Mitochondrial Reactive Oxygen Species. Contribution to Oxidative Stress and Interorganellar Signaling

The inner membrane of a plant mitochondrion contains the mitochondrial electron transport chain (mtETC), consisting of protein complexes that use an energy source-derived reductant to form a proton gradient across the membrane. This proton gradient drives ATP synthesis, a primary mitochondrial function. Ultimately, electrons in the mtETC are transferred through one of two pathways from ubiquinone to a terminal oxidase, where oxygen is reduced to water. Specifically, electrons pass through either the “standard” cytochrome pathway, which includes two sites of proton pumping downstream of the ubiquinone pool, to cytochrome c oxidase (COX), or the alternative pathway, which consists of the cyanide-insensitive alternative oxidase (AOX) and has no proton-pumping sites after the ubiquinone pool (Finnegan et al., 2004). Because the mtETC harbors electrons with sufficient free energy to directly reduce molecular oxygen, it is considered the unavoidable primary source of mitochondrial reactive oxygen species (mtROS) production, a necessary accompaniment to aerobic respiration. Formation of mtROS takes place under normal respiratory conditions but can be enhanced in response to a range of abnormal conditions, including exposure to biotic and abiotic stresses. The marked reactivity of ROS toward biological molecules, including lipids, proteins, and nucleic acids, requires multiple mechanisms for keeping mtROS levels under control, including pathways that attenuate mtROS formation in response to imposed stresses and protective, antioxidant enzyme systems. However, when mtROS formation exceeds normal levels despite the operation of these protective mechanisms, there are downstream consequences for the cell, including altered gene expression and even programmed cell death (PCD). Not simply agents of damage, mtROS also play roles in the signaling required to bring about these changes. The cell ultimately must strike a balance between the level of ROS required to elicit an appropriate response to a changing condition while at the same time keeping ROS levels sufficiently low to prevent large-scale cellular damage. On the other hand, cells must be able to determine a mtROS-initiated, extreme measure at the cellular level is appropriate for the good of the plant as a whole (e.g. the hypersensitive response to pathogen attack). This article provides an overview of our current understanding of plant mtROS. While much remains to be established, mtROS clearly play important roles in the responses of plants to the variety of environmental conditions they experience on a regular basis, as well as more extreme environmental stresses. The known sites of mtROS production in the mtETC are complexes I and III, where superoxide anion (O2 −) is formed and in turn is reduced by dismutation to H2O2 (Raha and Robinson, 2000; Møller, 2001; Sweetlove and Foyer, 2004). H2O2, a compound of relatively low toxicity, can react with reduced Fe2+ and Cu+ to produce highly toxic hydroxyl radicals and, being uncharged, can also penetrate membranes and leave the mitochondrion (Grene, 2002; Sweetlove and Foyer, 2004). The ubisemiquinone intermediate formed at complexes I and III is the principal electron donor to oxygen, although other complex I sites are also potential donors (Raha and Robinson, 2000; Sweetlove and Foyer, 2004). Thus, the overall reduction level of the mitochondrial ubiquinone pool will be the primary determinate of mtROS output (Sweetlove and Foyer, 2004). The amount of ROS produced by mitochondria and the fraction of total cellular ROS that come from mitochondria are difficult to determine, in part because ROS levels in general are difficult to measure accurately (Veljovic-Jovanovic et al., 2002; Halliwell and Whiteman, 2004). For isolated mitochondria and submitochondrial particles, ROS evolution varies with conditions but falls within the wide range of 0.2 to 30.0 nmol min−1 mg protein−1 for either H2O2 or superoxide (studies compiled in Møller, 2001; Popov et al., 2003). While we are unaware of any quantitative measurements of ROS production by mitochondria in vivo, estimates (Foyer and Noctor, 2003) indicate that the in situ level of mtROS evolution normally will be considerably less than that of chloroplasts or peroxisomes in the light due to the operation of photosynthesis and photorespiration. However, in the dark or in non-green tissues, mitochondria will be a major source of ROS (e.g. Puntarulo et al., 1988). Production of mtROS will increase if the rate of electrons leaving the mtETC through the terminal oxidases is slowed and/or the rate of electron input increases in excess of the ability of the two respiratory pathways to process the electrons, leading to an overreduced ubiquinone pool. This principle has been demonstrated in isolated mitochondria, where rates of H2O2 and superoxide generation show a substrate-dependent increase upon the addition of specific inhibitors of either the cytochrome pathway or AOX (Popov et al., 1997; Møller, 2001). Mitochondria in situ show the same effect. Studies using whole cells have shown increased ROS production specifically from mitochondria in the presence of the cytochrome pathway inhibitor antimycin A (AA; Maxwell et al., 1999; Yao et al., 2002). Of primary interest in mitochondrial research is the likelihood that naturally occurring physiological and environmental conditions encountered by plants can give rise to an overreduced ubiquinone pool and concomitant increased mtROS production. For example, the endogenous signaling molecule nitric oxide (NO) is an inhibitor of COX, but not AOX (Millar and Day, 1996), at concentrations achieved in vivo during normal seedling development (Caro and Puntarulo, 1999). Therefore, NO production could lead to increased mtROS formation. Many stresses cause oxidative damage in plant tissues, and increases in mtROS resulting from mtETC perturbations have been implicated as at least partly responsible for the damage and plant responses observed in several of these cases, including chilling (Prasad et al., 1994a, 1994b; Purvis et al., 1995), salt stress (Hernández et al., 1993; Mittova et al., 2003), and phosphate deficiency (Juszczuk et al., 2001; Parsons et al., 1999; Malusà et al., 2002). The preceding examples together with other studies strongly suggest that mtROS are involved in the responses of plants to stresses, as well as other plant processes. This means that plants are able to detect changes in mtROS output against a background of ROS production from other sources. Because mtROS output is predicted to be relatively constant during the course of a day/night cycle (Foyer and Noctor, 2003; Sweetlove and Foyer, 2004), changes in mtROS levels, even if low in magnitude, could be distinguished and registered by local detection mechanisms. The concept of local detection is consistent with accumulating evidence that the specific source of ROS is important in determining appropriate cellular responses (Dutilleul et al., 2003; Laloi et al., 2004; Clifton et al., 2005). A change in mtROS level is likely to have a consequence, including direct damage if levels increase (in which case damaged molecules could be part of signaling) or/and as early participants themselves in a signaling pathway(s). Therefore, as discussed below, mitochondria must be capable of controlling their ROS levels under normal conditions, detoxifying excess ROS, repairing oxidative damage arising from ROS formation, and modulating mtROS production appropriately for signaling. Peroxidation of mitochondrial membrane polyunsaturated fatty acids is initiated by the abstraction of a hydrogen atom by ROS, especially by hydroxyl radicals. This leads to the formation of cytotoxic lipid aldehydes, alkenals, and hydroxyalkenals (HAEs), such as the much-studied 4-hydroxy-2-nonenal (HNE) and malondialdehyde. Inhibition of the mtETC with AA can generate mitochondrial HAEs to levels similar to those generated by general oxidative stress through chemical treatments such as H2O2 or menadione (a compound that causes superoxide production; Sweetlove et al., 2002; Winger et al., 2005). Once formed, lipid peroxidation products can cause cellular damage by reacting with proteins, other lipids, and nucleic acids. Key oxylipins and smaller, lipid-derived reactive electrophile species may also be produced from lipid peroxidation (Alméras et al., 2003), but, to our knowledge, there is no direct evidence of these compounds being produced in plant mitochondria from oxidative stress. Proteins can be damaged and/or inhibited by oxidative conditions in several ways, including: (1) direct oxidation of amino acids by ROS, such as the oxidation of Cys residues to form disulfide bonds, oxidation of Met residues to form Met sulfoxide, and oxidation of Arg, Lys, Pro, and Thr residues, which creates carbonyl groups in the side chains (Berlett and Stadtman, 1997; Dean et al., 1997); (2) oxidation that breaks the peptide backbone (Dean et al., 1997); (3) reactions with lipid peroxidation products (such as HNE); (4) reactions with reactive nitrogen species that are formed by reaction of NO with ROS (Sakamoto et al., 2003); and (5) direct ROS interaction with metal cofactors, illustrated by the TCA cycle iron-sulfur enzyme aconitase, which is sensitive to H2O2 (Verniquet et al., 1991) and superoxide (Flint et al., 1993). Proteomics approaches have been undertaken to determine the damaging effects of oxidative stress on mitochondrial proteins (Sweetlove et al., 2002; Kristensen et al., 2004; Taylor et al., 2005). These studies primarily used chemical or environmental stresses to impose general, non-mitochondria-specific oxidative conditions, though some studies also employed AA (see below), which causes mtROS production specifically. Proteins that accumulate but that are degraded (as determined by identification of fragments) as a result of high ROS levels imposed by addition of H2O2 or menadione were identified (Sweetlove et al., 2002). Oxidatively damaged mitochondrial proteins of the of ATP and of the TCA mitochondrial proteins by with H2O2 were with which with carbonyl groups resulting from oxidation of amino acids et al., 2004). mitochondrial proteins were of these proteins were those observed to be damaged by oxidative stresses (Sweetlove et al., 2002; Taylor et al., 2005). such not the mitochondrial it is that several important mitochondrial proteins are damaged by general oxidative stresses and could be damaged by mtROS. for proteins degraded during more general oxidative treatments were also degraded AA (Sweetlove et al., that mtROS specifically can damage mitochondrial of mitochondria with or causes superoxide formation in chloroplasts and or or of plants leads to formation of a of the of several mitochondrial including enzyme in the (a TCA cycle and (Millar and 2000; Taylor et al., 2005). it has not been that the damage is from mtROS specifically or mitochondrial lipid peroxidation products produced during these stresses, of the mtETC result in levels et al., Winger et al., 2005). These indicate that oxidative conditions due to production of mtROS could cause damage to proteins through formation of HAEs that react with the can also form with and residues, altered enzyme 2003). of these reactions is likely the cause of of AOX et al., 2005). While one of AOX is to prevent mtROS formation, it may cells to an extreme stress that the of mtROS and of a more extreme such as (see While oxidative damage to proteins under conditions, it has also been shown to be a normal part of et al., 2004). protein oxidation has been to have a in controlling the course of during where the mitochondrial ATP one of the proteins et al., 2005). is mtROS can also cell through oxidative radicals are highly reactive and can damage and mitochondrial which cells to et al., 2000; et al., 2005). damage to which can be by mtROS the course of the of causes mitochondrial and can to and and Robinson, 2000; et al., 2004; 2005). For damage from oxidative stress mtETC by and effects on mitochondrial have been in other (Raha and Robinson, 2000; et al., 2005). However, there is a of such studies using plant the plants could the consequences of damage by that damaged or On the other hand, there could be an of in that are in cells of that from these tissues, though we are not of any studies prevent the effects of damage in plant cells is the of in mitochondrion (e.g. et al., et al., The of can determine levels of gene expression and et al., and, could the of the effects of by the of mitochondria in plant cells from and to from damaged mitochondrial and their et al., 2005). a low rate of in mitochondria to cause an of in mitochondrial at an rate to the rate of is that can on a time of of of and these not consequences of potential damage in cells by oxidative stress. mitochondria can superoxide production from the mtETC by two mechanisms that to the ubiquinone pool reduction level The is not inhibited by the proton gradient across the inner membrane and can when the cytochrome pathway is (Finnegan et al., 2004). AOX to a ubiquinone pool reduction by Purvis and and is by studies with with a cytochrome pathway inhibitor et al., operation of AOX mtROS production Popov et al., 1997; Maxwell et al., 1999). expression of alternative mitochondrial which not increased AOX expression et al., 2005). of these as an alternative to complex I and could mtROS production. The protein also is in the inner mitochondrial membrane. by a proton across the membrane and of the mtETC and 2004; et al., 2004). in isolated mitochondria ROS formation et al., et al., On the other hand, ROS are required for et al., 2000; et al., 2003), a direct being et al., 2004). This ROS by is in to the of ROS on of AOX to oxidative stress can the protein the form et al., 1999; and AOX et al., 2005). Therefore, while AOX and may to mtROS production, may be able to when ROS levels Once superoxide has been generated from the the in superoxide to H2O2, is by mitochondrial superoxide et al., Møller, 2001). The presence of though in mitochondria, not to be (Sweetlove and Foyer, 2004). H2O2 is by other mechanisms in plant of these is the mitochondrial cycle et al., 1997; et al., 2003). H2O2 to using which is by the of and which reduced in the reduction reduced also could reduce mitochondrial These use reduced or with as reductant which in turn are reduced by and (Sweetlove and Foyer, 2004). mitochondrial has been identified by and et al., 2004; et al., and plant mitochondria (Sweetlove and Foyer, 2004). Ultimately, the electrons for these are from mitochondrial and a high level of of these could overall mitochondrial and even cellular addition to directly detoxifying ROS, these and other enzyme together with their electron may be involved in repairing lipid peroxidation damage and some of protein For example, can by it to the and there is evidence for at least one in plant mitochondria et al., 2004). The could also reduce lipid et al., 2004). although oxidative damage to proteins is and in protein mitochondria a protein disulfide together with and can reduce resulting from protein oxidation (Sweetlove et al., 2002). remains to be determined these mechanisms in plant mitochondria to oxidative damage. Mitochondria can their oxidative through as is the for synthesis, which two of the TCA cycle that are known to by oxidative stress. that show increased damage and H2O2 levels, because less is generated in the mitochondria than is to conditions et al., 2003; and 2004). is the of the of which the mtETC et al., 1999; et al., 2000; et al., 2003). on the mtETC could with effects on mitochondrial and cellular balance (Millar et al., 2003), as by et al., 2005). The antioxidant of plant mitochondria could mtROS to other cellular through their effects on the overall and reduction of and in the For example, and expression of plant and are of cellular and (Foyer et al., 1997; and Foyer, et al., 2002). also is a potential of stress signaling (Foyer et al., and is involved in of enzyme et al., 2005). can also rates of ROS evolution in the The mtETC is as for excess reductant produced by the light reactions of reductant levels from the the ability of the not mtROS production likely but ROS production could also increase due to an reduced reaction et al., 2004; et al., 2004). Therefore, mitochondrial antioxidant are to ROS and and cellular (Foyer et al., 1997; and Foyer, mitochondrial can cause altered gene expression through which is to as mitochondrial This has been in cell and plants and 2004; and 2004). are several examples in which gene expression altered in plants by mitochondrial by or expression of mitochondrial proteins et al., 2001; et al., 2002; et al., 2005). not directly demonstrated in these there is evidence for the of mtROS in plant Inhibition of the cytochrome pathway by AA in production of mtROS et al., 1999; Yao et al., and altered gene expression et al., 1997; et al., 2001; et al., 2002; Maxwell et al., 2002). The direct of mtROS in in plants is strongly by the reduction of gene observed when are to AA et al., 1999). by of gene from cytochrome pathway the of ROS in it not mtROS leave the mitochondria and gene expression changes. of the two ubisemiquinone sites of complex III is to the mitochondrial a for superoxide production to the 2001). of H2O2 from mitochondria at specific sites et al., in response to AA also suggest that direct effects of mtROS the mitochondria, such as of gene expression are and that may be through specific or clearly not in the that plant mtROS are at specific from AA of cells can be inhibited by inhibitors of the et al., and superoxide has been directly leaving isolated mitochondria by an et al., 2003). However, changes in the mitochondria initiated by increased mtROS may produce from local (in or to ROS detection mechanisms that are to the has been discussed and 2004), any of the known signaling pathways or pathways to be could be involved in a to the are those signaling that are likely involved in oxidative stress and/or signaling. potential signaling proteins were to mitochondria et al., and could be involved as local detection mechanisms for increased mtROS and of signaling to the the in plant mtETC are of (1) proteins involved in mitochondrial reactions other than those in the cytochrome pathway, including AOX and alternative (2) antioxidant such as and a and (3) proteins for plant against biotic and abiotic stresses et al., 1997; et al., 2001; Maxwell et al., 2002; Clifton et al., 2005). least for the is likely the result of increased at a et al., et al., 2005). plants that a mtETC due to in the mitochondrial a cellular must be gene expression is part of altered expression of proteins, and antioxidant et al., 2002; et al., 2003; et al., 2004). Thus, of mtETC in altered gene plants in complex I increased et al., but cellular H2O2 levels, likely due to the observed increased antioxidant enzyme expression and These plants also increased levels of and (Dutilleul et al., but no change in and (Dutilleul et al., 2003) they have altered to to altered mitochondrial but are altered et al., 1997; et al., 2005). Because these plants have an it is not signaling from the mitochondria is responsible for changes in gene it is that increased mtROS production and/or by mitochondria to signaling (Dutilleul et al., 2005). mtROS could from mitochondria and to other of signaling by ROS at other in the cell (such as the et al., and which could result in altered gene is an important part of plant responses to stresses and includes the hypersensitive response to early cellular for process is an increase in ROS production due to membrane oxidase et al., 2003). mitochondrial responses to are similar to those of mitochondria and a of cytochrome and a in ATP production and 2000; et al., 2002; et al., 2002; and 2004). in mtROS production are with these a result of the perturbations by the mtETC et al., 1999; Yao et al., 2002; and 2004). while an may in mitochondrial it that of mitochondrial and the concomitant mtROS that is generated are necessary for for from using of plant that mitochondrial and mtROS formation. is a protein of the hypersensitive addition to cells in increased mtROS and cytochrome c and 2004), consistent with the of mtROS in signaling. Mitochondria are the cellular to show including of the mitochondrial when to the The mitochondrial response well other cellular responses are observed and 2004). during the course of on mtROS were produced and, when their production reduced by ROS were et al., 2002). evidence for a direct of mtROS in signaling from with cells with reduced levels of These protective pathway, were much more to and 2002; et al., but were partly when with an antioxidant and 2002). plants AOX produced upon pathogen et al., 2002). Thus, mtROS to be an important in signaling. of mitochondrial can also bring about changes leading to similar to those for pathogen The cytochrome pathway inhibitor AA in with the of mitochondrial membrane of mitochondrial H2O2 from specific and these effects were by ROS et al., that mtROS in as with were the AA gene expression in which were inhibited by an inhibitor of the mitochondrial et al., between altered gene and addition to mitochondrial of the mtETC by or by and by pathogen similar gene evidence of similar signaling occurring in of these by with by of the mtETC by mitochondrial et al., 2002; and 2004; et al., 2004). the of the whole response to AA is similar to the responses to pathogen et al., 2001). Thus, pathogen and mtETC have similar cellular which that pathways are may be that signaling is a of the responses of plants to a variety of stresses that mitochondrial function. ROS may as in oxygen the of it and 2005). of ROS under oxygen may there is evidence for the of ROS even under oxygen deficiency and a for ROS in the stress (e.g. et al., 2001; et al., 2002). is within of oxygen to the rise in that changes in gene expression et al., and et al., 2000; et al., 2004). of mtROS levels by AOX is likely to be an important early preceding the of to much for oxygen et al., than that of et al., is likely to be inhibited by even a in oxygen and has been shown to be under et al., 2003). AOX may be under conditions (e.g. with a increase in mtROS levels 2003). ROS in mitochondria can (e.g. et al., and is a for rise in et al., 2002). are by even a in oxygen (e.g. and Thus, an of mtROS due to the of AOX may be the for the of and changes in gene expression under oxygen NO to be involved in the and the pathways of NO production are in mitochondria and 2005). The pathway is under oxygen et al., leading to NO (e.g. et al., 2003). NO COX, an increase in mtROS production even under and NO a rise in et al., 2004), either directly or on mtROS Thus, by the production of mtROS as well as increase in may as a of oxygen from mitochondria to the addition to signaling the of NO can mitochondrial damage and cell death et al., 2005). as a NO can as an antioxidant under conditions, during cell death (e.g. et al., 2002; and 2005). of NO and superoxide can also lead to the generation of under is a and that membranes than et al., can several mitochondrial and, cell death in plants and 2001; 2003); although cell death not to result in cells (e.g. et al., 2004). ROS are known to be important signaling molecules in plants et al., 2001; et al., 2002; Laloi et al., 2004). ROS levels, antioxidant levels, and are by plant including within is important for plant of balance are likely and the plant to changes in ROS levels of expression of that to by antioxidant compounds and protective and is known about increased mtROS production is to the Because ROS are involved in of plant responses to stresses, there must be detection mechanisms that levels, and even of ROS must of the current leads to that the of mitochondrial perturbations may not be predicted due to the variety of potential mitochondrial including mtROS production, as well as other and changes that may be arising in the cell These responses may under some stresses and lead to a For example, a stress may the mtETC mtROS production of a sufficient level to cytochrome c and On the other hand, treatments or the same treatments in plant species may cause mitochondria to with such as signaling for changes in gene expression to and of with the by other of of the signaling pathways will likely complex the extreme case of high mtROS production, when cells a mitochondria likely plants important to their in such as pathogen such an extreme be under mtROS can cause in cell because antioxidant ultimately electrons from such as which of will be to determine the roles of mtROS and the potential consequences of altered mtROS levels, such as production of other reactive molecules and in in plant and stress to those could not be due to of and