Role of reactive oxygen species and isoflavonoids in soybean resistance to the attack of the southern green stink bug.

Southern green stink bugs (Nezara viridula L.) are one of the major pests in many soybean producing areas. They cause a decrease in yield and affect seed quality by reducing viability and vigor. Alterations have been reported in the oxidative response and in the secondary metabolites in different plant species due to insect damage. However, there is little information available on soybean-stink bug interactions. In this study we compare the response of undamaged and damaged seeds by Nezara viridula in two soybean cultivars, IAC-100 (resistant) and Davis (susceptible), grown under greenhouse conditions. Pod hardness, H2O2 generation, enzyme activities in guaiacol peroxidase (GPOX), catalase (CAT) and superoxide dismutase (SOD) as well as lipoxygenase expression and isoflavonoid production were quantified. Our results showed a greater resistance of IAC-100 to pod penetration, a decrease in peroxide content after stink bug attack, and higher GPOX, CAT and SOD activities in seeds due to the genotype and to the genotype-interaction with the herbivory treatment. Induction of LOX expression in both cultivars and higher production of isoflavonoids in IAC-100 were also detected. It was then concluded that the herbivory stink bug induces pathways related to oxidative stress and to the secondary metabolites in developing seeds of soybean and that differences between cultivars hold promise for a plant breeding program.

Erratum to "Improvement of growth and yield of soybean plants through the application of non-thermal plasmas to seeds with different health status" [Heliyon Volume 5, Issue 4, April 2019, e01495].

[This corrects the article DOI: 10.1016/j.heliyon.2019.e01495.].

Improvement of growth and yield of soybean plants through the application of non-thermal plasmas to seeds with different health status.

Soybean (Glycine max (L.) Merrill) is a globally important crop, providing oil and protein. Diaporthe/Phomopsis complex includes seed-borne pathogens that affect this legume. Non-thermal plasma treatment is a fast, cost-effective and environmental-friendly technology. Soybean seeds were exposed to a quasi-stationary (50 Hz) dielectric barrier discharge plasma operating at atmospheric pressure air. Different carrying gases (O2 and N2) and barrier insulating materials were used. This work was performed to test if the effects of non-thermal plasma treatment applied to healthy and infected seeds persist throughout the entire cycle of plants. To this aim, lipid peroxidation, activity of catalase, superoxide dismutase and guaiacol peroxidase, vegetative growth and agronomic traits were analysed. The results here reported showed that plants grown from infected seeds did not trigger oxidative stress due to the reduction of pathogen incidence in seeds treated with cold plasma. Vegetative growth revealed a similar pattern for plants grown from treated seeds than that found for the healthy control. Infected control, by contrast, showed clear signs of damage. Moreover, plasma treatment itself increased plant growth, promoted a normal and healthy physiological performance and incremented the yield of plants. The implementation of this technology for seeds treatment before sowing could help reducing the use of agrochemicals during the crop cycle.

Enhancement of ionizing radiation response by histamine in vitro and in vivo in human breast cancer.

The radioprotective potential of histamine on healthy tissue has been previously demonstrated. The aims of this work were to investigate the combinatorial effect of histamine or its receptor ligands and gamma radiation in vitro on the radiobiological response of 2 breast cancer cell lines (MDA-MB-231 and MCF-7), to explore the potential molecular mechanisms of the radiosensitizing action and to evaluate the histamine-induced radiosensitization in vivo in a triple negative breast cancer model. Results indicate that histamine significantly increased the radiosensitivity of MDA-MB-231 and MCF-7 cells. This effect was mimicked by the H1R agonist 2-(3-(trifluoromethyl)phenyl)histamine and the H4R agonists (Clobenpropit and VUF8430) in MDA-MB-231 and MCF-7 cells, respectively. Histamine and its agonists enhanced radiation-induced oxidative DNA damage, DNA double-strand breaks, apoptosis and senescence. These effects were associated with increased production of reactive oxygen species, which correlated with the inhibition of catalase, glutathione peroxidase and superoxide dismutase activities in MDA-MB-231 cells. Histamine was able also to potentiate in vivo the anti-tumoral effect of radiation, increasing the exponential tumor doubling time. We conclude that histamine increased radiation response of breast cancer cells, suggesting that it could be used as a potential adjuvant to enhance the efficacy of radiotherapy.

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.

Heme oxygenase-1 overexpression fails to attenuate hypertension when the nitric oxide synthase system is not fully operative.

Heme oxygenase (HO) is an enzyme that is involved in numerous secondary actions. One of its products, CO, seems to have an important but unclear role in blood pressure regulation. CO exhibits a vasodilator action through the activation of soluble guanylate cyclase and the subsequent production of cyclic guanosine monophosphate (cGMP). The aim of the present study was to determine whether pathological and pharmacological HO-1 overexpression has any regulatory role on blood pressure in a renovascular model of hypertension. We examined the effect of zinc protoporyphyrin IX (ZnPP-IX) administration, an inhibitor of HO activity, on mean arterial pressure (MAP) and heart rate in sham-operated and aorta-coarcted (AC) rats and its interaction with the nitric oxide synthase (NOS) pathway. Inhibition of HO increased MAP in normotensive rats with and without hemin pretreatment but not in hypertensive rats. Pretreatment with NG-nitro-L-arginine methyl ester blocked the pressor response to ZnPP-IX, suggesting a key role of NOS in the cardiovascular action of HO inhibition. In the same way, AC rats, an experimental model of hypertension with impaired function and low expression of endothelial NOS (eNOS), did not show any cardiovascular response to inhibition or induction of HO. This finding suggests that eNOS was necessary for modulating the CO response in the hypertensive group. In conclusion, the present study suggests that HO regulates blood pressure through CO only when the NOS pathway is fully operative. In addition, chronic HO induction fails to attenuate the hypertensive stage induced by coarctation as a consequence of the impairment of the NOS pathway.

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.

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.

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.

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.

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.

The effect of nitric oxide on heme oxygenase gene expression in soybean leaves.

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.

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.

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.

Heme oxygenase exerts a protective role against oxidative stress in soybean leaves.

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.

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.