Dihydroquercetin (DHQ, taxifolin) is a plant flavonoid that exhibits antioxidant and cytoprotective properties in animal and human cells. Its effect on programmed cell death (PCD), respiration, photosynthesis, and the formation of reactive oxygen species (ROS) in plant cells has been shown. At concentrations of 0.1–3 mM, DHQ suppressed KCN-induced PCD in guard cells of pea leaf epidermis in the light or in the dark, which was determined by the destruction of cell nuclei. In pea leaf cuttings, DHQ had no effect on respiration, as measured by O2 consumption in the dark. DHQ did not affect photosynthetic O2 evolution by leaf cuttings with p-benzoquinone and ferricyanide as electron acceptors nor on the consumption of oxygen as a result of the oxidation of ascorbate and N,N,N′,N′-tetramethyl-p-phenylenediamine and the reduction of methyl viologen in the light in PS I. DHQ inhibited menadione-induced ROS formation in pea leaf epidermis, which was detected by fluorescence 2′,7′-dichlorofluorescein. DHQ itself had no effect on H2O2 in solution, but it utilized hydrogen peroxide in combination with peroxidase. The antioxidant effect of DHQ and protection of plant cells from death may be due to its suitability as a peroxidase substrate.

In vitro redox properties of the green tea component epigallocatechin gallate (EGCG) and its effect on pea plant cells were investigated. EGCG was found to exhibit both pro- and antioxidant properties. In solutions, EGCG was oxidized by oxygen at physiological (slightly alkaline) pH values with the generation of O 2 –• and H 2 O 2 , the reaction being slowed down by a decrease in the medium pH. On the other hand, EGCG functioned as an electron donor for peroxidase, resulting in the H 2 O 2 utilization. EGCG suppressed respiration, reduced mitochondrial transmembrane potential difference and inhibited electron transfer in the photosynthetic electron transport chain in pea leaf cells (leaf cuttings and epidermis). Among components of the photosynthetic redox chain, Photosystem II was the least sensitive to the EGCG action. In the epidermis, EGCG reduced the rate of reactive oxygen species formation that was induced by NADH. EGCG at the concentrations from 10 μM to 1 mM suppressed the KCN-induced death of guard cells in the epidermis, which was determined from the destruction of cell nuclei. EGCG at a concentration of 10 mM disrupted the barrier function of the guard cell plasma membrane, increasing its permeability to propidium iodide.

Chitosan modified with a (2-hydroxy-3-trimethylammonium) propyl group and gallic acid residue, or quaternized chitosan with gallic acid (QCG), was synthesized. Antioxidant properties of the produced QCG have been investigated. Peroxidase in combination with NADH and salicyl hydroxamate (SHAM) caused consumption of oxygen and production of H2O2 in aqueous solution as a result of O2 reduction in the peroxidase–oxidase reactions. The rates of O2 consumption and H2O2 generation were reduced in the presence of QCG. The antioxidant propyl gallate (PG) and superoxide dismutase (SOD) had the same effect, but not the quaternized chitosan (QC) without gallic acid. The effect of chitosan derivatives on the production of reactive oxygen species (ROS) in the cells of pea leaf epidermis and on the cell death detected by the destruction of cell nuclei, was investigated. QCG, QC, and SOD had no effect, while PG decreased the rate of ROS generation in the cells of the epidermis, which was induced by NADH with SHAM or by menadione. QCG and QC prevented destruction of the guard cell nuclei in the pea leaf epidermis that was caused by NADH with SHAM or by KCN. SOD had no effect on the destruction of nuclei, while the effect of PG depended on the inducer of the cell death. Suppression of the destruction of guard cell nuclei by chitosan derivatives was associated not with their antioxidant effect, but with the disruption of the plasma membrane of the cells. The results obtained have shown that QCG exhibits antioxidant properties in solutions, but does not prevent generation of ROS in the plant cells. The mechanism of antioxidant effect of QCG is similar to that of PG and SOD.

The effect of low molecular weight (MW) chitosans with a MW of 5, 6, 10, 25, 45 kDa and a deacetylation degree (DD) of 85–99% obtained by chemical depolymerization and enzymatic hydrolysis on the cells of the epidermis of pea leaves was analyzed. Low MW chitosans induced guard cells plasma membrane damage, which was estimated from the change in its permeability for the fluorescent dye propidium iodide, and programmed death of epidermal cells determined from destruction of cell nuclei. These effects are similar to those of commercial high MW chitosan with a MW of 290 kDa, a DD of 90%, which was manifested at higher concentrations than with low MW chitosan. The destructive effect of chitosan on the cells was predominantly DD-dependent, and it was not produced at concentrations up to 10 μg/ml inclusive. Low MW chitosan enhanced the generation of reactive oxygen species in the epidermis, which was detected by monitoring the fluorescence of 2',7'-dichlorofluorescein. The antioxidants nitroblue tetrazolium and propyl gallate inhibited, and Н 2 О 2 accelerated this process.

The effects of superoxide dismutase (SOD) inhibitors, diethyldithiocarbamate (DDC), triethylenetetramine (trien), and their combination with glucose on cells of the epidermis from pea leaves of different age (rapidly growing young leaves and slowly growing old leaves) was investigated. DDC and trien caused death of the guard cells as determined by destruction of their nuclei. Glucose did not affect destruction of the nuclei induced by SOD inhibitors in the cells from old leaves, but intensified it in the cells from young leaves. 2-Deoxyglucose, an inhibitor of glycolysis, and propyl gallate, SOD-mimic and antioxidant, suppressed destruction of the nuclei that was caused by SOD inhibitors and glucose in cells of the epidermis from the young, but not from the old leaves. Glucose and trien stimulated, and propyl gallate reduced generation of reactive oxygen species (ROS) in the pea epidermis as determined by the fluorescence of 2′,7′-dichlorofluorescein (DCF). Carbonyl cyanide m-chlorophenylhydrazone (CCCP), a protonophoric uncoupler of oxidative and photosynthetic phosphorylation, suppressed the DCF fluorescence in the guard cells. Treatment of the cells with CCCP followed by its removal with washing increased destruction of the nuclei caused by SOD inhibitors and glucose. In young leaves, CCCP was less effective than in old ones. The findings demonstrate the effects of SOD inhibitors and glucose on the cell death and generation of ROS and could indicate glycolysis-dependent ROS production.

The effect of low molecular weight chitosans with a molecular weight (MW) of 5, 6, 10, 25, and 45 kDa and a deacetylation degree (DD) of 85–99% obtained by chemical depolymerization and enzymatic hydrolysis on the cells of the epidermis of pea leaves was analyzed. Low molecular weight chitosans induced damage to guard cells' plasma membrane, which was estimated from the change in its permeability for the fluorescent dye propidium iodide, and programmed death of epidermal cells determined from the destruction of cell nuclei. These effects are similar to those of commercial high molecular weight chitosan with an MW of 290 kDa and a DD of 90%, which was manifested at higher concentrations than with low molecular weight chitosan. The destructive effect of chitosan on the cells was predominantly DD-dependent, and it was not produced at concentrations up to 10 μg/mL inclusive. Low molecular weight chitosan enhanced the generation of reactive oxygen species in the epidermis, which was detected by monitoring the fluorescence of 2′,7′-dichlorofluorescein. The antioxidants nitroblue tetrazolium and propyl gallate inhibited this process, while Н2О2 accelerated it.

Damage to the plasma membrane (PM) of cells in pea leaf epidermis, determined by its permeability to propidium iodide (PI), which binds to DNA of cell nuclei, and programmed cell death (PCD) detected by the destruction of cell nuclei was investigated. PM of the epidermal cells in the isolated epidermis was permeable to PI (it stained their nuclei). PM of the guard cells did not allow PI to pass through. KCN, an inducer of PCD, caused the destruction of the both epidermal and guard cell nuclei. KCN-Induced destruction of guard cell nuclei was accompanied by the penetration of PI into the cells. The polycation chitosan at a concentration of 0.1 mg/ ml caused the destruction of the epidermal cell nuclei, but at a concentration of 1 mg/ ml, the permeability of the guard cell PM for PI staining their nuclei was induced. Other polycations (cytochrome c , polylysine, polyethylenimine and protamine) also caused staining of the guard cell nuclei by PI. Polyanions (polyacrylic acid, dextran and heparin) initiated the destruction of cell nuclei, which was accompanied by the penetration of PI into cells. Detergents Triton X-100 and lauryldimethylamine- N-oxide produced the permeability of the guard cell PM for PI and prevented the destruction of the nuclei that was induced by KCN. Treatment of the epidermis with Triton X-100 (for 2 h with its subsequent washing) increased the destruction of the guard cell nuclei that was caused by KCN. Polycations polyethyleneimine and protamine were prevented, while chitosan, cytochrome c , and polylysine, on the contrary, enhanced KCN-induced destruction of the guard cell nuclei. The data obtained shows that the destruction of cell nuclei upon induction of cell death with KCN or polyanions is accompanied by damage to PM (producing its permeability for PI). Damage to PM caused by detergents or polycations prior to cell treatm by KCN, can prevent or, on the contrary, intensify the destruction of cell nuclei.

Damage to the plasma membrane (PM) of cells in pea leaf epidermis determined by its permeability to propidium iodide (PI), which binds to DNA of cell nuclei, and programmed cell death (PCD) detected by the destruction of cell nuclei were investigated. PM of the epidermal cells in the isolated epidermis was permeable to PI (it stained their nuclei). PM of the guard cells did not allow PI to pass through. KCN, an inducer of PCD, caused the destruction of both epidermal and guard cell nuclei. KCN-induced destruction of guard cell nuclei was accompanied by the penetration of PI into the cells. The polycation chitosan at a concentration of 0.1 mg/mL caused the destruction of the epidermal cell nuclei, but the permeability of the guard cell PM for PI staining their nuclei was induced at a concentration of 1 mg/mL. Other polycations (cytochrome c, polylysine, polyethylenimine, and protamine) also caused staining of the guard cell nuclei by PI. Polyanions (polyacrylic acid, dextran, and heparin) initiated the destruction of cell nuclei, which was accompanied by the penetration of PI into cells. Detergents Triton X-100 and lauryldimethylamine-N-oxide produced the permeability of the guard cell PM for PI and prevented the destruction of the nuclei that was induced by KCN. Treatment of the epidermis with Triton X-100 (for 2 h with its subsequent washing) increased the destruction of the guard cell nuclei that was caused by KCN. Polycations polyethyleneimine and protamine were prevented, while chitosan, cytochrome c, and polylysine, on the contrary, enhanced KCN-induced destruction of the guard cell nuclei. The obtained data shows that the destruction of cell nuclei upon induction of cell death with KCN or polyanions is accompanied by damage to PM (producing its permeability for PI). Damage to PM caused by detergents or polycations prior to cell treatment by KCN can prevent or, on the contrary, intensify the destruction of cell nuclei.

This work focuses on the effect of mitochondria-targeted quinones (SkQs) on plants. SkQs with antioxidant properties are accumulated in the mitochondria of pea cells and suppress the generation of reactive oxygen species. At nanomolar concentrations, SkQs prevented the death of pea leaf epidermal or guard cells caused by chitosan, bacterial lipopolysaccharide or KCN. The protective effect of SkQs was removed by a protonophoric uncoupler. SkQs at micromolar concentrations inhibited the O2 evolution by illuminated chloroplasts and stimulated the respiration of mitochondria. SkQs slowed down the senescence and the death of Arabidopsis thaliana leaves and improved the wheat crop structure.

Salicylhydroxamic acid (SHAM), an alternative oxidase inhibitor of plant mitochondria, enhances the NADH-oxidase activity in mitochondrial and chloroplast suspensions obtained from pea roots or leaves, respectively. This reaction is inhibited by the washing of mitochondria or chloroplasts and is observed in supernatants after the removal of the organelles by centrifugation. The reaction is sensitive to CN– and to antioxidant propyl gallate. The NADH oxidation is also enhanced by 2,4-dichlorophenol or phenol, but not salicylic acid. The acceleration of NADH oxidation by phenolic compounds is observed with presence of commercial horseradish peroxidase and is connected with the involvement of these compounds in NADH-dependent peroxidase reaction. SHAM and 2,4-dichlorophenol significantly enhance the destruction of nuclei in guard cells of pea leaf epidermis caused by the generation of reactive oxygen species during the oxidation of exogenous NADH by apoplastic peroxidase.

Salicylhydroxamic acid (SHAM), an inhibitor of the alternative oxidase in plant mitochondria, accelerated the NADH-oxidase activity in suspensions of mitochondria and chloroplasts, obtained by their isolation from the roots or leaves of pea, respectively. The reaction was suppressed by washing mitochondria and chloroplasts. It also proceeded in supernatants where the organelles were removed by centrifugation. The reaction was sensitive to CN– and propyl gallate, an antioxidant. In addition to SHAM, NADH oxidation was stimulated by 2,4-dichlorophenol or phenol, but not by salicylic acid. The acceleration of NADH oxidation by the phenolic compounds occurred in the presence of commercial horseradish peroxidase. It is due to the involvement of these compounds in the NADH-dependent peroxidase reaction. 2,4-Dichlorophenol and SHAM enhanced significantly destruction of nuclei in guard cells of the epidermis from pea leaves induced by generation of reactive oxygen species under oxidation of exogenous NADH by means of the apoplastic peroxidase.

Controlled cell death, along with cell proliferation and differentiation, is essential for the life-sustaining activities of an organism. Programmed cell death (PCD) is involved in individual developmental program and the maintenance of tissue homeostasis. It is also prerequisite for the operation of the immunity system and interactions with abiogenic factors. PCD in animals and humans is sufficiently well understood. Less information is available concerning the mechanisms of cell death in plants. However, the data obtained up to now suggest that the signal-transducing pathways in animal, plants, and fungi are general. This paper discusses the PSD-triggering signals in plants and the systems involved in their transduction. Mitochondria perform an important function in the apoptosis of animal cells. Chloroplasts are structurally, functionally, and phylogenetically related to mitochondria, and they are apparently involved in plant PCD.

Plastoquinone bound with decyltriphenylphosphonium cation (SkQ1) penetrating through the membrane in nanomolar concentrations inhibited H2O2 generation in cells of epidermis of pea seedling leaves that was detected by the fluorescence of 2′,7′-dichlorofluorescein. Photosynthetic electron transfer in chloroplasts isolated from pea leaves is suppressed by SkQ1 at micromolar concentrations: the electron transfer in chloroplasts under the action of photosystem II or I (with silicomolybdate or methyl viologen as electron acceptors, respectively) is more sensitive to SkQ1 than under the action of photosystem II + I (with ferricyanide or p-benzoquinone as electron acceptors). SkQ1 reduced by borohydride is oxidized by ferricyanide, p-benzoquinone, and, to a lesser extent, by silicomolybdate, but not by methyl viologen. SkQ1 is not effective as an electron acceptor supporting O2 evolution from water in illuminated chloroplasts. The data on suppression of photosynthetic O2 evolution or consumption show that SkQ1, similarly to phenazine methosulfate, causes conversion of the chloroplast redox-chain from non-cyclic electron transfer mode to the cyclic mode without O2 evolution. Oxidation of NADH or succinate in mitochondria isolated from pea roots is stimulated by SkQ1.

Chitosan (partially deacetylated chitin), a component of fungal cell walls, caused epidermal cell (EC) death in the leaves of pea (Pisum sativum L.) and tobacco Nicotiana tabacum or Nicotiana benthamiana detected by destruction of cell nuclei. The mitochondria-targeted quinone SkQ1 prevented the destruction of EC nuclei induced by chitosan. Chitosan increased and SkQ1 suppressed the activity of protein kinases in N. benthamiana and P. sativum and eliminated the effect of chitosan. Chitosan induced the generation of reactive oxygen species (ROS) in the guard cells (GC) of pea plants. Treatment with chitosan or H2O2 did not cause destruction of GC nuclei; however, it resulted in disruption of the permeability barrier of the plasma membrane detected by propidium iodide fluorescence. Treatment with bacterial lipopolysaccharide but not peptidoglycan caused destruction of pea EC nuclei, which was prevented by SkQ1. Leaves of tobacco plants containing the N gene responsible for resistance to tobacco mosaic virus (TMV) were infiltrated with Agrobacterium tumefaciens cells. These cells contained a genetic construct with the gene of the helicase domain of TMV replicase (p50); its protein product p50 is a target for the N-gene product. As a result, the hypersensitive response (HR) was initiated. The HR manifested itself in the death of leaves and was suppressed by SkQ3. Treatment of tobacco epidermal peels with the A. tumefaciens cells for the p50 gene expression stimulated the destruction of EC nuclei, which was inhibited by SkQ1 or SkQ3. The p50-lacking A. tume-faciens cells did not induce the destruction of EC nuclei. The protective effect of mitochondria-targeted antioxidants SkQ1 and SkQ3 demonstrates the involvement of mitochondria and their ROS in programmed cell death caused by pathogen elicitors.

Исследование проведено на пленках эпидермиса листьев гороха (Pisum sativum L.), представляющих собой монослой из клеток двух типов – замыкающих клеток устьиц (устьичных клеток – УК), содержащих хлоропласты и митохондрии, и основных эпидермальных клеток (ЭК), содержащих только митохондрии. В качестве индукторов программируемой клеточной смерти использовали KCN, разрушающий ядра УК и ЭК, и хитозан, разрушающий только ядра ЭК. AgNO3 (10 мкМ) стимулировал CN--индуцированное разрушение ядер УК и ЭК и подавлял хитозан-индуцированное разрушение ядер ЭК. Разрушение ядер УК, индуцированное CN-, происходило в аэробных условиях и предотвращалось в анаэробных условиях. Разрушение ядер УК, индуцированное комбинацией (CN- + Ag+), происходило и в аэробных, и в анаэробных условиях и не тормозилось антиоксидантами. Среди испытанных катионов металлов (Ag+, Hg2+, Fe2+, Fe3+, Cu2+, Mn2+) Ag+ наиболее эффективен в стимуляции CN--индуцированного разрушения ядер УК. Полумаксимальные концентрации Ag+ и Hg2+ составляли 4–5 мкМ. УК в пленках эпидермиса, обработанных хитозаном, были проницаемы для пропидия йодистого, но ядра УК, в отличие от ядер ЭК, не разрушались при воздействии хитозаном. Сделан вывод, что Ag+ пригоден как акцептор электронов при фотосинтетическом переносе электронов в хлоропластах из листьев гороха, тормозит выделение О2 хлоропластами, обработанными феррицианидом или кремнемолибдатом как акцепторами электронов, и поглощение О2 при переносе электронов от пары (аскорбат + N,N,N,N-тетраметил-п-фенилендиамин) на метилвиологен, подавляет образование активных форм кислорода в УК и ЭК.

We investigated epidermal peels from the leaves of pea (Pisum sativum L.) consisting of a monolayer of the cells of two types: stomatal guard cells (GC) with chloroplasts and mitochondria and basic epidermal cells (EC) containing only mitochondria. As inducers of programmed cell death, we used KCN destroying the nuclei in GC and EC and chitosan that destroys nuclei only in EC. AgNO3 (10 μM) stimulated the destruction of nuclei in GC and EC induced by CN− and suppressed chitosan-induced destruction of nuclei in EC. The destruction of nuclei in GC induced by CN− occurred under aerobic conditions and was prevented under anaerobiosis. The destruction of nuclei in GC induced by (CN− + Ag+) occurred both under aerobic and anaerobic conditions and was not suppressed by antioxidants. Among the tested cations of metals (Ag+, Hg2+, Fe2+, Fe3+, Cu2+, and Mn2+), Ag+ turned out to be the most efficient in respect to the stimulation of cyanide-induced destruction of nuclei in GC. Half-maximum concentrations of Ag+ and Hg2+ were equal to 4–5 μM. In epidermal peels treated with chitosan, GC were permeable to propidium iodide; however, the nuclei in GC (in contrast to EC) were not destructed in the presence of chitosan. It was concluded that Ag+, acting as an electron acceptor during photosynthetic electron transfer in the chloroplasts from pea leaves, impeded the O2 evolution by the chloroplasts treated with ferricyanide or silicomolybdate as electron acceptors, impeded the consumption of O2 in the course of electron transfer from the (ascorbate + N,N,N′,N′-tetramethyl-p-phenylenediamine) to methylviologen and suppressed the production of reactive oxygen species (ROS) in GC and EC.