Plant Physiol. (1998) 118: 599-607

The Role of the Alternative Oxidase in Stabilizing the in Vivo Reduction State of the Ubiquinone Pool and the Activation State of the Alternative Oxidase

Frank F. Millenaar^*, Joris J. Benschop, Anneke M. Wagner, and Hans Lambers

Department of Plant Ecology and Evolutionary Biology, Utrecht University Graduate School of Experimental Plant Science, Sorbonnelaan 16, 3584 CA Utrecht, The Netherlands (F.F.M., J.J.B., H.L.); Department of Molecular Cell Physiology, Vrije Universiteit, De Boelelaan 1087, 1081 HV Amsterdam, The Netherlands (A.M.W.); and Department of Plant Science, Faculty of Agriculture, The University of Western Australia, Nedlands WA 6907, Australia (H.L.)

	ABSTRACT

Top Abstract Introduction Methods Results Discussion References

A possible function for the alternative (nonphosphorylating) pathway is to stabilize the reduction state of the ubiquinone pool (Q_r/Q_t), thereby avoiding an increase in free radical production. If the Q_r/Q_t were stabilized by the alternative pathway, then Q_r/Q_t should be less stable when the alternative pathway is blocked. Q_r/Q_t increased when we exposed roots of Poa annua (L.) to increasing concentrations of KCN (an inhibitor of the cytochrome pathway). However, when salicylhydroxamic acid, an inhibitor of the alternative pathway, was added at the same time, Q_r/Q_t increased significantly more. Therefore, we conclude that the alternative pathway stabilizes Q_r/Q_t. Salicylhydroxamic acid increasingly inhibited respiration with increasing concentrations of KCN. In the experiments described here the alternative oxidase protein was invariably in its reduced (high-activity) state. Therefore, changes in the reduction state of the alternative oxidase cannot account for an increase in activity of the alternative pathway upon titration with KCN. The pyruvate concentration in intact roots increased only after the alternative pathway was blocked or the cytochrome pathway was severely inhibited. The significance of the pyruvate concentration and Q_r/Q_t on the activity of the alternative pathway in intact roots is discussed.

	INTRODUCTION

Top Abstract Introduction Methods Results Discussion References

The Cyt pathway and the alternative pathway constitute the respiratory electron-transport pathways of plant mitochondria. In contrast to the Cyt pathway, beyond the branch point (ubiquinone), the alternative pathway does not contribute to the generation of a proton-motive force. The AOX protein is found in every examined plant species and in every plant organ, and the genes encoding AOX have regions that are very conserved (Vanlerberghe and McIntosh, 1997), suggesting that the alternative pathway plays a vital role in plant functioning. However, a clearly identified function for the alternative pathway has been documented only once to our knowledge (in thermogenic flowers; Meeuse, 1975).

Purvis and Shewfelt (1993) and Wagner and Wagner (1995) speculated that the alternative pathway helps to stabilize Q_r/Q_t. Q_r is a common substrate for both respiratory pathways. It has been suggested that high Q_r/Q_t levels promote free radical formation when the Cyt pathway is inhibited or restricted; respiration via the alternative pathway might then help to maintain Q_r/Q_t at a low level.

Although there is a linear relationship between the rate of mitochondrial respiration and the rate of radical formation (Puntelarulo et al., 1991; Leprince et al., 1994), radical formation is not directly connected to O₂ consumption, because uncouplers increase radical formation only to a minor extent (Chance et al., 1977; Leprince et al., 1994) and may even decrease it (Liu and Huang, 1996). Rather, radical formation is linked to the relative reduction state of the respiratory chain (Forman and Boveris, 1982). The addition of uncoupler enhances respiration but not Q_r/Q_t (Wagner and Wagner, 1995). Radical formation increases if the appropriate inhibitors (Purvis et al., 1995) are used to block one or more respiratory pathways (Chance et al., 1977; Forman and Boveris, 1982, and refs. therein; Rich and Bonner, 1987). However, when the transmembrane potential increases, the production of radicals and H₂O₂ increase as well (Liu and Huang, 1996), so it is reasonable to assume that the formation of radicals increases with an increase in Q_r/Q_t.

If Q_r/Q_t is stabilized by the alternative pathway, then the Q_r/Q_t should be less stable if the alternative pathway is blocked (with SHAM) than when it is not blocked. To determine if Q_r/Q_t is stabilized by the alternative pathway in vivo, we titrated root respiration of Poa annua (L.) with KCN (an inhibitor of the Cyt pathway) in the absence or presence of SHAM. We used a range of KCN concentrations to achieve no inhibition, a small inhibition, or full inhibition of the Cyt pathway.

On the basis of data on isolated mitochondria and kinetic modeling (Wagner and Krab, 1995) it can be expected that the alternative pathway stabilizes Q_r/Q_t in vivo; however, this hypothesis remains to be proven.

In the recent past our understanding of the mechanisms that account for activation of the alternative pathway in isolated mitochondria has increased dramatically. We now know that the alternative pathway is more active when the AOX protein becomes reduced or when specific organic acids, e.g. pyruvate, are present in sufficiently high concentrations (Umbach and Siedow, 1993; Umbach et al., 1994; Hoefnagel et al., 1995; Millar et al., 1996). If and how the activity of the alternative pathway is controlled in vivo is still entirely unknown.

To determine the activation state of the alternative pathway in intact roots, we measured the concentration of the activator pyruvate and the reduction state of the AOX protein in the roots that were used in the titration experiments.

	MATERIALS AND METHODS

Top Abstract Introduction Methods Results Discussion References

Plant Material and Growth Conditions

2

1

Respiration of Intact Roots

Measurements of Pyruvate, Ethanol, and Lactate in Intact Roots

AOX Protein

Measurement of Ubiquinone Reduction Levels in Intact Roots

The extracted ubiquinones were resuspended with a glass rod in 75 µL of N₂-purged ethanol and analyzed by HPLC (HP 1050 series, Hewlett-Packard, Amstelveen, the Netherlands). A reversed-phase Lichrosorb 5 RP 18 column (Chrompack, Bergen op Zoom, The Netherlands) with an ethanol-methanol mixture (starting with 10 min in 20% ethanol, and then through a gradient to 70% ethanol at 40 min as the mobile phase at 1 mL min¹) was used. Detection was performed at 290 and 275 nm for Q_r and oxidized ubiquinone, respectively. Commercially obtained Q₁₀ and Q₉ were used as standards (Sigma and Fluka). The extinction of Q_r measured at 290 nm was multiplied by 3.56 according to the method of Crane (1963) because of the lower extinction coefficient for Q_r compared with oxidized ubiquinone. The ubiquinone measurements were made with a recovery for Q₁₀ of 93% (n = 4); the Q₁₀ was added to the sample just after grinding.

Isolation of Mitochondria from Roots

	RESULTS

Top Abstract Introduction Methods Results Discussion References

To study the effects of a (partial) inhibition of the Cyt pathway on the levels of Q_r/Q_t with or without the alternative pathway operating, we first examined the KCN concentrations at which the Cyt pathway is inhibited and electrons are diverted to the alternative pathway.

O₂ uptake in intact roots was measured at a range of KCN concentrations in the absence or presence of SHAM. The rate of root respiration in the absence of SHAM was unaffected by KCN concentrations lower than 2 µM and was 4.4 nmol O₂ g¹ FM s¹ (Fig. 1). The respiration decreased by 35% in the presence of 100 to 400 µM KCN. SHAM (3 mM) alone significantly decreased the rate of respiration by 11% (P = 0.013). In the presence of SHAM, KCN concentrations between 0.6 and 25 µM inhibited respiration significantly more, up to 75%. Therefore, in the absence of SHAM, the alternative pathway apparently takes over an increasing number of electrons from the increasingly inhibited Cyt pathway.

View larger version (12K): [in this window] [in a new window]   Figure 1. O2 consumption (percent of control) by intact roots (detached from the plant) of P. annua plotted against the KCN concentration (note the logarithmic scale). The following KCN concentrations were used: 0 (control), 0.25, 0.63, 1, 2, 4, 25, 40, and 400 µM. The error bars indicate the SD. , Measurements made in the absence of SHAM; , measurements made in the presence of SHAM (3 mM). The number of replicates was at least three and these were from different plants and plant batches. The control respiration was 4.4 nmol O2 g1 FM s1.

At KCN concentrations exceeding 25 µM, the difference in inhibition of respiration with and without SHAM was constant, with an average of 49%.

To determine if the presence of an alternative pathway acting as an overflow for an inhibited Cyt pathway can stabilize Q_r/Q_t, we determined Q_r/Q_t at different KCN concentrations in the absence and presence of SHAM.

In intact roots of P. annua we found mainly Q₉ (67%), as has been found for many other species (Threlfall and Whistance, 1970), some Q₈ (23%), and almost no Q₁₀ (<10%). The present results for Q_r/Q_t are for Q₉ only, which had an average concentration of 4.4 ± 0.6 nmol g¹ FM. Ribas-Carbo et al. (1995) showed that different ubiquinones have the same redox behavior in isolated mitochondria.

To determine if the alternative pathway stabilizes Q_r/Q_t, we looked for an extra increase in Q_r/Q_t, which would be expected when the alternative pathway is inhibited by SHAM. By measuring Q_r/Q_t at different KCN concentrations, we determined if an increase in Q_r/Q_t coincided with an increase in activity of the alternative pathway. Q_r/Q_t was constant (44% ± 3%) up to a KCN concentration of 0.63 µM with or without SHAM (Fig. 2). Between 0.63 and 2 µM KCN, Q_r/Q_t increased much more in the presence of SHAM than in its absence. Between 2 and 25 µM KCN, the increase in Q_r/Q_t was the same (12%) in the absence and presence of SHAM. With increasing inhibition of the Cyt pathway, the alternative pathway became increasingly engaged in respiration. An increase in the activity of the alternative pathway might be the result of an increased substrate concentration (Q_r). However, AOX might also be activated when the Cyt pathway is inhibited. Such an activation could be the result of an increase in pyruvate concentration and/or in the reduction state of the protein.

View larger version (12K): [in this window] [in a new window]   Figure 2. Qr/Qt (percent) in intact roots (detached from the plant) of P. annua plotted against the KCN concentration (note the logarithmic scale). The following KCN concentrations were used: 0 (control), 0.25, 0.63, 1, 2, 4, 25, 40, and 400 µM. The error bars indicate the SD. , Measurements made in the absence of SHAM; , measurements made in the presence of SHAM (3 mM). The number of replicates was at least three and these were from different plants and plant batches.

The pyruvate concentration in the roots was constant (58 ± 7 nmol g¹ FM) up to a KCN concentration of 0.63 µM with or without SHAM (Fig. 3). Up to a KCN concentration of 4 µM (without SHAM), the pyruvate concentration did not change, whereas after the addition of SHAM, it increased significantly (P < 0.05) to 206 nmol g¹ FM. At the two highest KCN concentrations there was no further effect of SHAM on the pyruvate concentration.

View larger version (14K): [in this window] [in a new window]   Figure 3. Pyruvate concentration (nanomoles per gram FM) in intact roots (detached from the plant) of P. annua 10 to 15 min after the inhibitors were applied plotted against the KCN concentration (note the logarithmic scale). The following KCN concentrations were used: 0 (control), 0.25, 0.63, 1, 2, 4, 25, 40, and 400 µM. The error bars indicate the SD. , Measurements made in the absence of SHAM; , measurements made in the presence of SHAM (3 mM). The number of replicates was at least three and these were from different plants and plant batches.

We also determined the reduction state of the AOX protein in vivo in a tissue extract without the intermediate step of isolating mitochondria. No changes in the reduction state of the protein upon addition of KCN or SHAM were observed. The protein was invariably almost completely in its reduced (higher-activity) state (Figs. 4 and 5). The reduced AOX protein gave one prominent and three minor bands around 35 kD. The oxidized dimer gave two bands around 66 kD. The difference between the intensity of the reduced (monomer) and oxidized (dimer) bands was at least 50-fold. However, when we first isolated mitochondria from the roots and then assayed AOX, the oxidized bands were more abundant than the reduced forms. In lanes with a mixture of isolated mitochondria and whole-root extract the oxidized bands were clearly detectable (data not shown). We conclude that the absence of visible oxidized bands of AOX on our gels was not an artifact, but was caused by the virtual absence of oxidized protein in intact P. annua roots.

View larger version (38K): [in this window] [in a new window]   Figure 4. Western blot of AOX detected with monoclonal antibodies of whole-root extracts of P. annua at different concentrations of KCN in the presence or absence of SHAM (3 mM). Lane 1, 0 µM KCN without SHAM; lane 2, 0 µM KCN + SHAM; lane 3, 0.63 µM KCN without SHAM; lane 4, 0.63 µM KCN + SHAM; lane 5, 4 µM KCN without SHAM; lane 6, 4 µM KCN + SHAM; lane 7, 25 µM KCN without SHAM; lane 8, 25 µM KCN + SHAM.

View larger version (61K): [in this window] [in a new window]   Figure 5. Relative levels of reduced AOX protein from whole-root extracts of P. annua at different concentrations of KCN in the presence (cross-hatched columns) or absence (open columns) of SHAM (3 mM), with the control (0 KCN without SHAM) as 100%. The error bars indicate the SD. The number of replicates was at least three and these were from different plants and plant batches.

Figure 6 shows the root respiration in the absence and presence of SHAM (Fig. 6A) and pyruvate concentrations (Fig. 6B) plotted against Q_r/Q_t, calculated from Figures 1-3. Both O₂ uptake and pyruvate concentration were dependent on Q_r/Q_t and were unaffected by the presence of SHAM. The increase in Q_r/Q_t was obtained because the respiration was increasingly blocked by inhibitors. Because at every point a steady state was reached at which the rate of ubiquinone oxidation by definition equaled the rate of ubiquinone reduction, the curve shown in Figure 6B represents the kinetics of the combined actions of the several dehydrogenases operating in vivo: when Q_r/Q_t increases, the dehydrogenases are less able to donate electrons to the Q_r/Q_t pool. With the dehydrogenases becoming less active, the concentration of respiratory substrates (pyruvate) increases (compare Fig. 6, A and B).

View larger version (15K): [in this window] [in a new window]   Figure 6. Respiration (A; percent of control) and pyruvate concentration (B; nanomoles per gram FM) plotted against Qr/Qt in roots of P. annua. , Measurements made in the absence of SHAM (3 mM); , measurements made in the presence of SHAM. The error bars indicate the SD. The number of replicates was at least three. The data were extracted from Figures 1-3.

There was no significant change in the concentration of lactate with SHAM (235 ± 44, 240 ± 48, and 189 ± 87 nmol g¹ FM for 0, 4, and 100 µM KCN, respectively; mean values ± SD; n = 3) or ethanol (9.9 and 9.0 nmol g¹ FM for 0 and 100 µM KCN, respectively) 15 min after the addition of inhibitors.

	DISCUSSION

Top Abstract Introduction Methods Results Discussion References

A Role for the Alternative Pathway in the Stabilization of Q_r/Q_t in Intact Roots

We conclude from the data presented in Figures 1 and 2 that, especially at low KCN concentrations (up to 4 µM), when respiration is inhibited by 20%, Q_r/Q_t increases by almost 20% when SHAM is present, but only by approximately 8% when the alternative pathway is able to accept electrons. In addition, O₂ uptake proceeds at a faster rate when the alternative pathway participates in respiration. Because a high Q_r/Q_t favors the formation of free radicals, it is feasible that engaging the AOX when the Cyt pathway is inhibited is an important mechanism to prevent such a potentially harmful situation. With increasing KCN concentrations, Q_r/Q_t further increases, also in the absence of SHAM, suggesting that the AOX is not able to fully buffer a considerable inhibition of the Cyt pathway.

Activity of the Alternative Pathway in Intact Roots

At KCN concentrations greater than 25 µM, SHAM inhibited O₂ uptake, with an average inhibition of 48%, whereas Q_r/Q_t was similar in the absence and presence of SHAM. This is explained by the relationship between O₂ uptake and Q_r/Q_t as plotted in Figure 6B. At a high Q_r/Q_t, a considerable decrease in respiration no longer results in a further increase in Q_r/Q_t (Fig. 7).

View larger version (16K): [in this window] [in a new window]   Figure 7. Hypothetical reducing (curved line) and oxidizing (straight lines) pathway activities against Qr/Qt. In a steady state, the reducing activity (combined dehydrogenases) is by definition equal to the oxidizing activity (Cyt and alternative pathway). If 50% of the oxidizing pathways is inhibited, the other 50% will become more active (long arrow) because of the increase in Qr/Qt, and the inhibition will be underestimated. If subsequently 75% of the oxidizing pathways is inhibited, the 25% that is left becomes only slightly more active (short arrow) because of the steep slope of the reducing pathways and therefore the estimation of the inhibition becomes more accurate.

When the Cyt pathway is partially inhibited by low KCN concentrations (<0.60 µM), a small (11%) but significant inhibition of O₂ uptake by SHAM was observed in intact roots of P. annua. If this inhibition represented the participation of the alternative pathway, it is expected that at any of those low KCN concentrations, Q_r/Q_t would be higher in the presence of SHAM than in its absence (Wagner and Krab, 1995). However, no difference was observed in Q_r/Q_t with or without SHAM, suggesting that the alternative pathway does not contribute to respiration under these conditions. If, on the other hand, the inhibition were caused by a nonspecific effect on the reducing side of ubiquinone, a change in Q_r/Q_t would be expected: a shift to a more oxidized situation. However, Q_r/Q_t remained unchanged, so SHAM probably inhibited a nonmitochondrial component of O₂ uptake.

The Mechanisms Accounting for Increased AOX Activity

In vitro the AOX can become more active by increasing the concentration of its substrate (Q_r), by reduction of the AOX protein to its higher-activity configuration (Umbach and Siedow, 1993; Umbach et al., 1994), or by increasing the concentration of some -keto acids, e.g. pyruvate (Umbach et al., 1994; Hoefnagel et al., 1995; Millar et al., 1996). It is unknown if and to what extent these mechanisms occur in intact tissues. In vivo the amount of AOX protein might also change over time; however, because our experiments lasted only 10 to 15 min, we can exclude such an effect of protein synthesis.

Activation of the AOX by the Reduction State of AOX

The AOX protein of P. annua roots was much more oxidized in the same experiments when mitochondria were isolated before the AOX protein measurements (data not shown). The oxidized form of the protein is clearly detectable in isolated mitochondria and in samples with a mixture of isolated mitochondria with whole-root extracts. This indicates that in our experiments, the primary antibodies recognized both the oxidized and the reduced forms of the protein, as has been found previously in comparable experiments (e.g. Umbach and Siedow, 1993). The only difference between the AOX protein measurements in intact plant material and those in isolated mitochondria was the isolation of the mitochondria before immunodetection. Therefore, our results show that the AOX protein becomes more oxidized during isolation of the mitochondria material, as observed previously by Umbach and Siedow (1997). Estimations of the reduction state of the AOX protein in vivo using isolated mitochondria are therefore not suitable for obtaining information on the reduction state of the protein in intact plants.

Activation of the AOX by Pyruvate

If the alternative pathway were activated by increased concentrations of pyruvate, the concentrations inside the mitochondria (Umbach and Siedow, 1996) should change in a range around the half-maximum pyruvate stimulation. Values for the half-maximum pyruvate stimulation vary between different studies, from 128 µM in mitochondria isolated from potato tubers (Wagner et al., 1995) to 500 µM in mitochondria isolated from tobacco leaves (Vanlerberghe et al., 1995). Values for half-maximum pyruvate stimulation were determined by adding pyruvate to intact mitochondria. The binding site of pyruvate on the AOX is at the matrix side of the mitochondria (Umbach and Siedow, 1996). The pyruvate concentration outside the mitochondria may not be representative of the concentration inside the mitochondria at the activation site because of a low capacity of the mitochondrial pyruvate transporter and the production of pyruvate by malic enzyme from malate inside the mitochondria, especially at pH values around 6.5 (Millar et al., 1996).

Zang et al. (1996) found a half-maximum pyruvate stimulation of 400 µM for purified AOX protein from Arum lily and soybean. However, in these studies the protein was isolated from the mitochondrial membranes, which does not necessary represent the situation when the protein is still in a membrane, e.g. due to conformational changes.

Millar et al. (1996) used inside-out mitochondrial particles to measure the half-maximum pyruvate stimulation and found a value of less then 4 µM (in sweet potato and soybean). Finnegan et al. (1997) found comparable half-maximum pyruvate stimulation: 4.5 µM in cotyledons and 51 µM in roots of soybean inside-out mitochondrial particles. This is presumably the most appropriate method for determining the half-maximum pyruvate stimulation, because it gives the pyruvate concentration at the site where it reacts with the AOX protein to stimulate its activity.

The measured pyruvate concentrations are values pertaining to the whole tissue and were 58 ± 7 nmol g¹ FM for the control plants and increased to 250 nmol g¹ FM at the highest KCN concentrations (Fig. 3). These concentrations are similar to those found for other plants (e.g. carrot roots, 39 nmol g¹ FM [Kato-Noguchi, 1996]; petunia cell suspensions, 100 nmol g¹ FM [Wagner and Wagner, 1997]; and 60, 32, and 67 nmol g¹ FM for roots of spinach, bean, and wheat, respectively [Day and Lambers, 1983]). During hypoxia, the pyruvate concentration in barley roots increases from 60 to 120 nmol g¹ FM (Good and Muench, 1993). In tobacco cell suspensions, the pyruvate concentration increases upon the addition of antimycin from 100 to 550 nmol g¹ FM (assuming a dry matter percentage of 10%) in less than 1 h (Vanlerberghe et al., 1997).

If we estimate the concentration of pyruvate in the cytoplasm we might be able to conclude if it is in the range of the half-maximum pyruvate stimulation. The mitochondrial volume is about 0.2% to 0.7% of the total cell volume (Douce, 1985). The volume of the mitochondria is small in comparison with that of the whole cell; therefore, it is impossible to separate the pyruvate concentration inside the mitochondria from whole-tissue data. If the pyruvate is equally distributed over the mitochondria, cytosol, and vacuole, then the concentration is 60 µM, which is 1.2 to 15 times as high as the half-maximum pyruvate stimulation of 50 and 4 µM found by Finnegan et al. (1997). If the pyruvate concentration in the vacuole is 0 (which it probably is), then the concentration in the cytoplasm (10% of cell volume) will be about 10 times as high, i.e. 600 µM (12-150 times as high as the half-maximum pyruvate stimulation of 50 and 4 µM). If the pyruvate concentration in the mitochondria is at least equal to that in the cytosol, then the AOX is always fully activated by pyruvate. The end product of glycolysis in plants is not only pyruvate but also a substantial amount of malate (Day and Hanson, 1977). Malate by itself cannot be the substrate for the citric acid cycle, but needs to be converted into pyruvate via malic enzyme. The pyruvate concentration inside the mitochondria may be even higher than that in the cytosol.

Increase in AOX Activity Caused by an Increase in Q_r/Q_t

If we consider data from the literature that describe the relationship between Q_r/Q_t and O₂ uptake via the alternative pathway, a nonlinear relationship is often found when the oxidized form is measured in the absence of pyruvate. If the AOX protein is reduced, the relationship often approaches linearity, except for the first approximately 10% of the O₂ uptake (nanomoles of O₂ per milligram of protein per minute). If a straight line is fitted through these points, an average slope of 143% ± 39% (with pyruvate) and 208% ± 96% (without pyruvate) AOX activity (% Q_r/Q_t)¹ is found (Umbach et al., 1994; Day et al., 1995; Hoefnagel et al., 1995; Hoefnagel and Wiskich, 1996; Millar et al., 1996, 1997). The first nonlinear part was ignored if necessary, and the values were recalculated to percentages to compare the different respiration rates. Millar et al. (1996) found even steeper slopes using inside-out submitochondrial particles, 476% AOX activity (% Q_r/Q_t)¹ in the presence of pyruvate.

If the Q_r/Q_t increases by 10% (on a 0%-100% scale), then the activity of the alternative pathway increases by 14%, and if the active ubiquinone pool is 50% of the Q_t pool, then a change in Q_r/Q_t of 10% results in an increase of 28% in alternative pathway activity.

The alternative pathway can stabilize Q_r/Q_t because of its steep kinetics, even though Q_r/Q_t cannot be kept absolutely constant; if Q_r/Q_t increases marginally, the alternative pathway rapidly becomes more active and thereby prevents a further increase in Q_r/Q_t.

Kinetic Properties of Mitochondrial Dehydrogenases

Vanlerberghe et al. (1997) showed that if there is an imbalance between oxidation of organic acids (production of NADH) and the activity of the electron-transport pathway (production of NAD⁺), pyruvate will accumulate, which may result in fermentation under aerobic conditions. In roots of P. annua there was no accumulation of ethanol or lactate, and therefore fermentation did not occur, probably because the respiration measurements lasted only 10 to 15 min. In roots of maize (Wignarajah and Greenway, 1976), barley, and rice (Wignarajah et al., 1976), the maximum activity of alcohol dehydrogenase and especially pyruvate decarboxylase is low and increases upon anoxia. These enzymes probably have to be synthesized first in the roots of P. annua before lactate/ethanol accumulates, and that takes longer than 15 min.

The alternative pathway can avoid fermentation because it can prevent an increase in Q_r/Q_t and therefore prevent the dehydrogenases from becoming less active, so pyruvate accumulates.

	CONCLUDING REMARKS

Our results show that the alternative pathway can stabilize Q_r/Q_t in roots of P. annua (Figs. 1 and 2) when the Cyt pathway is restricted by KCN. By stabilizing Q_r/Q_t, an increase in the production of radicals and fermentation products can be prevented. In this way potential cell damage is avoided.

The increased activity of the alternative pathway as a result of KCN inhibition of the Cyt pathway is not caused by a further reduction of the AOX protein (Fig. 5); almost all of the AOX is already in its reduced state in the intact P. annua roots in the absence of inhibitors. A small change in Q_r/Q_t has a large effect on the activity of the alternative pathway. Therefore, the alternative pathway stabilizes Q_r/Q_t. The role of pyruvate in the increased activity of the alternative pathway is not entirely clear from our results, but the pyruvate concentration always seemed to be higher than the half-maximum pyruvate stimulation.

	FOOTNOTES

   Received February 27, 1998; accepted July 9, 1998.

	ABBREVIATIONS

Abbreviations: AOX, alternative oxidase. FM, fresh mass. Q_r, reduced ubiquinone. Q_r/Q_t, reduction state of the ubiquinone pool. Q_t, total ubiquinone. SHAM, salicylhydroxamic acid.

	LITERATURE  CITED

Top Abstract Introduction Methods Results Discussion References

Atkin OK, Villar R, Lambers H (1995) Partitioning of electrons between the cytochrome and alternative pathways in intact roots. Plant Physiol 108: 1179-1183 [Abstract]

Chance B, Boveris A, Oshino N (1977) Alcohol and Aldehyde Metabolizing Systems. Academic Press, New York, pp 261-274

Crane FL (1963) The coenzyme Q group (ubiquinones). Prog Chem Fats Other Lipids 7: 267-289

Day DA, Hanson JB (1977) Pyruvate and malate transport and oxidation in corn mitochondria. Plant Physiol 59: 630-635 [Abstract/Free Full Text]

Day DA, Krab K, Lambers H, Moore AL, Siedow JN, Wagner AM, Wiskich JT (1996) The cyanide-resistant oxidase. To inhibit or not to inhibit, that is the question. Plant Physiol 110: 1-2 [ISI][Medline]

Day DA, Lambers H (1983) The regulation of glycolysis and electron transport in roots. Plant Physiol 58: 155-160

Day DA, Whelan J, Millar AH, Siedow JN, Wiskich JT (1995) Regulation of the alternative oxidase in plants and fungi. Aust J Plant Physiol 22: 497-509

Douce R (1985) Mitochondria in Higher Plants: Structure, Function, and Biogenesis. Academic Press, New York, pp 4-7

Elthon TE, Nickels RL, McIntosh L (1989) Monoclonal antibodies to the alternative oxidase of higher plant mitochondria. Plant Physiol 89: 1311-1317 [Abstract/Free Full Text]

Finnegan PM, Whelan J, Millar AH, Zhang Q, Smith MK, Wiskich JT, Day DA (1997) Differential expression of the multigene family encoding the soybean mitochondrial alternative oxidase. Plant Physiol 114: 455-466 [Abstract]

Forman HJ, Boveris A (1982) Superoxide radical and hydrogen peroxide in mitochondria. In Free Radicals in Biology, Vol V. Academic Press, New York, pp 65-90

Good AG, Muench DG (1993) Long-term anaerobic metabolism in root tissue. Plant Physiol 101: 1163-1168 [Abstract]

Hoefnagel MHN, Millar AH, Wiskich JT, Day DA (1995) Cytochrome and alternative respiratory pathways compete for electrons in the presence of pyruvate in soybean mitochondria. Arch Biochem Biophys 318: 394-400 [CrossRef][ISI][Medline]

Hoefnagel MHN, Wiskich JT (1996) Alternative oxidase activity and the ubiquinone redox level in soybean cotyledon and Arum spadix mitochondria during NADH and succinate oxidation. Plant Physiol 110: 1329-1335 [Abstract]

Kato-Noguchi H (1996) Effects of citrate on respiratory gas exchange and metabolism in carrot root tissues. Phytochemistry 45: 225-227 [CrossRef]

Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685 [CrossRef][Medline]

Leprince O, Atherton NM, Deltour R, Hendry GAF (1994) The involvement of respiration in free radical processes during loss of desiccation tolerance in germinating Zea mays L. Plant Physiol 104: 1333-1339 [Abstract]

Liu SS, Huang JP (1996) Coexistence of a "reactive oxygen cycle" with the "Q-cycle" in the respiratory chain: a hypothesis for generating, partitioning, and function of superoxide in mitochondria. In Proceedings of International Symposium on Natural Antioxidants: Molecular Mechanisms and Health Effects. AOCS Press, Champaign, IL, pp 513-529

Meeuse BJD (1975) Thermogenic respiration in aroids. Annu Rev Plant Physiol 26: 117-126 [ISI]

Millar AH, Atkin OK, Lambers H, Wiskich JT, Day DA (1995) A critique of the use of inhibitors to estimate partitioning of electrons between mitochondrial respiratory pathways in plants. Physiol Plant 95: 523-532 [CrossRef]

Millar AH, Finnegan PM, Whelan J, Drevon JJ, Day DA (1997) Expression and kinetics of the mitochondrial alternative oxidase in nitrogen-fixing nodules of soybean roots. Plant Cell Environ 20: 1273-1282 [CrossRef]

Millar AH, Hoefnagel MHN, Day DA, Wiskich JT (1996) Specificity of the organic acid activation on the alternative oxidase in plant mitochondria. Plant Physiol 111: 613-618 [Abstract]

Poorter H, Remkes C (1990) Leaf area ratio and net assimilation rate of 24 wild species differing in relative growth rate. Oecologia 83: 553-559 [CrossRef][ISI]

Puntelarulo S, Galleano M, Sanchez RA, Boveris A (1991) Superoxide anion and hydrogen peroxide metabolism in soybean embryonic axes during germination. Biochim Biophys Acta 1074: 277-283 [Medline]

Purvis AC, Shewfelt RL (1993) Does the alternative pathway ameliorate chilling injury in sensitive plant tissue? Physiol Plant 88: 712-718 [CrossRef]

Purvis AC, Shewfelt RL, Gegogeine JW (1995) Superoxide production by mitochondria isolated from green bell pepper fruit. Physiol Plant 94: 743-749 [CrossRef]

Ribas-Carbo M, Wiskich JT, Berry JA, Siedow JN (1995) Ubiquinone redox behavior in plant mitochondria during electron transport. Arch Biochem Biophys 317: 156-160 [CrossRef][Medline]

Rich PR, Bonner WD (1987) The sites of superoxide anion generation in higher plant mitochondria. Arch Biochem Biophys 188: 206-213

Threlfall DR, Whistance GR (1970) Biosynthesis of ubiquinone: a search for polyprenyl phenol and quinone precursors. Phytochemistry 9: 355-359 [CrossRef]

Umbach AL, Siedow JN (1993) Covalent and noncovalent dimers of the cyanide-resistant alternative oxidase protein in higher plant mitochondria and their relationship to enzyme activity. Plant Physiol 103: 845-854 [Abstract]

Umbach AL, Siedow JN (1996) The reaction of the soybean cotyledon mitochondrial cyanide-resistant oxides with sulfhydryl reagents suggests that -keto acid activation involves the formation of a thiohemiacetal. J Biol Chem 271: 25019-25026 [Abstract/Free Full Text]

Umbach AL, Siedow JN (1997) Changes in the redox state of the alternative oxidase regulator sulfhydryl/disulfide system during mitochondrial isolation: implications for inferences of activity in vivo. Plant Sci 123: 19-28 [CrossRef]

Umbach AL, Wiskich JT, Siedow JN (1994) Regulation of alternative oxidase kinetics by pyruvate and intermolecular disulfide bond redox status in soybean seedling mitochondria. FEBS Lett 348: 181-184 [CrossRef][ISI][Medline]

van den Bergen CWM, Wagner AM, Krab K, Moore AL (1994) The relationship between electron flux and the redox poise of the quinone pool in plant mitochondria: interplay between quinol-oxidizing and quinone-reducing pathways. Eur J Biochem 226: 1071-1078 [Medline]

Vanlerberghe GC, Day DA, Wiskich JT, Vanlerberghe AE, McIntosh L (1995) Alternative oxidase activity in tobacco leaf mitochondria. Plant Physiol 109: 353-361 [Abstract]

Vanlerberghe GC, McIntosh L (1997) Alternative oxidase: from gene to function. Annu Rev Plant Physiol Plant Mol Biol 48: 703-734 [CrossRef][ISI]

Vanlerberghe GC, Vanlerberghe AE, McIntosh L (1997) Molecular genetic evidence of the ability of alternative oxidase to support respiratory carbon metabolism. Plant Physiol 113: 657-661 [Abstract]

Wagner AM, Bergen van CWM, Wincencjusz H (1995) Stimulation of the alternative pathway by succinate and malate. Plant Physiol 108: 1035-1042 [Abstract]

Wagner AM, Krab K (1995) The alternative respiration pathway in plants. Role and regulation. Plant Physiol 95: 318-325 [CrossRef]

Wagner AM, Wagner MJ (1995) Measurements of in vivo ubiquinone reduction levels in plant cells. Plant Physiol 108: 277-283 [Abstract]

Wagner AM, Wagner MJ (1997) Changes in mitochondrial respiratory chain components of petunia cells during culture in the presence of antimycin A. Plant Physiol 115: 617-622 [Abstract]

Wignarajah K, Greenway H (1976) Effect of anaerobiosis on activities of alcohol dehydrogenase and pyruvate decarboxylase in roots of Zea mays. New Phytol 77: 575-584

Wignarajah K, Greenway H, John CD (1976) Effect of waterlogging on growth and activity of alcohol dehydrogenase in barley and rice. New Phytol 77: 585-592

Zhang Q, Hoefnagel MHN, Wiskich JT (1996) Alternative oxidase from Arum and soybean: its stabilization during purification. Physiol Plant 96: 551-558 [CrossRef]

Copyright Clearance Center:   0032-0889/98/118//09
 
© 1998 American Society of Plant Physiologists