Strawberry Fruit Oxalate Oxidase — Detection, Purification, Characterization and Physiological Role

A soluble oxalate oxidase activity has been detected in homogenate of ripened fruits of strawberry (Fragaria ananassa), as confirmed by the stoichiometric relationship between the disappearance of oxalate and utilization of dissolved O₂, and generation of H₂O₂. The enzyme was purified up to apparent homogeneity and had a Mr of 119 kDa with two identical subunits. K_m for oxalate was found to be 1.67×10^?3 M, and V_max of 0.741 mmoles ml^?1min^?1. It retained 76% of its initial activity, when heated at 60°C for 30 min. The enzyme was found to be glycoprotein in nature. The significant increase in the enzyme activity of ripened fruits compared to that in pre-ripened fruit, and decrease in oxalate level (?0.927 correlation with oxalate oxidase) with advancement of ripening indicated the physiological role of enzyme in fruit ripening.     

Determination of oxalate in plant tissues with oxalate oxidase prepared from wheat

A method for determination of oxalate with oxalate oxidase (OxO, EC 1.2.3.4) prepared from wheat bran, is based on specific oxidation of oxalate to produce H₂O₂. The H₂O₂ formed was colorimetrically determined using horseradish peroxidase-catalyzed oxidation of 4-aminoantipyrine and N,N-dimethylaniline by H₂O₂. The new method was tested on rice, buckwheat, soybean and oxalis leaves, showing it is precise, sensitive, inexpensive, highly reproducible and simple to perform. Good agreement could be obtained between this method and the HPLC.     

Ethanol increases sensitivity of oxalate oxidase assays and facilitates direct activity staining in SDS gels

Oxalate oxidase, and H₂O₂-generating enzyme, has been characterized from several plants, and is widely used for clinical detection of oxalate. Using a germin-like oxalate oxidase from barley leaves, we have developed and optimized novel methods for measuring oxalate oxidase activity. As oxalate oxidase is SDS-tolerant, its activity can be detected directly in SDS-PAGE gels in the presence of ethanol. This ethanol-dependent method is a hundred times more sensitive than the current methods. Furthermore, ethanol also improves the sensitivity of oxalate oxidase assays performed in solution. We found at least a 10-fold increase in sensitivity in comparison to a current method. The assay in solution is, in addition, useful for detection of oxalate. This elevation in sensitivity may be due to the immobilization of the enzyme in protein precipitates as a result of the treatment with ethanol.     

Changes in the isozyme composition of antioxidant enzymes in response to aminotriazole in leaves ofArabidopsis thaliana

The changes in isozyme profiles of catalase (CAT), peroxidase (POD), ascorbate peroxidase (APX), and glutathione reductase (GR) during severe deactivation of total CAT activity by aminotriazole (AT) treatment were investigated in the leaves ofArabidopsis thaliana (Columbia ecotype) in relation to H₂O₂-mediated oxidative stress. In spite of striking deactivation of total CAT activity by 0.1 mM AT, there were no significant differences in H₂O₂ levels or total leaf soluble protein contents including a Rubisco in both the control and AT-treated leaves. On the other hand, one specific protein band (molecular mass, 66 kD) was observed on the SDS-gel from leaf soluble proteins whose staining intensity was strikingly enhanced by AT treatment for 6 h. However, this band disappeared at 12 h. In the native-gel assays of CAT, POD, APX and GR isozymes, AT remarkably inhibited the expression of the CAT1 isozyme with no effects on CAT2 and CAT3, and generally had no effect on POD isozyme profiles. However, AT stimulated the intensity of activities of pre-existing APX1 and GR1 isozymes. In particular, it induced a new synthesis of one GR isozyme. Therefore, these results collectively suggest that a striking deactivation of total CAT activity by AT inA. thaliana leaves largely results from the suppression of CAT1 isozyme, and that APX1, GR1, and a newly synthesized GR isozyme could complement the role of CAT1 to metabolize H₂O₂ into non-toxic water.     

Specific and constitutive expression of oxalate oxidase during the ageing of leaf sheaths of ryegrass stubble

Changes in the activity of oxalate oxidase (OxO) and of the concentrations of oxalate and H₂O₂ were investigated during the ageing of leaf sheaths of ryegrass (Lolium perenne L.) stubble. The accumulation of H₂O₂ during ageing coincides with the increases of both oxalate level and OxO activity. Western and Northern blot analyses using protein and RNA extracts of the different categories of leaf sheaths suggested that OxO gene expression, as well as Ca-oxalate synthesis, are crucial events of ageing for leaf sheaths. Immunocytochemistry experiments have revealed that OxO, which is an extracellular enzyme, is nearly always present in the parenchymatous cells surrounding the vascular bundles and in the cells of the lower epidermis. Overall, results suggest that in ryegrass that synthesizes both Ca-oxalate and OxO, the production of H₂O₂ and Ca²⁺ during ageing of stubble might be involved in the constitutive defences against pathogens, thus allowing the phloem mobilization of nutrient reserves from the leaf sheaths towards elongating leaf bases of ryegrass.     

Salicylic and Jasmonic acids in regulation of the proantioxidant state in wheat leaves infected by <Emphasis Type="Italic">Septoria nodorum</Emphasis> Berk

Influence of mediators of the signal systems of salicylic (SA) and jasmonic (JA) acids and their mixture on reactive oxygen species’ (ROS) (superoxide radical and O₂^·− H₂O₂) generation and activity of oxidoreductases (oxalate oxidase, peroxidase and catalase) in leaves of wheat Triticum aestivum L. infected by Septoria leaf blotch pathogen Septoria nodorum Berk has been studied. Presowing treatment of seeds by SA and JA decreased the development rate of fungus on wheat leaves. SA provided earlier inductive effect on production of O₂^·− and H₂O₂ compared with JA. The protective effect of the salicylic and jasmonic acids against Septoria leaf blotch pathogen was caused by activation of oxalate oxidase, induction of anion and cation peroxidases, and decrease of catalase activity. Ability of compounds to stimulate ROS in the plant tissues can be used as criteria for evaluation of immune-modulating activity of new substances for protection of the plants.     

Exogenous calcium affects nitrogen metabolism in root-zone hypoxia-stressed muskmelon roots and enhances short-term hypoxia tolerance

We investigated the effects of short-term root-zone hypoxic stress and exogenous calcium application or deficiency in an anoxic nutrient solution on nitrogen metabolism in the roots of the muskmelon cultivar Xiyu No. 1. Seedlings grown in the nutrient solution under hypoxic stress for 6 d displayed significantly reduced plant growth and soluble protein concentrations. However, NO₃⁻ uptake rate and activities of nitrate reductase and glutamate synthase were significantly increased. We also found higher amounts of nitrate, ammonium, amino acids, heat-stable proteins, polyamines, H₂O₂, as well as higher polyamine oxidase activity in the roots. In comparison to the reactions seen under hypoxic stress, exogenous calcium application led to a marked increase in plant weights, photosynthesis parameters, NO₃⁻ uptake rate and contents of nitrate, ammonium, amino acids (e.g., glutamic acid, proline, glycine, cystine, γ-aminobutyric acid), soluble and heat-stable proteins, free spermine, and insoluble bound polyamines. Meanwhile, exogenous calcium application resulted in significantly increased activities for nitrate reductase, glutamine synthetase, and glutamate synthase but decreased activities for diamine and polyamine oxidase, as well as lower H₂O₂ content in roots during exposure to hypoxia. However, calcium deficiency in the nutrient solution decreased plant weight, photosynthesis parameters, NO₃⁻ reduction, amino acids (e.g., alanine, aspartic acid, glutamic acid, γ-aminobutyric acid), protein, all polyamines except for free putrescine, and the activities of glutamate synthase and glutamine synthetase. Additionally, there was an increase in the NO₃⁻ uptake rate, polyamine oxidase activity and H₂O₂ contents under hypoxia-Ca. Simultaneously, exogenous calcium had little effect on nitrate absorption and transformation, photosynthetic parameters, and plant growth under normoxic conditions. These results suggest that calcium confers short-term hypoxia tolerance in muskmelon, most likely by promoting nitrate uptake and accelerating its transformation into amino acids, heat-stable proteins or polyamines, as well as by decreasing polyamine degradation in muskmelon seedlings.     

The effect of 1-methylcyclopropene on the components of pro- and antioxidant systems of wheat and the development of defense reactions in fungal pathogenesis

The effect of 1-methylcyclopropene (1-MCP), which inhibits the reception of ethylene, on the following has been studied: hydrogen peroxide generation, oxalate oxidase activity, peroxidase activity, catalase activity, and lignin accumulation in infected leaves of soft spring wheat (Triticum aestivum L.) cultivars that differ in their resistance to the leaf blotch disease, caused by the hemibiotrophic fungus Septoria nodorum Berk. A decrease in the development of leaf blotch in wheat leaves under the influence of 1-MCP was, on one hand, followed by an inhibition of catalase activity; on the other hand, it was accompanied by an increase in oxalate oxidase and peroxidase activity, as well as an accumulation of H₂O₂ in tissues and lignin in the infected zone. The role of the ethylene reception system in the defense response of plants to infection with a hemibiotrophic pathogen, that causes leaf blotch disease, is discussed.     

Salicylic acid induces resistance to <Emphasis Type="Italic">Septoria nodorum</Emphasis> berk. in wheat

The effect of salicylic acid (SA) on oxalate oxidase and peroxidase activities and hydrogen peroxide (H₂O₂) production in leaf cells has been studied in wheat of the susceptible cultivar Zhnitsa infected by Septoria nodorum, a pathogen of wheat leaf blotch. The results show that fungal hyphae spread into interstices between mesophyll cells and that infected tissues contain H₂O₂. Treatment with SA results in enhanced H₂O₂ production in mesophyll cells, which is due to activation of oxalate oxidase and peroxidase in the cell wall. It is proposed that the modulating effect of SA on oxidoreductase activities is involved in the induction of protective response to fungal infection in wheat plants.     

OsHK3 is a crucial regulator of abscisic acid signaling involved in antioxidant defense in rice

In this study, the role of the rice(Oryza sativa L.)histidine kinase Os HK3 in abscisic acid(ABA)-induced antioxidant defense was investigated. Treatments with ABA, H2O2,and polyethylene glycol(PEG) induced the expression of Os HK3 in rice leaves, and H2O2 is required for ABA-induced increase in the expression of Os HK3 under water stress. Subcellular localization analysis showed that Os HK3 is located in the cytoplasm and the plasma membrane. The transient expression analysis and the transient RNA interference test in rice protoplasts showed that Os HK3 is required for ABA-induced upregulation in the expression of antioxidant enzymes genes and the activities of antioxidant enzymes. Further analysis showed that Os HK3 functions upstream of the calcium/calmodulin-dependent protein kinase Os DMI3 and the mitogen-activated protein kinase Os MPK1 to regulate the activities of antioxidant enzymes in ABA signaling. Moreover, Os HK3was also shown to regulate the expression of nicotinamide adenine dinucleotide phosphate oxidase genes, Osrboh B and Osrboh E, and the production of H2O2 in ABA signaling. Our data indicate that Os HK3 play an important role in the regulation of ABA-induced antioxidant defense and in the feedback regulation of H2O2 production in ABA signaling.     

Influence of Ca<Superscript>2+</Superscript> ions on metabolism of active oxygen species in wheat calli cocultured with the bunt pathogen <Emphasis Type="Italic">Tilletia caries</Emphasis>

The effect of Ca²⁺ on morphophysiological parameters of wheat calli (Triticum aestivum L.) infected by the bunt pathogen Tilletia caries, in particular on the level of active oxygen species, activity of oxalate oxidase, peroxidase, and catalase is investigated. The concentration of O²⁻, H₂O₂, and activity of oxidoreductases (oxalate oxidase, peroxidase, and catalase) depended on the content of Ca²⁺ in the culture medium of calli. The increase of the concentration of Ca²⁺ ions in the culture medium led to forming of calli with high structure, induction of activity of oxalate oxidase and of some isoperoxidase, and to accumulation of active oxygen species. These changes contributed to inhibition of development of the fungus. So this dependence confirm the role of calcium as the intermediant in biochemical reactions related to the formation of the protective response of plant cells to biotic stress.     

Hydrogen peroxide is involved in methyl jasmonate-induced senescence of rice leaves

The role of H₂O₂ in the senescence of detached rice leaves induced by methyl jasmonate (MJ) was investigated. MJ treatment resulted in H₂O₂ production in detached rice leaves, which was prior to the occurrence of leaf senescence. Dimethylthiourea, a chemical trap of H₂O₂, was observed to be effective in inhibiting MJ‐induced senescence and MJ‐increased malondialdehyde (MDA) content in detached rice leaves. Diphenyleneiodonium chloride (DPI) and imidazole (IMD), inhibitors of NADPH oxidase, prevented MJ‐induced H₂O₂ production, suggesting that NADPH oxidase is a H₂O₂‐generating enzyme in MJ‐treated detached rice leaves. DPI and IMD also inhibited MJ‐promoted senescence and MJ‐increased MDA content in detached rice leaves. Phosphatidylinositol 3‐kinase inhibitors wortmannin (WM) or LY 294002 (LY) inhibited MJ‐induced H₂O₂ production and senescence of detached rice leaves. Exogenous H₂O₂ reversed the inhibitory effect of WM or LY. In terms of leaf senescence, it was observed that rice seedlings of cultivar Taichung Native 1 (TN1) are jasmonic acid (JA)‐sensitive and those of cultivar Tainung 67 (TNG67) are JA‐insensitive. On treatment with JA, H₂O₂ accumulated in the leaves of TN1 seedlings but not in the leaves of TNG67. Evidence was also provided to show that MJ‐induced H₂O₂ production in detached rice leaves is abscisic acid (ABA)‐independent. Ethylene action inhibitor, silver thiosulfate, was observed to inhibit MJ‐ and ABA‐induced H₂O₂ production and senescence of detached rice leaves, suggesting that the action of MJ and ABA is ethylene‐dependent.     

Involvement of copper amine oxidase (CuAO)-dependent hydrogen peroxide synthesis in ethylene-induced stomatal closure in Vicia faba

Ethylene promotes stomatal closure via inducing hydrogen peroxide (H₂O₂) generation. H₂O₂ can be catalytically synthesized by several enzymes in plants. Here, by means of stomatal bioassay, the analysis of enzyme activity and using laser-scanning confocal microscopy based on the H₂O₂-sensitive probe 2′,7′-dichlorodihydrofluorescein diacetate (H₂DCF-DA), the roles of copper amine oxidase (CuAO) in ethylene-induced H₂O₂ production in guard cells and stomatal closure in Vicia faba L. were investigated. 1-aminocyclopropane-1-carboxylic acid (ACC), an immediate precursor of ethylene synthesis, and ethylene gas significantly activated CuAO in intercellular washing fluid from leaves, the production of H₂O₂ in guard cells, and stomatal closure. These effects of ACC and ethylene gas were largely prevented by both aminoguanidine and 2-bromoethylamine, which are irreversible inhibitors of CuAO. Among major catalyzed and metabolized products of CuAO, only H₂O₂ could markedly promote stomatal closure and evidently reversed the effect of CuAO inhibitor on stomatal closure by ACC and ethylene gas. The data described above show that CuAO-mediated H₂O₂ production is involved in ethylene-induced stomatal closure.     

The generation of H2O2 in the xylem of Zinnia elegans is mediated by an NADPH-oxidase-like enzyme

The nature of the enzymatic system responsible for the generation of H₂O₂ in the lignifying xylem of Zinnia elegans (L.) was studied using the starch/KI method for monitoring H₂O₂ production and the nitroblue tetrazolium method for monitoring superoxide production. The results showed that lignifying xylem tissues are able to accumulate H₂O₂ and to sustain H₂O₂ production. Hydrogen peroxide production in the xylem of Z. elegans was sensitive to pyridine, imidazole, quinacrine and diphenylene iodonium, which are inhibitors of phagocytic plasma-membrane NADPH oxidase. The sensitivity of H₂O₂ production to the inhibitor of phospholipase C, neomycin, and to the inhibitor of protein kinase, staurosporine, and its reversion by the inhibitor of protein phosphatases, cantharidin, pointed to the analogies existing between the mechanism of H₂O₂ production in lignifying xylem and the oxidative burst observed during the hypersensitive plant cell response. A further support for the participation of an NADPH-oxidase-like activity in H₂O₂ production in lignifying xylem was obtained from the observation that areas of H₂O₂ production were superimposed on areas producing superoxide anion, the suspected product of NADPH oxidase, although attempts to demonstrate the existence of superoxide dismutase activity in intercellular washing fluid from Z. elegans were unsuccessful. Even so, the levels of NADPH-oxidase-like activity in microsomal fractions, and of peroxidase in intercellular washing fluids, are consistent with a role for NADPH oxidase in the delivery of H₂O₂ which may be further used by xylem peroxidases for the synthesis of lignins. This hypothesis was further confirmed through a direct histochemical probe based on the H₂O₂-dependent oxidation of tetramethylbenzidine by xylem cell wall peroxidases. These results are the first evidence for the existence of an NADPH oxidase responsible for supplying H₂O₂ to peroxidase in the lignifying xylem of Z. elegans. Received: 6 February 1998 / Accepted: 14 August 1998     

Salicylic acid increases tolerance to oxidative stress induced by hydrogen peroxide accumulation in leaves of cadmium-exposed flax (Linum usitatissimum L.)

The aim of this study is to investigate the impacts of exogenous salicylic acid (SA) pretreatments on hydrogen peroxide (H₂O₂) accumulation, protein oxidation, and H₂O₂-scavenging enzymes in leaves of Cd-treated flax seedlings. Cd-enhanced H₂O₂ levels were related to increased activities of guaiacol peroxidase (POX, EC 1.11.1.7) and ascorbate peroxidase (APX, EC 1.11.1.11), and were independent of changes in catalase (CAT, EC 1.11.1.6) and superoxide dismutase (SOD, EC 1.15.1.1) activities. In control flax seedlings, exogenous SA pretreatments inhibited the activity of CAT, resulted in an enhanced production of H₂O₂ suggesting that SA requires H₂O₂ to initiate an oxidative stress. However, although leaves of Cd-free flax seedlings pretreated with SA accumulated in vivo H₂O₂ by 1.2-fold compared with leaves of Cd-only exposed ones; the damage to growth and proteins after the exposure to Cd was significantly less, indicating that SA can regulate the Cd-induced oxidative stress. Moreover, the Cd-treated seedlings primed with SA exhibited a higher level of total antioxidant capacities and increased activities of H₂O₂-detoxifying enzymes.     

Orchestration of hydrogen peroxide and nitric oxide in brassinosteroid‐mediated systemic virus resistance in Nicotiana benthamiana

Brassinosteroids (BRs) play essential roles in modulating plant growth, development and stress responses. Here, involvement of BRs in plant systemic resistance to virus was studied. Treatment of local leaves in Nicotiana benthamiana with BRs induced virus resistance in upper untreated leaves, accompanied by accumulations of H₂O₂ and NO. Scavenging of H₂O₂ or NO in upper leaves blocked BR‐induced systemic virus resistance. BR‐induced systemic H₂O₂ accumulation was blocked by local pharmacological inhibition of NADPH oxidase or silencing of respiratory burst oxidase homolog gene NbRBOHB, but not by systemic NADPH oxidase inhibition or NbRBOHA silencing. Silencing of the nitrite‐dependent nitrate reductase gene NbNR or systemic pharmacological inhibition of NR compromised BR‐triggered systemic NO accumulation, while local inhibition of NR, silencing of NbNOA1 and inhibition of NOS had little effect. Moreover, we provide evidence that BR‐activated H₂O₂ is required for NO synthesis. Pharmacological scavenging or genetic inhibiting of H₂O₂ generation blocked BR‐induced systemic NO production, but BR‐induced H₂O₂ production was not sensitive to NO scavengers or silencing of NbNR. Systemically applied sodium nitroprusside rescued BR‐induced systemic virus defense in NbRBOHB‐silenced plants, but H₂O₂ did not reverse the effect of NbNR silencing on BR‐induced systemic virus resistance. Finally, we demonstrate that the receptor kinase BRI1(BR insensitive 1) is an upstream component in BR‐mediated systemic defense signaling, as silencing of NbBRI1 compromised the BR‐induced H₂O₂ and NO production associated with systemic virus resistance. Together, our pharmacological and genetic data suggest the existence of a signaling pathway leading to BR‐mediated systemic virus resistance that involves local Respiratory Burst Oxidase Homolog B (RBOHB)‐dependent H₂O₂ production and subsequent systemic NR‐dependent NO generation.     

Senescence of leaf sheaths of ryegrass stubble: changes in enzyme activities related to H2O2 metabolism

Changes in the levels of reactive oxygen species (O2^.-, H₂O₂), and of activities of enzymes involved in their detoxification were investigated during senescence of leaf sheaths of ryegrass stubble. The accumulation of H₂O₂ in the medium leaf sheaths coincided with a drop in the levels of total glutathione, of pyridine nucleotides and of activities of monodehydroascorbate reductase and dehydroascorbate reductase. Conversely, a paradoxical increase in the ascorbate/ascorbate plus dehydroascorbate ratio was observed, which appears to be inconsistent with H₂O₂ accumulation. Our results suggest that oxalate might be an essential source of H₂O₂ in senescent leaf sheaths, and that oxalate oxidase might be involved in the defence of foliar tissue against pathogens during the progress of senescence. Moreover, it is assumed that glucid catabolism of the ryegrass stubble might be a starting point of a metabolic drain leading to ascorbate, then to oxalate during the late phase of leaf sheath senescence.