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246 BIOCHEMICAL SOCIETY TRANSACTIONS The ‘Uniqueness’ of Plant Mitochondria J. M. PALMER Department of Botany, Imperial College of Science and Technology, Prince Consort Road, London S W7 2BB, U.K. The accepted biochemical view of the mammalian mitochondrion is that it functions as the powerhouse of the cell. It is equipped with a linear respiratory chain that is tightly coupled to synthesis of ATP, which is the intermediate needed to run the energy economy of the cell. It is not thought to have any other major metabolic role in the cell. For many years plant mitochondria were thought to be very similar to mammalian mitochondria. However, it is becoming increasingly apparent that mitochondria isolated from plants and some micro-organisms have a considerably more complex respiratory chain, which may enable them to fulfil a more complex metabolic function in the cell. It is these differences that form the basis of the ‘uniqueness’ that is the subject of the present review. Here I shall limit my discussion to what I consider are the major differences. These are (a) the characteristics of the cyanide-resistant oxidase, which is not uniquely associated with plant mitochondria and is frequently encountered in microbial mitochondria; ( b ) the organization of the NADH dehydrogenase system, which is a less-well-documented area; nevertheless it raises important issues concerning the regulation of electron flux along branched pathways and the metabolic significance of the mitochondrion in the cell; and finally (c) the nature of the enzymes responsible for the oxidation of malate, which differ from those present in mammalian mitochondria by enabling malate oxidation to proceed in the presence of oxaloacetate. This complex system for the oxidation of malate makes it possible to postulate that the tricarboxylic acid cycle in plant mitochondria can oxidize to completion any intermediate of the cycle without the necessity to supply pyruvate from glycolysis. Thus the tricarboxylic acid cycle in plants is a more versatile system than is its mammalian counterpart. Cyanide-resistant respiration Progress in elucidating the molecular basis of cyanide-resistant respiration in plant mitochondria has been frustratingly slow. The alternative oxidase is found in different amounts in many different types of plant mitochondria. Mitochondria isolated from storage tubers of Solanum tuberosum (potatoes) or Helianthus tuberosus (Jerusalem artichokes) are very sensitive to inhibition by cyanide, which is rather surprising, since oxygen uptake in the intact tubers is stimulated by gaseous cyanide. The fate of the cyanide-resistant oxygen uptake on isolation of the mitochondria is not known; it is possible that it may not have been originally associated with the mitochondria or that the resistant oxidase may have been inactivated during the isolation of the organelles. It has been suggested that the loss of the cyanide-resistant oxidase is caused by the destruction of phospholipids caused by the release of phospholipases during the isolation procedure (Solomos, 1977). Mitochondria isolated from tubers of Ipomoea batatas (sweet potatoes) or Manihot esculenta (cassava) have about equal concentrations of both the cyanide-sensitive and -resistant oxidases, whereas mitochondria isolated from the mature spadix of Arum maculatum (cuckoo pint) have ten times more cyanide-resistant oxidase than cytochrome oxidase. The spadix of the Arum is a very interesting tissue; its physiological role is to become thermogenic for a limited period during the life of the flower; the heat releases amines that attract flies involved in the pollination process. Mitochondria isolated from this tissue are capable of very high rates of oxygen uptake, in the order of 4000nmol/min per mg of protein, most of which is mediated by the cyanide-resistant oxidase. Little is definitely known about the characteristics of this alternative cyanide-resistant oxidase. Many theories have been proposed and none has stood the test of time. It was first thought that the oxidase had a high K,,, for oxygen, and on this basis it was suggested that the oxidase may be a flavoprotein (James & Beevers, 1950). The K,,, 1979 BIOCHEMICAL REVIEWS 247 measurements were subsequently shown to be incorrect, owing to problems of oxygen accessibility to the oxidase, and the idea that the oxidase was a flavoprotein has dropped from favour. However, it has been frequently observed that mitochondria that contain significant amounts of the alternative oxidase also contain significantly higher amounts of flavoprotein; it is also clear that the induction of cyanide resistance in microorganisms alters the spectral behaviour of the flavoproteins (Lloyd, 1974) ; and finally, kinetic measurements show that there is a pool of flavoprotein that could act as the oxidase (Erecinska & Storey, 1970). A second theory was that the autoxidation of cytochrome b could mediate the oxidase; initial kinetic experiments showed that, in the presence of cyanide, there was a species of cytochrome 6 that could fulfil this role. However, when antimycin A was added, it prevented the redox changes associated with cytochrome b without having any inhibitory effect on the cyanide-resistant oxidase (Bendall & Bonner, 1971). In recent years the most significant observation relating to the operation of the alternative oxidase has been that it can be specifically inhibited by substituted benzhydroxamic acids (Schonbaum et al., 1971). Since these compounds can chelate iron, much effort has been expended to prove that the oxidase is an iron-sulphur protein. This theory has not met with any success, and direct examination of Arum mitochondria with e.p.r. techniques has failed to show any unique ironsulphur centre that could be a candidate for the oxidase (Cammack & Palmer, 1977). These e.p.r. studies showed, however, that Arum mitochondria did differ from animal mitochondria by having very weak e.p.r. signals that could be attributed to centres N1-4 of the NADH dehydrogenase and very strong signals associated with centres S1-3 of the succinic dehydrogenase. Therefore at present we have no definitive evidence concerning the nature of the oxidase. Even simple facts such as the nature of the products formed when the oxidase interacts with oxygen remain to be clarified, although recent studies appear to confirm that water is the first detectable product (Huq & Palmer, 1978a); this information is significant when speculating on the nature of the oxidase. Some progress has been made concerning the relationship between the alternative oxidase and the conventional respiratory chain. Kinetic measurements (Storey, 1976) show that at least a portion of the ubiquinone pool acts as if it were mediating electron flux to the alternative oxidase. Rich & Moore (1976) have produced evidence that an e.p.r. signal at g = 1.98, resulting from spin-spin interaction between two ubisemiquinone radicals, is closely associated with the alternative oxidase, and have proposed that the cyanide-resistant oxidase acts by oxidizing the ubiquinol to the ubisemiquinone. More recently it has been possible to extract the quinones from freeze-dried mitochondria and demonstrate that cyanide-resistant oxidation ceases until ubiquinone is re-incorporated (Huq & Palmer, 19786). The elucidation of the mechanism by which the reduced quinones are oxidized remains a prize to be won. Recently a quinol oxidase with the correct properties and specific activities to be the alternative oxidase has been solubilized from Arum mitochondria in my laboratory (Huq & Palmer, 1978c) and by Rich & Bonner (1978). We have partially purified and characterized our preparation and find it contains a fluorescent compound, that is probably a flavoprotein, and significant quantities of copper. It is not clear whether these components are involved in mediating electron flux between the quinol and oxygen. It is noteworthy that quinols can naturally interact with molecular oxygen, and it is possible that the oxidase could be a protein with no redox component that provides a favourable environment to enhance the rate of autoxidation. However, it seems likely that the nature of the oxidase will be resolved in the near future. From the discussion so far it may be concluded that the alternative oxidase may simply operate as a branch from the conventional respiratory chain at the level of ubiquinone. However, data are accumulating to suggest that the system may not be this simple. In the aroid spadices, which use the cyanide-resistant oxidase to produce heat, all substrates appear to have equal access to the oxidase. However, in mitochondria from non-thermogenic tissue, such as Triticum vulgare (wheat) roots, Ipomoea batatas and Munihot esculenta, it appears that, whereas electrons from endogenous NADH can have access to the cyanide-resistant oxidase, electrons from exogenous VOl. 7 248 BIOCHEMICAL SOCIETY TRANSACTIONS NADH do not have access to this oxidase (Huq & Palmer, 19786). It is thought that electrons from endogenous and exogenous NADHJoin the main chain at the level of the quinone. If there is a single homogeneous pool of quinone receiving electrons from the various dehydrogenases and distributing them to the different oxidases, it is difficult to see, if using the foregoing model, how electrons emanating from exogenous NADH can be excluded from the cyanide-resistant oxidase. In order to achieve this discrimination, more than one functional pool of quinone must be present. Experimental evidence is now available to suggest that the quinone associated with the cyanide-resistant oxidase is more readily extracted with pentane than is the quinone associated with the cytochrome c oxidase (Huq & Palmer, 19786). This would be consistent with the existence of two functional pools of ubiquinone. If this should prove to be correct, then the alternative oxidase would branch from the conventional respiratory chain before the quinone pool. There is evidence to support the speculation that the two respiratory systems do not necessarily branch from each other but represent two parallel chains which can in vitro, under appropriate conditions of phosphate potential and inhibitors, communicate with each other. There is little evidence to show that, in mitochondria from higher plants, electrons must traverse the first site of oxidative phosphorylation before becoming available to the cyanide-resistant oxidase. Lips & Biale (1 966) observed that amytal, normally considered to be an inhibitor of the NADH dehydrogenase, failed to inhibit respiration in Persea gratissima (avocado) tissue and proposed that two parallel pathways of electron transport existed ;one was resistant to amytal, antimycin A and cyanide and was unable to support oxidative phosphorylation; the other chain had conventional sensitivity to inhibitors and was coupled to the synthesis of three molecules of ATP. Treatments that induce cyanide-resistant oxidation also increase the activity of piericidin-resistant NADH dehydrogenase (Cammack & Palmer, 1973). If the two oxidases represent the terminal components of two parallel chains rather than the end of two branches with a common origin, then it becomes necessary to re-examine the mechanism by which electron flux to the two oxidase systems is regulated. The study by Bahr & Bonner (1973) represents the only viable model for regulation and visualizes that the alternative electron-transport sequence branches from, and is in rapid equilibrium with, the conventional electron-transport chains as shown in Scheme 1. In this scheme the alternative electron-transport sequence branches from the conventional respiratory chain at the unidentified component B, and the first component of the alternative sequence, represented by A, has a standard redox potential approximately 50 mV more negative than that of component B. Under normal State-3 conditions, component B would be partially reduced and capable of acting as a donor to the cytochrome system, whereas component A, in equilibrium with B, would be much less reduced and consequently would fail to act as an efficient donor to the alternative oxidase. According to the scheme proposed by Bahr & Bonner (1973) the alternative oxidase only works when the cytochrome c oxidase is saturated, which only occurs under State-4 conditions or in the presence of inhibitors of the conventional oxidase. Under these conditions compound B becomes very reduced and consequently compound A becomes more Succinate --f Fp -B A c--, cyt CI alt oxidase t , - cyt c c+ cyt u C, cyt u3 -----+ o2 O2 Scheme 1. Possible relationship between the conventional and alternative oxidases in plant mitochondria The horizontal axis represents an approximate measure of standard redox potential, hence A is more reducing than B. Abbreviations used: Fp, flavoprotein; cyt, cytochrome; alt, alternative. 1979 249 BIOCHEMICAL REVIEWS Step (I) NADH --+ F p + Fe-S -+ Q + cyt b -+ cyt c t ---+ cyt c -+ cyt u -+ cyt u3 -+ O2 I \ ll /I 1I i l Step(2) NADH + F p ____+ Q + alt oxidase ’ 0 2 Scheme 2. Possible organization of electron transport as two parallel steps Step (1) is stimulated by AMP, and step ( 2 ) inhibited by AMP (Sotthibandhu &Palmer, 1975). Q , quinone; other abbreviations as in Scheme 1. reduced and can then donate electrons to the cyanide-resistant oxidase. There is some doubt as to whether the energy charge becomes high enough in intact plant cells to impose State-4 control, and the concentration of cyanide in higher-plant cells, even in tissues containing cyanogenic glucosides, is not thought to be sufficient to inhibit cytochrome oxidase. Therefore, it is not certain whether the control mechanisms described by Bahr & Bonner (1973) can play a role in uiuo. If the respiratory system is organized as two parallel chains, as shown in Scheme 2, then the control system outlined by Bahr & Bonner (1973) would not apply. It is, however, possible to speculate that factors such as AMP, which regulate electron flux through the different dehydrogenases (Palmer & Coleman, 1974; see also below), may influence which oxidase is used. NA DH dehydrogenase system Plant mitochondria also differ from their mammalian counterpart in having a more complex NADH dehydrogenase system. These differences are illustrated by the observations that (a) plant mitochondria can oxidize exogenous NADH and (b) the oxidation of endogenous NADH is only partially inhibited by rotenone or piericidin (Palmer & Coleman, 1974). Intact mammalian mitochondria cannot oxidize exogenous NADH because the NADH molecule cannot traverse the inner membrane. This compartmentation allows the nicotinamide nucleotide in the cytosol and mitochondria1 matrix to adopt different redox poises. The ability of plant mitochondria to oxidize exogenous NADH was originally thought to be due to the NADH entering the matrix space and being oxidized by the internal dehydrogenase. This apparent ‘leakiness’ was thought to be due to damage induced by the rigorous grinding techniques used in isolating the organelles. However, evidence suggests that plant mitochondria are relatively tough structures capable of withstanding far greater osmotic stress than are their mammalian counterparts. The osmotic stability may be related to the large amounts of polyuronides associated with the outer membrane (Mannella & Bonner, 1975). It is now generally agreed that exogenous and endogenous NADH are oxidized by different NADH dehydrogenases and that the enzyme responsible for the oxidation of exogenous NADH may have its active site located on the outer surface of the inner membrane (Palmer & Coleman, 1974). However, the existence of an NADH dehydrogenase associated with the outer membrane has hampered research into the precise location of the external dehydrogenase associated with the inner membrane. A similar NADH dehydrogenase is known to exist in mitochondria from the fungus Neurospora crassa (Von Jajow & Klingenberg, 1970). The external dehydrogenase feeds electrons into ubiquinone without involving the piericidin A-sensitive site of the respiratory chain or the first site of oxidative phosphorylation. The metabolic significance of this external dehydrogenase capable of oxidizing cytosolic NADH is not known. At first sight it appears to be an undesirable feature, and it seems reasonable to expect that the electron flux through this dehydrogenase would be closely regulated; little evidence is available on how this may be achieved. The oxidation of external NADH appears to be dependent on the presence of bivalent cations, particularly calcium, and is inhibited by bivalent-cation VOl. 7 250 BIOCHEMICAL SOCIETY TRANSACTIONS chelators such as EGTA and citrate (Palmer & Coleman, 1974). The mechanism by which calcium stimulates the electron flux remains a mystery. It is therefore possible, but not certain, that the distribution of bivalent cations in the cell may be involved in the regulation of dehydrogenase activity. Results have been obtained (R. C. Cowley & J. M. Palmer, unpublished work) that show that the oxidation of succinate takes precedence over the oxidation of exogenous NADH. Thus the addition of succinate to mitochondria oxidizing exogenous NADH severely curtails the NADH oxidase. This is clearly a potential control mechanism, although the molecular basis of the interaction between internal and external dehydrogenases remains unsolved. The NADH dehydrogenase responsible for the oxidation of endogenous NADH appears to be more complex than its enzymic counterpart in mammalian mitochondria. The most obvious manifestation of this phenomenon is the observation that inhibitors such as rotenone or piericidin A, which inhibit the oxidation of endogenous NADH in animal mitochondria by interacting with the iron-sulphur centres of the dehydrogenase, only cause a partial inhibition in the plant system. By measuring the efficiency of ATP synthesis it is apparent that the piericidin-resistant pathway is not coupled to the first site of ATP synthesis, whereas the piericidin-sensitive component does result in ATP synthesis in this region of the chain. The components involved in catalysing this inhibitor-resistant non-phosphorylating oxidation of endogenous NADH are not yet known. It seems clear, however, that some system must exist to regulate the flux of electrons through the two internal NADH dehydrogenase systems. Early observations seem to suggest that under normal State-3 respiratory conditions the nonphosphorylating dehydrogenase plays only a small role, since the oxidation of NAD+linked substrates can be shown to be coupled to ATP synthesis at three sites. However, as soon as piericidin A is added, only two sites of ATP remain functional. The electron flux through the two internal NADH dehydrogenases appears to be under the control of the adenine nucleotides in the cytosol. This conclusion was reached when investigating why plant mitochondria did not oxidize NAD+-linked substrates rapidly in the presence of weak acid uncouplers (Sotthibandhu & Palmer, 1975). The results clearly showed that adenine nucleotides, and particularly AMP, were necessary in order to obtain maximum rates of oxidation in the presence of uncoupling agents. AMP stimulated electron flux from NADH to cytochrome b via a piericidin A-sensitive pathway. It was also apparent that the AMP did not have to enter the mitochondrion in order to bring about the stimulation. This appears to be a potential control mechanism, whereby an increase in AMP in the cytosol results in the stimulation of the internal NADH dehydrogenase coupled to ATP synthesis. The mechanism by which external AMP stimulates the oxidation of internal NADH is at present speculative, but presumably it occurs as the result of AMP binding to the outer loop of the proton-pumping NADH dehydrogenase. The physiological significance of the multiple systems for oxidizing NADH is not understood. It has already been suggested in the present review that the piericidin A-sensitive phosphorylating dehydrogenase may be associated with the cyanidesensitive oxidase, whereas the non-phosphorylating dehydrogenase, which works best in the absence of AMP, is associated with the cyanide-resistant oxidase providing a totally non-phosphorylating pathway for the oxidation of NADH when the energy charge is high. It must be emphasized that this is speculation, and further experimental justification is necessary. Oxidation of malate There is evidence that the different NADH dehydrogenases described above may be closely associated with the NAD+-linked dehydrogenases responsible for the oxidation of malate. Malate oxidation in plant mitochondria is a much more complex process than in mammalian mitochondria, in which oxidation is achieved by a single enzyme, malate dehydrogenase, and continual oxidation of malate depends on the removal of oxaloacetate, which is a product of the reaction. It has long been observed 1979 BIOCHEMICAL REVIEWS 25 1 that plant mitochondria were capable of oxidizing malate without the need to remove oxaloacetate, indicating that plant mitochondria either have a system for removing the oxaloacetate or do not produce oxaloacetate as a product of malate oxidation. It is now clear that both mechanisms are involved, and malate is oxidized by the malate dehydrogenase and an NAD+-linked ‘malic’ enzyme to produce oxaloacetate and pyruvate respectively, which then give rise to citrate. The ability to produce pyruvate from malate in the matrix of the mitochondrion makes it possible to completely oxidize any tricarboxylic acid-cycle acid without the necessity to supply acetyl-CoA to complete the cycle (Palmer, 1976). This scheme has many attractions, since large quantities of organic acids are known to be stored in plant tissue. It also has the disadvantage that it could allow the oxidation of the catalytic pool of acids necessary for the operation of the tricarboxylic acid cycle, and would also keep the concentration of oxaloacetate very low. The concentration of this acid is believed to be an important factor in integrating the activity of catabolic and anabolic metabolism. Factors that determine the relative contribution of the malate dehydrogenase and ‘malic’ enzyme to the overall rate of malate oxidation are not fully understood. There is, however, evidence to suggest that although both enzymes are located inside the inner membrane of the mitochondrion, they are spatially separate from each other (Brunton & Palmer, 1973). This can be shown by using exogenous oxaloacetate to oxidize endogenous NADH. Plant mitochondria are readily permeable to added oxaloacetate, which, if added at a suitable concentration, will enter the matrix and cause the malate dehydrogenase to run in reverse; this reaction oxidizes any NADH being produced more readily than does the respiratory chain. Hence when oxaloacetate is added to mitochondria oxidizing NAD+linked substrates, there is an immediate and essentially complete inhibition of oxygen uptake until all the oxaloacetate has been removed. This inhibition was observed with all substrates except malate, which continued to supply NADH to the respiratory chain in the presence of oxaloacetate, clearly indicating spatial separation of the malate dehydrogenase and ‘malic’ enzyme. The evidence (Brunton & Palmer, 1973) clearly suggests that the NADH produced by malate dehydrogenase and ‘malic’ enzyme appears to be oxidized by different NADH dehydrogenase systems. The NADH produced by the ‘malic’ enzyme appears to be oxidized by the NADH dehydrogenase closely associated with the piericidin A-sensitive component and coupled phosphorylation, whereas the NADH from the malate dehydrogenase appears more readily accessible to the piericidin A-resistant, non-phosphorylating dehydrogenase. This possible association of the malate oxidoreductases with different NADH dehydrogenases provides a potential physiological role for the multiple dehydrogenase systems and a regulating mechanism for malate oxidation via the two enzymes involved. The physiological importance of the complex respiratory chain and tricarboxylic acid cycle known to exist in plant mitochondria can only be the subject of speculation. It seems clear, however, that plant mitochondria have a complex respiratory chain that can by-pass all the sites of ATP synthesis and a tricarboxylic acid cycle that can oxidize to completion a variety of compounds other than pyruvate. The combination of these two abilities would result in a system that could metabolize carbon compounds through the mitochondria1 enzyme system much more rapidly than the turnover of the ADP/ATP pool. This is clearly important in thermogenesis, but it could also be significant in photosynthetic tissue, where photosynthesis maintains a high energy charge, or in tissue in which carbon metabolism is very rapid, particularly when one form is being converted into another, when substrate-level phosphorylation may maintain a high energy charge. Bahr, J. T. & Bonner, W. D. (1973) J . Biol. Chem. 248,3446-3450 Bendall, D. S . & Bonner, W. D. (1971) Plant Physiol. 47,236-245 Brunton, C . J. & Palmer, J. M. (1973) Eur. J . Biochem. 39,283-291 Cammack, R. & Palmer, J. M. (1973) Ann. N . Y.Acad. Sci. 222,16-23 Cammack, R. & Palmer, J. M. (1977) Biochem. J . 166,347-355 Erecinska, M. & Storey, B. T. (1970) Plant Physiol. 46, 618-624 VOl. 7 252 BIOCHEMICAL SOCIETY TRANSACTIONS Huq, S. &Palmer, J. M. (1978~)Plant Sci. Lett. 11, 351-358 Huq, S. &Palmer, J. M. (19786) in Plant Mitochondria(Ducet,G. &Lance, C.,eds.), pp. 225-232, Elsevier/North-Holland Biomedical Press, Amsterdam Huq, S. L Palmer, J. M. (1978~)FEBSLett. 95,217-220 James, W. 0. & Beevers, H. (1950) New Phytol. 49,353-374 Lips, S . H. & Riale, J. B. (1966) Plant Physiol. 41,797-802 Lloyd, D. (1974) The Mitochondria of Microorganisms pp. 137-158, Academic Press, London Mannella, C. A. & Bonner, W. D. (1975) Biochim. Biophys. Acta 413,213-225 Palmer, J. M. (1976) Annu. Rev. Plant Physiol. 27, 133-157 Palmer, J. M. & Coleman, J. 0. D. (1974) Horizons Biochem. Biophys. 1,220-260 Rich, P. R. & Bonner, W. D. (1978) in Plant Mitochondria (Ducet, G.& Lance, C., eds.), pp. 61-68, Elsevier/North-Holland Biomedical Press, Amsterdam Rich, P. R. & Moore, A. L. (1976) FEBS Leti. 65,339-344 Schonbaum, G. R.,Bonner, W. D.,Storey,B. T. &Bahr, J. T. (1971)PIantPhysiol. 44,115-128 Solomos, T. (1977) Annu. Rev. Plant Physiol. 28,279-297 Sotthibandhu, R. & Palmer, J. M. (1975) Biochem. J. 152,637-645 Storey, B. T. (1976) Plant Physiol. 58,521-525 Von Jagow, G. & Klingenberg, M. (1970) Eur. J. Biochem. 12,583-592 1979