Plant Physiol. (1998) 116: 173-181

Oxidative Damage in Pea Plants Exposed to Water Deficit or Paraquat¹

Iñaki Iturbe-Ormaetxe, Pedro R. Escuredo, Cesar Arrese-Igor, and Manuel Becana^*

Departamento de Nutrición Vegetal, Estación Experimental de Aula Dei, Consejo Superior de Investigaciones Científicas, Apdo 202, 50080 Zaragoza, Spain (I.I.-O., P.R.E., M.B.); and Departamento de Ciencias del Medio Natural, Universidad Pública de Navarra, 31006 Pamplona, Spain (C.A.-I.)

	ABSTRACT

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The application of a moderate water deficit (water potential of 1.3 MPa) to pea (Pisum sativum L. cv Lincoln) leaves led to a 75% inhibition of photosynthesis and to increases in zeaxanthin, malondialdehyde, oxidized proteins, and mitochondrial, cytosolic, and chloroplastic superoxide dismutase activities. Severe water deficit (1.9 MPa) almost completely inhibited photosynthesis, decreased chlorophylls, -carotene, neoxanthin, and lutein, and caused further conversion of violaxanthin to zeaxanthin, suggesting damage to the photosynthetic apparatus. There were consistent decreases in antioxidants and pyridine nucleotides, and accumulation of catalytic Fe, malondialdehyde, and oxidized proteins. Paraquat (PQ) treatment led to similar major decreases in photosynthesis, water content, proteins, and most antioxidants, and induced the accumulation of zeaxanthin and damaged proteins. PQ decreased markedly ascorbate, NADPH, ascorbate peroxidase, and chloroplastic Fe-superoxide dismutase activity, and caused major increases in oxidized glutathione, NAD⁺, NADH, and catalytic Fe. It is concluded that, in cv Lincoln, the increase in catalytic Fe and the lowering of antioxidant protection may be involved in the oxidative damage caused by severe water deficit and PQ, but not necessarily in the incipient stress induced by moderate water deficit. Results also indicate that the tolerance to water deficit in terms of oxidative damage largely depends on the legume cultivar.

	INTRODUCTION

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In natural conditions crops are often exposed to various environmental stresses that decrease production. At the whole-plant level, the effect of stress is usually perceived as a decrease in photosynthesis and growth, and is associated with alterations in C and N metabolism. At the molecular level, the negative effect of stress on leaves may be in part a consequence of the oxidative damage to important molecules, as a result of the imbalance between production of activated O₂ and antioxidant defenses (Foyer et al., 1994).

This hypothesis is very plausible because chloroplasts are a major source of activated O₂ in plants (Asada and Takahashi, 1987; Foyer et al., 1994), and because antioxidants, which may play a critical role in preventing oxidative damage, are greatly affected by environmental stress (Bowler et al., 1994). In chloroplasts the superoxide radical (O₂) is produced by photoreduction of O₂ at PSI and PSII, and singlet O₂ is formed by energy transfer to O₂ from triplet excited state chlorophyll (Asada and Takahashi, 1987). H₂O₂ can originate, in turn, from the spontaneous or enzyme-catalyzed dismutation of O₂. Other subcellular compartments of leaves, such as peroxisomes and mitochondria, are also potential generators of O₂ and H₂O₂, mainly as a consequence of electron transport and enzymic reactions (Del Rio et al., 1992). Fortunately, in optimal conditions leaves are rich in antioxidant enzymes and metabolites and can cope with activated O₂, thus minimizing oxidative damage. Antioxidants include ASC, GSH, carotenoids, -tocopherol, SOD, catalase, and the enzymes of the ASC-GSH cycle (Foyer et al., 1994). Antioxidant metabolites are present in chloroplasts at very high concentrations (10-20 mm ASC and 1-4 mm GSH), and, apart from their obvious role as enzyme substrates, they can react chemically with almost all forms of activated O₂ (Halliwell and Gutteridge, 1989). The importance of ASC and GSH as antioxidants is underlined by the results of a recent study showing that overexpression of GR in chloroplasts doubles the concentrations of ASC and GSH in leaves and confers increased resistance to oxidative stress (Foyer et al., 1995).

An aspect frequently overlooked in studies on free radicals and antioxidants is the pro-oxidant properties of Fe. The so-called "catalytic Fe" catalyzes the decomposition of H₂O₂ and lipid hydroperoxides to hydroxyl and alkoxyl radicals, respectively. Both free radicals are extremely cytotoxic and promote lipid peroxidation (Halliwell and Gutteridge, 1989). Lipid peroxidation is commonly taken as an indicator of oxidative stress. Lipid peroxides are quantified by the TBA test, which is easy to perform and allows the results to be conveniently expressed as TBARS. However, results from the TBA test need to be compared with more specific assays. Because the hydroxyl radical and other highly reactive species can oxidize proteins in addition to lipids, the suitability of lipid peroxidation for diagnosing oxidative stress can be tested by measuring protein oxidation in leaves exposed to adverse conditions.

In this paper we report the results of detailed measurements of the physiological status, oxidant damage, catalytic Fe, and antioxidant enzymes and metabolites of pea (Pisum sativum L.) leaves exposed to two types of stress of agronomic interest, water deprivation and PQ treatment. Two intensities of water stress were applied for comparison with our previous work using a different pea cultivar (Moran et al., 1994), and PQ, a potent herbicide that exacerbates O₂ radical production, was used as a model to study oxidative stress (Asada and Takahashi, 1987; Bowler et al., 1994), especially those aspects for which little or no information is currently available.

	MATERIALS AND METHODS

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Plant Material and Stress Treatments

2

1

Physiological measurements and harvest of leaves for all treatments were made when plants were at the late-vegetative growth stage. Leaves to be used for biochemical determinations were frozen in liquid N₂ immediately after harvest and stored at 80°C until required for analyses.

Water Deficit

PQ

2

1

Control (C₁) for Water Deficit

Control (C₂) for PQ

2

1

Physiological Parameters

Catalase, Enzymes of the ASC-GSH Cycle, and Related Metabolites

ASC was extracted from 0.5 g of leaves with 5 mL of 5% (w/v) metaphosphoric acid and quantified by its ability to reduce Fe³⁺ at acidic pH (Law et al., 1983). GSH and GSSG were extracted from 0.5 g of leaves with 5 mL of 5% (w/v) sulfosalicylic acid. The homogenate was filtered and centrifuged, and the concentrations of GSH plus GSSG and GSSG were determined in two aliquots of the supernatant essentially by the method of Law et al. (1983). Pyridine nucleotides were extracted from 30 mg of leaves with 1 mL of 0.1 m NaOH (reduced forms) or 5% (w/v) TCA (oxidized forms) (Gogorcena et al., 1995) and were quantified by an enzymatic-cycling method (Matsumura and Miyachi, 1980).

SOD: Activity, Isoforms, and Subcellular Location

Leaf organelles were purified by differential and Percoll density-gradient centrifugation, according to published protocols (Palma et al., 1986; Sandalio et al., 1987; Struglics et al., 1993). The pellet obtained after the initial centrifugation at 2,200g for 5 min was resuspended in wash medium and layered on top of a discontinuous Percoll gradient (Sandalio et al., 1987). The gradient was centrifuged at 13,000g for 35 min, and the two higher-density bands containing chloroplasts were recovered (Palma et al., 1986). The supernatant obtained after the initial centrifugation was centrifuged again at 2,200g for 5 min. The pellet was discarded and the supernatant was centrifuged at 12,000g for 15 min. The final pellet was resuspended in wash medium, applied to a Percoll gradient, and centrifuged as before (Sandalio et al., 1987). The bands corresponding to mitochondria and peroxisomes were recovered, and the mitochondria were repurified on a second Percoll gradient as described by Struglics et al. (1993).

All organelles banded in the gradients at their expected equilibrium densities (Palma et al., 1986; Sandalio et al., 1987; Struglics et al., 1993). Isolated organelles were washed twice and broken by resuspension in SOD extraction medium. After clearing the samples by centrifugation, the composition of SOD isoforms was determined by native PAGE as indicated above. Purity of organelles was assessed using the following markers: chlorophyll for chloroplasts (Lichtenthaler, 1987), Cyt c oxidase for mitochondria (Schnarrenberger et al., 1971), and catalase for peroxisomes (Aebi, 1984).

Catalytic Fe

Oxidative Damage of Lipids and Proteins

After the reaction was stopped by cooling the samples in an ice bath, two protocols were followed. For determination of TBARS, the chromogen formed was extracted by adding 1.5 mL of 1-butanol, the tubes were vigorously shaken, the organic (upper) phase was separated by low-speed centrifugation, and the absorbance of TBARS was read at 532 nm. For MDA determination, the chromogen was extracted by adding 0.4 mL of 1-butanol. The organic phase was obtained as before, the process was repeated, and the pooled organic phase was evaporated under N₂ and kept at 80°C until use. The samples were resuspended in 100 µL of HPLC solvent, and an aliquot of 20 µL was immediately injected. The HPLC equipment (Waters) included a photodiode-array detector (model 996) controlled by Millennium software. The (TBA)₂-MDA adduct was resolved on an Ultrasphere C₁₈ column (5 µm, 25 cm × 4.6 mm; Beckman) and was eluted with 5 mm potassium phosphate buffer (pH 7.0) containing 15% acetonitrile and 0.6% tetrahydrofuran (Draper et al., 1993). The flow rate was 1 mL min¹ and detection was at 532 nm.

Calibration curves were made using 1,1,3,3-tetraethoxy-propane (Sigma) in the range of 0 to 2 nmol (TBARS) and 0 to 1 nmol (MDA). Tetraethoxypropane is stoichiometrically converted into MDA during the acid-heating step of the assay. Recovery experiments were performed by adding 0.25 to 2 nmol (for TBARS) or 0.2 to 1 nmol (for MDA) of tetraethoxypropane at the moment of extraction and by taking into account the amounts of endogenous TBARS.

Proteins were extracted from 0.5 g of leaves as described in detail (Moran et al., 1994), and oxidative damage was quantified as total protein carbonyl content by reaction with 2,4-dinitrophenylhydrazine after the removal of possible contaminating nucleic acids with 1% (w/v) of streptomycin sulfate (Levine et al., 1990).

	RESULTS

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Photosynthesis, Water Status, and Pigment Composition

A further decrease of _w to 1.9 MPa (D₂ plants) led to a reduction in leaf water content to only 78% and to the almost complete suppression of net photosynthesis. This was accompanied by decreases of 92% in stomatal conductance and of 84% in transpiration. Other general indicators of leaf activity, such as dry weight, soluble protein, chlorophylls a and b, -carotene, neoxanthin, and lutein, were reduced by 21 to 38% (Tables I and II). The effect of severe water deficit was more pronounced on the xanthophyll cycle components. In leaves of D₂ plants the contents of antheraxanthin and zeaxanthin increased 2.1- and 5.7-fold, respectively, at the expense of violaxanthin, which decreased by 61%. Hence, the epoxidation index declined to 0.57 in D₂ plants.

The same parameters were also measured in plants of the same age sprayed with PQ 6 h before sampling to assess the damage inflicted on the photosynthetic machinery. Leaves of PQ plants had no measurable photosynthesis and contained only 78% water, exactly the same as for D₂ plants (Table I). Treatment with PQ had no effect on leaf dry weight but lowered the contents of soluble protein, chlorophylls a and b, -carotene, neoxanthin, and lutein by 20 to 30%, and that of violaxanthin by 72%. In contrast, the content of antheraxanthin was doubled and that of zeaxanthin reached 0.46 µg cm² (Table II). Consequently, the epoxidation index declined from 0.97 to 0.56 in PQ-treated plants.

Antioxidant Enzymes

Five SOD isoforms were separated in extracts of pea leaves by nondenaturing PAGE (Fig. 1). These isoforms were identified and labeled by increasing mobility as Mn (no inhibition by KCN or H₂O₂), Fe-1, and Fe-2 (no inhibition by KCN but inhibited by H₂O₂), and CuZn-1 and CuZn-2 (inhibited by both KCN and H₂O₂). Densitometric analysis of activity gels showed that the Mn, Fe-1, Fe-2, CuZn-1, and CuZn-2 isoforms accounted for approximately 60, 6, 2, 7, and 25% of total SOD activity, respectively. In an attempt to localize the Fe-SOD isoforms at the subcellular level, leaves were fractionated using Percoll-density gradients. Cross-contamination between organelles was evaluated using marker molecules. Chloroplasts were contaminated by <2% with mitochondria and peroxisomes, and mitochondria were contaminated by <15% with chloroplasts and peroxisomes. Comparison of SOD composition in the leaf extract, the mitochondria, and the two fractions of chloroplasts indicated that the Mn and CuZn-2 isoforms are located in the mitochondria and chloroplasts, respectively, and strongly suggested that the CuZn-1 isoform, which is most abundant in the unfractionated soluble extract, is located in the cytosol but also in the mitochondria (Fig. 2). All of these results are consistent with earlier studies on SOD subcellular localization in pea leaves (Palma et al., 1986; Sandalio et al., 1987; Sen Gupta et al., 1993).

View larger version (30K): [in this window] [in a new window]   Figure 1. Separation on nondenaturing activity gels of SOD isoforms from pea leaves subjected to water deficit or treated with PQ. In each case 45 µg of protein per lane was loaded. Identification of SOD isoforms was performed by pre-incubation of gels with 3 mm KCN or 5 mm H2O2 for 60 min prior to activity staining. Plant treatments are as described in Table I.

View larger version (40K): [in this window] [in a new window]   Figure 2. Subcellular localization of SOD isoforms in pea leaves. E, Whole-leaf extract; Chl1 and Chl2, fractions 1 and 2 of chloroplasts; and M, mitochondria. Lanes were loaded with 40 to 60 µg of protein.

Very recently, Donahue et al. (1997) found two Fe-SODs in pea leaves, although these activities were only unmasked when gels were preincubated with KCN to inhibit the CuZn-SODs. In our case, the Fe-SODs exhibited the expected relative mobilities between those of Mn- and CuZn-SODs (Figs. 1 and 2). At least one Fe-SOD is located in the chloroplasts (Fig. 2), which is also in agreement with previous work on Fe-SOD localization in other higher plants (Bowler et al., 1994). We could not detect Fe-SOD in mitochondria (Fig. 2) or in peroxisomes (data not shown).

According to the isoform patterns observed for each treatment, the 27% increase of total SOD activity in D₁ plants can be ascribed to the increase in the Mn- and CuZn-SODs. In D₂ plants total SOD activity, as well as the activities of the CuZn-isoforms, declined back to control or lower levels, but Mn-SOD activity further increased (Fig. 1; Table III). The overall decrease of total SOD activity in PQ plants may result from the inhibition of Fe-SODs (Fig. 1) and CuZn-SODs. The inhibition of both CuZn-SODs was observed in some leaf samples (data not shown) but not in others (Fig. 1).

Antioxidant Metabolites and Nucleotides

Leaves of PQ plants had 87% less ASC, 73% less GSH, and 43% more GSSG than C₂ plants (Table IV). Consequently, the GSH/GSSG ratio declined from 14.2 to 2.65, and the GSH/(GSH plus GSSG) ratio from 93 to 73%. The effect of PQ on the pyridine nucleotide content was surprisingly variable. Leaves exposed to the herbicide had 46% less NADP⁺ and 90% less NADPH than untreated leaves, whereas the levels of NAD⁺ and NADH were 60% greater (Table IV).

Catalytic Fe and Oxidative Damage of Biomolecules

View larger version (22K): [in this window] [in a new window]   Figure 3. Catalytic Fe (A), oxidized lipids (B), and oxidized proteins (C) in pea leaves subjected to water deficit or treated with PQ. Catalytic Fe is expressed as A532 because actual concentrations are dependent on the assay conditions and on the form in which Fe is present (free or bound to chelates). In our conditions (25-µL samples, incubation at 37°C for 120 min), 0.2 absorbance units correspond to approximately 1 µm of catalytic Fe. The content of lipid peroxides is expressed as nanomoles of TBARS () or MDA () per gram dry weight, and that of oxidized proteins as micromoles of total carbonyl groups per gram dry weight. Values are means ± se of six to nine independent plant samples. Plant treatments and statistical analysis for each parameter are as described in Table I.

Lipid peroxidation in leaves was measured spectrophotometrically as the content of TBARS and by HPLC as the content of MDA (Fig. 3B). For a closer comparison, we used in both cases identical extraction medium containing butyl hydroxytoluene to avoid artifactual formation of peroxides during tissue grinding. The same extracts were also used in the two assays. Recovery of standard MDA added to plant tissue prior to extraction was 88% for the TBARS method and 99% for the HPLC method. According to the TBA test, leaves of C₁ and D₁ plants had approximately 150 nmol of MDA equivalents per gram dry weight, but this value was 23% greater in D₂ plants. Using the same units, levels of TBARS in PQ plants were not found to differ significantly from those of C₂ plants (Fig. 3B).

The specific quantification of MDA by HPLC allowed us to ascertain that MDA is a major product of lipid peroxidation in pea leaves and to eliminate possible interferences with other TBARS. Identification of the peak of the (TBA)₂MDA adduct was based on the retention time and visible spectrum, which were obtained on-line with the photodiode-array detector (Fig. 4). Both parameters were identical to those of the adduct made from authentic MDA. Levels of MDA were substantially lower than those of TBARS for all treatments. The content of MDA per gram dry weight of leaves in D₁ and D₂ plants was 24 and 45% greater than in C₁ plants, but there was no difference between that of PQ and C₂ plants (Fig. 3B).

View larger version (17K): [in this window] [in a new window]   Figure 4. Analysis of MDA in pea leaves by HPLC with diode-array detection. The chomatogram was obtained at 532 nm and visible spectra (400-600 nm) were recorded on-line during the HPLC analysis. Inset, Visible spectrum of the (TBA)2-MDA complex at the retention time (approximately 6.7 min) of the peak, showing a peak at 532 nm and a shoulder (sh) at 495 nm.

The amount of oxidatively modified proteins was estimated by the carbonyl assay and expressed on a dry weight (Fig. 3C) or a protein basis, showing a similar trend in both cases for all plant treatments. The leaf content of oxidized proteins per gram dry weight increased by 36, 67, and 30% in D₁, D₂, and PQ plants compared with the controls. The corresponding increases per milligram of protein were 47, 135 and 80%. These observations show that the damage to leaf proteins increases with stress because there is a reduction in the soluble protein content of leaves upon exposure to water deficit or PQ (Table I).

	DISCUSSION

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The application of a moderate water stress to pea plants inhibited photosynthesis by 75%, which was probably attributable at least in part to CO₂ limitation by stomatal closure because there were large decreases in stomatal conductance and transpiration (Table I), but no changes or only small changes in pigments other than those of the xanthophyll cycle (Table II). In contrast, the application of a severe water stress caused the virtual disappearance of photosynthesis, a marked increase in antheraxanthin and zeaxanthin, and a substantial reduction in all of the other pigments. In this case, the inhibition of photosynthesis was probably due to both CO₂ limitation and damage of the photosynthetic apparatus. This damage may arise from the lowering of antioxidant protection (Tables III and IV) and from an insufficient capacity of D₂ plants to dissipate excess excitation energy through the xanthophyll cycle. The sustained increase of zeaxanthin in D₁ and D₂ plants is consistent with previous reports on Nerium oleander and cotton plants, and is associated with a decrease in photochemical efficiency; the increase in zeaxanthin also confirms that this pigment is a sensitive indicator of plant stress (Demmig-Adams and Adams, 1993; Smirnoff, 1993). Likewise, the treatment with PQ caused almost identical decreases in photosynthesis, water content, and soluble protein to those of D₂ plants, as well as the accumulation of similarly high levels of antheraxanthin and zeaxanthin (Tables I and II).

In D₁ plants there were already symptoms of moderate oxidative stress, such as an increase in total and CuZn-SOD activity and in the contents of oxidized proteins and MDA (Table III; Figs. 1 and 3C). Leaves of D₁ plants retained most of their antioxidant capacity and had virtually no catalytic Fe, which may explain why oxidative damage in D₁ plants was incipient compared with D₂ plants. Thus, in D₂ and PQ plants there was a substantial decrease in antioxidant defenses along with an increase in catalytic Fe and protein damage (Tables III and IV; Fig. 3, A and C).

There were several indications that the functioning of the ASC-GSH cycle was limited or even impaired in D₂ and PQ plants. In both cases there was a shortage of photosynthetic NADPH, which is required for GR activity (Fig. 5). Total APX activity and ASC content showed an identical moderate reduction (40%) in D₂ plants; both parameters decreased in parallel, but to a much larger extent (83-87%), in PQ plants (Tables III and IV). The identical decline in APX activity and ASC induced by severe water deficit or PQ strongly suggests that the loss in enzyme activity can be ascribed at least in part to substrate depletion (Fig. 5). The decrease of ASC in D₂ and PQ plants may be due to its direct destruction by O₂ and derived species, but also to the consumption of ASC for zeaxanthin synthesis and tocopherol regeneration (Smirnoff, 1993). Another factor indicating a limited operation of the ASC-GSH cycle in D₂ and PQ plants is the large decreases in GSH (70%) and GR activity (52%). The decrease of GSH in PQ plants may be explained in part by oxidation of GSH to GSSG by activated O₂, whereas in D₂ plants the decrease is probably a result of the inhibition of GSH synthesis, a phenomenon consistent with the general inhibition of protein synthesis and high turnover rates that occur following the gradual imposition of water stress (Fig. 5). The lack of GSSG accumulation in D₂ plants is also consistent with the observation that GSH oxidation does not occur in plants until the loss of water content is very near the limit of survival (Smirnoff, 1993).

View larger version (21K): [in this window] [in a new window]   Figure 5. Scheme representing the possible sequence of events leading to oxidative damage in pea leaves subjected to severe water stress or treated with PQ.

Water deficit and PQ are known to enhance O₂ production in the chloroplasts (Asada and Takahashi, 1987; Price et al., 1989). In this respect, the induction of chloroplastic and cytosolic CuZn-SODs in D₁ plants (Fig. 1) may be interpreted as a response to augmented O₂ generation in both cellular compartments. On the other hand, the inhibition of chloroplast Fe-SOD but not of the corresponding CuZn-SOD in PQ plants (Fig. 1) suggests a greater lability of the former to PQ-imposed stress. In this scenario Fe-SOD would be inhibited by PQ but CuZn-SOD activity would be maintained or inhibited, depending on the level of PQ reaching the chloroplasts. On the other hand, Mn-SOD activity was not inhibited in PQ plants (Fig. 1), and this could reflect sustained O₂ production in mitochondria. The pool of NAD⁺ plus NADH increased by 60% in PQ plants, but the NAD⁺/NADH ratio remained constant (Table IV). This suggests that PQ does not affect mitochondrial respiration, in agreement with Tsang et al. (1991).

The toxicity caused by the increased generation of O₂ and H₂O₂ may be further aggravated by the accumulation of catalytic Fe at moderate levels in D₂ plants and at high levels in PQ plants (Fig. 3A). This catalytic Fe may be released from Fe-proteins such as phytoferritin, which occurs in the chloroplasts (Briat et al., 1995) following their oxidative attack by O₂ and/or H₂O₂. Catalytic Fe may interact in turn with O₂ and H₂O₂ through a Haber-Weiss reaction to yield hydroxyl radicals (Fig. 5). The large increase of catalytic Fe in PQ plants further strengthens the hypothesis that the toxicity of PQ is mediated by Fe. Indirect support for this hypothesis comes from the observations that there is generation of hydroxyl radicals in PQ plants (Babbs et al., 1989) and that pretreatment of pea plants with the Fe chelator desferrioxamine lessens PQ-induced damage (Zer et al., 1994).

Finally, it is clear that the response of antioxidants to a water deficit depends on the severity of stress and on the species and age of plants (for example, see Smirnoff and Colombé, 1988; Tanaka et al., 1990; Mittler and Zilinskas, 1992, 1994). But there is also a differential sensitivity of cultivars to water deficit with respect to the induction of oxidative stress. These differences can be illustrated by comparison with our previous work (Moran et al., 1994). At the age and intensity of stress equivalent to D₁ plants, pea cv Frilene showed a reduction of 72 to 85% in catalase, DR, and GR activities, and increases of 180 to 240% in oxidized lipids and proteins (Moran et al., 1994). In cv Lincoln, however, the same activities decreased by <13% (Table III) and the increases in oxidative damage ranged from 0 to 36% (Fig. 3, B and C). In cv Frilene low-molecular-mass Fe (used as an estimate of catalytic Fe) increased 2.4-fold with water deficit, whereas in cv Lincoln catalytic Fe was not detectable. These observations indicate that, at the biochemical level, cv Lincoln is more tolerant to water deficit than is cv Frilene; the decrease in antioxidant protection and the increase in oxidative damage are closely related; and the accumulation of catalytic Fe is not the only factor likely to be involved in the initiation of oxidative damage, despite the requirement of a catalytic metal for protein oxidation (Stadtman, 1992). The last contention is substantiated by the fact that lipid peroxidation increases in leaves or other plant tissues subjected to water deficit (Moran et al., 1994; Gogorcena et al., 1995; this work) but not to other types of stress, despite the increase in catalytic Fe (Escuredo et al., 1996; Gogorcena et al., 1997). However, the duration of exposure to catalytic Fe may be an important factor. Lipids and proteins of leaves were probably exposed to catalytic Fe for longer periods in D₂ plants (days) than in PQ plants (hours). This may have contributed to the build-up of oxidized lipids and proteins in D₂ plants, especially if water stress decreases the efficiency in the mechanisms involved in the repair (de novo synthesis) and/or the degradation (lipases and proteases) of damaged molecules.

	FOOTNOTES

   Received May 22, 1997; accepted September 19, 1997.

	ABBREVIATIONS

Abbreviations: APX, ascorbate peroxidase. ASC, ascorbate. CuZn-, Fe-, or Mn-SOD, SODs containing Cu and Zn, Fe, or Mn as metal cofactors. DR, dehydroascorbate reductase. GR, glutathione reductase. GSSG, oxidized glutathione. MDA, malondialdehyde. MR, monodehydroascorbate reductase. PQ, paraquat. SOD, superoxide dismutase. TBA, 2-thiobarbituric acid. TBARS, 2-thiobarb-ituric acid-reactive substances. _w, water potential.

	ACKNOWLEDGMENTS

We thank Roger B. Austin for helpful comments on the manuscript. Thanks are also due to Manuel A. Matamoros for help with organelle purification, Gloria Rodríguez for growing the plants, and María A. Gracia for HPLC analyses of photosynthetic pigments at Consejo Superior de Investigaciones Científicas (Zaragoza, Spain).

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