Factors Affecting the Enhancement of Oxidative Stress Tolerance in Transgenic Tobacco Overexpressing...

Plant Physiol. (1995) 107: 737-750

Factors Affecting the Enhancement of Oxidative Stress Tolerance in Transgenic Tobacco Overexpressing Manganese

Superoxide Dismutase in the Chloroplasts'

Luit Slooten*, Katelijne Capiau, Wim Van Camp, Marc Van Montagu, Chris Sybesma, and Dirk lnzé

Vrije Universiteit Brussel, Laboratorium voor Biophysica, Pleinlaan 2, B- I 050 Brussels, Belgium (L.S., K.C., C.S); and Laboratorium voor Genetika (W.V.C. M.V.M.) and Laboratoire associé d'lnstitut National de Ia Recherche

Agronomique (D.I.), Universiteit Gent, K.L. Ledeganckstraat 35, B-9000 Gent, Belgium

Two varieties of tobacco (Nicofiana fabacum var PBD6 and var SR1) were used to generate transgenic lines overexpressing Mn- superoxide dismutase (MnSOD) in the chloroplasts. The overexpressed MnSOD suppresses the activity of those SODs (endogenous MnSOD and chloroplastic and cytosolic Cu/ZnSOD) that are prominent in young leaves but disappear largely or completely during aging of the leaves. The transgenic and control plants were grown at different light intensities and were then assayed for oxygen radical stress tolerance in leaf disc assays and for abundance of antioxidant enzymes and substrates in leaves. Transgenic plants had an enhanced resistance to methylviologen (MV), compared with control plants, only after growth at high light intensities. In both varieties the activities of FeSOD, ascorbate peroxidase, dehydroascorbate reductase, and monodehydroascorbate reductase and the concentrations of glutathione and ascorbate (all expressed on a chlorophyll basis) increased with increasing light intensity during growth. Most of these components were correlated with MV tolerance. It i s argued that SOD overexpression leads to enhancement of the tolerance to MV-dependent oxidative stress only i f one or more of these components is also present at high levels. Furthermore, the results suggest that in var SR1 the overexpressed MnSOD enhances primarily the stromal antioxidant system.

The photosynthetic electron transport chain in higher plant chloroplasts contains, at the acceptor side of PSI, a number of auto-oxidizable enzymes. For example, Fd in the reduced state can react with oxygen, yielding the superoxide anion radical (OJ (Furbank and Badger, 1982; Asada and Takahashi, 1987), and it has been shown that superoxide is also generated in the aprotic membrane interior (Elstner and Frommeyer, 1978; Takahashi and Asada, 1988). Superoxide does not seem to be particularly toxic, although it does inactivate a number of metal-containing enzymes such as Fd-linked nitrate reductase, catalase, and peroxidases (reviewed by Asada and Takahashi, 1987). However, superoxide will dismutate nonenzymically and

This work was supported by grant No. 2-91-2-21-700-5 (VLAB/067) from the Flemish Ministry of Economic Affairs and grant No. 3.0104.90 from the Belgian National Fund for Scientific Research ("0). D.I. is a Research Director of the Institut Na- tional de la Recherche Agronomique (France).

* Corresponding author: e-mail lslooten8vnet3.vub.ac.be; fax 32-2-6293389.

73 7

enzymically (see below) into hydrogen peroxide and oxygen. The hydrogen peroxide can react in turn with superoxide in what is known as a metal-catalyzed Haber-Weiss reaction. This yields the hydroxyl radical (OH.), one of the most reactive species known to chemistry. In chloroplasts, the Fe atoms of Fd seem to fulfill the role of metal catalysts (Elstner and Konze, 1974; Elstner et al., 1978). The hydroxyl radical can also be formed from hydrogen peroxide and reduced Fd (Hosein and Palmer, 1983; Bowyer and Camil- leri, 1985). The hydroxyl radical can initiate self-propagat- ing peroxidative reactions leading to the destruction of membrane lipids and of DNA, and hence to extensive tissue damage (see Bowler et al., 1992, for review). In addition, there is some evidence that part of the superoxide generated in illuminated chloroplasts diffuses toward the thylakoid lumen. Because of the low pH in the lumen during illumination, the superoxide can be protonated in that compartment, yielding the perhydroxyl radical (HO, ). Unlike the superoxide anion, the perhydroxyl radical can initiate lipid peroxidation directly (reviewed by Asada and Takahashi, 1987). The conditions leading to this type of damage will be referred to as oxidative stress.

Two key enzymes for oxygen radical detoxification in the chloroplast are SOD (EC 1.15.1.1) and APx (EC 1.11.1.11). SOD catalyzes the dismutation of two molecules of superoxide into oxygen and hydrogen peroxide. APx reduces hydrogen peroxide to water, with ascorbate as electron donor. SODs are classified, according to their metal cofac- tor, as FeSOD, MnSOD, or Cu/ZnSOD. Chloroplasts generally contain Cu/ZnSOD and, in a number of plant species, FeSOD (Bowler et al., 1992). Whereas the distribution of SODs in the chloroplast is not known, APx was recently found to occur in a stromal and a membrane-bound form (Miyake and Asada, 1992, 1994). It was proposed that the re-reduction of the reaction product of APx, monodehydroascorbate, proceeds along different pathways depending on the type of APx involved. The reaction product of stromal APx would be reduced by NADPH, either directly

Abbreviations: APx, ascorbate peroxidase; DHAR, dehydroascorbate reductase; GR, glutathione reductase; MDHAR, monodehydroascorbate reductase; MV, methylviologen; aI1, quantum efficiency for exciton trapping by the PSII reaction center in dark- adapted material; o,, standard deviation of the mean; SOD, superoxide dismutase.

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738 Slooten et al. Plant Physiol. Vol. 107, 1995

or via glutathione. The enzymes taking part in this so- called ascorbate-glutathione cycle (Foyer and Halliwell, 1976; Nakano and Asada, 1981) are GR (EC 1.6.4.2), DHAR (EC 1.8.5.11, and MDHAR (EC 1.6.5.4). The latter three are stromal enzymes (see Asada and Takahashi [1987] and Halliwell i19871 for reviews). By contrast, the reaction product of membrane-bound APx would be re-reduced directly by the photosynthetic electron transport chain (Miyake and Asada, 1992). In either case the end result is "pseudo-cyclic electron flow" involving both photosystems, but without net oxygen evolution. Experiments with "0 indicated that the occurrence of pseudocyclic electron flow is largely confined to conditions in which assimilatory electron flow is inhibited (Marsho et al., 1979).

In tobacco, MnSOD occurs in the mitochondria, FeSOD in the chloroplasts, and Cu/ZnSOD in the chloroplasts as well as in the cytosol (Bowler et al., 1992). To assess the role of SOD in oxidative stress tolerance we have, as we reported earlier, generated transgenic plants of Nicotiana tabacum var PBD6 that express elevated levels of MnSOD, derived from Nicotiana plumbaginifolia, either in the chloroplasts (ChlSOD plants) or in the mitochondria (MitSOD plants) (Bowler et al., 1991). The influence of the overexpressed enzyme on the resistance of the plants against oxidative stress was investigated in model systems in which leaf discs were floated on aqueous solutions of MV. This water- and lipid-soluble compound is reduced in the light by the photosynthetic electron transport chain, but it is also (although less rapidly) reduced in the dark by other as yet unidentified sources. When reduced MV reacts with oxygen, it yields superoxide. The ensuing oxygen radical damage was estimated from ion leakage out of the leaf discs, or from decreases in activity of the reaction center of PSII. In light-incubation experiments, only ChlSOD plants were significantly more resistant to MV than control plants. In dark-incubation experiments, both ChlSOD and MitSOD plants were significantly more resistant to MV than control plants (Bowler et al., 1991; Slooten et al., 1992). In addition, the ChlSOD plants exhibited an up to 3- or 4-fold increase in tolerance to ozone compared with control plants (Van Camp et al., 1994). Meanwhile, other groups succeeded in enhancing the stress tolerance of plants by SOD overexpression. Thus, alfalfa (Medicago sativa) transformed with the same construct that we used for the ChlSOD plants was more tolerant to freezing stress (McKersie et al., 1993). Potato plants (Solanum tuberosum) overexpressing chloroplast Cu/ZnSOD from tomato had an increased tolerance to MV (Perl et al., 1993), and overexpression of chloroplastic Cu/ZnSOD from pea in the chloroplasts of N. tabacum rendered the plants more tolerant to MV as well as to photoinhibition (Sen Gupta et al., 1993a, 1993b).

The present work grew out of observations suggesting, first, that plants grown at high light were more tolerant to MV than plants grown at low light and, second, that overexpression of MnSOD in the chloroplasts enhanced the tolerance to MV only when the plants were grown at high- light intensities. Both observations could be explained by considering that in the chloroplast antioxidant system, SODs are one link in a chain of enzymes. We hypothesized

that overexpression of SOD would enhance thc oxidative stress tolerance only when other antioxidant enzymes or substrates did not limit the oxygen-radical-scat enging capacity. Growth at high-light intensities would enhance expression of these other enzymes or substrates, thus increasing the tolernnce to MV and, in addition, a1 owing the overexpressed SOD to cause a further enhancement of the oxidative-stress tolerance.

To test this hypothesis we grew transgenic and nontransgenic plants at different light intensities and measured the tolerance to MV as well as the abundance of enzymes and substrates known to be involved in oxygen re dica1 scavenging. In addition, we correlated the observetl MV toler- ante with the abundance of a11 antioxidant enzymes and substrates investigated. Such correlations were 2xpected to yield clues as to which enzymes or substrates w 2re limiting the oxygen-radical-scavenging capacity under 3ur growth conditions. This may serve as a guideline for selecting enzymes that should be overexpressed in order to enhance the oxidative-stress tolerance of the plants.

MATERIALS AND METHODS

Plant Material and Growth Conditions

Two varieties of Nicotiana tabacum were Ujed for the genetic transformations: var PBD6 and var Petit Havana SR1. Transgenic PBD6 plants overexpressing MnSOD in the chloroplasts were described by Bowler et al. (1991), and the SR1 transgenics were obtained in a similar way. Briefly, the coding sequence of the mature, mitochondiial MnSOD of Nicotiana plumbaginifolia was coupled in-fríime behind the chloroplast transit sequence of the pea Rubisco (for details, see Bowler et al., 1991). Under the control of the cauliflower mosaic virus 355 promoter, this construct al- lows overproduction of MnSOD in the chloroplasts. Pri- mary transformants, selected on kanamycin, w ere assayed for MnSOD activity. Transformants overexpressing Mn- SOD to high levels and containing a single lacus T-DNA insertion were made homozygous. One homozygous line was used for future research. The transgenics overproducing MnSOD in chloroplasts will be referred to as ChlSOD- PBD6 and ChlSOD-SR1.

The plants were grown on peat-based compcst and were given fertilizer once a week. Most of the plants were grown at light intensities (between 400 and 700 nm) of approximately 40, 90, or 135 pmol m-* s-' in a 12-11 light/l2-h dark cycle, with day and night temperatures of about 22 and 16"C, respectively. Illumination was provided by halo- gen-metal vapor discharge lamps with a daylight spectrum (HQI/T-D, Osram, München, Germany), except at the lowest light intensity, for which cool-white and red (Fluora, Osram) fluorescent tubes were used. Unless indicated otherwise, the experiments were carried out with the seventh to ninth leaf from the top at 10 to 13 weeks after sowing. One series of experiments was carried out with plants grown at 800 pmol m-' s-'; for these expuiments the seedlings were transferred to a growth chamber at 2 weeks after sowing. In the growth chamber they rectzived a 16-h light/8-h dark cycle with day and night temperatures of 25




Mn Superoxide Dismutase-Overproducing Tobacco 739

and 22"C, respectively. During the first 6 d the light intensity was gradually raised from 360 to 800 kmol mP2 s-I. The assays were carried out 15 to 18 d after transfer to the growth chamber.

Assessment of Oxidative-Stress Tolerance

The oxidative-stress tolerance of the plants was assessed as described previously (Bowler et al., 1991). Briefly, leaf discs were incubated overnight in the dark at room temperature with water or an aqueous solution of MV. They were then illuminated for 2 h at 30 kmol m-' s-' provided by cool-white fluorescent tubes and were subsequently incubated for another 20 h at 28°C. The MV-dependent oxygen radical damage was estimated first from ion leakage out of the leaf discs, due to destruction of membrane lipids. Ion leakage was measured as an increase in the conductance of the floating solution. In addition, we measured the MV-dependent decrease in activity of the reaction center of PSII. As a measure of this activity we used a,,. The fluorescence measurements were made with a PAM fluorometer (Walz, Effeltrich, Germany). The average values of three to four leaf discs at each MV concentration (including zero) were used to calculate the MV-induced increase in conductance of the floating solution or the MV-induced decrease in aI1. The decrease in aI1 was expressed as a percentage of the QII value in leaf discs without MV.

Alternatively, leaf discs floating on water at 4°C were either kept in the dark or illuminated with white light at 360 pmol m-' s-' for 4 h. Following this treatment the leaf discs were dark-incubated at room temperature for 1 h, and thereafter the light-induced damage was estimated from the decrease in the activity of PSII, as above.

Biochemical Assays

All biochemical assays were done with leaf disc extracts prepared by homogenization in ice-cold mortars. Debris was pelleted by centrifugation at 40,OOOg for 30 min. The activity of APx was determined according to Chen and Asada (1989), that of GR according to Schaedle and Bassham (1977), that of DHAR according to Asada (1984), and that of MDHAR according to Polle et al. (19901, with minor modifications. For these enzymes, 1 unit of activity equals 1 pmol/min. The extraction buffer for these assays was as described by Chen and Asada (1989). Total ascorbate was determined according to Okamura (1980), and total glutathione was determined according to Griffith (1980). For these assays the leaf discs were extracted as described by Tanaka et al. (1985).

SOD activity was determined first in a solution assay, and second after an activity staining following electro- phoresis on nondenaturing polyacrylamide gels (Bowler et al., 1991). The gels were stained by illuminating them for exactly 10 min in the assay mixture described by Beauchamp and Fridovich (1973) under weak light (30 pmol m-'s-') provided by two fluorescent tubes. After staining, the gels were scanned with a one-dimensional scanning densitometer. The relative contribution of each of

the SOD isoforms to the overall activity was determined from the contribution of the area under the corresponding peak of the densitogram to the total area. The overall SOD activity in leaf disc extracts was determined in the solution assay with the same assay mixture. In the solution assay, 1 unit of SOD activity causes a half-maximal inhibition of the rate of light-induced, riboflavin-mediated reduction of ni- troblue tetrazolium. In both assays the activity was measured with and without 2 mM KCN, which causes a virtually complete inhibition of Cu/ZnSOD (Geller and Winge, 1984).

Chl was determined according to Arnon (19491, as mod- ified by Lichtenthaler (1987). All assays were done within 5 d with the same leaf. Results are usually presented as mean ? a,. a, equals the population SD divided by Jn, where n is the number of experiments. The significance of the difference between means was assessed with a one-sided t test.

RESULTS

Control Experiments on MV-lnduced Oxygen Radical Damage

As reported earlier, prolonged dark incubation of leaf discs on aqueous solutions of MV can cause significant oxygen radical damage (Bowler et al., 1991). However, under the conditions used in the present experiments the damage was dependent mainly on light-induced electron transport. This is demonstrated in Figure 1, which shows the effect of DCMU, an inhibitor of photosynthetic electron transport acting at the leve1 of QA (a plastoquinone molecule bound to the reaction center of PSII). Without DCMU, illumination in the presence of MV caused a strong decrease in PSII activity (as estimated from @,,) and a pronounced increase in the conductance of the floating solution. Both effects were almost completely prevented by the inclusion of DCMU in the incubation mixture. This indicates that under the present assay conditions the damage arose within the chloroplast and then spread outward, causing damage to the cell membranes and ion leakage. As expected, DCMU had no protective effect when it was added immediately after illumination of the MV-treated leaf discs (not shown).

Oxidative Stress Tolerance in Transgenic and Nontransgenic Plants in Relation to Light lntensity Received during Growth

In most of the remaining experiments, the resistance against MV was tested using a MV concentration of 2 PM. At this concentration large differences in MV sensitivity were observed between plants grown at light intensities of 40 and 135 pmol m-'s-' (see below). However, leaf discs from plants grown at 800 pmol m-' s-' were virtually insensitive to 2 p~ MV. These leaf discs were accordingly treated with 5 to 7 p~ MV. Figure 2 shows that the damage induced by 2 p~ MV was less extensive in plants grown at higher light intensities. This was the case in var PBD6 (Fig. 2, A and B), as well as in vai SR1 with plants grown at light intensities ranging from 40 to 135 pmol m-'s-' (Fig. 2, C





Figure 1. DCMU prevents MV-mediated oxygen radical damage. Data were obtained with nontransgenic PBD6. The experiment was carried out as described in "Material5 and Meth- ods," except that the MV concentration was varied. N.A., No further additions. Where indicated, DCMU (20 PM) was added along with o DCMU MV. Error bars indicating population SD for four replicas are shown only if their magnitude ex- O 1 2 3 4 5 6 2 3 4 5 6

N.A.

5 - : 100 - 5

I - DCMU __. O

ceeds that of the symbols.

and D). ChlSOD-PBD6 plants were on average more tolerant of MV than control plants, independent of the light intensity received during growth. However, the differences between transgenic and control plants were more significant after growth at higher light intensities. In addition, the attenuation of MV-induced ion leakage in the transgenic plants (Fig. 2A) was more extensive and more significant than the attenuation of MV-induced decrease in mI1 (Fig. 2B). In ChlSOD-SR1 plants, the transgenic MnSOD had no positive effect at a11 on the average MV-induced decrease in mI1 (Fig. 2D). However, it did attenuate the MV-induced ion leakage (Fig. 2C), but only when the plants were grown at relatively high light intensities; in fact, the differences between transgenic and control plants were significant only when the plants were grown at 800 pmol m-'s-'. Taken together, these data indicate that the protection provided by overexpressed MnSOD against MV-dependent oxidative stress is not a static characteristic, but is dependent on (i.e. is positively correlated with) light intensity received during growth. This extends our earlier finding that the protection provided by overexpressed MnSOD against MV-dependent oxidative stress increased with increasing age of the leaf and the plant (Bowler et al., 1991).

With var PBD6 grown at 135 pmol m-* s-', as well as with var SR1 grown at 135 or 800 pmol m-' s-', we found that the transgenic MnSOD had no detectable effect on the

Figure 2. MV-dependent conductance increase A

extent of photoinhibition induced by illuminalion at 4°C (not shown).

Abundance of SODs in Transgenic and Nontransgenic Plants in Relation to Light lntensity Received during Crowth

In leaf extracts from nontransgenic plants of var SR1 and var PBD6, the total SOD activity was on tlie average around 50 units/g fresh weight, fairly independent of the light intensity received during growth. The activity in transgenic plants of var SRl and var PBD6 was on the average approximately 40 and 100% higher, rctspectively, than in the nontransgenic plants (data not shown). The difference between the two varieties was not dite to different levels of transgenic MnSOD activity, but rather to a differential suppression of endogenous activities in the transgenic plants of var SR1 and var PBD6, as will be detailed below.

Figure 3 shows some densitometer scans of SOD activity gels obtained with leaf extracts from var PB136. Similar patterns were obtained with extracts from v2.r SR1 (not shown). In the nontransgenic plants, four peaEa were observed after staining without KCN (left, dashed line). These peaks represent mitochondrial MnSOD (band 1); a nonre- solved band representing chloroplastic FeSOD ,and cytoso-

PBD6 B PBD6 (A-and C) and MV-dependent decrease in @,, (B and D) in transgenic (black bars) and control plants (gray bars) of var PBD6 (A and B) and var SR1 (C and D) grown at different light intensities, as indicated along the x-axes. The conductance increase is expressed in microsiemens cm-' (g fresh weight)-' (mean and a,, n = 7-9). The numbers in the figure indicate the probability (in percent) that the difference be-

o 8 Zo0

2 i ls0

8 0 7 -

tween the means of transgenic and control plants is significant (one-sided t test).

C SRI D SRI 80

3 60 c

s .- e 4 0 57

% - 20

O 0 t

40 90 135 800 Light intensity (pmioI,m-Z.s-~) Light intensily (pmo1.m-2.s-I)





I 1 2 3 4 5 1 2 3 I Figure 3. Banding pattern of SOD activity gels obtained with whole- leaf extracts from transgenic (solid lines) and nontransgenic (dashed lines) var PBD6. The gels were stained in the absence (left) or presence (right) of KCN. The bands migrate to the right. For more details, see text.

lic Cu/ZnSOD (band 3); and two forms of chloroplastic Cu/ZnSOD (bands 4 and 5; Bowler et al., 1991). The transgenic plants contain a strong additional band representing the overexpressed MnSOD (band 2). Preincubation with KCN prior to staining caused a complete inactivation of Cu/ZnSOD (right), so that band 3 now represents only FeSOD. In each experiment the total area under the peaks of the densitogram was plotted against the activity in the solution assay. This yielded a straight line passing through the origin, indicating that the total area under the peaks was proportional to the total activity (not shown). This allowed us to estimate the activity of the individual SOD species as outlined in ”Materials and Methods.”

Below, the activities of cytosolic Cu/ZnSOD and endogenous, mitochondrial MnSOD are expressed in units/g fresh weight. However, unless indicated otherwise, the activities of the SOD species known to be located in the chloroplasts (transgenic MnSOD, FeSOD, and chloroplastic Cu/ZnSOD) were expressed in units/mg Chl. This was more relevant to our interest in the chloroplastic defense mechanisms against oxidative stress.

On a Chl basis, the activity level of the overexpressed MnSOD in ChlSOD-PBD6 plants was about the same as in ChlSOD-SR1 plants, fairly independent of the light intensity received during growth (Fig. 4A). The total activity of the chloroplastic SOD species was around 2.5 to 3.5 times

higher in the transgenic plants than ip the control plants (Fig. 4B).

In nontransgenic plants the average activity of FeSOD increased with increasing light intensity received during growth, at least above a threshold value of 90 pmol m-’ s-* (Fig. 5, A and C). We did not observe consistent differences in FeSOD activity between transgenic and control plants.

In var SR1 (but not in var PBDG) the activity level of chloroplastic Cu/ZnSOD tended to decrease with increasing light intensity received during growth (Fig. 5, B and D). In both varieties the activity of chloroplastic Cu/ZnSOD tended to be lower in transgenic plants than in nontransgenic plants. The largest and most significant differences between transgenic and control plants with respect to activities of chloroplastic Cu/ZnSOD were observed in var SR1. However, the results were somewhat variable in this respect and depended on the light intensity received during growth, although not in a consistent manner when the two varieties are compared.

The activity of cytosolic Cu/ZnSOD (in units/g fresh weight) in nontransgenic plants was not significantly dependent on the light intensity received during growth, as shown in Figure 6, A and C. However, in var SR1 this activity was consistently and considerably lower in the transgenic plants than in the control plants. With var PBD6 the same trend was observed, but in general to a lesser extent.

The activity of endogenous, mitochondrial MnSOD was usually low, and it was often absent, so that the errors on the average values were relatively large. Nevertheless, it can be seen from Figure 6, B and D, that its activity tended to be lower in transgenic than in nontransgenic plants, especially in var SRI. In nontransgenic plants the activity of mitochondrial MnSOD varied rather irregularly with light intensity received during growth.

In summary, the activities of the endogenous MnSOD and Cu/ZnSODs were generally lower in transgenic plants than in nontransgenic plants. There were no indications of a positive correlation between the average abundances of the various endogenous SOD species and tolerance to MV- dependent oxidative stress (Fig. 2).

40 90 135 40 90 135 800 40 90 135 40 90 135 800 Light intensity (pmo1.m-2,s-1) Light lntensity (pmol.m-?s-I)

Figure 4. A, Activity of transgenic MnSOD in plants grown at different light intensities. The error bars indicate a, ( n = 8-9, except for SR1 grown at 800 pmol m-* s-’, where n = 3). The data were obtained with the leaves used for the experiments shown in Figure 2. The data shown in Figures 4 to 6 were obtained with the same extracts. B, Relative increase in total chloroplastic SOD activity in plants grown at different light intensities. The ordinate shows the ratio between transgenic and control plants with respect to total chloroplastic SOD activity, as measured in leaf extracts.





Figure 5 . Activity of chloroplastic FeSOD (A A PBD6 and C) and chloroplastic Cu/ZnSOD (B and D) in control and transgenic plants of var PBD6 (A and E) and var SR1 (C and D) Rrown at different T - light intensities, as indicated along the x-axes. Activities are expressed in units (mg Chl)-’. For further details see legend to Figure 4A.

C SR1

Abundance of SODs in Relation to Age of the Leaves

The above data indicate that transgenic MnSOD tends to suppress the expression of the endogenous MnSOD and Cu/ZnSODs. These are precisely the SODs for which the activity is high in young leaves but disappears or declines upon maturation of the leaf. An example is shown in Figure 7. At 10 or 11 weeks after sowing, chloroplastic Cu/ZnSOD and endogenous, mitochondrial MnSOD activity were present only in the immature top leaves. These enzymes declined to very low or undetectable levels upon maturing of the leaves. Cytosolic Cu/ZnSOD activity like

Light intensity &mol.m-z.s-l)

B PBD6

D SRl

I 7RANSGENIC m CONTROL

Light intensity (pmcll.m-?s-l)

wise decreased strongly with increasing leaf a?,e, although it did not disappear completely. In 13-week-old plants, chloroplastic Cu/ZnSOD activity was very low even in the top leaves, which had reached approximatelj, their final size by that time. By contrast, chloroplastic FeSOD re- mained present at a relatively constant level, independent of leaf age or position. The data presented in the previous and following sections were obtained with the seventh to ninth leaf from the top at 10 to 13 weeks after sowing (see ”Materials and Methods”). This would correspond, for example, with leaves 9 to 11 (counted from the base) of the plant represented in the middle pane1 of Figure 7.

A PBD6 Figure 6 . Activity of cytosolic Cu/ZnSOD (A and C) and endogenous MnSOD (B and D) in control and transgenic plants of var PBD6 (A and B) and var SRI (C and D) grown at different light intensities, as indicated along the x-axes. Activities are expressed in units (g fresh weight)-‘. For further details see legend to Fig- ure 4A.

C SRI

LigM intensity (pmo1.m-2,s-1)

B PBD6

T 68 I

D SRl

Wd CONTROL

Light intensity (pmo1.m-2,s-I)





30

P g 20 4

10

o 4 6 7 9 11 13 15 16

40, 30] llweeks

.- b 2 20 I

10

o 4 6 7 9 11 13 15 16

2 3 10 a

o 4 6 7 9 11 13 15 16 Leaf number

Figure 7. SOD activities in whole-leaf extracts from a nontransgenic plant of var SR1 grown at 135 pmol m-* s-’. The age of the plant, in weeks after sowing, i s indicated within each panel. The leaves were numbered starting from the base of the stem. Activities are expressed in units (mg Chl)-’ for FeSOD and chloroplastic (chl) Cu/ZnSOD and in units (g fresh weight)-’ for MnSOD and cytosolic (cyt) Cu/ZnSOD.

Abundance of Other Antioxidant Enzymes and Substrates in Relation to Light lntensity Received during Growth

The data concerning other enzymes and substrates involved in oxygen-radical scavenging have been summa- rized in Tables I and 11. We did not find any difference between transgenic and control plants with respect to abundances of APx, DHAR, GR, MDHAR, ascorbate, or

glutathione. Therefore, Tables I and I1 show the pooled data from both transgenic and control plants.

In var PBD6 the average abundance of most of the enzymes and substrates was, on a Chl basis, about the same in plants grown at a light intensity of 45 or 90 pmol m-’ s-l (Table I). The only exception was DHAR, which exhibited, on a Chl basis, an activity about 70% higher after growth at 90 pmol m-’ s-’. However, plants grown at 135 Fmol m-* s-’ exhibited an increase in a11 components, compared with plants grown at 90 pmol m-’ s-’. Ascor- bate exhibited the strongest increase (90-100%), followed by APx and glutathione (40-50%), and DHAR, GR, and MDHAR (20-30% increase).

In var SR1, the average abundance (on a Chl basis) of DHAR, MDHAR, and ascorbate was higher in plants grown at 90 pmol m-’s-’ than in plants grown at 40 pmol m-2 s-l (Table 11). The increases amounted to approximately 80% for DHAR and 25% for total ascorbate and MDHAR. The abundance of APx, GR, and glutathione was, on a Chl basis, about the same in plants grown at these two light intensities. Plants grown at 135 pmol m-‘ s-’ exhibited a further increase in a11 components except DHAR, compared with plants grown at 90 pmol m-’ s-’. The increases amounted to around 60% for glutathione, GR, and MDHAR, and 40 to 50% for APx and ascorbate. Addi- tional increases in the concentration of ascorbate and the activity of APx, DHAR, and MDHAR were observed after growth at 800 pmol m-’s-’. Thus, in general, there was a fair correspondence between the increase in abundance of components involved in oxygen-radical scavenging and the decrease in sensitivity to MV when the plants were grown at increasing light intensities.

Correlation of MV Tolerance with Abundance of Antioxidant Enzymes and Substrates

More direct evidence for correlations between enzyme or substrate concentrations and tolerance to MV was sought from scattergrams in which the abundance of a given enzyme or substrate was plotted against MV-induced damage in the same leaf. This was done with a data base comprising a11 experiments in which oxygen radical dam-

Table 1. Abundances o f enzymes and substrates in whole leaf extracts from nontransgenic and transgenic plants o f var PBD6, in relation to light intensity received during growth

used for the experiments shown in Figure 2, mean t o, (n). Enzyme activities are expressed in units (mg Chl)-’ and substrate concentrations in pmol (mg Chl)-’. The data were obtained with the leaves

Abundance Enzvme or Substrate

At 40 pmol m-‘ s-‘ At 90 pmol m-2 5 - l At 135 pnol m-’ 5 - l

Enzymes APx 7.01 t 0.57 (12) 6.89 t 0.39 (18) 10.5 t 1.2 (12) DHAR 0.57 t 0.05 (12) 0.99 t 0.06 (18) 1.19 2 0.13 (12) CR 0.14 t 0.01 (12) 0.15 t 0.01 (18) 0.20 t 0.03 (12) MDHAR 0.23 +- 0.02 (12) 0.18 t 0.02 (18) 0.23 t 0.03 (12)

Ascorbate 0.41 2 0.05 (16) 0.43 t 0.04 (18) 0.80 t 0.04 (16) G I utath ione 0.11 2 0.02 (14) 0.10 +- 0.01 (18) 0.14 +- 0.01 (14)

Substrates




744 Slooten et ai. Plant Physiol. Vol. 107, 1995

Table II. Abundances o f enzymes and substrates in whole leaf extracts from nontransgenic and transgenic plants o f var S R I , in rolation to light intensity received during growth

used for the experiments shown in Figure 2, mean ? uhil (n). Enzyme activities are expressed in units (mg Chl)-' and substrate conceritrations in pmol (mg Chl)-'. The data were obtained with the leaves

Enzyme or Substrate

Enzyme APx DHAR GR MDHAR

Su bstrate Ascorbate G I utath ione

Abundance

At 90 pmol m-' sC1 At 135 pmol m-* 5 - l At 800 pmol m-* s - l _____ At 40 pmol m-* s-'

7.86 2 1.31 (13) 7.80 t 0.54 (15) 11.1 t 0.8 (18) 18.5 t :!.O (6) 0.78 2 0.05 (13) 1.38 t 0.22 (15) 1.25 t 0.07 (18) 2.72 t 0.39 (6) 0.19 t 0.03 (13) 0.19 t 0.02 (15) 0.31 t 0.02 (18) 0.26 t 0.04 (6) 0.34 t 0.03 (13) 0.43 t 0.05 (15) 0.68 2 0.1 1 (18) 0.99 rt 0.27 (6)

0.37 t 0.04 (15) 0.47 rt 0.02 (17) 0.67 2 0.04 (18) 1.09 t 0.08 (4) 0.11 t 0.02 (13) 0.10 2 0.01 (15) 0.16 2 0.02 (17) nda

a nd, Not determined.

age had been elicited with 2 p~ MV. To avoid ambiguities we restricted our analysis to groups of data in which the range of both the Chl concentrations and the fresh weights were limited in such a way that neither of these exhibited a significant correlation with MV-induced damage.

For the calculation of the correlation coefficients it was assumed that the MV-induced damage (conductance increase or decrease in QII) was linearly dependent on the abundance of the enzyme or substrate under consideration. The correlation coefficient can be thought of as a measure of the goodness of fit of the data with this model. Its value ranges from O (no correlation) to +1 or -1 (perfect correlation), the sign depending on the slope of the regression line. In our case, protective effects give rise to regression lines with negative slopes, and hence to negative correlation coefficients. We also calculated the probability that the

regression coefficient (the slope of the regression line) has the indicated sign, i.e. that it differs from zero (in a one- sided t test). For the sake of clarity, entries in the tables are confined to enzymes or substrates for which the absolute value of the correlation coefficient was at least 0.35, and for which, in addition, the correlation was significant at the 95% leve1 (P > 0.95).

The results obtained with nontransgenic ancl transgenic plants are shown in Tables I11 and IV, respectively. We arrived at these results by using pools of data obtained with plants grown at two "adjacent" light intensities: either 40 and 90, or 90 and 135 pmol m-' s-'. Furthermore, enzyme and substrate levels were correlated either with MV-induced increase in conductance or with FdV-induced decrease in aI1. This yielded four types of correlations, which will be denoted as correlation types 1 to 4 for ease of

Table 111. Correlation coefficients for linear correlations o f enryme activities and substrate concentrations with MV-induced coriductance increase and MV-induced decrease in

The data were obtained with leaves from plants grown at the indicated light intensity (expressed in pmol m-' s-'). From the available data, selections were made in which the range of fresh weights and Chl contents of the leaf discs was limited such that there were no significant correlation between Chl content or fresh weight with sensitivity to MV. The number of experiments used for the regression analys s is indicated. In the regression analysis, enzyme activities were expressed in units (mg Chl)-' and substrate concentrations in pmol (mg Chl)-'. Only cytosolic CuRnSOD was expressed in units (g fresh weight)-' . Only those correlation coefficients are shown that were significantly diffe,.ent from zero in a one-sided t test (P > 0.95; with four exceptions, P > 0.97). Correlations that are illustrated in Figure 8 are indicated. Cond. Incr., Conductance increase.

in nontransgenic plants

Correlation Type

1 7 7 4

Parameter Cond. Incr. Cond. Incr. A@,, li@,l

Light intensity 40 + 90 90 + 135 40 + 90 90 + 135 Var PBD SR1 PBD SR1 PBD SR1 PBD SR1 n 13 8 1 2 14 13 8 12 14 FeSOD -0.52 -0.68a Chloroplastic Cu/ZnSOD 0.76 0.80 APx -0.66a -0.57 -0.60 DHAR -0.52 MDHAR -0.55 Ascorbate -0.60 -0.70 -0.66" -0.60 -0.63 -0.55 C I utathione -0.83 -0.64 -0.47 -0.79 -0.57 Chl -0.23 -0.33 -0.33 -0.11 -0.29 0.1 7 -0.24 -0.07 Fresh weight -0.14 -0.24 -0.25 -0.31 0.21 -0.28 -0.26 -0.31

a See Figure 8.





90 and 135

3 100

U

V 6

Ascorbate (pmoUmg chlorophyll)

90 and 135

E

a,

3 100 a P

7 9 11 APx (Ulmg chlorophyll)

B 120 SRI 90 and 135 , . I 40

0'; 2 3 4 5 6 7 8 9 ' FeSOD (Uhg chlorophyll)

D I O O r tr. SRI 40 and 90

e 2

t

O ' " ' ' I 2 16 tr. MnSOD (Ulrng chlorophyll)

reference. The two bottom lines of Tables I11 and IV are controls indicating that within each group of experiments, the Chl content and the fresh weight of the leaf discs were at most only weakly correlated with MV-induced damage. These correlations were a11 insignificant.

In nontransgenic plants (Table III) ascorbate and glutathione were negatively correlated with MV-induced damage in a11 types of correlations. In other words, these substrates seemed to exert control on the extent of MV- induced decrease in QI1 and/or MV-induced increase in conductance in plants grown at light intensities ranging from 40 to 135 pmol m-' s-'. Such correlations were observed in var SR1 as well as in var PBDG. An example is shown in Figure 8A. Furthermore, FeSOD and APx were negatively correlated with MV-induced damage in var SRl as well as in var PBD6. Examples are shown in Figure 8, B and C, respectively. DHAR and MDHAR were negatively correlated with MV-induced damage in var SR1 only. In MDHAR this extends the observation that its activity in var PBD6 was almost independent of the light intensity received during growth (Table I). By contrast, the sensitivity to MV decreased strongly with increasing light intensity received during growth (Fig. 2). FeSOD, DHAR, and MDHAR yielded significant results only in type 2 correlations; APx yielded significant results only in type 2 and type 4 correlations. In other words, the activities of FeSOD, DHAR, and MDHAR seemed to exert control only on the extent of MV-induced ion leakage in plants grown at relatively high light intensities; in addition, APx seemed to exert control on the extent of MV-induced decrease in al1 in such plants. In FeSOD (Fig. 5, A and C) and APx (Tables I and 11) the observed correlations agree with the observation that the activity of these enzymes increased after growth at light intensities above 90 pmol m-'s-'.

The activities of cytosolic Cu/ZnSOD and GR were not significantly correlated with MV-induced damage. This extends the observation that the activity of these enzymes

Figure 8. Correlations between the abundance of antioxidants and MV-induced oxygen radical damage in control plants (A-C) and transgenic plants (D). The MV concentration was 2 p ~ . T h e variety and the light intensity received during growth (in pmol m-'s-') are indicated in each panel. In A to C, the conductance increase is expressed in microsiemens cm-' (g fresh weight)-'. In D, the decrease in @,, was expressed as percentage of the @,, value in leaf discs without MV.

was not or was only weakly dependent on the light intensity received during growth (Fig. 6; Tables I and 11). Fur- thermore, the activity of chloroplastic Cu/ZnSOD was always positively correlated with MV-induced damage. This is in line with the fact that in var SR1 the activity of this enzyme decreased with increasing light intensity received during growth (Fig. 5, B and D).

The relatively strong type 1 and type 3 correlations for glutathione and ascorbate in var SR1 and PBD6 (Table 111) could not have been predicted from the average values in Tables I and 11; the ascorbate and glutathione levels were on the average the same, or nearly the same, in plants grown at 40 and 90 pmol m-'s-'. Yet this is not a contra- diction, since correlations between two quantities yield more information than can be extracted from average values of a single quantity. However, it is not yet clear why significant type 1 and type 3 correlations showed up only in the substrates ascorbate and glutathione and not in the enzymes.

The results obtained with transgenic plants are shown in Table IV. In transgenic plants of var SR1 the activity of overexpressed MnSOD was significantly correlated with MV-induced damage in a11 types of correlations. An example is shown in Figure 8D. In addition, severa1 enzymes exhibited significant correlations with MV-induced decrease in @,, in plants grown at relatively low light intensities (correlation type 3) in contrast to nontransgenic plants. In the transgenic plants of var PBD6, none of the listed components exhibited significant correlations with MV-induced damage when the plants were grown at relatively low light intensities (type 1 and 3 correlations). Un- fortunately, it was not possible to define a sufficiently large group of data from plants grown at relatively high light intensities (type 2 and 4 correlations) that met the requirement that there be no significant correlation of the Chl content or the fresh weight of the leaf discs with MV- induced damage.





Table IV. Correlation coefficients for linear correlations of enzyme activiiies and substrate concentrations with MV-induced conciuctance increase and MV-induced decrease in a,, in transgenic plants of var SR1

See legend to Table 111. Cond. Incr., Conductance increase. Parameter

Cond. Incr. Cond. Incr. A@,l A % Light intensity 40 + 90 90 + 135 40 + 90 90 + 135 n 11 12 11 'I 2 Transgenic MnSOD -0.61 -0.64 -0.78a -0.59 FeSOD -0.64 -0.58 -0.67 Chloroplastic Cu/ZnSOD 0.61 0.58 0.59 APx -0.55 -0.62 - 3.66 DHAR -0.57 MDHAR -0.60 -0.51 Ascorbate -0.60 Glutathione -0.74 -0.70 -0.74 Chl -0.33 -0.28 -0.33 -0.23 Fresh weight -0.06 -0.36 0.06 -0.23

a See Figure 8.

DISCUSSION

Severa1 groups have generated transgenic plants that overexpress SODs in order to enhance tolerance to oxidative stress. These attempts have been successful to various degrees. Overexpression of chloroplastic Cu/ZnSOD from Petunia hybrida in tobacco (N. tabacum) did not increase the tolerance of the plants to MV (Tepperman and Dunsmuir, 1990) or ozone (Pitcher et al., 1991). Tomato plants (Lyco- persicon esculentum) transformed with the same construct did not exhibit an increased resistance to chilling-induced photoinhibition (Tepperman and Dunsmuir, 1990). How- ever, overexpression of a mitochondrial MnSOD from N. plumbaginifolia in the chloroplasts of N. tabacum resulted in an increased tolerance of the plants to MV (Bowler et al., 1991) as well as to ozone (Van Camp et al., 1994). Alfalfa plants ( M . sativa) transformed with the same construct were more tolerant to freezing stress (McKersie et al., 1993). Potato plants (S. tuberosum) overexpressing Cu/ZnSOD from tomato had an increased tolerance to MV (Perl et al., 1993); and overexpression of chloroplastic Cu/ZnSOD from pea in the chloroplasts of N. tabacum rendered the plants more tolerant to MV as well as to photoinhibition (Sen Gupta et al., 1993a). The present results confirm that overexpression of mitochondrial MnSOD in the chloroplasts of tobacco enhanced the resistance of the plants to MV-dependent, light-induced oxidative stress. However, the degree to which the MV tolerance is enhanced is strongly dependent on the growth conditions of the plants. This is a factor not taken into account in previous studies, and it may be partly responsible for the different results obtained by various workers, at least with regard to enhancement of MV tolerance.

It has become clear that there are two major mechanisms of photoinhibition: acceptor-side and donor-side photoinhibition. The latter type occurs when electron donation to the PSII reaction center is inefficient; it has been demonstrated only in subcellular systems, but it might occur in vivo as a consequence of acceptor-side photoinhibition (see Prasil et al. [1992] for review). It is currently thought that

acceptor-side photoinhibition involves the generation of singlet oxygen following inactivation of the boimd electron acceptor QA (Barber and Andersson, 1992; Vass et al., 1992). On the other hand, superoxide radical:; have been implicated in the generation of chilling-induced photoinhibition in subcellular systems (Richter et al.. 1990), and unspecified oxygen free radicals different froni singlet oxygen were iinplicated in the light-dependent Aegradation of the 32-kD protein of the PSII reaction center (Sopory et al., 1990). Furthermore, a mutant of Conyza bomriensis with elevated levels of SOD, APx, and GR was reported to exhibit an enhanced tolerance to photoinhibition (Jansen et al., 1989). In our hands the overexpressed MnSOD did not provide any significant protection against chilling-induced photoinhibition, as determined from measureinents of (not shown). Thus, if superoxide anion radicds are at a11 involved in the generation of chilling-induced photoinhibition, they are not accessible to the overexpressed Mn- SOD. This is in contrast with data from Sen Gupta et al. (1993a), who found that transgenic tobacco plants overexpressing chloroplastic Cu/ZnSOD from pea were more resistant to chilling-induced photoinhibition, (3s well as to MV, than control plants.

Abundance of SODs and the Effect of Transgeiiic MnSOD on Activities of Other Antioxidant Enzymes

The activities of cytosolic and chloroplastic Cu/ZnSOD and of mitochondrial MnSOD were, in general, lower in transgenic (MnSOD-overexpressing) plants t han in nontransgenic plants, especially in var SR1 (Figs. E; and 6). This suggests that overexpression of MnSOD some how leads to suppression of the activity of cytosolic and chloroplastic Cu/ZnSOD and of mitochondrial MnSOD. Sen Gupta et al. (1993a), who designed experiments in which chloroplastic Cu/ZnSOD from pea was overexpressed ir. the chloroplasts of tobacco, noted an even stronger decline in the activity of the endogenous chloroplastic Cu/i:nSOD in the transgenic plants.




M n Superoxide Dismutase-Overproducing Tobacco 747

Chloroplastic Cu/ZnSOD is especially prominent in young, expanding leaves. It shares this characteristic with endogenous, mitochondrial MnSOD and (to a lesser extent) with cytosolic Cu/ZnSOD (Fig. 7). The reason for this may be that in tobacco these three SODs are associated mainly with dark metabolism. Young, expanding leaves exhibit a high metabolic activity, which presumably entails an increased risk of oxygen radical formation. It remains to be determined whether chlororespiration (a cyanide-sensitive respiratory pathway in chloroplasts, branching off from the photosynthetic pathway at the level of the plastoquinone pool) is involved in the putative increased rate of oxygen radical formation in young leaves. Garab et al. (1989) found evidence for the occurrence of this pathway in tobacco, especially in young leaves. This might explain the high activity of chloroplastic Cu/ZnSOD in young tobacco leaves. The fact that the initially high activity of the Cu/ ZnSODs and of endogenous MnSOD decreases to much lower or even undetectable levels when the leaves mature may indicate that these enzymes have a relatively high turnover rate.

It may be hypothesized that the expression of the endogenous Mn- and Cu/ZnSODs is induced by superoxide radicals, or by a more mobile "signal molecule" produced by reaction with superoxide. A decline in the production of superoxide, due to a decreased metabolic activity in mature leaves, would limit the expression of these SODs. Overex- pression of MnSOD in the chloroplasts would likewise reduce the concentration of the signal molecule and would thus bring about a similar decrease in the expression of the Cu/ZnSODs and of endogenous MnSOD. This would im- ply that the hypothetical signal molecule cannot be hydrogen peroxide, because the concentration of that component would be higher in transgenic than in nontransgenic plants. It must be noted, however, that Tsang et al. (1991), who studied induction of SODs in N. plumbaginifolia at the mRNA level, observed that stress conditions can be imposed leading only to induction of cytosolic Cu/ ZnSOD (e.g. during illumination at high temperatures). Combined with our data this suggests that different mechanisms for induction of SODs may exist, depending on the developmental stage of the leaf and on the type of imposed stress.

The abundance of FeSOD is not strongly dependent on leaf age or position (Fig. 7) but increases with increasing light intensity received during growth, at least above a threshold value of 90 Fmol m-' s-' (Fig. 5). Tsang et al. (1991) interpreted their results as indicating that light- dependent oxidative stress causes induction of FeSOD. This is not inconsistent with our data, although we found no clear evidence for the involvement of superoxide in the induction of FeSOD. If this were the case, one would expect that overexpression of MnSOD would also suppress the induction of FeSOD. This was not consistently observed (Fig. 5).

The activities of APx, DHAR, GR, and MDHAR were not affected by the overexpression of MnSOD. As for APx, this observation is at variance with data from Sen Gupta et al. (1993b), who noted a 3-fold increase in activity of APx in

transgenic tobacco plants overexpressing SOD in the chloroplasts. However, these authors used chloroplastic Cu/ ZnSOD instead of MnSOD, which was used in this study. We found that transgenic plants overexpressing FeSOD in the chloroplasts of var SR1 did not show a change in APx activity either (our unpublished data). The question of how two functionally similar, although structurally distinct, enzymes can have such dissimilar effects on the activity of other enzymes is interesting but as yet unanswered.

Enhancement of M V Tolerance by Overexpressed MnSOD

The overexpressed MnSOD did not significantly enhance the tolerance of the plants to MV when the plants were grown at low-light intensities (40 or 90 pmol m-* s-'). A simple explanation would be that in plants grown at low- light intensities, the oxygen-radical-scavenging capacity is limited by the low concentrations of other components involved in oxygen-radical scavenging. Under those conditions overexpression of MnSOD would not have much effect. The increase in the concentration of these other components, in response to higher-light intensities received during growth, would enable MnSOD to exert a beneficia1 effect on the MV tolerance. The data shown in Figure 2 and Tables I and I1 are in agreement with this notion. In var PBD6 most enzymes and substrates known to be involved in oxygen-radical scavenging were present at relatively high levels only in plants grown at 135 pmol m-2 s-l (Table I). The effect of overexpressed MnSOD on the tolerance to MV was significant only in these plants (Fig. 2). In var SR1, plants grown at 90 pmol mP2 s-' had somewhat elevated levels of ascorbate, DHAR, and MDHAR, compared with plants grown at 40 pmol m-' s-' (Table II), but this was not accompanied by an increase in the protection provided by overexpressed MnSOD against MV. Apparently, raising the levels of these components was not sufficient to increase the protection provided by MnSOD against MV. An additional increase of these, as well as other, components occurred after growth at 135 pmol m-' s-' (Table 111, and in these plants the transgenic MnSOD did have a positive, albeit weak, effect on MV tolerance (Fig. 2). Plants grown at 800 pmol m-'s-' exhibited an even greater increase in the levels of most antioxidant enzymes and substrates compared with plants grown at 135 pmol m-'s-' (Table 11), with the exception of GR and chloroplastic and cytosolic Cu/ZnSOD, none of which were correlated with MV tolerance (see below). In agreement with this, MnSOD overexpression caused a more substantial enhancement of MV tolerance in plants grown at 800 pmol m-' s-l than in plants grown at 135 pmol mP2 s-'. In conclusion, our results suggest that enhancement of the oxidative-stress tolerance by overexpression of MnSOD requires that other antioxidant enzymes and substrates be present at relatively high levels. In the present experiments this requirement was met by growing the plants at high-light intensities.

As mentioned in the introduction, transgenic plants of var PBD6, overexpressing MnSOD in the chloroplasts, also exhibited an enhanced ozone tolerance. Interestingly, the decrease in ozone injury observed in these plants was





likewise light dependent, ranging between 45 and 75%. The largest decrease was observed after acclimatization (4 d) and ozone treatment (7 d) at high-light intensities (Van Camp et al., 1994). The decrease in MV-induced ion leakage that we observed in var PBD6 after growth at 135 pmol

(around 60%, Fig. 2A) was within the range ob- m-z s-l

served by Van Camp et al. (1994).

Differential Attenuation of Two Types of MV-lnduced Oxygen-Radical Damage by Overexpressed MnSOD

Taking average values, we found that in transgenic plants of var SR1, the enhancement of MV tolerance was expressed only in the conductance measurements (Fig. 2C) and not in the fluorescence measurements (Fig. 2D). One might argue that the MV-induced decrease in aI1 arises because reduced MV, after having received an electron from PSI, occasionally diffuses toward the reaction center of PSII and inactivates it without involvement of superoxide. SOD would be unable to attenuate this type of damage. However, this argument can be dismissed on several grounds. First, MnSOD overexpressed in the chloroplasts of var PBD6 caused a pronounced and significant attenuation of the MV-induced decrease in QII in at least two independent transgenic lines (Bowler et al., 1991; Slooten et al., 1992). To a lesser extent this was also the case in a third transgenic line that was used in the present work. Second, overexpression of chloroplastic FeSOD from Arabidopsis thaliana in the chloroplasts of var SRl attenuated the decrease in QII as well as the ion leakage induced by MV (data not shown). This indicates that these two types of damage are both due to superoxide but can be differentially attenuated, depending on the identity of the overexpressed enzyme as well as on the target variety. Interestingly, experiments currently in progress suggest that in transgenic plants of var SR1, the overexpressed FeSOD has a higher membrane affinity than the overexpressed MnSOD (our unpublished data).

The major site of electron donation to MV has been shown to be the Fe-S center B in PSI (Fujii et al., 1990). From there the oxygen radicals must propagate, on the one hand out of the chloroplasts and to the cell membrane, resulting in ion leakage, and on the other hand to the reaction center of PSII, resulting in loss of QI1. From Figure 2 it appears that in var SRl the overexpressed MnSOD interferes more extensively with the former pathway of damage propaga- tion than with the latter. According to recent data, the chloroplasts possess two separate oxygen-radical-scavenging systems: a stromal system comprising APx, DHAR, GR, and MDHAR, and a membrane-bound system that is functionally linked to PSI and comprises at least .APx (Miyake and Asada, 1992, 1994). It seems reasonable to speculate that the stromal system intercepts the oxygen radicals spreading outward, thus reducing ion leakage, but has little influence on the spreading of oxygen radicals along or within the membrane, leading to PSII inactivation. The membrane-bound system, which is linked to PSI, would intercept the toxic oxygen species very close to the site of formation, and hence would prevent both kinds of damage. To our knowledge there are no data available concerning

the spatial distribution of either endogenous 01' transgenic SODs within the chloroplast. However, to enhance the scavenging capacity of the membrane-bound system, the transgenic SOD must probably meet two conditions: it must be membrane bound and it must be bocnd in close association with PSI. We speculate that in var SRl, at least the second condition is not sufficiently met, siich that the overexpressed MnSOD enhances primarily t he oxygen- radical-scavenging capacity of the stromal sysi em.

Correlation of Abundance of Antioxidants with M V Tolerance

The abundances of FeSOD, DHAR, and MIIHAR were correlated predominantly with attenuation of IvlV-induced ion leakage. The location of FeSOD in the chioroplasts is not known, but DHAR and MDHAR are stronial enzymes (Asada and Takahashi, 1987). APx, on the other hand, occurs in the chloroplasts in a membrane-bourtd as well as in a stromal form, the membrane-bound form being functionally linked to PSI (Miyake and Asada, 1992,1994). This enzyme was, at least in var SRl, correlated Mith attenuation of MV-induced ion leakage as well as MV-induced loss of QII. AI1 this supports our interpretation, given in the preceding paragraph, that in var SRl the o1,erexpressed MnSOD enhances primarily the oxygen-radica -scavenging capacity of the stromal system.

In transgenic plants of var SRl the activity of overexpressed MnSOD was significantly correlated with attenuation of MV-induced damage under a11 conditi ons studied. This was surprising because, at least in plants Srown at the two lowest light intensities, transgenic plants were on the average no more tolerant of MV, and sometimes less tolerant of MV, than control plants (Fig. 2). This suggests in fact that low levels of overexpressed MnSOD è ecreased the tolerance of the leaf discs to MV, and that Iiigher levels increased this tolerance. As outlined previous Ly (Bowler et al., 1991), the reason may be that during illumination of MV-treated leaf discs, low levels of MnSOD ,ire sufficient to allow generation of H,O, at fairly high rates but are not sufficient to deplete the superoxide that arise 5 from reoxi- dation of photoreduced MV. Under those conditions, H,O, and 0,- may react together in a metal-catalyzed Haber- Weiss reaction. This would result in the forriation of the hydroxyl radical, OH (see introduction), and in enhancement rather than attenuation of MV-induced tlamage. This deleterious effect could be prevented either b y an increase in the leve1 of overexpressed MnSOD or FeSOD (resulting in more complete depletion of O J or by an increase in the rate of H,O, scavenging. This may explain why in transgenic plants of var SRl several enzymes exhibited significant correlations with attenuation of MV-induced decrease in aI1 in plants grown at relatively low-light intensities (correlation type 3), in contrast to nontransgmic plants.

Chloroplastic CuZnSOD was always negttively correlated with MV tolerance, in some cases significantly so (Tables 111 and IV). Apparently the leaves wii h higher MV tolerance were physiologically somewhat older and had lost more of their chloroplastic Cu/ZnSOD tl- an the leaves with lower MV tolerance. The activities of cytosolic Cu/





ZnSOD and GR were not significantly correlated with MV- induced damage, in spite of the fact that a severalfold variation occurred in the activity of these enzymes in individual experiments (similar to the variations shown in Fig. 8). This could mean either that these enzymes are not involved in oxygen-radical scavenging or, more likely, that they are involved but do not limit the oxygen-radical- scavenging capacity under the growth and assay conditions used in this work. As for GR, this is at variance with the results of Aono et al. (1993), who reported that tobacco plants overexpressing GR in the chloroplasts exhibited an increased tolerance to MV, as judged by visual inspection of MV-induced pigment bleaching. It may be noted, however, that GR overexpression in poplar chloroplasts resulted in an increased size of the glutathione pool in the leaves (Foyer et al., 1994). It is not clear to what extent this effect may have been responsible for the results obtained by Aono et al. (1993).

CONCLUDINC REMARKS

It cannot be excluded that other factors besides the components studied here affect the tolerance of the leaves to MV. Consequently, the correlations discussed above cannot be used as evidence for causal relationships. On the other hand, such relationships may exist. It has been shown in maize inbreeds that lines that have elevated levels of both SOD and GR exhibit an increased tolerance to drought as well as to MV (Malan et al., 1990). A mutant of Conyza bonariensis with elevated levels of SOD, APx, and GR had an increased tolerance to MV (Shaaltiel and Gressel, 1986) as well as to photoinhibition (Jansen et al., 1989). The present results suggest, as indicated above, that overexpression of SOD resulted in an increased tolerance to MV only if other components were also present at high levels. The correlation studies suggest that APx, DHAR, and MDHAR are among those components. Therefore, it will be of interest to attempt to further enhance the oxidative- stress tolerance of the plants by overexpressing, in addition to SOD, one or more of the latter three enzymes. The strongest case in this respect is that for APx: for this enzyme, correlations with MV tolerance were observed in both varieties, SR1 and PBD6. The correlations of the ascorbate and glutathione pools with MV tolerance, observed in both tobacco varieties, suggest that it will also be of interest to enlarge the pools of these components. In the case of poplar it has been shown that enlarged pools of glutathione (Foyer et al., 1994) occur after overexpression of GR in the chloroplasts.

ACKNOWLEDCMENT

The skillful technical assistance of S. Vandenbranden is grate- fully acknowledged.

Received August 22, 1994; accepted November 14, 1994. Copyright Clearance Center: 0032-0889/95/107/0737/14,

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Factors Affecting the Enhancement of Oxidative Stress Tolerance in Transgenic Tobacco Overexpressing...

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