Using two cultivars of Pisum sativum L. with different sensitivity to NaCl, the effect of long-term (15 d) NaCl (70 mM) treatments on the activity and expression of the foliar ascorbate-glutathione cycle enzymes, superoxide dismutase isozymes and their mRNAs was evaluated and related to their ascorbate and glutathione contents. High-speed supernatant (soluble) fractions, enriched for cytosolic components of the antioxidant system, were used. In this fraction from the NaCl-tolerant variety (cv Granada), the activities of ascorbate peroxidase (APX), glutathione reductase (GR), monodehydroascorbate reductase (MDHAR), Mn-superoxide dismutase (Mn-SOD) and dehydroascorbate reductase (DHAR) increased, while CuZn-SOD activity remained constant. In the NaClsensitive plants (cv Challis), salinity did not produce significant changes in APX, MDHAR and GR activities. Only DHAR activity was induced in cv Challis, whereas soluble CuZn-SOD activity decreased by about 35%. Total ascorbate and glutathione contents decreased in both cultivars, but the decline was greater in NaCl-sensitive plants. This difference between the two cultivars was more pronounced when the transcript levels of some these enzymes were examined. Transcript levels for mitochondrial Mn-SOD, chloroplastic CuZn-SOD and phospholipid hydroperoxide glutathione peroxidase (PHGPX), cytosolic GR and APX were strongly induced in the NaCl-tolerant variety but not in the NaCl-sensitive variety. These data strongly suggest that induction of antioxidant defences is at least one component of the tolerance mechanism of peas to long-term salt-stress.

l h e presence of the enzymes of the ascorbate-glutathione cycle was investigated in mitochondria and peroxisomes purified from pea (Pisum sativum L.) leaves. AI1 four enzymes, ascorbate peroxidase (APX; EC 1.1 1.1.1 l ) , monodehydroascorbate reductase (EC 1.6.5.4), dehydroascorbate reductase (EC 1.8.5.1), and glutathione reductase (EC 1.6.4.2), were present in mitochondria and peroxisomes, as well as i n the antioxidants ascorbate and glutathione. l h e activity of the ascorbate-glutathione cycle enzymes was higher in mitochondria than in peroxisomes, except for APX, which was more active in peroxisomes than in mitochondria. lntact mitochondria and peroxisomes had no latent APX activity, and this remained i n the membrane fraction after solubilization assays with 0.2 M KCI.Monodehydroascorbate reductase was highly latent in intact mitochondria and peroxisomes and was membrane-bound, suggesting that the electron acceptor and donor sites of this redox protein are not on the externa1 side of the mitochondrial and peroxisomal membranes. Dehydroascorbate reductase was found mainly in the soluble peroxisomal and mitochondrial fractions. Clutathione reductase had a high latency in mitochondria and peroxisomes and was present i n the soluble fractions of both organelles. I n intact peroxisomes and mitochondria, the presence of reduced ascorbate and glutathione and the oxidized forms of ascorbate and glutathione were demonstrated by high-performance liquid chromatography analysis. l h e ascorbate-glutathione cycle of mitochondria and peroxisomes could represent an important antioxidant protection system against H,O, generated in both plant organelles.

This review deals with the adaptive mechanisms that plants can implement to cope with the challenge of salt stress. Plants tolerant to NaCl implement a series of adaptations to acclimate to salinity, including morphological, physiological and biochemical changes. These changes include increases in the root/canopy ratio and in the chlorophyll content in addition to changes in the leaf anatomy that ultimately lead to preventing leaf ion toxicity, thus maintaining the water status in order to limit water loss and protect the photosynthesis process. Furthermore, we deal with the effect of salt stress on photosynthesis and chlorophyll fluorescence and some of the mechanisms thought to protect the photosynthetic machinery, including the xanthophyll cycle, photorespiration pathway, and water-water cycle. Finally, we also provide an updated discussion on salt-induced oxidative stress at the subcellular level and its effect on the antioxidant machinery in both salt-tolerant and salt-sensitive plants. The aim is to extend our understanding of how salinity may affect the physiological characteristics of plants.

No abstract

The present work describes, for the first time, the changes that take place in the leaf apoplastic antioxidant defenses in response to NaCl stress in two pea (Pisum sativum) cultivars (cv Lincoln and cv Puget) showing different degrees of sensitivity to high NaCl concentrations. The results showed that only superoxide dismutase, and probably dehydroascorbate reductase (DHAR), were present in the leaf apoplastic space, whereas ascorbate (ASC) peroxidase, monodehydroascorbate reductase (MDHAR), and glutathione (GSH) reductase (GR) seemed to be absent. Both ASC and GSH were detected in the leaf apoplastic space and although their absolute levels did not change in response to salt stress, the ASC/dehydroascorbate and GSH to GSH oxidized form ratios decreased progressively with the severity of the stress. Apoplastic superoxide dismutase activity was induced in NaCl-treated pea cv Puget but decreased in NaCl-treated pea cv Lincoln. An increase in DHAR and GR and a decrease in ASC peroxidase, MDHAR, ASC, and GSH levels was observed in the symplast from NaCl-treated pea cv Lincoln, whereas in pea cv Puget an increase in DHAR, GR, and MDHAR occurred. The results suggest a strong interaction between both cell compartments in the control of the apoplastic ASC content in pea leaves. However, this anti-oxidative response does not seem to be sufficient to remove the harmful effects of high salinity. This finding is more evident in pea cv Lincoln, which is characterized by a greater inhibition of the growth response and by a higher rise in the apoplastic hydrogen peroxide content, O 2 .Ϫ production and thiobarbituric acid-reactive substances, and CO protein levels. This NaCl-induced oxidative stress in the apoplasts might be related to the appearance of highly localized O 2 .Ϫ /H 2 O 2 -induced necrotic lesions in the minor veins in NaCl-treated pea plants. It is possible that both the different anti-oxidative capacity and the NaCl-induced response in the apoplast and in the symplast from pea cv Puget in comparison with pea cv Lincoln contributes to a better protection of pea cv Puget against salt stress. NaCl stress is a major factor limiting crop production because it affects almost all plant functions (Bohnert and Jensen, 1996). Therefore, it is important to understand how plants respond and adapt to such stress. Adaptation of the plant cells to high salinity involves osmotic adjustment and the compartmentation of toxic ions, whereas an increasing body of evidence suggests that high salinity also induces oxidative stress (Hernández et al., 1993(Hernández et al., , 1995(Hernández et al., , 1999Gosset et al., 1996;Gó mez et al., 1999;Savouré et al., 1999). Therefore, antioxidant resistance mechanisms may provide a strategy to enhance salt tolerance, and processes underlying antioxidant responses to salt stress must be clearly understood. In previous studies, we have suggested a pivotal role for subcellular compartmentation in antioxidant defense mechanisms under stress conditions, including senescence and NaCl stress (Jiménez...

In pea (Pisum sativum L.) plants the effect of short-term salt stress and recovery on growth, water relations and the activity of some antioxidant enzymes was studied. Leaf growth was interrupted by salt addition. However, during recovery, growth was restored, although there was a delay in returning to control levels. Salt stress brought about a decrease in osmotic potential and in stomatal conductance, but at 48 h and 24 h post-stress, respectively, both parameters recovered control values.In pea leaves, a linear increase in the Na + concentration was observed in salt treated plants. In the recovered plants, a slight reduction in the Na + concentration was observed, probably due to a dilution effect since the plant growth was restored and the total Na + content was maintined in leaves after the stress period.A significant increase of SOD activity occurred after 48 h of stress and after 8hof the recovery period (53% and 42%, respectively), and it reached control values at 24 h post-stress. APX activity did not change during the stress period, and after only 3 8h post-stress it was increased by 48% with respect to control leaves. GR showed a 71% increase after 24h of salt stress and also a significant increase was observed in the recovered plants. A strong increase of TBARS was observed after 8h of stress (180% increase), but then a rapid decrease was observed during the stress period.Surprisingly, TBARS again increased at 8h post-stress (78% increase), suggesting that plants could perceive the elimination of NaCl from the hydroponic cultures as another stress during the first hours of recovery.These results suggest that short-term NaCl stress produces reversible effects on growth, leaf water relations and on SOD and APX activities. This work also suggests that both during the first hours of imposition of stress and during the first hours of recovery an oxidative stress was produced.

The present work describes, for the first time, the changes that take place in the leaf apoplastic antioxidant defenses in response to NaCl stress in two pea (Pisum sativum) cultivars (cv Lincoln and cv Puget) showing different degrees of sensitivity to high NaCl concentrations. The results showed that only superoxide dismutase, and probably dehydroascorbate reductase (DHAR), were present in the leaf apoplastic space, whereas ascorbate (ASC) peroxidase, monodehydroascorbate reductase (MDHAR), and glutathione (GSH) reductase (GR) seemed to be absent. Both ASC and GSH were detected in the leaf apoplastic space and although their absolute levels did not change in response to salt stress, the ASC/dehydroascorbate and GSH to GSH oxidized form ratios decreased progressively with the severity of the stress. Apoplastic superoxide dismutase activity was induced in NaCl-treated pea cv Puget but decreased in NaCl-treated pea cv Lincoln. An increase in DHAR and GR and a decrease in ASC peroxidase, MDHAR, ASC, and GSH levels was observed in the symplast from NaCl-treated pea cv Lincoln, whereas in pea cv Puget an increase in DHAR, GR, and MDHAR occurred. The results suggest a strong interaction between both cell compartments in the control of the apoplastic ASC content in pea leaves. However, this anti-oxidative response does not seem to be sufficient to remove the harmful effects of high salinity. This finding is more evident in pea cv Lincoln, which is characterized by a greater inhibition of the growth response and by a higher rise in the apoplastic hydrogen peroxide content, O 2 .Ϫ production and thiobarbituric acid-reactive substances, and CO protein levels. This NaCl-induced oxidative stress in the apoplasts might be related to the appearance of highly localized O 2 .Ϫ /H 2 O 2 -induced necrotic lesions in the minor veins in NaCl-treated pea plants. It is possible that both the different anti-oxidative capacity and the NaCl-induced response in the apoplast and in the symplast from pea cv Puget in comparison with pea cv Lincoln contributes to a better protection of pea cv Puget against salt stress. NaCl stress is a major factor limiting crop production because it affects almost all plant functions (Bohnert and Jensen, 1996). Therefore, it is important to understand how plants respond and adapt to such stress. Adaptation of the plant cells to high salinity involves osmotic adjustment and the compartmentation of toxic ions, whereas an increasing body of evidence suggests that high salinity also induces oxidative stress (Hernández et al., 1993(Hernández et al., , 1995(Hernández et al., , 1999Gosset et al., 1996;Gó mez et al., 1999;Savouré et al., 1999). Therefore, antioxidant resistance mechanisms may provide a strategy to enhance salt tolerance, and processes underlying antioxidant responses to salt stress must be clearly understood. In previous studies, we have suggested a pivotal role for subcellular compartmentation in antioxidant defense mechanisms under stress conditions, including senescence and NaCl stress (Jiménez...

This review deals with the adaptive mechanisms that plants can implement to cope with the challenge of salt stress. Plants tolerant to NaCl implement a series of adaptations to acclimate to salinity, including morphological, physiological and biochemical changes. These changes include increases in the root/canopy ratio and in the chlorophyll content in addition to changes in the leaf anatomy that ultimately lead to preventing leaf ion toxicity, thus maintaining the water status in order to limit water loss and protect the photosynthesis process. Furthermore, we deal with the effect of salt stress on photosynthesis and chlorophyll fluorescence and some of the mechanisms thought to protect the photosynthetic machinery, including the xanthophyll cycle, photorespiration pathway, and water-water cycle. Finally, we also provide an updated discussion on salt-induced oxidative stress at the subcellular level and its effect on the antioxidant machinery in both salt-tolerant and salt-sensitive plants. The aim is to extend our understanding of how salinity may affect the physiological characteristics of plants.