Essay 12.7 Reactive Oxygen Species (ROS) and Plant Respiration

Essay 12.7 Reactive Oxygen Species (ROS) and Plant Respiration

Ian M. Møller, Department of Molecular Biology and Genetics, Science and Technology, Aarhus University, Slagelse, Denmark

July 2013


The production of ROS is an unavoidable consequence of aerobic respiration. When the terminal oxidases—cytochrome c oxidase and the alternative oxidase—react with oxygen, four electrons are transferred and water is the product. However, occasionally oxygen can react with other electron transport components. Here, only one electron is transferred, and the result is the superoxide anion, O2–. In plant tissues it has been estimated that 1–2 percent of oxygen consumption leads to superoxide formation (Puntarulo et al. 1988). Through a variety of reactions, superoxide leads to the formation of hydrogen peroxide, hydroxyl radicals, and other ROS—all of which can cause damage in various ways. Protein side chains are modified, and even the backbone can be broken. For instance, unsaturated fatty acids in membrane lipids are attacked and peroxide derivatives are formed, eventually leading to breakage of the fatty acid backbone. Finally, ROS can react with DNA and cause mutations (Halliwell and Gutteridge 2007, Møller et al. 2007).

From the foregoing it is clear that there is a need to limit the production of ROS, to detoxify ROS once formed, and to repair damage caused by ROS (Table 1). However, when plants are stressed, the steady-state level of ROS usually increases, and it has been hypothesized that ROS (specifically, hydrogen peroxide) might also act as a messenger that turns on stress-related genes (Figure 1). This could happen via amplitude- and frequency-modulated waves or pulses of ROS (Vestergaard et al. 2012).

Table 1 Strategy for the Defense Against Oxidative Stress in (Plant) Mitochondria
Note: Not all of the listed mechanisms and enzymes have been firmly established to be present in plant mitochondria (see text for a critical evaluation).
ETC, electron transport chain; MnSOD, Mn-superoxide dismutase
Source: Modified from Møller 2001.

Figure 1   Sequence of events involving plant mitochondria in response to stress. ETC, electron transport chain; AOX, alternative oxidase. (From Møller 2001; and based on Wagner, 1995 and Wagner and Krab, 1995.)

Relatively little is known about ROS turnover in plant mitochondria, so the following discussion will lean heavily on knowledge gained from experiments with mammalian mitochondria (see Figure 1).

Plant Mitochondria Produce ROS but Have Mechanisms for Minimizing This Production

In mammalian mitochondria, complexes I and III are the main sites of ROS production (Brand 2010). In complex III, superoxide is formed via the reduction of oxygen by the semiquinone (Turrens 2003; see Web Topic 12.2). In plant mitochondria, complexes I and III certainly contribute to ROS (Møller 2001). However, until the possible contribution by the rotenone-insensitive NAD(P)H dehydrogenases (see Web Topic 12.3) has been carefully tested, we do not have the complete picture.

ROS production is highest in both mammalian and plant mitochondria under conditions in which the electron transport chain is relatively reduced. This means that any mechanism that can minimize this reduction will limit ROS production. Thus, activation of both the alternative oxidase and the uncoupling protein causes a marked reduction in ROS production (Purvis 1997, Maxwell et al. 1999, Camacho et al. 2004; see Web Topic 12.3) probably through a signal transduction pathway like that shown in Figure 1. Whether activation of NDin(NADH) will limit ROS production at complex I, as would be expected (see Web Topic 12.3), has not yet been tested.

Another way to prevent overreduction of the electron transport chain—and thus ROS production—is to maintain the mitochondria in State 3 by immediately regenerating ADP from ATP produced by the mitochondria. This is done efficiently by transferring the terminal phosphate group on ATP to glucose by means of a hexokinase associated with the mitochondria (Camacho-Pereira et al. 2009).

Plant Mitochondria Have Ways of Detoxifying ROS

Superoxide is converted into hydrogen peroxide by superoxide dismutase, an enzyme found in all mitochondria. Since hydrogen peroxide is also a ROS, this does not solve the problem, and hydrogen peroxide must also be detoxified (Figure 2). In fact, hydrogen peroxide can be converted into the highly reactive hydroxyl radical by unprotected (or poorly liganded) metal ions in the so-called Fenton reaction, which then causes oxidative damage (Kell 2010; Tan et al. 2010; Møller et al. 2011; Keunen et al. 2011).

There are five potential enzymes (or enzyme systems) for removing hydrogen peroxide (see Figure 2), but at present we do not know which one is active in plant mitochondria.

  • Catalase is normally considered to be a peroxisomal enzyme, but it has been found in heart mitochondria as well. Catalase activity is also found in plant mitochondria, but proof that it is not a contaminant is lacking.
  • The ascorbate/glutathione cycle comprises four enzymes and two low-molecular-weight compounds: ascorbate (vitamin C) and glutathione (a sulfhydryl-containing tripeptide). It is the main ROS-detoxifying system in the chloroplast, but it has also been reported to be present in plant mitochondria (Jimenez et al. 1997, 1998; Chew et al. 2003).
  • Thioredoxin, a small sulfhydryl-containing protein, and thioredoxin reductase regulate the activity of several Calvin cycle enzymes in the chloroplast stroma (see Web Topic 8.6). Several mitochondrial proteins able to interact with, and possibly be regulated by, thioredoxin have been identified (Balmer et al. 2004). Specifically, thioredoxin has been shown to be involved in the regulation of the alternative oxidase (Gelhaye et al. 2004; see Web Topic 12.3). Both thioredoxin and thioredoxin reductase also have the ability to reduce hydrogen peroxide.
  • Peroxiredoxin, another small sulfhydryl-containing protein, is involved in detoxification of ROS such as hydrogen peroxide and various other peroxides. Arabidopsis plants in which the mitochondrial peroxiredoxin has been knocked out are more sensitive to strong abiotic stresses than are wild-type plants (Finkemeier et al. 2005).
  • The main hydrogen peroxide-removing enzyme in mammalian mitochondria is glutathione peroxidase, which uses reduced glutathione as a direct source of reducing equivalents. Although reported to be present in plants, this enzyme has not yet been found in plant mitochondria.

Figure 2   Summary of the enzyme systems possibly involved in ROS detoxification in plant mitochondria. ETC, electron transport chain; ICDH, isocitrate dehydrogenase; MnSOD, Mn-superoxide dismutase; Trx, thioredoxin. (From Møller 2001)  Not shown is peroxiredoxin, which can also detoxify hydrogen peroxide using glutathione and, ultimately, NADPH, as sources of reducing equivalents.

The last four hydrogen peroxide-removing enzymes use sulfhydryl groups as donors of reducing equivalents and, in all four cases, these sulfhydryl groups are regenerated by reduction with NADPH. This is a good example of the many uses of NADPH in mitochondria (Møller and Rasmusson 1998).

ROS Can Modify and Damage Lipids, Proteins, and DNA

Peroxidation of polyunsaturated fatty acids by a ROS attack can lead to chain breakage and shortening, which will increase membrane fluidity and permeability. When isolated mammalian mitochondria are exposed to oxidative stress, the membrane phospholipid diphosphatidylglycerol is damaged, presumably via peroxidation of the polyunsaturated fatty acids 18:2 and 18:3, which are the main fatty acids in this lipid. The damage to diphosphatidylglycerol causes an inhibition of cytochrome c oxidase, which appears to require this phospholipid for activity (Paradies et al. 2000).

Proteins can be modified/damaged by ROS either through direct chemical interaction or indirectly, involving end products of lipid peroxidation. A number of amino acids can be modified; for example, cysteine can be oxidized to cystine, and both proline and arginine are converted to glutamyl semialdehyde. Such modifications can affect the function of proteins. In some cases, the damaged amino acids are repaired in situ, whereas in other cases, the entire protein is removed and degraded (Dean et al. 1997; Møller and Kristensen 2004; Møller et al. 2007). A number of oxidized proteins have been identified in isolated plant mitochondria, presumably because they are particularly susceptible to oxidative damage in vivo (Kristensen et al. 2004; Møller and Kristensen 2006). We still do not understand the metabolic significance of this oxidation.

Breakdown products of lipid peroxidation, notably 4-hydroxy-2-nonenal (HNE), affect several mitochondrial processes. Decarboxylating dehydrogenases, such as glycine decarboxylase, are inhibited by HNE because it specifically binds to, and inactivates, lipoic acid, an essential cofactor for these enzymes (Millar and Leaver 2000). HNE inhibits the alternative oxidase, so an increasing proportion of the enzyme may become inactivated during oxidative stress. The induction of alternative oxidase gene expression during stress might therefore be necessary to maintain the activity of the enzyme in the face of increasing inactivation rather than to upregulate its activity (Winger et al. 2005). Interestingly, HNE stimulates the uncoupling protein (Smith et al. 2004), which will prevent overreduction of the electron transport chain and thus lower ROS production. The sensitivity of the alternative oxidase to HNE might explain the presence of both of these energy-wasteful enzymes in plant mitochondria (see Web Topic 12.3).

One of the theories of ageing is that oxidatively modified proteins accumulate over time. This does not appear to be the case in Arabidopsis, where the amount of proteins with free carbonyl groups increases during the vegetative phase, but decreases dramatically during the flowering and senescence phases (Johansson et al. 2004).

Finally, ROS can cause mutations in mtDNA. During aging in mammals, mutations in mtDNA accumulate faster than in nuclear DNA, possibly because mtDNA is closer to the site of ROS synthesis. However, plant mtDNA does not have a particularly high rate of mutation; in fact, rearrangements are more common. Little is known about ROS-induced DNA modifications in plant mitochondria.

As mentioned in the introduction, hydrogen peroxide could be a messenger from the mitochondria to the nucleus. If formed in the matrix, the hydrogen peroxide would have to cross the inner membrane and this might happen via aquaporins, channel-forming proteins (Bienert et al. 2007). However, ROS-modified fatty acids or proteins or their fragments could also act as messengers (Møller and Sweetlove 2010).


Balmer, Y., Vensel, W. H., Tanaka, C. K., Hurkman, W. J., Gelhaye, E., Rouhier, N., Jacquot, J. P., Manieri, W., Schürmann, P., Droux, M., and Buchanan, B. B. (2004) Thioredoxin links redox to the regulation of fundamental processes of plant mitochondria. Proc. Natl. Acad. Sci. USA 101: 2642–2647.

Bienert, G.P., Møller, A.L.B., Kristiansen, K.A., Schulz, A., Møller, I.M., Schjoerring, J.K., Jahn, J.P. 2007. Specific aquaporins facilitate the diffusion of hydrogen peroxide across membranes. J. Biol. Chem. 282: 1183-1192.

Brand, M.D. (2010) The sites and topology of mitochondrial superoxide production. Exp. Gerontol. 45: 466–472.

Camacho, A., Moreno-Sanchez, R., and Bernal-Lugo, I. (2004) Control of superoxide production in mitochondria from maize mesocotyls. FEBS Lett. 570: 52–56.

Camacho-Pereira, J., Meyer, L. E., Machado, L. B., Oliveira, M. F., and Galina, A. (2009) Reactive oxygen species production by potato tuber mitochondria is modulated by mitochondrially bound hexokinase activity. Plant Physiol. 149: 1099–1110.

Chew, O., Whelan, J., and Millar, A. H. (2003) Molecular definition of the ascorbate-glutathione cycle in Arabidopsis mitochondria reveals dual targeting of antioxidant defenses in plants. J. Biol. Chem. 278: 46869–46877.

Dean, R. T., Fu, S., Stocker, R., and Davies, M. J. (1997) Biochemistry and pathology of radical-mediated protein oxidation. Biochem. J. 324: 1–18.

Finkemeier, I., Goodman, M., Lamkemeyer, P., Kandlbinder, A., Sweetlove, L. J., and Dietz, K.-J. (2005) The mitochondrial type II peroxiredoxin F is essential for redox homeostasis and root growth of Arabidopsis thaliana under stress. J. Biol. Chem. 280: 12168–12180.

Gelhaye, E., Rouhier, N., Gérard, J., Jolivet, Y., Gualbeto, J., Navrot, N., Ohlsson, P.-I., Wingsle, G., Hirasawa, M., Knaff, D. B., Wang, H., Dizengremel, P., Meyer, Y., and Jacquot, J.-P. (2004) A specific form of thioredoxin h occurs in plant mitochondria and regulates the alternative oxidase. Proc. Natl. Acad. Sci. USA 101: 145445–14550.

Halliwell, B., and Gutteridge, J. M. C. (2007) Free Radicals in Biology and Medicine, 3rd Ed., Oxford University Press, Oxford.

Jimenez, A., Hernández, J. A., del Rio, L. A., and Sevilla., F. (1997) Evidence for the presence of the ascorbate-glutathione cycle in mitochondria and peroxisomes of pea leaves. Plant Physiol. 114: 275–84.

Jimenez, A., Hernández, J. A., Pastori, G., del Rio, L. A., and Sevilla, F. (1998) Role of the ascorbate-glutathione cycle of mitochondria and peroxisomes in the senescence of pea leaves. Plant Physiol. 118: 1327–1335.

Johansson, E., Olsson, O., and Nyström, T. (2004) Progression and specificity of protein oxidation in the life cycle of Arabidopsis thaliana. J. Biol. Chem. 279: 22204–22208.

Kell, D. B. (2010) Towards a unifying, systems biology understanding of large-scale cellular death and destruction caused by poorly liganded iron: Parkinson’s, Huntington’s, Alzheimer’s, prions, bactericides, chemical toxicology and others as examples. Arch. Toxicol. 84: 825–889.

Keunen, E., Remans, T., Bohler, S., Vangronsveld, J., and Cuypers, A. (2011) Metal-induced oxidative stress and plant mitochondria. Int. J. Mol. Sci. 12: 6894–6918.

Kristensen, B. K., Askerlund, P., Bykova, N. V., Egsgaard, H., and Møller, I. M. (2004) Identification of oxidised proteins in the matrix of rice leaf mitochondria by immunoprecipitation and two-dimensional liquid chromatography-tandem mass spectrometry. Phytochemistry 65: 1839–1851.

Maxwell, D. P., Wang, Y., and McIntosh, L. (1999) The alternative oxidase lowers mitochondrial reactive oxygen production in plant cells. Proc. Natl. Acad. Sci. USA 96: 8271–8276.

Millar, A. H., and Leaver, C. J. (2000) The cytotoxic lipid peroxidation product, 4-hydroxy-2-nonenal, specifically inhibits decarboxylating dehydrogenases in the matrix of plant mitochondria. FEBS Lett. 481: 117–121.

Møller, I. M. (2001) Plant mitochondria and oxidative stress: Electron transport, NADPH turnover and metabolism of reactive oxygen species. Annu. Rev. Plant Physiol. Plant Mol. Biol. 52: 561–591.

Møller, I. M., and Kristensen, B. K. (2004) Protein oxidation in plant mitochondria as a stress indicator. Photochem. Photobiol. Sci. 3: 730–735.

Møller, I. M., and Kristensen, B. K. (2006) Protein oxidation in plant mitochondria detected as oxidized tryptophan. Free Radical Biol. Med. 40: 430–435.

Møller, I. M., and Rasmusson, A. G. (1998) The role of NADP in the mitochondrial matrix. Trends Plant Sci. 3: 21–27.

Møller, I. M., and Sweetlove, L. J. (2010) ROS signalling – Specificity is required. Trends Plant Sci. 15: 370–374

Møller, I. M., Jensen, P. E., and Hansson, A. (2007) Oxidative modifications to cellular components in plants. Annu. Rev. Plant Biol. 58: 459–481.

Møller, I. M., Rogowska-Wrzesinska, A., and Rao, R. S. P. (2011) Protein carbonylation and metal-catalyzed oxidation in a cellular perspective. J. Proteomics 74: 2228–2242.

Paradies, G., Petrosillo, G., Pistolese, M., and Ruggiero, F. M. (2000) The effect of reactive oxygen species generated from the mitochondrial electron transport chain on the cytochrome c oxidase activity and on the cardiolipin content in bovine heart submitochondrial particles. FEBS Lett. 466: 323–326.

Puntarulo, S., Sanchez, R. A., and Boveris, A. (1988) Hydrogen peroxide metabolism in soybean embryonic axes at the onset of germination. Plant Physiol. 86: 626–630.

Purvis, A. C. (1997) Role of the alternative oxidase in limiting superoxide production by plant mitochondria. Physiol. Plant. 100: 165–170.

Smith, A.M.O., Ratcliffe, R.G., and Sweetlove, L.J. 2004. Activation and function of mitochondrial uncoupling proteinin plants. J. Biol. Chem. 279, 51944–51952.Tan, Y.-F., O’Toole, N., Taylor, N. L., and Millar, A. H. (2010) Divalent metal ions in plant mitochondria and their role in interactions with proteins and oxidative stress-induced damage to respiratory function. Plant Physiol. 152: 747–761.

Turrens, J. F. (2003) Mitochondrial formation of reactive oxygen species. J. Physiol. 555: 335–344.

Vestergaard, C. L., Flyvbjerg, H., and Møller, I. M. (2012) Intracellular signalling by diffusion – Can waves of hydrogen peroxide transmit intracellular information in plant cells? Front. Plant Sci. 3: 295. doi: 10.3389/ fpls.2012.00295

Wagner, A. M. (1995) A role for active oxygen species as second messengers in the induction of alternative oxidase gene expression in Petunia hybrida cells. FEBS Lett. 368: 339–342.

Wagner, A. M., and Krab, K. (1995) The alternative respiration pathway in plants: Role and regulation. Physiol. Plant. 95: 318–325.

Winger, A. M., Millar, A. H., and Day, D. A. (2005) Sensitivity of plant mitochondrial terminal oxidases to the lipid peroxidation product 4-hydroxy-2-nonenal (HNE). Biochem. J. 387: 865–870.