The antioxidant performance of a sterically hindered bisphenol antioxidant, 4,4'-bis(2,6-di-tert-butylphenol), in model reactions of accelerated aging of carbon-chain polymers (polypropylene, isoprene rubber) was studied. This antioxidant was examined by differential scanning calorimetry and Wallace plasticity measurements in comparison with known commercial phenolic and amine stabilizers. High performance of 4,4'-bis(2,6-di-tert-butylphenol) as antioxidant was revealed.

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.

Tetraphenylphosphonium (TPP+) and tetramethylrhodamine ethyl ester (TMRE+) cations used as transmembrane carriers of ubiquinone (MitoQ) and plastoquinone (SkQ, SkQR) in mitochondria prevented at nanomolar concentrations the chitosanor H2O2-induced destruction of the nucleus in epidermal cells of epidermis isolated from pea leaves. The protective effect of the cations was potentiated by palmitate. Penetrating anions of tetraphenylboron (TB−) and phenyl dicarbaundecaborane also displayed protective effects at micromolar concentrations; the effect of TB− was potentiated by NH4Cl. It is proposed that the protective effect of the penetrating cations and anions against chitosan is due to suppression of the generation of reactive oxygen species in mitochondria as a result of the protonophoric effect of the cations plus fatty acids and the anions plus NH 4 + . Phenol was suitable as the electron donor for H2O2 reduction catalyzed by horseradish peroxidase, preventing the destruction of cell nuclei. The penetrating cations and anions, SkQ1, and SkQR1 did not maintain the peroxidase or peroxidase/oxidase reactions measured by their suitability as electron donors for H2O2 reduction or by the oxidation of exogenous NADH.

Ubiquinone or plastoquinone covalently linked to synthetic decyltriphenylphosphonium (DTPP+) or rhodamine cations prevent programmed cell death (PCD) in pea leaf epidermis induced by chitosan or CN−. PCD was monitored by recording the destruction of cell nuclei. CN− induced the destruction of nuclei in both epidermal cells (EC) and guard cells (GC), whereas chitosan destroyed nuclei in EC not in GC. The half-maximum concentrations for the protective effects of the quinone derivatives were within the pico- and nanomolar range. The protective effect of the quinones was removed by a protonophoric uncoupler and reduced by tetraphenylphosphonium cations. CN−-Induced PCD was accelerated by the tested quinone derivatives at concentrations above 10−8–10−7 M. Unlike plastoquinone linked to the rhodamine cation (SkQR1), DTPP+ derivatives of quinones suppressed menadione-induced H2O2 generation in the cells. The CN−-induced destruction of GC nuclei was prevented by DTPP+ derivatives in the dark not in the light. SkQR1 inhibited this process both in the dark and in the light, and its effect in the light was similar to that of rhodamine 6G. The data on the protective effect of cationic quinone derivatives indicate that mitochondria are involved in PCD in plants.

The effect of Ca2+ on programmed death of guard cells (GC) and epidermal cells (EC) determined from destruction of the cell nucleus was investigated in epidermis of pea leaves. Ca2+ at concentrations of 1–100 μM increased and at a concentration of 1 mM prevented the CN—induced destruction of the nucleus in GC, disrupting the permeability barrier of GC plasma membrane for propidium iodide (PI). Ca2+ at concentrations of 0.1–1 mM enhanced drastically the number of EC nuclei stained by PI in epidermis treated with chitosan, an inducer of programmed cell death. The internucleosomal DNA fragmentation caused by CN− was suppressed by 2 mM Ca2+ on 6 h incubation, but fragmentation was stimulated on more prolonged treatment (16 h). Presumably, the disruption of the permeability barrier of plasma membrane for PI is not a sign of necrosis in plant cells. Quinacrine and diphenylene iodonium at 50 μM concentration prevented GC death induced by CN− or CN− + 0.1 mM Ca2+ but had no influence on respiration and photosynthetic O2 evolution in pea leaf slices. The generation of reactive oxygen species determined from 2′,7′-dichlorofluorescein fluorescence was promoted by Ca2+ in epidermal peels from pea leaves.

Addition of chitosan or H2O2 caused destruction of nuclei of epidermal cells (EC) in the epidermis isolated from pea leaves. Phenol, a substrate of the apoplastic peroxidase-oxidase, in concentrations of 10−10–10−6 M prevented the destructive effect of chitosan. Phenolic compounds 2,4-dichlorophenol, catechol, and salicylic acid, phenolic uncouplers of oxidative phosphorylation pentachlorophenol and 2,4-dinitrophenol, and a non-phenolic uncoupler carbonyl cyanide m-chlorophenylhydrazone, but not tyrosine or guaiacol, displayed similar protective effects. A further increase in concentrations of the phenolic compounds abolished their protective effects against chitosan. Malate, a substrate of the apoplastic malate dehydrogenase, replenished the pool of apoplastic NADH that is a substrate of peroxidase-oxidase, prevented the chitosan-induced destruction of the EC nuclei, and removed the deleterious effect of the increased concentration of phenol (0.1 mM). Methylene Blue, benzoquinone, and N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD) capable of supporting the optimal catalytic action of peroxidase-oxidase cancelled the destructive effect of chitosan on the EC nuclei. The NADH-oxidizing combination of TMPD with ferricyanide promoted the chitosan-induced destruction of the nuclei. The data suggest that the apoplastic peroxidase-oxidase is involved in the antioxidant protection of EC against chitosan and H2O2.

Chitosan, CN−, or H2O2 caused the death of epidermal cells (EC) in the epidermis of pea leaves that was detected by monitoring the destruction of cell nuclei; chitosan induced chromatin condensation and marginalization followed by the destruction of EC nuclei and subsequent internucleosomal DNA fragmentation. Chitosan did not affect stoma guard cells (GC). Anaerobic conditions prevented the chitosan-induced destruction of EC nuclei. The antioxidants nitroblue tetrazolium or mannitol suppressed the effects of chitosan, H2O2, or chitosan + H2O2 on EC. H2O2 formation in EC and GC mitochondria that was determined from 2′,7′-dichlorofluorescein fluorescence was inhibited by CN− and the protonophoric uncoupler carbonyl cyanide m-chlorophenylhydrazone but was stimulated by these agents in GC chloroplasts. The alternative oxidase inhibitors propyl gallate and salicylhydroxamate prevented chitosan- but not CN−-induced destruction of EC nuclei; the plasma membrane NADPH oxidase inhibitors diphenylene iodonium and quinacrine abolished chitosan- but not CN−-induced destruction of EC nuclei. The mitochondrial protein synthesis inhibitor lincomycin removed the destructive effect of chitosan or H2O2 on EC nuclei. The effect of cycloheximide, an inhibitor of protein synthesis in the cytoplasm, was insignificant; however, it was enhanced if cycloheximide was added in combination with lincomycin. The autophagy inhibitor 3-methyladenine removed the chitosan effect but exerted no influence on the effect of H2O2 as an inducer of EC death. The internucleosome DNA fragmentation in conjunction with the data on the 3-methyladenine effect provides evidence that chitosan induces programmed cell death that follows a combined scenario including apoptosis and autophagy. Based on the results of an inhibitor assay, chitosan-induced EC death involves reactive oxygen species generated by the NADPH oxidase of the plasma membrane.

Destruction of guard cell nuclei in epidermis isolated from leaves of pea, maize, sunflower, and haricot bean, as well as destruction of cell nuclei in leaves of the aquatic plants waterweed and eelgrass were induced by cyanide. Destruction of nuclei was strengthened by illumination, prevented by the antioxidant α-tocopherol and an electron acceptor N,N,N′,N′-tetramethyl-p-phenylenediamine, and removed by quiacrine. Photosynthetic O2 evolution by the leaf slices of a C3 plant (pea), or a C4 plant (maize) was inhibited by CN− inactivating ribulose-1,5-bisphosphate carboxylase, and was renewed by subsequent addition of the electron acceptor p-benzoquinone.