Anton Novacky

The initiation of membrane lipid peroxidation during bacteria-induced hypersensitive reaction

Abstract The initiation of lipid peroxidation reported previously may be initiated by free radicals or lipoxygenase. Levels of the free radicals can be affected by activity of protective enzymes (such as catalase and Superoxide dismutase), levels of antioxidants and rate of free radical production. We followed and compared the activity of lipoxygenase, catalase and superoxide dismutase in cucumber cotyledons infiltrated with: (1) Pseudomonas syringae pv. pisi (incompatible pathogen); (2) Pseudomonas syringae pv. lachrymans (compatible pathogen); and (3) Pseudomonas fluorescens (saprophyte). The only difference in enzyme activity was an increase in lipoxygenase activity in cotyledons infiltrated with Pseudomonas syringae pv. pisi . To assess the possibility of free radical initiation of lipid peroxidation we infiltrated cotyledons with P. syringae pv. pisi plus free radical scavengers (superoxide dismutase, catalase, a -tocopherol-acetate and dipyridamole) and followed membrane alteration (assessed by electrolyte leakage and fluorescein accumulation). Also, these results were compared with the results of experiments in which the free radical scavengers and paraquat (which is proposed to initiate lipid peroxidation via the production of superoxide radicals) were infiltrated together. We observed a significant reduction of membrane alteration induced by P. syringae pv. pisi or paraquat by addition of superoxide dismutase. We suggest that lipid peroxidation mediated membrane alteration induced by P. syringae pv. pisi is initiated by free radicals and the superoxide radical is a likely candidate. We propose that the increase in lipoxygenase activity may be a response to fatty acids released by free radical attack of membrane lipids during lipid peroxidation.

Phosphate uptake inLemna gibba G1: energetics and kinetics

Phosphate uptake was studied by determining [32P]phosphate influx and by measurements of the electrical membrane potential in duckweed (Lemna gibba L.). Phosphate-induced membrane depolarization (ΔEm) was controlled by the intracellular phosphate content, thus maximal ΔEm by 1 mM H2PO4-was up to 133 mV after 15d of phosphate starvation. The ΔEm was strongly dependent on the extracellular pH, with a sharp optimum at pH 5.7. It is suggested that phosphate uptake is energized by the electrochemical proton gradient, proceeding by a 2H+/H2PO4-contransport mechanism. This is supported also by the fusicoccin stimulation of phosphate influx. Kinetics of phosphate influx and of ΔEm, which represent mere plasmalemma transport, are best described by two Michaelis-Menten terms without any linear components.

Nitrate-dependent membrane potential changes and their induction in Lemna Gibba G 1

Abstract The electrical membrane potential (Em) of Lemna gibba cells was recordered during addition of nitrate. Transient nitrate-induced depolarisation in nitrogen-starved and induced plants was 55–70 mV in the dark, but only 20–30 mV in the light. Depolarisation was strongly enhanced by nitrate induction; it was pH-dependent and showed a saturation at 0:2 mM nitrate; it was iependent of K+. Plants with a low Em showed no depolarisation or even slight hyperpolarisation upon nitrate addition. The data suggest an H+/NO3− cotransport mechanism for nitrate uptake.

Membrane potential changes during transport of hexoses in Lemna gibba G1

The membrane potential (pd) of duck weed (Lemna gibba G1) proved to be energy dependent. At high internal ATP levels of 74 to 105 nmol ATP g-1 FW, pd was between -175 and -265 mV. At low ATP levels of 23 to 46 nmol ATP g-1 FW, pd was low, about -90 to -120 mV at pH 5.7, but -180 mV at pH 8. Upon addition of glucose in the dark or by light energy the low pd recovered to the high values. The active component of the pd was depolarized by the addition of hexoses in the dark and in the light. Hexose-dependent depolarization of the pd (=Δ pd) followed a saturation curve similar to active hexose influx kinetics. Depolarization of the pd recovered in the dark even in the presence of the hexoses and with a 10fold enhancement in the light. Depolarization and recovery could be repeated several times with the same cell. Glucose uptake caused a maximum depolarization of 133 mV, fructose uptake half that amount, sucrose had the same effect as glucose. During 3-O-methylglucose and 2-deoxyglucose uptake the depolarizing effect was only slightly lower. The pd remained unchanged in the presence of mannitol. The glucose dependent Δ pd and especially the rate of pd recovery proved to be pH-dependent between pH 4 and pH 8. It was independent of the presence of 1 mM KCl. Although no Δ pH could be measured in the incubation medium, these results can be best explained by a H+-hexose cotransport mechanism powered by active H+ extrusion at the plasmalemma.

Rapid and transient activation of a myelin basic protein kinase in tobacco leaves treated with harpin from Erwinia amylovora

Harpins are bacterial protein elicitors that induce hypersensitive response-like necrosis when infiltrated into nonhost plants such as tobacco (Nicotiana tabacum L.) (Z.-M. Wei, R.J. Laby, C.H. Zumoff, D.W. Bauer, S.Y. He, A. Collmer, S.V. Beer [1992] Science 257: 85–88). Activity of a 49-kD Mg2+-dependent and Ca2+-independent kinase in tobacco leaves increased 50-fold 15 min after infiltration of harpin from Erwinia amylovora (harpinEa). Much less pronounced and more transient activation was detected in water-infiltrated leaves. Biochemical characteristics of the harpinEa-activated protein kinase (HAPK) activity are consistent with those of the mitogen-activated protein kinase family. HAPK is cytosolic and phosphorylates myelin basic protein on serine/threonine residues. Treatment with a protein tyrosine phosphatase completely eliminated HAPK activity, suggesting that tyrosine phosphorylation is required for posttranslational activation. Sustained HAPK activation after cycloheximide treatment implies that HAPK may be negatively regulated by a translation-dependent mechanism. The extracellular Ca2+ chelator EGTA or the protein kinase inhibitor K252a, infiltrated in planta together with harpinEa, partially blocked HAPK activation. The Ca2+-channel blocker La3+ had no effect on HAPK activation, suggesting that phosphorylation events precede and/or do not depend on the entry of extracellular Ca2+ into the cell. These results suggest that early signal transduction events during harpinEa- induced hypersensitive response elicitation depend in part on the activation of HAPK.

Electrical Potentials of Plant Cell Walls in Response to the Ionic Environment

Electrical potentials in cell walls (ψWall) and at plasma membrane surfaces (ψPM) are determinants of ion activities in these phases. The ψPM plays a demonstrated role in ion uptake and intoxication, but a comprehensive electrostatic theory of plant-ion interactions will require further understanding of ψWall. ψWall from potato (Solanum tuberosum) tubers and wheat (Triticum aestivum) roots was monitored in response to ionic changes by placing glass microelectrodes against cell surfaces. Cations reduced the negativity of ψWall with effectiveness in the order Al3+ > La3+ > H+ > Cu2+ > Ni2+ > Ca2+ > Co2+ > Cd2+ > Mg2+ > Zn2+ > hexamethonium2+ > Rb+ > K+ > Cs+ > Na+. This order resembles substantially the order of plant-root intoxicating effectiveness and indicates a role for both ion charge and size. Our measurements were combined with the few published measurements of ψWall, and all were considered in terms of a model composed of Donnan theory and ion binding. Measured and model-computed values for ψWall were in close agreement, usually, and we consider ψWall to be at least proportional to the actual Donnan potentials. ψWall and ψPM display similar trends in their responses to ionic solutes, but ions appear to bind more strongly to plasma membrane sites than to readily accessible cell wall sites. ψWall is involved in swelling and extension capabilities of the cell wall lattice and thus may play a role in pectin bonding, texture, and intercellular adhesion.

Active hexose uptake in Lemna gibba G1

Growth of autotrophically growing duck-weeds (Lemna gibba L., G1) was stimulated by sucrose. The rate of respiration increased when plants had been grown on sucrose (8.7 μmol O2 g-1 fresh weight (FW) h-1) and was reduced after growth without sucrose in the dark or under longday conditions (2.5 μmol O2 g-1 FW h-1). Photosynthesis was induced already by low light intensities (0.1 klx).Short-time application of glucose or sucrose stimulated respiration in proportion to the hexose uptake rate. Sucrose is probably not taken up as the disaccharide. The transported sugar species after addition of sucrose are its hexose moieties produced by the high activity of the cell wall invertase. Fructose stimulated to a lesser extent; mannitol induced no enhancement; 2-deoxyglucose slightly inhibited O2 uptake. After mild carbon starvation of the plants the uptake of glucose and 3-O-methylglucose proceeded without any lag phase, with similar saturation kinetics in both cases. The initial uptake rate at substrate saturation was 2.6 μmol glucose g-1 FW h-1 in the dark. Light stimulated hexose uptake by 2 to 3 times. The results show that Lemna gibba has an energy-dependent constitutive system for hexose uptake.

Suppression of the bacterially induced hypersensitive reaction by cytokinins

Abstract Development of the bacterially induced hypersensitive reaction was overcome when tobacco leaves were pretreated with 5 × 10−5 m -kinetin, or other compounds with cytokinin activity, 48 h before inoculation with Pseudomonas pisi. Bacterial growth in the treated tissue was not affected. Developmental stage of the treated leaves was critical for demonstrating the protective effect of cytokinin. Only older leaves approaching senescence exhibited this effect. Development of symptoms of wildfire disease caused by Pseudomonas tabaci were delayed by cytokinin treatment, but 72 h after inoculation both water (control) and cytokinin-treated leaves exhibited the same degree of necrosis.

Biological activity of harpin produced by Pantoea stewartii subsp. stewartii

Pantoea stewartii subsp. stewartii causes Stewarts wilt of sweet corn. A hypersensitive response and pathogenicity (Hrp) secretion system is needed to produce water-soaking and wilting symptoms in corn and to cause a hypersensitive response (HR) in tobacco. Sequencing of the hrp cluster revealed a putative harpin gene, hrpN. The product of this gene was overexpressed in Escherichia coli and shown to elicit the HR in tobacco and systemic resistance in radishes. The protein was designated HrpN(Pnss). Like other harpins, it was heat stable and protease sensitive, although it was three- to fourfold less active biologically than Erwinia amylovora harpin. We used antibodies to purified HrpN(Pnss) to verify that hrpN mutants could not produce harpin. This protein was secreted into the culture supernatant and was produced by strains of P. stewartii subsp. indologenes. In order to determine the importance of HrpN(Pnss) in pathogenesis on sweet corn, three hrpN::Tn5 mutants were compared with the wild-type strain with 50% effective dose, disease severity, response time, and growth rate in planta as parameters. In all tests, HrpN(Pnss) was not required for infection, growth, or virulence in corn or endophytic growth in related grasses.

Effect of abscisic acid on membrane potential and transport of glucose and glycine in Lemna gibba G1.

The membrane potential of Lemna gibba G1 was measured with a microelectrode; glucose and glycine uptake were measured with 14C-labeled substances. The membrane potential was increased by 85 mV on the average, after the plants had been pretreated with 10 μM abscisic acid (ABA) for more than 30 min. This effect is not linked to the endogenous level of soluble sugars. The concentration of these soluble sugars was increased to more than 200% by pretreatment of the plants with ABA, however, the respiration of the plants was not affected. ABA stimulated uptake of glucose and glycine. Glucose- and glycine-dependent depolarization and repolarization of the membrane was altered: depolarization was less and repolarization was slower; during uptake of glycine, the first typical phase of repolarization was suppressed. The data suggest that ABA interferes with the primary steps of substrate uptake.