Current studies, particularly in Arabidopsis, have demonstrated that mutants deficient in cytosolic ascorbate peroxidases (APXs) are susceptible to the oxidative damage induced by abiotic stress. In contrast, we demonstrate here that rice mutants double silenced for cytosolic APXs (APx1/2s) up-regulated other peroxidases, making the mutants able to cope with abiotic stress, such as salt, heat, high light and methyl viologen, similar to non-transformed (NT) plants. The APx1/2s mutants exhibited an altered redox homeostasis, as indicated by increased levels of H2O2 and ascorbate and glutathione redox states. Both mutant and NT plants exhibited similar photosynthesis (CO2 assimilation and photochemical efficiency) under both normal and stress conditions. Overall, the antioxidative compensatory mechanism displayed by the mutants was associated with increased expression of OsGpx genes, which resulted in higher glutathione peroxidase (GPX) activity in the cytosolic and chloroplastic fractions. The transcript levels of OsCatA and OsCatB and the activities of catalase (CAT) and guaiacol peroxidase (GPOD; type III peroxidases) were also up-regulated. None of the six studied isoforms of OsApx were up-regulated under normal growth conditions. Therefore, the deficiency in cytosolic APXs was effectively compensated for by up-regulation of other peroxidases. We propose that signalling mechanisms triggered in rice mutants could be distinct from those proposed for Arabidopsis.

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RESUMOEstudou-se o efeito da aplicação exógena de peróxido de hidrogênio (H 2 O 2 ) na aclimatação ao estresse salino com base nas alterações fotossintéticas, indicadores de estresse oxidativo e atividade de enzimas antioxidativas em folhas de plantas de arroz. Utilizaram-se duas diferentes concentrações de H 2 O 2 (1 e 10 M) para o pré-tratamento, concentrações essas aplicadas na solução nutritiva dois dias antes da indução do estresse salino (100 mM de NaCl). A limitação fotossintética e estomática resultante da exposição ao NaCl foi amenizada quando as plantas foram pré-tratadas com 1 M de H 2 O 2 . Plantas expostas ao pré-tratamento com H 2 O 2 e expostas ao NaCl apresentaram dano de membrana menor quando comparadas com as plantas submetidas ao NaCl isoladamente. O conteúdo de TBARS e H 2 O 2 foi reduzido sensivelmente nas plantas pré-tratadas com 1 M de H 2 O 2 e expostas ao NaCl em relação às que não foram pré-tratadas. O sistema antioxidativo enzimático nas plantas expostas ao NaCl foi induzido principalmente quando ocorreu o pré-tratamento com 1 M de H 2 O 2 . Os dados sugerem que exposição prévia ao H 2 O 2, pode resultar numa aclimatação mais efetiva às condições de estresse salino. Palavras-chave: peróxido de hidrogênio, estresse oxidativo, salinidadeSalt stress acclimation in rice plants induced by H 2 O 2 pretreatment ABSTRACT The effect of exogenous application of hydrogen peroxide (H 2 O 2 ) in the acclimation to salt stress was studied on the basis of photosynthetic changes, indicators of oxidative stress and activity of antioxidant enzymes in leaves of rice plants. Two different concentrations of the H 2 O 2 (1 and 10 M) were used for the pre-treatment, these concentrations were applied in the nutrient solution two days before the induction of salt stress (100 mM NaCl). The photosynthetic and stomatal limitations after exposure to NaCl were alleviated when the plants were pretreated with 1 M H 2 O 2 . Plants pretreated with H 2 O 2 and submitted to salt stress showed membrane damage lower in compared to plants exposed to NaCl alone. TBARS and H 2 O 2 content was reduced appreciable in plants pretreated with 1 M H 2 O 2 and exposed to NaCl in relation to not pretreated. The enzymatic antioxidative system in plants exposed to NaCl was mainly induced when there was the 1 M H 2 O 2 pretreatment. Data suggest that an anticipated exposure to H 2 O 2 may result in more effective acclimation to salt stress.

Soil salinity is an important abiotic stress worldwide, and salt-induced oxidative stress can have detrimental effects on the biological nitrogen fixation. We hypothesized that co-inoculation of cowpea plants with Bradyrhizobium and plant growth-promoting bacteria would minimize the deleterious effects of salt stress via the induction of enzymatic and non-enzymatic antioxidative protection. To test our hypothesis, cowpea seeds were inoculated with Bradyrhizobium or co-inoculated with Bradyrhizobium and plant growth-promoting bacteria and then submitted to salt stress. Afterward, the cowpea nodules were collected, and the levels of hydrogen peroxide; lipid peroxidation; total, reduced and oxidized forms of ascorbate and glutathione; and superoxide dismutase, catalase and phenol peroxidase activities were evaluated. The sodium and potassium ion concentrations were measured in shoot samples. Cowpea plants did not present significant differences in sodium and potassium levels when grown under non-saline conditions, but sodium content was strongly increased under salt stress conditions. Under non-saline and salt stress conditions, plants co-inoculated with Bradyrhizobium and Actinomadura or co-inoculated with Bradyrhizobium and Paenibacillus graminis showed lower hydrogen peroxide content in their nodules, whereas lipid peroxidation was increased by 31% in plants that were subjected to salt stress. Furthermore, cowpea nodules co-inoculated with Bradyrhizobium and plant growth-promoting bacteria and exposed to salt stress displayed significant alterations in the total, reduced and oxidized forms of ascorbate and glutathione. Inoculation with Bradyrhizobium and plant growth-promoting bacteria induced increased superoxide dismutase, catalase and phenol peroxidase activities in the nodules of cowpea plants exposed to salt stress. The catalase activity in plants co-inoculated with Bradyrhizobium and Streptomyces was 55% greater than in plants inoculated with Bradyrhizobium alone, and this value was remarkably greater than that in the other treatments. These results reinforce the beneficial effects of plant growth-promoting bacteria on the antioxidant system that detoxifies reactive oxygen species. We concluded that the combination of Bradyrhizobium and plant growth-promoting bacteria induces positive responses for coping with salt-induced oxidative stress in cowpea nodules, mainly in plants co-inoculated with Bradyrhizobium and P. graminis or co-inoculated with Bradyrhizobium and Bacillus.

The biochemical mechanisms underlying the involvement of cytosolic ascorbate peroxidases (cAPXs) in photosynthesis are still unknown. In this study, rice plants doubly silenced in these genes (APX1/2) were exposed to moderate light (ML) and high light (HL) to assess the role of cAPXs in photosynthetic efficiency. APX1/2 mutants that were exposed to ML overexpressed seven and five proteins involved in photochemical activity and photorespiration, respectively. These plants also increased the pheophytin and chlorophyll levels, but the amount of five proteins that are important for Calvin cycle did not change. These responses in mutants were associated with Rubisco carboxylation rate, photosystem II (PSII) activity and potential photosynthesis, which were similar to non-transformed plants. The upregulation of photochemical proteins may be part of a compensatory mechanism for APX1/2 deficiency but apparently the finer-control for photosynthesis efficiency is dependent on Calvin cycle proteins. Conversely, under HL the mutants employed a different strategy, triggering downregulation of proteins related to photochemical activity, Calvin cycle and decreasing the levels of photosynthetic pigments. These changes were associated to strong impairment in PSII activity and Rubisco carboxylation. The upregulation of some photorespiratory proteins was maintained under that stressful condition and this response may have contributed to photoprotection in rice plants deficient in cAPXs. The data reveal that the two cAPXs are not essential for photosynthesis in rice or, alternatively, the deficient plants are able to trigger compensatory mechanisms to photosynthetic acclimation under ML and HL conditions. These mechanisms involve differential regulation in protein expression related to photochemistry, Calvin cycle and photorespiration.