Aurenivia Bonifacio

Role of peroxidases in the compensation of cytosolic ascorbate peroxidase knockdown in rice plants under abiotic stress

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.

The knockdown of chloroplastic ascorbate peroxidases reveals its regulatory role in the photosynthesis and protection under photo-oxidative stress in rice

The inactivation of the chloroplast ascorbate peroxidases (chlAPXs) has been thought to limit the efficiency of the water-water cycle and photo-oxidative protection under stress conditions. In this study, we have generated double knockdown rice (Oryza sativa L.) plants in both OsAPX7 (sAPX) and OsAPX8 (tAPX) genes, which encode chloroplastic APXs (chlAPXs). By employing an integrated approach involving gene expression, proteomics, biochemical and physiological analyses of photosynthesis, we have assessed the role of chlAPXs in the regulation of the protection of the photosystem II (PSII) activity and CO2 assimilation in rice plants exposed to high light (HL) and methyl violagen (MV). The chlAPX knockdown plants were affected more severely than the non-transformed (NT) plants in the activity and structure of PSII and CO2 assimilation in the presence of MV. Although MV induced significant increases in pigment content in the knockdown plants, the increases were apparently not sufficient for protection. Treatment with HL also caused generalized damage in PSII in both types of plants. The knockdown and NT plants exhibited differences in photosynthetic parameters related to efficiency of utilization of light and CO2. The knockdown plants overexpressed other antioxidant enzymes in response to the stresses and increased the GPX activity in the chloroplast-enriched fraction. Our data suggest that a partial deficiency of chlAPX expression modulate the PSII activity and integrity, reflecting the overall photosynthesis when rice plants are subjected to acute oxidative stress. However, under normal growth conditions, the knockdown plants exhibit normal phenotype, biochemical and physiological performance.

Cytosolic APX knockdown rice plants sustain photosynthesis by regulation of protein expression related to photochemistry, Calvin cycle and photorespiration

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.

Silenced rice in both cytosolic ascorbate peroxidases displays pre-acclimation to cope with oxidative stress induced by 3-aminotriazole-inhibited catalase.

The maintenance of H2O2 homeostasis and signaling mechanisms in plant subcellular compartments is greatly dependent on cytosolic ascorbate peroxidases (APX1 and APX2) and peroxisomal catalase (CAT) activities. APX1/2 knockdown plants were utilized in this study to clarify the role of increased cytosolic H2O2 levels as a signal to trigger the antioxidant defense system against oxidative stress generated in peroxisomes after 3-aminotriazole-inhibited catalase (CAT). Before supplying 3-AT, silenced APX1/2 plants showed marked changes in their oxidative and antioxidant profiles in comparison to NT plants. After supplying 3-AT, APX1/2 plants triggered up-expression of genes belonging to APX (OsAPX7 and OsAPX8) and GPX families (OsGPX1, OsGPX2, OsGPX3 and OsGPX5), but to a lower extent than in NT plants. In addition, APX1/2 exhibited lower glycolate oxidase (GO) activity, higher CO2 assimilation, higher cellular integrity and higher oxidation of GSH, whereas the H2O2 and lipid peroxidation levels remained unchanged. This evidence indicates that redox pre-acclimation displayed by silenced rice contributed to coping with oxidative stress generated by 3-AT. We suggest that APX1/2 plants were able to trigger alternative oxidative and antioxidant mechanisms involving signaling by H2O2, allowing these plants to display effective physiological responses for protection against oxidative damage generated by 3-AT, compared to non-transformed plants.

Compostos nitrogenados e carboidratos em sorgo submetido à salinidade e combinações de nitrato e amônio

The objective of this study was to evaluate the effects of combinations of nitrate and ammonium in the medium, on the accumulation of nitrogen compounds and carbohydrates, in the presence and absence of salinity in sorghum plants. CSF 20 genotype sorghum seeds, were grown in the greenhouse and supplied with a nutritive solution at three different concentrations of nitrate and ammonium (100% NO3-: 0% NH4+, 75% NO3-: 25% NH4+ e 0% NO3-: 100% NH4+) both in the presence and absence of NaCl. Salinity reduced the nitrate concentration in both leaves and stalks in treatments at 100:0 and 75:25. The accumulation of ammonium was higher in those plants, in both leaves and stalk, in the presence of NaCl. The levels of free amino acids in the leaves showed no change, whereas those in the stalk increased in the presence of salt and with the increase of ammonium. The concentration of free proline in both leaves and stalk was higher in plants under salinity. The total nitrogen content was higher in the leaves without, however, showing much change in the presence of salt. The concentration of total soluble carbohydrates was higher in those plants treated with salt, except in the stalk 0:100. Sucrose levels in the leaves were lower in the presence of NaCl and the increase in ammonium, while they were higher in the stalk at 75:25 NaCl. The starch content did not alter much with the different levels of nitrogen and salinity. It is concluded that NO-3 and NH4+ combinations in the medium affect the accumulation of nitrogen compounds and carbohydrates in the leaves and stalks of sorghum plants, both in the presence and absence of salinity.

Changes induced by co-inoculation in nitrogen–carbon metabolism in cowpea under salinity stress

To mitigate the deleterious effects of abiotic stress, the use of plant growth-promoting bacteria along with diazotrophic bacteria has been increasing. The objectives of this study were to investigate the key enzymes related to nitrogen and carbon metabolism in the biological nitrogen fixation process and to elucidate the activities of these enzymes by the synergistic interaction between Bradyrhizobium and plant growth-promoting bacteria in the absence and presence of salt stress. Cowpea plants were cultivated under axenic conditions, inoculated with Bradyrhizobium and co-inoculated with Bradyrhizobium sp. and Actinomadura sp., Bradyrhizobium sp. and Bacillus sp., Bradyrhizobium sp. and Paenibacillus graminis, and Bradyrhizobium sp. and Streptomycessp.; the plants were also maintained in the absence (control) and presence of salt stress (50 mmolL−1 NaCl). Salinity reduced the amino acids, free ammonia, ureides, proteins and total nitrogen content in nodules and increased the levels of sucrose and soluble sugars. The co-inoculations responded differently to the activity of glutamine synthetase enzymes under salt stress, as well as glutamate synthase, glutamate dehydrogenase aminating, and acid invertase in the control and salt stress. Considering the development conditions of this experiment, co-inoculation with Bradyrhizobium sp. and Bacillus sp. in cowpea provided better symbiotic performance, mitigating the deleterious effects of salt stress.

Azospirillum sp. as a Challenge for Agriculture

Several processes mediated by soil microorganisms play an important role in nutrient cycling. One such process is biological nitrogen fixation (BNF) by representatives of various bacterial phylogenetic groups, which are called diazotrophs. Most studies of the Azospirillum-plant association have been conducted on cereals and grasses. Currently, 17 species of Azospirillum have been described. However, a great diversity of these bacteria continues to be revealed, and little is known of the potential applications of the many species that have been described. The Azospirillum-plant association begins with the adsorption and adherence process of these bacteria in roots. Involved in these processes is the recognition of bacterial polysaccharides by the host plant, a step that is necessary in successfully forming a positive relationship between roots and Azospirillum. The presence of Azospirillum in the rhizosphere can minimize the susceptibility to diseases caused by plant pathogens. Furthermore, the ability to produce phytohormones, mainly auxins (indole-3-acetic acid) and other molecules from secondary metabolism has been suggested to underlie the growth response to inoculation by Azospirillum species. These positive aspects of Azospirillum colonization in the roots are also responsible for the alleviation of plant stress. For all of the above-mentioned reasons, Azospirillum are also widely used as commercial inoculants, resulting in a significant economic impact in crop yields in many countries. In fact, solid and liquid formulations containing Azospirillum are marketed in various countries, such as Brazil, Argentina, Mexico, Italy, France, Belgium, Africa, Germany, Pakistan, Uruguay, India and the USA. In addition, new formulations containing Azospirillum, such as polymeric inoculants (alginate, agar, chitosan and gum), are already used for the improvement of many crops. This chapter summarizes the positive effects of Azospirillum-plant interactions and their biological importance for the improvement of agriculture worldwide.

Symbiotic performance, nitrogen flux and growth of lima bean ( Phaseolus lunatus L.) varieties inoculated with different indigenous strains of rhizobia

Legumes, such as lima bean, can form symbiotic relationships with rhizobia and can therefore grow in nitrogen-poor soils. Lima bean varieties in symbiosis with indigenous rhizobia were evaluated for their symbiotic performance over three harvest periods. Four indigenous rhizobial strains (ISOL-16, ISOL-18, ISOL-19 or ISOL-35) were isolated from soil samples in lima bean fields and used separately to inoculate two lima bean varieties (‘boca de moça’ and ‘branca’). Uninoculated, unfertilized plants were used as controls, and uninoculated, nitrogen-supplied plants were used as the nitrogen control. All inoculated plants and the uninoculated control were cultivated using nitrogen-free nutrient solutions in the greenhouse. As expected, the uninoculated plants did not develop nodules on their root systems and were inferior in all evaluated parameters. The lima bean nodulated by Bradyrhizobium exhibited growth variables, nodules parameters, and nitrogen flux values superior to those of plants inoculated with Rhizobium. The highest total chlorophyll values were recorded in lima bean inoculated with Bradyrhizobium, confirming that these plants had the greenest leaves and likely have superior photosynthetic efficiency – a hypothesis supported by the greater growth exhibited by these plants. Nitrogen fixation efficiency was superior in lima bean nodulated by Bradyrhizobium, indicating that this microbe possesses a greater ability to fix nitrogen and provide it continuously to the host plant. The symbiosis between lima bean and Bradyrhizobium sp. ISOL-18 displayed the best values with respect to carbon and nitrogen flow. We conclude that Bradyrhizobium is the most effective at establishing an efficient and successful symbiotic relationship with lima bean and emphasize the potential value of Bradyrhizobium strains as inoculants in lima bean cultivation.

Antioxidant response of cowpea co-inoculated with plant growth-promoting bacteria under 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.