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First published online December 24, 2008; 10.1104/pp.108.129247 Plant Physiology 149:1099-1110 (2009) © 2009 American Society of Plant Biologists
Reactive Oxygen Species Production by Potato Tuber Mitochondria Is Modulated by Mitochondrially Bound Hexokinase Activity1Instituto de Bioquímica Médica, Programa de Biofísica e Bioquímica Celular and Programa de Biologia Molecular e Biotecnologia, Universidade Federal do Rio de Janeiro, Laboratório de Bioenergética e Fisiologia Mitochondrial, Cidade Universitária, Rio de Janeiro 21941–590, Brazil
Potato tuber (Solanum tuberosum) mitochondria (PTM) have a mitochondrially bound hexokinase (HK) activity that exhibits a pronounced sensitivity to ADP inhibition. Here we investigated the role of mitochondrial HK activity in PTM reactive oxygen species generation. Mitochondrial HK has a 10-fold higher affinity for glucose (Glc) than for fructose (KMGlc = 140 µM versus KMFrc = 1,375 µM). Activation of PTM respiration by succinate led to an increase in hydrogen peroxide (H2O2) release that was abrogated by mitochondrial HK activation. Mitochondrial HK activity caused a decrease in the mitochondrial membrane potential and an increase in oxygen consumption by PTM. Inhibition of Glc phosphorylation by mannoheptulose or GlcNAc induced a rapid increase in H2O2 release. The blockage of H2O2 release sustained by Glc was reverted by oligomycin and atractyloside, indicating that ADP recycles through the adenine nucleotide translocator and F0F1ATP synthase is operative during the mitochondrial HK reaction. Inhibition of mitochondrial HK activity by 60% to 70% caused an increase of 50% in the maximal rate of H2O2 release. Inhibition in H2O2 release by mitochondrial HK activity was comparable to, or even more potent, than that observed for StUCP (S. tuberosum uncoupling protein) activity. The inhibition of H2O2 release in PTM was two orders of magnitude more selective for the ADP produced from the mitochondrial HK reaction than for that derived from soluble yeast (Saccharomyces cerevisiae) HK. Modulation of H2O2 release and oxygen consumption by Glc and mitochondrial HK inhibitors in potato tuber slices shows that hexoses and mitochondrial HK may act as a potent preventive antioxidant mechanism in potato tubers.
Production of reactive oxygen species (ROS) is an unavoidable consequence of aerobic respiration (Chance et al., 1979  ). The mitochondrial electron transport system (ETS) is the major site of ROS production in mammalian and nonphotosynthesizing plant cells (Puntarulo et al., 1991; Halliwell and Gutteridge, 2007). Depending on the mitochondrial respiratory states, a small portion of the consumable oxygen is partially reduced to generate ROS (Skulachev, 1996; Liu, 1997; Turrens, 1997; Møller, 2001; Considine et al., 2003; Smith et al., 2004). In plants, the monoelectronic reduction of oxygen by ETS leads to the production of superoxide radicals (O2·–) that can be dismutated by SOD, producing hydrogen peroxide (H2O2), and further decomposed by catalase and/or ascorbate-glutathione peroxidase cycles (Møller, 2001). An imbalance between the ROS production and antioxidant defenses can lead to an oxidative stress condition. Increased levels of ROS may be a consequence of the action of plant hormones, environmental stress, pathogens, or high levels of sugars and fatty acids (Bolwell et al., 2002; Couée et al., 2006; Gechev et al., 2006; Liu et al., 2007; Rhoads and Subbaiah, 2007). These conditions may lead to storage deterioration or impairment of seedling growth decreasing on crop yield. To avoid the harmful accumulation of ROS or to fine tune the steady-state levels of ROS, various enzymatic systems control the rate of ROS production in mitochondria (Schreck and Baeuerle, 1991; Møller, 2001).
Mitochondrial ROS production is highly dependent on the membrane potential (
The metabolism of free hexoses begins by their phosphorylation in a reaction catalyzed by the hexokinase (HK):
HK is a ubiquitous enzyme found in many organisms. In plants, the binding mechanism of HK to the outer mitochondrial membrane is not fully established, but some reports indicate that it may differ considerably from those properties described for mammal cells (Dry et al., 1983
In mammals, HK types I and II are associated with the mitochondrial outer membrane through the voltage-dependent anion channel (VDAC) and adenine nucleotide transporter (ANT). These associations were found in tissues with a high energy demand, such as heart, brain, and tumor cells (Arora and Pedersen, 1988
Recently, our group demonstrated that mt-HK activity plays a key preventive antioxidant role by reducing mitochondrial ROS generation through a steady-state ADP recycling mechanism in rat brain neurons. The mitochondrial ADP recycling leads to a decrease in the
Although plant HK is recognized to fulfill a catalytic function, the role of mt-HK activity in the regulation of both mitochondrial respiration and ROS production in plants is unknown. Recently, an authentic HK activity was detected in PTM (Graham et al., 2007
An Authentic HK Activity Is Associated with PTM
Previous studies have shown that HK is bound to mitochondria in mammalian tissues, in different plant species (Galina et al., 1995
Kinetic Properties of Potato Tuber mt-HK and Dependence of Activity on Oxidative Phosphorylation The next step was to evaluate the activity of mt-HK after addition of ATP and Glc using intact, Percoll-purified PTM (Fig. 1 ). The mt-HK activity was detected immediately after addition of 1 mM ATP and 5 mM Glc (Fig. 1A, traces 1 and 2), but was progressively inhibited by more than 90% after 2 min. However, the mt-HK activity could be restored by the inclusion of succinate, an oxidizable substrate that supports the conversion of ADP to ATP during oxidative phosphorylation, indicating that inhibition observed for mt-HK was due to the ADP accumulated by the mt-HK reaction (Fig. 1A, traces 1 and 2). The mt-HK sensitivity to ADP inhibition was confirmed when 0.3 mM ADP was added to the reaction medium prior to starting the mt-HK reaction with ATP plus Glc (Fig. 1A, trace 3). The mt-HK reaction rate in this case was much lower than that observed in the first 2 min in the absence of ADP (Fig. 1A, trace 1). The addition of succinate reestablished the maximal rate of mt-HK activity (Fig. 1A, trace 3). The authenticity of mt-HK activity was confirmed when its specific competitive inhibitor, 50 mM mannoheptulose (MHP), was added to the reaction medium (Fig. 1A, trace 2). No oxidative phosphorylation-supported mt-HK activity was observed in the presence of oligomycin (a specific inhibitor of F0F1ATPase), atractyloside (a specific inhibitor of adenine-nucleotide translocator ANT), or in the absence of succinate (data not shown). In addition, these oxidative phosphorylation inhibitors had no effect on PTM mt-HK activity (data not shown).
Figure 1B shows the ADP dependence for mt-HK inhibition using Glc or Fru as substrate. ADP caused a half inhibition of mt-HK activity in the micromolar range with Glc (IC50 = approximately 40–70 µM), but much more ADP was needed to cause a similar degree of inhibition (IC50 = approximately 400–700 µM) with Fru as substrate. This result suggests that the affinity of mt-HK for Glc and Fru is different and the inhibitory activity of ADP depends on the substrate used. In fact, the affinity of mt-HK from PTM was higher for Glc (KM 0.14 mM) than for Fru (KM 1.4 mM). The mt-HK activity measured with saturating concentrations of hexoses was practically the same (Fig. 1C).
The experiments shown in Figure 1 indicated that PTM respiration is important to maintain the mt-HK activity free of the ADP inhibition. In the next set of experiments, we evaluated the effect of the mt-HK activity on several mitochondrial functional parameters (Figs. 2
, 3
, and 4
). During PTM respiration, the addition of small amounts of ADP (0.15 mM) accelerated the rate of O2 consumption (state 3; Fig. 2A). When all the ADP was converted to ATP, the respiration rate decreased, returning to the resting state (state 4), but a further addition of Glc permanently stimulated the O2 consumption (Fig. 2A). Based on previous data from our group (da-Silva et al., 2004
It has been established that mitochondrial H2O2 formation is strongly dependent on high   m values (Korshunov et al., 1997). In Figure 3 we observed substantial H2O2 release after induction of respiration by succinate and ATP. The addition of 1 mM ADP strongly reduced H2O2 release, showing that a small decrease in m, due to activation of state 3 respiration, is the main mechanism by which mitochondrial H2O2 release is prevented (Fig. 3, left trace). A similar blockage in H2O2 release was observed when ADP was replaced by 5 mM Glc (Fig. 3, right trace). This was reversed by either mt-HK inhibitors such as MHP (Fig. 3) or N-acetylglucosamine (NAG) (Fig. 4C) by oxidative phosphorylation poisons such as oligomycin or atractyloside, two inhibitors that promote an increase in m in the presence of oxidizable substrates (Figs. 2, and 4, A and B). These experiments demonstrate that mt-HK activity is able to modulate the m and H2O2 release in PTM by an ADP-recycling mechanism through ANT/F0F1ATP synthase activities. The regulation of H2O2 release from PTM by mt-HK is attained regardless if the source of ATP for the mt-HK is external (Fig. 4B) or from oxidative phosphorylation (Fig. 4A).
The mt-HK activity and the rate of H2O2 release by PTM were measured simultaneously with increasing amounts of Glc (Fig. 5A ). We observed an inverse relation between mt-HK activity and mitochondrial H2O2 release. The IC50 to inhibit H2O2 release was approximately 20 µM Glc (Fig. 5A). The dependence on mt-HK activity to abrogate the H2O2 release was confirmed in Figure 5B, when 25 mM NAG, a competitive inhibitor of mt-HK was included in the assay medium. Under this condition, the IC50 for Glc to inhibit H2O2 release rose to about 800 µM. Importantly, a half-maximum inhibition of the mt-HK activity was able to maintain the rate of H2O2 release at about 15% of its maximal value (Fig. 5C).
Comparison of the Preventive Antioxidant Role of mt-HK and PUMP Activities in PTM
The rate of H2O2 release, oxygen consumption, and
In an attempt to compare the impact of mt-HK activity with that of PUMP activation on the bioenergetic parameters evaluated above, we next measured the effect of increasing amounts of LA, either with (Fig. 6B) or without fatty acid-free bovine serum albumin (faf-BSA; a protein that sequesters LA; Fig. 6C). The IC50 for LA to inhibit H2O2 release was about 30 µM in the presence of faf-BSA (Fig. 6B, white triangles). Under this specific condition mt-HK activation exerted an even more powerful effect on H2O2 release than did PUMP activation (Fig. 6C, black triangle). The IC50 for LA to inhibit H2O2 release when faf-BSA was omitted from the reaction medium (Fig. 6C) was about 4 µM, confirming the ability of this fatty acid to activate PUMP. In this condition, 30 µM LA almost completely blocked H2O2 release, similar to mt-HK activation (Fig. 6C, white and black triangles).
To evaluate whether a specific location of mt-HK on mitochondria is required to prevent H2O2 release we next measured the rate of H2O2 release in PTM using two alternative hexose substrates, Glc or Fru at 0.5 mM (Fig. 7A ). Mt-HK activity using Fru as substrate is much lower when compared with Glc, as expected from the apparent affinities of these two substrates (Fig. 1C). In fact, when 0.5 mM Fru was added to the reaction mixture, a negligible effect was observed on the rate of H2O2 release, contrasting with the strong reduction in the rate of H2O2 release when 0.5 mM Glc was used as substrate (Fig. 7A). Using Fru as a substrate, we only observed a decrease in the rate of H2O2 release when the hexose concentration was increased to 10 mM (Fig. 7A). Regardless of the substrate used, oligomycin promptly increased the H2O2 release by impairing the ADP recycling activity mediated by mt-HK.
In contrast to PTM mt-HK activity, soluble yeast (Saccharomyces cerevisiae) HK exhibits a high affinity for Fru with an apparent Km of 0.7 mM (Avigad and Englard, 1968 ; Bernard, 1975). Taking advantage of this difference in kinetic properties between the two enzymes, we next compared the effect of soluble yeast HK activation, with that of activating the endogenous PTM mt-HK by using Fru as substrate and measuring the rate of H2O2 release. The rationale was that at 0.5 mM Fru, PTM mt-HK would not be active, thus not producing ADP, but addition of soluble yeast HK, which has a higher affinity for Fru, would promptly catalyze this reaction. Thus, under this condition, the ADP produced from the HK reaction comes only from the exogenous HK activity. Clearly, increasing amounts of the soluble yeast-HK decreased the rate of H2O2 release in PTM (Fig. 7A). On comparing at the levels of soluble yeast HK activity required to impair H2O2 release in PTM with the activity of endogenous mt-HK, we observed a difference of at least two orders of magnitude (Fig. 7B). This result led us to conclude that specific location of mt-HK at the outer PTM membrane is absolutely essential to improve ADP delivery for the oxidative phosphorylation, decreasing PTM H2O2 release.
Potato tuber slices incubated with Glc showed a rate of oxygen consumption 1.6-fold higher than those without Glc or with Suc (Fig. 8A ). The potato tuber slices treated with FCCP showed oxygen consumption 2.2-fold higher than that observed for the control (Fig. 8A). To ascertain whether these increased rates in O2 consumption were related to the rate of H2O2 release, we measured the accumulation of H2O2 in potato tuber slices medium. The response was the opposite to that observed for the rate of respiration by potato tuber slices in each condition (Fig. 8, B and C). The rate H2O2 release in disc slices was 3-fold lower when the slices were incubated with 5 mM Glc compared to the rate measured in the absence or in the presence of Suc, a nonphosphorylatable sugar for mt-HK (Fig. 8B, black circles and white triangles against white circles). When H2O2 release by potato tuber slices was measured in the presence of Glc and 50 mM NAG, an inhibitor of mt-HK, the rate of H2O2 release tended to be higher than that measured only in the presence of Glc (Fig. 8B, black triangles). This result is in accordance with those shown in Figures 3, 4, and 5 using isolated PTM. The H2O2 release measured in the presence of FCCP and without Glc was as low as that recorded for the potato slices incubated with 5 mM Glc without FCCP (Fig. 8B, white squares). These results indicate that the preventive effects of Glc on H2O2 release mediated by the mt-HK activity observed in isolated PTM are working in a similar manner in the potato tuber slices and are associated with increased O2 consumption.
Changes in free hexose levels by carbohydrate starvation and reverse feeding lead to variations in the respiration rate in heterotrophic plant tissues (Brouquisse et al., 1991 ; Dieuaide et al., 1992). These free hexose fluctuations may correspond to certain environmental or developmental conditions where sugar availability may be limiting (Morrell and ap Rees, 1986; Geigenberger and Stitt, 1993; Renz and Stitt, 1993; Couée et al., 2006) and are associated with ROS generation.
High Glc levels are toxic in several models because of increased release of ROS by Glc autooxidation and metabolism (Couée et al., 2006
In this work we observed mt-HK activity (Table I) in isolated PTM, in agreement with a recent report (Graham et al., 2007
As previously observed for maize mt-HK, mt-HK activity from PTM is also much more sensitive to ADP inhibition in the micromolar range when the substrate is Glc than with Fru (Fig. 1B; Galina et al., 1995
Because atractyloside and oligomycin impair the decrease of
Besides the classical antioxidant enzymes (superoxide dismutase, catalase, and ascorbate-glutathione peroxidase), two systems have been identified as participants in controlling pro- and antioxidant balance in plants, the alternative oxidase (AOX) and the uncoupling protein (PUMP). Ultimately, these two systems work to decrease the long-lived ubisemiquinone concentration which, in turn, is capable of directly reducing O2 (Skulachev, 1996
The effect of Glc in decreasing the H2O2 release by potato tuber slices and in isolated PTM could be due to a tightly bound mt-HK that guides the ADP delivery to F0F1ATP synthase via ANT in an efficient channeling to the mitochondrial matrix (Fig. 7). Almost two orders of magnitude more activity from an unbound HK form than from mt-HK is needed to reduce the rate of H2O2 release in isolated PTM (Fig. 7B). These data suggest that the access to ADP is substantially increased by mt-HK in PTM and in potato tuber slices. Several lines of evidence indicate that, in addition to its activity, the localization of mt-HK is relevant for mitochondrial respiration (Moore and Jöbsis, 1970
Comparison of ADP-recycling activity of mt-HK with the activity of StUCP as preventive antioxidant systems in PTM (Fig. 6) shows that mt-HK is able to decrease the
These similarities between mt-HK and StUCP in the response with regard to ROS formation may indicate that potato tubers have complementary mechanisms against oxidative damage induced by respiration in heterotrophic plant tissues. In conditions of higher oxidative metabolism fueled by hexoses leading to increased ROS formation, the mt-HK activity would play a predominant role as a preventive mechanism. On the other hand, when the rate of fatty acid β-oxidation is increased, the UCP becomes the main mechanism to prevent the accumulation of ROS. In plant tissues, we cannot exclude the possible operation of AOX in detoxifying the formation of superoxide anions. However, in potato tuber under no abiotic stress, the AOX activity levels are low (Calegario et al., 2003
Thus, besides its involvement in general plant sugar metabolism, in Glc sensing (Rolland et al., 2006 ), and in the regulation of programmed cell death (Kim et al., 2006), we propose that mt-HK plays a specific role in generating ADP to support oxidative phosphorylation, thereby avoiding an ATP synthesis-related limitation of respiration and subsequent H2O2 release in plants.
Chemicals and Biological Materials ADP, ATP, FCCP, horseradish peroxidase, rotenone, safranine O, MHP, NAG, yeast (Saccharomyces cerevisiae) HK, faf-BSA, β-NAD+, LA, oligomycin, and G6PDH from Leuconostoc mesenteroides were purchased from Sigma-Aldrich. Percoll was from Amersham Biosciences. Amplex Red was purchased from Invitrogen. All other reagents were analytical grade. Potato tubers (Solanum tuberosum) were purchased from a local supermarket.
PTM were obtained as previously described (Neuburger et al., 1982
The activity of mt-HK was determined by a coupled assay according to Galina et al. (1995) G6PDH activity was assayed in a reaction medium containing 50 mM Tris-HCl buffer, pH 7.4, 6 mM MgCl2, 0.1% (v/v) Triton X-100, and 0.5 mM β-NADP+. The reaction was started by adding 1 mM G6P, and G6PDH activity was determined by measuring the absorption of β-NADPH formation at 340 nm. F0F1ATPase activity was determined by measuring the release of Pi from ATP in two different reaction media: (1) in the absence or (2) in the presence of 5 mM NaN3. Both contained 20 mM Tris-HCl pH 8.0, 5 mM MgCl2, 2 mM ATP, and 1 µM FCCP. The reaction was started by the addition of PTM protein and the difference between the activities in media 1 and 2 was considered as an authentic F0F1ATPase. The PTM protein concentration varied from 0.06 to 0.1 mg/mL.
The kinetic parameters were estimated by nonlinear regression analysis applied to the Michaelis-Menten equation using the program package supplied by Origin software (Galina et al., 1999
Oxygen uptake was measured in an oximeter fitted with a water-jacket Clark-type electrode (Yellow Springs Instruments Co., model 5300) or in Oxytherm system for photosynthesis and respiration measurements in liquid phase (Hansatech Instruments).The PTM (0.2 mg/mL) were incubated with 1 to 1.5 mL of the standard respiration buffer containing 0.3 M mannitol, 10 mM Tris-HCl pH 7.2, 3 mM MgSO4, 10 mM NaCl, 5 mM KH2PO4, 0.3 mM β-NAD+, and 0.1% (v/v) faf-BSA. The cuvette was closed immediately before starting the experiments. Respiratory control ratio values were obtained with isolated PTM, after complex I inhibition by 1 µM rotenone and complex II activation by 10 mM succinate. Other additions are indicated in the figure legends.
The
The H2O2 released from PTM was determined by the Amplex Red oxidation method, as previously described (Smith et al., 2004
Small square pieces were cut (8 mm diameter, 2 mm thickness) perpendicular to the stolon-apex axis of potato tuber (Tiessen et al., 2002
The protein concentration was determined as described by Lowry et al. (1951)
Data were plotted with Origin 7.0 and analyzed by one-way ANOVA and a posteriori Tukey's test. P values < 0.05 were considered statistically different.
We are grateful to Dr. Martha Sorenson for kind and valuable comments in improving the article. This work is dedicated to Leopoldo de Meis in honor of his 70th birthday. Received September 3, 2008; accepted December 15, 2008; published December 24, 2008.
1 This work was supported by grants from the Conselho Nacional de Desenvolvimento Científico e Tecnológico, Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro. 
2 These authors contributed equally to the article. The author responsible for the distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Antonio Galina (galina{at}bioqmed.ufrj.br). www.plantphysiol.org/cgi/doi/10.1104/pp.108.129247 * Corresponding author; e-mail galina{at}bioqmed.ufrj.br.
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), 10 mM succinate (Succ) or 50 mM MHP was added to the respiration medium. In trace 3, 0.3 mM ADP was included before starting the mt-HK reaction with ATP and Glc. In trace 2, the reaction course was as in trace 1, but when indicated MHP was added. B, Inhibition of the mt-HK activity by ADP in the presence of 5 mM Glc (black circles) or Fru (white circles). The maximal activity (Vo/Vi) of 1.0 was 160 ± 23 (mean ± SE, n = 5) for Glc or 107 ± 6 (mean ± SE, n = 4) for Fru (nmol hexose-6-P min–1 mg ptn–1). In C, the Glc (black circles) dependence for mt-HK activity revealed a KM of 148 ± 12 mM. The Fru (white circles) dependence revealed a KM of 1,375 ± 380 mM. The Vmax was 150 or 121 nmol G6P min–1 mg ptn–1 for Glc or Fru, respectively. The figure shows a representative experiment. The mean values ± SE are from at least four independent preparations.
J, Maia Ide G, Arruda P, Cuccovia IM, Chaimovich H (2006) Plant uncoupling mitochondrial proteins. Annu Rev Plant Biol 57: 383–404