JXB Advance Access originally published online on November 28, 2003
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Journal of Experimental Botany, Vol. 55, No. 394, pp. 1-10, January 1, 2004
© 2004 Oxford University Press
Plant Carbon-Nitrogen Interactions from Rhizospheres to Planet |
Targets of stress-induced oxidative damage in plant mitochondria and their impact on cell carbon/nitrogen metabolism
Received 28 April 2003; Accepted 26 June 2003
Biochemistry and Molecular Biology, School of Biomedical and Chemical Sciences, Faculty of Life and Physical Sciences, The University of Western Australia, 35, Stirling Highway, Crawley, WA 6009, Australia
* To whom correspondence should be addressed. Fax: +61 8 93801148. E-mail: hmillar{at}cyllene.uwa.edu.au
Abbreviations: HNE, 4-hydroxy-2-nonenal; AOS, active oxygen species; GDC, glycine decarboxylase; MDA, malondialdehyde; Fe-S, iron-sulphur.
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Abstract |
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Plant mitochondria link the cellular processes of carbon and nitrogen metabolism through the tricarboxylic acid cycle and the photorespiratory cycle. Environmental stresses lead to damage of specific mitochondrial targets through the direct action of reactive oxygen species and indirect action of lipid peroxidation products. Uncovering the extent of this damage, the exact sites of damage and the mechanisms of avoidance and/or repair remains a largely unresearched challenge for plant scientists. Damage to Fe-S centres and proteins containing lipoic acid moieties appear to predominate in current reports. Substantial evidence that both TCA cycle and photorespiratory capacity of mitochondria are sensitive sites for damage is highlighted and the implications for mitochondrial-dependent carbon and nitrogen metabolism are discussed.
Key words: Active oxygen species, carbon metabolism, environmental stress, glycine decarboxylase, lipid peroxidation, nitrogen metabolism, plant mitochondria.
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Introduction |
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Mitochondria contain biochemical pathways and components which link the cellular processes of carbon and nitrogen metabolism in plants (Fig. 1). The tricarboxylic acid cycle (TCA cycle) links both carbon and nitrogen metabolism by the oxidation of organic acids from glycolysis and the export of either
-ketoglutarate directly or citrate, which can be converted to -ketoglutarate in the cytosol via cytosolic isoforms of aconitase and isocitrate dehydrogenase, as carbon skeletons for amino acid synthesis (Hodges, 2002). The components of the photorespiratory cycle in mitochondria decarboxylate and deaminate glycine to produce serine which feeds into the resynthesis of phosphoglycerate for the Calvin cycle. This process also releases ammonia that becomes a source for nitrogen assimilation, yielding amino acids in plants. Both the TCA and photorespiratory cycles produce the bulk of NADH in mitochondria required for fuelling respiratory electron transport and thus ATP production. A disruption in the normal function of the mitochondria will, therefore, have serious consequences for plant carbon and nitrogen metabolism and cellular biosynthetic reactions.
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Active oxygen species (AOS) are able to damage proteins, lipids and nucleic acids in plants and thus their production and removal must be strictly controlled (Mittler, 2002; Møller, 2001; Noctor and Foyer, 1998). In animal cells, mitochondria are major sites of AOS formation and major targets of AOS-induced damage (see reviews by Skulachev, 1996; Kowaltowski and Vercesi, 1999). In plant cells, especially in photosynthetic cells, the plastids and peroxisomes are also likely to produce large quantities of AOS, but mitochondrial enzymes are nonetheless susceptible to this and their response may affect leaf metabolism significantly. Respiration of isolated plant mitochondria can be disrupted by the generation of AOS (Verniquet et al., 1991) or the production of toxic lipid peroxidation end-products following AOS accumulation (Millar and Leaver, 2000; Taylor et al., 2002). In vivo, a range of biotic and abiotic stresses can elevate AOS levels in plants due to perturbations of metabolism in organelles and the generation of AOS in defence responses through NADPH oxidases, cell wall peroxidases and amine oxidases (Dat et al., 1998; Van Camp et al., 1998). The aim of this review is to highlight research on the molecular targets of accumulated AOS, and their downstream toxic products, in mitochondria and to consider the implications of this damage for nitrogen and carbon metabolism.
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Environmental stress and active oxygen species |
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 TopAbstractIntroduction Environmental stress and active... Direct inhibition of...Lipid peroxidationNew lipid peroxidation targets...Mechanisms to repair or...Implications of mitochondrial...References |
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A wide range of different environmental conditions can induce stresses, which significantly alter plant metabolism, growth and development and, at their extremes, ultimately lead to plant death. These abiotic stresses include drought, high salinity, extremes of temperature (both high and low), heavy metals, ultraviolet radiation, nutrient deprivation, high light stress, and hypoxia (Dat et al., 2000; Noctor and Foyer, 1998). Plants are unable to escape exposure to these environmental extremes and, therefore, have developed defence responses in order to survive. The signal transduction pathways that elicit these responses, or even the way in which plants perceive these environmental stresses, are not well understood (Braam et al., 1997; Mittler, 2002). Environmental stresses have often been linked to AOS production and AOS-induced damage in plants (Dat et al., 2000). To understand this link, the mechanism of AOS accumulation and the degree to which stresses differ from one another must be considered, and it must be acknowledged that AOS targets can be, on the one hand, antioxidant defences and, on the other, sensitive sites of damage.
Common mechanisms or complex networks?
Although the physical nature of environmental stress conditions varies greatly, a number of common responses have been shown to exist and sites of damage are often the same. For example, mild heat treatment of tomato plants confers resistance to subsequent oxidative stresses, and vice versa, and this has been shown to be correlated with the induction of a series of heat shock proteins in both cases (Banzet et al., 1998). Thermotolerance induced in mustard seedlings by both heat acclimation and salicylic acid treatment lead to the same modulations in H2O2 and catalase levels (Dat et al., 1998). Trehalose, thought only to provide tolerance to plants exposed to drought by acting as a compatible solute, was recently found to confer tolerance to a range of other environmental stresses (Garg et al., 2002). In a recent survey of 7000 Arabidopsis genes in response to cold, drought and high salt treatments, a significant number of changes in expression observed were common to more than one stress (Seki et al., 2002). Such studies indicate that plants may employ some common strategies for responding to a number of different conditions. The inhibition and rapid turnover of the D1 protein in chloroplasts in response to a wide range of environmental stresses (Aro et al., 1993), and of glycine decarboxylase (GDC) to herbicide application, drought and low temperatures (Taylor et al., 2002), further suggests that different stresses can have similar effects on specific molecular targets. The identification of such common induction responses and common damage sites has been the focus of much research to date (for reviews see Dat et al., 2000; Mittler, 2002; Pastori and Foyer, 2002).
Care must be taken, however, not to assume that all important responses are common and neglect individual responses. This is highlighted by the example of the response of enzymes of the ascorbate–glutathione cycle, which increased activity during high salinity (Hernandez et al., 1999) and decreased activity during drought (Iturbe-Ormaetxe et al., 1998). Kliebenstein et al. (1998) also identified seven different SOD isoforms in Arabidopsis and showed that these were differentially regulated by light, ozone, and ultraviolet-B irradiation. Recently, Kreps et al. (2002) exposed Arabidopsis seedlings to chilling, salt and osmotic stresses, and found that, of the 2409 genes induced over all treatments, many were stress-specific 3 h after exposure, and this proportion increased 27 h following exposure. Also, in the microarray study by Seki et al. (2002), in which common responses were observed, many stress-specific responses were observed between drought, cold and high-salinity treatments. Such studies act as a timely warning against generalizing from results of studies that use only a single treatment and/or single cell type or species, and which measure only a few parameters. Thus, while there are common elements in the response of plants to different stresses, specific responses may be as important, or more important, than the generalized responses in allowing plants to recover from stresses. The degree of importance of specific and common responses in stress phenotypes and in stress-tolerance traits is still largely unresolved in the view of the authors.
Basis of AOS accumulation
It is now widely accepted that most environmental stresses lead to the accumulation of AOS such as OH·, H2O2, O2·– and O12 in plant cells (Dat et al., 2000; Mittler, 2002; Noctor and Foyer, 1998). This accumulation has a number of implications for biological processes within the plant as a whole and also within mitochondria. Most research on AOS accumulation in plants to date, has focused on the roles of antioxidant defence systems in alleviating the accumulation of AOS. Largely validating this focus, over-expression of certain antioxidant enzymes has clearly been shown to enhance yield and survival under some environmental stresses (Alscher et al., 2002; McKersie et al., 1996; Van Camp et al., 1996). Further, mutants with suppressed ascorbate are hypersensitive to stresses (Conklin et al., 2000) while paradoxically increasing glutathione levels appears by unknown mechanisms to decrease stress tolerance (Creissen et al., 1999). Much less attention has been paid so far to the reactions of AOS and its products with cellular components, especially to sites of action outside the chloroplast.
The exact mechanisms that facilitate AOS accumulation during environmental stress are not entirely clear. However, AOS production through disruption or inhibition of the mitochondrial electron transfer chain (Hernandez et al., 1993; Kowaltowski and Vercesi, 1999; Lam et al., 2001; Skulachev, 1996) or photosynthetic apparatus (Aro et al., 1993; Noctor and Foyer, 1998) are major factors. AOS are also produced at a number of other sites in plant cells, such as glycolate oxidase and oxalate oxidase, to fulfil various biological functions (Huckman and Tanaka, 1996; Noctor and Foyer, 1998). AOS produced at lower levels by cell wall NADPH oxidase, peroxidases, amino oxidases or flavin-containing oxidases, may form part of a defence strategy against invading pathogens or, when produced at very low levels, act as signalling molecules (Dat et al., 1998; Delledonne et al., 1998; Van Camp et al., 1998; Vera-Estrella et al., 1992). Thus, plants, like other organisms, have learnt to live with AOS and there is a delicate balance between AOS production and AOS scavenging. During environmental stress the plant can ‘lose control’ over this balancing act. Loss of control often involves a combination of increasing AOS production and limited energy resources to replenish defence mechanisms, such as reductant for antioxidants, leading to these defences being overwhelmed and ultimately resulting in AOS accumulation.
Critical damage or cunning decoy?
Once accumulation of AOS occurs, damage to cellular components begins. This includes direct inhibition of enzymes by AOS, protein oxidation reactions, membrane lipid peroxidation yielding toxic products, and oxidative DNA and RNA damage (Elstner, 1982). The impact of AOS on mtDNA is well documented in animals and humans, where it leads to a number of different diseases and may contribute to ageing (Wallace, 1999). Such damage is also likely to occur in plant mitochondria, but this area has received little attention to date.
When attempting to identify the targets of AOS or lipid peroxidation product damage, it is a challenge to distinguish between targets whose damage causes a large decrease in biological efficiency, and therefore has a high cost to the cell, and those that may be acting as sacrificial components or even scavengers of these toxic compounds. It has generally been assumed that small molecular mass targets (e.g. ascorbate, glutathione, tocopherols) are the sacrificial components, and large molecular mass molecules (e.g. proteins, DNA) are targets for damage (Elstner, 1982; Noctor and Foyer, 1998). But this clear-cut distinction may not always be the case, and it would be well to keep an open mind on this issue. Lipoic acid has been claimed to be an antioxidant, possibly a sacrificial component in plant cells as a free molecule (Navari-Izzo et al., 2002). However, when incorporated into the active site of enzymes where it carries out major metabolic functions, this same molecule is widely considered a site of damage (Humphries et al., 1998; Millar and Leaver, 2000; Taylor et al., 2002). Another antioxidant enzyme susceptible to oxidative damage is peroxiredoxin, which can be inactivated by high concentrations of peroxides via a mechanism thought to be important in allowing the enzyme to distinguish between AOS signalling and oxidative stress situations (Rabilloud et al., 2002; Wood et al., 2003). These enzymes are known in plant mitochondria (Sweetlove et al., 2002; Horling et al., 2003), but detailed analysis of their functions have yet to be undertaken.
An understanding of the targets of damage and their significance as points of AOS damage or as AOS sinks, will be essential to allow the breeding and possible engineering of plants to make them more robust in surviving exposure to environmental stress.
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Direct inhibition of mitochondrial enzymes |
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A number of mitochondrial proteins have been shown to be inhibited or degraded by AOS exposure in mammals. These include the mitochondrial NADH dehydrogenase (complex I), succinate dehydrogenase (complex II), and ATP synthase (complex V) (Zhang et al., 1990). As far as is known, there is only one reported example of direct inhibition of a plant mitochondrial protein by AOS through a known mechanism; mitochondrial aconitase (EC 4.2.1.3 [EC] ) (Verniquet et al., 1991). Aconitase catalyses the reversible hydration of cis-aconitate to either citrate or isocitrate and is a component of the TCA cycle. Verniquet et al. (1991) showed that H2O2 was able to inhibit citrate-stimulated O2 consumption in potato mitochondria, but that O2 consumption could be recovered following the addition of isocitrate. They also demonstrated similar inhibition following exposure of the isolated enzyme, and changes in EPR spectra of aconitase indicated modification of the 4Fe–4S cluster. They also noted no significant effects on the rates of succinate, NADH and 2-oxoglutarate-dependent respiration in the presence of H2O2 and concluded that aconitase is the major intramitochondrial target for inactivation by H2O2. Their data further suggest that other likely targets of direct inhibition by AOS are proteins containing Fe–S clusters, due to the high reactivity of AOS with Fe2+ found in these proteins. In a proteomic study of the impact of oxidative stress on Arabidopsis mitochondria (Sweetlove et al., 2002), decreased abundances of a series of specific proteins were found. These included aconitase, Fe–S centres of the NADH dehydrogenase (complex I) and core subunits of ATP synthase. These data are clearly in line with the inhibitions reported in mammalian mitochondria in response to oxidative stress (Zhang et al., 1990). The induction of a mitochondrial protease specific for oxidatively modified proteins has been documented in mammals (Marcillat et al., 1988). Preliminary evidence for the presence of a similar protease has been presented for plant mitochondria (Sweetlove et al., 2002). Such a proteolytic mechanism might indicate that longer-term losses in activity are due to degradation of modified proteins rather than direct oxidative inactivation of enzymatic function.
The direct effects of AOS on proteins observed to date, both in mammalian mitochondria and in the limited examples that there are in plants, have usually involved the direct application of vastly different concentrations of AOS. Some of these effects may have little physiological significance and it is important to distinguish these from the effects of physiological concentrations of AOS. However, this proves very difficult in the plant context, as few reliable measurements of steady-state AOS concentrations in plant tissues have been reported and thus the likely range of physiologically significant concentrations is largely unknown (as recently reviewed by Dat et al., 2000; Møller, 2001).
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Lipid peroxidation |
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Lipid peroxidation is broadly defined as the oxidative deterioration of polyunsaturated lipids, but in a mitochondrial context it principally refers to the polyunsaturated fatty acids of membrane lipids such as linoleic acid, linolenic acid and arachidonic acid, which yield various cytotoxic aldehydes, alkenals and hydroxyalkenals. Three different mechanisms are able to induce lipid peroxidation: autoxidation (Halliwell and Gutteridge, 1989), photo-oxidation (Aro et al., 1993), and enzyme catalysis via lipo- or cyclo-oxygenases (Feussner and Wasternack, 2002; Montillet et al., 2002). While lipoxygenase-mediated peroxidation and photo-oxidation are considered very significant pathways in chloroplasts, due to the absence of photosensitizers or lipoxygenase activity (Siedow and Girvin, 1980) in mitochondria, free radical autoxidation is the primary route of lipid peroxidation. The mechanism of such autoxidation is outlined below.
The interaction of AOS, specifically OH· (which is sufficiently reactive to facilitate autoxidation) with polyunsaturated fatty acids initiates lipid peroxidation (Fig. 2). First-chain initiation involves the attack of OH· on the methylene (-CH2-) of a polyunsaturated fatty acid, to abstract a hydrogen atom. This abstraction of an H from -CH2- group leaves behind an unpaired electron on the carbon (-CH-). The presence of a double bond in the fatty acid weakens the C-H bonds on the carbon atom adjacent to the double bond and so makes removal of the H easier. The carbon radical tends to be stabilized by a molecular rearrangement to form a conjugated diene. These can undergo various reactions, the most common being, in aerobic conditions, to combine with O2, giving rise to a (1st) peroxyl radical (CHOO·). Propagation of the chain reaction continues by the (1st) peroxy radical abstracting an H from another adjacent polyunsaturated fatty acid. The carbon radical (CH·) formed can react with O2 to form another (2nd) peroxyl radical and so the chain reaction of lipid peroxidation continues. The initial (1st) peroxyl radical combines with the hydrogen it had removed to yield a lipid hydroperoxide (Fig. 2).
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These hydroperoxides are then degraded non-enzymatically to yield carbonyl compounds, many of which are aldehydes (Noordermeer et al., 2000; Schneider et al., 2001). This decomposition is enhanced by the presence of metal ions, such as Fe2+ (Poli and Schaur, 2000). In plants the 3Z-alkenals are then oxygenated to form 4-hydroxy-2-alkenals by a non-enzymatic process (Noordermeer et al., 2000). These 4-hydroxy-2-alkenals such as 4-hydroxy-2-nonenal (HNE) and 4-hydroxy-2-hexanal (HHE) are examples of lipid peroxidation products, which have toxic effects on both plant and animal cells. Probably the most cytotoxic and the most studied lipid peroxidation end-product is HNE.
HNE (4-hydroxy-2-nonenal)
HNE was first discovered in 1964 in natural fats and was thought to be a by-product of the autoxidation of unsaturated fatty acids (Schauenstein, 1967). However, it is also potentially able to undergo a number of reactions with proteins, phospholipids and nucleic acids. Since its discovery it has been shown to accumulate in mammalian cells under both normal and stress conditions (Esterbauer et al., 1991) and in plants during the oxidative burst (Deighton et al., 1999). In mammals, it has been implicated as being causally involved in the pathogenesis of a number of inflammatory and degenerative diseases (Poli and Schaur, 2000). Increased steady-state levels of HNE have been detected in a wide range of human diseases including Alzheimer’s disease, Parkinson’s disease, rheumatoid arthritis, deep venous thrombosis, diabetes mellitus, and mitochondrial complex 1 deficiency (Poli and Schaur, 2000).
HNE modifies lipoic acid moieties
HNE has been shown to inhibit the activities of potato mitochondrial pyruvate dehydrogenase (PDC) and 2-oxoglutarate dehydrogenase (OGDC) (Millar and Leaver, 2000) via the modification of the lipoic acid residues found on the E2 subunits of these enzymes. This modification by HNE results in the formation of an HNE-Michael adduct (Fig. 3), which no longer allows the normal function of the essential E2 catalytic subunit. With these initial observations in plant mitochondria, the effect of HNE on the most abundant lipoic acid-containing protein in plants, the glycine decarboxylase complex (GDC) was also examined. In photosynthetic tissues this enzyme can account for up to 50% of matrix protein, and is responsible for the most prominent metabolic activity in the mitochondria of illuminated leaves, photorespiration (Douce et al., 2001). GDC is a multienzyme complex composed of four component enzymes, the P-protein, H-protein, T-protein, and L-protein, and is responsible for the conversion of glycine produced in the peroxisome to serine in the mitochondria, during the operation of the photorespiratory cycle (Douce et al., 2001). The H-protein plays a pivotal role as a mobile substrate which commutes between the other subunits allowing its lipoic acid ‘arm’ to visit the active sites of the other three components (Vauclare et al., 1996). It was shown that HNE inhibits glycine-dependent respiration of isolated pea leaf mitochondria and that the site of inhibition is the H-protein. The H-protein subunit of GDC was substantially more sensitive to modification than the lipoic acid-containing subunits of related 2-oxo acid dehydrogenases (Taylor et al., 2002). A substantial decline in GDC activity was also observed following the induction of in vivo oxidative stress, using the herbicide paraquat. A similar result was also observed when plants were exposed to the environmental stresses of drought and chilling. Taken together, the results obtained suggest that environmental stress leads directly to lipid peroxidation, the products of which can then inhibit mitochondrial function and, in particular, GDC. This photorespiratory enzyme is thus a major target for oxidative damage in mitochondria in leaves (Taylor et al., 2002).
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Other targets for HNE
Recent research has also identified a wider range of proteins, or pathways that are damaged or inhibited by HNE. In some cases, lipoic acid moieties are not involved and HNE acts directly by covalent modification of amino acid residues such as Cys, Lys, His, Ser, and Tyr (Esterbauer et al., 1991) or acts non-covalently as a substrate analogue of other aldehydes. Cytochrome c oxidase is inhibited by binding of HNE to histidine residues near the functional core of this enzyme (Chen et al., 1998). In plants, NAD-malic enzyme is inhibited irreversibly by HNE, and it has been proposed that modification of a critical cysteine residue near the active site might be responsible (Millar and Leaver, 2000). HNE inhibition of mitochondrial aldehyde-dependent enzymes, the class 2 aldehyde dehydrogenase and the succinic semialdehyde dehydrogenase, have also been reported in mammals (Nguyen and Picklo, 2003). These are examples of HNE acting as a competitive inhibitor, preventing breakdown of the other aldehydes. Does this mean that all proteins with Cys, Lys, His, Ser, and Tyr in crucial places in their active sites are potential targets for HNE? In vitro, if applied HNE levels are high enough, this is likely to be the case. Much work still needs to be done to define the degree of susceptibility of different sites in vivo to (largely unknown) endogenous HNE concentrations.
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New lipid peroxidation targets in plant mitochondria |
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TopAbstractIntroductionEnvironmental stress and active...Direct inhibition of...Lipid peroxidationNew lipid peroxidation targets...Mechanisms to repair or...Implications of mitochondrial...References |
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As the list of protein targets for HNE steadily grows in mammalian mitochondrial systems, it is imperative to consider that many more plant targets remain unidentified. To date, only four plant mitochondrial enzymes have been experimentally shown to be inhibited by HNE. All of these are involved in the major metabolic pathways, one in photorespiration, and the others in the TCA cycle. Their inhibition will directly affect the activity of the mitochondrial electron transfer chain through depletion of matrix NADH pools. But how about identifying more targets and understanding their significance, and how can it be known that HNE is the only or indeed the major lipid peroxidation cytotoxin in plants?
Identifying new targets
It is possible to continue to rely on chance observations in the course of other work. For example, it has recently been observed that component(s) of the protein import machinery of plant mitochondria, yet to be identified, are inhibited during environmental stress. This may be another example of a target of HNE, and this possibility is currently being investigated (Taylor et al., 2003). Alternatively, the impact of oxidative and environmental stress on mitochondrial protein composition can be studied more widely. Sweetlove et al. (2002) showed, using Arabidopsis cell cultures under chemically induced oxidative stress, that an increase in the presence of lipid peroxidation end-products occurred. They also observed that a significant number of mitochondrial protein subunits were degraded by these stresses. These subunits were identified by IEF/SDS-PAGE 2D gel separation and mass spectrometry. Those identified included expected components such as pyruvate and 2-oxoglutarate dehydrogenases, aconitase and complex I Fe–S centres, but also unexpected components such as succinyl CoA ligase, methylmalonate semialdehyde dehydrogenase, fumarase, and GABA aminotransferase. Also identified were a number of proteins which increased during these stresses, including a thioredoxin-dependent peroxidase, a thioredoxin reductase-dependent protein disulphide isomerase and a glutathione-S-transferase. These may represent targets of damage, responses to stress, or detoxification systems. A similar study is being undertaken in pea mitochondria during environmental stresses in order to identify key responsible proteins in a background where lipoic acid modifications have already been extensively studied (NL Taylor, DA Day, AH Millar, unpublished data). Alternatively, direct searches might be made for HNE modified targets. The advances in immunopurification and mass spectrometry characterization allow both the purification and identification of targets as well as the localization of the exact sites of modification. Recently, the use of anti-HNE antibodies immobilized on CNBr-activated sepharose allowed the selective enrichment of HNE-adducted peptides in biological samples. These samples were then tryptic digested and analysed by electrospray ionization mass spectrometry, allowing the identification of these modified peptides at a substantially lower detection limit than previously possible (Fenaille et al., 2002).
Identifying new cytotoxic lipid peroxidation products
While HNE is probably the most studied lipid peroxidation end-product, a number of other end-products have also been shown to be cytotoxic through inhibitory effects on proteins and biochemical pathways. These include the 4-hydroxyalkenals of various chain lengths such as 4-hydroxy-pentanal, which inhibits mammalian glucose-6-phosphate, succinate dehydrogenase, RNA synthesis, and phosphate transport. Other 4-hydroxyalkenals including 4-hydroxy-hexanal and 4-hydroxy-octanol have been shown to have genotoxic effects and to inhibit anaerobic glycolysis. Also, malondialdehyde (MDA), a commonly studied marker of oxidative stress, and acrolein, both produced from the peroxidation of polyunsaturated fatty acids, have been shown to have various DNA damaging effects (Esterbauer et al., 1991). MDA can also modify proteins by Schiff base addition (Fenaille et al., 2002). The impact of MDA in plants is largely unresearched. There is also the possibility of other, as yet undetermined, products of lipid peroxidation that accumulate during stress and may inhibit proteins or disrupt biochemical processes within plant mitochondria. Recently, another lipid peroxidation product was described for the first time: 4-oxo-2-nonenal was identified as a major product of lipid peroxidation in the presence of low Fe2+ concentrations (Lee and Blair, 2000). The cytotoxic effects of this compound are yet to be examined. Analysing which products of lipid peroxidation predominate in plant mitochondria under in vivo conditions remains a challenge.
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Mechanisms to repair or prevent lipid peroxidation damage. |
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With all the potential damage occurring to plant proteins, it seems only logical that there must be mechanisms present either to prevent this type of damage occurring or to repair damage once it has occurred. A series of different enzymes has been proposed as HNE-detoxifying systems and a range of repair options for HNE-induced damage have been highlighted.
Aldehyde dehydrogenases
The discovery, in 1996, of the nuclear restorer gene of maize T cytoplasm CMS as an aldehyde dehydrogenase (Cui et al., 1996) has excited researchers to consider that restoration may be due to the alleviation of lipid peroxidation stress induced by URF13 accumulation in the T cytoplasm background (Liu and Schnable, 2002; Møller, 2001). If true, such aldehyde dehydrogenases might be part of the plant mitochondrial–lipid peroxidation product detoxifying system. Such dehydrogenases have been found in proteome studies of pea, rice and Arabidopsis mitochondria (Bardel et al., 2002; Heazlewood et al., 2003; Kruft et al., 2001; Millar et al., 2001). However, recent work by the Schnable laboratory has largely dismissed the suggestion that T cytoplasm pollen abortion is due to elevated lipid peroxidation (Liu and Schnable, 2002). While the restoring aldehyde dehydrogenase (rf2a) does appear to use HNE as a substrate, this is a very slow reaction and the dehydrogenase is much more active against other classes of aldehydes (Liu and Schnable, 2002).
NADPH-dependent quinone oxidoreductase and aldo-keto reductase
In plants, two other pathways of HNE metabolism have been identified, which may prevent its accumulation and subsequent damage to proteins, although to date neither has been localized to mitochondria. The first of these is a NADPH:quinone oxidoreductase called P1-
-crystallin (P1-ZCr), which is able to catalyse the reduction of 2-alkenals by -ß-hydrogenation (Mano et al., 2002). The highest specific activity of this enzyme is for HNE (kcat=88 s–1 and Km=13.4 µM), but it also reacts with HHE (kcat=42 s–1 and Km=145 µM) and acrolein (kcat=40 s–1 and Km=4.65 mM) (Mano et al., 2002). The second is an NADPH-dependant aldo-keto reductase identified by Oberschall et al. (2000). They isolated a gene MsALR from alfalfa encoding an aldose/aldehyde reductase that reduces the aldehyde. This enzyme exhibited characteristics homologous to the human enzyme involved in HNE metabolism. However, it has a significantly lower kcat (8.9 s–1) and higher Km (740 µM) than P1-ZCr. This indicates P1-ZCr may have a greater role in HNE metabolism than the MsALR gene product.
Alcohol dehydrogenase and glutathione-S-transferase
In mammals, two additional mechanisms have been identified for HNE detoxification, one an oxidation of the aldehyde by an alcohol dehydrogenase (Sellin et al., 1991) and the second, the conjugation of the aldehyde to GSH by the GST A4-4 (Hubatsch et al., 1998). While there is evidence for enzymes from both these general classes in plant mitochondrial preparations, there is no evidence yet that they are capable of detoxifying HNE.
Repairing HNE damage
Once damage has occurred mechanisms may exist to repair damage, avoiding costly resynthesis of whole polypeptides in order to repair single residue defects. For example, there may be enzymes which can remove HNE once it has attached to Cys, Lys, His, Ser or Tyr residues. In the case of lipoic acid moieties, there may be a lipoate protein lyase which is able to remove damaged moieties, regenerating the apoprotein and allowing reattachment of a unmodified lipoate by the established lipoate transferase pathway (Gueguen et al., 2000). As far as is known, none of these proposed enzymes have been identified in plants or animals.
Alternatively, damaged proteins could simply be targeted for degradation and new protein synthesized. However, recently it was demonstrated that the mammalian 26s proteasome responsible for the degradation of abnormal proteins, including proteins with carbonyl residues generated by oxidative stress, is itself readily inhibited by HNE. Inhibition of the proteasome increases the levels of ubiquinated and carbonylated proteins and was implicated in rendering cells sensitive to oxidative stress and apoptotic death (Hyun et al., 2002). In plants, inhibition of the 26s proteasome has very recently been shown to lead to an accumulation of ubiquinated proteins and induction of programmed cell death, however, a direct link to HNE inhibition still awaits to be elucidated (Kim et al., 2003). Clearly, a pathway for HNE damage repair is likely to exist in eukaryotes, but the mechanism is still unresolved.
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Implications of mitochondrial oxidative damage for integrated cellular carbon and nitrogen metabolism |
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The sensitivity of key mitochondrial enzymes to damage by AOS and lipid peroxidation products has the potential to impair primary metabolism significantly. This is an aspect of environmental stress that has been highlighted by the work of some, especially with regard to photorespiration (Wingler et al., 2000; Bauwe and Kolukisaoglu, 2003), but that deserves broader attention from plant researchers. The data presented here would suggest a slowing of both photorespiration and nitrogen assimilation during environmental stress that will have immediate direct effects on photosynthetic rate and efficiency and longer-term implications for plant nutrition. Further, the replenishment of the cellular antioxidant machinery is driven by reductant in the form of NAD(P)H and the availability of amino acids for glutathione synthesis. Losses of TCA cycle and GDC will impact on both of these, at least at the level of the mitochondrion. Deficits in all of these areas are common features of the phenotype of environmentally stressed plants (for review see Vierling and Kimpel, 1992; Bohnert and Sheveleva, 1998; Dat et al., 2000). Uncovering the significance of the mitochondrial involvement in these phenotypes and further uncovering plant pathways of repair and avoidance of protein damage will provide tools for enhancing plant performance through the manipulation of mitochondrial function in the future.
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Acknowledgements |
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AHM and DAD acknowledge funding from the Australian Research Council Discovery Program. NLT acknowledges a University Postgraduate Scholarship from the University of Western Australia.
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