JXB Advance Access originally published online on February 13, 2004
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Journal of Experimental Botany, Vol. 55, No. 397, pp. 605-612, March 1, 2004
© 2004 Oxford University Press
Cell and Molecular Biology, Biochemistry and Molecular Physiology |
The effect of Botrytis cinerea infection on the antioxidant profile of mitochondria from tomato leaves
Received 20 August 2003; Accepted 20 November 2003
bieta Ku
niak*
odowska
Department of Plant Physiology and Biochemistry, University of 
ód, 90-237 ód, Banacha 12/16, Poland
* To whom correspondence should be addressed. E-mail: elkuz{at}biol.uni.lodz.pl
Abbreviations: AA, reduced ascorbate; APX, ascorbate peroxidase (EC 1.11.1.11); AOS, activated oxygen species; DHA, dehydroascorbate; DHAR, dehydroascorbate reductase (EC 1.8.5.1); GLDH, L-galactono-
-lactone dehydrogenase (EC 1.3.2.3); GR, glutathione reductase (EC 1.6.4.2); GSH, reduced glutathione, GSH-Px, glutathione peroxidase (EC 1.11.1.9), GSSG, oxidized glutathione; GST, glutathione transferase (EC 2.5.1.18); MDHAR, monodehydroascorbate reductase (EC 1.6.5.4); SOD, superoxide dismutase (EC 1.15.1.1).
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Abstract |
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Infection of tomato leaves with the necrotrophic fungus Botrytis cinerea resulted in substantial changes in enzymatic and non-enzymatic components of the ascorbate–glutathione cycle as well as in superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), glutathione transferase (GST), and L-galactono-
-lactone dehydrogenase (GLDH) activities. In the initial phase of the 5 d experiment CuZn SOD was the most rapidly induced isoform (up to 209% of control), whereas later on its activity increase was not concomitant with the constant total SOD enhancement. Starting from the second day B. cinerea infection diminished the mitochondrial antioxidant capacity by decreasing activities of ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR) as well as declining ascorbate and glutathione contents. This was accompanied by dehydroascorbate (DHA) and oxidized glutathione (GSSG) accumulation that resulted in ascorbate and glutathione redox ratios decreases. The strongest redox ratio decline of 29% for ascorbate and of 34% for glutathione was found on the 3rd and 2nd days, respectively. Glutathione reductase (GR) induction (185% of control 2 d after inoculation) was insufficient to overcome the decreased antioxidant potential of glutathione. Changes in the ascorbate pool size were closely related to the activity of L-galactono--lactone dehydrogenase (GLDH). The activities of two glutathione-dependent enzymes: GSH-Px and GST were increased from day 1 to day 4. These results demonstrated that in B. cinerea–tomato interaction mitochondria could be one of the main targets for infection-induced oxidative stress.
Key words:
: Ascorbate–glutathione cycle, L-galactono--lactone dehydrogenase, glutathione-dependent enzymes.
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Oxidative stress resulting from the imbalance between enhanced activated oxygen species (AOS) production and detoxification is a common feature of a variety of environmental stresses including pathogen attack (Foyer et al., 1994; Wojtaszek, 1997; Smirnoff, 1998). All cellular compartments have mechanisms protecting them from the deleterious effects of AOS generation. In plant mitochondria, assumed to be one of the major sites of AOS production, SODs localized in the intermembrane space (CuZn SOD) and in the matrix (Mn SOD) form the first line of the antioxidative defence protecting against O2.– (Millar et al., 2001; Møller, 2001). It has not been fully elucidated how the resulting H2O2 is scavenged in mitochondria. A prominent role in the antioxidative defence mechanism in plant cells has been attributed to the ascorbate–glutathione cycle. It involves the oxidation and re-reduction of ascorbate and glutathione through the action of four enzymes constituting the cycle: APX, MDHAR, DHAR, and GR. The ascorbate–glutathione cycle, considered to be the major H2O2 detoxification system in chloroplasts, has recently been reported to be operative in mitochondria and peroxisomes as well (Jiménez et al., 1997; Mittova et al., 2000). Moreover, it has been shown that GLDH catalysing the final step of ascorbate biosynthesis in plant cells is localized in mitochondria, additionally indicating that the function of mitochondria is important for the cellular antioxidative defence system (Bartoli et al., 2000). GLDH resides in the inner mitochondrial membrane and uses cytochrome c as an electron acceptor, hence the final oxidation of L-galactono-
-lactone to ascorbate is coupled to the mitochondrial electron transport chain between complexes III and IV (Bartoli et al., 2000). The contribution of the mitochondrial antioxidative system to abiotic stress responses has been well documented (Hernández et al., 1993; Peckmann and Herpich, 1998; Mittova et al., 2000). The question arises whether the mitochondrial antioxidative system is involved in the defence against pathogen attack. Unfortunately, there is scarce information on its possible role in plant–pathogen interaction (Chivasa and Carr, 1998; Garmier et al., 2002). This aspect is important considering the recent results indicating the central regulatory function of mitochondria under stress conditions (for a review see Jones, 2000).
To characterize the involvement of the mitochondrial AOS-scavenging system in biotic stress defence, the response of SOD, as well as the ascorbate–glutathione cycle components, induced after Botrytis cinerea infection, has been studied in mitochondria from tomato leaves. The changes of GLDH, as well as GSH-Px and GST, have also been investigated. The present work is the next step towards a long-term goal to elucidate the role of compartment-specific ascorbate–glutathione cycle components in biotic stress defence using tomato–Botrytis cinerea interaction as a model experimental system. This knowledge could allow insights into the molecular mechanisms of plant response to infection-induced oxidative stress.
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Materials and methods |
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Plant material and B. cinerea infection
Tomato (Lycopersicon esculentum Mill) plants cv ‘Perkoz’ were grown as described earlier (Ku
niak and Skodowska, 2001). At the age of 6 weeks, plants were inoculated by an inoculation solution containing B. cinerea conidia (2x 106 spores ml–1), 5 mM glucose, and 2.5 mM KH2PO4. The inoculation solution was applied on both leaf surfaces using a soft brush. After inoculation, plants were kept at 100% relative humidity to ensure spore germination. Analyses were performed 1, 2, 3, 4, and 5 d after inoculation.
Isolation of leaf mitochondria
Mitochondria were isolated from the first, second, and third true leaves by differential and Percoll density-gradient centrifugation. Leaves (40 g) without the main midribs were cut into pieces, chilled in an ice bath for 20 min, and homogenized in a blender for 2x15 s in an ice-cold isolation medium (1:5 w/v). The medium (pH 7.4) contained 20 mM TRIS-HCl, 0.3 M mannitol, 1 mM EDTA, 10 mM sodium ascorbate, 0.1% bovine serum albumin, and 2 mM MgCl2. The homogenates were filtered through four layers of Miracloth and centrifuged at 200 g for 20 min to remove chloroplasts. The supernatants were centrifuged again at 10 000 g for 10 min to recover the mitochondrial preparation. The pellet was gently resuspended in a medium containing 0.3 M mannitol, 0.05% bovine serum albumin, 1 mM EDTA, 10 mM TRIS-HCl, 1 mM sodium ascorbate, and 0.15 M sucrose (pH 7.2) and separated on a continuous Percoll gradient as described by Struglics et al. (1993). All operations were carried out at 0–4 °C. In order to determine ascorbate content, the buffers lacking sodium ascorbate were used.
Organelle intactness and enzyme latency
The quality of mitochondrial preparations has been evaluated by measurement of marker enzyme activities and examined with an electron microscope. The integrity of the outer mitochondrial membrane was estimated from the activity of succinate:cytochrome c oxidoreductase (EC 1.3.99.1
[EC]
) and NADH:cytochrome c oxidoreductase (EC 1.6.99.3
[EC]
) in the presence or absence of antimycin A as described by Douce et al. (1972). The activities of both enzymes were assayed for mitochondria in the isotonic medium and the mitochondrial extract. Organelle intactness was calculated according to the formula of Burgess (Burgess et al., 1985). The calculated intactness was about 80% for mitochondrial preparations obtained from control and B. cinerea-infected leaves. The latency values for APX and GR were 82% and 98%, respectively. The activities of glucose-6-phosphate dehydrogenase (EC 1.1.1.49
[EC]
), glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.9
[EC]
), fumarase (EC 4.2.1.2
[EC]
), and catalase (EC 1.11.1.6
[EC]
), being specific marker enzymes for cytosol, chloroplast stroma, mitochondria, and peroxisomes, respectively, were measured according to the previously published protocol (Kuniak and Skodowska, 2001). In purified mitochondria obtained from both control and inoculated leaves, the activities of glucose-6-phosphate dehydrogenase, and glyceraldehyde-3-phosphate dehydrogenase were negligible, indicating the high purity of the collected fraction. This fraction contained 4.5% of total catalase activity on a protein basis and 1.4% of the total chlorophyll content thus the cross-contamination of mitochondria with peroxisomes and chloroplasts was also negligible.
Enzyme assays and determination of antioxidants
The mitochondrial pellet was homogenized in 0.05 M ice-cold potassium phosphate buffer (pH 7.0) containing 1 M NaCl, 1 mM EDTA, 1% (w/v) polyvinylpyrrolidone and the supernatant was used for determination of SOD, APX, MDHAR, DHAR, GLDH, GSH-Px, and GST activities, as well as glutathione content. For the determination of ascorbate, the pellet was homogenized in 6% (w/v) ice-cold trichloroacetic acid. The assays of APX, DHAR, GR, GSH-Px, GST, ascorbate, and glutathione were run as described in detail previously (Kuniak and Skodowska, 1999, 2001). SOD activity was determined by the method of Minami and Yoshikawa (1979) with 50 mM TRIS-cacodylic sodium salt buffer pH 8.2 containing 0.1 mM EDTA, 1.4% (v/v) Triton X-100, 0.055 µM NBT, 16 µM pyrogallol, and enzyme extract. The concentration of the reduced form of NBT was measured at 540 nm. The activity unit (50% inhibition) was defined according to McCord and Fridovich (1969). For the assay of total SOD activity the extract was used, whereas CuZn SOD activity was measured by the same method following an extraction by shaking the extract with a chloroform and ethanol mixture (1:2 v/v). The activity of MDHAR was measured following the oxidation rate of NADH at 340 nm (
=6.2 mM–1 cm–1) in a reaction mixture containing 0.1 mM TRIS-HCl buffer pH 7.2, 0.2 mM NADH, 2 mM ascorbic acid, and the mitochondrial extract. The reaction was started by adding 1 unit of ascorbate oxidase (Fluka) to generate a saturating concentration of monodehydroascorbate radical. The rate of monodehydroascorbate-independent NADH oxidation (without ascorbate and ascorbate oxidase) was subtracted. L-galactono--lactone dehydrogenase activity was measured as described by De Gara et al. (2000) by following the reduction of cytochrome c at 550 nm (=21.1 mM–1 cm–1) in a reaction mixture containing 0.1 M TRIS-HCl buffer pH 8.0, 60 µM cytochrome c, 2 mM L-galactono--lactone, and enzyme extract.
Other determinations
The protein content was determined according to Bradford (1976) using bovine serum albumin as a standard.
Statistical analysis
The significance of differences between mean values was determined by a non-parametric Mann–Whitney Rank Sum Test. Differences at P <0.05 were considered significant. All mean values (n=4–6) are presented ±SD.
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Results |
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The first macroscopic infection symptoms in the form of localized, brownish necrotic lesions were visible 2–3 d after the inoculation of tomato leaves with the necrotrophic B. cinerea spores. Later on, a moderate number of spreading lesions developed.
Ascorbate and glutathione pools
A temporary decrease in the mitochondrial AA content following B. cinerea inoculation was observed (Table 1). The decline occurred from day 1 to day 3 and was most pronounced 2 d and 3 d after inoculation (71% and 36% of the control, P <0.05, respectively). Thereafter there was a rise in AA content to the uninfected control levels. The total ascorbate content in mitochondria isolated from inoculated leaves followed similar changes. Conversely, DHA content increased from day 1 onward and was higher throughout the entire period of investigation, except for the 3rd day (Table 1). The maximum accumulation of DHA, up to 130% (P <0.05) of control, was found 2 d after inoculation. The significantly lower (P <0.05) redox state of the ascorbate pool observed 2 d and 3 d after inoculation was the consequence of increasing DHA content and/or decreasing AA level.
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A strong reduction of GSH and total glutathione pools was found following B. cinerea inoculation (Table 2). Starting from day 2 after inoculation, the GSH level declined strongly from 3.29 nmol mg–1 protein (43% of control) down to 1.70 nmol mg–1 protein (29% of control) by day 5. The changes in total glutathione content showed a similar pattern. The GSSG concentration was continuously higher compared with the control, except on the 5th day after inoculation (Table 2). In mitochondria isolated from the inoculated leaves, GSSG accounted for 14–34% of the total glutathione pool, whereas, in the controls, less than 10% of glutathione was detected as GSSG. This resulted in an inoculation-dependent decrease in the GSH/total glutathione ratio down to 0.66 (P <0.05), starting from day 2.
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Enzyme activities
The inoculation significantly induced mitochondrial SOD activity (Fig. 1). The total SOD activity began to increase 1 d after inoculation and afterwards it remained, on average, 66% higher than in the control mitochondria. In the initial phase of the experiment the mitochondrial CuZn SOD was the most rapidly induced isoform (209% of control 1 d after inoculation, P <0.05). Later, however, CuZn SOD activity increase was not concomitant with the total SOD induction. Moreover, in mitochondria isolated from control leaves, besides limited fluctuations in total SOD activity, a gradual age-dependent increase in CuZn SOD activity was observed. In control mitochondria, the activity of this isoform accounted for 54–77% of the total SOD activity over the 5 d time-course of the experiment.
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B. cinerea infection resulted in a decrease in the activities of mitochondrial enzymes involved in ascorbate metabolism, namely: APX, MDHAR, and DHAR, as well as GLDH, but the rate and timing of the changes were different for each of them. In the case of APX the strongest activity decline of 48% (P <0.05), found 3 d after inoculation, was followed by a recovery to 90% and 80% of control on the 4th and 5th days, respectively (Fig. 2). In mitochondria isolated from infected leaves, the pattern of APX activity changes was similar to that observed for AA, and the maximum decrease in this enzyme activity coincided with the lowest AA concentration. The activities of MDHAR and DHAR, enzymes responsible for keeping ascorbate in the reduced form, decreased gradually, starting from the 1st day after inoculation (Fig. 2). The activity of MDHAR showed the most marked decrease 4 d after inoculation (63% of control, P <0.05) before the accumulation of DHA reached the highest level. B. cinerea infection substantially decreased mitochondrial DHAR activity. However, it was significantly decreased (60% of control, P <0.05) only 3 d after inoculation, although a similar rate of decline was maintained until the end of the experiment.
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During the period examined, B. cinerea infection markedly affected the activity of GLDH (Fig. 3). GLDH activity began to decrease 1 d after inoculation and a maximum decline to 36% (P <0.05) of the control values was found on the 2nd and 3rd days. However, after 3 d its activity gradually recovered, reaching 81% of control 5 d after inoculation.
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By contrast with the ascorbate-metabolizing enzymes, B. cinerea inoculation led to a noticeable induction of GR, GSH-Px, and GST (Fig. 4). Markedly increased GR activity, up to 185% (P <0.05) of control, was observed by day 3. Thereafter it declined and remained, on average, 23% below control levels. The activity of mitochondrial GSH-Px was increased following inoculation, except for a 40% decrease (P <0.05) found on the 5th day. The GSH-Px activity started to rise 1 d after inoculation and it reached 198% (P <0.05) and 177% (P <0.05) of the untreated controls after 3 d and 4 d, respectively. The activity of GST was significantly increased (P <0.05) in the range of 137–144% of the control values. This effect was observed from day1 to day 4.
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Discussion |
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It was found that the mitochondrial SOD underwent the strongest B. cinerea-induced changes. Infection brought about a significant increase in total SOD activity, but the specific isoforms were differentially induced as disease development proceeded. The initial CuZn SOD induction ceased and an increase in other isoform activities, presumably Mn SOD, was observed. This is in agreement with results indicating the importance of high mitochondrial Mn SOD activity under severe abiotic stress (Hernández et al., 1993; McKersie et al., 1996; Yu et al., 1999), probably because, unlike CuZn SOD, it is not inactivated by its reaction product. In addition, the expression of CuZn SOD encoding the cytosolic isoform was shown to be induced by GSH whereas GSSG had no effect (Hérouart et al., 1993). Thus, the decrease in GSH content and GSSG accumulation found in the course of this analysis could be unfavourable to CuZn SOD induction. However, as SOD transforms one AOS into another a simultaneous induction of components of the antioxidative network dealing with H2O2 is needed to prevent oxidative damage. In mitochondria from the inoculated leaves a considerable decrease in the APX/total SOD ratio, ranging from 41% to 66%, was observed. The maximum decrease was concomitant with the appearance of spreading lesions. Although this ratio does not represent the stoichiometry between H2O2 removal and production, the results indicate that the protection enzymes were differentially affected and that H2O2 accumulation could be favoured. It is supported by studies on H2O2 generation in the same pathosystem carried out in the authors’ laboratory which revealed a significant H2O2 accumulation both in the cytosolic and apoplastic fractions. A considerable increase in cytosolic H2O2 was found between 5 h and 24 h after inoculation and apoplastic H2O2 generation, as judged by NADH peroxidase activity, was enhanced in B. cinerea-inoculated leaves between 24 h and 72 h (Patykowski and Urbanek, 2003).
APX is considered to be one of the major H2O2-decomposing enzymes in plant cells. In previous studies, transient increases in both cytosolic and chloroplastic APX activities resulting from B. cinerea infection were found (Kuniak and Skodowska, 1999, 2001). However, in the mitochondrial fraction a continuous decrease in APX activity was observed in the inoculated tomato leaves. The reduced H2O2-scavenging capacity of APX could be the consequence of a diminished AA concentration. This suggestion is supported by the observation that, in the inoculated leaves, the time-dependent fluctuations in APX activity level and AA concentration were correlated, both reaching their minimum values on the 3rd day. Similarly, a decline in APX activity due to a decrease in ascorbate content was found in Phaseolus vulgaris after Zn application and in senescing spinach leaves (Hodges and Forney, 2000; Cuypers et al., 2001). Moreover, Conklin et al. (1997) have shown that in the Arabidopsis thaliana vtc1 mutant, which is deficient in ascorbate biosynthesis, APX activity was lower than in the wild type. However, stress responses of plant cells have been found to be associated not only with H2O2 but also with NO synthesis (Neill et al., 2002). The latter has been shown to inhibit APX (Clark et al., 2000) and to induce the expression of GR and GST during plant–pathogen interaction (Delledonne et al., 1998).
With regard to the ascorbate pool it is generally determined by its rates of synthesis, regeneration, and breakdown. Although very little is known about the control of ascorbate synthesis or pool size, a feedback inhibition of its biosynthesis has been postulated. For example, AA synthesis was inhibited by an increased AA pool size in the embryogenic axes of pea seedlings (Pallanca and Smirnoff, 2000). These results concerning the close correlation between the activity of GLDH and AA content imply a similar mechanism regulating AA biosynthesis. However, recent genetic studies have highlighted the importance of GDP-mannose pyrophosphorylase in controlling the ascorbate biosynthesis pathway (Conklin et al., 1999; Keller et al., 1999), suggesting that GLDH is not a rate-limiting step in this process. Furthermore, as mitochondria have been shown to be very sensitive to oxidative inhibition of function (Sweetlove et al., 2002) and recent evidence has suggested a physical and functional link between complex I and ascorbate synthesis (Millar et al., 2003), these results can also be indicative of ascorbate biosynthesis decline resulting from the oxidative impairment of respiration.
B. cinerea inoculation also increased DHA content. Regarding the declining APX activity, the accumulation of DHA was not the result of APX action but rather of the AOS scavenging properties of AA and its turnover perturbations. It is believed that, in plant cells, the AA oxidation products are enzymatically reduced by the action of ascorbate–glutathione cycle enzymes: MDHAR and DHAR (Shalata and Tal, 1998; Mittova et al., 2000), but the involvement of other mechanisms has been also documented (Smirnoff and Wheeler, 2000). It was found that the accumulation of DHA coincided with a suppressed capacity to regenerate ascorbate by MDHAR and DHAR over the period studied. The results are in agreement with the hypothesis that the activities of MDHAR and DHAR correspond to limitation of DHA formation rather than to ascorbate pool replenishment (Arrigoni, 1994). Moreover, as ROS-scavenging enzymes: CuZn SOD and APX as well as DHAR show sensitivity to oxidative stress (Shigeoka et al., 2002), it cannot be excluded that their decreased activities resulted from enzyme inactivation.
B. cinerea infection promoted marked changes in the glutathione pool. Unlike the reversible decrease in AA level, a constant GSH decline was observed. May et al. (1996) demonstrated that, in the A. thaliana mutant, the depletion in glutathione by 70% did not alter its response to pathogens, but to ensure the antioxidative defence the ascorbate level was significantly increased. A likely compensatory role could be proposed for increasing AA content observed 4 d and 5 d after inoculation in this study. Moreover, the induction of GSH-Px, participating in the reduction of lipid peroxides and other hydroperoxides by GSH, may also contribute to the decrease in the glutathione pool. The marked increase in GSH-Px activity seems to reflect the necessity for eliminating the products of lipid peroxidation resulting from B. cinerea-imposed oxidative stress as early as 1 d after inoculation. Taking into account the changes in APX and GSH-Px activities, the latter could also be interpreted as a compensatory mechanism for the reduced APX activity, designed to minimize the potential AOS-mediated damage in mitochondria from the inoculated leaves. Moreover, GSSG was found to accumulate in mitochondria from the inoculated leaves, indicating that GR induction was insufficient to reconvert GSSG to GSH and to overcome the decreased antioxidant potential of glutathione. According to Noctor et al. (2002) the decrease in GSH/GSSG ratio is linked to H2O2 accumulation and change in the intracellular glutathione status is the primary signal responsible for the increase in phytoalexins following pathogen attack. Moreover, the pro-oxidant state resulting from reduced GSH content is believed to trigger GST gene expression (Vranova et al., 2002). Sweetlove et al. (2002) pointed out that GST, an enzyme generally considered to be non-mitochondrial, was induced in Arabidopsis mitochondria in response to H2O2 or menadione treatment-generated oxidative stress and it could be used as an early marker of biotic and abiotic stresses. In contrast to the weak response of cytosolic and chloroplastic GST found in previous studies (Kuniak and Skodowska, 1999, 2001) the mitochondrial GST was significantly increased starting from the 1st day of the experiment, confirming the suggestion that mitochondria could be key organelles in tomato–B. cinerea interaction.
It is of interest to note that GSH and AA pools as well as the ascorbate-related enzymatic reactions were heavily suppressed when the spreading lesions started to develop. Similarly, in B. cinerea-infected A. thaliana leaves a massive depletion of AA level occurred before visible infection (Muckenschnabel et al., 2002) and a diminished antioxidative capacity prior to the appearance of disease symptoms was reported for TMV-infected tobacco leaves (Fodor et al., 1997).
In general, the present results reflect the oxidative effects of B. cinerea infection at the level of the mitochondrion. Regarding the intensity of changes, it could be hypothesized that, at the subcellular level, the mitochondrion could be one of the main targets for B. cinerea-induced oxidative stress. By contrast with the well-known plant–biotrophic pathogen interactions, very little is known about the oxidative metabolism in the plant responses to pathogens that do not induce HR responses such as necrotrophic fungi. Hence the reduction of the mitochondrial antioxidant capacity found in the present study could be interpreted as: (a) an aspect of defence strategy aimed at restricting fungal growth, assuming direct toxic effects of accumulated AOS or their signalling functions or (b) a less efficient AOS-scavenging mechanism favouring infection progress. The latter is in agreement with recent reports that pathogenesis of some necrotrophic pathogens, for example, B. cinerea relies on high concentrations of AOS (Govrin and Levine, 2000).
Based on our results the success of the pathogen is, at least partly, related to depletion of the antioxidant capacity at the cellular and subcellular levels (Kuniak and Skodowska, 1999, 2001). As the ascorbate–glutathione cycle-related enzymes were differentially affected by B. cinerea inoculation, indicating possible functional differences in enzymes located in respective organelles, a massive decrease in glutathione content was found in all cellular compartments studied so far. So it seems to be a common feature of infection process studied at different levels (Kuniak and Skodowska, 1999, 2001). By contrast, in French bean leaves inoculated with B. cinerea, glutathione was insensitive to the infection process (Muckenschnabel et al., 2001). It appears, therefore, that the redox balance-related events during the infection process could be species-specific or different compensatory mechanisms could be activated to cope with the changing ascorbate and glutathione redox status. The seemingly contradictory data obtained from different pathosystems reflect the difficulty in creating a generalized model of the role of AOS and antioxidants in plant resistance/susceptibility to necrotrophic pathogens.
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Acknowledgements |
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The authors are grateful to Professor Barbara Gabara (Department of Plant Cytology and Cytochemistry, University of
ód) for electron microscope analysis of the organelle fractions quality. This work was partly supported by University of ód grants No 505/469 and 505/470.
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