Stress-Induced Changes in Ubiquinone ... - Bioscience Reports

Bioscience Reports, Vol. 21, No. 3, June 2001 ( 2001)

Stress-induced Changes in Ubiquinone Concentration and Alternative Oxidase in Plant Mitochondria Vasily N. Popov,1 Albert C. Purvis,2 Vladimir P. Skulachev,3 and Anneke M. Wagner4,5 Receiûed April 16, 2001 We have investigated the influence of stress conditions such as incubation at 4°C and incubation in hyperoxygen atmosphere, on plant tissues. The ubiquinone (Q) content and respiratory activity of purified mitochondria was studied. The rate of respiration of mitochondria isolated from cold-treated green bell peppers (Capsicum annuum L) exceeds that of controls, but this is not so for mitochondria isolated from cold-treated cauliflower (Brassica oleracea L). Treatment with high oxygen does not alter respiration rates of cauliflower mitochondria. Analysis of kinetic data relating oxygen uptake with Q reduction in mitochondria isolated from tissue incubated at 4°C (bell peppers and cauliflowers) and at high oxygen levels (cauliflowers) reveals an increase in the total amount of Q and in the percentage of inoxidizable QH2 . The effects are not invariably accompanied by an induction of the alternative oxidase (AOX). In those mitochondria where the AOX is induced (cold-treated bell pepper and cauliflower treated with high oxygen) superoxide production is lower than in the control. The role of reduced Q accumulation and AOX induction in the defense against oxidative damage is discussed. KEY WORDS: Alternative oxidase; cold; oxygen, plant mitochondria; stress; superoxide radical; ubiquinone ABBREVIATIONS: AOX, alternative oxidase; MDA, malondialdehyde; Q, ubiquinone; QH2 , ubiquinol; ROS, reactive oxygen species; PVP, polyvinylpyrrolidone; ECL, enhanced chemiluminescence; TCA, trichloroacetic acid; BHAM, benzohydroxamate

INTRODUCTION Ubiquinine (Q) plays a central role in the plant mitochondrial electron transport chain. It is reduced to ubiquinol (QH2) by complexes I and II and by an external NAD(P)H dehydrogenase. QH2 can be oxidized by the cytochrome pathway and 1

Department of Plant Physiology and Biochemistry, Voronezh State University, Voronezh 394693, Russia. 2 Department of Horticulture, University of Georgia, Tifton, GA 31793, USA. 3 Department of Bioenergetics, Belozersky Institute for Physical and Chemical Biology, Moscow M.V. Lomonosov State University, Moscow 119899, Russia. 4 Department of Molecular Cell Physiology, IMBW, Vrije Universiteit, De Boelelaan 1087, 1081 HV Amsterdam, The Netherlands. 5 To whom all correspondence should be addressed. E-mail: [email protected] 369 0144-8463兾01兾0600-0369$19.50兾0  2001 Plenum Publishing Corporation

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the alternative pathway. The relative distribution of electrons between the two pathways is regulated by the Q兾QH2 ratio (Dry et al., 1989; Van den Bergen et al., 1994). In addition to its role as substrate for the mitochondrial respiratory oxidases, QH2 may act as a scavenger of reactive oxygen species (ROS) in general, and the superoxide radical, in particular, which is produced during mitochondrial electron transport in plant (Rich and Bonner, 1978) and animal cells (Boveris and Chance, 1973). For this function, both the redox state and the size of Q pool are important (Beyer, 1992). In this respect, it is interesting to note that a considerable part of the Q in plant mitochondria often remains reduced in the absence of substrate. In a previous paper we reported that both the total Q content and the relative amount of the inoxidizable part of the Q pool increased during cold-storage of bell peppers (Wagner and Purvis, 1998). Both phenomena were also observed in mitochondria isolated from Petunia hybrida cell suspensions cultured in the presence of antimycin A, an inhibitor of the cytochrome pathway (Wagner and Wagner, 1997), suggesting that it is not the cold treatment per se, but the stress condition that causes these responses in bell pepper mitochondria. The synthesis of antioxidants can be regarded as an indirect line of defense against oxidative damage of the mitochondrial lipids, proteins and especially DNA, caused by ROS. Direct defense mechanisms include a lowering of the oxygen concentration in the cell (Skulachev, 1996). A decrease in the oxygen concentration can be accomplished by uncoupled and non-coupled respiration (Bertsova et al., 1997; Korshunov et al., 1997). The branched electron transport chain of plant mitochondria contains a number of components that enable non-coupled respiration: a nonphosphorylating rotenone-insensitive NADH dehydrogenase, an exogenous NAD(P)H dehydrogenase and the alternative oxidase (AOX). When these components act together, all three energy-coupling sites of the main respiratory chain are bypassed. The result is a high rate of respiration and a decrease of the oxygen concentration in mitochondria and thus a decrease in the amount of ROS. In addition, the membrane potential decreases which also leads to a lowering of ROS production (Skulachev, 1996; Korshunov et al., 1997). Previous work by our groups suggests that the AOX may indeed play an important role in limiting ROS production in plants and can therefore be considered as a defense of plant mitochondria against oxidative damage (Purvis and Shewfelt, 1993; Popov et al., 1997; Purvis, 1997; Wagner and Moore, 1997). This suggestion is supported by the observation that situations leading to oxidative stress (wounding, chilling, osmotic stress, antimycin A treatment) often induce AOX activity (Purvis and Shewfelt, 1993; Wagner, 1995; Wagner and Wagner, 1997). Moreover, treatments with H2O2 can lead to an expression of AOX protein (Wagner, 1995). Recently, Parsons et al. (1999) reported that the induction of AOX inhibits excessive ROS production in phosphate-limited tobacco cells. The question then arises whether the increase in AOX is co-ordinately related to the increase in Q content that is also observed in stress conditions (Wagner and Wagner, 1997, Wagner and Purvis, 1998). To test this hypothesis, we investigated whether a cold treatment induced an increase in both (reduced) Q and AOX in mitochondria isolated from cauliflower (inflorescence) and bell pepper fruits. In addition, these parameters were

Stress and Plant Mitochondria

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also measured in mitochondria that were isolated from cauliflowers that had been incubated in elevated oxygen levels to induce oxygen stress. The reasons for using bell peppers and cauliflowers were merely practical. Both cauliflowers and bell peppers are good sources for obtaining large amounts of mitochondria and, even more important, the mitochondrial preparations have a low SOD-insensitive rate of epinephrine oxidation (no interfering substances like phenoloxidases), enabling accurate measurements of superoxide production. The consequences of the increases in the total Q and the inoxidizable Q-pools for the kinetics of Q-oxidizing pathways are also the subject of this study. In many studies of these kinetic parameters, Q reduction is measured with a Q-electrode (see e.g. Dry et al., 1989). With that method, however, it is not possible to estimate the amounts of total Q and the Q that is reduced in the absence of substrate. In the present study we used Q extraction (Van den Bergen et al., 1994) to determine the sizes of the total Q and the inoxidizable Q pools in bell pepper and cauliflower mitochondria. We also demonstrate the importance of knowing the size of the inoxidizable Q pool for the determination of kinetic parameters. MATERIALS AND METHODS Intact, freshly harvested, field grown cauliflowers (inflorescences of Brassica oleracea L) and freshly harvested green bell pepper fruits (Capsicum annuum L), cultivated in green houses, were obtained from local markets and were placed in the dark, either at room temperature or at 4°C, or at room temperature in a desiccator which was flushed with pure oxygen twice daily. The oxygen concentration in the desiccator was monitored with an oxygen electrode (Yellow Springs Instruments Co. Inc., USA) and remained between 50% and 60% throughout the time of incubation. Humidity of atmosphere was between 60% and 70% during all treatments. Isolation of Mitochondria and Respiratory Measurements Mitochondria were isolated using a Percoll self-generated gradient (Wagner, 1995). 500 g of cauliflower or bell pepper tissue was homogenized in a Braun multipress MP50. The homogenization medium contained 20 mM HEPES (pH 7.2), 350 mM mannitol, 1 mM EDTA, 2 mM cysteine, 0.7% PVP and 0.5 mg−1 ml −1 BSA. Rates of respiration were measured with an oxygen electrode (Yellow Springs Instruments Co. Inc., USA) at 25°C in 1 ml of incubation medium, containing 20 mM HEPES (pH 7.2), 350 mM mannitol, 1 mM potassium phosphate, 5 mM MgCl2 and approximately 0.5 mg of mitochondrial protein. As a substrate for respiration 10 mM succinate was supplied along with 0.1 mM ATP to ensure complete activation of succinate dehydrogenase. The cytochrome pathway was inhibited with 0.5 mM KCN. Measurement of Superoxide Production by Mitochondria Superoxide production was monitored in an Aminco SPF 500 Dual Wavelength spectrophotometer as the SOD-sensitive part of epinephrine oxidation at

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480A579 nm in the same incubation medium as used for oxygen consumption measurements, but with the addition of 1 mM epinephrine (Purvis et al., 1995). Measurement of Q Concentration and Q Reduction Extraction, HPLC separation, and determination of reduced and oxidized Q from isolated mitochondria was performed as described previously (Wagner and Wagner, 1995). For each extract, 0.5 to 1 mg of mitochondrial protein was used. SDS-PAGE and Immunoblotting Mitochondrial protein (100 µg) was solubilized in sample buffer (312 mM Tris [pH 6.8], 10% [w兾v] SDS, 10% [v兾v] glycerol, 0.002% [w兾v] bromophenol blue and 100 mM dithiothreitol (DTT) and boiled for 1–2 min. The mitochondrial samples were subjected to SDS-polyacrylamide electrophoresis (10% gel), followed by western blotting (Wagner, 1995). Antibodies developed against the AOX protein of Sauromatum guttatum (generously supplied by Dr. T. Elthon) were used at dilutions of 1 :1000. Visualization was with the ECL chemiluminescent reagent system (Amersham). Measurements of Malondialdehyde Production The concentrations of malondialdehyde (MDA) in isolated mitochondria were detected immediately after isolation by using thiobarbituric acid. Mitochondria (0.5– 1.0 mg mitochondrial protein in reaction medium), were added to 2 ml of a mixture of 0.25% thiobarbituric acid and 10% TCA and heated at 95°C for 30 min. After cooling, the absorbance was measured at 532 nm. The MDA molar extinction coefficient of 1.56B105 mol −1 cm−1 was used to calculate the concentration of MDA. Protein Determination The protein concentration in the mitochondrial preparations was determined with the BCA Protein Assay reagent A (Pierce, Rockford, IL), with BSA as a standard. RESULTS Effects of Cold and High Oxygen on Ubiquinone Contents and Kinetic Parameters of Ubiquinone Oxidizing Pathways The kinetics of the oxidizing pathways (total respiration via both the cytochrome pathway and the alternative pathway) were obtained by measuring the relationship between Q reduction and oxygen uptake with succinate (in the absence or presence of various concentrations of malonate) in mitochondria from bell peppers and cauliflowers which had been incubated under various conditions (Fig. 1A


Fig. 1. Relationship between oxygen uptake with succinate (10 mM) as the substrate and relative Q reduction in mitochondria from bell peppers (A) and cauliflowers (B) treated under various conditions. Respiratory rates were varied by titrating with malonate. Data are combined results of 3–6 separate experiments. ●, mitochondria isolated from tissue incubated in air at room temperature; 䊊, mitochondria isolated from tissue incubated at 4°C; ■, mitochondria isolated from tissue incubated at elevated oxygen levels.

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and B). Maximal respiration with succinate as substrate was slightly higher in mitochondria isolated from bell peppers stored at 4°C for 4 days than in control mitochondria, but the relative Q reduction at maximal respiratory rates was not different between the two treatments (the latter has been reported before, Wagner and Purvis, 1998). The shape of the line describing the relationship between Q reduction and respiratory rates was linear for control mitochondria, but non-linear for mitochondria from cold-treated bell peppers, suggesting considerable engagement of the AOX in total oxygen uptake (Dry et al., 1989, Van den Bergen et al., 1994). The intercepts of the best fit lines with the X-axis were also different. At a respiratory rate of zero, a much greater portion of the Q pool was still reduced in the mitochondria from cold-treated tissue than in the control (Fig. 1A; Tables 1 and 2; see also Wagner and Purvis, 1998). In contrast, there was no significant difference in maximal respiration with succinate as the substrate between mitochondria isolated from control cauliflower and cauliflower stored at 4°C for 2 days (Fig. 1B; Table 2). Maximal Q reductions, however, were somewhat higher in mitochondria from cold-treated tissue than control tissue. The intercepts of the best fit lines with the X-axis were also different. At a respiratory rate of zero, a larger portion of the Q pool was still reduced in the mitochondria from cold-treated tissue than in the control (Fig. 1B; see also Table 1), with the result that the slopes of the best fit lines were almost equal. Prolonging the cold treatment did not cause any additional changes (data not shown). In order to discriminate between the effects of cold and stress in general, cauliflowers were treated for 2 days at 4°C or with 50–60% oxygen to induce oxidative stress. The MDA content in mitochondria (measured immediately after isolation) was 1.1 nmol · mg prot−1 in mitochondria from control tissue and 2.3 and 3.1 nmol · mg prot−1 in mitochondria from cold-treated and oxygen-treated tissues, respectively, indicating that treatment with elevated oxygen levels was indeed stressful (mean values of 3 separate experiments, in each experiment a two- and threefold difference was observed between control and cold and oxygen treated tissue, respectively). Table 1. Concentrations of Total Q, Q Reduced in the Absence of Substrate (‘‘Inoxidizable Q’’), and Q Active in Respiration (Calculated from Total Q and % Reduction in the Absence of Substrate) in Mitochondria Isolated from Green Bell Peppers and Cauliflowers after Various Treatments. Data are Given as Mean ValuesJSD. Number of Separate Experiments in Brackets Species and treatment Bell pepper control 4°C, 4 days Cauliflower control 4°C, 2 days high oxygen, 2 days

Total Q Inoxidizable Q Q active in respiration nmol · mg prot−1 (%) nmol · mg prot−1 1.59J0.21 (3) 3.09J0.58 (3)

33.9J4.0 (3) 55.0J8.1 (3)

1.09 1.39

1.01J0.04 (8) 1.38J0.11 (8)

30.3J5.3 (8) 42.9J3.9 (8)

0.709 0.865

1.74J0.29 (6)

53.5J6.1 (8)

0.809


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A non-linear relationship exists between oxygen uptake and Q reduction in mitochondria from cauliflowers that had been incubated at elevated oxygen levels, similar to cold-treated bell peppers, suggesting an increased participation of AOX in total respiration (Fig. 1B). Maximal respiration (in the absence of malonate), however, was not significantly different between the treatments, but the relative Q reduction at the highest respiratory rates was somewhat higher in the mitochondria from oxygen-treated tissue. After oxygen treatment the relative amount of Q that was reduced in the absence of substrate (respiratory rate of zero) increased to 50% and also, as in bell pepper, the total amount of Q increased (Fig. 1B and Table 1). Consequences of an Increase in Reduced Q for the Interpretation of Kinetic Parameters of the QH2-Oxidizing Pathways When a treatment leads to a larger total amount of Q and a relatively larger inoxidizable Q pool, the question arises whether it is justifiable to compare effects of treatments on kinetic parameters by expressing the redox state of the Q-pool as percent reduction of total Q, as is usually done and as we have in Fig. 1. If indeed a considerable portion of the Q pool is not participating in respiratory electron transport, we need to reassess the data and calculate the actual amount of Q engaged in electron transport for each treatment. In Fig. 2, respiratory rates in bell pepper and cauliflower mitochondria are expressed per nmol QH2 participating in electron transport (see Table 1). It is obvious that the conclusions drawn from Fig. 2 are quite different from those in Fig. 1. Although the shapes of the curves describing the relationships between respiratory rates and Q reduction do not change, when the respiration rates are plotted against nmoles QH2 per mg protein participating in electron transport (Fig. 2A), the conclusion that at low Q reduction levels a lower respiration rate is found in mitochondria isolated from cold-treated bell peppers than in control mitochondria is obviously not correct. When respiration is plotted against the actual concentration of Q (Fig. 2B), the slopes of the lines representing the relationship between respiratory rates and Q reduction in mitochondria from control and cold-treated cauliflower are no longer the same. Induction of AOX The non-linear relationship between Q reduction and oxygen uptake after a cold treatment in bell pepper and an oxygen treatment in cauliflower suggests considerable engagement of the AOX. In the past, the extent of the engagement of AOX in oxygen uptake was measured by using inhibitors of AOX, such as BHAM. Unfortunately, it is now clear that this method does not give reliable results, due to competition between the AOX and the cytochrome pathways (Wagner and Krab, 1995). The suggestion that AOX is more active after cold treatment of bell peppers and high oxygen treatment of cauliflowers, however, is supported by the fact that in both kinds of mitochondria an increase in the total amount of AOX protein is observed in western blots (Fig. 3). Accordingly, cyanide-resistant respiration increased (as reported before, Wagner and Purvis, 1998) in mitochondria isolated from cold-treated bell peppers

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Fig. 2. Relationship between oxygen uptake with succinate (10 mM) as the substrate and the concentration of reduced Q, actually participating in electron transport (see Table 1) in mitochondria from bell peppers (A) and cauliflowers (B) treated under various conditions. Data from Fig. 1. ●, mitochondria isolated from tissue incubated in air at room temperature; 䊊, mitochondria isolated from tissue incubated at 4°C; ■, mitochondria isolated from tissue incubated at elevated oxygen levels.


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Fig. 3. Alternative oxidase amounts in mitochondria from cauliflower (left) and green bell pepper (right). C, control; OX, after incubation at high oxygen; 4C, after incubation at 4°C. Immunoblots from 10% SDS gels were probed for the alternative oxidase protein using AOX monoclonal antibodies. Electrophoresis took place under reducing conditions in the presence of 100 mM DTT. The amount of protein loaded for all lanes was 100 µg.

and in cauliflower mitochondria from oxygen-treated, but not cold-treated cauliflowers (Table 2). In cauliflowers incubated at 4°C, no changes in amounts of AOX protein were observed (Fig. 3). Since an increase in AOX and an increase in reduced Q can be regarded as protective measures of the plant against damage by ROS production during electron transport, we compared superoxide production by mitochondria of cold-treated cauliflowers and bell peppers with that in control plants. Superoxide production was unchanged in mitochondria isolated from cauliflowers after cold treatment, in contrast to the decrease found in cold-treated peppers. Superoxide production slightly decreased after incubation of cauliflower in a 50% oxygen atmosphere (Table 2). DISCUSSION AND CONCLUSIONS The observed increase in Q content in these mitochondria support a role for Q in an antioxidative defense mechanism. It was shown before that ubiquinones could Table 2. Total Respiration and CN-resistant Respiration, Production of Superoxide and MDA Content in Mitochondria Isolated from Green Bell Peppers and Cauliflowers after Various Treatments. Respiration was Measured with Succinate as the Substrate. Data are Given as Mean ValuesJSD. Number of Separate Experiments in Brackets. n.d., Not Determined Total respiration nmol · min−1 · mg prot−1

CN-resistant respiration nmol · min−1 · mg prot−1

O·2− production nmol. min−1 mg prot−1

MDA content nmol. mg prot−1

Bell pepper control 4°C, 4 days

83J6.8 (3) 127J28.1 (3)

23J5.2 (3) 86J11.1 (3)

32J7.6 (3) 10J4.0 (3)

n.d. n.d.

Cauliflower control 4°C, 2 days 50% oxygen, 2 days

207J14.0 (12) 218J19.7 (12) 199J15.4 (12)

35J5.1 (12) 24J3.0 (12) 78J6.1 (12)

21J1.4 (8) 20J1.6 (8) 15J0.8 (6)

1.1J0.1 (3) 2.0J0.2 (3) 3.2J0.2 (3)

Species and treatment

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play an antioxidant role in mitochondria (Beyer, 1992). We suggest that the synthesis and accumulation of reduced Q, inaccessible for the components of the mitochondrial respiratory chain, could serve in the scavenging of the superoxide radical. From the results obtained with mitochondria isolated from cold-treated cauliflowers, it can be concluded that an increase in total Q and inoxidizable Q is not invariably accompanied by an induction of AOX. The reason why no increase in AOX was observed during the cold treatment of cauliflower is not clear. While in many plants AOX increases in the cold, the opposite has also been reported for potato tuber discs (Hemrika-Wagner et al., 1982). A change in storage temperature of potato from 8°C to room temperature leads to an increase in the content of inoxidizable Q, but not to the induction of AOX (data not shown). In those mitochondria where AOX was induced (cold-treated bell pepper and cauliflower treated with elevated oxygen levels) superoxide production was also lower than the control. Activity of AOX also lowers the membrane potential which in turn has been shown to lower ROS production in animal mitochondria (Skulachev, 1996; Korshunov et al., 1997). Initially, it appears that an increase in total Q and inoxidizable Q has little effect on ROS production in cold-treated cauliflowers, compared to control. However, the method used to determine ROS (epinephrine oxidation) measures ROS outside the mitochondria, but it is possible that at least part of the ROS is produced and remains inside the mitochondria. The method used did not allow us to determine if and how much of the ROS produced is scavenged by reduced Q. The location of the inoxidizable Q pool, especially, is also crucial for its scavenging activity. The nature and the location of the inoxidizable Q are presently under study by our groups and the control and regulation of ROS production in plant mitochondria will be the subject of future studies. Regardless of the outcome of these studies, it is clear from a comparison of Fig. 1 and Fig. 2 that correctly to interpret kinetic data, one needs to know both the size of the total Q pool and the size of the inoxidizable part, in order to know the size of the Q pool active in electron transport. The Q electrode alone is inadequate for such measurements, but the extraction method we used here is very time-consuming. The best procedure, therefore, is a combination of both: extraction of samples incubated without substrate to provide the size of the inoxidizable Q pool and the total concentration of Q, together with fast titration experiments with the Q electrode.

ACKNOWLEDGMENTS V.N.P. was supported by a FEBS Fellowship and RFBR (grant 01-04-48431); A.C.P. acknowledges the USDA National Research Initiative Competitive Grants Program (grant # 96-35100-3225); A.M.W. and V.P.S. acknowledge the financial support of N.W.O. (Russia兾Netherlands research grant #047-006-005).

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