Depolarization of Cell Membrane Potential during Trans-Plasma Membrane Electron Transfer to Extracellular Electron Acceptors in Iron-Deficient Roots of Phaseolus vulgaris L

Transfer of electrons from the cytosol of bean (Phaseolus vulgaris L.) root cells to extracellular acceptors such as ferricyanide and Fe^IIIEDTA causes a rapid depolarization of the membrane potential. This effect is most pronounced (30-40 millivolts) with root cells of Fe-deficient plants, which have an increased capacity to reduce extracellular ferric salts. Ferrocyanide has no effect. In the state of ferricyanide reduction, H⁺ (1H⁺/2 electrons) and K⁺ ions are excreted. The reduction of extracellular ferric salts by roots of Fe-deficient bean plants is driven by cellular NADPH (Sijmons, van den Briel, Bienfait 1984 Plant Physiol 75: 219-221). From this and from the membrane potential depolarization, we conclude that trans-plasma membrane electron transfer from NADPH is the primary process in the reduction of extracellular ferric salts.     

Effects of Ca(NO<Subscript>3</Subscript>)<Subscript>2</Subscript> stress on oxidative damage,antioxidant enzymes activities and polyamine contents in roots of grafted and non-grafted tomato plants

The effects of Ca(NO₃)₂ stress on biomass production, oxidative damage, antioxidant enzymes activities and polyamine contents in roots of grafted and non-grafted tomato plants were investigated. Results showed that when exposed to 80 mM Ca(NO₃)₂ stress, the biomass production reduction in non-grafted plants was more significant than that of grafted plants. Under Ca(NO₃)₂ stress, superoxide anion radical (O₂•⁻) producing rate, hydrogen peroxide (H₂O₂) and malondialdehyde (MDA) contents of non-grafted plants roots were significantly higher than those of grafted plants, however, nitrate (NO₃ ⁻), ammonium (NH₄ ⁺) and proline contents, superoxide dismutase (SOD, EC1.15.1.1), peroxidase (POD, EC1.11.1.7), catalase (CAT, EC1.11.1.6) and arginine decarboxylase (ADC, EC 4.1.1.19) activities of grafted plants roots were significantly higher than those of non-grafted plants. Regardless of stress, free, conjugated and bound polyamine contents in roots of grafted plants were significantly higher than those of non-grafted plants. The possible roles of antioxidant enzymes, prolines and polyamines in adaptive mechanism of tomato roots to Ca(NO₃)₂ stress were discussed. Gu-Wen Zhang and Zheng-Lu Liu contributed equally to this work.     

Cytosolic NADPH is the electron donor for extracellular fe reduction in iron-deficient bean roots

Pyridine nucleotides were determined in lateral roots of iron-deficient and iron-sufficient Phaseolus vulgaris L. cv Prelude. In iron-deficient plants, total NADP per gram fresh weight and the NADPH/NADP⁺ ratio were twice the values found in iron-sufficient plants. The NADPH/NADP⁺ ratio in iron-deficient plants was considerably lowered after a 2 minute incubation in 1 millimolar ferricyanide. Total NAD was not influenced by growth conditions and was mainly present in oxidized form.

These results indicate that NADPH is the electron donor for the high Fe^III reduction activity found in iron-deficient roots, a process that is part of the Fe-uptake mechanism.

     

Effects of various inhibitors on in vivo reduction by Plantago lanceolata L. roots

Roots of Plantago lanceolata L. showed an iron stress-induced increase in the rates of electron transport to the extracytoplasmatic acceptors FeEDTA and ferricyanide. No significant changes in the reduction of hexachloroiridate were observed with respect to the iron-nutritional status of the plants. The reduction activity of iron-deficient roots was inhibited by the translation inhibitor cycloheximide (CHM) and the amino acid analog p-fluorophenylalanine (FPA). In both cases, the reduction of FeEDTA and ferricyanide was affected to a different extent, providing evidence for enzyme heterogeneity. Resupply of FeEDTA to iron-deficient plants resulted in a qualitatively similar pattern of decrease in FeEDTA and ferricyanide reduction rates, although a longer time period was required for the decrease of the redox activity by iron resupply compared to the effect of inhibitors of protein synthesis.Inhibitors of the plasma membrane (PM)-bound H⁺-ATPase decreased the FeEDTA reduction activity of iron-deficient plants. In contrast, the reduction of ferricyanide and hexachloroiridate was not inhibited. Oxidation of ferrocyanide occurs in both iron-deficient and iron-sufficient plants at comparable rates. The reaction was decreased by the H⁺-ATPase inhibitor orthovanadate.The results are interpreted in terms of a simultaneous action of distinct redox systems in iron-deficient roots. The role of proton extrusion in the regulation of iron stress-induced electron transport is discussed.     

Oxidative Stress Induced in Sunflower Seedling Roots by Aqueous Dry Olive-Mill Residues

The contamination of soils with dry olive-mill residue can represent a serious problem as being an environmental stressor in plants. It has been demonstrated that inoculation of aqueous extract of olive oil-mill residue (ADOR) with saprobe fungi removes some phenolic compounds. In this paper we studied the effect of ADOR uninoculated or inoculated with saprobe fungi in sunflower seedling roots. The germination and root growth, O₂·^- generation, superoxide dismutase (SOD) and extracellular peroxidases (EC-POXs) activities, and the content of some metabolites involved in the tolerance of stress were tested. The roots germinated in ADOR uninoculated show a decrease in meristem size, resulting in a reduction of the root length and fresh weight, and in the number of layers forming the cortex, but did not alter the dry weight, protein and soluble amino acid content. ADOR caused the decreases in O₂·^- generation and EC-POX′s activities and protein oxidation, but enhanced SOD activity, lipid peroxidation and proline content. Fluorescence imaging showed that ADOR induced O₂·^- and H₂O₂ accumulation in the roots. The increase in SOD and the decrease in EC-POX′s activities might be involved in the enhancement of H₂O₂ content and lipid peroxidation. Control roots treated with ADOR for 10 min show an oxidative burst. Roots germinated in ADOR inoculated with saprobe fungi partially recovered normal levels of ROS, morphological characteristics and antioxidant activities. These results suggested that treatment with ADOR caused a phytotoxic effect during germination inducing an oxidative stress. The inoculation of ADOR with saprobe fungi limited the stress.     

Effect of zinc nutritional status on activities of superoxide radical and hydrogen peroxide scavenging enzymes in bean leaves

The effect of varied zinc (Zn) supply on the activities of superoxide dismutase (SOD), ascorbate (AsA) peroxidase, glutathione (GSSG) reductase, catalase and guaiacol peroxidase was studied in leaves of bean (Phaseolus vulgaris) plants grown for 15 days in nutrient solution. Zinc deficiency severely decreased plant growth and the leaf concentrations of soluble protein and chlorophyll. Resupply of Zn to deficient plants for up to 72h restored protein concentrations more rapidly than chlorophyll and plant growth. With the exception of guaiacol peroxidase, the activities of all enzymes were significantly decreased by Zn deficiency, in particular GSSG reductase and SOD. Within 72h of resupplying Zn to deficient plants, the enzyme activities reached the level of the Zn sufficient plants. The results indicate severe impairment in the ability of Zn-deficient leaves to enzymically scavenge O₂ ^- and H₂O₂. Consequences and reasons of this impairment are discussed in terms of photooxidation of chloroplast pigments and inhibition of the biosynthesis of the related scavenger enzyme proteins.     

Reactive oxygen species production and antioxidative defense in pea (Pisum sativum L.) root nodules after short-term aluminum treatment

Pea plants (Pisum sativum L.) were treated with 50???M aluminum chloride at pH 4.5 for 2 or 24?h at room temperature. Following treatment, root nodule Al uptake, the generation of reactive oxygen species (ROS, O ₂ ^?· and H₂O₂), and the activities of the antioxidant enzymes catalase (CAT), superoxide dismutase (SOD) and peroxidase (POX) were investigated. Aluminum accumulation was found chiefly in the apoplast of the nodule cortex, endodermis and meristem, while the formation of peroxide was detected in the nodule cortex, infection threads and bacteroidal tissue. Further, there were increased levels of superoxide in the meristem and bacteroidal tissue. The activity of SOD (EC 1.15.1.1) and POX (EC 1.11.1.7) increased in the Al-treated nodules and the roots of pea plants, whereas CAT (EC 1.11.1.6) activity decreased. The Al absorbed by the nodules induced ROS production. The POX and SOD are important ROS-scavengers in Al-stressed nodules.     

Recovery of bean plants from boron-induced oxidative damage by zinc supply

The effects of zinc on growth, boron uptake, lipid peroxidation, membrane permeability (MP), lypoxygenase (LOX) activity, proline and H₂O₂ accumulation, and the activities of major antioxidant enzymes (superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX)) in bean plants were investigated under greenhouse conditions. Treatments consisted of control, 20 mg/kg B, and 20 mg/kg B plus 20 mg/kg Zn. When the plants were grown with 20 mg/kg Zn, B toxicity was less severe. Zinc supplied to soil counteracted the deleterious effects of B on root and shoot growth. Excess B significantly increased and Zn treatment reduced B concentrations in shoot and root tissues. Applied Zn increased the Zn concentration in the roots and shoots. While the concentrations of H₂O₂ and proline were increased by B toxicity, their concentrations were decreased by Zn supply. Boron toxicity increased the MP, malondialdehyde content, and LOX activity in excised bean leaves. Applied Zn significantly ameliorated the membrane deterioration. Compared with control plants, the activity of SOD was increased while that of CAT was decreased and APX remained unchanged in B-stressed plants. However, application of Zn decreased the SOD and increased the CAT and APX activities under toxic B conditions. It is concluded that Zn supply alleviates B toxicity by preventing oxidative membrane damage. Published in Russian in Fiziologiya Rastenii, 2009, Vol. 56, No. 4, pp. 555–562. This text was submitted by the authors in English.     

Cholic acid changes defense response to oxidative stress in soybean induced by <Emphasis Type="Italic">Aspergillus niger</Emphasis>

The oxidative stress and antioxidant systems in soybean leaves and roots infected with plant pathogen Aspergillus niger were studied following treatment with different concentrations of cholic acid. Several oxidative stress parameters were analyzed: production of superoxide (O₂ ^·−) and hydroxyl radicals (^·OH), lipid peroxidation (LP), and superoxide dismutase (SOD; EC 1.15.1.1) activity, as well as the content of reduced glutathione (GSH). Results showed that inoculation with A. niger led to the increase of O₂ ^·− production and GSH quantities in leaves and ^·OH in roots. The highest activity of SOD occured in infected plants treated with cholic acid in concentrations of 40 and 60 mg L⁻¹ which ultimately led to a decrease in O₂ ^·− production. Inoculation with Aspergillus in combination with elevated cholic acid concentrations also increased ^·OH production which is correlated with increased LP. These results may support the idea of using cholic acid as an elicitor to trigger hypersensitive response in plant cells. Use of cholic acid may also actively contribute to soybean plants defense response against pathogen attack.     

Interrelationships between trans-Plasma Membrane Electron/Proton Transfer Stoichiometry, Organic Acid Metabolism, and Nitrate Reduction in Dwarf Bean (Phaseolus vulgaris)

Iron deficiency in dwarf bean (Phaseolus vulgaris L.) induces an increased activity of a system in the rhizodermal cells, which reduces extracellular ferric salts, and an active proton efflux from the roots, which is coupled to accumulation of citrate and malate in the roots and subsequent export of these compounds in the xylem. During reduction of extracellular ferricyanide by Fe-deficient plants, the stoichiometry of electron transport to proton efflux is 2e⁻/1H⁺, and citrate and malate levels in the roots are strongly decreased. Reduction of ferricyanide by Fe-sufficient plants has no influence on root and shoot levels of citrate and malate, but in such plants the process is characterized by a e⁻/H⁺ efflux stoichiometry close to unity. Apparently, organic acid metabolism and transport are closely associated with the e⁻/H⁺ efflux ratio. To assess the significance of organic acid metabolism as one of the direct intracellular components of the induced unbalanced e⁻/H⁺ efflux by roots, we studied NO₃⁻ reduction in shoots and roots of Fe-deficient and Fe-sufficient plants. Nitrate reductase activity in the roots was positively correlated with the level of citrate and malate, whereas the enzyme activity in the leaves responded positively to the import of these organic acid anions.     

Short-term effect of elevated CO2 concentration and high irradiance on the antioxidant enzymes in bean plants

The effect of short-term exposure to elevated CO₂ concentration and high irradiance on the activity of superoxide dismutase (SOD), ascorbate peroxidase (APX), guaiacol peroxidases (GPX) and catalase (CAT), and on the extent of the lipid peroxidation was studied in bean (Phaseolus vulgaris L.) plants. Plants were exposed for 4 d (8 h a day) to irradiance of 100 (LI) or 1000 (HI) μmol m⁻² s⁻¹ at ambient (CA, 350 μmol mol⁻¹) or elevated (CE, 1300 μmol mol⁻¹) CO₂ concentration. Four-day exposure to CE increased the leaf dry mass in HI plants and RuBPC activity and chlorophyll content in LI plants. Total soluble protein content, leaf dry matter and RuBPC activity were higher in HI than in LI plants, although the HI and CE increased the contents of malonyldialdehyde and H₂O₂. Under CA, exposure to HI increased the activity of APX and decreased the total SOD activity. Under CE, HI treatment also activated APX and led to reduction of both, SOD and GPX, enzymes activities. CE considerably reduced the CAT activity at both irradiances, possibly due to suppressed rate of photorespiration under CE conditions.     

Excess copper induces accumulation of hydrogen peroxide and increases lipid peroxidation and total activity of copper–zinc superoxide dismutase in roots of Elsholtzia haichowensis

The effects of excess copper (Cu) on the accumulation of hydrogen peroxide (H₂O₂) and antioxidant enzyme activities in roots of the Cu accumulator Elsholtzia haichowensis Sun were investigated. Copper at 100 and 300 μM significantly increased the concentrations of malondialdehyde and H₂O₂, and the activities of catalase (E.C. 1.11.1.6), ascorbate peroxidase (E.C. 1.11.1.11), guaiacol peroxidase (GPOD, E.C. 1.11.1.7) and superoxide dismutase (SOD, E.C. 1.15.1.1). Isoenzyme pattern and inhibitor studies showed that, among SOD isoforms, only copper–zinc superoxide dismutase (CuZn–SOD) increased. Excess Cu greatly increased the accumulation of superoxide anion (O₂ ^·−) and H₂O₂ in E. haichowensis roots. This study also provides the first cytochemical evidence of an accumulation of H₂O₂ in the root cell walls as a consequence of Cu treatments. Experiments with diphenyleneiodonium as an inhibitor of NADPH oxidase, 1,2-dihydroxybenzene-3,5-disulphonic acid as an O₂ ^·− scavenger, and N-N-diethyldithiocarbamate as an inhibitor of SOD showed that the source of H₂O₂ in the cell walls could partially be NADPH oxidase. The enzyme can use cytosolic NADPH to produce O₂ ^·−, which rapidly dismutates to H₂O₂ by SOD. Apoplastic GPOD and CuZn–SOD activities were induced in roots of E. haichowensis with 100 μM Cu suggesting that these two antioxidant enzymes may be responsible for H₂O₂ accumulation in the root apoplast.     

Arsenic-induced root growth inhibition in mung bean (<Emphasis Type="Italic">Phaseolus aureus</Emphasis> Roxb.) is due to oxidative stress resulting from enhanced lipid peroxidation

Arsenic (As) toxicity and its biochemical effects have been mostly evaluated in ferns and a few higher plants. In this study, we investigated the effect of As (10.0 and 50.0 μM) on seedling growth, root anatomy, lipid peroxidation (malondialdehyde and conjugated dienes), electrolyte leakage, H₂O₂ content, root oxidizability and the activities of antioxidant enzymes in mung bean (Phaseolus aureus Roxb.). Arsenic significantly enhanced lipid peroxidation (by 52% at 50.0 μM As), electrolyte leakage and oxidizability in roots. However, there was no significant change in H₂O₂ content. Arsenic toxicity was associated with an increase in the activities of superoxide dismutase (SOD), guaiacol peroxidase (GPX) and glutathione reductase (GR). In response to 50.0 μM As, the activities of SOD and GR increased by over 60% and 90%, respectively. At 10.0 μM As, the activity of ascorbate peroxidase (APX) increased by 83%, whereas at 50.0 μM it declined significantly. The catalase (CAT) activity, on the other hand, decreased in response to As exposure, and it corresponded to the observed decrease in H₂O₂ content. We conclude that As causes a reduction in root elongation by inducing an oxidative stress that is related to enhanced lipid peroxidation, but not to H₂O₂ accumulation.     

Plasma membrane-bound NADH: Fe3+-EDTA reductase and iron deficiency in tomato (Lycopersicon esculentum). Is there a Turbo reductase?

The properties of NADH-dependent Fe³⁺-EDTA reductase in plasma membranes (PM) from roots of iron-deficient and -sufficient tomato plants [Lycopersicon esculentum L. (Mill.) cv. Abunda] were examined. Iron deficiency resulted in a 3-fold increase of in vivo root iron-chelate reductase activity with a K_m (Fe³⁺-EDTA) of 230 μM. In purified root PM, average specific activities of ferric chelate reductase of 410 and 254 nmol Fe (mg protein)^?1 min^?1 were obtained for iron-deficient and -sufficient plants, respectively. In both cases, the PM-bound activity showed a pH optimum at pH 6.8. Activity depended on NADH and not on NADPH and on the presence of detergent. The activity was inhibited 40-50% by superoxide dismutase (EC 1.15.1.1) and ca 30% by oxygen. Kinetic analysis of the membrane-bound enzyme revealed a K_m (Fe³⁺-EDTA) of ca 200 μM for both iron-stressed and -sufficient plants. For NADH, K_m values around 230 μM were obtained. The ferric chelate reductase could be solubilised from salt-washed PM with Triton X-100 at a protein:detergent ratio of 1:2.8 (w/w). The Triton-soluble fraction revealed one enzyme-stained band in native polyacrylamide electrophoresis. Although the membranes showed no nitrate reductase (NR; EC 1.6.6.1) activity, anti-spinach NR immunoglobulin G (IgG) recognized a 54 kDa band both in the PM and the Triton-soluble fraction, but not in the enzymatically active material obtained from the native gel. No evidence could be found for the synthesis of a new, biochemically distinct PM-bound ferric chelate reductase under iron deficiency, which might be identified as the so-called Turbo reductase. It is concluded that iron deficiency in tomato induces increased expression of a ferric chelate reductase in root PM, which is already present in iron-sufficient plants and probably also in plants, which do not contain the Turbo reductase, like the grasses. The iron reductase is not identical with the recently reported PM-associated nitrate reductase.     

Antioxidant enzyme activities and isozyme pattern in hairy roots and regenerated tobacco plants

Hairy root disease is caused by infection of wounded higher plants with Agrobacterium rhizogenes. Transformation of tissues or plants with A. rhizogenes, as well as transformation with rol genes, in addition to hairy roots, may produce alterations in the plant secondary metabolism. H₂O₂ and other ROS are involved as signals in secondary metabolite production pathways and play a key role in plant defense reactions. In this work the effects of A. rhizogenes rol genes on nicotine content, antioxidant enzymes activity, H₂O₂ production, the pattern of peroxidase (POX) and superoxide dismutase (SOD) isozymes in hairy roots and regenerated Nicotiana tabacum plants were studied. The rise in SOD and POX activities in the transformed lines TRa and TRb and the resulting regenerated plants and a decreased level of H₂O₂ in them as compared with the untransformed lines indicates that rol gene expression decreases H₂O₂ level probably by increasing production of antioxidant enzymes. A decreased H₂O₂ content in TRc line, in spite of similarity of antioxidant enzyme activity as compared to normal roots, indicates that rol genes activate other mechanisms except SOD and POX enzymes for reducing H₂O₂.     

Oxidative damages induced by short-term exposure to cadmium in bean plants: Protective role of salicylic acid

The role of salicylic acid (SA) in alleviating cadmium (Cd) toxicity was investigated in a hydroponic cultivation system. Short-term exposure of bean (Phaseolus vulgaris) plants to 20 μM Cd inhibited biomass production and intensively increased accumulation of Cd in both roots and leaves. At leaf level, Cd significantly decreased mineral ions, chlorophyll and carotenoids concentrations. Concomitantly, Cd enhanced electrolyte leakage, H₂O₂ content and lipid peroxidation as indicated by malondialdehyde (MDA) accumulation. SA pretreatment decreased the uptake and the transport of Cd, alleviated the Cd-induced inhibition of nutrient absorption and led to a significant increase of chlorophyll and carotenoid content. SA application alleviated the oxidative damages as evidenced by the lowered H₂O₂ and MDA content. SA particularly induced an increase in both CAT and APX activities accompanied by a significant reduction in SOD and POD activities. As important antioxidants, ascorbate and glutathione contents in bean leaves exposed to cadmium were significantly decreased by SA treatment. These results reveal the potentiating effect of salicylic acid in regulating cadmium induced oxidative stress in bean plants.     

Rhizosphere acidification as a response to iron deficiency in bean plants

Iron deficiency in higher plants causes accumulation of salts of organic acids in the roots, the most characteristic being citrate. We show that citrate and malate accumulate in beans (Phaseolus vulgaris L. var Prélude), not because of a lack of the iron-containing enzyme aconitase (EC 4.2.1.3), but in close coupling to the extrusion of protons during rhizosphere acidification, one of the `Fe-efficiency' reactions of dicotyledonous plants. When proton excretion is induced in roots of control bean plants by addition of fusicoccin, only malate, not citrate, is accumulated. We propose that iron deficiency induces production of organic acids in the roots, which in beans leads to both proton excretion and an increased capacity to reduce ferric chelates via the induced electron transfer system in the root epidermis cells.     

Impact of SOD in eNOS uncoupling: a two-edged sword between hydrogen peroxide and peroxynitrite

In endothelial cell dysfunction, the uncoupling of eNOS results in higher superoxide (O₂^??) and lower NO production and a reduction in NO availability. Superoxide reacts with NO to form a potent oxidizing agent peroxynitrite (ONOO^?) resulting in nitrosative and nitroxidative stresses and dismutates to form hydrogen peroxide. Studies have shown superoxide dismutase (SOD) plays an important role in reduction of O₂^?? and ONOO^? during eNOS uncoupling. However, the administration or over-expression of SOD was ineffective or displayed deleterious effects in some cases. An understanding of interactions of the two enzyme systems eNOS and SOD is important in determining endothelial cell function. We analyzed complex biochemical interactions involving eNOS and SOD in eNOS uncoupling. A computational model of biochemical pathway of the eNOS-related NO and O₂^?? production and downstream reactions involving NO, O₂^??, ONOO^?, H₂O₂ and SOD was developed. The effects of SOD concentration on the concentration profiles of NO, O₂^??, ONOO^? and H₂O₂ in eNOS coupling/uncoupling were investigated. The results include (i) SOD moderately improves NO production and concentration during eNOS uncoupling, (ii) O₂^?? production rate is independent of SOD concentration, (iii) Increase in SOD concentration from 0.1 to 100 μM reduces O₂^?? concentration by 90% at all [BH₄]/[TBP] ratios, (iv) SOD reduces ONOO^? concentration and increases H₂O₂ concentration during eNOS uncoupling, (v) Catalase can reduce H₂O₂ concentration and (vi) Dismutation rate by SOD is the most sensitive parameter during eNOS uncoupling. Thus, SOD plays a dual role in eNOS uncoupling as an attenuator of nitrosative/nitroxidative stress and an augmenter of oxidative stress.     

The effect of zinc stress combined with high irradiance stress on membrane damage and antioxidative response in bean seedlings

Zinc (Zn) is a necessary element for plants, but excess Zn can be detrimental. The effect of Zn and high irradiance (HI) stress on the growth, lipid peroxidation (MDA), membrane permeability (EC), hydrogen peroxide (H₂O₂) accumulation, non-enzymatic antioxidants like proline accumulation and ascorbic acid (AsA) and the activities of major antioxidant enzymes (superoxide dismutase, SOD; peroxidase, POX; polyphenol oxidase, PPO) of bean leaves were investigated under controlled growth conditions. The root length was not reduced at excess Zn level. Application of Zn significantly increased Zn concentration in the leaves of bean plants. Under Zn and HI stress, the Zn-deficient and Zn-excess conditions significantly increased the EC, MDA and H₂O₂ content of excised leaves of bean. The SOD activity was found to be increased significantly in both Zn-deficiency and Zn-excess leaves under Zn and HI stress. Under both Zn and HI stress conditions, the antioxidant enzyme activities; POX, PPO and the non-enzymatic antioxidants, AsA and proline accumulation were found to be significantly increased in the Zn-excess leaves which showed that the bean plant had the ability to tolerate the excess level of Zn and HI stress. A significant increase in MDA, H₂O₂, and EC with a simultaneous decrease in the antioxidant enzyme activities under Zn-deficiency compared to Zn-sufficient condition shows the inefficiency of the bean plant in response to Zn deficiency. To the best of our knowledge, this is the first report on the effect of Zn stress combined with HI stress in bean plant.     

Superoxide reduction as a mechanism of ascorbate-stimulated oxygen uptake by isolated chloroplasts

Addition of 1m$\text{M}$ ascorbate to isolated chloroplasts with methyl viologen (MV) as electron acceptor trebled the rate of oxygen uptake and decreased the $\text{ADP}\text{O}$ ratio to a third of that with no ascorbate present. These effects of ascorbate were reversed by superoxide dismutase (SOD), which in the absence of ascorbate had little effect on O₂ uptake or $\text{ADP}\text{O}$ ratio. A chloroplast-associated SOD activity equivalent to 500 units/mg chlorophyll was detected. The effects of ascorbate and SOD on O₂ uptake were similar in both coupled and uncoupled chloroplasts. The results are consistent with the hypothesis that ascorbate stimulates O₂ uptake by reduction of superoxide, which is formed by autoxidation of the added electron acceptor (MV), and which dismutates in the absence of ascorbate. Ascorbate does not seem to stimulate O₂ uptake by replacing water as the photosystem II donor.