Journal of Experimental Botany, Vol. 53, No. 372, pp. 1255-1272,
May 15, 2002
© 2002 Oxford University Press
Original Papers |
Reactive oxygen species, antioxidant systems and nitric oxide in peroxisomes
1 Departamento de Bioquímica, Biología Celular y Molecular de Plantas, Estación Experimental del Zaidín, CSIC, Apartado 419, E-18080 Granada, Spain
2 Departamento de Agroecología y Protección Vegetal, Estación Experimental del Zaidín, CSIC, Apartado 419, E-18080 Granada, Spain
3 Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias Experimentales, Universidad de Jaén, Paraje ‘Las Lagunillas' s/n, E-23071 Jaén, Spain
Received 10 July 2001; Accepted 7 January 2002
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Abstract |
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 Top Abstract IntroductionProduction of oxygen radicals...Antioxidant systems in...Peroxisomes and nitric oxideConcluding remarksReferences |
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Peroxisomes are subcellular organelles with an essentially oxidative type of metabolism. Like chloroplasts and mitochondria, plant peroxisomes also produce superoxide radicals (
) and there are, at least, two sites of superoxide generation: one in the organelle matrix, the generating system being xanthine oxidase, and another site in the peroxisomal membranes dependent on NAD(P)H. In peroxisomal membranes, three integral polypeptides (PMPs) with molecular masses of 18, 29 and 32 kDa have been shown to generate radicals. Besides catalase, several antioxidative systems have been demonstrated in plant peroxisomes, including different superoxide dismutases, the ascorbate–glutathione cycle, and three NADP-dependent dehydrogenases. A CuZn-SOD and two Mn-SODs have been purified and characterized from different types of peroxisomes. The four enzymes of the ascorbate–glutathione cycle (ascorbate peroxidase, monodehydroascorbate reductase, dehydroascorbate reductase, and glutathione reductase) as well as the antioxidants glutathione and ascorbate have been found in plant peroxisomes. The recycling of NADPH from NADP+ can be carried out in peroxisomes by three dehydrogenases: glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and isocitrate dehydrogenase. In the last decade, different experimental evidence has suggested the existence of cellular functions for peroxisomes related to reactive oxygen species (ROS), but the recent demonstration of the presence of nitric oxide synthase (NOS) in plant peroxisomes implies that these organelles could also have a function in plant cells as a source of signal molecules like nitric oxide (NO•), superoxide radicals, hydrogen peroxide, and possibly S-nitrosoglutathione (GSNO). Key words: Antioxidants, ascorbate–glutathione cycle, nitric oxide, peroxisomes, ROS.
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Introduction |
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TopAbstractIntroductionProduction of oxygen radicals...Antioxidant systems in...Peroxisomes and nitric oxideConcluding remarksReferences |
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The first description of peroxisomes was carried out in 1954 in the course of electron microscopy studies in mouse kidney tubules (Rhodin, 1954
) and these organelles were designated as ‘microbodies’, a morphological name not implying any biochemical function. But it was De Duve at the beginning of the 1960s who carried out the biochemical characterization of peroxisomes from mammalian tissues which led to their recognition as distinct cell organelles (De Duve et al., 1960). These authors, by using differential and density-gradient centrifugation techniques, achieved the differentiation of the microbody fraction from lysosomes, microsomes, and mitochondria. By using combined biochemical and morphological methods the identity of microbodies and peroxisomes was unequivocally demonstrated. The presence of hydrogen peroxide-producing enzymes, like urate oxidase and D-amino acid oxidase, and the hydrogen peroxide-scavenging catalase in microbodies, led De Duve to propose the term ‘peroxisomes’ for these organelles (De Duve and Baudhuin, 1966). In the following years, these organelles were found to be present in almost all eukaryotic cells. Peroxisomes are organelles with diameters ranging from 0.1–1.7 µm, bounded by a single membrane, and contain a coarsely granular or fibrillar matrix that occasionally has amorphous or paracrystalline inclusions (Tolbert, 1981; Huang et al., 1983). From a physiological and biochemical viewpoint, peroxisomes can be broadly defined as ubiquitous subcellular organelles containing as basic enzymatic constituents catalase and H2O2-producing flavin oxidases (De Duve and Baudhuin, 1966; Tolbert, 1981; Huang et al., 1983). Although some authors postulated the presence of nucleic acids in peroxisomes (Gerhardt and Beevers, 1969; Ching, 1970; Osumi and Kazawa, 1978) today it is widely admitted that these organelles, unlike mitochondria and chloroplasts, do not have a genetic system and all their proteins are nuclear-encoded, synthesized on free polyribosomes and imported post-translationally into peroxisomes (Huang et al., 1983; Lazarow and Fujiki, 1985; Reddy et al., 1996; Kunau, 1998; Olsen, 1998; López-Huertas and Baker, 1999). Electron micrographs of plant peroxisomes from four different origins, including watermelon, pea, pepper, and olive, are presented in Fig. 1.
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Initially, it was thought that the main function of peroxisomes was the removal by catalase of toxic hydrogen peroxide generated in the peroxisomal respiratory pathway by different oxidases. Peroxisomal oxidases are mainly flavoproteins, with the exception of urate oxidase, and include D-amino acid oxidase, acyl-CoA oxidase, glycolate oxidase,
-hydroxy acid oxidase, and glutaryl-CoA oxidase, among others, depending on the tissue origin (Huang et al., 1983; Angermüller, 1989).
In recent years, it has been demonstrated that peroxisomes are involved in a range of important cellular functions in almost all eukaryotic cells (Fahimi and Sies, 1987; Van den Bosch et al., 1992; Masters and Crane, 1995; Olsen, 1998; Reddy et al., 1996; Subramani, 1998; Tabak et al., 1999). These organelles have an essentially oxidative type of metabolism and can carry out different metabolic pathways depending on their source. Table 1 shows different functions that have been described so far for peroxisomes in plant and fungal cells. In plants, there are several types of peroxisomes which are specialized in certain metabolic functions. Glyoxysomes are specialized peroxisomes, occurring in the storage tissues of oilseeds, that contain the fatty acid ß-oxidation and glyoxylate cycle enzymes to convert the seed reserve lipids into sugars which are used for germination and plant growth (Tolbert, 1981; Beevers, 1982; Huang et al., 1983). Leaf peroxisomes are specialized peroxisomes present in photosynthetic tissues that carry out the major reactions of photorespiration (Tolbert, 1980; Huang et al., 1983; Douce and Heldt, 2000). Another type of specialized peroxisomes are root-nodule peroxisomes from certain tropical legumes, in which the synthesis of allantoin, the major metabolite for nitrogen transport within these plants, is carried out (Schubert, 1986). The main metabolic processes responsible for the generation of H2O2 in different types of peroxisomes are the photorespiratory glycolate oxidase reaction, the ß-oxidation of fatty acids, the enzymatic reaction of flavin oxidases, and the disproportionation of superoxide radicals (Huang et al., 1983; del Río et al., 1992, 1996).
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Studies on the compartmentation of the photorespiratory pathway in peroxisomes have led to the proposal that, in these organelles, unlike mitochondria and chloroplasts, the compartmentation of peroxisomal metabolism is, in major part, not caused by the boundary membrane but by the specific structure of the protein matrix. Apparently, the enzymes of the photorespiratory pathway are arranged in the peroxisomal matrix in the form of multienzyme complexes that allow efficient metabolite channelling (Douce and Heldt, 2000
; Reumann, 2000), and transfer of metabolites proceeds across the peroxisomal membrane by porin-like channels (Reumann, 2000; Corpas et al., 2000).
A characteristic property of peroxisomes is their metabolic plasticity since their enzymatic content can vary depending on the organism, cell/tissue-type and environmental conditions (Van den Bosch et al., 1992; Mullen and Trelease, 1996). In animals, a variety of xenobiotics, mainly including hypolipidemic drugs, and certain herbicides and phthalate-ester plasticizers, induce in liver the proliferation of the peroxisomal population as well as the activity of the H2O2-producing acyl-CoA oxidase (Reddy et al., 1987, 1996). In mussels, the proliferation of peroxisomes has been proposed as a specific biomarker of pollution by xenobiotics in marine and estuarine environments (Cancio and Cajaraville, 2000). In some yeast species, peroxisomes may be induced by growth on methanol, alkanes, fatty acids, D-alanine, and cadmium (Huang et al., 1983; Chen et al., 1995; Van der Klei and Veenhuis, 1997). In plants, the cellular proliferation of peroxisomes has also been reported both under natural and abiotic stress conditions. Table 2 illustrates the cases and the agents responsible for the induction of peroxisomes in different plant species. Very recently, the induction of peroxisome biogenesis genes (PEX) by H2O2 has been demonstrated in both plant and animal cells, indicating that the signal molecule H2O2 is responsible for the proliferation of peroxisomes (López-Huertas et al., 2000). According to these authors, in plant wounding and pathogen attack a proliferation of peroxisomes takes place and, probably, this also happens in other stress situations that lead to an increased generation of H2O2 (López-Huertas et al., 2000). However, it must be taken into account that quantitative evidence for the proliferation of the peroxisomal population, i.e. counting of the number of organelles per cell section of tissue, has only been reported in plants treated with isoproturon (de Felipe et al., 1988), clofibrate (Palma et al., 1991), cadmium (Romero-Puertas et al., 1999) and during senescence of pea leaves (Pastori and del Río, 1994).
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An example of the inducible nature of peroxisomal metabolism is the light-induced transition of glyoxysomes, the specialized peroxisomes of oilseeds, to leaf-type peroxisomes (Huang et al., 1983
; Masters and Crane, 1995; Mullen and Trelease, 1996). Likewise, during plant senescence and under the effect of abiotic stress by cadmium the reverse process is observed, that is the metabolic conversion of leaf peroxisomes into glyoxysomes (De Bellis et al., 1990; Landolt and Matile, 1990; Nishimura et al., 1993, 1996; Vicentini and Matile, 1993; Pastori and del Río, 1994; del Río et al., 1998b, 2000). In these metabolic transitions of peroxisomes, endogenous proteases could be involved in the turnover of peroxisomal proteins. In plant peroxisomes, the presence of exo- and endo-proteolytic activity was reported for the first time in pea leaves, and an exo-peptidase was characterized as a leucine aminopeptidase, belonging to the family of the serine-proteinases (Corpas et al., 1993a). The different proteases characterized thus far in leaf peroxisomes are shown in Table 3. An increase in the total endo-proteolytic activity and in the number of endoprotease isoenzymes was found in peroxisomes purified from senescent pea leaves in comparison with peroxisomes from young leaves (Distefano et al., 1997). In pea plants grown with cadmium an increase in the activity of four endoproteases of leaf peroxisomes was recently found (McCarthy et al., 2001). In general, there are data that suggest the involvement of peroxisomal endoproteases in a regulated modification of proteins in this organelle (Distefano et al., 1999).
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In recent years, different experimental evidence has suggested the existence of cellular functions for leaf peroxisomes related to reactive oxygen species which are summarized in Table 4
. These ROS-related roles add to the other well-established functions known for peroxisomes from plant cells. In this review, the production of reactive oxygen species (ROS), the different antioxidant systems and the generation of nitric oxide in peroxisomes will be analysed in the context of these new ROS-mediated functions of plant peroxisomes.
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Production of oxygen radicals in peroxisomes |
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TopAbstractIntroductionProduction of oxygen radicals...Antioxidant systems in...Peroxisomes and nitric oxideConcluding remarksReferences |
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In plant cells, the production of ROS has been shown in chloroplasts, mitochondria, the plasma membrane and the apoplastic space (Elstner, 1991
; Asada, 1994; Bolwell, 1999). Peroxisomes also produce superoxide radicals () as a consequence of their normal metabolism. In peroxisomes from pea leaves by biochemical and EPR methods the existence of, at least, two sites of generation was demonstrated: one in the organelle matrix, in which the generating system was identified as xanthine oxidase (XOD), and another site in the peroxisomal membranes dependent on NAD(P)H (Sandalio et al., 1988; del Río et al., 1989, 1992, 1998a). Xanthine oxidase catalyses the oxidation of xanthine and hypoxanthine to uric acid and is a well-known producer of superoxide radicals (Fridovich, 1986). The presence of xanthine and uric acid, substrate and product, respectively, of the xanthine oxidase reaction, as well as allantoin, the product of the urate oxidase reaction, were detected in leaf peroxisomes by HPLC analysis (Corpas et al., 1993b). The occurrence of xanthine, uric acid, and allantoin in leaf peroxisomes indicate a role for these organelles in the catabolism of xanthine produced as a result of the turnover of nucleotides, RNA and DNA in leaf cells (Corpas et al., 1993b; del Río et al., 1998a).
On the other hand, in the other peroxisomal site of production, the peroxisomal membranes, a small electron transport chain similar to that reported in peroxisomal membranes from castor bean endosperm (Fang et al., 1987) appears to be involved. This electron-transport chain is composed of a flavoprotein NADH:ferricyanide reductase of about 32 kDa and a Cyt b (measured as NADH:CCRase) (Fang et al., 1987). The integral peroxisomal membrane polypeptides (PMPs) of pea leaf peroxisomes were identified by SDS–PAGE (López-Huertas et al., 1995) and recently three of these membrane polypeptides, with molecular masses of 18, 29, and 32 kDa have been characterized and demonstrated to be responsible for generation (López-Huertas et al., 1997, 1999). The properties and possible identities of these polypeptides are shown in Table 5.
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The main producer of superoxide radicals in the peroxisomal membranes was the 18 kDa PMP which was proposed to be a cytochrome, possibly belonging to the b-type group, and uses NADH as electron donor (López-Huertas et al., 1997
). The PMP32 is also NADH-dependent for superoxide production and, on the basis of its biochemical and immunochemical properties, very probably corresponds to the monodehydroascorbate reductase (MDHAR) (López-Huertas et al., 1999) whose activity was previously detected in pea leaf peroxisomal membranes (Jiménez et al., 1997). This indicates the participation of MDHAR in production by peroxisomal membranes. Very recently, it has been reported that chloroplast MDHAR mediates the production of in spinach thylakoid membranes (Miyake et al., 1998). This shows that the chloroplastic and peroxisomal membrane-bound MDHARs share the property of being producers, although by different mechanisms. In chloroplast thylakoid membranes, radicals are generated via O2 reduction by MDHAR photoreduced at photosystem I (Miyake et al., 1998), whereas in peroxisomal membranes radicals are produced by MDHAR (PMP32) previously reduced by NADH (López-Huertas et al., 1999). The third -generating polypeptide, PMP29, is strictly dependent on NADPH as electron donor, is able to reduce cytochrome c, and could be related to the peroxisomal NADPH:cytochrome P-450 reductase.
Superoxide production by peroxisomal membranes may be an obligatory consequence of NADH re-oxidation by the peroxisomal electron-transport chain, in order to regenerate NAD+ to be re-utilized in peroxisomal metabolic processes (del Río et al., 1990, 1992; del Río and Donaldson, 1995). Under normal metabolic conditions, the production by peroxisomal membranes is not dangerous to the cell, which is adequately protected against these radicals. However, under certain conditions of plant stress, the release of peroxisomal membrane-generated radicals into the cytosol can be enhanced (del Río et al., 1996), producing cellular oxidative stress situations mediated by reactive oxygen species. Since radicals have a short half-life under physiological conditions and are rapidly converted into H2O2 and O2, the final result of these stress conditions will be an increase of H2O2 in the cell (del Río et al., 1996). In recent years, H2O2 has been described as a diffusible transduction signal in different physiological processes and plant stress, leading to the induction of genes encoding different cellular protectants. Some functions that have been reported for H2O2 as a signal molecule are shown in Table 6.
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In mammalian and yeast peroxisomes there is very little information on the production of oxygen radicals. In mouse peroxisomes, a 20 kDa membrane protein (Mpv17) has been reported to produce ROS. This peroxisomal membrane protein was identified by biochemical and genetic means, and is thought to be involved in kidney diseases like the glomerulosclerosis and nephrotic syndrome (Zwacka et al., 1994
). In Arabidopsis thaliana, a peroxisomal membrane protein (PMP22) was identified and characterized at the molecular level and had a 55% similarity with mammalian Mpv17 (Tugal et al., 1999). However, the generation of ROS by this Arabidopsis PMP has not been studied.
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Antioxidant systems in peroxisomes |
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TopAbstractIntroductionProduction of oxygen radicals...Antioxidant systems in...Peroxisomes and nitric oxideConcluding remarksReferences |
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Superoxide dismutases
Superoxide dismutases (SODs; EC 1.15.1.1) are a family of metalloenzymes that catalyze the disproportionation of
radicals into H2O2 and O2, and play an important role in protecting cells against the toxic effects of superoxide radicals produced in different cellular compartments (Fridovich, 1986; Elstner, 1991; Halliwell and Gutteridge, 2000). SODs are distributed in different cell loci, mainly chloroplasts, cytosol, and mitochondria (Fridovich, 1986; del Río et al., 1992; Bowler et al., 1994; Halliwell and Gutteridge, 2000), but the presence of SOD in peroxisomes was demonstrated for the first time in plant tissues (del Río et al., 1983; Sandalio et al., 1987). Since then, the occurrence of SODs in isolated plant peroxisomes has been reported in at least nine different plant species (Table 7). And in five of these plants the presence of SOD in peroxisomes has been confirmed by immunogold electron microscopy using an antibody prepared against peroxisomal CuZn-SOD from watermelon (Sandalio et al., 1997; Corpas et al., 1998a; Valderrama et al., unpublished results).
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Results obtained on the presence of SOD in plant peroxisomes were, years later, extended to human and animal cells, which were found to contain CuZn-SOD in peroxisomes and, more recently, Mn-SOD as well (Table 7
). In mammals, a role for peroxisomes in oxidative stress-induced cellular pathophysiology has been proposed (Singh, 1996). In yeast cells, the gene of a human CuZn-SOD (SOD1) was expressed and immunocryoelectron microscopy of cells showed that human CuZn-SOD was accumulated in peroxisomes (Keller et al., 1991). In an ectomycorrhizal fungus, the gene encoding a Mn-SOD was cloned, and the protein was found to contain a C-terminal peroxisomal localization peptide (PTS1) and lack an N-terminal mitochondrial transit peptide (Jacob et al., 2001). This putative peroxisomal Mn-SOD appears to be involved in the cellular response of the fungus to cadmium stress (Jacob et al., 2001).
Three SODs of peroxisomal origin have been purified and characterized, a CuZn-SOD and a Mn-containing SOD from watermelon cotyledons (Bueno et al., 1995; Pastori et al., 1996) and a Mn-SOD from pea leaves (Palma et al., 1998). The molecular properties determined for these peroxisomal SODs and their intraorganellar localizations are summarized in Table 8. On the basis of determination of SOD latency in intact organelles and by solubilization assays with 0.2 M KCl and 0.1 M sodium carbonate, a Mn-SOD was found to be present in the external side of the membrane of peroxisomes from watermelon cotyledons (Sandalio and del Río, 1988; Sandalio et al., 1997) (Table 8). A Mn-SOD was also localized in membranes of peroxisomes isolated from castor bean endosperm, and this isozyme was removed from the membranes by washing with 0.1 M sodium carbonate (del Río and Donaldson, 1995). Therefore, Mn-SOD appears to be a peripheral protein in those peroxisomal membranes, and this localization is pending to be ratified by immunoelectron microscopy using the specific antibody to Mn-SOD.
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Ascorbate–glutathione cycle
The ascorbate–glutathione cycle, also called Foyer–Halliwell–Asada cycle, is an efficient way for plant cells to dispose of H2O2 in certain cellular compartments where this metabolite is produced and no catalase is present (Halliwell and Gutteridge, 2000). This cycle makes use of the non-enzymic antioxidants ascorbate and glutathione in a series of reactions catalysed by four antioxidative enzymes and has been demonstrated in chloroplasts, cytosol, and root nodule mitochondria (Foyer and Mullineaux, 1994). In peroxisomes purified from pea leaves, the presence of the four enzymes of the ascorbate–glutathione cycle, ascorbate peroxidase (APX; EC 1.11.1.11), monodehydroascorbate reductase (MDHAR; EC 1.6.5.4), dehydroascorbate reductase (DHAR; EC 1.8.5.1), and glutathione reductase (GR; EC 1.6.4.2) was demonstrated (Jiménez et al., 1997). Likewise, in intact peroxisomes, the presence of reduced ascorbate (ASC) and glutathione (GSH), and their oxidized forms, dehydroascorbate (DHA) and GSSG, respectively, was demonstrated by HPLC analysis (Jiménez et al., 1997). The presence of the ascorbate–glutathione cycle enzymes has also been reported recently in peroxisomes of leaf and root cells of two species of tomato (Mittova et al., 2000). The intraperoxisomal distribution of the four enzymes was studied by determining enzyme activity latency in intact organelles and by solubilization assays with 0.2 M KCl. On the basis of the results obtained, a model for the function of the ascorbate–glutathione cycle in leaf peroxisomes is shown in Fig. 2. DHAR and GR were found in the soluble fraction of peroxisomes, whereas APX activity was bound to the cytosolic side of the peroxisomal membrane. By Western blot analysis of peroxisomal membranes with a specific antibody against pumpkin APX, an integral membrane polypeptide of 35 kDa was identified as the APX of pea leaf peroxisomes (López-Huertas et al., 1999). These results agree with recent findings of an APX isoenzyme in membranes of pumpkin and cotton peroxisomes (Yamaguchi et al., 1995; Bunkelmann and Trelease, 1996). The substrate specificity of the membrane-bound peroxisomal APX from pea leaves was recently studied. The APX specific activity was much higher with pyrogallol than with ascorbate and the enzyme was more sensitive to incubation with Triton X-100 than the mitochondrial APX (Jiménez et al., 1998a). By native-PAGE of peroxisomal membranes no APX activity could be detected probably due to enzyme inactivation. The low stability of this enzyme in the presence of Triton and after native-PAGE suggests that peroxisomal APX is more related to chloroplast APX than to mitochondrial and cytosolic APXs detected in pea leaves (Jiménez et al., 1998a). cDNAs encoding peroxisomal APX have been isolated from cotton (Bunkelmann and Trelease, 1996), Arabidopsis (Zhang et al., 1997; PM Mullineaux, personal communication), and spinach (Ishikawa et al., 1998). The deduced amino acid sequence of peroxisomal APX has a high degree of identity with cytosolic APX, but it has a C-terminal amino acid extension containing a single, putative membrane-spanning region (Mullen et al., 1999).
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Two peroxisomal proteins from human and murine origin (HsPMP20 and MmPMP20) that exhibit antioxidant activity in vitro were characterized (Yamashita et al., 1999
). The PMP20 proteins were initially characterized in yeast and defined as membrane proteins (Garrard and Goodman, 1989). However, further research demonstrated that the PMP20 was released from the peroxisomal membrane, contained PTS1 sequences and was shown to be present in the peroxisomal matrix by immunocytochemistry (see references in Yamashita et al., 1999). In spite of this evidence, these proteins are still termed peroxisomal membrane proteins (PMPs). The human HsPMP20 and murine MmPMP20 proteins can remove H2O2 by its thiol-peroxidase activity and have been proposed as novel members of the Ahp/thiol-specific antioxidant family, which might play a protecting role against oxidative stress in peroxisomes (Yamashita et al., 1999).
Another peroxidase of putative peroxisomal localization could be thioredoxin-dependent peroxidase (peroxiredoxin). This enzyme has not been directly localized in peroxisomes, but an E. coli recombinant thioredoxin-dependent peroxidase has been recently characterized and was found to have a high homology with the C-terminal sequence of the peroxisomal protein PMP20 from Candida boidini (Verdoucq et al., 1999). Additionally, in the human and rat protein AOEB166, a novel member of the mammalian peroxiredoxin family, the amino acid analysis of their C-terminal domains revealed the existence of a peroxisomal targeting sequence (PTS1) (Knoops et al., 1999). The presence of peroxiredoxins in peroxisomes would supply these organelles with another antioxidant enzyme system which would join catalase and the ascorbate–glutathione cycle in the control of the peroxisomal level of H2O2.
MDHAR was also localized in the peroxisomal membranes and was highly latent in intact peroxisomes (Fig. 2). It has been proposed that the trans-membrane protein MDHAR can oxidize NADH on the matrix side of the peroxisomal membrane and transfer the reducing equivalents as electrons to the acceptor monodehydroascorbate on the cytosolic side of the membrane (Luster and Donaldson, 1987; Bowditch and Donaldson, 1990). In this process, molecular O2 could also act as an electron acceptor, with the concomitant formation of (López-Huertas et al., 1999).
The presence of APX and MDHAR in leaf peroxisomal membranes suggests a dual complementary function in peroxisomal metabolism of these membrane-bound antioxidant enzymes. The first role could be to reoxidize endogenous NADH to maintain a constant supply of NAD+ for peroxisomal metabolism (Fig. 2), an idea that was originally proposed for the membrane-bound NADH dehydrogenase of glyoxysomes from castor bean endosperm (Fang et al., 1987; Luster and Donaldson, 1987; Bowditch and Donaldson, 1990). A second function of the membrane antioxidative enzymes could be to protect against H2O2 leaking from peroxisomes, particularly when the catalase activity of peroxisomes is depressed and, as a result of it, the endogenous level of H2O2 is enhanced. Hydrogen peroxide can easily permeate the peroxisomal membrane, and an important advantage of the presence of APX in the membrane would be the degradation of leaking H2O2, as well as the H2O2 that is being continuosly formed by dismutation of the generated in the NADH-dependent electron transport system of the peroxisomal membrane (Fig. 2). It should be taken into account that although catalase in the peroxisomal matrix decomposes most of the H2O2 produced in these organelles, the catalase affinity for H2O2 is relatively low (Halliwell, 1974). However, at lower levels of H2O2 the ascorbate peroxidase activity of the ascorbate–glutathione cycle can scavenge H2O2 more efficiently than catalase owing to its much lower Km (Nakano and Asada, 1981). This membrane scavenging of H2O2 could prevent an increase in the cytosolic H2O2 concentration during normal metabolism and under certain plant stress situations, when the level of H2O2 produced in peroxisomes can be substantially enhanced (del Río et al., 1992, 1996).
NADP-dependent dehydrogenases
The reduced coenzyme NADPH is a basic electron donor in many biosynthetic and detoxification reactions of living cells. The main cellular sources of NADPH are the dehydrogenases of the pentose phosphate pathway, glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH), the malic enzyme, isocitrate dehydrogenase (ICDH), and chloroplastic ferredoxin-NADP+ reductase.
In recent years, different studies have shown that G6PDH has a relevant role in the mechanism of protection against oxidative stress of bacteria, yeast and mammalian cells (for references see Corpas et al., 1998b). In plant cells, NADPH has an important role in the protection systems against oxidative stress due to its involvement in the ascorbate–glutathione cycle of chloroplasts (Foyer and Halliwell, 1976; Asada, 1994). This evidence has supported the notion of G6PDH as an antioxidative enzyme which can be included in the group of catalase, SOD, APX and GR/peroxidase (Martini and Ursini, 1996). In higher plants, only two isoforms of G6PDH have been reported, which are localized in the cytosol and the plastidic stroma (Schnarrenberger et al., 1995; Fickenscher and Scheibe, 1986).
In a recent work carried out in our laboratory, the presence of three NADP-dehydrogenases in leaf peroxisomes purified from pea plants was investigated. The dehydrogenases included: G6PDH, 6PGDH, and ICDH. The only NADP-dependent dehydrogenase that had been previously detected in peroxisomes was the ICDH present in trace amounts in spinach leaf peroxisomes (Tolbert, 1981) and in castor bean glyoxysomes (Donaldson, 1982). These three dehydrogenases were found to be present in the matrix of leaf peroxisomes and showed a typical Michaelis–Menten kinetic saturation curve (Table 9) (Corpas et al., 1998b, 1999). The possible occurrence of the malic enzyme in pea leaf peroxisomes was investigated, but no traces of this NADP-dehydrogenase were found in peroxisomes (FJ Corpas et al., unpublished results).
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By isoelectric focusing four isoforms of ICDH, three isoforms of G6PDH, and one isoform of 6PGDH were detected in peroxisomal matrices (Corpas et al., 1998
b, 1999). The presence of G6PDH and ICDH in leaf peroxisomes was also checked by immunoblot analysis of peroxisomal matrices with antibodies against yeast G6PDH and pea leaf ICDH. Single cross-reactivity bands of 56 kDa and 46 kDa were detected for G6PDH and ICDH, respectively (Fig. 3). The localization of G6PDH and ICDH in pea leaf peroxisomes was also studied by immunogold electron microscopy using antibodies against yeast G6PDH and pea ICDH (Fig. 3). After embedding, electron microscopy immunogold labelling confirmed that G6PDH was localized in the peroxisomal matrices, with an average number of gold particles of 5.1, and also in chloroplasts (both thylakoid membranes and stroma) and cytosol (Corpas et al., 1998b). The proportion of G6PDH in peroxisomes agrees with that reported for cellular fractions of mouse kidney, where 10% of the total G6PDH activity was also found in peroxisomes (Patel et al., 1987). ICDH was also localized in the peroxisomal matrices, as well as in mitochondria, chloroplasts and cytosol, but the highest density of immunogold particles was found in peroxisomes and mitochondria (Corpas et al., 1999) (Fig. 3).
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The effect of plant senescence on the kinetic properties of the NADP-ICDH in pea leaf peroxisomes was analysed. No significant changes were observed in the specific activity or Vmax values of ICDH in the two experimental situations. However, in peroxisomes from senescent leaves, the Km of ICDH decreased almost 11-fold. This kinetic behaviour resulted in a catalytic efficiency approximately 12 times higher for peroxisomal ICDH from senescent leaves (Corpas et al., 1999
). However, the protein levels of ICDH in peroxisomes were not altered during senescence. The physiological significance of the change in the Km of peroxisomal ICDH during senescence is probably double: first, to compete with isocitrate lyase, an enzyme of the glyoxylate cycle which is present in peroxisomes from senescent leaves, for the intracellular pool of isocitrate; and second, to provide a higher and constant supply of NADPH in order to eliminate, by the ascorbate–glutathione cycle, the excess of H2O2 produced during senescence when catalase activity decreases dramatically (Pastori and del Río, 1997).
The presence in peroxisomes of the two oxidative enzymes of the pentose phosphate pathway, G6PDH and 6PGDH, and ICDH implies that these organelles have the capacity to reduce NADP+ to NADPH for its re-utilization in peroxisomal metabolism. However, the production of NADPH by peroxisomal ICDH is 13- and 5-fold higher than that due to peroxisomal G6PDH and 6PGDH, respectively (Corpas et al., 1999). This fact, together with the observed fine control of ICDH during senescence, strongly indicates the importance of this enzyme in the enzymatic systems of NADPH recycling. The occurrence of NADP(H) in castor bean peroxisomes has been demonstrated (Donaldson, 1982). NADPH is necessary for the function of the NADPH:cytochrome P-450 reductases, whose presence has been detected in the membranes of castor bean peroxisomes (Alani et al., 1990). On the other hand, one of the -generating polypeptides of peroxisomal membranes mentioned above, the PMP29, was clearly dependent on NADPH and was also able to reduce cytochrome c with NADPH as electron donor (López-Huertas et al., 1999).
NADPH has been reported to protect catalase from oxidative damage (Kirkman et al., 1999). This cofactor tightly binds to mammalian catalase and avoids the H2O2-dependent conversion of the enzyme to an inactive state (compound II). But, additionally, NADPH may lead to the reduction of oxidizing states and internal groups of catalase other than the intermediate (Kirkman et al., 1999). NADPH is also required for the maintenance of the ascorbate–glutathione cycle because it is necessary for the glutathione reductase activity which recycles reduced glutathione (GSH) from its oxidized form (GSSG) in order to be used for the scavenging of H2O2 in the cycle (Foyer and Halliwell, 1976; Halliwell and Gutteridge, 2000). Therefore, the three NADP-dependent dehydrogenases of peroxisomes (G6PDH, 6PGDH and ICDH) represent a very efficient system to recycle NADPH for its re-utilization in the peroxisomal metabolism, particularly for the functioning of the ascorbate–glutathione cycle, which together with catalase represent an important antioxidant protection system against H2O2 generated in peroxisomes. An additional potential function for NADPH in peroxisomes could be related to the mechanism of protein import into these organelles. It was shown that the NADPH to NADP+ ratio is important in peroxisomal protein import (Pool et al., 1998).
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Peroxisomes and nitric oxide |
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TopAbstractIntroductionProduction of oxygen radicals...Antioxidant systems in...Peroxisomes and nitric oxideConcluding remarksReferences |
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Nitric oxide (NO•) is a widespread intracellular and intercellular messenger in vertebrates with a broad spectrum of regulatory functions in the central nervous, cardiovascular, and immune systems (Moncada et al., 1991
; Knowles and Moncada, 1994). As a consequence of the physiological importance of the free radical NO•, numerous studies have been focused on the enzyme responsible for its endogenous production. Nitric oxide synthase (NOS; EC 1.14.13.39) catalyses the oxygen- and NADPH-dependent oxidation of L-arginine to NO• and citrulline in a complex reaction requiring FAD, FMN, and tetrahydrobiopterin, and in some cases also calcium and calmodulin (Knowles and Moncada, 1994). NOSs have been characterized as functional homodimers with a subunit of 
130 kDa that contain iron-protoporphyrin IX, flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN), and tetrahydrobiopterin (BH4) as prosthetic groups (Knowles and Moncada, 1994; McMillan et al., 1996). Three NOS isoforms have been identified in mammalian tissues: neuronal NOS (nNOS or type I), inducible NOS (iNOS or type II), and endothelial NOS (eNOS or type III). While types I and II are present in the soluble fraction, type III has been shown to be membrane-associated and none of the isoforms has a strict tissue-specific expression pattern (Sessa et al., 1992; McMillan et al., 1996). In organelles from animal systems, as far as is known, the presence of NOS activity has only been detected so far in mitochondria of rat liver (Ghafourifar and Richter, 1997).
Research on NO• in plants has gained considerable attention in recent years and there is increasing evidence of a role of this molecule in plant growth and development (Leshem et al., 1998; Leshem and Haramaty, 1996; Ribeiro et al., 1999). Ya'acov Leshem, the pioneer in the study of nitric oxide in plants, has proposed that NO• should be classified as a phytohormone which may function as a gaseous endogenous plant growth regulator (Leshem, 2000). Moreover, different studies have suggested that, in plants, nitric oxide could also function as a signal molecule in the transduction pathway leading to the induction of defence responses against pathogens and in damage leading to cell death (Delledonne et al., 1998; Van Camp et al., 1998; Bolwell, 1999; Durner and Klessig, 1999; McDowell and Dangl, 2000; Grant and Loake, 2000; Leshem, 2000; Pedroso et al., 2000).
In plant extracts, NOS-like activity has been detected during the interaction of Rhizobium–legumes (Cueto et al., 1996) and fungi–plants (Ninnemann and Maier, 1996), in soybean cell suspensions and Arabidopsis (Delledonne et al., 1998), and in tobacco leaves (Durner et al., 1998). The presence of NOS in the cytosol and nucleus of maize cells has also been reported (Ribeiro et al., 1999).
Very recently, the presence of NOS in subcellular fractions of pea leaves was studied. Mitochondria and peroxisomes were purified from pea leaves by differential and sucrose density-gradient centrifugation. The assay of NOS activity throughout the gradient fractions by the citrulline assay showed an activity peak in the peroxisomal fractions of 170 pmol of L-[3H]citrulline min-1 mg-1 protein coinciding with the maximum of catalase activity in the gradient (Barroso et al., 1999). However, no NOS activity was detected in mitochondria. The specific activity of the peroxisomal NOS (perNOS) was reduced more than 70% in the absence of calcium and was strictly dependent on NADPH. The effect of seven archetype NOS inhibitors, including specific and unspecific inhibitors of different types of NOS isoforms was assayed. The results indicated a clear inhibition of the perNOS activity of 59–100%, L-aminoguanidine being the most effective inhibitor. The production of citrulline was specifically due to a NOS because a specific antibody against iNOS inhibited this reaction by more than 80% (Barroso et al., 1999).
In the subcellular fractions of pea leaves the presence of NOS was also studied by Western blot analysis, and in intact leaves by electron microscopy immunogold-labelling, using an antibody against the peptide PT387 from the C terminus (Ac-Cys-residues 1131–1144) of the murine iNOS (Barroso et al., 1999). The electron microscopy immunolocalization of NOS demonstrated the presence of the enzyme in the matrix of peroxisomes and also in chloroplasts (Fig. 4) (Barroso et al., 1999). However, no immunogold labelling of NOS was detected in mitochondria. This contrasts with results obtained in mammalian tissues where NOS activity was found in mitochondria isolated from rat liver (Ghafourifar and Richter, 1997). The immunoblot analysis of the peroxisomal fractions revealed an immunoreactive band of about 130 kDa (Fig. 4), which is in the same molecular mass range described for most iNOS and eNOS (for references see Barroso et al., 1999). Immunoreactive bands with similar mobility were obtained with the hepatic iNOS from lipopolysaccharide-induced rat and murine macrophage lysates used as positive controls (Fig. 4, lanes 2 and 3). A summary of the biochemical properties of NOS from pea leaf peroxisomes is presented in Table 10.
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Therefore, the biochemical, immunological, and immunocytochemical data provide evidence that plant peroxisomes contain nitric oxide synthase activity (perNOS) which is calcium-dependent, constitutively expressed, and immunorelated with the mammalian iNOS (Barroso et al., 1999
).
The presence of NOS in peroxisomes suggests that these organelles are a cellular source of NO• and also indicates possible interactions of NOS with other components of the metabolism of reactive oxygen species of leaf peroxisomes. A hypothetical model of this interaction is presented in Fig. 5. The NADP-dependent dehydrogenases found in the peroxisomal matrix (Corpas et al., 1998b, 1999) could provide the necessary NADPH for the NOS reaction. But inside peroxisomes NO• could react with radicals generated in the peroxisomal matrix by xanthine oxidase (Sandalio et al., 1988; del Río et al., 1992) to form the powerful oxidant peroxynitrite which (according to Sakuma et al., 1997) could regulate the conversion of xanthine dehydrogenase into the superoxide-generating xanthine oxidase. Besides, the information available on the proteolytic activity of peroxisomes from pea leaves suggests that peroxisomal endoproteases could, potentially, carry out the partial proteolysis which results in the irreversible conversion of xanthine dehydrogenase into xanthine oxidase (Distefano et al., 1999). On the other hand, it is known that NO• in the presence of O2 can react with reduced glutathione (GSH) to form S-nitrosoglutathione (GSNO), a reactive nitrogen oxide species (Wink et al., 1996). As GSH occurs in leaf peroxisomes (Jiménez et al., 1997) and NO• can be formed in these organelles (Barroso et al., 1999), GSNO could be generated inside peroxisomes. In animal systems, GSNO has been reported to function as an intercellular and an intracellular NO• carrier, and in plants GSNO was found to be a powerful inducer of defence genes (Durner et al., 1998). As hypothesized previously (Durner and Klessig, 1999), GSNO could function as a long-distance signal molecule, transporting glutathione-bound NO• throughout the plant. In this mechanism, leaf peroxisomes could participate through the endogenous production of GSNO which could diffuse to the cytosol.
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NO• could also diffuse through the peroxisomal membrane, reacting with
produced in the cytosolic side of the membrane (del Río et al., 1998a), thus generating the oxidant peroxynitrite in the cytosol. However, a modulation by NO• of the endogenous antioxidant enzymes of peroxisomes and the fatty acid ß-oxidation enzymes cannot be ruled out. In animal systems, catalase and glutathione peroxidase activity are down-regulated by NO•, whereas the activity of the peroxisomal ß-oxidation is enhanced by NO•. This has been proposed as the mechanism responsible for the H2O2-associated cytoxicity of NO• in peroxisomes, cytoplasm and mitochondria of animal cells (Dobashi et al., 1997).
Under normal physiological conditions, the production by peroxisomes of the ROS H2O2 and should be adequately controlled by catalase and ascorbate peroxidase, and SOD, respectively, which are present in peroxisomes. However, catalase is known to be inactivated by light and different stress conditions that suppress protein synthesis (Schäfer and Feierabend, 2000), and in these conditions an increase in the peroxisomal generation of H2O2 and can take place (del Río et al., 1992, 1996). Superoxide radicals are known to inhibit catalase activity (Kono and Fridovich, 1982) and very recently it has been reported in tobacco plants that NO• and peroxynitrite inhibit catalase and APX activity, the two major H2O2-scavenging enzymes of plant peroxisomes (Clark et al., 2000). In addition, in animal cells an enhanced synthesis of nitric oxide was found to increase the peroxisomal H2O2-producing ß-oxidation. Taken together, these data mean that if, under any type of plant biotic and/or abiotic stress an induction of the peroxisomal production of and NO• radicals takes place, this can lead to the inhibition of catalase and APX activities and, possibly to an increase of the H2O2 level from the enhanced fatty acid ß-oxidation. This breakdown of the peroxisomal antioxidant defences would originate an overproduction of H2O2 in peroxisomes and a toxic situation to the plant cell.
The rate of ROS and NO• generation in plant cells can have an ambivalent effect. A high cellular production of these active molecules can bring about extensive oxidative damage (Halliwell and Gutteridge, 2000). On the contrary, numerous studies carried out in recent years have suggested that low levels of NO• and ROS, like H2O2 and , are involved as signal mole