RESUMEN
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.
Asunto(s)
Glycine max/química , Hemo Oxigenasa (Desciclizante)/metabolismo , Ácidos Indolacéticos/química , Estrés Oxidativo , Antioxidantes/metabolismo , Ácido Ascórbico/metabolismo , Sequías , Glutatión/metabolismo , Ácidos Indolacéticos/metabolismo , Peroxidación de Lípido , Óxido Nítrico/metabolismo , Polietilenglicoles/química , Glycine max/enzimología , Estrés Fisiológico , Sustancias Reactivas al Ácido Tiobarbitúrico/metabolismoRESUMEN
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.
Asunto(s)
Ácido Aminolevulínico/farmacología , Antioxidantes/farmacología , Catalasa/metabolismo , Glycine max/metabolismo , Hemo-Oxigenasa 1/metabolismo , Ácido Aminolevulínico/análisis , Biliverdina/farmacología , Frío , Estrés Oxidativo , Hojas de la Planta/química , Glycine max/efectos de los fármacos , Agua/análisisRESUMEN
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.
Asunto(s)
Angiotensina II/fisiología , Cardiomegalia/etiología , Hipertensión Renovascular/complicaciones , Estrés Oxidativo , Animales , Presión Sanguínea , Catalasa/metabolismo , Glutatión/análisis , Hipertensión Renovascular/metabolismo , Losartán/farmacología , Masculino , NADPH Oxidasas/metabolismo , Ratas , Ratas Wistar , Superóxido Dismutasa/metabolismoRESUMEN
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.
Asunto(s)
Cadmio/farmacología , Catalasa/genética , Glycine max/enzimología , Hemo Oxigenasa (Desciclizante)/genética , Estrés Oxidativo , Proteínas de Plantas/genética , Raíces de Plantas/enzimología , Antioxidantes/metabolismo , Catalasa/metabolismo , Inducción Enzimática , Regulación de la Expresión Génica de las Plantas , Hemo Oxigenasa (Desciclizante)/metabolismo , Proteínas de Plantas/metabolismo , Raíces de Plantas/efectos de los fármacos , Glycine max/anatomía & histología , Glycine max/efectos de los fármacosRESUMEN
Heme oxygenase (HO, EC 1.14.99.3) catalyzes the oxidative conversion of heme to biliverdin IXalpha with the concomitant release of carbon monoxide and iron. Recently, HO has been involved in the protection against oxidative stress in plants. The fact that nitric oxide (NO), an endogenous signaling molecule in animals and plants mediates responses to abiotic and biotic stresses, prompted us to study whether this molecule could modulate HO-1 gene transcription. To fulfill this objective leaves of soybean (Glycine max L.) plants were stimulated with Cd, employing an acute intoxication model. Cadmium caused dehydration, chlorophyll loss and ion leakage. Semi-quantitative RT-PCR analysis showed no augmentation of HO-1 transcript levels with respect to controls. Pretreatment with 100 microM sodium nitroprussiate (SNP), a well-known NO donor, prevented the effects caused by Cd. When the HO-1 mRNA levels were analyzed, a significant augmentation (54%) was observed with respect to Cd-treated plants. On the other hand, 50 or 300 microM SNP did not fully prevent the effects elicited by Cd. When HO-1 transcript levels were analyzed, no significant enhancement or a down-regulation was observed. The potassium salt of 2-(4-carboxylphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO), a specific NO scavenger, arrested NO-mediated protective effects against to Cd-induced oxidative damage. These data provide an understanding of one of the possible roles that NO can play against an oxidative insult. NO is cytoprotective depending on its concentration, and it was further demonstrated that this protection could be, at least in part, mediated by an enhancement of HO-1 mRNA, as it happens with genes associated with the antioxidant defense system.
Asunto(s)
Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Glycine max/efectos de los fármacos , Hemo Oxigenasa (Desciclizante)/genética , Óxido Nítrico/farmacología , Hojas de la Planta/efectos de los fármacos , Hojas de la Planta/enzimología , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Glycine max/enzimologíaRESUMEN
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.
Asunto(s)
Ácido Aminolevulínico/metabolismo , Cadmio/farmacología , Regulación Enzimológica de la Expresión Génica , Antioxidantes/metabolismo , Cadmio/metabolismo , Cadmio/toxicidad , Glutatión/metabolismo , Estrés Oxidativo , Hojas de la Planta/metabolismo , Raíces de Plantas , Plantas/metabolismo , S-Adenosilmetionina/metabolismo , Glycine max , Superóxido Dismutasa/metabolismo , Sustancias Reactivas al Ácido Tiobarbitúrico/metabolismoRESUMEN
Ultraviolet-B (UV-B) radiation has a negative impact on plant cells, and leads to the generation of reactive oxygen species (ROS). Heme oxygenase (HO, EC 1.14.99.3) plays a protective role against oxidative stress in mammals, but little is known about this issue in plants. Here, we report for the first time the response of HO in leaves of soybean (Glycine max L.) plants subjected to UV-B radiation. Under 7.5 and 15 kJ m(-2 )UV-B doses, HO, catalase (CAT, EC 1.11.1.6) and ascorbate peroxidase (APX, EC 1.11.1.11) activities were increased and the production of thiobarbituric acid reactive substances (TBARS) regain control values after 4 h of plant recuperation. Treatment with 30 kJ m(-2) UV-B provoked a decrease in these antioxidant enzyme activities. Immunoblot analysis showed a 4.3 and 3.7-fold increase in HO-1 protein expression after irradiation with 7.5 and 15 kJ m(-2), respectively. HO-1 transcript levels were enhanced (up to 77%) at these doses, as assessed by semi-quantitative RT-PCR. These data demonstrated that increased HO activity was associated with augmented protein expression and transcript levels. Plants pre-treated with the antioxidant ascorbic acid did not show the UV-B-induced up-regulation of HO-1 mRNA, but hydrogen peroxide treatment could mimic this reaction. Our results indicate that HO is up-regulated in a dose-depending manner as a mechanism of cell protection against oxidative damage and that such response occurred as a consequence of HO-1 mRNA enhancement involving ROS.
Asunto(s)
Glycine max/efectos de la radiación , Hemo Oxigenasa (Desciclizante)/efectos de la radiación , Especies Reactivas de Oxígeno/efectos de la radiación , Rayos Ultravioleta , Regulación hacia Arriba , Ascorbato Peroxidasas , Ácido Ascórbico/farmacología , Catalasa/metabolismo , Catalasa/efectos de la radiación , Expresión Génica , Hemo Oxigenasa (Desciclizante)/metabolismo , Peróxido de Hidrógeno/farmacología , Peroxidación de Lípido/efectos de la radiación , Peroxidasas/metabolismo , Peroxidasas/efectos de la radiación , Hojas de la Planta/metabolismo , Hojas de la Planta/efectos de la radiación , Especies Reactivas de Oxígeno/metabolismo , Glycine max/metabolismo , Factores de Tiempo , Transcripción Genética/efectos de los fármacosRESUMEN
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.
Asunto(s)
Antioxidantes/farmacología , Glycine max/química , Hemo Oxigenasa (Desciclizante)/farmacología , Raíces de Plantas/química , Antioxidantes/metabolismo , Biliverdina/farmacología , Cloruro de Cadmio/antagonistas & inhibidores , Cloruro de Cadmio/farmacología , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Enzimas/efectos de los fármacos , Enzimas/metabolismo , Glutatión/efectos de los fármacos , Hemo Oxigenasa (Desciclizante)/antagonistas & inhibidores , Hemo Oxigenasa (Desciclizante)/metabolismo , Hemo-Oxigenasa 1 , Raíces de Plantas/efectos de los fármacos , Raíces de Plantas/enzimología , Protoporfirinas/farmacología , Especies Reactivas de Oxígeno/metabolismo , Glycine max/efectos de los fármacos , Glycine max/enzimología , Sustancias Reactivas al Ácido Tiobarbitúrico/metabolismoRESUMEN
We have previously demonstrated that the induction of heme oxygenase-1 (EC 1.14.99.3) plays a protective role for mammalian cells against oxidative stress. Here, we investigated for the first time the possible role of heme oxygenase-1 as an antioxidant defense in leaves of soybean plants. Treatment with 200 microM Cd during 48 h caused a 70% increase in thiobarbituric acid reactive substances, whereas GSH decreased 67%, guaiacol peroxidase and total superoxide dismutase also inhibited 49% and 46%, respectively. Two hundred micromolar of Cd produced the overexpression of heme oxygenase-1, as well as a 4.5-fold enhancement of its activity. Administration of biliverdin partially prevented the effects caused by Cd. Pretreatment with Zn protoporphyrin IX, a potent inhibitor of heme oxygenase, expectedly decreased heme oxygenase-1 activity to half. When the inhibitor was given before Cd, it completely prevented the enzyme induction increasing the levels of oxidative stress parameters. Collectively, these results indicated that although plant heme oxygenases share little homology to heme oxygenases from non-plant species, they also play an important protective role against oxidative cell damage.
Asunto(s)
Antioxidantes/metabolismo , Glycine max/enzimología , Hemo Oxigenasa (Desciclizante)/metabolismo , Estrés Oxidativo/fisiología , Hojas de la Planta/enzimología , Especies Reactivas de Oxígeno/metabolismo , Biliverdina/farmacología , Cadmio/farmacología , Relación Dosis-Respuesta a Droga , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Hemo Oxigenasa (Desciclizante)/antagonistas & inhibidores , Oxidación-Reducción , Estrés Oxidativo/efectos de los fármacos , Hojas de la Planta/efectos de los fármacos , Glycine max/efectos de los fármacosRESUMEN
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.
Asunto(s)
Ácido Aminolevulínico/toxicidad , Antioxidantes/farmacología , Bilirrubina/farmacología , Hígado/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , 5-Aminolevulinato Sintetasa/metabolismo , Amoníaco-Liasas/antagonistas & inhibidores , Animales , Bilirrubina/administración & dosificación , Catalasa/metabolismo , Inducción Enzimática , Femenino , Glutatión/metabolismo , Glutatión Peroxidasa/metabolismo , Hemo Oxigenasa (Desciclizante)/metabolismo , Peroxidación de Lípido/efectos de los fármacos , Hígado/enzimología , Porfobilinógeno Sintasa/antagonistas & inhibidores , Ratas , Ratas Wistar , Superóxido Dismutasa/metabolismoRESUMEN
The in vivo effect of the known herbicide, paraquat, on both hepatic oxidative stress and heme metabolism was studied. A marked increase in lipid peroxidation and a decrease in reduced glutathione (GSH) content were observed 1 h after paraquat administration. The activity of liver antioxidant enzymes, superoxide dismutase, catalase and glutathione peroxidase was decreased 3 h after paraquat injection. Heme oxygenase-1 induction started 9 h after treatment, peaking at 15 h. delta-aminolevulinic acid synthase induction occurred once heme oxygenase had been enhanced, reaching its maximum (1.5-fold of control) at 16 h. delta-aminolevulinic acid dehydratase activity was 40% inhibited at 3 h showing a profile similar to that of GSH, while porphobilinogenase activity was not modified along the whole period of the assay. Administration of alpha-tocopherol (35 mmol/kg body weight) 2 h before paraquat treatment entirely prevented the increase in thiobarbituric acid reactive substances (TBARS) content, the decrease in GSH levels as well as heme oxygenase-1 and delta-aminolevulinic acid synthase induction. This study shows that oxidative stress produced by paraquat leads to an increase in delta-aminolevulinic acid synthase and heme oxygenase-1 activities, indicating that the herbicide affects both heme biosynthesis and degradation.
Asunto(s)
5-Aminolevulinato Sintetasa/efectos de los fármacos , Hemo Oxigenasa (Desciclizante)/efectos de los fármacos , Herbicidas/toxicidad , Hígado/enzimología , Paraquat/toxicidad , 5-Aminolevulinato Sintetasa/metabolismo , Animales , Antioxidantes/metabolismo , Enzimas/efectos de los fármacos , Enzimas/metabolismo , Femenino , Glutatión/metabolismo , Hemo/metabolismo , Hemo Oxigenasa (Desciclizante)/metabolismo , Hemo-Oxigenasa 1 , Hígado/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Ratas , Ratas Wistar , Sustancias Reactivas al Ácido Tiobarbitúrico/metabolismo , alfa-Tocoferol/farmacologíaRESUMEN
The effect of two different doses of angiotensin-(1-7) and angiotensin II on the oxidative stress generation was analyzed in rat kidney. Animals were injected intraperitoneally with a single dose of angiotensin-(1-7) or angiotensin II (20 or 50 nmol/kg body weight) and killed 3 h after injection. Production of thiobarbituric acid reactive substances (TBARS), measured as indicator of oxidative stress induction, was significantly increased in rat kidney after Ang-(1-7) administration up to 30% and 50% over controls, at 20 and 50 nmol/kg, respectively. Reduced glutathione (GSH), the most important soluble antioxidant defense in mammalian cells, showed a significant decrease of 13% and 20% at 20 and 50 nmol/kg of angiotensin-(1-7), respectively. When the antioxidant enzyme activities were determined, it was found that catalase activity was not altered by the assayed angiotensin-(1-7) doses while superoxide dismutase and glutathione peroxidase activities were significantly reduced by injection of 20 nmol/kg (34% and 13%, with respect to controls) and 50 nmol/kg of angiotensin-(1-7) (54% and 22%, respectively). In contrast, angiotensin II injections did not produce significant changes neither in TBARS levels nor in soluble and enzymatic defense parameters at the two doses used in this work. These results suggest that angiotensin-(1-7) is undoubtedly related to oxidative stress induction.