Location and effects of long-term NaCl stress on superoxide dismutase and ascorbate peroxidase isoenzymes of pea (Pisum sativum cv. Puget) chloroplasts

The present work describes the intrachloroplast localization and the changes that took place in the thylakoid and stroma-located superoxide dismutases (SOD, EC 1.15.1.1) and ascorbate peroxidases (APX, EC 1.11.1.11), in response to long-term NaCl stress in Pisum sativum L. cv. Puget plants. Native PAGE using high chloroplast protein concentrations pointed to the presence of the two main Fe-SODs, together with CuZn-SODs, both in thylakoids and in the stroma. Western blot and immunogold labelling using the antibodies against chloroplastic Fe-SOD from Nuphar luteum also confirmed the chloroplastic localization of a Fe-SOD. Thylakoidal Fe-SOD activity was induced by a NaCl concentration as low as 70 mM, while CuZn-SOD was induced at 90 mM, although in severe stress conditions (110 mM) both activities were similar to the levels at 90 mM NaCl. NaCl stress also induced stromatic Fe-SOD and CuZn-SOD activities, although these inductions only started at higher NaCl concentration (90 mM) and were significant at 110 mM NaCl. The increase in activity of both Fe-SODs was matched by an increase in Fe-SOD protein. Chloroplastic APX isoenzymes behaved differently in thylakoids and stroma in response to NaCl. A significant increase of stromal APX occurred at 70 mM, whereas the thylakoidal APX activity was significantly and progressively lost in response to NaCl stress (70-110 mM). A significant increase in the H2O2 content of chloroplasts during stress and a reduction in the ascorbate level at 90 mM NaCl also took place, although the oxidized ascorbate pool at the highest NaCl concentration did not show significant changes. These results suggest that the loss of thylakoidal APX may be an important factor in the increase in chloroplastic H2O2, which also results from the increased thylakoid and stroma-located Fe-SOD and CuZn-SOD activities. This H2O2 may be involved in the induction of stromal APX. The up-regulation of the above enzymes in the described stress conditions would contribute to the adaptation of cv. Puget plants to moderate NaCl stress.