Karina B. Balestrasse

Effect of cadmium stress on nitrogen metabolism in nodules and roots of soybean plants

The nitrogen metabolism of soybean (Glycine max L.) nodules and roots was studied in plants subjected to two different concentrations (50 and 200 μM) of CdCl2. Nitrogenase activity was decreased in nodules treated with 200 μM Cd2+. In 50 μM Cd2+-treated plants, NH4+ content showed similar values to controls in nodules, but increased by 55% in roots. However, after treatment with 200 μM Cd2+, NH4+ levels increased in both tissues. Glutamate (Glu) and protein contents remained unaltered in nodules treated with 50 μM Cd2+, while at the higher Cd2+ concentration both were decreased. Nevertheless, polyamine content was increased at the two Cd2+ concentrations. In roots, Glu, polyamine and protein levels were significantly diminished at 50 and 200 μM CdCl2. For nitrogen-assimilation enzymes, glutamate dehydrogenase activity was moderately increased in nodules and roots following the lower Cd2+ treatment, though at the higher Cd2+ concentration root enzyme activity returned to control levels. An impressive increase in enzyme activity was found in nodules. In roots, the glutamine synthetase / glutamate synthase pathway was decreased at the two Cd2+ concentrations, though in nodules it was diminished only at 200 μM Cd2+. No changes in protease activity were found in the two tissues treated with 50μMCd2+. However, at 200 μM Cd2+, nodule and root protease activities decreased and increased, respectively. These results suggest that, in general, treatment with Cd2+ affects nitrogen assimilation and metabolism to a greater extent in soybean roots than in nodules.

Response of antioxidant defence system in soybean nodules and roots subjected to cadmium stress

The antioxidant defence system of soybean (Glycine maxL.) nodules and roots was studied in plants subjected to three different concentrations (50, 100 and µ200 M ) of CdCl 2 . Cadmium (Cd)-induced oxidative stress was determined by lipid peroxidation, which remained unaltered in nodules and roots treated with 50 µM Cd(II). No changes were observed in nodules treated with 100 M Cd(II), while a 20% increase was found in roots and 200 µM Cd(II) produced an increase of about 55% in both tissues. The soluble antioxidant defence, reduced glutath one(GSH) and the corresponding reduced/oxidised glutathione (GSH/GSSG) ratio showed different behaviour in both tissues, decreasing in roots with 100 and 200 µM Cd(II). No changes were observed in the GSH/GSSG ratio in nodules under the three Cd treatments. However, ascorbate content and ascorbate/dehydroascorbate (As/DAs) ratio were diminished in nodules and roots subjected to the three Cd concentrations. Regarding the antioxidant enzymes, it was found that, except for catalase, a general decrease in nodule enzyme activities was produced only under the 200 µM Cd(II) treatment. Nevertheless, root enzymatic antioxidant defences showed significant increments in L -ascorbate peroxidase (APOX), dehydroascorbate reductase (DHAR) and glutathione reductase (GR) activities under lower Cd treatment, while the enzyme activities decreased with the two higher concentrations of CdCl 2 . These results suggest that soybean roots were more affected than nodules by Cd treatments, although the higher Cd concentration produced oxidative stress and deleterious effects in antioxidant defence system in both tissues.

Nitric oxide synthase-like dependent NO production enhances heme oxygenase up-regulation in ultraviolet-B-irradiated soybean plants

Heme oxygenase (HO) has antioxidant properties and is up-regulated by reactive oxygen species (ROS) in ultraviolet-B-irradiated soybean plants. This study shows that nitric oxide (NO) protects against oxidative damage and that nitric oxide synthase (NOS)-like activity is also required for HO-1 induction under UV-B radiation. Pre-treatments with sodium nitroprussiate (SNP), a NO-donor, prevented chlorophyll loss, H(2)O(2) and O(2)(*-) accumulation, and ion leakage in UV-B-treated plants. HO activity was significantly enhanced by NO and showed a positive correlation with HO-1 transcript levels. In fact, HO-1 mRNA levels were increased 2.1-fold in 0.8 mM SNP-treated plants, whereas subsequent UV-B irradiation augmented this expression up to 3.5-fold with respect to controls. This response was not observed using ferrocyanide, a SNP inactive analog, and was effectively blocked by 2-(4-carboxyphenil)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO), a specific NO-scavenger. In addition, experiments carried out in the presence of N(G)-nitro-L-arginine methyl ester (L-NAME) or tungsten, well-known inhibitors of NOS and nitrate reductase, showed that NOS is the endogenous source of NO that mediates HO-1 expression. In summary, we found that NO is involved in the signaling pathway leading to HO-1 up-regulation under UV-B, and that a balance between NO and ROS is important to trigger the antioxidant response against oxidative stress.

The role of 5-aminolevulinic acid in the response to cold stress in soybean plants

In this study, the possibility of enhancing cold stress tolerance of soybean plants (Glycine max L.) by exogenous application of 5-aminolevulinic acid (ALA) was investigated. ALA was added to the Hoagland solution at various concentrations ranging from 0 to 40 μM for 12 h. After ALA treatment, the plants were subjected to cold stress at 4°C for 48 h. ALA at low concentrations (5-10 μM) provided significant protection against cold stress compared to non-ALA-treated plants, enhancing chlorophyll content (Chl) as well as relative water content (RWC). Increase of thiobarbituric acid reactive species (TBARS) levels was also prevented, whereas exposure to higher ALA concentrations (15-40 μM) brought about a dose dependent increase of these species, reaching a maximum of 117% in plants pre-treated with 40 μM ALA compared to controls. ALA pre-treatment also enhanced catalase (CAT) and heme oxygenase-1 (HO-1) activities. These findings indicate that HO-1 acts not only as the rate limiting enzyme in heme catabolism, but also as an antioxidant enzyme. The highest cold tolerance was obtained with 5 μM ALA pre-treatment. Results show that ALA, which is considered as an endogenous plant growth regulator, could be used effectively to protect soybean plants from the damaging effects of cold stress by enhancing the activity of heme proteins, e.g., catalase (CAT) and by promoting heme catabolism leading to the production of the highly antioxidant biliverdin and carbon monoxide, without any adverse effect on the plant growth.

Involvement of heme oxygenase as antioxidant defense in soybean nodules

Objective: We have previously demonstrated that the inducible form of heme oxygenase plays a critical role in protecting against oxidative stress in mammals. To gain further insight into the functions of this enzyme in plants, we have tested its activity and expression in soybean nodules subjected to cadmium (Cd) stress. Materials and methods: Four-weeks-old soybean nodulated plants were treated with different cadmium chloride concentrations (0, 50 and 200 μM) during 48 h. Oxidative stress parameters such as TBARS content, GSH levels and antioxidant enzyme activities were measured as well as heme oxygenase activity and expression. Besides, the effect of biliverdin and Zn-protophorphyrin IX were analized. Results: Treatment with 200 μM Cd during 48 h caused a 67% increase in TBARS content, whereas GSH decreased 44%, and total superoxide dismutase, gluthatione reductase and guaiacol peroxidase were also inhibited 54, 20 and 60%, respectively. A total of 200 μM Cd produced the overexpression of heme oxygenase-1, as well as a 10-fold enhancement of its activity. Co-administration of biliverdin (10 μM) completely prevented the effects caused by Cd. Treatment with Zn protoporphyrin IX, a strong inhibitor of heme oxygenase, expectedly decreased heme oxygenase-1 activity to half. When the inhibitor was given together with Cd, completely prevented the enzyme induction and oxidative stress parameters were significantly enhanced. Conclusion: Taking together, these results are indicating that heme oxygenase plays a protective role against oxidative cell damage in soybean nodules.

Oxidation of the enzymes involved in nitrogen assimilation plays an important role in the cadmium-induced toxicity in soybean plants

Cadmium causes oxidative damage and hence affects nitrogen assimilation. In the present work we tested the relationship between the inactivation of the enzymes involved in nitrogen assimilation pathway (glutamine synthetase (GS)/glutamate synthase (GOGAT)) and the protein oxidation in nodules of soybean (Glycine max L.) plants under Cd2+ stress. Therefore, the effect of Cd2+ and reduced gluthatione (GSH) on GS and GOGAT activities, and protein abundance and oxidation were analyzed. Under the metal treatment, amino acids oxidative modification occurred, evidenced by the accumulation of carbonylated proteins, especially those of high molecular weight. When Cd2+ was present in the nutrient solution, although a decrease in GS and GOGAT activities was observed (17 and 52%, respectively, compared to controls), the protein abundance of both enzymes remained similar to control nodules. When GSH was added together with Cd2+ in the nutrient medium, it protected the nodule against Cd2+ induced oxidative damage, maintaining GS and GOGAT activities close to control values. These results allow us to conclude that the inactivation of the nitrogen assimilation pathway by Cd2+ in soybean nodules is due to an increment in GS and GOGAT oxidation that can be prevented by the soluble antioxidant GSH.

Signal transduction pathways and haem oxygenase induction in soybean leaves subjected to salt stress

Abstract We have previously demonstrated that the induction of haem oxygenase-1 (EC 1.14.99.3) plays a protective role for soybean plants against cadmium and UV-B stress. Here, we have investigated the possible signal transduction pathways involved in haem oxygenase-1 induction in leaves of soybean plants subjected to salt stress. Treatment with 100 mM NaCl during 48 h increased thiobarbituric acid reactive substances by 30%, whereas GSH decreased by 50%, with respect to controls. These effects were prevented by pre-incubation with diphenyleneiodonium (DPI; an NADPH oxidase inhibitor), [1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ; a guanylate cyclase inhibitor) or LaCl3 (calcium channel blocker). NaCl at 100 mM produced in situ accumulation of H2O2 and O2•−, which were also prevented by DPI, ODQ or LaCl3. Moreover, salt-induced haem oxygenase-1 activity was also totally abolished by pretreatment with the different inhibitors. These results clearly demonstrated that the signal transduction pathways involved in oxidative stress triggered by salt stress were similar to those implicated in haem oxygenase-1 induction, and provide additional information suggesting that haem oxygenase might play a key role in the antioxidative protection machinery of higher plants.

Angiotensin II Regulates Cardiac Hypertrophy via Oxidative Stress but Not Antioxidant Enzyme Activities in Experimental Renovascular Hypertension

The aim of this study was to provide new insights into the role of angiotensin II and arterial pressure in the regulation of antioxidant enzyme activities in a renovascular model of cardiac hypertrophy. For this purpose, aortic coarcted rats were treated with losartan or minoxidil for 7 days. Angiotensin II induced cardiac hypertrophy and oxidative stress via Nox4, p22phox and p47phox, which are components of the NAD(P)H oxidase. Antioxidant enzymes were regulated by arterial pressure and were not implicated in cardiac hypertrophy. Heme oxygenase-1, the rate-limiting enzyme in heme catabolism, behaved as a catalase and glutathione peroxidase, and is regulated by arterial pressure. In summary, the present report indicates that cardiac hypertrophy, induced by renovascular hypertension, depends on angiotensin II through reactive oxygen species and is not prevented by the action of antioxidant enzymes.

Nitric oxide induces specific isoforms of antioxidant enzymes in soybean leaves subjected to enhanced ultraviolet-B radiation

Antioxidant enzymes play a key role in plant tolerance to different types of stress, including ultraviolet-B (UV-B) radiation. Here we report that nitric oxide (NO) enhances antioxidant enzymes gene expression and increases the activity of specific isoforms protecting against UV-B radiation. Pre-treatments with sodium nitroprussiate (SNP), a NO-donor, prevented lipid peroxidation, ion leakage and H2O2 and superoxide anion accumulation in leaves of UV-B-treated soybean plants. Transcripts levels of superoxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase (APX) were significantly induced by SNP. These data correlated with the enhancement of particular antioxidant enzyme isoforms, such as one CAT isoform and two APX isoforms. Moreover, SNP induced the expression of three new isoforms of SOD, identified as Mn-SOD subclass. Further results showed that total activities of SOD, CAT and APX significantly increased by 2.2-, 1.8- and 2.1-fold in SNP-treated plants compared to controls, respectively. The protective effect of SNP against UV-B radiation was negated by addition of the specific NO scavenger cPTIO, indicating that NO released by SNP mediates the enhancement of antioxidant enzymes activities. In conclusion, NO is involved in the signaling pathway that up-regulates specific isoforms of antioxidant enzymes protecting against UV-B-induced oxidative stress.

Heme Oxygenase Contributes to Alleviate Salinity Damage in Glycine max L. Leaves.

Plants are frequently subjected to different kinds of stress, such as salinity and, like other organisms, they have evolved strategies for preventing and repairing cellular damage caused by salt stress. Glycine max L. plants were subjected to different NaCl concentrations (0–200 mM) for 10 days. Treatments with 100 and 200 mM NaCl induced ion leakage and lipid peroxidation augmentation, loss in chlorophyll content, and accumulation of O2 •− and H2O2. However, 50 mM NaCl did not modify these parameters, which remains similar to control values. Catalase, superoxide dismutase, and heme oxygenase (HO-1) activities and gene expressions were increased under 100 mM NaCl, while no differences were observed with respect to controls under 50 mM salt. Treatment with 200 mM NaCl caused a diminution in the enzyme activities and gene expressions. Results here reported let us conclude that HO also plays a leading role in the defense mechanisms against salinity.