JXB Advance Access originally published online on March 26, 2004
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Journal of Experimental Botany, Vol. 55, No. 399, pp. 1105-1113, May 1, 2004
© 2004 Oxford University Press
Plants and the Environment |
Salinity up-regulates the antioxidative system in root mitochondria and peroxisomes of the wild salt-tolerant tomato species Lycopersicon pennellii
Received 1 October 2003; Accepted 26 January 2004
1 Albert Katz Department of Dryland Biotechnologies, Jacob Blaustein Institute for Desert Research, Ben-Gurion University of the Negev, Sede Boqer Campus 84990, Israel
2 Department of Life Science, Ben-Gurion University of the Negev, PO Box 653, Beer Sheva 84105, Israel
3 The National Institute of Biothechnology in the Negev, Ben-Gurion University of the Negev, PO Box 653, Beer Sheva 84105, Israel
* To whom correspondence should be addressed. Fax: +972 8 6596742. E-mail: michagu{at}bgumail.bgu.ac.il
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
|---|
The effect of salinity on the antioxidative system of root mitochondria and peroxisomes of a cultivated tomato Lycopersicon esculentum (Lem) and its wild salt-tolerant related species L. pennellii (Lpa) was studied. Salt stress induced oxidative stress in Lem mitochondria, as indicated by the increased levels of lipid peroxidation and H2O2. These changes were associated with decreased activities of superoxide dismutase (SOD) and guaiacol peroxidases (POD) and contents of ascorbate (ASC) and glutathione (GSH). By contrast, in mitochondria of salt-treated Lpa plants both H2O2 and lipid peroxidation levels decreased while the levels of ASC and GSH and activities of SOD, several isoforms of ascorbate peroxidase (APX), and POD increased. Similarly to mitochondria, peroxisomes isolated from roots of salt-treated Lpa plants exhibited also decreased levels of lipid peroxidation and H2O2 and increased SOD, ascorbate peroxidase (APX), and catalase (CAT) activities. In spite of the fact that salt stress decreased activities of antioxidant enzymes in Lem peroxisome, oxidative stress was not evident in these organelles.
Key words: Ascorbate, ascorbate–glutathione cycle enzymes, glutathione, mitochondria, oxidative stress, peroxisomes, roots, salinity, tomato.
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Plant roots are directly exposed to biotic and abiotic stresses which are known to induce oxidative stress. Among the abiotic stresses, salinity and drought are the most sever factors limiting plant productivity (Boyer, 1982; Pitman and Läuchli, 2002). Plants are equipped with an array of non-enzymic scavengers and antioxidant enzymes that act in concert to alleviate cellular damage under oxidative stress conditions (for a review see Foyer and Noctor, 2000). Superoxide dismutase (SOD) reacts with the superoxide radical at almost diffusion-limited rates to produce H2O2 (Scandalios, 1997). Root mitochondria, like those of leaves (Møller, 2001; del Rio et al., 2003), contain MnSOD (Malecka et al., 2001). H2O2, produced by SOD and some oxidases, is scavenged by peroxidases, especially ascorbate peroxidase (APX), and catalase (CAT). CAT has been found predominantly in leaf peroxisomes where it functions chiefly to remove H2O2 formed in photorespiration or in ß-oxidation of fatty acids in the glyoxysomes (Dat et al., 2000). However, CAT activity was also found in the roots (Corpas et al., 1999; Shalata et al., 2001). APX which uses ASC as a reductant in the first step of the ascorbate–glutathione cycle is the most important plant peroxidase in H2O2 detoxification (Foyer and Halliwell, 1976; Noctor and Foyer, 1998). In addition to APX, guaiacol-dependent peroxidase (POD) is also involved in the scavenging of soluble hydroperoxides in plants (Otter and Polle, 1994), and its presence has been reported in maize root mitochondria by Sukalovic and Vuletic (2003). ROS is scavenged also non-enzymatically by hydrophilic antioxidants, such as ascorbate (ASC) and glutathione (GSH), and these antioxidants were detected in bean and tomato roots (Cuypers et al., 2000; Shalata et al., 2001, respectively).
Various abiotic stresses such as heavy metals (Rucinska et al., 1999), hypoxia (Biemelt et al., 2000), chilling (Queiroz et al., 1998), silicon treatment (Liang et al., 2003), and salinity (Lee et al., 2001; Khan et al., 2002) were shown to alter the root antioxidant system activity. Several studies have demonstrated that salt-tolerant species increase their antioxidant enzyme activities and antioxidant contents in response to salt stress, while salt-sensitive species failed to do so (Lopez et al., 1996; Meneguzzo et al., 1999; Shalata et al., 2001; Jbir et al., 2001). Using DNA microarray methodology Kawasaki et al. (2001) have identified the salt-induced up-regulation of several antioxidant genes in rice roots. However, in all the above studies organellar antioxidative system(s) were overlooked.
In addition to chloroplasts (Foyer and Noctor 2000), mitochondria (Møller, 2001) and peroxisomes (Corpas et al., 2001; del Rio et al., 2002) are also considered to be major sites of ROS generation. However, the role of the mitochondrial and peroxisomal antioxidant system in the protection of the root against oxidative stress has hardly been investigated. Evidence is presented here that the wild salt-tolerant species Lpa is capable of up-regulating its root mitochondrial and peroxisomal antioxidant system in response to salt stress, while Lem fails to do so.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Plant material
Plants of the cultivated tomato Lycopersicon esculentum Mill. cultivar M82 (Lem) and its wild salt-tolerant relative L. pennellii accession Atico (Lpa) were grown in a greenhouse with day/night temperature of 30/20 °C and light intensity, at noon, ranging from 700 to 1000 µmol m–2 s–1. The plants were grown hydroponically, six plants per container of 4.0 l of aerated Hoagland’s solution. Salt treatment started at the stage of about four true leaves by increasing the NaCl concentration by 25 mM d–1 to a final concentration of 100 mM.
Organelle isolation
Mitochondria and peroxisomes were isolated from roots of control and salt-stressed plants (14 d after the completion of salt treatment) and purified by differential and sucrose density-gradient centrifugation as described by Mittova et al. (2000). Intactness of both organelles was determined as described earlier (Mittova et al., 2000). As in an earlier study (Mittova et al., 2000), the contamination of mitochondria and peroxisomes by plastids was about 2%, of mitochondria by peroxisomes 10%, and of peroxisomes by mitochondria 4%.
Organelle fractionation
Purified organelles were loaded on Sephadex G-25 columns (1 ml), pre-equilibrated with 20 mM HEPES-KOH buffer (pH 7.2). The void volume of each column was collected by centrifugation (700 g for 5 min; Kubota 2010). This procedure osmotically shocked the organelles as judged by loss of intactness. The recovery of organellar enzymes was estimated by determination of marker enzyme activities before and after the gel filtration step. The Sephadex G-25 eluted organellar proteins were resuspended (1:2 v/v) in a medium containing 50 mM HEPES-KOH, (pH 7.2), 1 mM EDTA, 2 mM ASC, and 10% (v/v) glycerol. The preparations were kept on ice for 40 min, vortexed every 10 min, and then centrifuged at 150 000 g for 30 min in a Kontron TBF 80.2 rotor. The distribution of the enzyme activities between the matrix and membrane fractions was estimated after releasing the soluble fraction by osmotic shock. The membrane fraction (150 000 g pellet) was then resuspended in the above buffer with/without 0.2 M KI in order to release peripheral and loosely bound proteins.
Lipid peroxidation
Lipid peroxidation was determined as the amount of malondialdehyde (MDA, 
=155 mM–1 cm–1), a product of lipid peroxidation, according to Draper and Hadley (1990). MDA content was determined in the pellets (150 000 g for 30 min) after osmotic shock of mitochondria and peroxisomes, in order to minimize reaction of thiobarbituric acid with oxidized products of amino acids, including proline (Gutteridge and Halliwell, 1990).
H2O2 content
H2O2 content was determined according to Wolff (1994). The assay is based on ferrous ion oxidation in the presence of the ferric ion indicator xylenol orange.
Enzyme assays
Total superoxide dismutase (SOD; EC 1.15.1.1
[EC]
.), ascorbate peroxidase (APX; EC 1.11.1.11
[EC]
), monodehydroascorbate reductase (MDHAR; EC 1.6.5.4
[EC]
), dehydroascorbate reductase (DHAR; EC 1.8.5.1
[EC]
), glutathione reductase (GR; EC 1.6.4.2
[EC]
), and catalase (CAT; EC 1.11.1.6
[EC]
) were assayed as described previously (Mittova et al., 2000). The activities of CuZn- and Fe-SODs were sequentially subtracted from the total SOD activity by using 3 mM KCN or 5 mM H2O2, respectively (Yu and Rengel, 1999). Glutathione peroxidase (GPX; EC 1.11.1.9
[EC]
) was determined following Drotar et al. (1985) using H2O2 as a substrate.
Native gel electrophoresis and isozyme staining
Mitochondria and peroxisomes were disrupted in medium containing 50 mM HEPES (pH 7.5), 5 mM ascorbate, 2 mM EDTA, 1 mM MgCl2, 1 mM MnCl2, and 1% (w/v) CHAPSO. The disrupted organelles were kept on ice for 1 h and vortexed every 10 min. Proteins were separated on 10% non-denaturating PAGE (Laemmli, 1970). APX activity was detected according to Mittler and Zilinkas (1993) and non-specific peroxidase (POD) according to Fieldes and Gerhardt (1998).
Ascorbate and glutathione determination
Reduced and total ascorbate were determined according to Law et al. (1983). Reduced and total glutathione were determined according to Anderson et al. (1992).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
H2O2 and lipid peroxidation levels
The inherent H2O2 content was the same in root mitochondria of both species, however, under salt treatment it increased (by 90%) in Lem mitochondria and decreased (by 45%) in those of Lpa. Concomitantly, under these conditions, H2O2 content decreased (by 20%) in Lpa peroxisomes and remained at the control level in those of Lem (Fig. 1). The inherent lipid peroxidation level of root mitochondria was higher in Lpa (Fig. 1). In response to salinity it decreased somewhat in the Lpa mitochondria whereas it increased in those of Lem (by 90%). By contrast, the inherent level of peroxisomal lipid peroxidation was the same in both species, while under salinity it was not affected in Lem and modestly decreased in Lpa peroxisomes.
|
Activities of antioxidant enzymes
SOD and APX activities increased (by 40% and 90%, respectively) in root mitochondria of salt-treated Lpa plants while MDHAR, GPX, and GR activities remained unchanged and that of DHAR even decreased (Fig. 2). By contrast, in the case of Lem, APX, DHAR, GR, and GPX activities remained at the control level under salt-stress conditions and those of SOD and MDHAR decreased (by 20% and 40%, respectively).
|
In response to salinity, SOD and APX activities slightly decreased in Lem root peroxisomes, while a more profound decrease (by 40%) was observed in MDHAR and DHAR activities (Fig. 3). Catalase activity, however, was not affected by salinity. On the other hand, SOD, APX, MDHAR, and CAT activities increased (by 140%, 140%, 80%, and 170%, respectively) in root peroxisomes of salt-treated Lpa plants. Salt treatment did not affect GR activity in root peroxisomes of either species (Fig. 3). It should be noted that, in contrast to mitochondria, GPX activity could not be detected in peroxisomes of both species.
|
Activities of SOD-type, APX and POD isozymes
The activities of the various SOD types were distinguished using KCN and H2O2 as inhibitors (Table 1). In both species, the mitochondrial SOD activity was comprised only of MnSOD. Peroxisomal SOD activity was comprised mostly of matrix CuZnSOD activity in Lem roots, but of matrix MnSOD in Lpa (Table 1). The distribution of SOD-type activities between the membrane and soluble fractions of mitochondria and peroxisomes from control and salt-treated Lem and Lpa plants are given in Table 1. In mitochondria of both species MnSOD was found to be mainly soluble and its activity decreased (by 20%) in Lem and increased (by 40%) in Lpa in response to salinity. Matrix CuZnSOD, the only detectable SOD isozyme in Lem peroxisomes, was not affected by salinity while the matrix MnSOD activity, the most abundant Lpa peroxisomal SOD type, increased (by 80%) in response to salinity. Interestingly, FeSOD activity was restricted to Lem leaf peroxisomes and was not detected in those of Lpa (Mittova et al., 2003).
|
Two APX isozymes were identified on an activity gel in root mitochondria and peroxisomes of both species (Fig. 4A). The prominent band, with the highest electrophoretic mobility, was assigned as matrix APX and the lower mobility band was assigned as membrane-bound (data not shown). Decreased activities of both the matrix and membrane-bound isozymes were observed in mitochondria and peroxisomes of salt-stressed Lem plants. By contrast, the activities of both mitochondrial APX isozymes increased in salt-stressed Lpa plants. In the peroxisomes of this species, only the matrix APX isozyme activity increased (Fig. 4A).
|
Native gel activity revealed five POD bands in protein preparations of mitochondria isolated from control Lem. In response to salinity, four of these bands were below the detection limit. Unlike in Lem, only two POD activity bands were detected in mitochondria isolated from control Lpa roots. However, the activity of these two PODs increased (Fig. 4B) and an additional two POD activity bands were detected in response to salinity. While POD activity was not detected in Lem peroxisomes, a single band could be observed in Lpa. Since the electrophoretic mobility of this band was similar to that of one of the mitochondrial PODs, the possibility that this band is a mitochondrial contaminant could not be excluded (Fig. 4B).
Intraorganellar distribution of the ascorbate–glutathione cycle enzymes
The effect of salinity on the distribution of intraorganellar activity, matrix and membrane-bound was studied in both species and is expressed as the specific activity (i.e. the abundance of a given enzyme activity relative to the total protein content of that fraction). Most of the mitochondrial APX activity in both species was recovered as soluble.
In response to salinity, the specific activities of both matrix and membrane-bound mitochondrial APX increased in Lpa (by 50% and 30%, respectively) while in those of Lem no significant salt effect was observed (Table 2). The findings that in Lpa mitochondria both matrix and membrane-bound APX activities increased are in agreement with the data obtained by native PAGE analysis (Fig. 4). However, such an agreement was not found in the case of Lem. This can be explained by the fact that enzyme activity, measured by gel staining methods, may not always bear a linear quantitative relationship to the amount of protein loaded.
|
In response to salinity, the mitochondrial matrix MDHAR activity of Lem decreased (by 40%) while that of Lpa was not significantly affected. Salinity also affected the ratio between the matrix to membrane-bound MDHAR activity, decreasing in Lem and increasing in Lpa. The matrix DHAR activity increased (by 40%) in mitochondria of salt-stressed Lem plants and decreased (by 60%) in those of Lpa (Table 2). It should be noted that the activities of membrane-bound MDHAR and DHAR isozymes were not affected by salinity in mitochondria of either species.
Both the matrix and membrane-bound APX isozyme activities decreased (by about 25%) in Lem peroxisomes in response to salinity and increased in those of Lpa (by 120% and 20%, respectively) (Table 3). Under these conditions, the matrix MDHAR activity decreased (by 20%) in Lem peroxisomes and increased (by 45%) in those of Lpa. The activity of peroxisomal membrane-bound MDHAR was not affected by salinity in either species. The matrix DHAR activity decreased (by 30%) in peroxisomes of salt-treated Lem plants and remained at the control level in those of Lpa. The activity of peroxisomal membrane-bound DHAR was not affected by salinity in either species. In an earlier study (Mittova et al., 2000), evidence was brought that GR activity was predominantly present in the soluble fraction of root mitochondria and peroxisomes of both species. In the present study it was found that also under salinity, the bulk of GR activity was localized to the soluble fractions of the root mitochondria and peroxisomes of both species (data not shown).
|
Ascorbate and glutathione contents
In Lem mitochondria the inherent ASC content was higher (by 30%) than that of Lpa, while inherent GSH content was similar (Table 4). ASC content decreased and that of DHA increased (by 20% and 120%, respectively) in mitochondria of salt-treated Lem plants. By contrast, in those of Lpa, ASC content increased (by 110%) and DHA content decreased (by 40%). As a result, ASC/DHA ratio decreased (by 60%) in Lem and increased (by 200%) in Lpa mitochondria. The salt-dependent decrease in mitochondrial GSH content in Lem resulted in a decrease (by 60%) of the GSH/GSSG ratio, in contrast, GSH content and GSH/GSSG ratio increased (by 50% and 20%, respectively) in mitochondria of salt-stressed Lpa plants
|
The inherent ASC and GSH contents were comparable in Lem and Lpa peroxisomes (Table 4). Peroxisomal ASC/DHA ratio decreased (by 80%) in salt-stressed Lem plants. This was the result of the decrease in ASC content (by 50%) and increase in that of DHA (by 100%). In peroxisomes of salt-treated Lpa plants ASC/DHA ratio increased (by 130%) as a result of decreased (by 50%) DHA content. Salinity did not affect GSH/GSSG ratio in Lem peroxisomes and decreased it (by 20%) in those of Lpa.
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Mitochondria
This study has revealed that salt-induced oxidative stress indeed occurs in Lem root mitochondria as indicated by their increased levels of H2O2 and lipid peroxidation (Fig. 1). It has further demonstrated that such oxidative stress was effectively alleviated in Lpa root mitochondria (Fig. 1). The increased H2O2 content in the case of Lem, probably resulted from a salt-induced increase in the rate of O2– production, considered to be the main precursor of mitochondrial H2O2 (Møller, 2001). However, information about the effect of salinity on ROS production in root mitochondria is lacking. Increased leaf mitochondrial H2O2 content in response to salinity was reported for pea (Hernández et al., 1993; Gómez et al., 1999) and tomato (Mittova et al., 2003). Treatment of pea plants with hexavalent chromium increased the generation of O2– radicals by root mitochondria at the ubiquinone–cytochrome b site of complex III (Dixit et al., 2002). In Lem root mitochondria, similar to the leaf mitochondria (Mittova et al., 2003), the increased H2O2 content was accompanied by decreased activity of SOD (H2O2 producer) while the activities of enzymes involved in H2O2 detoxification (APX and GPX) remained at the control level or even decreased (POD) (Figs 2, 4). Since both ASC and GSH contents decreased in mitochondria of salt-stressed Lem plants (Table 4) it is suggested that, as in the case of leaf mitochondria of this species (Mittova et al., 2003), the increased H2O2 content can be attributed to non-enzymatic reduction of superoxide by ASC and GSH. In agreement, decreased MDHAR activity and increased DHA content were observed under these conditions (Table 4). The observed alleviation of oxidative stress in Lpa root mitochondria can be attributed to salt-induced increased activities of the mitochondrial SOD and APX. Similarly, salt-induced increased activities of these enzymes were found in leaf Lpa mitochondria (Mittova et al., 2003). However, the finding that, in these root Lpa organelles, the salt-dependent increase in APX activity, both the matrix and membrane-bound isozymes, (Table 2) was higher than that of SOD (Table 1) indicates that under salinity the rate of H2O2 detoxification is higher than that of its production..
Mitochondrial H2O2 can probably diffuse through the mitochondrial membrane into the cytosol (Boveris, 1984). This is supported by the recent findings that, in E. coli, membrane permeability to H2O2 is substantial but not unlimited (Seaver and Imlay, 2001) and that H2O2 probably uses water channels to permeate the cell membrane (Henzler and Steudle, 2000) and hence may be subjected to some control. Gómez et al. (1999) proposed that such mitochondrial H2O2 leakage is favoured under salt-stress conditions. The salt-induced increase in membrane-bound APX activity (Fig. 4; Table 2) may, therefore, be important for lowering the H2O2 and lipid peroxidation levels observed in Lpa root mitochondria. In addition, the finding that salt stress increased the activity of two POD isozymes in these organelles (Fig. 4) indicates that PODs are involved in the control of mitochondrial H2O2 content, as was recently suggested by Sukalovic et al. (2003) for mitochondria of maize roots. In contrast to leaf mitochondria where salt-induced GPX activity was observed (Mittova et al., 2003), GPX activity was unaffected by salinity in root mitochondria of Lpa (Fig. 2), suggesting that, in these organelles, APX and POD are the main H2O2 scavengers.
Both ASC and GSH contents increased in mitochondria of salt-treated Lpa plants (Table 4) indicating that non-enzymatic scavenging of H2O2 also contribute to the salt-induced decrease in H2O2 content. Interestingly, the increased levels of these antioxidants were not accompanied by increased activities of MDHAR, DHAR, and GR, enzymes that are involved in their regeneration (Fig. 2; Table 4). The increased content of these antioxidants can therefore be explained by either increased biosynthesis and/or import into the mitochondria. The last step of ASC biosynthesis occurs in mitochondria and is catalysed by 
-galactonolactone dehydrogenase (GLDH) (Bartoli et al., 2000). Therefore, the possibility that, in Lpa root mitochondria, GLDH activity is up-regulated by salinity awaits further clarification.
Peroxisomes
This study has thrown some light on the, as yet, little explored field of root peroxisomal ROS metabolism. In both tomato species, SOD and the enzymes of the ascorbate–glutathione cycle were localized to the matrix of the root peroxisomes. This emerges from the finding that activities of these enzymes were released into the medium by hypotonic shock and that KI treatment failed to release enzyme activity further (Table 3). These results are in contrast to the situation with other leaf and root organelles where membrane-bound isoezyme activities have been reported (Jiménez et al., 1997; Mittova et al. 2002a, b, 2003). Based on the results of the present study, it is therefore likely that metabolic processes responsible for ROS production in the peroxisomes are localized in their matrix. However, as far as the authors are aware, there is no information about the mechanism of ROS production in root peroxisomes.
The activity of SOD decreased and the activities of H2O2 detoxifying enzymes (APX and CAT) decreased and remained unchanged (Table 3 and Fig. 3, respectively) in root peroxisomes of salt-stressed Lem plants (Fig. 1). In agreement, in these organelles H2O2 content did not change. Similar responses to salinity were exhibited by Lem leaf peroxisomes (Mittova et al., 2003), although in these organelles lipid peroxidation increased, whereas in the root peroxisomes it remained unchanged.
By contrast to Lem, decreased H2O2 and lipid peroxidation levels were found in peroxisomes of salt-treated Lpa plants (Fig. 1). These responses to salinity were, at least partially, the result of differential increased activities of APX and CAT over that of SOD (Fig. 3). Such a differential increase in the activities of APX and CAT was recently reported by Mittova et al. (2003) in leaf peroxisomes of salt-treated Lpa plants.
It is possible that the decreased MDHAR activity in peroxisomes of salt-stressed Lem plants (Fig. 3) is responsible for the lower capacity of these organelles to regenerate ASC under these conditions, as reflected by their increased DHA content (Table 4). By contrast, MDHAR activity increased in peroxisomes of salt-treated Lpa plants (Fig. 3) concomitant with the decreased DHA content. Similarly, increased MDHAR activity was found in leaf peroxisomes of salt-treated Lpa plants while in those of Lem it slightly decreased (Mittova et al., 2003). Interestingly, the activities of matrix DHAR and membrane-bound MDHAR in these peroxisomes were not affected by salinity, while that of the matrix MDHAR increased (Table 3). Taken together, these results suggest that regeneration of ASC in peroxisomes of salt-treated Lpa plants occurs in the matrix via MDHAR activity.
Different SOD types were detected in the root peroxisomes of the two species: in Lem, SOD activity was comprised of matrix CuZnSOD (mainly the matrix isozyme) while that of Lpa was comprised of both MnSOD (mainly the matrix isozyme) and CuZnSOD (exclusively the membrane-bound isozyme) (Table 1). It is suggested that the matrix SOD functions as the main detoxifier of O2– produced by xanthine oxidase activity (Sandalio and del Rio, 1988), while the membrane-bound SOD scavenges O2– generated by the membranal NAD(P)H-dependent O2– production site described by Lopez-Huertas et al. (1999). The finding that matrix MnSOD activity increased in root peroxisomes of salt-treated Lpa plants, suggests that under salinity the matrix production of O2– exceeds that of the membrane. By contrast with CuZnSOD, MnSOD is insensitive to inhibition by H2O2 (Kanematsu and Asada, 1994; Scandalios, 1997). It is possible that the inherent MnSOD activity observed only in Lpa peroxisomes enables these organelles to cope better with a high H2O2 content (Fig. 1). Under salinity, however, excessisive H2O2 produced by the matrix MnSOD is efficiently scavenged by the matrix APX and CAT, as these activities were differentially increased in these organelles (Fig. 3; Table 3).
Recently, there has been some evidence for salt-induced increased activities of key Lpa’s antioxidative enzymes including SOD, APX, and MDHAR in leaf and root chloroplasts/plastids (Mittova et al., 2002a, b) and leaf and root mitochondria and peroxisomes (Mittova et al., 2003, and this work, respectively). This was correlated with decreased levels of the oxidative stress indicators such as H2O2 and lipid peroxidation in these organelles. By contrast, salt-induced increased activity of theses enzymes was not observed in Lem, and as a result oxidative stress was imposed as judged by the increased contents of H2O2 and lipid peroxidation in its organelles. This differential response of the organellar antioxidative systems of the two tomato species to salt stress may suggest that the corresponding orthologue antioxidative genes of Lem are not up-regulated by salinity. To address this hypothesis, the cloning of several Lpa and Lem orthologues of the apx gene family has currently been initiated.
|
Acknowledgements |
|---|
This work was supported by the Dr Herman Kessel Research Fund, in memory of Mr CJJ Van Kensbury. VM is a J Blaustein fellowship incumbent. We thank Professor L Reinhold for critical reading of the manuscript and many useful comments.
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Anderson JV, Chevone BI, Hess JL. 1992. Seasonal variation in the antioxidant system of eastern white pine needles. Plant Physiology 98, 501–508.
Bartoli CG, Pastori GM, Foyer CH. 2000. Ascorbate biosynthesis in mitochondria is linked to the electron transport chain between complexes III and IV. Plant Physiology 123, 335–343.
Biemelt S, Keetman U, Mock HP, Grimm B. 2000. Expression and activity of isoenzymes of superoxide dismutase in wheat roots in response to hypoxia and anoxia. Plant, Cell and Environment 23, 135–144.
Boveris A. 1984. Determination of the production of superoxide radicals and hydrogen peroxide in mitochondria. Methods in Enzymology 105, 429–435.[Web of Science][Medline]
Boyer JS. 1982. Plant productiviy and the environment. Science 218, 443–448.
Corpas FJ, Barroso JB, del Rio LA. 2001. Peroxisomes as a source of reactive oxygen species and nitric oxide signal molecules in plant cells. Trends in Plant Science 6, 145–150.[CrossRef][Web of Science][Medline]
Corpas FJ, Palma JM, Sandalio LM, Lopez-Huertas E, Romero-Puertas MC, Barroso JB, Del Rio LA. 1999. Purification of catalase from pea leaf peroxisomes: identification of five different isoforms. Free Radical Research 31, Supplement, S235–S241.
Cuypers A, Vangronsveld J, Clijsters H. 2000. Biphasic effect of copper on the ascorbate–glutathione pathway in primary leaves of Phaseolus vulgaris seedlings during the early stages of metal assimilation. Physiologia Plantarum 110, 512–517.[CrossRef]
Dat J, Vandenabeele S, Vranova E, Van Montagu M, Inzé D, Van Breusegem F. 2000. Dual action of the active oxygen species during plant stress responses. Cellular and Molecular Life Sciences 57, 779–795.[CrossRef][Web of Science][Medline]
del Rio LA, Corpas FJ, Sandalio LM, Palma JM, Gomez M, Barroso JB. 2002. Reactive oxygen species, antioxidant systems and nitric oxide in peroxisomes. Journal of Experimental Botany 53, 1255–1272.
del Rio LA, Sandalio LM, Altomare DA, Zilinskas BA. 2003. Mitochondrial and peroxisomal manganese superoxide dismutase: differential expression during leaf senescence. Journal of Experimental Botany 54, 923–933.
Dixit V, Pandey V, Shyam R. 2002. Chromium ions inactivate electron transport and enhance superoxide generation in vivo in pea (Pisum sativum L. cv. Azad) root mitochondria. Plant, Cell and Environment 25, 687–693.[CrossRef]
Draper HH, Hadley M. 1990. Malondialdehyde determination as index of lipid peroxidation. Methods in Enzymology 186, 421–431.[Medline]
Drotar A, Phelps P, Fall R. 1985. Evidence for glutathione peroxidase activities in cultured plant cells. Plant Physiology 42, 35–40.
Fieldes MA, Gerhardt KE. 1998. Flax guaiacol peroxidases can be used to illustrate the possibility of misinterpreting the effects of stress on the activity of developmentally regulated enzymes. Plant Science 132, 89–99.[CrossRef]
Foyer CH, Halliwell B. 1976. Presence of glutathione and glutathione reductase in chloroplasts: a proposed role in ascorbic acid metabolism. Planta 133, 21–25.[CrossRef][Web of Science]
Foyer CH, Noctor G. 2000. Oxygen processing in photosynthesis: regulation and signalling. New Phytologist 146, 359–388.[CrossRef][Web of Science]
Gómez JM, Hernández JA, Jiménez A, del Rio LA, Sevilla F. 1999. Differential response of antioxidative system of chloroplasts and mitochondria to long-term NaCl stress of pea plants. Free Radicals Research 31, 11–18.
Gutteridge JMC, Halliwell B. 1990. The measurement and mechanism of lipid peroxidation in biological systems. Trends in Biochemical Science 15, 129–135.
Henzler T, Steudle E. 2000. Transport and metabolic degradation of hydrogen peroxide in Chara corallina: model calculations and measurements with the pressure probe suggest transport of H2O2 across water channels. Journal of Experimental Botany 51, 2053–2066.
Hernández JA, Corpas FJ, Gómez M, del Rio LA, Sevilla F. 1993. Salt-induced oxidative stress mediated by activated oxygen species in pea leaf mitochondria. Physiologia Plantarum 89, 103–110.[CrossRef]
Jbir N, Chaibi W, Ammar S, Jemmali A, Ayadi A. 2001. Root growth and lignification of two wheat species differing in their sensitivity to NaCl, in response to salt stress. Comptes rendus hebdomadaire des séances de l’Academie des sciences 324, 863–868.
Jiménez A, Hernández JA, del Río LA, Sevilla F. 1997. Evidence for the presence of the ascorbate–glutathione cycle in mitochondria and peroxisomes of pea leaves. Plant Physiology 114, 275–284.[Abstract]
Kanematsu S, Asada K. 1994. Superoxide dismutase. In: Fukui T, Soda K, ed. Molecular aspects of enzyme catalysis. Tokyo: Kondansha Ltd., 191–210.
Kawasaki S, Borchert C, Deyholos M, Wang H, Brazille S, Kawai K, Galbraith D, Bohnert HJ. 2001. Gene expression profiles during the initial phase of salt stress in rice. The Plant Cell 13, 889–905.
Khan MH, Singha KLB, Panda SK. 2002. Changes in antioxidant levels in Oryza sativa L. roots subjected to NaCl-salinity stress. Acta Physiologiae Plantarum 24, 145–148.
Laemmli AK. 1970. Cleavage of structural protein during the assembly of the head bacteriophage T4. Nature 227, 680–685.[CrossRef][Medline]
Law MY, Charles SA, Halliwell B. 1983. Glutathione and ascorbic acid in spinach (Spinacia oleracea) chloroplasts. Biochemical Journal 210, 899–903.[Web of Science][Medline]
Lee DH, Kim YS, Lee CB. 2001. The inductive responses of the antioxidant enzymes by salt stress in the rice (Oryza sativa L.). Journal of Plant Physiology 158, 737–745.[CrossRef]
Liang YC, Chen Q, Liu Q, Zhang WH, Ding RX. 2003. Exogenous silicon (Si) increases antioxidant enzyme activity and reduces lipid peroxidation in roots of salt-stressed barley (Hordeum vulgare L.). Journal of Plant Physiology 160, 1157–1164.[CrossRef][Web of Science][Medline]
Lopez F, Vansuyt G, Casse-Delbart F, Fourcroy P. 1996. Ascorbate peroxidase activity, not the mRNA level, is enhanced in salt-stressed Raphanus sativus plants. Physiologia Plantarum 97, 13–20.[CrossRef]
Lopez-Huertas E, Corpas FJ, Sandalio LM, Del Rio LA. 1999. Characterization of membrane polypeptides from pea leaf peroxisomes involved in superoxide radical generation. Biochemical Journal 337, 531–536.
Malecka A, Jarmuszkiewicz W, Tomaszewska B. 2001. Antioxidative defense to lead stress in subcellular compartments of pea root cells. Acta Biochimia Polonica 48, 687–698.
Meneguzzo S, Navari-Izzo F, Izzo R. 1999. Antioxidative responses of shoots and roots of wheat to increasing NaCl concentrations. Journal of Plant Physiology 155, 274–280.
Mittler R, Zilinskas BA. 1993. Detection of ascorbate peroxidase activity in native gels by inhibition of the ascorbate–dependent reduction of nitroblue tetrazolium. Analytical Biochemistry 212, 540–546.[CrossRef][Web of Science][Medline]
Mittova V, Guy M, Tal M, Volokita M. 2002a. Response of the cultivated tomato and its wild salt-tolerant relative Lycopersicon pennellii to salt-dependent oxidative stress: increased activities of antioxidant enzymes in root plastids. Free Radicals Research 36, 195–202.
Mittova V, Tal M, Volokita M, Guy M. 2002b. Salt stress induces up-regulation of an efficient chloroplast antioxidant system in the salt-tolerant wild tomato species Lycopersicon pennellii but not in the cultivated species. Physiologia Plantarum 115, 393–400.[CrossRef][Medline]
Mittova V, Tal M, Volokita M, Guy M. 2003. Up-regulation of the leaf mitochondrial and peroxisomal antioxidative systems in response to salt-induced oxidative stress in the wild salt-tolerant tomato species Lycopersicon pennellii. Plant, Cell and Environment 26, 845–856.[CrossRef][Medline]
Mittova V, Volokita M, Guy M, Tal M. 2000. Activities of SOD and the ascorbate–glutathione cycle enzymes in subcellular compartments in leaves and roots of the cultivated tomato and its wild salt-tolerant relative Lycopersicon pennellii. Physiologia Plantarum 110, 42–51.[CrossRef]
Møller IM. 2001. Plant mitochondria and oxidative stress: electron transport, NADPH turnover, and metabolism of reactive oxygen species. Annual Review of Plant Physiology and Plant Molecular Biology 52, 561–591.[CrossRef][Web of Science][Medline]
Noctor G, Foyer CH. 1998. Ascorbate and glutathione: keeping active oxygen under control. Annual Review of Plant Physiology and Plant Molecular Biology 49, 249–279.[CrossRef][Web of Science]
Otter T, Polle A. 1994. The influence of apoplastic ascorbate on the activities of cell wall-associated peroxidase and NADH oxidase in needles of Norway spruce (Picea abies L). Plant Cell Physiology 35, 1231–1238.
Pitman MG, Läuchli A. 2002. Global impact of salinity and agricultural ecosystyems. In: Läuchli A, Lüttge U, eds. Salinity. environment—plants, molecules, Kluwer Academic Publishers, 3–20.
Queiroz CGS, Alonso A, Mares-Guia M, Magalhaes AC. 1998. Chilling-induced changes in membrane fluidity and antioxidant enzyme activities in Coffea arabica L. roots. Biologia Plantarum 41, 403–413.[CrossRef]
Rucinska R, Waplak S, Gwozdz E. 1999. Free radical formation and activity of antioxidant enzymes in lupin roots exposed to lead. Plant Physiology and Biochemistry 37, 187–194.
Sandalio LM, del Rio LA. 1988. Intraorganellar distribution of superoxide dismutase in plant peroxisomes (glyoxisomes and lef peroxisomes). Plant Physiology 88, 1215–1218.
Scandalios JG. 1997. Molecular genetics of superoxide dismutase in plants. In: Scandalios JG, ed. Oxidative stress and the molecular biology of antioxidant defenses. New York: Cold Spring Harbor Laboratory Press, 527–568.
Seaver LC, Imlay JA. 2001. Hydrogen peroxide fluxes and compartmentalization inside growing Escherichia coli. Journal of Bacteriology 183, 7182–7189.
Shalata A, Mittova V, Volokita M, Guy M, Tal M. 2001. Response of the cultivated tomato and its wild salt-tolerant relative Lycopersicon pennellii to salt-dependent oxidative stress: the root antioxidative system. Physiologia Plantarum 112, 487–494.[CrossRef][Medline]
Sukalovic VHT, Vuletic M. 2003. The characterization of peroxidases in mitochondria of maize roots. Plant Science 164, 999–1007.[CrossRef]
Wolff S. 1994. Ferrous ion oxidation in presence of ferric ion indicator xylenol orange for measurement of hydroperoxides. Methods in Enzymology 233, 182–189.
Yu Q, Rengel Z. 1999. Drought and salinity differentially influence activities of superoxide dismutases in narrow-leafed lupines. Plant Science 142, 1–11.[CrossRef]
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  M. Alvarez, D. Huygens, C. Fernandez, Y. Gacitua, E. Olivares, I. Saavedra, M. Alberdi, and E. Valenzuela Effect of ectomycorrhizal colonization and drought on reactive oxygen species metabolism of Nothofagus dombeyi roots Tree Physiol, August 1, 2009; 29(8): 1047 - 1057. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Stepien and G. N. Johnson Contrasting Responses of Photosynthesis to Salt Stress in the Glycophyte Arabidopsis and the Halophyte Thellungiella: Role of the Plastid Terminal Oxidase as an Alternative Electron Sink Plant Physiology, February 1, 2009; 149(2): 1154 - 1165. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Yoshimura, A. Masuda, M. Kuwano, A. Yokota, and K. Akashi Programmed Proteome Response for Drought Avoidance/Tolerance in the Root of a C3 Xerophyte (Wild Watermelon) Under Water Deficits Plant Cell Physiol., February 1, 2008; 49(2): 226 - 241. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. A. del Rio, L. M. Sandalio, F. J. Corpas, J. M. Palma, and J. B. Barroso Reactive Oxygen Species and Reactive Nitrogen Species in Peroxisomes. Production, Scavenging, and Role in Cell Signaling Plant Physiology, June 1, 2006; 141(2): 330 - 335. [Full Text] [PDF]
|
|
|
|
 |
|
 J. M Palma, A. Jimenez, L. M Sandalio, F. J Corpas, M. Lundqvist, M. Gomez, F. Sevilla, and L. A del Rio Antioxidative enzymes from chloroplasts, mitochondria, and peroxisomes during leaf senescence of nodulated pea plants J. Exp. Bot., May 1, 2006; 57(8): 1747 - 1758. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Tirajoh, T. S. T. Aung, A. B. McKay, and A. L. Plant Stress-responsive {alpha}-dioxygenase expression in tomato roots J. Exp. Bot., February 1, 2005; 56(412): 713 - 723. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||