Nitric oxide is a signaling molecule of plants under stressful conditions, and also this molecule can have a toxic effect. This study focused on investigation of a dose-dependent effect of the nitric oxide donor, sodiumμ2-dithiosulphate-tetranitrosyl diferrate tetrahydrate, on the structural state of mitochondrial membranes of epicotyls of pea seedlings. Treatment of mitochondria with 10–8 M of this drug led to thermo-induced structural transitions within the temperature range from 15°C to 21°C and from 30°C to 36°C in the lipid regions, and within the temperature range from 12°C to 15°C and from 27°С to 33°С in preprotein regions of membranes. In the lipid regions of the membranes, the compound exhibited antioxidant activity, leading to accumulation of long unsaturated fatty acids. Treatment of mitochondria with 10–4 M sodium-μ2-dithiosulphatetetranitrosyl diferrate tetrahydrate most likely led to a significant increase of the lipid peroxidation level and membrane lipid phase fluidity.

Исследовали влияние предобработки 0.1 мкМ мелатонином и кратковременного обезвоживания набухающих семян на водный дефицит, уровень ПОЛ и дыхание зародышей, а также последующий рост эпикотилей проростков гороха (Pisum sativum L.) и окислительную активность выделенных из них митохондрий. Две группы зародышей одинакового возраста, но отличающихся по весу и по стадии развития исследовали отдельно: набухшие зародыши, но не прорвавшие оболочки семени (группа 1) и проклюнувшиеся зародыши (группа 2). Уровень водного дефицита зародышей 1 и 2 группы в отсутствие доступа воды повышался на 8.7 и 6.9% соответственно, что оказалось чувствительным для дыхательной активности зародышей. Было показано, что зародыши группы 2, прорвавшие оболочку семени и вступившие в контакт с кислородом воздуха, переживали резкий подъем дыхательной активности, который был сопряжен с повышением уровня ПОЛ, регистрируемого по накоплению продуктов, реагирующих с тиобарбитуровой кислотой. Кратковременное обезвоживание тормозило дыхание зародышей этой группы, связанное с активностью цитохромного пути, но не сопровождалось возникновением окислительного стресса, а напротив, снижало уровень ПОЛ. У набухших, но не прорвавших оболочку семени зародышей группы 1 также отсутствовал окислительный стресс при обезвоживании. Предобработка мелатонином существенно повышала активность цитохромного пути дыхания у обеих групп зародышей, как в контроле, так и у подвергнутых обезвоживанию семян. У митохондрий, выделенных из эпикотилей 5-дневных проростков, выросших из семян, предобработанных мелатонином и подвергнутых обезвоживанию, наблюдалась активация окисления НАД-зависимого субстрата (малата), как в контрольных, так и в стрессовых условиях. Также было показано, что предобработка мелатонином стимулировала рост эпикотилей проростков группы 2 во всех вариантах опытов, тогда как у проростков группы 1 только после действия обезвоживания. Сделан вывод, что предобработка семян мелатонином стимулировала процесс дыхания зародышей за счет активации цитохромного пути, а также активировала окисление малатамитохондриями эпикотилей проростков и действовала как стимулятор роста эпикотилей после обезвоживания. Впервые показано, что кратковременное обезвоживание тканей зародыша может не сопровождаться окислительным стрессом.

The present study has been carried out to investigate changes in the structural characteristics of mitochondrial membranes isolated from epicotyls of pea seedlings of variety Nemchinovskiy 100 under the influence of different concentrations of NaHS. It was shown that treatment of pea seeds with NaHS at doses of 2∙10–4 and 5∙10–6 M resulted in the shift of thermally-induced structural transitions toward the region of lower temperatures and an increase in the microviscosity of both lipid and preprotein regions of mitochondrial membrane due to a decrease in the volume of their crystallization matrix. The presence of exogenous NaHS or endogenous hydrogen sulfide can enhance the activity of antioxidant defense enzymes and lead to accumulation of osmolytes. As a result, the microviscosity of the lipid bilayer could increase. To test this hypothesis, the effect of resveratrol, an antioxidant agent, on microviscosity of the lipid bilayer of epicotyl mitochondria of pea seedlings was investigated using normal pea seeds treated with resveratrol with or without pre-treatment with NaHS. Additionally, bioenergetic characteristics of mitochondria were studied. The results obtained confirmed that hydrogen sulfide has an effect on the structural characteristics of mitochondrial membranes through the activation of antioxidant enzymes and accumulation of osmolytes.

Effect of pretreatment with 0.1 µM melatonin and brief dehydration of swelling seeds on water deficit, LP level, respiration of embryos, subsequent growth of epicotyls, and oxidative activity of mitochondria isolated from them were investigated in the seedlings of pea (Pisum sativum L.). Two groups of embryos of the same age but differing by weight and the stage of development were examined separately: swollen embryos that so far did not break the seed coat (group one) and embryos that have broken through the seed coat (group two). Without water supply, water deficit in the embryos from groups one and two increased by 8.7 and 6.9

The possibility of using antioxidants-derivatives of 3-hydroxypyridine: N-acetylcysteinate 2-ethyl-6-methyl-3-hydroxypyridine and carnitinate 2-ethyl-6-methyl-3-hydroxypyridine as plant growth regulators investigated on the pea seedlings mitochondria (Pisum sativum L.) Flora 2 cultivar. The influence of water deficiency (WD) and 3-HP on the functional state of mitochondria was studied. Water deficiency activated lipid peroxidation and led to mitochondrial swelling. An increase in LPO intensity was associated with significant changes in the content of C18 fatty acids The content of linoleic and linolenic acids in the mitochondrial membranes of seedlings decreased by 12.5 and 22.3

An Erratum to this paper has been published: https://doi.org/10.1134/S1021443723330026

Изучено действие растительного антиоксиданта ресвератрола на структуру мембран митохондрий, выделенных из проростков гороха Pisum sativum L . , подвергнутых воздействию теплового стресс-фактора. В качестве структурных характеристик мембран использовали температурную зависимость микровязкости. Микровязкость определяли методом электронного парамагнитного резонанса спиновых зондов. Тепловой шок приводил к уменьшению кристалличности мембран митохондрий. Показано, что сверхмалые дозы ресвератрола возвращают структуру липидного бислоя митохондрий после воздействия теплового шока до состояния, свойственного нативным растениям.

The interaction of antioxidants, plant polyphenol resveratrol and nitric oxide donor, sulfur nitrosyl iron complex with thiosulfate Na2[Fe2(S2O3)2(NO)4]2 · 4H2O (TNIC-thio), and their combined action on the mitochondria of epicotyls of pea seedlings in vitro were studied. The antioxidant activity of resveratrol at a concentration of 10–6 M partially compensated for the toxic effect of TNIC-thio at high concentrations, which was most likely due to the manifestation of the pro-oxidant properties of high concentrations of the nitric oxide donor. The effect of resveratrol at concentrations of 10–6 and 10–8 M on the membranes of mitochondria isolated from the epicotyls of pea seedlings in the presence of 10–8 M TNIC-thio led to a violation of the regulation system of lipid peroxidation of membranes, which caused antioxidant stress. The effect of resveratrol at a dose of 2 × 10–5 M was of a dual nature and practically did not affect the structural state of mitochondrial membranes.

The effect of the antioxidant resveratrol on the structure of mitochondrial membranes isolated from Pisum sativum L. pea germ exposed to a heat stress factor is studied. The temperature dependence of microviscosity is used as the structural characteristics of the membranes. The microviscosity is determined by the method of electron spin resonance (ESR). Heat stress leads to a decrease in the crystallinity of mitochondrial membranes. It is shown that ultralow doses (ULDs) of resveratrol restore the structure of the lipid bilayer of the mitochondria after exposure to heat stress (HS) to a state similar to intact plants.

Оксид азота (NO) является сигнальной молекулой клетки при стрессе. Известно, что воздействие стрессовых факторов может не только задерживать рост всего растения, листьев, корней, но и прорастание семян, а также скорость роста проростков. NO участвует в процессах адаптации, позволяя растению защищать себя от стресс-факторов путем предотвращения дисфункции митохондрий. Однако, о защитных механизмах оксида азота известно мало. Натрий-μ2-дитиосульфатотетранитозилдиферрат тетрагидрат (ТНКЖ-тио) - кристаллический водорастворимый донор NO. Nitric oxide (NO) is a cell signaling molecule during stress. It is known that the impact of stress factors can not only retard the growth of the whole plant, leaves, roots, but also the germination of seeds, as well as the growth rate of seedlings. NO is involved in adaptation processes, allowing the plant to protect itself from stress factors by preventing mitochondrial dysfunction. However, little is known about the protective mechanisms of nitric oxide. Sodium-μ2-dithiosulfatetetranitosyl diferrate tetrahydrate (TNIC-thio) is a crystalline water-soluble NO donor.

The possibility of using the NO sodium μ2-dithiosulfato-tetranitrosyldiferrate tetrahydrate (TNIC-thio) as a drug preventing mitochondrial dysfunction under stress was studied. The properties of TNIC-thio were studied on the mitochondria of 5-day-old etiolated pea seedlings. The functional state of mitochondria was determined under conditions of heat shock (HS) and treatment of seedlings with TNIC-thio. Heat shock activated lipid peroxidation and led to swelling of mitochondria and changes in the content of C18 fatty acids (FAs): the C18 FA unsaturation index in mitochondrial membranes decreased from 1.42 ± 0.01 to 1.22 ± 0.01. Changes also took place in the C20 FA content: the 20:3ω6 content decreased by almost 43.6%, and the 20:0 content decreased by 1.3 times. The change in the pool of unsaturated FAs affected the bioenergetic characteristics of mitochondria: an almost 1.5-fold drop in the rates of oxidation of NAD-dependent oxidation substrates was shown. Treatment of pea seeds with 10 –6 M TNIC-thio was accompanied by prevention of lipid peroxidation (LPO), prevention of changes in the composition of FA membranes and mitochondrial morphology, and restoration of the bioenergetic characteristics of these organelles. The drug could help maintain the functional state of mitochondria under stress by preventing LPO. It perhaps resulted in the preservation of the efficiency of energy processes in the cell, which probably increased the organism’s resistance to changing environmental conditions.

The present study was designed to investigate the antistress properties of the nitric oxide donor: the ferric-thiosulfate complex of sodium μ2-dithiosulfate-tetranitrosyl diferrate tetrahydrate Na2[Fe2(S2O3)2(NO)4]2 · 4H2O (TNIC-thio), in particular, to assess the effects of different concentrations of the donor on the functional state of mitochondria isolated from pea seedlings. Incubation of mitochondria with 10–4 M of the drug led to a sharp decrease in the microviscosity of a freestanding lipid bilayer. This is attributable to a rise in the content of unsaturated fatty acids in the lipid fraction of mitochondrial membranes. At the same time, the drug uncoupled the processes of oxidation and phosphorylation. Treatment of mitochondria with 10–8 M TNIC-thio brought about an increase in the content of unsaturated C20 fatty acids (by 25–45

The effect of stress (water deficiency, high-temperature stress) and nitric oxide donor sodium μ2-dithiosulphate-tetranitosyldiferrate tetrahydrate Na2 [Fe2 (S2O3)2 (NO)4]2 × 4H2O (TNIC-thio) on the fatty acid composition and bioenergetic characteristics of 5-day etiolated pea seedling mitochondria was studied. Stressful effects caused the activation of LPO in the mitochondrial membranes. At the same time, significant changes occurred in the content of C18 and C20 fatty acids (FA). A decrease in the content of linoleic and linolenic acids, one of the main FA components of cardiolipin in higher plants, apparently caused a decrease in the maximum rates of oxidation of NAD-dependent substrates. The. treatment of pea seeds with 10-6M TNIC-thio prevented the activation of LPO, changes in the fatty acid composition of mitochondrial membranes, and contributed to the preservation of the bioenergetic characteristics of these organelles. By preventing the decline in energy metabolism, TNIC-thio probably has adaptogenic properties, that were also reflected in physiological parameters, namely, the growth of seedlings. Treatment of pea seeds and seedlings with the studied preparation prevented inhibition of root and shoot growth in conditions of water deficiency. Based on the data obtained, it can be concluded that the protective properties of TNIC-thio are due to its antioxidant activity.

Изучено действие антиоксиданта ресвератрола в различных концентрациях на микровязкость мембран митохондрий, выделенных из клеток листьев гороха. Показано, что ресвератрол в физиологических (5·10-6 М) и сверхмалых (5·10-14 М) дозах изменял микровязкость и структуру липидного бислоя мембран. По-видимому, данный антиоксидант путем изменения структуры липидного окружения мембранных белков изменяет их активность и таким образом влияет на активность и функции митохондрий. В областях доз между физиологическими и сверхмалыми не было обнаружено никаких изменений, т.е. наблюдалась «мертвая зона». Кроме того, было выявлено, что ресвератрол в физиологических концентрациях сдвигает термоиндуцированный структурный переход в область более низких температур, что может мешать нормальной регуляции естественных процессов.

In this study, the effects of different concentrations of antioxidant resveratrol on microviscosity of mitochondrial membranes isolated from pea leaves were investigated. It was shown that resveratrol in physiological (5 × 10 –6 M) and ultralow (5 × 10 –14 M) doses changed the microviscosity and altered the structure of the membrane lipid bilayer. This antioxidant is thought to affect the activity of membrane proteins due to altered structures of lipid molecules that surround them and, in turn, influence mitochondrial activity and function. No change was found in the range between physiological and ultralow doses, which is a “dead” zone. Moreover, resveratrol in physiological concentrations was found to shift thermally induced structural transition to the lower temperature region; therefore, it can interfere with the normal regulation of natural processes

The possibilities of using natural resveratrol polyphenol as an adaptogen were investigated by means of atomic force microscopy (AFM) and fluorometry. The study was carried out on mitochondria of pea seedlings (Pisum sativum L., Flora 2 variety). The protective properties of the drug were studied using models of mitochondrial “aging” and water deficiency. Incubation of isolated mitochondria in a hypotonic medium (the aging process) activated lipid peroxidation (POL): the fluorescence intensity of POL products increased compared to the control and led to an increase in the size of AFM images of mitochondria, which indicated swelling of the organelles. Resveratrol at a concentration of 10−6 M reduced the fluorescence intensity of lipid peroxidation products almost to control values, as well as preventing mitochondrial swelling. In vivo experiments were carried out in a water deficiency model. Pea seeds previously soaked in water or treated with resveratrol were subjected to water deficiency. Water-deficiency conditions caused an increase in lipid peroxidation of the mitochondrial membranes of pea seedlings, led to swelling of mitochondria and inhibition of the biogenesis of these organelles as compared to the control. Resveratrol treatment of pea seeds (3 × 10−4 M) reduced the number of swollen mitochondria in relation to the control, activated the biogenesis of these organelles, and normalized POL. It was hypothesized that the protective effect of the studied drug is due its antioxidant properties and AFM and fluorometry methods can be used to screen the protective properties of biologically active substances.

The antiradical properties and biological activity of natural polyphenol—resveratrol—are studied. The chemiluminescence method recorded high values of the antiradical activity of this drug. Using the model system, it is shown that the drug in the concentration range of 10–5  to 10–12 M prevented the activation of lipid peroxidation (LPO) in the mitochondrial membranes of pea seedlings, which could indicate the presence of antistress properties in this preparation, which are studied under conditions of water deficiency, which causes LPO and leads to mitochondrial swelling. Resveratrol seed treatment (3 × 10–5 M) under these conditions leads to a decrease in the number of swollen mitochondria and the appearance of small mitochondria, which, perhaps, is a sign of the activation of mitochondrial biogenesis. Resveratrol, which prevents LPO, helps maintain the functional state of mitochondria and activates the energy metabolism by increasing their number, which is reflected in the physiological parameters: the drug prevents a decrease in the growth rate of pea seedlings in conditions of water deficiency. Based on the data obtained, it is assumed that the drug has antistress properties in the concentration range in which it exhibits an antioxidant effect.

The influence of nitric oxide donor - iron-sulfur-nitrosyl complex with thiosulfate Na-2[Fe-2 (S2O3)(2) (NO)(4)](2) center dot 4H(2)O (TNIC-thio) - on lipid peroxidation (LPO) in mitochondrial membranes of 5-day etiolated pea seedlings (Pisum sativum L.) in the model system of "aging" of mitochondria and in the conditions of water deficiency was investigated. In addition, the effect of TNIC-thio on the expression of the sofiA gene of the DNA repair SOS response in E. coli PQ37 cells was studied. TNIC-thio in the concentration range of 10(-5)-10(-9) M reduced the intensity of LPO to control values in the model system of "aging" mitochondria, which may indicate the presence of anti-stress properties of the drug. In the conditions of water deficiency the fluorescence intensity of LPO products in the membranes of the mitochondria increased twofold. At the same time, the treatment of pea seeds with 3 x 10(-5) or 3 x 10(-6) M TNIC-thio prevented activation of LPO under conditions of water deficiency. In addition, the treatment of E. coli cells with this drug increased 2.7-fold the expression of gene sfiA of SO S-regulon under heat stress, which also pointed to the adaptogenic properties of the drug. Protective properties of TNIC-thio were also exhibited with respect of physiological indicators, namely, growth of seedlings. The treatment of pea seeds and seedlings with TNIC-thio prevented the inhibition of root and shoot growth under conditions of water deficiency. It is proposed that protective properties of TNIC-thio are related with its ability to prevent LPO.