Peroxidase Activity and Isoperoxidase Pattern in Tobacco Leaves Infected with Tobacco Necrosis Virus and Other Viruses Inducing Necrotic and Non-Necrotic Alterations

The level of peroxidase activity was greatly enhanced in tobacco leaves infected by tobacco necrosis virus (TNV) and other viruses which induce necrotic symptoms (TMV, ToMV and PVY^N). The intensity was related to the age of the leaves infected: absent or neglible in mature leaves and very pronounced in young growing infected leaves. On the contrary, changes in peroxidase activity were negligible when the infection was provoked by viruses which do not produce necrotic reactions (TMV and PVY^O). Analysis of the peroxidase isoenzymes, pattern in tobacco leaves infected by TNV and other necrosis-inducing viruses revealed in all cases, a slight increase in anionic (pl 3.5–3.7) and a considerable increase in moderately anionic isoenzymes particularly the pl 4.6 isoenzyme which in TNV and PVY^N-infected leaves reached levels up to 21 and 72 times the healthy control values. A considerable increase in the cationic (pl9.3–8.8) isoenzymes and the appearance of one moderately cationic isoenzyme (pl 8.2) was also detected. In leaf extracts from-virus-infected tobacco leaves with nonnecrotic response, no, or negligible alterations on the isoenzyme pattern were detected. However, infection by a fungal parasite (Erisyphe cichoracearum), which established a fully compatible, non-necrotic, interaction with tobacco leaves, like the necrosis-inducing viruses, changed the isoperoxidase pattern. The data suggest the necrotic alterations and associated changes in the peroxidase activity and isoperoxidase pattern in virus-infected leaves are not clearly related.     

Involvement of plasma membrane calcium influx in bacterial induction of the k/h and hypersensitive responses in tobacco

An early event in the hypersensitive response of tobacco to Pseudomonas syringae pv syringae is the initiation of a K⁺/H⁺ response characterized by specific plasma membrane K⁺ efflux, extracellular alkalinization, and intracellular acidification. We investigated the role of calcium in induction of these host responses. Suspension-cultured tobacco cells exhibited a baseline Ca²⁺ influx of 0.02 to 0.06 micromole per gram per hour as determined from ⁴⁵Ca²⁺ uptake. Following bacterial inoculation, uptake rates began to increase coincidently with onset of the K⁺/H⁺ response. Rates increased steadily for 2 to 3 hours, reaching 0.5 to 1 micromole per gram per hour. This increased Ca²⁺ influx was prevented by EGTA and calcium channel blockers such as La³⁺, Co²⁺, and Cd²⁺ but not by verapamil and nifedipine. Lanthanum, cobalt, cadmium, and EGTA inhibited the K⁺/H⁺ response in both suspension-cultured cells and leaf discs and prevented hypersensitive cell death in leaf discs. We conclude that increased plasmalemma Ca²⁺ influx is required for the K⁺/H⁺ and hypersensitive responses in tobacco.     

Protective effects of complementary Ca2+ on low-light-induced oxidative damage in tall fescue

Low-light (LL) intensity is a primary abiotic stressor that negatively influences turf grass quality. In the present experiment, we studied the effect of exogenous Ca²⁺ (0, 10, 50, 100, and 200 mM) on the antioxidant system, the accumulation of MDA and proline, the content of photosynthetic pigments in plant leaves in order to investigate whether exogenous Ca²⁺ treatment improves LL tolerance in tall fescue (Festuca arundinacea Schreb.). We have found that LL significantly reduced a number of growth parameters (plant height, leaf width, leaf fresh weight, root fresh weight, leaf dry weight, and root dry weight), chlorophyll (Chl) a and Chl b contents, and carotenoid (Car) levels, while considerably enhancing electrolyte leakage (EL), MDA accumulation, calcium (Ca²⁺) concentration, and generation of reactive oxygen species (ROS), such as hydrogen peroxide (H₂O₂) and superoxide radical (O ₂ ^·? ). Moreover, LL significantly induced the activities of antioxidant enzymes, such as peroxidase (POD) and catalase (CAT), and slightly increased the activity of superoxide dismutase (SOD) in tall fescue leaves. In contrast, POD and SOD activities declined considerably while CAT activity significantly increased in plant roots under LL stress. The application of 50 mM Ca²⁺ significantly improved the aforementioned growth parameters, the content of photosynthetic pigments, and further enhanced the activities of POD, SOD, and CAT, but decreased electrolyte leakage and MDA and H₂O₂ levels in the leaves and roots of tall fescue under LL stress. These results suggest that Ca²⁺ is likely involved in a resistance to LL by regulating antioxidant enzyme action in tall fescue leaves and roots.     

An Ethylene Biosynthesis-Inducing Endoxylanase Elicits Electrolyte Leakage and Necrosis in Nicotiana tabacum cv Xanthi Leaves

We have previously demonstrated that a protein purified from xylan-induced culture filtrates of Trichoderma viride contains β-1,4-endoxylanase activity and induces ethylene biosynthesis in tobacco (Nicotiana tabacum cv Xanthi) leaf discs. When the ethylene biosynthesis-inducing xylanase (EIX) was applied to cut petioles of detached tobacco leaves, it induced ethylene biosynthesis within 1 hour and extensive electrolyte leakage and necrosis were observed in tobacco leaf tissue within 5 hours. Ethylene-pretreatment (120 microliters per liter ethylene for 14 hours) of tobacco leaves enhanced ethylene biosynthesis in response to EIX by more than threefold and accelerated development of cellular leakage and necrosis. In intact plants, similar symptoms could be induced in leaves that were distant from the point of the enzyme application. The evidence suggests that EIX is translocated via the vascular system and elicits plant responses similar to those observed in a hypersensitive response.     

Effects of salicylate on stomatal resistance in detached tobacco leaves

Salicylate administered to detached tobacco (Nicotiana tabacum L.) leaves kept in the light rapidly induced an increase in stomatal resistance. This effect was not the result of water stress. Both the concentration of salicylate and the duration of the application to leaves affected the extent of the stomatal response. Application of salicylate for short periods showed that, once the stomatal response started, it was maintained for long period of time in the absence of further supply. K⁺ or Ca²⁺ were able to lower the stomatal resistance induced by salicylate, but only when both the ions were administered together with salicylate the stomatal closure was prevented. The results suggest that salicylate elicits some alterations interfering with the control of, stomatal movements, possibly affecting the integrity of cell membranes.     

The Respiratory Burst and Electrolyte Leakage Induced by Sulfhydryl Blockers in Egeria densa Leaves Are Associated with H2O2 Production and Are Dependent on Ca2+ Influx

In leaves of Egeria densa Planchon, N-ethylmaleimide (NEM) and other sulfhydryl-binding reagents induce a temporary increase in nonmitochondrial respiration (ΔQO₂) that is inhibited by diphenylene iodonium and quinacrine, two known inhibitors of the plasma membrane NADPH oxidase, and are associated with a relevant increase in electrolyte leakage (M. Bellando, S. Sacco, F. Albergoni, P. Rocco, M.T. Marré [1997] Bot Acta 110: 388–394). In this paper we report data indicating further analogies between the oxidative burst induced by sulfhydryl blockers in E. densa and that induced by pathogen-derived elicitors in animal and plant cells: (a) NEM- and Ag⁺-induced ΔQO₂ was associated with H₂O₂ production and both effects depended on the presence of external Ca²⁺; (b) Ca²⁺ influx was markedly increased by treatment with NEM; (c) the Ca²⁺ channel blocker LaCl₃ inhibited ΔQO₂, electrolyte release, and membrane depolarization induced by the sulfhydryl reagents; and (d) LaCl₃ also inhibited electrolyte leakage induced by the direct infiltration of the leaves with H₂O₂. These results suggest a model in which the interaction of sulfhydryl blockers with sulfhydryl groups of cell components would primarily induce an increase in the Ca²⁺ cytosolic concentration, followed by membrane depolarization and activation of a plasma membrane NADPH oxidase. This latter effect, producing active oxygen species, might further influence plasma membrane permeability, leading to the massive release of electrolytes from the tissue.     

Reduced external calcium or sodium stimulates calcium influx in Pelvetia eggs

The effect of external calcium and sodium ion concentrations on the calcium fluxes on the Pelvetia fastigiata De Toni egg was measured. Decreasing external [Ca²⁺] greatly increased the permeability of the eggs to Ca²⁺; at 1 mM external Ca²⁺ this permeability was 60 times as great as it was at the normal [Ca²⁺] of 10 mM. Lowering the external [Na⁺] also increased Ca²⁺ influx; at 2 mM Na⁺, the Ca²⁺ influx was 2–3 times as great as it was at the normal [Na⁺] if choline was used as a Na⁺ substitute. Lithium was less effective as a Na⁺ substitute in increasing Ca²⁺ influx. The extra Ca²⁺ influx in low [Na⁺] seemed to be dependent on internal [Na⁺]. The Ca²⁺ efflux increased transiently and then declined in low Na⁺ media.     

Systemic Acquired Resistance Induced in Tobacco Plants by Localized Virus Infection Does Not Operate Against Challenging Viruses That Infect Systemically

Localized infections produced by tobacco necrosis virus (TNV) or tomato mosaic virus (ToMV) in White Burley tobacco induced a systemic acquired resistance in upper, uninoculated leaves. This resistance was effective against challenge infection by TNV or ToMV but not by potato virus Y, necrotic strain (PVYⁿ), tobacco mosaic virus (TMV) or tobacco rattle virus (TRV), viruses giving systemic infections. Systemic acquired resistance against TNV or ToMV was expressed as a reduction in lesion size but not in viral antigen content of the resulting necrotic local lesions. The acquisition of resistance was concurrent with an increased capacity of the resistant leaves to convert 1-aminocyclopropane-1-carboxylic acid into ethylene. Systemic acquired resistance was ineffective to contrast or minimize in whatever way the systemic challenge infection produced by PVY^N, TMV or TRV. Severity of symptoms and virus multiplication did not differ in resistant leaves from controls. This result does not allow any optimistic promise on possible application of the systemic acquired resistance against severe viral diseases of crops.     

Role of calcium in nitric oxide-induced programmed cell death in tobacco protoplasts

We tried to determine the mechanisms by which Ca²⁺ mediated NO-induced programmed cell death (PCD) in tobacco protoplasts. Treatment of tobacco protoplasts with the NO donor sodium nitroprusside (SNP) resulted in a rapid [Ca²⁺]_cyt accumulation and decrease in mitochondrial membrane potential (ΔΨ_m) before the appearance of PCD. NO-induced PCD could be largely prevented not only by NO scavenger c-PTIO, but also by EGTA (Ca²⁺ chelator), LaCl₃ (Ca²⁺-channel blocker) or CsA (a specific mitochondrial permeability transition pore inhibitor, which also inhibit Ca²⁺ cycling by mitochondria). All results suggested that NO-induced PCD is mediated through mitochondrial pathway and regulated by Ca²⁺.     

Effect of Age on Adenosine Triphosphatase Activity from Tobacco Chloroplasts

Adenosine triphosphatase activity of tobacco leaf chloroplasts in the dark was measured, using leaves of different age as determined by the position of the leaves along the stem. The activity of the chloroplast preparations strongly decreased with age, regardless of the addition of Mg²⁺ or Ca²⁺. Opposite effects of Mg²⁺ and Ca²⁺ on the activity of the chloroplasts were noted in experiments where different ratios of Mg²⁺/Ca²⁺ were applied. They were related to the age of the leaves, Ca²⁺ strongly stimulated the activity of the preparations from old leaves but was practically without effect in young, just expanded leaves. Mg²⁺ slightly stimulated the activity from old leaves while it invariably inhibited the hydrolytic activity of preparations from young leaves.     

Leakage from egg phosphatidylcholine vesicles induced by Ca2+ and alcohols

The results shown in this paper indicate that the permeability properties of egg yolk phosphatidylcholine sonicated vesicles as detected by the leakage of carboxy fluorescein changes according to the Ca²⁺ content. Vesicles containing Ca²⁺ show a higher rate of leakage than those containing Na⁺ solutions in response to the increase of Ca²⁺ concentration in the outer solution. The results are interpreted in terms of the rigidity promoted by Ca²⁺ and are compared to those obtained with long and short chain alcohols.     

The role of calcium ions in the permeability changes produced by external ATP in transformed 3T3 cells

External ATP causes a rapid increase in passive permeability to nucleotides and phosphate esters in transformed cell lines, such as 3T6 mouse fibroblasts. However, untransformed lines, such as 3T3, do not show a similar sensitivity to external ATP. Ca²⁺ inhibits permeabilization, but only at concentrations approaching those of external ATP. In contrast, La³⁺ and Tb³⁺ inhibit ATP-dependent permeabilization at one-fifth the concentration of external ATP. Considering reports that lanthanides can substitute for calcium ion in many enzymatic reactions, often with a higher affinity, it would appear that Ca²⁺ plays a specific role in the maintenance of a passive membrane permeability barrier and in opposing the effects of external ATP.Other data suggest a regulatory role for the Ca²⁺-calmodulin complex in the permeabilization process. Trifluoperazine, chlorpromazine and W-7, compounds which inhibit cellular functions dependent on the Ca²⁺-calmodulin complex, are able to enhance the effect of external ATP. Thus, a dramatic stimulation of nucleotide permeability occurs with concentrations of external ATP and inhibitor that are ineffective when added alone. Calmodulin antagonists and low concentrations of external ATP increased membrane permeability to Na⁺ and K⁺ as was previously shown for permeabilization with ATP alone. Earlier studies have shown that energy inhibitors which reduce intracellular ATP levels greatly increase the sensitivity of transformed cells to external ATP. However, the Ca²⁺-calmodulin antagonists used in the present study exert their effects at concentrations which do not alter intracellular ATP levels.     

Effects of calcium on potassium and water transport in human erythrocyte ghosts

Bulk water transport in reconstituted ghosts is statistically comparable to that in the parent red cells, and is unaffected by incorporation of Ca²⁺ over the range of 0.01 to 1 mM. Brief exposure of ghosts to p-chloromercuribenzene sulfonate results in a supression of osmotic water flow but leaves K⁺ permeability unchanged. Incorporation of p-chloromercuribenzene sulfonate provokes extremely rapid K⁺ loss which can be counteracted by simultaneous inclusion of Ca²⁺.Erythrocyte ghosts, when prepared with a small amount of Ca²⁺, demonstrate recovery of normal impermeability to choline, sucrose, Na⁺ and inulin and have an improved K⁺ retention over Ca²⁺-free preparations.The rate of passive transport of K⁺ from unwashed erythrocyte ghosts was measured during the initial few minutes of efflux. The initial rates vary in a bimodal fashion with the concentration of Ca²⁺ incorporated at the time of hemolysis. In low concentrations (0.01–0.1 mM), Ca²⁺ protects the K⁺ barrier while at higher concentrations (0.1–1.0 mM) it provokes a K⁺ leakage ranging from 7 to 50 times the normal rate of passive K⁺ loss. The Ca²⁺-induced K⁺ leak is thus a graded response rather than a discrete membrane transport state. The transition from a Ca²⁺-protected to a Ca²⁺-damaged membrane occurs upon an increase in Ca²⁺ concentration of less than 50 μmoles/l.     

Nystatin effects on cellular calcium in Saccharomyces cerevisiae

The primary effects of nystatin, a polyene antibiotic, on the yeast Saccharomyces cerevisiae were investigated. Though K⁺ leakage was observed shortly after the addition of nystatin, Ca²⁺ leakage was delayed 2–3 h after its application and it occurred only at an acidic pH and in the absence of K⁺, Na⁺ or Mg²⁺ from the medium. However, within 4 min after application nystatin induced a passive influx of Ca²⁺ into the cells even at a concentration of 1 μM in the medium. These results led to the conclusion that the primary membranal lesion induced by nystatin is not restricted to monovalent cations but is also manifested by increased permeability to Ca²⁺. The delayed leakage of Ca²⁺ is explained by the assumption that the bulk of cellular calcium is sequestered so that the concentration of free Ca²⁺ in the cytoplasm is very low. The sequestered calcium may be liberated 2–3 h after the addition of nystatin as a consequence of secondary damage to the cells such as intracellular acidification and loss of cations.     

Calcium-salinity interactions affect ion transport in Chara corallina

Detached internodes of Chara corallina survived in solutions containing 100 mol m^?3 NaCl when the external concentration of Ca²⁺ was greater than 1 mol m^?3. Na⁺ influx was roughly proportional to external Na⁺ up to 100 mol m^?3 NaCl. Na⁺ influx involved two components: a Ca²⁺-insensitive influx which allowed the passage of Na⁺ independently of external Ca²⁺; and a Ca²⁺-inhibitable mechanism where Na⁺ influx was inversely proportional to external Ca²⁺. The Ca²⁺-inhibitable Na⁺ influx was similar to the Ca²⁺-inhibitable K⁺ influx. Mg²⁺ and Ba²⁺ were able to substitute for Ca²⁺ in partially inhibiting Na⁺ influx in the absence of external Ca²⁺. The effect of Ca²⁺ appears specific to Na⁺ and K⁺ influx since the effects of a Ca²⁺-free solution on the influx of some other cations, anions and neutral compounds is small. It is suggested that Na⁺ influx via the Ca²⁺-inhibitable mechanism represents Na⁺ leakage through K⁺ channels and that cell death at high salinity occurs due to a cytotoxic Na⁺ influx via this mechanism.     

Can Salicylic Acid Affect the Intercellular Transport of the Tobacco Mosaic Virus by Changing Plasmodesmal Permeability?

We studied the effects of salicylic acid (SA) on the plasmodesmal permeability as evaluated by the tobacco mosaic virus (TMV) spreading in tobacco Nicotiana glutinosaleaves, where TMV induces necrotic lesions. When leaves were treated with SA simultaneously with their viral inoculation, SA retarded the development of necrotic lesions and reduced their number. When inoculated leaves were kept on the SA solution at an elevated temperature (31°C) for a short period of time, the size of the necrotic lesions, which developed after leaf transfer to room temperature, was decreased. SA stimulated the formation of rapid callose involved in the control of the plasmodesmal permeability, which was assessed from fluorescence after tissue staining with Aniline Blue. On the basis of these data, we suggest that SA suppressed TMV spreading in the inoculated tobacco leaves by reducing the plasmodesmal permeability.     

The mechanism of vanadium action on selective K+-permeability in human erythrocytes

Low concentrations of chelating agents such as EDTA prevent the air oxidation of vanadyl (VO²⁺, +4 oxidation state) to vanadate (VO₃^?, +5 oxidation state). Under these conditions, the ionophore A23187 mediates the rapid entry of vanadyl into human erythrocytes. In the presence of A23187, vanadyl at concentrations in excess of EDTA gives rise to a dramatic increase in K⁺ permeability, which is very similar to the Gardos Ca²⁺-induced K⁺ permeability increase with respect to ion selectivity, response to inhibitors, effects of pH, and stimulation by external K⁺. In ultrapure media with very low Ca²⁺, however, vanadyl has no effect on K⁺ permeability. These experiments suggest that Ca²⁺ is displaced from EDTA by vanadyl and then enters the cell via A23187 where it triggers the increase in K⁺ permeability. This hypothesis is confirmed by experiments demonstrating that vanadyl does displace Ca²⁺ from EDTA. Vanadate, an inhibitor of Ca²⁺-ATPase, causes a selective increase in K⁺ permeability in metabolically depleted cells, but the increase is abolished by low concentrations of EDTA, indicating that this effect is also due to entry of extracellular Ca²⁺. Earlier observations of effects of vanadyl and vanadate on erythrocyte K⁺ permeability can thus be explained on the basis of inhibition of the Ca²⁺ pump by vanadium, leading to an increase in intracellular Ca²⁺ concentration.     

Ca2+ control of electrolyte permeability in plasma membrane vesicles from cat pancreas

Summary The influence of Ca²⁺ and other cations on electrolyte permeability has been studied in isolated membrane vesicles from cat pancreas.Ca²⁺ in the micromolar to millimolar concentration range, as well as Mg²⁺, Sr²⁺, Mn²⁺ and La³⁺ at a tested concentration of 10^–4 m, increased Na⁺ permeability when applied at the vesicle inside. When added to the vesicle outside, however, they decreased Na⁺ permeability. Ba²⁺ was effective from the outside but not from the vesicle inside.When Ca²⁺ was present at both sides of the membrane, Na⁺ efflux was not affected as compared to that in the absence of Ca²⁺. Monovalent cations such as Rb⁺, Cs⁺, K⁺, Tris⁺ and choline⁺ decreased Na⁺ permeability when present at the vesicle outside at a concentration range of 10 to 100mm. Increasing Na⁺ concentrations from 10 to 100mm at the vesicle inside increased Na⁺ permeability.The temperature dependence of Na⁺ efflux revealed that the activation energy increased in the lower temperature range (0 to 10°C) when Ca²⁺ was present at the outside or at both sides, but not when present at the vesicle inside only or in the absence of Ca²⁺.The results suggest that the Ca²⁺ outside effect is due to binding of calcium to negatively charged phospholipids with a consequent reduction of both fluidity and Na⁺ permeability of the membrane. The Ca²⁺-inside effect most likely involves interaction with proteins with consequent increase in Na⁺ permeability.The data are consistent with current hypotheses on secretagogue-induced fluid secretion in acinar cells of the pancreas according to which secretagogues elicit NaCl and fluid secretion by liberating Ca²⁺ from cellular membranes and by stimulating Ca²⁺ influx into the cell. The increased intracellular Ca²⁺ concentration in turn increases the contraluminal Na⁺ permeability which leads to NaCl influx. The luminal sodium pump finally transports Na⁺ ions into the lumen.