Comparative biochemical characterization of peroxidases (class III) tightly bound to the maize root cell walls and modulation of the enzyme properties as a result of covalent binding.

Comparative biochemical characterization of class III peroxidase activity tightly bound to the cell walls of maize roots was performed. Ionically bound proteins were solubilized from isolated walls by salt washing, and the remaining covalently bound peroxidases were released, either by enzymatic digestion or by a novel alkaline extraction procedure that released covalently bound alkali-resistant peroxidase enzyme. Solubilized fractions, as well as the salt-washed cell wall fragments containing covalently bound proteins, were analyzed for peroxidase activity. Peroxidative and oxidative activities indicated that peroxidase enzymes were predominately associated with walls by ionic interactions, and this fraction differs from the covalently bound one according to molecular weight, isozyme patterns, and biochemical parameters. The effect of covalent binding was evaluated by comparison of the catalytic properties of the enzyme bound to the salt-washed cell wall fragments with the corresponding solubilized and released enzyme. Higher thermal stability, improved resistance to KCN, increased susceptibility to H2O2, stimulated capacity of wall-bound enzyme to oxidize indole-3-acetic acid (IAA) as well as the difference in kinetic parameters between free and bound enzymes point to conformational changes due to covalent binding. Differences in biochemical properties of ionically and covalently bound peroxidases, as well as the modulation of the enzyme properties as a result of covalent binding to the walls, indicate that these two fractions of apoplastic peroxidases play different roles.

Plasma membrane-associated malate dehydrogenase of maize (Zea mays L.) roots: native versus recombinant protein.

Malate dehydrogenase (MDH, EC 1.1.1.37) is involved in several cellular processes including plant development, nutrient uptake and oxidative stress. Evidence for a plasma membrane-associated MDH has been presented for maize (Zea mays L.) roots. In the present study isoenzymes of MDH were purified from highly enriched plasma membrane preparations of maize and compared with soluble isoenzymes (Km, pH optima, pI and molecular masses). Modified SDS-PAGE analyses revealed monomers of 41 kDa for membrane-associated MDH, whereas monomers (35 kDa) and dimers (70 kDa) were detected for soluble isoenzymes. Membrane-associated MDH of cauliflower (Brassica oleracea L.) inflorescences and spinach (Spinacia oleracea L.) leaves showed molecular masses similar to the membrane-associated MDH of maize. The specific maize MDH involved was identified by mass spectrometry (ESI-QTOF-MS/MS, MALDI-TOF-MS). The corresponding gene was cloned and the protein was characterised after heterologous expression in Escherichia coli. Enzyme kinetics and properties of the recombinant and native proteins were compared. The function of thiol groups and the presence of disulphide bonds were analysed by the effect of N-ethylmaleimide, diagonal electrophoresis and labelling. Semiquantitative reverse transcription polymerase chain reaction of maize root transcripts demonstrated a constitutive expression of the gene encoding plasma membrane-associated MDH.

Cell wall-associated malate dehydrogenase activity from maize roots.

Isolated cell walls from maize (Zea mays L.) roots exhibited ionically and covalently bound NAD-specific malate dehydrogenase activity. The enzyme catalyses a rapid reduction of oxaloacetate and much slower oxidation of malate. The kinetic and regulatory properties of the cell wall enzyme solubilized with 1M NaCl were different from those published for soluble, mitochondrial or plasma membrane malate dehydrogenase with respect to their ATP, Pi, and pH dependence. Isoelectric focusing of ionically-bound proteins and specific staining for malate dehydrogenase revealed characteristic isoforms present in cell wall isolate, different from those present in plasma membranes and crude homogenate. Much greater activity of cell wall-associated malate dehydrogenase was detected in the intensively growing lateral roots compared to primary root with decreased growth rates. Presence of Zn(2+) and Cu(2+) in the assay medium inhibited the activity of the wall-associated malate dehydrogenase. Exposure of maize plants to excess concentrations of Zn(2+) and Cu(2+) in the hydroponic solution inhibited lateral root growth, decreased malate dehydrogenase activity and changed isoform profiles. The results presented show that cell wall malate dehydrogenase is truly a wall-bound enzyme, and not an artefact of cytoplasmic contamination, involved in the developmental processes, and detoxification of heavy metals.

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.

Effectiveness of phenoxyl radicals generated by peroxidase/H2O2-catalyzed oxidation of caffeate, ferulate, and p-coumarate in cooxidation of ascorbate and NADH.

The rate of ascorbate and nicotinamide adenine dinucleotide plus hydrogen (NADH) cooxidation (i.e., their nonenzymic oxidation by peroxidase/H2O2-generated phenoxyl radicals of three hydroxycinnamates: caffeate, ferulate and p-coumarate) was studied in vitro. The reactions initiated by different sources of peroxidase (EC 1.11.1.7) [isolates from soybean (Glycine max L.) seed coat, maize (Zea mays L.) root-cell wall, and commercial horseradish peroxidase] were monitored. Native electrophoresis of samples and specific staining for peroxidase activity revealed various isoforms in each of the three enzyme sources. The peroxidase sources differed both in the rate of H2O2-dependent hydroxycinnamate oxidation and in the order of affinity for the phenolic substrates. The three hydroxycinnamates did not differ in their ability to cooxidize ascorbate, whereas NADH cooxidation was affected by substitution of the phenolic ring. Thus, p-coumarate was more efficient than caffeate in NADH cooxidation, with ferulate not being effective at all. Metal ions (Zn2+ and Al3+) inhibited the reaction of peroxidase with p-coumarate and affected the cooxidation rate of ascorbate and the peroxidase reaction in the same manner with all substrates used. However, inhibition of p-coumarate oxidation by metal ions did not affect NADH cooxidation rate. We propose that both the ascorbate and NADH cooxidation systems can function as mechanisms to scavenge H2O2 and regenerate phenolics in different cellular compartments, thus contributing to protection from oxidative damage.

The coexistence of the oxidative and reductive systems in roots: the role of plasma membranes.

Different components of the plasma membrane bound and associated redox system, which participate in the energy transfer from the predominantly reducing intercellular environment to the extracellular oxidizing environment, are reviewed. Special attention is given to plant root cells. An analysis of the plasma membrane-associated redox components, such as the cytochromes, quinones, and different types of oxidoreductases (dehydrogenases, oxidases, peroxidases, and superoxide dismutases), is made, as well as their coupling with naturally occurring extracellular substrates, such as oxygen and its reactive forms, phenols, ascorbate, nitrate, ferric ion, and organic acids. The participation of different free radical species in most of the plasma membrane-bound redox reactions is documented.

Detection of oxygen-centered radicals using EPR spin-trap DEPMPO: the effect of oxygen.

Studies of the ability of the EPR spin trap DEPMPO to detect both superoxide and hydroxyl radicals produced by systems in vitro and in vivo are presented. Experiments using free radical-generating systems confirmed the suitability of the EPR spin trapping technique but also revealed the existence of an undesirable conversion of DEPMPO/OOH into DEPMPO/OH adducts. The rate of conversion decreases with oxygenation, and the production of oxygen-centered radicals increases. However, this property of DEPMPO does not have a significant influence on its ability to independently detect radicals produced by plant plasma membranes. Since the adduct conversion appears to be rather slow compared to radical generation, we conclude that the DEPMPO spin-trap can be efficiently used for detection of oxygen-centered radicals produced by systems in vivo, as demonstrated for isolated plasma membranes.

Oxygen radicals produced by plant plasma membranes: an EPR spin-trap study.

Plant plasma membranes are known to produce superoxide radicals, while the production of the hydroxyl radical, previously detected in complex plant tissues, is thought to occur in the cell wall. The mechanism of production of superoxide radicals by plant plasma membranes is, however, under dispute. It is shown, using electron paramagnetic resonance spectroscopy with a 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide spin-trap capable of differentiating between radical species, that isolated purified plasma membranes from maize roots produce hydroxyl radicals besides superoxide radicals. The results argue in favour of superoxide production through an oxygen and diphenylene iodonium-sensitive, NADH-dependent superoxide synthase mechanism, as well as through other unidentified mechanism(s). The hydroxyl radical is produced by an oxygen-insensitive, NADH-stimulated mechanism, which is enhanced in membranes in which the superoxide synthase is incapacitated by substrate removal or inhibition.