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Lipid–protein microdomains (presumably rafts) of the plasmalemma isolated from the beetroots subjected to hyperosmotic stress and hypoosmotic stress were studied. In these microdomains, the variations in the composition of total lipids, sterols, and fatty acids were observed. These variations differed under hypo- and hyperosmotic types of stress. We presumed that such variations were bound up with different strategies, which are probably related to protecting the cell from osmotic stress. One of the protection tendencies might be related, in our opinion, to credible growth of the content of such lipids as sterols and sterol esters, which are considered as raft-forming. Under osmotic stress, these lipids can contribute to the formation of both new raft structures and new membrane contacts of plasmalemma with intracellular organelles. Another protection tendency may be bound up with the redistribution of membrane phospholipids and phosphoglycerolipids possibly to stabilize the membrane’s lamellar structure, which is ensured by credible growth of the content of such lipids as phosphatidylcholines, phosphatidylinositols, and digalactosyldiacylglycerol. The participation of lipid–protein microdomains in the adaptive mechanisms of plant cells may, in our opinion, also be bound up with the redistribution of membrane sterols, which (redistribution) in a number of variants may provoke credible growth in the content of cholesterol or “anti-stress” sterols (campesterol and stigmasterol). So, according to our results, the variations in the content of the plasmalemma lipid–protein microdomains take place under osmotic stress. These variations may influence the functioning of plasmalemma and take part in the adaptive mechanisms of plant cells.

We analyzed the distribution of the ordering parameter of raft-like microdomains in the mitochondrial membranes of halophytes, which differ in the type of salt tolerance: Salicornia perennans, Halocnemum strobilaceum, and Artemisia santonica. The packing density of the membrane lipids (lipid order) was assessed by measuring generalized polarization (GP) of the Laurdan fluorescence. In the mitochondrial membrane material of S. perennans, H. strobilaceum, and A. santonica, in the range of 15 and 25% of the sucrose density gradient, the GP distributions contain 4–6 components presumably related to the raft structures. Lipids isolated from ordered domains are enriched in sterols, cerebrosides, and saturated fatty acids; the quantitative content of raft-forming lipids depends on the salt tolerance strategy of halophytes.

The effects of tonoplast and rafts isolated from vacuolar membranes on the import of DNA into mitochondria of potato tubers (Solanum tuberosum) were studied. It was shown that the addition of rafts isolated from the tonoplast to mitochondria increased 8-fold the activity of the DNA import into mitochondria as compared to the import in the untreated mitochondria.

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 f luorescence 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.Род Rhodococcus объединяет полиморфные неспорообразующие грамположительные бактерии, относящиеся к классу Actinobacteria. Rhodococcus вместе с Mycobacterium и Corynebacterium входят в группу Mycolata. Благодаря относительно высокой скорости роста и способности к образованию биопленок, Rhodococcus являются удобной моделью для изучения действия биологически активных соединений на патогенные Mycolata. Ранее было показано, что колхицин угнетал образование биопленок у P. carotovorum ВКМ В-1247 и R. quingshengii ВКМ Ac-2784D. Целью настоящей работы было изучение действия колхицина на жирнокислотный состав и микровязкость мембран Rhodococcus qingshengii ВКМ Ac-2784D для понимания механизма действия этого алкалоида на бактериальную клетку. В качестве положительного контроля использовали нистатин, снижающий микровязкость мембран. Установлено, что колхицин в концентрациях 0.01 и 0.03 г/л и нистатин (0.03 г/л) не оказали существенного влияния на выживаемость R. qingshengii ВКМ Ac-2784D, культивируемого в забуференном физиологическом растворе глюкозы (ЗФРГ). Однако колхицин (0.03 г/л) значительно угнетал образование биопленки. Клетки Rhodococcus, культивируемые в течение суток в ЗФРГ с колхицином, приоб- ретали округлую форму. Колхицин в концентрации 0.01 г/л вызывал увеличение жирных кислот C16:1(n-7), С17:0, C20:1(n-9) и C21:0. Микровязкость мембраны отдельных клеток распределялась от максимально низких до максимально высоких значений показателя обобщенной поляризации флуоресценции лаурдана (GP), что свидетельствует о разнообразии адаптационных ответов на этот алкалоид. При более высокой концентрации колхицина (0.03 г/л) в мембранах клеток R. qingshengii ВКМ Ac-2784D увеличивалось содержание насыщен- ных жирных кислот и падало – разветвленных. Это способствовало увеличению микровязкости мембраны, что подтверждается данными по GP. Таким образом, колхицин индуцирует перестройку клеточной мембраны Rhodococcus, вероятно, в сторону увеличения ее микровязкости, что может быть одной из причин негативного действия колхицина на образование биопленок R. qingshengii ВКМ Ac-2784D.

The article dwells upon identifying the effect of cadmium on the roots of beetroot. The exposure effects of various concentrations of cadmium were studied at different levels of the plant organization (tissue pieces, organelles, membrane vesicles). The effect was noted only at a concentration of 100 μm. The negative effect of cadmium on the roots tissues of beetroot appeared with an increase in permeability and a decrease in the stability of cell membranes due to a change in the composition of fatty acids of membrane lipids and an increase in oxidation processes. The effect of cadmium in model experiments on the activity of the proton pumps of the vacuolar membrane has been evaluated. The pumps provide for the transport of heavy metals into the vacuole, which is one of the effective mechanisms for phytoremediation. The influence of cadmium in model experiments on the activity of the proton pump of a vacuolar membrane was evaluated. Under the influence of cadmium, a decrease in the activity of V-ATPase was observed, while the activity of V-PPase did not change. The results obtained complement our understanding of the damaging effects that occur in plant cells under cadmium stress.

Lipid-protein microdomains (presumably rafts) of the plasmalemma isolated from the beetroots subjected to hyperosmotic stress and hypoosmotic stress were studied. In these microdomains, the variations in the composition of total lipids, sterols, and fatty acids were observed. These variations differed under hypo- and hyperosmotic types of stress. We presumed that such variations were bound up with different strategies, which are probably related to protecting the cell from osmotic stress. One of the protection tendencies might be related, in our opinion, to credible growth of the content of such lipids as sterols and sterol esters, which are considered as raft-forming. Under osmotic stress, these lipids can contribute to the formation of both new raft structures and new membrane contacts of plasmalemma with intracellular organelles. Another protection tendency may be bound up with the redistribution of membrane phospholipids and phosphoglycerolipids possibly to stabilize the membrane's lamellar structure, which is ensured by credible growth of the content of such lipids as phosphatidylcholines, phosphatidylinositols, and digalactosyldiacylglycerol. The participation of lipid-protein microdomains in the adaptive mechanisms of plant cells may, in our opinion, also be bound up with the redistribution of membrane sterols, which (redistribution) in a number of variants may provoke credible growth in the content of cholesterol or "anti-stress" sterols (campesterol and stigmasterol). So, according to our results, the variations in the content of the plasmalemma lipid-protein microdomains take place under osmotic stress. These variations may influence the functioning of plasmalemma and take part in the adaptive mechanisms of plant cells.

The vacuolar membrane is an essential component in protecting the plant cell from stress factors. Different variations in the tonoplast lipid content, which depend on the type of stress, have been reviewed. The lipid content of vacuolar membranes of beet roots (Beta vulgaris L.) under hypoosmotic, hyperosmotic and oxidative types of stress has been studied. These types of stress induce variations in the content of almost all the classes of studied lipids (phospholipids, glycoglycerolipids, sterols and fatty acids). The variations, which are characteristic of a single stress, include the variations (i) in the content of individual glycoglycerolipids and in their total content, (ii) in the total content of sterols, and (iii) in the ratio of content of phosphatidylcholine/phosphatidylethanolamine in the scope of tonoplast phospholipids. Variations observed under all of the types of stress under scrutiny include (i) variations in the content of fatty acids of tonoplast lipids, (ii) some decrease in the content of phosphatidic acid and phosphatidylethanolamine, and (iii) variations in the content of individual sterols. Stigmasterol, campesterol, as well as the stigmasterol/sitosterol ratio increased in varying degrees under all of the types of stress. The most substantial variations have been observed in the content of sterols under abiotic stress. This is probably due to role of sterols in regulation of such membrane characteristics as permeability and microviscosity. In our opinion, sterols may represent one of the main components of tonoplast adaptive mechanisms.

Glycoglycerolipids (GL) of the red beet vacuolar membrane under osmotic and oxidative stresses have been investigated. Variations of the GL content under stress conditions might be indicators of an important role of these compounds in protective mechanisms. Changes of the GL levels and the digalactosyldiacylglycerols/monogalactosyldiacylglycerol (DGDG/MGDG) ratio in the vacuolar membrane under hypoosmotic and oxidative stresses corresponded mainly to those observed in other cell membranes under majority of stresses studied, namely, the ratio increased that contributed to membrane stabilization. The changes of the GL content of vacuolar membrane caused by hyperosmotic stress notably differed. The DGDG content and DGDG/MGDG ratio significantly decreased. These alterations did not necessarily result in a decrease in the membrane stability under hyperosmotic stress, since the content of MGDG involved in the formation of a hexagonal structure and capable of destabilizing lipid bilayer also decreased. The changes of the GL level in the vacuolar membrane under hyperosmotic stress can be due to an increased degradation or reduced biosynthesis of GL; these changes may represent yet another defense mechanism of a plant cell against stresses.

Vacuolar and plasma membranes were isolated by a detergent-free method from beet roots (Beta vulgaris L.), and were fractionated in a sucrose density gradient of 15–60% by high-speed centrifugation at 200,000×g during 18 h. The membrane material distributed over the sucrose density gradient was analyzed for the presence of lipids characteristic of raft structures in different zones of the gradient. The quantitative and qualitative content of lipids and sterols, and the composition of fatty acids were analyzed. Some membrane structures differing in their biochemical characteristics were revealed to be located in different zones of the sucrose gradient. The results of the analysis allowed us to identify three zones in the sucrose gradient after the vacuolar membrane fractionation and two zones in the plasma membrane where membrane structures, which may be defined as rafts for their lipid composition, were presented.

The composition of vacuolar membrane phospholipids in the taproot of red beet (Beta vulgaris L.), cv. Modana, was determined at normal conditions and under different types of stress (hypo- and hyperosmotic and oxidative stress). The experiments have shown that, among vacuolar membrane phospholipids in red beet taproot, phosphatidylcholines and phosphatidylethanolamines dominated and accounted for 70% of total phospholipids. It is interesting that the content of phosphatidic acid was high (20% of total phospholipids of the vacuolar membrane). Stress effects brought about changes in the composition of membrane phospholipids, which may be an element of phenotypic adaptation. Under hypoosmotic stress, reliable changes in the content of phosphatidic acid were observed, hyperosmotic stress was associated with changes in the level of phosphatidylcholines and phosphatidylinositols, and oxidative stress was notable for changes in the content of phosphatidylethanolamines and phosphatidylserines. The most significant changes were observed in the classes of phospholipids that may be involved in structural modification of membranes associated with transformation of their bilayer lamellar structure into hexagonal. These phospholipids comprise phosphatidic acid, phosphatidylcholines, and phosphatidylethanolamines. Revealed changes in the content of these phospholipids may alter the ratio between lamellar bilayer and nonbilayer hexagonal lipid structures in the vacuolar membrane and act as an important adaptation mechanism ensuring protection against stress.

Peculiar properties of morphological structures of organelle membranes were studied by fluorescent confocal microscopy. The list of objects in our experiments was represented by mitochondria, chloroplasts and vacuoles. During this study, identification of lipid microinclusions having the form of such lipid-protein structural microformations as lipid-protein microdomains, vesicles and membrane tubular structures (cytoplasmic transvacuolar strands and nanotubes) located in organelle membranes or bound up with them was conducted. Such membrane probes as laurdan, DPH, ANS and bis-ANS were used. Comparison of fluorescence intensity of these membrane probes was conducted. This investigation of the morphological properties of lipid-protein structural microformations was accompanied with analysis of 1) the phase state and 2) dynamics of microviscosity variations in the membrane elements of isolated plant cell organelles. Distributions of laurdan fluorescence generalized polarization (GP) values for the membrane on the whole and for the intensively fluorescing membrane segments were obtained. It was discovered that the microviscosity of intensively fluorescing membrane segments essentially differed from the microviscosity of the rest part of the membrane. In conclusion, some results of the study of peculiar properties of lipid-protein structural microformations related to the structure of organelle membranes and the discoveries made in this investigation are discussed.

To study the characteristics of the structure and the functional role of chloroplast membranes of halophytes with different salt tolerance strategy, raft structures were isolated. Lipid analysis data provided evidence for the presence of rafts in the chloroplast membranes. The discovered significant differences in the content of raft-forming lipids in different halophytes suggest a relationship between the functions of rafts and the mechanisms of salt tolerance in plants.

In the present work, we studied detergent-resistant membrane microdomains (DRM) of chloroplasts and mitochondria – organelles that provide photosynthesis and respiration in a plant cell. The objects of the study were euhalophyte Salicorniaperennans Willd., which relates to salt-accumulating plants and glycohalophyte Artemisia santonica L., which relates to salt-excluder plants. To get DRM, the chloroplast and mitochondria fractions were solubilised with a solution containing Triton X-100. The resulting material was introduced in sucrose gradient of 35–25–15–5% and centrifuged at 200000 g, 2 h. The presence of an opalescent detergent-resistant zone of membranes in 15% sucrose layer and a specific lipid composition of this zone were the signs of successful rafts obtaining of. The isolated DRM are sterol- and cerebroside-enriched (27–89% of the sum of membrane lipids) domains with a high degree of saturation of fatty acids composition (more than 50% of the sum). The main DRM-specific lipids of chloroplast of A. santonica glycohalophyte are cerebrosides, whereas those of S. perennans euhalophyte are sterols. The revealed differences in the composition of raft-forming lipids in chloroplast and mitochondria halophyte membranes, differing in the salt-resistance strategy, suggest the participation of rafts in salt-resistance mechanisms.

The presence of lipid-protein microdomains in biological membranes is considered proven in biological literature. The content of lipids and proteins in the microdomains depends on the membrane’s nature, as well as on the technique used for isolation of the membrane fractions. In the case of detergent technique (DT), the nativity of the microdomains (called rafts) is questioned. In this connection, in our studies isolation of tonoplast fractions was performed using not only by the well-known detergent technique but also by a milder detergent-free technique (DFT). The microdomains were identified in the fractions, and the comparative analysis of lipid content of tonoplast microdomains in the fractions isolated by the two techniques was conducted. It was demonstrated that microdomains isolated from tonoplast by DFT substantially differ in their lipid content from those isolated with the use of Triton X-100. This means that such microdomains cannot be qualified as rafts, despite the fact that they are located in the same opalescence zone of the sucrose density gradient, as the microdomains obtained from the fraction isolated with the use of Triton X-100 have a higher (than in the initial tonoplast) content of sterols and their esters. As far as the microdomains identified in the fraction obtained by DFT are concerned, we have revealed an unusual content of polar lipids, half of which is represented by the lipids (phosphatidylethanolamine, phosphatidic acid and monogalactosyldiglyceride) capable (under definite conditions and due to lipid polymorphism) of the formation of hexagonal structures, which are believed to participate in transport of water-soluble metabolites.

Results of investigation of peculiar properties related to the structure of plant cell vacuolar membranes are discussed. This investigation was conducted with the aid of confocal microscopy, which allowed us, in the process of scanning, to reveal nanotubes and vesicules in the preparations of isolated vacuoles. Such membrane nanotubes were observed both inside and outside the vacuoles, and, in the case of scanning intermittently at equal time intervals, transition of vesicules with a membrane nanotube. Furthermore, scanning of isolated vacuoles was conducted at various distances from the glass basement. Each time, in the upper part of an isolated vacuole lying on the basement, we observed a large segment of vacuolar membrane and registered the effect of highly intense fluorescing of some of the membrane segments. Distributions of laurdan fluorescence generalized polarization (GP) values for the vacuolar membrane on the whole and for the intensely fluorescing membrane segments have been obtained. It was discovered that the microviscosity of intensely fluorescing membrane segments essentially differs from the microviscosity of the rest part of the membrane.

ObjectiveThe membranes of plant and animal cells contain lipid-protein microdomains (LPMs) in their structure. We aimed to compare biochemical and biophysical characteristics of LPMs isolated by the two techniques from beet root tonoplast.MethodsFor the purposes of comparison the biochemical characteristics of LPMs the content of protein (Bradford’s method), the content of lipids (gravimetric method) were assessed. For the purpose of separating different kinds of lipids, one-dimensional thin layer chromatography (TLC), two-dimensional TLC and different specific systems of solvents were used. The content of fatty acids and sterols was determined by chromatography-mass spectrometry and gas-liquid chromatography-mass spectrometry correspondingly. In order to determine the orderings of the lipids in LPMs the laser scanning confocal fluorescence microscopy was used.ResultsSignificant differences were revealed as a result of investigation of biochemical and biophysical characteristics of LPMs. The total content of proteins was substantially higher (4 times as large) in LPMs identified by the detergent-free technique than in LPMs identified by the detergent technique. The orderings of lipids in LPMs isolated by detergent-free technique was substantially lower than that of LPMs isolated by detergent technique.ConclusionThese results may suggest an existence of several types of LPMs in tonoplast.

The experiments conducted on tonoplast of Beta vulgaris L. roots were performed to identify detergent-resistant lipid-protein microdomains (DRMs, interpreted as lipid rafts).The presence of DRMs can be found when dynamic clustering of sphingolipids, sterols, saturated fatty acids is registered, and the insolubility of these microdomains in nonionic detergents at low temperatures is proven. The elucidation of tonoplast microdomains has been based on results obtained with the aid of high-speed centrifuging in the sucrose gradient. The experiments have shown that tonoplast microdomains are rich in sphingolipids, free sterols and saturated fatty acids (such a lipid content is also typical of lipid-protein microdomains of other membranes), while only few phospholipids are present in tonoplast microdomains. The presence of microdomains has been confirmed by fluorescence and confocal microscopy using filipin and Laurdan as fluorescent probes. The experiments with Laurdan have shown that tonoplast microdomains are characterized by a high order compared to characteristics of the rest of the tonoplast. Thus, the presence of detergent-resistant lipid-protein microdomains in the tonoplast has been demonstrated.

Variations of the redox status is shown to inhibit the transport activity of tonoplast proton pumps at different stages of ontogenesis and under the conditions of hyperosmotic stress. However, the activity of H+-ATPase increased by 60% under hypoosmotic stress in the presence of GSH. The influence of nitric oxide on the transport activity of tonoplast proton pumps also depended on the redox status. In the case of change of the redox status, stimulating effect of nitric oxide turned inhibitory, except for simultaneous application of hypoosmotic stress and nitric oxide. In this case, stimulation of both proton pumps was observed and the activity of H+-ATPase increased in the presence of GSH, though the activity of H+-PPase increased in the presence of GSSG. This may explain the necessity of the presence in the vacuolar membrane of two proton pumps having similar functions.

Stability of plant vacuolar membranes (tonoplast), which underwent two types of osmotic stress and also the effect of redox agents (glutathione in its oxidized (GSSG) and reduced (GSH) forms) and nitric oxide (NO) under various pH values, has been investigated. The fatty-acid content of tonoplast has also been determined. It has been shown that stability of vacuoles decreased under hyperosmotic stress but not under hypoosmotic stress. These effects are unlikely caused by the fatty-acid content of vacuolar membranes. Stability of vacuoles changed under various redox conditions; this process was more intensive under the conditions of hypoosmotic stress in the experiments with oxidized glutathione. The influence of nitrogen oxide was different for various kinds of stress: stability of vacuoles decreased substantially under hypoosmotic stress, while under the conditions of hyperosmotic stress NO elevated the stability level of the vacuolar membranes.

The changes in transport activity of tonoplast proton pumps under the influence of exogenous NO donator and modulation of Ca2+ concentration jointly and separately were investigated at different stages of ontogenesis and under hyperosmotic stress. The results suggest that both exogenous NO donator and Ca2+ ions can influence the activity of transport processes related to tonoplast and this influence is especially evident in the period of growth and accumulation of metabolites. Under hyperosmotic stress, H+-pyrophosphatase plays a more important role than H+-ATPase: the activity of the former increases 2.3-fold compared to the control osmotic conditions, whereas the activity of H+-ATPase is practically unchanged. H+-pyrophosphatase was more responsive to the presence of exogenous NO donator and to variations in Ca2+ concentration. The effects of exogenous NO donator on tonoplast proton pumps depended on the concentration of Ca2+, which apparently can mediate NO action.

The effect of dihydroquercetin (DHQ) on proton pumps of the vacuolar membrane (H(+)-ATPase and H(+)-pyrophosphatase), slow vacuolar (SV) channel, lipid peroxidation, and stability of isolated vacuoles was studied. The results of experiments showed that DHQ affected active and passive transport systems of the vacuolar membrane. The mechanism of action of DHQ may be based on its combined effect on the sulfhydryl groups of proteins and the lipid component of the membrane. The strong stabilizing effect of DHQ on the membranes of isolated vacuoles may be associated not only with its antioxidant properties but also with changes in the membrane permeability affecting the ion channels.

120 The well known Singer–Nicholson model, according to which proteins “float” in a homogeneous lipid sea, has undergone significant changes. Today it is generally accepted that cell membranes are highly heterogeneous: they contain various lipid–protein structures that are involved in many metabolic pro cesses occurring in living cells [1]. According to the model of lipid–protein microdomains (rafts), which is currently most widespread, areas enriched in gly cosphingolipids, sterols, and lipids with saturated fatty acids occur around certain proteins; lipids in these areas are characterized by a denser packing of mole cules compared to the surrounding areas of mem branes [2]. The interest to rafts is determined by the increasing number of experimental data that confirm their involvement in vital cellular processes, such as intrac ellular signaling, endocytosis, apoptosis, sorting of proteins, as well as internalization of toxins, bacteria, viruses, etc. [3, 4]. In addition to specific lipid compo sition, these membrane domains are characterized by specific protein profiles. In plant plasma membrane rafts, P and V type ATPases, protein kinases, mono meric and heteromeric G proteins, 14 3 3 family proteins, aquaporins, etc. were identified [5]. Rafts have been found in the plasma membrane and in endoplasmic reticulum and Golgi membranes. Mitochondrial membranes do not contain rafts [6]. Data on the lipid composition of tonoplasts [7] suggest that plant vacuolar membranes may also contain pro tein–lipid rafts. In this study, we found lipid protein domains (rafts) in the vacuolar membrane. Experiments were performed with isolated vacu oles and membrane vesicles of dormant storage roots of the red beet (Beta vulgaris L.) variety Bordeaux. Vacuoles and tonoplast vesicles from the storage root tissue were isolated and purified as described in [8]. Sterol enriched microdomains (rafts) were detected by fluorescent microscopy using the sterol binding antibiotic filipin [9]. To ensure the binding of filipin to the vacuolar membrane, a suspension of isolated vac uoles in the isolation medium (0.8 M KCl, 20 mM EDTA, and 50 mM Na2HPO4 (pH 8.0)) was supple mented with filipin (0.2% solution in DMSO) to a final concentration of 5 μM. The suspension was incu bated at 20 ± 2°С for 30 min, washed in the same buffer, and analyzed under an Axio observer Z1 fluo rescence microscope (Carl Zeiss, Germany) equipped with an Axio Cam MRm monochrome digital camera and Axio Vision Rel.4.8 software package for captur Recognition of Lipid–Protein Rafts in Vacuolar Membrane

The effect of dihydroquercetin (DHQ) on proton pumps of the vacuolar membrane (H(+)-ATPase and H(+)-pyrophosphatase), slow vacuolar (SV) channel, lipid peroxidation, and stability of isolated vacuoles was studied. The results of experiments showed that DHQ affected active and passive transport systems of the vacuolar membrane. The mechanism of action of DHQ may be based on its combined effect on the sulfhydryl groups of proteins and the lipid component of the membrane. The strong stabilizing effect of DHQ on the membranes of isolated vacuoles may be associated not only with its antioxidant properties but also with changes in the membrane permeability affecting the ion channels.

We studied the dynamics of phytohormone levels (indole-3-acetic acid, abscisic acid, cytokinins, and gibberellin-like substances) in red beet (Beta vulgaris L.) roots at different developmental stages in comparison with the data on the dynamics of sugar accumulation in order to test possible hormonal regulation of sugar accumulation. The obtained data suggest the involvement of cytokinins and gibberellin-like substances in the control of sugar accumulation in the roots, while indole-3-acetic acid, abscisic acid, and gibberellin-like substances can control the outflow. The data on the dynamics of phytohormone levels shed light on their specific physiological role in red beet root development.

The effects of indole-3-acetic acid (IAA), abscisic acid (ABA), gibberellic acid (GA3) and kinetin on the hydrolytic activity of proton pumps (adenosine triphosphatase, H+-ATPase, pyrophosphatase, H+-PPase) of tonoplasts isolated from stored red beet (Beta vulgaris L. cv. Bordo) roots were studied. Results suggest that the phytohormones can regulate the hydrolytic activities of H+-ATPase and H+-PPase of the vacuolar membrane. Each of the proton pumps of the tonoplast has its own regulators in spite of similar localization and functions. IAA and kinetin seem to be regulators of the hydrolytic activity for H+-PPase whereas for H+-ATPase it may be GA3. Stimulation of enzyme activity by all hormones occurred at concentrations of 10−6 to 10−7M.

Effects of indoleacetic, abscisic, gibberellic acids and kinetin on the hydrolytic activity of the proton pumps H+-ATPase (EC 3.6.1.3) and H+-pyrophosphatase (EC 3.6.1.1) under non-optimal ionic conditions were studied in tonoplasts isolated from stored roots of red beet (Beta vulgaris L. cv. Bordo). We showed that ionic composition markedly affected the hormonal regulation. H+-pyrophosphatase was more sensitive to the ionic and hormonal regulation than H+-ATPase. Magnesium ions are essential for the maximal manifestation of hormonal stimulation.

The effect of low-intensity irradiation by a helium-neon laser on the hydrolytic activity of the vacuolar membrane proton pumps has been studied. The maximum effect was found for the 3 min irradiation from a close distance (0.3 m); moreover, the PPase activity increased by 33%, whereas the ATPase activity was inhibited by 44%. The effect described is suggested to be due to different conformations of the enzymes in the membrane and their different physiological roles.