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.

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...