures ranging from 6 to 24°C (squares or bars) and then allowed to recover in darkness for a further 2 h. Each point reflects a measurement on a different plant.

In contrast, when these experiments were conducted at low (1%) O₂, instead of air, the low-temperature-induced decrease in F_v/F_m ratios at high light was much less than that observed in air (Fig. 7B). In all cases, the decrease in the F_v/F_m ratio in these conditions was similar in all poplar lines (Fig. 7).

MV Treatments

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View larger version (18K): [in this window] [in a new window]   Figure 8. CO2-dependent O2 evolution by leaf discs from WT (white bars) and poplar overexpressing FeSOD (gray bars) incubated overnight in the presence of different concentrations of MV and then illuminated for 30 min in high light (1100 µmol m2 s1) and saturated CO2 (5%). Values are the mean ± SE of four separate experiments.

View larger version (16K): [in this window] [in a new window]   Figure 9. Dark-adapted Fv/Fm ratios measured on leaf discs from WT (white bars) and poplar overexpressing FeSOD (black bars) incubated overnight in the presence of different concentrations of MV and then illuminated for 2 h in high light (1100 µmol m2 s1) and saturating CO2 (5%). Leaf discs were then allowed to recover in darkness for 2 h prior to Fv/Fm measurement. The Fv/Fm values measured at the end of the overnight incubation in water or MV () were compared with Fv/Fm values measured after 2 h of dark adaptation following exposure to high light (bars). Values are means ± SE of six independent experiments.

	DISCUSSION

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Poplars overexpressing FeSOD in the chloroplasts showed greatly enhanced foliar FeSOD activity. Technical difficulties associated with producing intact chloroplasts from tree leaves at high yields prevented an estimation of the increase in SOD activity in the chloroplasts of the transformed plants. The FeSOD transformants had similar maximal extractable foliar activities of APX, MDHAR, and GR (Table I). At certain times of the year DHAR activity was 1.5 to 2 times higher in the leaves of the FeSOD transformants relative to those from untransformed controls. This increase in DHAR activity may be required to sustain cycling of oxidized ascorbate when flux through the ascorbate-glutathione cycle is increased in the FeSOD transformants relative to the untransformed controls, as discussed by Payton et al. (1997). Foliar H₂O₂, ascorbate, and glutathione contents were unchanged by FeSOD overexpression (Table II). Payton et al. (1997) observed an 80% increase in the ratio of GSSH to GSH in transformed cotton plants overexpressing a MnSOD in the chloroplasts, but the GSSH/GSH ratio was unchanged by FeSOD overexpression in poplar leaves. Similarly, FeSOD overexpression in poplar leaves did not lead to increased H₂O₂ contents or APX activities, as has been observed in other studies of SOD overexpression (Sen Gupta et al., 1993b; Prasad et al., 1994).

Increased chloroplastic FeSOD activity did not modify photosynthetic parameters such as net CO₂ uptake, F_v/F_m, F/F_m, or q_P over a broad PPFD range at ambient (280 µL L¹) Ci (Fig. 3A). Similar results were obtained for the light response curves of photosynthesis under nonphotorespiratory conditions (1% O₂; data not shown). When the CO₂ concentration of the surrounding air was varied between 30 to 1000 µL L¹ under 590 µmol m² s¹ PPFD at 21% O₂, no differences in net CO₂ uptake were observed. However, under these conditions, q_P was maintained at the same values with decreasing Ci in the FeSOD transformants, whereas a decline in q_P was observed in leaves from untransformed controls at Ci values below 200 µL L¹ (Fig. 3B). This suggests that primary PSII electron acceptors (such as Q_A) become progressively more reduced as CO₂ assimilation becomes limiting in untransformed poplar leaves.

In contrast, overreduction of Q_A was prevented under these conditions in the leaves of the FeSOD transformants even at very low Ci. Under conditions of diminished CO₂ reduction, the consumption of electrons by pathways other than the Calvin cycle can maintain noncyclic electron transport. One such pathway is the Mehler-peroxidase cycle (Schreiber and Neubauer, 1990). The addition of H₂O₂ to intact chloroplasts causes a rapid increase in q_P (Foyer et al., 1994), supporting the conclusion that H₂O₂ generation and degradation may act as an alternative electron sink maintaining the oxidized state of Q_A. This may be responsible for the observed sustained q_P values at low CO₂ concentrations in the leaves of FeSOD transformants. Whereas increased flux through the Mehler-peroxidase cycle may explain the q_P differences observed at low Ci values, maximum rates of photosynthetic O₂ reduction via the Mehler reaction are considered to be much less (approximately 10%) than the prevailing rates of CO₂ fixation in vivo (Robinson, 1988). It is important to note that measured F/F_m ratios (the relative measurements of electron flow) were not significantly increased in the FeSOD transformants compared with untransformed controls (Fig. 3B).

The differences in q_P values between FeSOD transformants and untransformed controls observed at low Ci in air were lost at low O₂ partial pressures (Fig. 4). In 1% O₂, a condition frequently used to suppress photorespiration, q_P decreased at Ci values below 150 µL L¹ in all plants. This could suggest that the reduction of O₂ by the Mehler-peroxidase reaction is also limited at 1% O₂ in poplar leaves. The apparent K_m for O₂ of the Mehler reaction is between 2 and 60 µM (Robinson, 1988), which corresponds to 0.15 to 5% O₂ in the air at 25°C. An alternative explanation is that electron transport is restricted to such a degree at both low CO₂ and O₂ that the Mehler reaction alone cannot maintain Q_A in an oxidized state. More detailed experiments with varying O₂ concentrations at limiting Ci values are required to characterize this phenomenon more clearly. Nevertheless, these results suggest that the Mehler reaction could provide an important mechanism for avoidance of Q_A overreduction in the FeSOD transformants in environmental conditions such as drought, which cause a decline in Ci due to stomatal closure. In support of this view, water-deficit tolerance was improved in transformed alfalfa overexpressing MnSOD in the chloroplast (McKersie et al., 1996).

By energy turnover in pseudocyclic electron transport the Mehler reaction may provide a residual flow of electrons, which could confer protection against photoinhibition in some circumstances. However, the Mehler reaction has the potential to damage the thylakoid system if superoxide and H₂O₂ are not removed efficiently by the antioxidant defense systems. The contribution of these effects to aerobic photoinhibition may be limited depending on the efficiency of scavenging reaction systems (Krause and Cornic, 1987). We are drawn to the conclusion that the rate of superoxide dismutation must limit the Mehler-peroxidase cycle in certain circumstances. In this work, photoinhibition was induced by high-light treatment and expressed by the decrease in the F_v/F_m ratio. Plants exposed to high light in air (21% O₂, 350 ppm CO₂) and decreasing temperatures showed marked decreases in F_v/F_m ratios (Fig. 7), indicative of the presence of photoinhibition. This decrease was largely unaffected by SOD overexpression in poplar leaves. Similarly, overexpression of FeSOD in tobacco did not confer tolerance to chilling-induced photoinhibition (Van Camp et al., 1996). In these conditions, endogenous SOD did not appear to be a limiting factor in protection against PSII damage. It is important to note, however, that FeSOD overexpressed in the stroma may not have immediate access to the superoxide anion ejected from the PSI complex on the thylakoid membrane.

High-light treatments imposed at 1% O₂ led to smaller decreases in F_v/F_m at 6°C than treatments performed in air (21% O₂; Fig. 7B), suggesting that O₂ indeed participates in the mechanism of photoinhibition in poplar leaves. Combined high-light and low-temperature treatments at 21% O₂ have also been found to cause more inhibition than similar treatments performed at 1% O₂ in other species (Powles et al., 1983). EPR signals provide direct evidence for the involvement of O₂ (most probably singlet oxygen) in the photoinhibitory destruction of PSII (Vass et al., 1992). Singlet oxygen is produced by direct energy transfer from the chlorophyll triplet state to O₂, in situations of overreduction of Q_A (Vass et al., 1992). Another explanation for the failure of FeSOD overexpression to protect against photoinhibition in these circumstances could be that the superoxide anion route may not represent a major pathway of PSII damage compared with that caused by singlet oxygen during high-light stress.

When MV is added to chloroplasts, it is univalently reduced by PSI to its cation radical, which rapidly donates electrons to O₂, producing the superoxide anion. CO₂-dependent O₂ evolution was completely abolished in poplar leaf discs treated with 1 µM MV in both transformed and untransformed plants (Fig. 8). F_v/F_m values of leaf discs after a high-light treatment in water were similar for all lines. In contrast, the F_v/F_m decrease found after a high-light treatment of discs incubated in 1 µM MV was less severe for FeSOD-overexpressing poplar than for untransformed plants (Fig. 9). In this situation, superoxide production probably far exceeded endogenous SOD capacity in untransformed control plants, whereas increased FeSOD activity in the transformants could effectively dismutate excess superoxide anion. FeSOD overexpression improved protection to PSII from damage, in agreement with the increased tolerance to MV-mediated oxidative stress observed previously in FeSOD-overexpressing tobacco (Van Camp et al., 1996).

Comparable decreases in F_v/F_m values after high-light treatment at 6°C (40% for WT) and after high-light treatment at 24°C in 1 µM MV-treated leaf discs (35% for WT) were observed in our experiments. The absence of marked differences in F_v/F_m ratios following chilling treatments, together with the less-accentuated decrease after the MV treatment in transformed plants compared with untransformed controls, suggests that these two treatments involve different mechanisms. A decrease in SOD activity at low temperatures could explain the absence of protection at 4°C. Although the temperature dependence of FeSOD from A. thaliana remains to be established, CuZnSOD from pea chloroplasts was more active at 10°C relative to higher temperatures (Burke and Oliver, 1992) and foliar SOD activities from two maize species measured at 5°C were similar to those maintained at 19°C (Jahnke et al., 1991). The location of FeSOD inside the chloroplast has not yet been elucidated and it could also explain these differences. The differential expression of chloroplastic CuZnSOD and FeSOD genes in tobacco suggests that these two isozymes could be involved in protecting different parts and/or processes within the chloroplast (Kurepa et al., 1997). Differences in the ability of different SOD isoforms to associate with the thylakoid membranes may also explain observed differences in the effectiveness of the overexpression of the MnSOD, CuZnSOD, and FeSOD isoforms in amelioration of protection of photosynthesis in stress conditions.

	FOOTNOTES

   Received October 22, 1997; accepted February 19, 1998.

	ABBREVIATIONS

Abbreviations: APX, ascorbate peroxidase. Ci, internal CO₂ concentration. DHAR, dehydroascorbate reductase. EPR, electron paramagnetic resonance. F, difference between maximal and steady-state chlorophyll a fluorescence measured at any point in time . F_m, maximal fluorescence measured in dark-adapted leaves. F_m, maximal fluorescence measured at any point in time in the light. F_v, variable fluorescence measured in dark-adapted leaves. F_v, variable fluorescence measured at any point in time. GR, glutathione reductase. MDHAR, monodehydroascorbate reductase. MV, methyl viologen. q_P, photochemical quenching. SOD, superoxide dismutase. rbcs, promoter for the small subunit of Rubisco. WT, untransformed poplars.

	ACKNOWLEDGMENT

We are thankful to Dirk Inzé for the generous gift of an A. thaliana FeSOD cDNA.

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