Mirjana Vuletić

Characterization of cell wall oxalate oxidase from maize roots

Oxalate oxidase activity was detected in the cell wall fraction isolated from maize roots (Zea mays L.). The enzyme was active at acidic pH with optimal activity at pH 3.2. It was thermally extremely stable and resistant to high salt concentration, SDS and pepsin. The enzyme activity was inhibited by sulphydryl reagents 2-mercaptoethanol (2-ME), N-ethyl maleimide (NEM) and dithiotreitol (DTT), but was insensitive to EDTA, KCN and metal ions. Measurements of enzyme activity were performed using colorimetric assay of H(2)O(2), as well as polarographic detection of O(2) consumption. Maximal activity was obtained with 5 mM oxalic acid for the colorimetric method, and 10 mM oxalic acid for the polarographic method. Both methods were applicable in oxalate oxidase characterization, the polarographic method being more suitable under conditions of H(2)O(2) interaction with some of the analyzed substances.

The characterization of peroxidases in mitochondria of maize roots

Abstract In the present study, we provide the evidence about the presence of peroxidases in mitochondria isolated from maize (Zea mays L.) roots with characteristics of both, peroxidase and oxidase activities. Oxidase activity, mediating reduction of oxygen to superoxide anion (O2− ) and H2O2 with NAD(P)H as a substrate, in the presence of MnCl2 and SHAM or p-coumaric acid, was demonstrated by using various known effectors and inhibitors of this reaction. Mitochondrial enzyme had some characteristics similar to MnCl2/SHAM-stimulated NAD(P)H oxidase from cell wall and plasma membrane. Peroxidatic activity was detected as NAD(P)H oxidation in the presence of exogenously added H2O2 and various phenolic compounds. This peroxidase dependent NAD(P)H oxidation via phenolics had similar characteristics with guaiacol peroxidase from the same mitochondrial preparation. Different effects of Cu2+, Fe2+ and phenolic compounds on oxidase and peroxidase reactions suggest that these two activities are catalyzed by two separate, well-coordinated isoenzymes. Their role in generation and removal of reactive oxygen species (ROS) in plant cell was proposed.

Differential response of antioxidative systems of maize (Zea mays L.) roots cell walls to osmotic and heavy metal stress.

An analysis of peroxidase and ascorbate oxidase activity, phenolic content and antioxidant capacity of isolated maize root cell walls was performed in controls and plants stressed with polyethylene glycol (PEG) or heavy metals, zinc or copper. Peroxidase activity (oxidative and peroxidative) was more pronounced in the ionic than in the covalent cell wall fraction. PEG induced an increase and Zn(2+) a decrease of both ionically bound peroxidase activities. In the covalent fraction, Cu(2+) decreased oxidative and increased peroxidative activity of peroxidase. Isoelectric focusing of ionically bound proteins and activity staining for peroxidase demonstrated increased intensities and appearance of new acidic isoforms, especially in Zn(2+) and PEG treatments. Most pronounced basic isoforms (pI ~ 7.5) in controls, decreased in intensity or completely disappeared in stressed plants. Ascorbate oxidase activity was significantly increased by PEG and decreased by Zn(2+) treatments, and highly correlated with peroxidase activity. Antioxidant capacity and total phenolics content increased in heavy metal-treated and decreased in PEG-treated plants. Analysis of individual phenolic components revealed p-coumaric and ferulic acids, as the most abundant, as well as ferulic acid dimers, trimers and tetramers in the cell walls; their quantity increased under stress conditions. Results presented demonstrate the existence of diverse mechanisms of plant response to different stresses.

The effects of manganese and copper in vitro and in vivo on peroxidase catalytic cycles.

Here we present the results of in vitro and in vivo studies of the influence of Mn²+ and Cu²+ on the peroxidative and oxidative catalytic functions of class III peroxidase. Complex peroxidase catalysis by intermediates generated in the reaction was analyzed by utilizing the activating effect of Mn²+ and the inhibitory effect of Cu²+ on the oxidative reaction in vitro. p-Coumaric acid was used as an enzyme substrate in the peroxidative reaction and as a cofactor in the oxidative reaction. In order to correlate the observed in vitro effects with the in vivo situation, we exposed maize plants to excess concentrations of Mn²+ and Cu²+ in the hydroponic solutions. Copper severely arrested plant growth, while manganese exerted no significant effect. The effects on peroxidase activity and isoforms profile of root soluble and cell wall bound fractions were studied. Inhibition of the peroxidase oxidative function by copper was reversible, localized in the cell wall, and accompanied by disappearance of some and appearance of new cationic isoforms. Copper-mediated changes were suppressed by the presence of manganese, although Mn²+ treatment per se did not affect the activity of the peroxidase enzyme. The results on the peroxidase activity in maize roots grown with excess Mn²+ and Cu²+ point to the coupling between the oxidative cycle, root growth and different peroxidase isoforms.

Antioxidative system in maize roots as affected by osmotic stress and different nitrogen sources

The activities of antioxidative enzymes and contents of proline and total phenolics were assayed in roots of two maize (Zea mays L.) genotypes grown in a medium containing nitrate (NO3−) or both nitrogen forms, nitrate and ammonium (NH4+/NO3−). An increase in the activities of class III peroxidases (POD), superoxide dismutase (SOD), ascorbate peroxidase (APX), ascorbate oxidase (AO) and proline content, and decrease in phenolic content were observed in NH4+/NO3− in comparison with NO3− grown plants. When polyethylene glycol (PEG) was added to both nitrogen treatments, the content of total phenolics and proline was increased, especially in NH4+/NO3− treatment. The PEG treatment decreased enzyme activities in NH4+/NO3− grown plants, but in NO3− grown plants activities of POD and SOD were increased, opposite to decreased APX and AO. Isoelectric focusing demonstrated increased activities of acidic POD isoforms in PEG treated NO3− grown plants, and lower activities of both, acidic and basic isoforms in NH4+/NO3−grown plants.

Properties of Maize Root Mitochondria from Plants Grown on Different Nitrogen Sources

Summary The role of maize ( Zea mays L. inbred line VA35) root mitochondria in nitrogen assimilation was investigated. Maize plants were grown for 2 weeks on a modified Knopp solution containing different forms of nitrogen: 10.9 mM nitrate or 10.9 mM nitrate + 7.2 mM ammonium. Mitochondria were isolated from root tissue and purified on a Percoll gradient. Glutamate dehydrogenase (GDH; EC 1.4.1.2), alanine aminotransferase (GPT; EC 2.6.1.2), NAD + -isocitrate dehydrogenase (NAD + -ICDH; EC 1.1.1.41), succinate dehydrogenase (SDH; EC 1.3.99.1) and malate dehydrogenase (MDH; EC 1.1.1.37) all showed increased specific activities in the case of mitochondria isolated from plants grown in the presence of ammonia. These results indicate the involvement of such mitochondria in ammonia assimilation by synthesizing glutamate in a reductive animation reaction catalyzed by glutamate dehydrogenase and enhanced TCA metabolism. Increased oxygen consumption rates of mitochondria isolated from ammonia grown plants was demonstrated in our experiments, supporting the occurrence of such a metabolic shift. The possibility that higher oxygen consumption rates of both respiratory pathways (phosphorylative and non-phosphorylative) with malate as substrate for oxidation result from stimulation of biosynthetic and catabolic functions of mitochondria from plants grown in the presence of ammonia is discussed.

Hydroquinone peroxidase activity of maize root mitochondria

Summary.The oxidation of hydroquinone with H2O2 in the presence of mitochondria isolated from maize (Zea mays L.) roots was studied. The results indicate that a reduced form of quinone may be a substrate of mitochondrial peroxidases. Specific activities in different mitochondrial isolates, the apparent Km for hydrogen peroxide and hydroquinone, and the influence of some known peroxidase inhibitors or effectors are presented. Zymographic assays revealed that all mitochondrial peroxidases, which were stained with 4-chloro-1-naphthol, were capable of oxidizing hydroquinone. A possible antioxidative role of hydroquinone peroxidase in H2O2 scavenging within the mitochondria, in cooperation with ascorbate or coupled with mitochondrial NAD(P)H dehydrogenases, is proposed.

Heterogeneity of Maize Root Mitochondria from Plants Grown in the Presence of Ammonium

Mitochondria isolated from root tissue of maize plants grown on a modified Knop solution containing 10.9 mM nitrate ± 7.2 mM ammonium were purified on the discontinuous Percoll density gradient with polyvinylpyrrolidone (PVP) added. The presence of PVP allowed separation of several mitochondrial fractions of a different density. Contrary to mitochondria isolated from plants grown in the presence of nitrate alone, revealing only two fractions, the mitochondria from NH4+/NO3−-plants were distributed in four fractions. Total amount of mitochondria, as well as specific activities of some nitrogen metabolism enzymes and tricarboxylic acid (TCA) cycle enzymes of all mitochondrial fractions, and respiratory activities of two lower density fractions isolated from plants grown on mixed nitrogen were higher in comparison to mitochondria from nitrate-grown plants.

The relationship between respiration rate and peroxidase activities in maize root mitochondria

In the present study we provide the evidence of different respiration rates and peroxidase activities in maize (Zea mays L.) mitochondria isolated from germinated seeds and roots of 2-week-old seedlings. The negative relationships between mitochondrial respiration rate measured with NADH as substrate and activities of peroxidases that oxidized NADH in both oxidative and peroxidative cycles were found. The possible role of peroxidase in the regulation of reactive oxygen species metabolism in expense of NADH oxidation was hypothesized.

The effect of metabolic inhibitors on the trans-root electrical potential difference of excised maize roots

Summary A comparative study of the effect of a number of metabolic inhibitors on the trans-root electrical potential difference (TRP) across excised maize roots ( Zea mays L.), and on their respiration rate, was performed in order to characterize in greater detail the possible generators contributing to the total electrical potential difference of the root system. The roots were treated with CCCP, NEM, PCMBS, KCN, SHAM, vanadate and DES at two pH values (4.5 and 7.0) and their effect on the TRP transients, sucrose-induced changes and oxygen consumption was analyzed. The results demonstrate that the TRP arising across excised roots is a complex resultant of at least two electrogenic processes producing current and transferring ions in different directions which are located at various sites in the root tissue. The results also argue in favour of the involvement of some other mechanism, besides the membrane H + -ATPases, in the generation of proton gradients and the TRP.