Microviscosity and lipid order are the main parameters characterizing the phase states of the membrane. Variations in microviscosity and lipid composition in a living cell may indicate serious disturbances, including various kinds of stress. In this work, the effect of hyperosmotic stress on the microviscosity of mitochondrial membranes was investigated, using potato (Solanum tuberosum L.) tuber mitochondria. The microviscosity of mitochondrial membranes isolated from check and stressed (9 days at 34–36 °C) tubers was estimated by determining the generalized polarization (GP) values using a Laurdan fluorescent probe in confocal microscopy studies. It was revealed that the GP distribution in mitochondria isolated from stressed tubers contained new component-characterizing membrane domains with an increased lipid order compared to the rest of the membrane. We have mapped the microviscosity of mitochondrial membranes for the first time and observed the dynamics of the membrane microviscosity of an individual mitochondrion. The hyperosmotic stress significantly influences the functional state of potato mitochondria, decreasing the substrate oxidation rate and respiratory control coefficient but increasing MitoTracker Orange fluorescence. Under hyperosmotic stress, the microviscosity of mitochondrial membranes changes, and membrane domains with increased lipid order are formed. The revealed changes open up prospects for further research on the participation of raft-like microdomains of mitochondria in plant resistance to stress factors.

The variations of the content of the main classes of lipids in the vacuolar membrane under oxidative stress have been investigated. The data obtained suggest the possibility of promoting the autophagy process. During autophagy, membrane contact sites, which are raft structures, have been established between tonoplast and autophagosomes. The results showed a significant increase in the content of raft-forming lipids (sterols, sphingolipids and lipids enriched with saturated fatty acids), what induce an elevation the number of membrane contact sites and activation of such a protective mechanism as autophagy under oxidative stress.

The study set out to investigate the effect of different concentrations of copper ions on the composition of phytosterols of the vacuolar membrane (tonoplast) of beetroot (Beta vulgaris L.). To characterise the stress caused by the action of copper ions, a conductometric study of the permeability of cell membranes and the level of lipid peroxidation was carried out. The results demonstrate an increase in these indicators in beetroot tissues. Among tonoplast phytosterols, β-sitosterol, stigmasterol, campesterol, and cholesterol were studied. In cell membranes, these phytosterols perform a structural function, contributing to lipid microdomain formation, influencing plant growth and development, and participating in plant responses to stress. The study of the effect of copper ions on tonoplast phytosterols showed most of them to be occupied by the free form. In the presence of 100 μM copper, a significant increase in campesterol content was detected. The ratio of stigmasterol/β-sitosterol and 24-methyl-/ ethylsterol, which may influence the stress response of plants, is an important indicator of plant membrane health. The observed increases in the ratios of stigmasterol/β-sitosterol and 24-methyl-/ethylsterols can be explained in terms of mechanisms for regulating the functioning of the tonoplast under stress caused by copper ions. Thus, the obtained results may indicate the participation of cell membranes, including tonoplast, in the mechanisms of adaptation of beetroot tissue cells to stress caused by copper ions.

Изучали действие различных концентраций Pb2+ (0.1 мкМ – 2 мМ) на работу вакуолярной Н+-пирофосфатазы корнеплодов столовой свеклы (Beta vulgaris L.). Установлено, что на уровне тканей в ответ на действие 1 мМ и 2 мМ ионов свинца происходило накопление продуктов перекисного окисления липидов (диеновых конъюгатов) и увеличение проницаемости клеточных мембран, что свидетельствует о развитии окислительного стресса. Оценку функционирования Н+-пирофосфатазы тонопласта проводили по уровню гидролитической и транспортной активности фермента. Наблюдалось стимулирование гидролиза субстрата, катализируемого Н+-пирофосфатазой, в присутствии низких концентраций Pb2+. Концентрации металла свыше 500 мкМ ингибировали гидролитическую активность пирофосфат-зависимого фермента. Однако уровень транспортной активности значимо стимулировался в присутствии 1 мМ Pb2+. Одновременно проводимый анализ качественного и количественного состава жирных кислот (ЖК) общих липидов тонопласта показал достоверно значимое снижение содержания олеиновой (С18:1(n-6)) и линолевой (С18:2(n-6)) ЖК при действии 1 мМ Pb2+. Ионы свинца в концентрации 1 мМ увеличивали сумму насыщенных ЖК. Установлено, что Pb2+ могут оказывать влияние на активность Н+-пирофосфатазы тонопласта и структуру клеточных мембран в зависимости от концентрации. Возможно, выявленные изменения являются одним из механизмов адаптации к условиям стресса, вызванного Pb2+.

The effect of different concentrations of copper ions (100 and 500 mu M) on the fatty acid composition of the vacuolar membrane lipids of beet root tissues ( Beta vulgaris L.) was studied. Exposure to 100 mu M copper led to an increase in the content of FAs by 57 mu g/mg of total lipids as compared to the control. Stearoyl desaturase (SDR) activity decreased from 0.87 in the control to 0.77 at 100 mu M copper. Exposure to 500 mu M copper caused an increase in the SDR activity up to 0.93, but the proportion of FAs decreased by 50 mu g/mg of total lipids compared with 100 mu M copper. In addition, there was an increase in the saturation of tonoplast lipids to 44 and 40% at 100 and 500 mu M Cu2+, respectively. The results suggest that FAs of tonoplast lipids are involved in the stress response mechanisms induced by excessive Cu2+ concentrations.

The effect of different concentrations of Pb2+ (0.1 µM−2 mM) on the functioning of vacuolar H+-pyrophosphatases of beetroot roots (Beta vulgaris L.) was studied. At the tissue level, in response to the action of 1 mM and 2 mM lead ions, there was an accumulation of lipid peroxidation products (diene conjugates) and an increase in the permeability of cell membranes, which indicates the development of oxidative stress. Evaluation of the functioning of H+-pyrophosphatases of tonoplast were determined according to the level of hydrolytic and transport activity of the enzyme. Stimulation of substrate hydrolysis catalyzed by H+-pyrophosphatase was observed in the presence of low concentrations of Pb2+. Metal concentrations above 500 μM inhibited the hydrolytic activity of the pyrophosphate-dependent enzyme. However, the level of transport activity was significantly stimulated in the presence of 1 mM Pb2+. Simultaneously, the conducted analysis of the qualitative and quantitative composition of fatty acids (FA) of total tonoplast lipids showed a reliably significant decrease in the content of oleic (C18:1(n-6)) and linoleic (C18:2(n-6)) FA under the action of 1 mM Pb2+. Lead ions at a concentration of 1 mM increased the amount of saturated fatty acids. It has been established that Pb2+ may affect the activity of H+-pyrophosphatases of tonoplast and the structure of cell membranes depending on concentration. It is possible that the identified changes are one of the mechanisms of adaptation to stress conditions caused by Pb2+.

An analysis of the distribution of the order parameter of lipids in the plasma-membrane and tonoplast fractions isolated from beetroot (Beta vulgaris L.) was carried out. The packing density (order) of membrane lipids was assessed based on measurements of the generalized polarization (GP) of Laurdan fluorescence. From two to four components were revealed in the distribution of GP values of plasma membrane fractions in zones of sucrose density gradient, and from two to six components were detected in the distribution of tonoplast GP fractions. According to the analysis of the distribution of the lipid ordering parameter, raft structures in the plasma membrane can be present in zones of 15 and 25

The influence of toxic concentrations of copper ions (100 and 500 μM) on the composition of sterols in the vacuolar membrane of beet roots was studied (Beta vulgaris L.). As a result of the studies, 12 compounds were identified in the free sterol (FS) fraction and 11 compounds in the sterol ester (ES) fraction. The ES contained compounds that were not found in the FS. Interestingly, the total content of these biologically active compounds increased at 500 µM Cu2+. A decrease in such triterpenes as lanosta-7,9(11)-diene-3β,18,20-triol, 3,18-diacetate, (20R)-(C34N54O5) was observed in FS and ES. In the FS fraction, the content of the compound 7,8-epoxylanostan-11-ol, 3-acetoxy- increased under stress, while its amount decreased in the ES. It has been established that the total content ∆5-sterols, under normal conditions and under stress, amounted to no more than 33

The influence of oxidative stress on the lipid composition of raft structures of vacuolar membranes isolated from Beta vulgaris L. beet roots was studied in order to clarify the role of these membrane structures in the adaptation mechanisms of the plant cell. Changes in the qualitative and quantitative composition of major lipids, sterols, and fatty acids resulting from stress were analyzed and compared with changes in lipids, the role of which has been reliably established in protecting cells from stress. Previously, the presence of three types of raft structures was shown in the vacuolar membrane. Under oxidative stress, variations took place in the composition of the lipids of these structures. The most significant of them, capable of influencing the protective mechanisms of the plant cell, were identified in raft microdomains of zone four of the sucrose gradient (35

The investigation of the lipid-protein microdomains of the plasmalemma isolated with the aid of the non-detergent technique in the zones of the sucrose density gradient after high-speed centrifugation from the tissue pieces of beet roots, which underwent oxidative stress, was conducted. The microdomains, whose lipid composition — according to the definition — allowed us to classify them as rafts, were studied. After the exposure to oxidative stress (100 mM hydrogen peroxide), the variations in the composition of membrane lipids bound up mainly with the elevations of the content of raft-forming lipids (sterols, sterol esters). Oxidative stress provoked redistribution in the composition of sterols, which led to an elevation in the content of campesterol and in the ratio of stigmasterol/sitosterol. Furthermore, the variations were registered in the content of phospholipids and phosphoglycerolipids, which are capable of stabilizing the lamellar structure of membranes. The results obtained allow one to assume that under the oxidative stress, variations in the composition of lipids in microdomains of the plasma membrane can take place. These variations may influence the functioning of the membranes, and the membranes may participate in the protection of the plant cell.

MAIN CONCLUSION:The comparison of the changes of the lipid content in plant cell boundary membranes demonstrates a substantial role of the vacuolar membrane in response to hyperosmotic stress. Comparison of variations in the lipid content of plant cell boundary membranes (vacuolar and plasma membranes) isolated from beet root tissues (Beta vulgaris L.) was conducted after the effect of hyperosmotic stress. Both types of membranes participate in the formation of protective mechanisms, but the role of the vacuolar membrane was considered as more essential. This conclusion was connected with more significant adaptive variations in the content and composition of sterols and fatty acids in the vacuolar membrane (although some of the adaptive variations, especially, in the composition of phospholipids and glycoglycerolipids were similar for both types of membranes). In the plasma membrane under hyperosmotic stress, the increase in the content of sphingolipids was noted that was not observed in the tonoplast.

Background. Presently, a selection of environmentally friendly technologies for pre-sowing treatment of wheat seeds is conducted in agriculture. Ozone, an allotropic form of oxygen, is highly reactive, acts as an effective insecticide, promotes seed improvement, and reduces soil contamination. The impact of ozone on seeds is one of the promising technologies to improve the quality of seedlings. It is known that the field germination of winter wheat may be less than 50%, depending on the environmental conditions, and therefore the quality of seedlings is very important. Purpose. To evaluate the effect of different ozone concentrations on morphological parameters and synthesis of fatty acids (FA) in winter wheat seedlings. Materials and methods. The object of the study was the seeds of soft winter wheat (variety Irkutskaya). The ozonation was conducted with ozone concentrations of 2, 4, 6 and 8 g/m3 during 15, 30, 45 and 60 min. The germination of treated and control seeds was carried out for three days in the dark in a thermostat (24±1°C). On the third day, the morphometric characteristics were measured. The extraction of lipids from shoots and roots was carried out according to the method of Bligh, Dyer [14]. The methyl esters of lipid fatty acids were analyzed by chromato-mass spectrometry. Statistical data processing was conducted with the use of R programming language and SigmaPlot v. 12.5. Results. Ozone in concentrations of 2, 4, 6 g/m3 has a stimulating effect on seedling germination, the length of the shoots and roots increases. At ozone concentration of 8 g/m3, the length of the shoots and roots decreases. There is a general trend in the content of fatty acids, in the shoots of seedlings there is some decrease in lipid unsaturation, and in the roots - some increase in lipid unsaturation. Conclusion. Ozone in concentrations up to 8 g/m3 is suitable for pre-sowing seed treatment and improves seedling performance.

-We compared oxidative and osmotic stress-induced changes in sterol content in plasma membranes and tonoplasts isolated from stored beet (Betavulgaris L.) roots. The most significant differences between the membranes under all studied stresses were observed in the cholesterol content: it decreased in the plasmalemma, but increased 4-6 times in the tonoplast. Similar changes were observed for other sterols, but in different ways under different types of stress. Particularly noticeable differences were noted under hyperosmotic stress. The increase in the content of sterols was much more pronounced in the vacuolar membrane compared to the plasmalemma. This observation allows us to conclude that tonoplast plays a more significant role in protection of the plant cell from stress compared to plasmalemma.

When studying the raft structures of wheat chloroplast membranes, previously identified in the 15% sucrose zone after high-speed centrifugation, an additional opalescence zone was found in the 5% sucrose region. Analysis of the composition of sterols and fatty acids of lipids in this zone in comparison with the zone of rafts and chloroplast membranes showed that raft structures may also be present in this zone. This suggests that wheat chloroplast membranes may contain two types of raft structures.

MAIN CONCLUSION:Variations in the content of tonoplast microdomains, isolated with the aid of a non-detergent technique, are induced by osmotic stress and may take part in plant cell adaptive mechanisms. Investigation of tonoplast microdomain lipids isolated with the aid of the non-detergent technique from beetroots (Beta vulgaris L.) subjected to either hyperosmotic or hypoosmotic stress was conducted. Earlier, an important role of tonoplast lipids in the protection of plant cells from stress was demonstrated (Ozolina et al. 2020a). In the present paper, we have put forward a hypothesis that lipids of microdomains of raft nature present in the tonoplast are responsible for this protective function. The variations in the content of lipids of the studied nondetergent-isolated microdomains (NIMs) under hyperosmotic and hypoosmotic stresses were different. Under hyperosmotic stress, in the scrutinized microdomains, some variations in the content of lipids were registered, which were characteristic of the already known protective anti-stress mechanisms. These variations were represented by an increase in sterols and polar lipids capable of stabilizing the bilayer structure of the membranes. The found variations in the content of sterols may be bound up with some intensification of the autophagy process under stress because sterols foster the formation of new membrane contacts necessary for this process. Under hypoosmotic stress, the pattern of redistribution of the lipids in the scrutinized membrane structures was different: the largest part of the lipids appeared to be represented by hydrocarbons, which fulfilled mainly a protective function in plants and could prevent the excess water influx into the vacuole. The results obtained not only demonstrate the possible functions of the vacuolar membrane microdomains but also put forward an assumption on the role of any membrane microdomain in the protection mechanisms of the plant cell.

The genus Rhodococcus includes polymorphic non-spore-forming gram-positive bacteria belonging to the class Actinobacteria. Together with Mycobacterium and Corynebacterium, Rhodococcus belongs to the Mycolata group. Due to their relatively high growth rate and ability to form biof ilms, Rhodococcus are a convenient model for studying the effect of biologically active compounds on pathogenic Mycolata. Colchicine was previously found to reduce biof ilm formation by P. carotovorum VKM B-1247 and R. qingshengii VKM Ac-2784D. To understand the mechanism of action of this alkaloid on the bacterial cell, we have studied the change in the fatty acid composition and microviscosity of the R. qingshengii VKM Ac-2784D membrane. Nystatin, which is known to reduce membrane microviscosity, is used as a positive control. It has been found that colchicine at concentrations of 0.01 and 0.03 g/l and nystatin (0.03 g/l) have no signif icant effect on the survival of R. qingshengii VKM Ac-2784D cultivated in a buffered saline solution with 0.5 % glucose (GBSS). However, colchicine (0.03 g/l) signif icantly inhibits biof ilm formation. Rhodococcus cells cultivated for 24 hours in GBSS with colchicine acquire a rounded shape. Colchicine at 0.01 g/l concentration increases C16:1(n-7), C17:0, C20:1(n-9) and C21:0 fatty acids. The microviscosity of the membrane of individual cells was distributed from the lowest to the highest values of the generalized laurdan f luorescence polarization index (GP), which indicates a variety of adaptive responses to this alkaloid. At a higher concentration of colchicine (0.03 g/l) in the membranes of R. qingshengii VKM Ac-2784D cells, the content of saturated fatty acids increases and the content of branched fatty acids decreases. This contributes to an increase in membrane microviscosity, which is conf irmed by the data on the GP fluorescence of laurdan. All of the above indicates that colchicine induces a rearrangement of the Rhodococcus cell membrane, probably in the direction of increasing its microviscosity. This may be one of the reasons for the negative effect of colchicine on the formation of R. qingshengii VKM Ac-2784D biof ilms.

The study of lipid rafts allowed us to take a new look at the morphology, organization and functioning of membranes, both of animal and plant origin. However, lipid rafts and their function in the cell membranes of plants are poorly understood in comparison with the membranes of animal cells. The protective function of the plant cell is of great importance for the body as a whole because plants lead an attached mode of life. To date, it is known that lipid rafts are involved in the membrane mechanisms of cell protection in response to negative effects. In this review, we summarized the literature data showing the participation of lipid rafts of plant membranes (plasmalemma, Golgi complex membranes, chloroplasts, mitochondria, and vacuoles) in the protective function of cells.