Deciphering lead tolerance mechanisms in a population of the plant species Biscutella auriculata L. from a mining area: Accumulation strategies and antioxidant defenses.

The uptake and distribution of Pb and the mechanisms involved in the metal tolerance have been investigated in a mine population of Biscutella auriculata. Seedlings were exposed to 125 µM Pb(NO3)2 for 15 days under semihydroponic conditions. The results showed an increase in the size of Pb-treated seedlings and symptoms of toxicity were not observed. ICP-OES analyses showed that Pb accumulation was restricted to root tissue. Imaging of Pb accumulation by dithizone histochemistry revealed the presence of the metal in vacuoles and cell wall in root cells. The accumulation of Pb in vacuoles could be stimulated by an increase in phytochelatin PC2 content. Pb did not promote oxidative damage and this is probably due the increase of antioxidative defenses. In the leaves, Pb produced a significant increase in superoxide dismutase activity, while in roots an increase in catalase and components of the Foyer- Halliwell-Asada cycle were observed. The results indicated that Biscutella auriculata has a high capacity to tolerate Pb and this is mainly due to a very efficient mechanism to sequester the metal in roots and a capacity to avoid oxidative stress. This species could therefore be very useful for phytostabilization and repopulation of areas contaminated with Pb.

Characterization of mechanisms involved in tolerance and accumulation of Cd in Biscutella auriculata L.

Biscutella auriculata L. is one of the rare species that is able to grow in a very contaminated mining area in Villamayor de Calatrava (Ciudad Real, Spain). In an effort to understand the mechanisms involved in the tolerance of this plant to high metal concentrations, we grew B. auriculata in the presence of 125 µM Cd(NO3)2 for 15 days and analysed different parameters associated with plant growth, nitric oxide and reactive oxygen species metabolism, metal uptake and translocation, photosynthesis rate and biothiol (glutathione and phytochelatins) content. Treatment with Cd led to growth inhibition in both the leaves and the roots, as well as a reduction of photosynthetic parameters, transpiration and stomatal conductance. The metal was mainly accumulated in the roots and in the vascular tissue, although most Cd was detected in areas surrounding their epidermal cells, while in the leaves the metal accumulated mainly in spongy mesophyll, stomata and trichrome. Based on the Cd bioaccumulation (5.93) and translocation (0.15) factors, this species denoted enrichment of the metal in the roots and its low translocation to the upper tissues. Biothiol analysis showed a Cd-dependent increase of reduced glutathione (GSH) as well as the phytochelatins (PC2 and PC3) in both roots and leaves. Cd-promoted oxidative damage occurred mainly in the leaves due to disturbances in enzymatic and nonenzymatic antioxidants, while the roots did not show significant damage as a result of induction of antioxidant defences. It can be concluded that B. auriculata is a new Cd-tolerant plant with an ability to activate efficient metal-sequestering mechanisms in the root surface and leaves and to induce PCs, as well as antioxidative defences in roots.

NADPH oxidases differentially regulate ROS metabolism and nutrient uptake under cadmium toxicity.

The role of NADPH oxidases under cadmium (Cd) toxicity was studied using Arabidopsis thaliana mutants AtrbohC, AtrbohD and AtrbohF, which were grown under hydroponic conditions with 25 and 100 µM Cd for 1 and 5 days. Cadmium reduced the growth of leaves in WT, AtrbohC and D, but not in AtrbohF. A time-dependent increase in H2 O2 and lipid peroxidation was observed in all genotypes, with AtrbohC showing the smallest increase. An opposite behaviour was observed with NO accumulation. Cadmium increased catalase activity in WT plants and decreased it in Atrboh mutants, while glutathione reductase and glycolate oxidase activities increased in Atrboh mutants, and superoxide dismutases were down-regulated in AtrbohC. The GSH/GSSG and ASA/DHA couples were also affected by the treatment, principally in AtrbohC and AtrbohF, respectively. Cadmium translocation to the leaves was severely reduced in Atrboh mutants after 1 day of treatment and even after 5 days in AtrbohF. Similar results were observed for S, P, Ca, Zn and Fe accumulation, while an opposite trend was observed for K accumulation, except in AtrbohF. Thus, under Cd stress, RBOHs differentially regulate ROS metabolism, redox homeostasis and nutrient balance and could be of potential interest in biotechnology for the phytoremediation of polluted soils.

Peroxisomes sense and respond to environmental cues by regulating ROS and RNS signalling networks.

BACKGROUND: Peroxisomes are highly dynamic, metabolically active organelles that used to be regarded as a sink for H2O2 generated in different organelles. However, peroxisomes are now considered to have a more complex function, containing different metabolic pathways, and they are an important source of reactive oxygen species (ROS), nitric oxide (NO) and reactive nitrogen species (RNS). Over-accumulation of ROS and RNS can give rise oxidative and nitrosative stress, but when produced at low concentrations they can act as signalling molecules. SCOPE: This review focuses on the production of ROS and RNS in peroxisomes and their regulation by antioxidants. ROS production is associated with metabolic pathways such as photorespiration and fatty acid ß-oxidation, and disturbances in any of these processes can be perceived by the cell as an alarm that triggers defence responses. Genetic and pharmacological studies have shown that photorespiratory H2O2 can affect nuclear gene expression, regulating the response to pathogen infection and light intensity. Proteomic studies have shown that peroxisomal proteins are targets for oxidative modification, S-nitrosylation and nitration and have highlighted the importance of these modifications in regulating peroxisomal metabolism and signalling networks. The morphology, size, number and speed of movement of peroxisomes can also change in response to oxidative stress, meaning that an ROS/redox receptor is required. Information available on the production and detection of NO/RNS in peroxisomes is more limited. Peroxisomal homeostasis is critical for maintaining the cellular redox balance and is regulated by ROS, peroxisomal proteases and autophagic processes. CONCLUSIONS: Peroxisomes play a key role in many aspects of plant development and acclimation to stress conditions. These organelles can sense ROS/redox changes in the cell and thus trigger rapid and specific responses to environmental cues involving changes in peroxisomal dynamics as well as ROS- and NO-dependent signalling networks, although the mechanisms involved have not yet been established. Peroxisomes can therefore be regarded as a highly important decision-making platform in the cell, where ROS and RNS play a determining role.

2,4-Dichlorophenoxyacetic acid promotes S-nitrosylation and oxidation of actin affecting cytoskeleton and peroxisomal dynamics.

2,4-Dichlorophenoxyacetic acid (2,4-D) is a synthetic auxin used as a herbicide to control weeds in agriculture. A high concentration of 2,4-D promotes leaf epinasty and cell death. In this work, the molecular mechanisms involved in the toxicity of this herbicide are studied by analysing in Arabidopsis plants the accumulation of reactive oxygen species (ROS) and nitric oxide (NO), and their effect on cytoskeleton structure and peroxisome dynamics. 2,4-D (23 mM) promotes leaf epinasty, whereas this process was prevented by EDTA, which can reduce ·OH accumulation. The analysis of ROS accumulation by confocal microscopy showed a 2,4-D-dependent increase in both H2O2 and O2·(-), whereas total NO was not affected by the treatment. The herbicide promotes disturbances on the actin cytoskeleton structure as a result of post-translational modification of actin by oxidation and S-nitrosylation, which could disturb actin polymerization, as suggested by the reduction of the F-actin/G-actin ratio. These effects were reduced by EDTA, and the reduction of ROS production in Arabidopsis mutants deficient in xanthine dehydrogenase (Atxdh) gave rise to a reduction in actin oxidation. Also, 2,4-D alters the dynamics of the peroxisome, slowing the speed and shortening the distances by which these organelles are displaced. It is concluded that 2,4-D promotes oxidative and nitrosative stress, causing disturbances in the actin cytoskeleton, thereby affecting the dynamics of peroxisomes and some other organelles such as the mitochondria, with xanthine dehydrogenase being involved in ROS production under these conditions. These structural changes in turn appear to be responsible for the leaf epinasty.

Regulation of epinasty induced by 2,4-dichlorophenoxyacetic acid in pea and Arabidopsis plants.

The herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) causes uncontrolled cell division and malformed growth in plants, giving rise to leaf epinasty and stem curvature. In this study, mechanisms involved in the regulation of leaf epinasty induced by 2,4-D were studied using different chemicals involved in reactive oxygen species (ROS) accumulation (diphenyleniodonium, butylated hydroxyanisole, EDTA, allopurinol), calcium channels (LaCl3), protein phosphorylation (cantharidin, wortmannin) and ethylene emission/perception (aminoethoxyvinyl glycine, AgNO3). The effect of these compounds on the epinasty induced by 2,4-D was analysed in shoots and leaf strips from pea plants. For further insight into the effect of 2,4-D, studies were also made in Arabidopsis mutants deficient in ROS production (rbohD, rbohF, xdh), ethylene (ein 3-1, ctr 1-1, etr 1-1), abscisic acid (aba 3.1), and jasmonic acid (coi 1.1, jar 1.1, opr 3) pathways. The results suggest that ROS production, mainly ·OH, is essential in the development of epinasty triggered by 2,4-D. Epinasty was also found to be regulated by Ca2+, protein phosphorylation and ethylene, although all these factors act downstream of ROS production. The use of Arabidopsis mutants appears to indicate that abscisic and jasmonic acid are not involved in regulating epinasty, although they could be involved in other symptoms induced by 2,4-D.

Oxidative stress and arsenic toxicity: role of NADPH oxidases.

The effect of arsenic (25 and 50 µM As for 1 and 5d) was analysed in wild type (WT) and Arabidopsis thaliana (L.) Heynh plants deficient in NADPH oxidase C (AtrbohC). The content of H(2)O(2) and malondialdehyde (MDA) increased with the As concentration, while the opposite effect was found for NO in WT and AtrbohC plants. The As treatment reduced catalase and increased glutathione reductase activities to the same extent in WT and AtrbohC plants, although the induction of all SOD isoforms (mainly CuZn-SODs) was observed in WT plants, the opposite effects being found in AtrbohC plants. Glycolate oxidase (H(2)O(2) producers) considerably increased with the concentration and time of treatment with As in WT and AtrbohC mutants. Arsenic induced the uptake and translocation of P, S, Cu, Zn, and Fe in WT plants, while in AtrbohC plants the opposite trend was noted and the uptake of As became considerably lower than in WT plants. These results suggest that As causes oxidative stress by inducing glycolate oxidase, while NADPH oxidase does not appear to participate in ROS overproduction but could be critical in regulating antioxidant defences as well as the transport and translocation of As and macro/micronutrients.

Characterisation and changes in the antioxidant system of chloroplasts and chromoplasts isolated from green and mature pepper fruits.

Purification and characterisation of pepper (Capsicum annuum L) chloroplasts and chromoplasts isolated from commercial green, red and yellow mature fruits were undertaken. Induction of the synthesis of several antioxidants in organelles isolated from mature fruits was found. The ultrastructure of organelles and the presence and activity of SOD isozymes and enzymes involved in the ASC-GSH cycle, together with the non-enzymatic antioxidant content and some oxidative parameters, were analysed. It was found that lipids, rather than proteins, seem to be a target for oxidation in the chromoplasts. The ascorbate and glutathione contents were elicited during differentiation of chloroplasts into chromoplasts in both red and yellow fruits. The activity of SOD and of components of the ASC-GSH cycle was up-regulated, suggesting that these enzymes may play a role in the protection of plastids and could act as modulators of signal molecules such as O(2) ( -) and H(2)O(2) during fruit maturation. The presence of an Mn-SOD in chromoplasts isolated from yellow pepper fruits was also investigated in terms of structural and antioxidant differences between the two cultivars.

Cadmium-induced changes in the growth and oxidative metabolism of pea plants.

The effect of growing pea (Pisum sativum L.) plants with CdCl(2) (0-50 microM) on different plant physiological parameters and antioxidative enzymes of leaves was studied in order to know the possible involvement of this metal in the generation of oxidative stress. In roots and leaves of pea plants Cd produced a significant inhibition of growth as well as a reduction in the transpiration and photosynthesis rate, chlorophyll content of leaves, and an alteration in the nutrient status in both roots and leaves. The ultrastructural analysis of leaves from plants grown with 50 microM CdCl(2), showed cell disturbances characterized by an increase of mesophyll cell size, and a reduction of intercellular spaces, as well as severe disturbances in chloroplast structure. Alterations in the activated oxygen metabolism of pea plants were also detected, as evidenced by an increase in lipid peroxidation and carbonyl-groups content, as well as a decrease in catalase, SOD and, to a lesser extent, guaiacol peroxidase activities. Glutathione reductase activity did not show significant changes as a result of Cd treatment. A strong reduction of chloroplastic and cytosolic Cu,Zn-SODs by Cd was found, and to a lesser extent of Fe-SOD, while Mn-SOD was only affected by the highest Cd concentrations. Catalase isoenzymes responded differentially, the most acidic isoforms being the most sensitive to Cd treatment. Results obtained suggest that growth of pea plants with CdCl(2) can induce a concentration-dependent oxidative stress situation in leaves, characterized by an accumulation of lipid peroxides and oxidized proteins as a result of the inhibition of the antioxidant systems. These results, together with the ultrastructural data, point to a possible induction of leaf senescence by cadmium.

Identification of porin-like polypeptide(s) in the boundary membrane of oilseed glyoxysomes.

A 36-kDa polypeptide of unknown function was identified by us in the boundary membrane fraction of cucumber seedling glyoxysomes. Evidence is presented in this study that this 36-kDa polypeptide is a glyoxysomal membrane porin. A sequence of 24 amino acid residues derived from a CNBr-cleaved fragment of the 36-kDa polypeptide revealed 72% to 95% identities with sequences in mitochondrial or non-green plastid porins of several different plant species. Immunological evidence indicated that the 36-kDa (and possibly a 34-kDa polypeptide) was a porin(s). Antiserum raised against a potato tuber mitochondrial porin recognized on immunoblots 34-kDa and 36-kDa polypeptides in detergent-solubilized membrane fractions of cucumber seedling glyoxysomes and mitochondria, and in similar glyoxysomal fractions of cotton, castor bean, and sunflower seedlings. The 36-kDa polypeptide seems to be a constitutive component because it was detected also in membrane protein fractions derived from cucumber leaf-type peroxisomes. Compelling evidence that one or both of these polypeptides were authentic glyoxysomal membrane porins was obtained from electron microscopic immunogold analyses. Antiporin IgGs recognized antigen(s) in outer membranes of glyoxysomes and mitochondria. Taken together, the data indicate that membranes of cucumber (and other oilseed) glyoxysomes, leaf-type peroxisomes, and mitochondria possess similar molecular mass porin polypeptide(s) (34 and 36 kDa) with overlapping immunological and amino acid sequence similarities.

Localization of nitric-oxide synthase in plant peroxisomes.

The presence of nitric-oxide synthase (NOS) in peroxisomes from leaves of pea plants (Pisum sativum L.) was studied. Plant organelles were purified by differential and sucrose density gradient centrifugation. In purified intact peroxisomes a Ca(2+)-dependent NOS activity of 5.61 nmol of L-[(3)H]citrulline mg(-1) protein min(-1) was measured while no activity was detected in mitochondria. The peroxisomal NOS activity was clearly inhibited (60-90%) by different well characterized inhibitors of mammalian NO synthases. The immunoblot analysis of peroxisomes with a polyclonal antibody against the C terminus region of murine iNOS revealed an immunoreactive protein of 130 kDa. Electron microscopy immunogold-labeling confirmed the subcellular localization of NOS in the matrix of peroxisomes as well as in chloroplasts. The presence of NOS in peroxisomes suggests that these oxidative organelles are a cellular source of nitric oxide (NO) and implies new roles for peroxisomes in the cellular signal transduction mechanisms.

Characterization of membrane polypeptides from pea leaf peroxisomes involved in superoxide radical generation.

The production of superoxide radicals (O2(-).) and the activities of ferricyanide reductase and cytochrome c reductase were investigated in peroxisomal membranes from pea (Pisum sativum L.) leaves using NADH and NADPH as electron donors. The generation of O2(-). by peroxisomal membranes was also assayed in native polyacrylamide gels using an in situ staining method with NitroBlue Tetrazolium (NBT). When peroxisomal membranes were assayed under native conditions using NADH or NADPH as inducer, two different O2(-).-dependent Formazan Blue bands were detected. Analysis by SDS/PAGE of these bands demonstrated that the NADH-induced NBT reduction band contained several polypeptides (PMP32, PMP61, PMP56 and PMP18, where PMP is peroxisomal membrane polypeptide and the number indicates molecular mass in kDa), while the NADPH-induced band was due exclusively to PMP29. PMP32 and PMP29 were purified by preparative SDS/PAGE and electroelution. Reconstituted PMP29 showed cytochrome c reductase activity and O2(-). production, and used NADPH specifically as electron donor. PMP32, however, had ferricyanide reductase and cytochrome c reductase activities, and was also able to generate O2(-). with NADH as electron donor, whereas NADPH was not effective as an inducer. The reductase activities of, and O2(-). production by, PMP32 were inhibited by quinacrine. Polyclonal antibodies against cucumber monodehydroascorbate reductase (MDHAR) recognized PMP32, and this polypeptide is likely to correspond to the MDHAR reported previously in pea leaf peroxisomal membranes.

Cadmium toxicity and oxidative metabolism of pea leaf peroxisomes.

The effect of growing pea plants with 50 microM CdCl2 on the activated oxygen metabolism was studied at subcellular level in peroxisomes isolated from pea leaves. Cadmium treatment produced proliferation of peroxisomes as well as an increase in the content of H2O2 in peroxisomes from pea leaves, but in peroxisomal membranes no significant effect on the NADH-dependent O2*- production was observed. The rate of lipid peroxidation of membranes was slightly decreased in peroxisomes from Cd-treated plants. This could be due to the Cd-induced increase in the activity of some antioxidative enzymes involved in H2O2 removal, mainly ascorbate peroxidase and glutathione reductase, as well as the NADP-dependent dehydrogenases present in these organelles. The activity of xanthine oxidase did not experiment changes by Cd treatment and this suggests that O2*- production in the peroxisomal matrix is not involved in Cd toxicity. This was supported by the absence of changes in plants treated with Cd in the Mn-SOD activity, responsible for O2*- removal in the peroxisomal matrix. Results obtained indicate that toxic Cd levels induce imbalances in the activated oxygen metabolism of pea leaf peroxisomes, but its main effect is an enhancement of the H2O2 concentration of these organelles. Peroxisomes respond to Cd toxicity by increasing the activity of antioxidative enzymes involved in the ascorbate-glutathione cycle and the NADP-dependent dehydrogenases located in these organelles.

Purification of catalase from pea leaf peroxisomes: identification of five different isoforms.

Catalase activity was analyzed in seven organs of pea (Pisum sativum L.) plants: leaves, seeds, flowers, shoots, whole fruits, pods and roots. Leaves showed the highest activity followed by whole fruits and flowers. Catalase was purified from pea leaf peroxisomes. These organelles were isolated from leaves by differential and sucrose density-gradient centrifugation, and catalase was purified by two steps involving anion exchange and hydrophobic chromatography using a Fast Protein Liquid Chromatography system. Pure catalase had a specific activity of 953 mmol H2O2 min(-1) mg(-1) protein and was purified 1000-fold, with a yield of about 19 microg enzyme per kg of pea leaves. Analysis by SDS-PAGE and immunoblot showed that the pea catalase was composed of subunits of 57 kDa. Ultraviolet and visible absorption spectra of the enzyme showed two absorption maxima at 252 and 400 nm with molar extinction coefficients of 2.14 x 10(6) and 7.56 x 10(6) M(-1) cm(-1), respectively. By isoelectric focusing (pH 5-7), five different isoforms were identified and designated as CAT1-5, with isoelectric points of 6.41, 6.36, 6.16, 6.13 and 6.09, respectively. All the catalase isoforms contained a subunit of 57 kDa. Post-embedment, EM immunogold labelling of catalase showed a uniform distribution of the enzyme inside the matrix and core of pea leaf peroxisomes.

A dehydrogenase-mediated recycling system of NADPH in plant peroxisomes.

The presence of the two NADP-dependent dehydrogenases of the pentose phosphate pathway has been investigated in plant peroxisomes from pea (Pisum sativum L.) leaves. Both enzymes, glucose-6-phosphate dehydrogenase (G6PDH; EC 1.1.1.49) and 6-phosphogluconate dehydrogenase (6PGDH; EC 1.1.1.44), were present in the matrix of leaf peroxisomes, and their kinetic properties were studied. G6PDH and 6PGDH showed a typical Michaelis-Menten kinetic saturation curve, and had specific activities of 12.4 and 29.6 mU/mg protein, respectively. The Km values of G6PDH and 6PGDH for glucose 6-phosphate and for 6-phosphogluconate were 107.3 and 10.2 microM, respectively. Dithiothreitol did not inhibit G6PDH activity. By isoelectric focusing of peroxisomal matrices, the G6PDH activity was resolved into three isoforms with isoelectric points of 5.55, 5.30 and 4.85. The isoelectric point of peroxisomal 6PGDH was 5.10. Immunoblot analyses of peroxisomal matrix with an antibody against yeast G6PDH revealed a single cross-reactive band of 56 kDa. Post-embedment, EM immunogold labelling of G6PDH confirmed that this enzyme was localized in the peroxisomal matrices, the thylakoid membrane and matrix of chloroplasts, and the cytosol. The presence of the two oxidative enzymes of the pentose phosphate pathway in plant peroxisomes implies that these organelles have the capacity to reduce NADP+ to NADPH for its re-utilization in the peroxisomal metabolism. NADPH is particularly required for the ascorbate-glutathione cycle, which has been recently demonstrated in plant peroxisomes [Jiménez, Hernández, del Río and Sevilla (1997) Plant Physiol. 114, 275-284] and represents an important antioxidant protection system against H2O2 generated in peroxisomes.

Superoxide radical generation in peroxisomal membranes: evidence for the participation of the 18 kDa integral membrane polypeptide.

Peroxisomes were isolated from pea (Pisum sativum L.) leaves and the peroxisomal membranes were purified by treatment with Na2CO3. The production of superoxide radicals (O2) induced by NADH was investigated in peroxisomal membranes from intact organelles incubated with proteases (pronase E and proteinase K). Under isoosmotic conditions, in the presence of pronase E, the production of O2-. radicals was inhibited by 80%. SDS-PAGE of peroxisomal membranes after protease treatment demonstrated a decrease in the 18-kDa PMP. This suggests that this polypeptide has a small fragment exposed to the cytosolic side of the peroxisomal membrane which is essential for O2-. production. The 18-kDa PMP was purified by preparative SDS-PAGE and in the reconstituted protein the NADH-driven production of O2-. radicals was investigated. The isolated polypeptide showed a high generation rate of superoxide (about 300 nmol O2-. x mg-1 protein x min-1) which was completely inhibited by 50 mM pyridine. The 18-kDa PMP was recognized by a polyclonal antibody against Cyt b5 from human erythrocytes. The presence of b-type cytochrome in peroxisomal membranes was demonstrated by difference spectroscopy. Results obtained show that in the NADH-dependent O2-. radical generating system of peroxisomal membranes, the 18-kDa integral membrane polypeptide, which appears to be Cyt b5, is clearly involved in superoxide radical production.

Immunocytochemical localization of copper,zinc superoxide dismutase in peroxisomes from watermelon (Citrullus vulgaris Schrad.) cotyledons.

In previous works using cell fractionation methods we demonstrated the presence of a Cu,Zn-containing superoxide dismutase in peroxisomes from watermelon cotyledons. In this work, this intracellular localization was evaluated by using western blot and EM immunocytochemical analysis with a polyclonal antibody against peroxisomal Cu,Zn-SOD II from watermelon cotyledons. In crude extracts from 6-day old cotyledons, analysis by western blot showed two cross-reactivity bands which belonged to the isozymes Cu,Zn-SOD I and Cu,Zn-SOD II. In peroxisomes purified by sucrose density-gradient centrifugation only one cross-reactivity band was found in the peroxisomal matrix which corresponded to the isozyme Cu,Zn-SOD II. When SOD activity was assayed in purified peroxisomes two isozymes were detected, Cu,Zn-SOD II in the matrix, and a Mn-SOD in the membrane fraction which was removed by sodium carbonate washing. EM immunocytochemistry of Cu,Zn-SOD on sections of 6-day old cotyledons, showed that gold label was mainly localized over plastids and also in peroxisomes and the cytosol, whereas mitochondria did not label for Cu,Zn-SOD.

Metabolism of oxygen radicals in peroxisomes and cellular implications.

Peroxisomes are subcellular respiratory organelles which contain catalase and H2O2-producing flavin oxidases as basic enzymatic constituents. These organelles have an essentially oxidative type of metabolism and have the potential to carry out different important metabolic pathways. In recent years the presence of different types of superoxide dismutase (SOD) have been demonstrated in peroxisomes from several plant species, and more recently the occurrence of SOD has been extended to peroxisomes from human and transformed yeast cells. A copper,zinc-containing SOD from plant peroxisomes has been purified and partially characterized. The production of hydroxyl and superoxide radicals has been studied in peroxisomes. There are two sites of O2- production in peroxisomes: (1) in the matrix, the generating system being xanthine oxidase; and (2) in peroxisomal membranes, dependent on reduced nicotinamide adenine dinucleotide (NADH), and the electron transport components of the peroxisomal membrane are possibly responsible. The generation of oxygen radicals in peroxisomes could have important effects on cellular metabolism. Diverse cellular implications of oxyradical metabolism in peroxisomes are discussed in relation to phenomena such as cell injury, peroxisomal genetic diseases, peroxisome proliferation and oxidative stress, metal and salt stress, catabolism of nucleic acids, senescence, and plant pathogenic processes.