New insights into the tonoplast architecture of plant vacuoles and vacuolar dynamics during osmotic stress

Background  

The vegetative plant vacuole occupies >90% of the volume in mature plant cells. Vacuoles play fundamental roles in adjusting cellular homeostasis and allowing cell growth. The composition of the vacuole and the regulation of its volume depend on the coordinated activities of the transporters and channels localized in the membrane (named tonoplast) surrounding the vacuole. While the tonoplast protein complexes are well studied, the tonoplast itself is less well described. To extend our knowledge of how the vacuole folds inside the plant cell, we present three-dimensional reconstructions of vacuoles from tobacco suspension cells expressing the tonoplast aquaporin fusion gene BobTIP26-1::gfp.     

Function of transport H+-ATPases in plant cell plasma and vacuolar membranes of maize under salt stress conditions and effect of adaptogenic preparations

Participations of electrogenic H+-pumps of plasma and vacuolar membranes represented by E1-E2 and V-type H+-ATPases in plant cell adaptation to salt stress conditions has been studied by determination of their transport activities. Experiments were carried out on corn seedlings exposed during 1 or 10 days at 0.1 M NaCl. Preparations Methyure and Ivine were used by seed soaking at 10(-7) M. Plasma and vacuolar membrane fractions were isolated from corn seedling roots. In variants without NaCl a hydrolytical activity of plasma membrane H+-ATPase was increased with seedling age and its transport one was changed insignificantly, wherease the response of the weaker vacuolar H+-ATPase was opposite. NaCl exposition decreased hydrolytical activities of both H+-ATPases and increased their transport ones. These results demonstrated amplification of H+-pumps function especially represented by vacuolar H+-ATPase. Both preparations, Methyure mainly, caused a further increase of transport activity which was more expressed in NaCl variants. Obtained results showed the important role of these H+-pumps in plant adaptation under salt stress conditions realized by energetical maintenance of the secondary active Na+/H+ -antiporters which remove Na+ from cytoplasm.     

Arbuscular mycorrhizal fungi influence growth, osmotic adjustment and photosynthesis of citrus under well-watered and water stress conditions

The influence of arbuscular mycorrhizal (AM) fungus Glomus versiforme on plant growth, osmotic adjustment and photosynthesis of tangerine (Citrus tangerine) were studied in potted culture under well-watered and water stress conditions. Seven-day-old seedlings of tangerine were transferred to pots containing Glomus versiforme or non-AMF. After 97 days, half of the seedlings were subject to water stress and the rest were well-watered for 80 days. AM colonization significantly stimulated plant growth and biomass regardless of water status. The soluble sugar of leaves and roots, the soluble starch of leaves, the total non-structural carbohydrates (NSC) of leaves and roots, and the Mg(2+) of leaves were higher in AM seedlings than those in corresponding non-AM seedlings. The levels of K(+) and Ca(2+) in leaves and roots were higher in AM seedlings than those in non-AM seedlings, but differences were only significant under water stress conditions. Moreover, AM colonization increased the distributed proportions of soluble sugar and NSC to roots. However, the proline was lower in AM seedlings compared with that in non-AM seedlings. AM seedlings had higher leaf water potential (Psi), transpiration rates (E), photosynthetic rates (Pn), stomatal conductance (g(s)), relative water content (RWC), and lower leaf temperature (Lt) than corresponding non-AM seedlings. This research also suggested that AM colonization improved the osmotic adjustment originating not from proline but from NSC, K(+), Ca(2+) and Mg(2+), resulting in the enhancement of drought tolerance.     

Growth and developmental responses of potato to osmotic stress under in vitro conditions

The effect of mannitol on different genotypes of potato was studied in callus and plantlet culture. In vitro responses of five potato genotypes with well-known field behaviour to water deficit were analysed. After a 4-week-long cultivation on media containing mannitol up to 0.8 M, different morpho-physiological parameters were determined and statistically analysed. The useful concentration of mannitol for in vitro screening the osmotic tolerance of different genotypes depended on the type of culture; it was 0.4 M in plantlet-test and 0.8 M in callus-test. In callus-test the relative increase of callus mass was a useful parameter for determination of osmotic tolerance of genotypes at cellular level. In plantlet culture, stress index calculated from the rate of surviving in vitro shoots, number and length of roots per surviving explant and the rate of rooted explants were applicable to determine three groups according to the tolerant, medium tolerant and sensitive categories in agreement with the field behaviour of these genotypes. Under in vitro stress conditions we were able to distinguish the examined genotypes with different drought tolerance.     

The structure of plant vacuolar membranes according to infrared spectroscopy

The structure of the vacuolar membrane (tonoplast) was studied in red beet roots by IR spectroscopy. The vacuolar membrane was shown to be composed of highly ordered lipids which form regions of free liquid lipid bilayer loosely bound to integral proteins. The prevalence of polar lipids in the tonoplast is responsible for the high elasticity and fluidity of the membrane. The presence of alpha-tocopherol in the tonoplast membrane accounts for a high antioxidant activity of the membrane. Integral proteins are immersed into the liquid matrix of the lipid bilayer to a different extent. Examination of the temperature effect on the kinetics of the hydrogen-deuterium exchange in integral membrane proteins showed that the efficient energy of the hydrogen exchange activation was 24 +/- 4 kcal/mol at 19-40 degrees C and increased to 54 kcal/mol at 40-50 degrees C because of the thermal denaturation of proteins. The secondary structure of integral membrane proteins is characterized by a high content of alpha-helices (53%) which decreased to 8% after the extraction of lipids.     

Levels of trehalose and glycogen inArthrobacter globiformis under conditions of nutrient starvation and osmotic stress

Cells ofArthrobacter globiformis grown in carbohydrate-rich media were found to contain large quantities of low-M_r carbohydrates (800 g/mg protein) and only small amounts of amino acids, in addition to high amounts of glycogen (2 mg/mg protein). At increasing osmotic values of the medium, low-M_r carbohydrate levels increased to 1300 g/mg protein. Low-M_r pools were extracted from the cells with hot 75% ethanol, and subjected to thin layer, gel and gas-liquid chromatography. They turned out to consist mainly of ,-trehalose. Levels of trehalose inArthrobacter cells have the tendency to remain constant, both during nutrient exhaustion (resulting in glycogen consumption), and on addition of excess of carbon source to the medium (resulting in an increased glycogen content of the cells). The stress-tolerant properties ofArthrobacter (resistance to nutrient starvation, desiccation and high salt concentration) are discussed with respect to the high glycogen and trehalose contents of the cells.     

Low aquaporin content and low osmotic water permeability of the plasma and vacuolar membranes of a CAM plant Graptopetalum paraguayense: comparison with radish.

Aquaporin facilitates the osmotic water transport across biomembranes and is involved in the transcellular and intracellular water flow in plants. We immunochemically quantified the aquaporin level in leaf plasma membranes (PM) and tonoplast of Graptopetalum paraguayense, a Crassulacean acid metabolism (CAM) plant. The aquaporin content in the Graptopetalum tonoplast was approximately 1% of that of radish. The content was calculated to be about 3 microg mg(-1) of tonoplast protein. The level of PM aquaporin in Graptopetalum was determined to be less than 20% of that of radish, in which an aquaporin was a major protein of the PM. The PM aquaporin was detected in the mesophyll tissue of Graptopetalum leaf by tissue print immunoblotting. The osmotic water permeability of PM and tonoplast vesicles prepared from both plants was determined with a stopped-flow spectrophotometer. The water permeability of PM was lower than that of the tonoplast in both plants. The Graptopetalum PM vesicles hardly showed water permeability, although the tonoplast showed a relatively high permeability. The water permeability changed depending on the assay temperature and was also partially inhibited by a sulfhydryl reagent. Furthermore, measurement of the rate of swelling and shrinking in different mannitol concentrations revealed that the protoplasts of Graptopetalum showed low water permeability. These results suggest that the low content of aquaporins in PM and tonoplast is one of the causes of the low water permeability of GRAPTOPETALUM: The relationship between the water-storage function of succulent leaves of CAM plants and the low aquaporin level is also discussed.     

Effects of mucilage on seed germination of the desert ephemeral plant Plantago minuta Pall. under osmotic stress and cycles of wet and dry conditions

To test the role of the seed mucilage of Plantago minuta Pall. in regulating germination under osmotic stress and cycles of hydration and dehydration, two experiments were carried out using seeds with intact mucilage and mucilage‐free seeds. In Experiment 1 seeds were immersed in a range of iso‐osmotic polyethylene glycol solutions (?1.15 to 0 MPa) for 14 days; any ungerminated seeds were transferred to deionized water to investigate the recovery germination. In Experiment 2 seeds were immersed in deionized water for 24 h, and were then incubated on filter paper for an additional 13 days to ensure complete desiccation before reimbibition to test the germination recovery percentage. Under mild osmotic stress (?0.73 to 0 MPa), the intact seeds with mucilage were shown to have higher germination rates than the mucilage‐free seeds, indicating that the mucilage led to a “fast sprouting” germination strategy under mild osmotic stress. However, when seeds were exposed to high osmotic stress (?1.15 MPa), the mucilage apparently slowed the germination rate, resulting in a “risk‐balancing” germination strategy. Extreme drought induced by polyethylene glycol solution and the desiccation pretreatment accelerated germination rates compared to non‐pretreated seeds; both germination potential and recovery percentage of the mucilage seeds were significantly higher than that of the mucilage‐free seeds. Our results revealed that the seed mucilage of P. minuta plays a crucial role in regulating seed germination rates and the germination strategies adopted by controlling seed water absorption when the seeds experience different osmotic stresses or alternating wet and dry conditions.     

High concentrations of the compatible solute glycinebetaine destabilize model membranes under stress conditions

Compatible solutes are accumulated by diverse organisms in response to environmental stresses such as drought, salt, or cold. Glycinebetaine (Bet) is such a solute that is accumulated by many plants and microorganisms to high concentrations under stress conditions. It is an osmoprotectant in bacteria and stabilizes both soluble and peripherally membrane-bound proteins in vitro. Here, the effects of Bet on the stability of model lipid membranes are compared to the effects of two other compatible solutes, sucrose and trehalose. Both in the presence of 1M NaCl and during freezing to -20 degrees C, Bet is highly destabilizing to liposomes containing nonbilayer lipids, while the disaccharides are either protective or, in some cases, much less destabilizing. The destabilizing effect of Bet is more pronounced in membranes containing the nonbilayer galactolipid monogalactosyldiacylglycerol from plant chloroplasts than in membranes containing the nonbilayer phospholipid phosphatidylethanolamine. The most dramatic differences between the sugars and Bet were observed in liposomes made from a combination of lipids resembling plant chloroplast thylakoid membranes. Measurements with the dye merocyanine 540 indicate that the water-membrane interface was affected in opposite directions by the presence of high concentrations of sucrose or Bet. The dynamics of the lipids, however, were not differentially affected by the solutes, making direct solute-lipid interactions an unlikely explanation for the different effects on stability. The data offer an explanation, why Bet at high concentrations achieved during exogenous feeding of leaf tissues can be detrimental to cellular stability and survival under stress, while bacterial membranes that contain phosphatidylethanolamine instead of monogalactosyldiacylglycerol, or cyanobacteria that contain highly saturated monogalactosyldiacylglycerol are less susceptible.     

Molecular and genetic aspects of plant responses to osmotic stress

Drought, high salinity and freezing impose osmotic stress on plants. Plants respond to the stress in part by modulating gene expression, which eventually leads to the restoration of cellular homeostasis, detoxification of toxins and recovery of growth. The signal transduction pathways mediating these adaptations can be dissected by combining forward and reverse genetic approaches with molecular, biochemical and physiological studies. Arabidopsis is a useful genetic model system for this purpose and its relatives including the halophyte Thellungiella halophila, can serve as valuable complementary genetic model systems.     

Photosynthesis of isolated chloroplasts and protoplasts under osmotic stress

1. Isolated intact spinach chloroplasts respond to changes of the sorbitol concentration of the suspending medium as near-perfect osmometers within a large range of osmotic potentials. Under isotonic conditions (=9–10 bar), their average osmotic volume is 24 m³ and the total volume 36 m³. The osmotic volume can be increased to 63 m³ by lowering the sorbitol concentration until a critical osmotic potential of =4 bar is reached. Below that value chloroplasts rupture. Between 10 bar and 4 bar, volume changes are reversible. 2. Increasing the chloroplast volume above 24 m³ causes inhibition of photosynthesis, with 50% inhibition occurring at an osmotic potential of =5–6 bar. This corresponds to an osmotic volume of 45–55 m³. Depending on the duration of hypotonic treatment, inhibition of photosynthesis is more or less reversible. 3. Between 4 and 10 bar, the chloroplast envelope exhibits a very low permeability for ferricyanide, many metabolites, and soluble stroma proteins. 4. Electron transport is not inhibited by swelling of chloroplasts. Also, the ATP/ADP-ratio remains unchanged. 5. The solute concentration in the chloroplasts appears to be optimal for photosynthesis at 10 bar. Increasing the chloroplast volume causes inhibition of photosynthesis by dilution effects.     

Interaction of Hsp90AA1 with phospholipids stabilizes membranes under stress conditions

During heat shock conditions, structural changes in cellular membranes may lead to cell death. Hsp90AA1 and other heat shock proteins involved in membranes are responsible for protecting membrane stabilization. However, the membrane binding mechanism of Hsp90AA1 remains largely uncharacterized. In this study, we showed Hsp90AA1 interacts with phospholipid membrane with high affinity. Using the depth-dependent fluorescence-quenching with brominated lipids, we found Hsp90AA1 penetrated 10.7?Å into the hydrocarbon core of the lipid bilayer. Circular dichroism spectra studies showed Hsp90AA1 lost part of its α-helical structures upon interaction with phospholipid membrane. By assessing binding properties of the three Hsp90AA1 domains, we found Hsp90AA1 interacted into the lipid bilayer mainly toward its C-terminus domain (CTD). Using scanning electron microscopy, we examined the protection on host cell membrane by overexpressing Hsp90AA1. The results indicated Hsp90AA1 or Hsp90AA1-CTD expressing E. coli cells exhibited better membrane integrity compared to the control after thermal treatment. The following liposome leakage assay suggested the protection of Hsp90AA1 might due to its stabilization of the membrane lipid. Collectively, the present study demonstrates Hsp90AA1 embeds into the lipid bilayer through its C-terminal domain and the Hsp90AA1-lipid association potentially has a significant function in keeping membranes stabilization during stress conditions.     

Nitric oxide is involved in light-specific responses of tomato during germination under normal and osmotic stress conditions

Background and Aims Nitric oxide (NO) is involved in the signalling and regulation of plant growth and development and responses to biotic and abiotic stresses. The photoperiod-sensitive mutant 7B-1 in tomato (Solanum lycopersicum) showing abscisic acid (ABA) overproduction and blue light (BL)-specific tolerance to osmotic stress represents a valuable model to study the interaction between light, hormones and stress signalling. The role of NO as a regulator of seed germination and ABA-dependent responses to osmotic stress was explored in wild-type and 7B-1 tomato under white light (WL) and BL. Methods Germination data were obtained from the incubation of seeds on germinating media of different composition. Histochemical analysis of NO production in germinating seeds was performed by fluorescence microscopy using a cell-permeable NO probe, and endogenous ABA was analysed by mass spectrometry. Key Results The NO donor S-nitrosoglutathione stimulated seed germination, whereas the NO scavenger 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO) had an inhibitory effect. Under WL in both genotypes, PTIO strongly suppressed germination stimulated by fluridone, an ABA inhibitor. The stimulatory effect of the NO donor was also observed under osmotic stress for 7B-1 seeds under WL and BL. Seed germination inhibited by osmotic stress was restored by fluridone under WL, but less so under BL, in both genotypes. This effect of fluridone was further modulated by the NO donor and NO scavenger, but only to a minor extent. Fluorescence microscopy using the cell-permeable NO probe DAF-FM DA (4-amino-5-methylamino-2',7'-difluorofluorescein diacetate) revealed a higher level of NO in stressed 7B-1 compared with wild-type seeds. Conclusions As well as defective BL signalling, the differential NO-dependent responses of the 7B-1 mutant are probably associated with its high endogenous ABA concentration and related impact on hormonal cross-talk in germinating seeds. These data confirm that light-controlled seed germination and stress responses include NO-dependent signalling.     

Higher plant antioxidants and redox signaling under environmental stresses

Main antioxidants in higher plants include glutathione, ascorbate, tocopherol, proline, betaine, and others, which are also information-rich redox buffers and important redox signaling components that interact with biomembrane-related compartments. As an evolutionary consequence of aerobic life for higher plants, reactive oxygen species (ROS) are formed by partial reduction of molecular oxygen. The above enzymatic and non-enzymatic antioxidants in higher plants can protect their cells from oxidative damage by scavenging ROS. In addition to crucial roles in defense system and as enzyme cofactors, antioxidants influence higher plant growth and development by modifying processes from mitosis and cell elongation to senescence and death. Most importantly, they provide essential information on cellular redox state, and regulate gene expression associated with biotic and abiotic stress responses to optimize defense and survival. An overview of the literature is presented in terms of main antioxidants and redox signaling in plant cells. Special attention is given to ROS and ROS-antioxidant interaction as a metabolic interface for different types of signals derived from metabolism and from the changing environment, which regulates the appropriate induction of acclimation processes or, execution of cell death programs, which are the two essential directions for higher plants.     

The action of adaptogenic preparations on vacuolar proton pumps activity in corn root cells under salt stress conditions

The function of two electrogenic H+ -pumps in plant vacuolar membrane (tonoplast) and their response to the salt stress conditions and adaptogenic preparations have been studied. Experiments were carried out on tonoplast fraction isolated from the roots of corn seedlings grown in water culture which were exposed at 7-day age in the presence of 0.1 M NaCl during 1 or 10 days. The role of every H+ -pump in potential generation was elucidated by assaying transport and hydrolytic activity of enzymes--V-type H+ -ATPase and H+ pyrophosphatase represented their mechanisms. It was found in the control variant that transport activity H+ -PPase exceeded considerably H+ -ATPase one in the tonoplast from 7-day seedlings. However this situation was changed in 18-day seedlings by H+ -ATPase transport activity increasing with age whereas its hydrolytic one was decreased. Both NaCI expositions caused the progressive decrease of transport and hydrolytic activity of H+ -PPase whereas H+ -ATPase responded to this factor by increasing transport activity, while its hydrolytic one fell. Bioactive preparations Methyure and Ivine (10(-7) M) used by seed soaking caused a further increase of H+ -ATPse transport activity especially in the presence of NaCl whereas H+ -PPase one was not changed. Methyure effect in these experiments was more pronounced. Obtained results demonstrated participation of tonoplast H+ -pumps in plant adaptation to NaCI which can be realized by amplification of Na+ -H(+) antiporter energization. An important role of vacuolar H+ -ATPase in growth and adaptation processes in plants has been proved.     

Immunolocalization of PIP aquaporins in protoplasts from the suspension culture of sugar beet mesophyll under isoosmotic conditions and osmotic stress

Protoplasts from the suspension culture of sugar beet (Beta vulgaris L.) mesophyll were found to change their volume in response to short-term osmotic stress. When the sorbitol concentration in the external medium was increased 1.5-fold (from 0.4 to 0.6 M) or decreased from 0.4 to 0.25 M, the volume of protoplasts decreased and increased, respectively, by 55–60%. These changes started immediately after the shift in osmoticum concentration and completed within 1–3 min. In the presence of an endocytosis marker FM1-43, its fluorescence increased conspicuously after replacement of isotonic medium with the hypotonic solution but did not change after the substitution with hypertonic medium. At the same time, the hypertonic shrinkage of protoplasts was accompanied by accumulation of fluorescent material in the periplasmic space. The western blot analysis with the use of immune serum for conservative sequence of PIP-type aquaporins revealed their presence in the plasmalemma and intracellular membranes. This conclusion was confirmed by indirect immunofluorescence microscopy: the membrane-bound secondary antibodies labeled with a fluorescent probe Alexa-Fluor 488 were distributed comparatively uniformly on the boundary between the plasmalemma and the protoplast internal compartment. As evident from micrographs of protoplasts exposed to the hypotonic treatment, the fluorescence was smoothly distributed over the plasmalemma after protoplast swelling but its intensity was not so bright. The protoplast shrinkage during the hypertonic treatment resulted in heterogeneous alternate distribution of fluorescent and transparent plasmalemma regions, the fluorescence of stained regions being very intense. The results are interpreted as the evidence that the short-term osmotic stress activates exo-and endocytosis. The migrating regions of the plasmalemma were depleted of PIP-type aquaporins; hence, the induction of osmotic stress has no effect on the amount of this type aquaporins in the plasma membrane.     

The influence of age of ewe on embryo mortality under heat stress conditions

Maiden and mature Merino ewes were subjected to continuous heat stress for the first 20 days of gestation.In both control and hotroom groups the age of the ewes had no apparent influence on the rate of embryonic mortality experienced, although heat stress gave rise to a highly significant (p < 0.001) increase in the overall embryonic mortality rate.

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Zusammenfassung Junge und erwachsene Merino Schafe wurden während der ersten zwanzig Tage der Trächtigkeit einer andauernden Hitzebelastung ausgesetzt.Sowohl in der Kontroll- wie in der belasteten Gruppe hatte das Alter der Schafe keinen nachweisbaren Einfluss auf die Absterberate der Embryos, obwohl die Hitzebelastung die allgemeine Absterberate von Embryos mit grosser Signifikanze (p < 0,001)anwachsen liess.

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Resume Des brebis et adultes de la race "Mérinos" furent exposées à une contrainte de chaleur constante durant les 20 premiers jours de leur gestation. Il n'a pas été possible de prouver que l'âge des brebis ait une influence sur le taux de mortalité des embryons ni dans le groupe des animaux exposés au chaud, ni dans celui servant au contrôle. La contrainte thermique a cependant fait augmenter de façon très significative (p < 0,001) le taux de mortalité des embryons.

     

Neosynthesis of cardiolipin in Rhodobacter sphaeroides under osmotic stress

The phospholipid composition of Rhodobacter sphaeroides cells resuspended in various hypertonic solutions has been examined by thin-layer chromatography and ESI mass spectrometry. R. sphaeroides responds to hyperosmotic stress by increasing the amount of cardiolipin in the membranes; this phenomenon occurs in spheroplasts also. Cardiolipin increases quickly and continuously during the time when the cells are resuspended in hypertonic medium. The optimum of stimulation of the neosynthesis of cardiolipin during osmotic stress was found to be at external 1 osm. ESI-MS analyses allowed the identification of two different cardiolipins in R. sphaeroides: the tetravaccenylcardiolipin ([M - H](-), m/z 1456.9) and the trivaccenylmonopalmitoylcardiolipin ([M - H](-), m/z 1430.0).