Comparison of the functions of plasma membrane and vacuolar membrane lipids in plant cell protection against hyperosmotic stress.

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.

Role of tonoplast microdomains in plant cell protection against osmotic stress.

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.

Non-detergent Isolation of Membrane Structures from Beet Plasmalemma and Tonoplast Having Lipid Composition Characteristic of Rafts.

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.

Variations in the content of tonoplast lipids under abiotic stress.

MAIN CONCLUSION: 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.

Tonoplast of Beta vulgaris L. contains detergent-resistant membrane microdomains.

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.