Evidence for a role of salicylic acid in the oxidative damage generated by NaCl and osmotic stress in Arabidopsis seedlings

Abstract

Previous studies have shown that salicylic acid (SA) is an essential component of the plant resistance to pathogens. We now show that SA plays a role in the plant response to adverse environmental conditions, such as salt and osmotic stresses. We have studied the responses of wild-type Arabidopsis and an SA-deficient transgenic line expressing a salicylate hydroxylase (NahG) gene to different abiotic stress conditions. Wild-type plants germinated under moderate light conditions in media supplemented with 100 mM NaCl or 270 mM mannitol showed extensive necrosis in the shoot. In contrast, NahG plants germinated under the same conditions remained green and developed true leaves. The lack of necrosis observed in NahG seedlings under the same conditions suggests that SA potentiates the generation of reactive oxygen species in photosynthetic tissues during salt and osmotic stresses. This hypothesis is supported by the following observations. First, the herbicide methyl viologen, a generator of superoxide radical during photosynthesis, produced a necrotic phenotype only in wild-type plants. Second, the presence of reactive oxygen-scavenging compounds in the germination media reversed the wild-type necrotic phenotype seen under salt and osmotic stress. Third, a greater increase in the oxidized state of the glutathione pool under NaCl stress was observed in wild-type seedlings compared with NahG seedlings. Fourth, greater oxidative damage occurred in wild-type seedlings compared with NahG seedlings under NaCl stress as measured by lipid peroxidation. Our data support a model for SA potentiating the stress response of the germinating Arabidopsis seedling.

Figure 1

Phenotype of wild-type and NahG seedlings germinated under different stress conditions. Seedlings were germinated on plates and either grown under light (approximately 39 μmol m⁻² s⁻¹) or in the dark. Photos of plates after 15 d are shown on left. The seedlings then were collected and weighed (right). The photographs shown are representative of three independent trials and the fresh weight values are the means of three different experiments (n = 50) ±se. Asterisks indicate that mean values are significantly different between wild type and NahG (P < 0.05). A, 100 mm NaCl in the light in a petri dish. B, 100 mm NaCl in the dark in a petri dish. C, 250 mm NaCl in the light in perlite. D, 270 mm mannitol in the light in a petri dish. E, 5 nm methyl viologen (MV) in the light in a petri dish.

Figure 2

The Arabidopsis ecotype Cvi-0 showed greater sensitivity to NaCl than Ler and NahG. Fresh weight of wild type, NahG, and Cvi-0 seedlings after growing in Murashige and Skoog media containing different NaCl concentrations. Seedlings were germinated and grown on plates under light (approximately 39 μmol m⁻² s⁻¹) and after 15 d the seedlings were collected and weighed. The experiment shown is representative of three independent trials (n = 50).

Figure 3

Protection against NaCl stress is increased in wild-type seedlings by GSH and ASA. Fresh weight of wild-type seedlings (white bars) and NahG seedlings (black bars) after growing in Murashige and Skoog media containing 100 mm NaCl and supplemented with 3 mm of reduced glutathione (GSH) or 2 mm of ASA. Seedlings were collected and weighted after 15 d. The values shown are the means of three independent experiments. Error bars indicate se (n = 30).

Figure 4

Lipid peroxidation is induced by NaCl only under light. TBARS content was determined as described in “Materials and Methods.” The values shown are the means of three independent experiments. Wild type, white bars; NahG, black bars. Asterisks indicate that mean values are significantly different between wild type and NahG (P < 0.05). Error bars indicate se (n = 50).

Figure 5

Effect of NaCl and SA on the expression of RD29A and PR1 in wild-type and NahG. Ten micrograms of total RNA from the wild-type and NahG seedlings was loaded per lane. Plants were grown in Murashige and Skoog media as a control (MS), treated with 1 mm SA, or treated with 200 mm NaCl as described in “Materials and Methods.”