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.

Indole acetic acid is responsible for protection against oxidative stress caused by drought in soybean plants: the role of heme oxygenase induction.

Objectives This study was focused on the role of indole acetic acid (IAA) in the defense against oxidative stress damage caused by drought in soybean plants and to elucidate whether heme oxygenase-1 (HO-1) and nitric oxide (NO) are involved in this mechanism. IAA is an auxin that participates in many plant processes including oxidative stress defense, but to the best of our knowledge no information is yet available about its possible action in drought stress. Methods To this end, soybean plants were treated with 8% polyethylene glycol (PEG) or 100 µM IAA. To evaluate the behavior of IAA, plants were pretreated with this compound previous to PEG addition. Lipid peroxidation levels (thiobarbituric acid reactive substances (TBARS)), glutathione (GSH) and ascorbate (AS) contents, catalase (CAT), superoxide dismutase (SOD), and guaiacol peroxidase (POD) activities were determined to evaluate oxidative damage. Results Drought treatment (8% PEG) caused a significant increase in TBARS levels as well as a marked decrease in the non-enzymatic (GSH and AS) and enzymatic (CAT, SOD, and POD) antioxidant defense systems. Pre-treatment with IAA prevented the alterations of stress parameters caused by drought, while treatment with IAA alone did not produce changes in TBARS levels, or GSH and AS contents. Moreover, the activities of the classical enzymes involved in the enzymatic defense system (SOD, CAT, and POD) remained similar to control values. Furthermore, this hormone could enhance HO-1 activity (75% with respect to controls), and this increase was positively correlated with protein content as well as gene expression. The direct participation of HO-1 as an antioxidant enzyme was established by performing experiments in the presence of Zn-protoporphyrin IX, a well-known irreversible inhibitor of this enzyme. It was also demonstrated that HO-1 is modulated by NO, as shown by experiments performed in the presence of an NO donor (sodium nitroprusside), an NO scavenger (2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide), or an NO synthesis inhibitor (N-nitro-l-arginine methyl ester, NAME). Discussion It is concluded that IAA is responsible, at least in part, for the protection against oxidative stress caused by drought in soybean plants through the modulation of NO levels which, in turn, enhances HO-1 synthesis and activity.

Symbiotic association between soybean plants and Bradyrhizobium japonicum develops oxidative stress and heme oxygenase-1 induction at early stages.

We have previously demonstrated that the induction of heme oxygenase-1 (HO-1) (EC 1.14.99.3) plays a protective role against oxidative stress in leaves and nodules of soybean plants subjected to cadmium, UV-B radiation, and salt stress. Here, we investigated HO-1, localization and their relationship with oxidative stress in different growth stages of soybean plants roots inoculated with Bradyrhizobium japonicum (3, 5, 7, 10, and 20 days post-inoculation) and nodules. After 7 days of inoculation, we observed a 70% increase in thiobarbituric acid-reactive substances that correlates with an enhancement in the gene expression of HO-1, catalase, and superoxide dismutase. Furthermore, the inhibition of HO-1 activity by Zn-protoporphyrin IX produced an increase in lipid peroxidation and a decrease in glutathione content suggesting that, in this symbiotic process, HO-1 may act as a signal molecule that protects the root against oxidative stress. We determined, for the first time, the tissular localization of HO-1 in nodules by electron-microscope examination. These results undoubtedly demonstrated that this enzyme is localized only in the plant tissue and its overexpression may play an important role as antioxidant defense in the plant. Moreover, we demonstrate that, in roots, HO-1 is induced by oxidative stress produced by inoculation of B. japonicum and exerts an antioxidant response against it.

Losartan exerts renoprotection through NAD(P)H oxidase downregulation in a renovascular model of hypertension.

This study was performed to provide insight into the regulatory role of angiotensin II and arterial pressure on the activity of antioxidant enzymes and oxidative stress generation in the hypertensive kidney from an experimental animal model of renovascular hypertension. Aortic coarcted and sham-operated rats received vehicle, losartan or minoxidil in their drinking water. After 7 d of treatment rats were sacrificed; hypertensive kidneys were excised, and the NAD(P)H oxidase subunits expression, TBARS production, glutathione level and the activity of heme oxygenase-1 and classical antioxidant enzymes, were evaluated. Losartan administration significantly reduced oxidative stress generation decreasing NAD(P)H oxidase expression, independently of the drop in arterial pressure. On the other hand, antioxidant enzymes were regulated by arterial pressure and they were not implicated in kidney protection against oxidative damage. Findings here reported strongly suggest that clinical therapeutics with the Ang II type 1 receptor blocker prevents oxidative stress generation and may attenuate the kidney oxidative damage in the renovascular hypertension. We hypothesize that the pathway followed by the Ang II blocker to achieve this renoprotection, though independent of the primary antioxidant enzymatic system, depends on NAD(P)H oxidase downregulation.

Inhibition of root growth and polyamine metabolism in sunflower (Helianthus annuus) seedlings under cadmium and copper stress.

Although sunflower is usually regarded as a highly tolerant crop, impairment of root growth at initial stages of plant development may result in poor crop establishment and higher susceptibility to pathogen attack. In order to evaluate if Cd2+ and Cu2+ may impact on sunflower germination and initial root development, a pot experiment under controlled conditions was carried out. Possible involvement of polyamine metabolism in sunflower response to these stressors was also investigated. Although Cd2+ and Cu2+ treatments affect neither seed germination nor radical emergence, sunflower seedlings grown in the presence of these heavy metals showed significant inhibition of root growth, being this inhibition greater for Cd2+. Both metals caused significant increases in proline contents at the highest concentrations tested (0.5 and 1 mM), and these increments were more pronounced for Cd2+ treatments, especially between days 3 and 10. Metals also increased putrescine (Put) contents at all concentrations assayed from the seventh day onward, causing no variations on this polyamine time-course pattern. Spermine and spermidine contents, however, were increased only by 1 mM Cd2+. Arginine decarboxylase seems to have been the enzyme responsible for Put increases under both metal treatments. This work demonstrates that initial root growth of sunflower seedlings may be significantly impaired in Cd2+ or Cu2+ contaminated soils. It also shows that polyamines are key biological compounds, which are probably involved in signaling pathways triggered under stress environmental conditions.

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, p22(phox) and p47(phox), 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.

Modifications in catalase activity and expression in developing sunflower seedlings under cadmium stress.

Catalase (CAT) dismutates the reactive oxygen species H2O2 into water and dioxygen and in plants; it is located in peroxisomes and glyoxysomes. In the present study, we investigated the effect of cadmium (a well-known oxidative stress inducer) on catalase in roots and cotyledons of developing sunflower seedlings, at 10 microM and 100 microM. Although germination was unaltered after 48 h of exposure to 100 microM Cd2+, root length was significantly reduced. CAT activity was also significantly reduced, but this activity was completely restored (10 microM treatment) or even enhanced (100 microM treatment) 24 h later. Although CAT protein abundance remained similar to control in roots and cotyledons of Cd-treated seedlings, cadmium produced CAT protein oxidation, indicating that the mechanism of CAT inactivation by Cd2+ involves oxidation of the protein structure. The transcripts of the four genes described for sunflower (CATA1 to CATA4) increased after cadmium treatment; CATA1 and CATA2 were the most overexpressed in cotyledon and root, respectively. The differential expression of catalase genes in sunflower seedlings under Cd stress might be related to the synthesis of CAT isoforms less sensitive to oxidation, which would prevent enzyme inactivation and H2O2 accumulation.

Heme oxygenase and catalase gene expression in nodules and roots of soybean plants subjected to cadmium stress.

Heme oxygenase (HO, EC 1.14.99.3) catalyses the oxidative conversion of heme to biliverdin IX alpha (BV) with the concomitant released of carbon monoxide and iron. Recently, plant HOs have been involved in the defence mechanism against oxidative stress. The goal of this study was to evaluate the time-course of HO-1 and catalase (CAT, EC 1.11.1.6) gene expressions in nodules and roots of soybean plants subjected to Cd treatment. No significant changes were observed up to 24 h. After 48 h of 200 microM Cd exposure, an up-regulation of HO-1 mRNA (110%) occurred in nodules. On the other hand, a down-regulation was found in roots (39%). While there was an augmentation in CAT transcript levels (30%) in nodules, an important diminution (52%) was evidenced in roots. Changes observed in gene expression were also found in protein levels and activities. These data suggest that an induction of CAT and HO-1 occurred in nodules as a response of cell protection against oxidative damage. However, after 72 h treatment, a down-regulation of HO-1 mRNA was found either in nodules or in roots (78% and 94%, respectively), while a similar response was evidenced for CAT (40% and 83%, respectively). These results are consistent with our previous findings suggesting that oxidative stress produced by Cd were more pronounced in roots than in nodules of soybean plants. Moreover, this behaviour could explain the major viability observed in nodules respect to roots, and provide a new insight into the processes involved in the antioxidant defence system in plant tissues.

Mechanism of CATA3 induction by cadmium in sunflower leaves.

One of the main antioxidant enzymes, catalase (CAT, EC 1.11.1.6), is capable of catalyzing the dismutation of H(2)O(2). This enzyme is involved in signal transduction pathway in plants, controlling the cellular level of this reactive oxygen species. Four different genes, CATA1-CATA4, were identified in Helianthus annuus L. cotyledons. Incubation of sunflower leaf discs with 300 and 500 microM CdCl(2) under light conditions increased CATA3 transcript level. However, it was not induced by Cd(2+) in etiolated plants. This Cd(2+)-induced increase was reverted by adding 10mM ascorbate. Treatments with 0.4 and 10 microM rose bengal (a generator of (1)O(2)) did not activate CATA3, but 10 microM methyl viologen (an enhancer of O(2)(-) production) and 10 mM H(2)O(2) increased its expression. In isolated chloroplasts, Cd(2+) and methyl viologen produced oxidation of the probe 2',7'-dichlorofluorescein diacetate indicating ROS formation. Besides, Cd(2+) treatment of leaf discs under light decreased CAT activity and increased carbonyl groups content, thus suggesting that enzyme inactivation could be due - in part - to a protein oxidation. These results indicate that light is involved in Cd(2+)-induced CATA3 enhancement, which leads to the synthesis of CAT isoforms less sensible to oxidation, and that chloroplast might be the main source of ROS responsible for this process.

20S proteasome and accumulation of oxidized and ubiquitinated proteins in maize leaves subjected to cadmium stress.

In order to examine the possible involvement of the 20S proteasome in degradation of oxidized proteins, the effects of different cadmium concentrations on its activities, protein abundance and oxidation level were studied using maize (Zea mays L.) leaf segments. The accumulation of carbonylated and ubiquitinated proteins was also investigated. Treatment with 50 microM CdCl(2) increased both trypsin- and PGPH-like activities of the 20S proteasome. The incremental changes in 20S proteasome activities were probably caused by an increased level of 20S proteasome oxidation, with this being responsible for degradation of the oxidized proteins. When leaf segments were treated with 100 microM CdCl(2), the chymotrysin- and trypsin-like activities of the 20S proteasome also decreased, with a concomitant increase in accumulation of carbonylated and ubiquitinated proteins. With both Cd(2+) concentrations, the abundance of the 20S proteasome protein remained similar to the control experiments. These results provide evidence for the involvement of this proteolytic system in cadmium-stressed plants.

Cadmium induced oxidative stress in soybean plants also by the accumulation of delta-aminolevulinic acid.

Cadmium toxicity has been extensively studied in plants, however its biochemical mechanism of action has not yet been well established. To fulfil this objective, four-weeks-old soybean nodulated plants were treated with 200 muM Cd(2+) for 48 h. delta-aminolevulinic acid dehydratase (ALA-D, E.C. 4.2.1.24) activity and protein expression, as well as delta-aminolevulinic acid (ALA) and porphobilinogen (PBG) concentrations were determined in nodules, roots and leaves. In vitro experiments carried out in leaves were performed using leaf discs to evaluate the oxidant and antioxidant properties of ALA and S-adenosyl-L: -methinone (SAM), respectively. Oxidative stress parameters such as thiobarbituric acid reactive substances (TBARS) and GSH levels as well as superoxide dismutase (SOD, E.C. 1.15.1.1), and guaiacol peroxidase (GPOX, E.C. 1.11.1.7) were also determined. Cadmium treatment caused 100% inhibition of ALA-D activity in roots and leaves, and 72% inhibition in nodules whereas protein expression remained unaltered in the three studied tissues. Plants accumulated ALA in nodules (46%), roots (2.5-fold) and leaves (104%), respect to controls. From in vitro experiments using leaf discs, exposed to ALA or Cd(2+), it was found that TBARS levels were enhanced, while GSH content and SOD and GPOX activities and expressions were diminished. The protective role of SAM against oxidative stress generated by Cd(2+) and ALA was also demonstrated. Data presented in this paper let us to suggest that accumulation of ALA in nodules, roots and leaves of soybean plants due to treatment with Cd(2+) is highly responsible for oxidative stress generation in these tissues.

Glutathione reductase activity and isoforms in leaves and roots of wheat plants subjected to cadmium stress.

The behavior of glutathione reductase (GR, EC 1.6.4.2) activity and isoforms were analyzed in wheat (Triticum aestivum L.) leaves and roots exposed to a chronic treatment with a toxic cadmium (Cd) concentration. A significant growth inhibition (up to 55%) was found in leaves at 7, 14 and 21 days, whereas roots were affected (51%) only after three weeks. Wheat plants grown in the presence of 100microM Cd showed a time-dependent accumulation of this metal, with Cd concentration being 10-fold higher in roots than in leaves. Nevertheless, lipid peroxidation was augmented in leaves in all experiments, but not in roots until 21 days. Cadmium treatment altered neither the GR activity nor the isoform pattern in the leaves. However, GR activity increased 111% and 200% in roots at 7 and 14 days, respectively, returning to control levels after 21 days. Three GR isoforms were found in roots of control and treated plants, two of which were enhanced by Cd treatment at 7 and 14 days, as assessed by activity staining on native gels. The changes in the isoform pattern modified the global kinetic properties of GR, thereby decreasing significantly (2.5-fold) the Michaelis constant (K(m)) value for oxidized glutathione. Isozyme induction was not associated with an enhancement of GR mRNA and protein expression, indicating that post-translational modification could occur. Our data demonstrated that up-regulation of GR activity by the induction of distinctive isoforms occurs as a defense mechanism against Cd-generated oxidative stress in roots.

Polyamines and heavy metal stress: the antioxidant behavior of spermine in cadmium- and copper-treated wheat leaves.

Polyamine metabolism, as well as spermine (Spm) antioxidant properties, were studied in wheat leaves under Cd2+ or Cu2+ stress. The oxidative damage produced by both metals was evidenced by an increased of thiobarbituric acid reactive substances (TBARS) and a significant decrease in glutathione under both metal treatments. Ascorbate peroxidase (APOX) and glutathione reductase (GR) activities were reduced by both metals to values ranging from 30% to 64% of the control values. Conversely, copper produced a raise in superoxide dismutase activity. The high putrescine (Put) content detected under Cd2+ stress (282% over the control) was induced by the increased activity of both enzymes involved in Put biosynthesis, arginine decarboxylase (ADC) and ornithine decarboxylase (ODC). However, only ODC activity was increased in wheat leaves subjected to Cu2+ stress, leading to a lower Put rise (89% over the controls). Spermidine (Spd) content was not affected by metal treatments, while Spm was significantly reduced. Pretreatment with Spm completely reverted the metals-induced TBARS increase whereas metals-dependent H2O2 deposition on leaf segments (revealed using diaminobenzidine), was considerably reduced in Spm pretreated leaf segments. This polyamine failed to reverse the depletion in APOX activity and glutathione (GSH) content produced by Cd2+ and Cu2+, although it showed an efficient antioxidant behavior in the restoration of GR activity to control values. These results suggest that Spm could be exerting a certain antioxidant function by protecting the tissues from the metals-induced oxidative damage, though this effect was not enough to completely avoid Cd2+ and Cu2+ effect on certain antioxidant enzymes, though the precise mechanism of protection still needs to be elucidated.

Behaviour of the anti-oxidant defence system and heme oxygenase-1 protein expression in fructose-hypertensive rats.

1. Addition of fructose to a rat diet for various periods of time leads to hypertension, hyperinsulinaemia and dyslipidaemia and provides a model for testing oxidative stress parameters in the animals. 2. In the present study, oxidative stress generation, the soluble and enzymatic defence system and heme oxygenase-1 (HO-1) protein expression were investigated in the heart, liver and kidney of rats fed fructose for a period of 1 or 8 months. 3. Compared with the control group, fructose-hypertensive rats showed increased in lipid peroxidation only in the heart after both 1 and 8 months of fructose treatment. Changes in the behaviour of the soluble and enzymatic defence system and HO-1 protein expression were different depending on the organ. Increased or unaltered activities of anti-oxidant enzymes were found in the liver and kidney, respectively. Induction of HO-1 prevented the generation of oxidative stress in the liver, where the activity of anti-oxidant defence enzymes was not reduced. Increased expression of HO-1 protein was not able to prevent the generation of oxidative stress in the heart, where fructose treatment diminished the activity of anti-oxidant enzymes. 4. The results of the present study demonstrate that upregulation of HO-1 may prevent the generation of oxidative stress only when the anti-oxidant defence system is still operative.

Effect of different metals on protease activity in sunflower cotyledons

Proteases are crucial for living cells and play a role in plant cell adaptation to environmental conditions. Oxidative stress produced oxidized proteins which are selectively degraded by proteases. To understand the role of proteolysis in response to metal stress, sunflower plants (a plant suitable for phytoremediation) were treated with 100 µM of CdCl2, CuCl2, AlCl3, CoCl2, PbCl2, CrCl3, NiCl2, HgCl2 or ZnCl2. Changes in protease activity, gelatinase profile and protein oxidation were examined in sunflower cotyledons. Our results indicate that this tissue has mainly acid proteases belonging to different classes. Although all metals (except Zn) increased protein oxidation (62, 57, 112, 74, 74, 68, 64 and 40 percent for Pb, Al, Ni, Cd, Hg, Co, Cr and Cu over the control), they altered proteolysis in different ways. Pb, Al and Ni treatment decreased protease activity 22, 28 and 30 percent respect to control while Cd and Hg increased this activity in 23 and 27 percent. In Zn, Cu and Co treatments protease activity remained similar to control treatment. These results indicate that different proteases are involved in plant defence against metal toxicity. However, the identification of specific oxidized proteins involved in this process and the metal effect on class specific proteases should provide greater information.

Proteolytic system in sunflower (Helianthus annuus L.) leaves under cadmium stress.

The effect of oxidative stress induced by cadmium on growth parameters and on the balance between protein synthesis and degradation was studied in sunflower (Helianthus annuus L.) leaves. Plants were germinated for 10 days and then transferred to hydroponic medium devoid (control) or containing 100, 200 and 300µM CdCl2. Analyses were performed between days 0 and 4 of Cd-treatment. All Cd(2+) concentrations significantly reduced leaf area and, fresh and dry weight, but leaf relative water content only decreased with 200 and 300µM Cd(2+). Control and treated plants had similar soluble protein content and showed the same rate of soluble protein labeling under the assay conditions. Although protease activity increased with cadmium treatment, proteasome activity was significantly inhibited. Expression of 20S proteasome remained similar to controls in cadmium treated plants. Cadmium caused an increase in ubiquitin-conjugated proteins and carbonyl groups content of treated plants, compared to control values. Cadmium induced an increase in protease specific activity; nevertheless, this increase was not relevant enough to avoid accumulation of oxidized proteins. Oxidation of proteins is one of the most important effects of cadmium treatment. The results presented here provide evidence for the role of the proteolytic system in sunflower plants subjected to cadmium stress.

Glutamine is highly effective in preventing in vivo cobalt-induced oxidative stress in rat liver.

AIM: To evaluate the in vivo effect of glutamine on cobalt-generated oxidative stress and (HO-1) induction in rat liver. METHODS: Fasted female Wistar rats received a single injection of cobalt chloride (375 micromol/kg body weight) and then were killed at different times. Lipid peroxidation and soluble and enzymatic antioxidant defense system (reduced glutathione (GSH), catalase (CAT), glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD)) were measured in liver homogenates. Ferritin and ferritin iron contents as well as heme oxygenase-1 (HO-1) activity and expression were also determined. The antioxidant properties of glutamine (Gln) were also evaluated. RESULTS: Cobalt chloride increased lipid peroxidation (50% over control values) 1 h after treatment. GSH reached a minimum at 3 h (40%) increasing thereafter. Twelve hours after CoCl2 injection, the antioxidant enzymes CAT, GSH-Px and SOD also diminished by about 30%. Heme oxygenase-1 induction was observed 6 h after treatment reaching a maximum value of 14-fold over the controls, 12 h after cobalt treatment. A 1.7-fold increase in ferritin and ferritin-bound iron 24 h after treatment were also obtained. Administration of glutamine (300 mg/kg body weight) by gavage 24 h before CoCl2 treatment entirely prevented the increase in thiobarbituric acid reactive substances (TBARS) content, the decrease in GSH levels, and partially reverted heme oxygenase-1 induction. CONCLUSION: These results suggested that a natural product such as glutamine prevents glutathione depletion and consequently heme oxygenase induction.

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 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 analyzed. RESULTS: Treatment with 200 microM 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 Cd produced the overexpression of heme oxygenase-1, as well as a 10-fold enhancement of its activity. Co-administration of biliverdin (10 microM) 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.

Bilirubin is highly effective in preventing in vivo delta-aminolevulinic acid-induced oxidative cell damage.

Delta-aminolevulinic acid (ALA), precursor of heme, accumulates in a number of organs, particularly in liver of patients with acute porphyrias or lead intoxication. This study characterizes the involvement of bilirubin as an antioxidant in a chronic intoxication with ALA. Female Wistar rats were injected intraperitoneally a daily dose of 40 mg ALA/body wt., during 10 days. A marked increase in lipid peroxidation and a decrease in GSH content were observed 24 h after the last injection of ALA. The activities of liver antioxidant enzymes, superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase were also diminished. ALA synthase (ALA-S) and heme oxygenase-1 were induced. Both ALA dehydratase (ALA-D) and porphobilinogenase (PBG-ase) activities were inhibited. Administration of bilirubin (5 mmol/kg body wt.) 2 h before ALA treatment entirely prevented the effects of ALA. Co-administration of ALA and Sn-protoporphyrin IX (Sn-PPIX; 100 microg/body wt., i.p.), a potent inhibitor of heme oxygenase, completely abolished its induction and provoked a marked decrease in liver GSH levels as well as an increase in lipid peroxidation. These results add further support to the proposal assigning bilirubin a key protective role against oxidative damage here induced by ALA.

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.