Characteristics of cellular membranes at rehydration of dehydrated yeast Saccharomyces cerevisiae

Summary This paper describes the characteristics of the structural and functional organization of cellular membranes rehydrated after dehydration of the yeast Saccharomyces cerevisiae. It was noted that dehydration and subsequent rehydration of yeast cells causes a considerable increase of cytoplasmic membrane permeability. Addition of CaCl₂, glucose and polyethyleneglycol to the rehydration medium caused a decrease in cell permeability, assessed as the losses of potassium ions, nucleotides, as well as the total losses of intracellular compounds. KCl had a positive effect only at concentrations above 10%. Yeast cells, dried to residual moisture lower than 20%, showed a decrease in membrane permeability as temperatures of the rehydration medium increased up to 38°–43°C. Upon reactivation of viable dehydrated cells in a nutrient medium, a reparation of the structural damages of various intracellular membranes takes place. It was established that at cell dehydration to residual moistures of 8%–12% all the free and a part of bound water is evaporated from cells.     

Changes of ergosterol content and resistance of population upon drying-rehydration of the yeast Saccharomyces cerevisiae

Summary An investigation was made of changes in ergosterol content of the yeast Saccharomyces cerevisiae upon drying and subsequent rehydration. It was established that drying increases, but rehydration diminishes ergosterol content in yeasts. A statistically reliable multiple correlation was established between the resistance of population to drying, decrease of ergosterol content and a diminishing degree of fatty acid unsaturation during dehydration of dry yeasts.     

Anhydrobiosis in yeast: is it possible to reach anhydrobiosis for yeast grown in conditions with severe oxygen limitation?

The yeast Saccharomyces cerevisiae was shown to be extremely sensitive to dehydration–rehydration treatments when stationary phase cells were subjected to conditions of severe oxygen limitation, unlike the same cells grown in aerobic conditions. The viability of dehydrated anaerobically grown yeast cells never exceeded 2 %. It was not possible to increase this viability using gradual rehydration of dry cells in water vapour, which usually strongly reduces damage to intracellular membranes. Specific pre-dehydration treatments significantly increased the resistance of anaerobic yeast to drying. Thus, incubation of cells with trehalose (100 mM), increased the viability of dehydrated cells after slow rehydration in water vapour to 30 %. Similarly, pre-incubation of cells in 1 M xylitol or glycerol enabled up to 50–60 % of cells to successfully enter a viable state of anhydrobiosis after subsequent rehydration. We presume that trehalose and sugar alcohols function mainly according to a water replacement hypothesis, as well as initiating various protective intracellular reactions.     

Controlling the membrane fluidity of yeasts during coupled thermal and osmotic treatments

Yeasts are often exposed to variations in osmotic pressure in their natural environments or in their substrates when used in fermentation industries. Such changes may lead to cell death or activity loss. Previous work by our team has allowed us to relate the mortality of cells exposed to a combination of thermal and osmotic treatments to leakage of cellular components through an unstable membrane when lipid phase transition occurs. In this study, yeast viability was measured after numerous osmotic and thermal treatments. In addition, the fluidity of yeast membranes was assessed according to a(w) and temperature by means of 1,6-diphenyl-1,3,5-hexatriene (DPH) anisotropy measurement. The results show that there is a negative correlation between the overall fluidity variation undergone by membranes during treatments and yeast survival. Using a diagram of membrane fluidity according to a(w) and temperature, we defined dehydration and rehydration methods that minimize fluidity fluctuations, permitting significantly increased yeast survival. Thus, such membrane fluidity diagram should be a potential tool for controlling membrane state during dehydration and rehydration and improve yeast survival. Overall fluidity measurements should now be completed by accurate structural analysis of membranes to better understand the plasma membrane changes occurring during dehydration and rehydration.     

Drying Increases Intracellular Partitioning of Amphiphilic Substances into the Lipid Phase : Impact on Membrane Permeability and Significance for Desiccation Tolerance

Previously we proposed that endogenous amphiphilic substances may partition from the aqueous cytoplasm into the lipid phase during dehydration of desiccation-tolerant organ(ism)s and vice versa during rehydration. Their perturbing presence in membranes could thus explain the transient leakage from imbibing organisms. To study the mechanism of this phenomenon, amphiphilic nitroxide spin probes were introduced into the pollen of a model organism, Typha latifolia, and their partitioning behavior during dehydration and rehydration was analyzed by electron paramagnetic resonance spectroscopy. In hydrated pollen the spin probes mainly occurred in the aqueous phase; during dehydration, however, the amphiphilic spin probes partitioned into the lipid phase and had disappeared from the aqueous phase below 0.4 g water g⁻¹ dry weight. During rehydration the probes reappeared in the aqueous phase above 0.4 g water g⁻¹ dry weight. The partitioning back into the cytoplasm coincided with the decrease of the initially high plasma membrane permeability. A charged polar spin probe was trapped in the cytoplasm during drying. Liposome experiments showed that partitioning of an amphiphilic spin probe into the bilayer during dehydration caused transient leakage during rehydration. This was also observed with endogenous amphipaths that were extracted from pollen, implying similar partitioning behavior. In view of the fluidizing effect on membranes and the antioxidant properties of many endogenous amphipaths, we suggest that partitioning with drying may be pivotal to desiccation tolerance, despite the risk of imbibitional leakage.     

Sequence of occurring damages in yeast plasma membrane during dehydration and rehydration: Mechanisms of cell death

Yeasts are often exposed to variations in osmotic pressure in their natural environments or in their substrates when used in fermentation industries. Such changes may lead to cell death or activity loss. Although the involvement of the plasma membrane is strongly suspected, the mechanism remains unclear. Here, the integrity and functionality of the yeast plasma membrane at different levels of dehydration and rehydration during an osmotic treatment were assessed using various fluorescent dyes. Flow cytometry and confocal microscopy of cells stained with oxonol, propidium iodide, and lucifer yellow were used to study changes in membrane polarization, permeabilization, and endocytosis, respectively. Cell volume contraction, reversible depolarization, permeabilization, and endovesicle formation were successively observed with increasing levels of osmotic pressure during dehydration. The maximum survival rate was also detected at a specific rehydration level, of 20 MPa, above which cells were strongly permeabilized. Thus, we show that the two steps of an osmotic treatment, dehydration and rehydration, are both involved in the induction of cell death. Permeabilization of the plasma membranes is the critical event related to cell death. It may result from lipidic phase transitions in the membrane and from variations in the area-to-volume ratio during the osmotic treatment.     

Achievement of rapid osmotic dehydration at specific temperatures could maintain high <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> viability

Various methods have been tried to prevent cell mortality during dehydration, but the reasons why microorganisms die when submitted to dehydration and rehydration are not well understood. The aim of this study was to further investigate the reasons for yeast mortality during dehydration. Osmotic dehydration and rehydration of Saccharomyces cerevisiae W303-1A were performed at different temperatures. Two different approaches were used: isothermic treatments (dehydration and rehydration at the same temperature), and cyclic treatments (dehydration at an experimental temperature and rehydration at 25 degrees C), with significant differences in viability found between the different treatments. Dehydration at lower and higher temperatures gave higher viability results. These experiments allowed us to propose a hypothesis that relates mortality to a high water flow through an unstable membrane during phase transition.     

Sequence of occurring damages in yeast plasma membrane during dehydration and rehydration: mechanisms of cell death

Yeasts are often exposed to variations in osmotic pressure in their natural environments or in their substrates when used in fermentation industries. Such changes may lead to cell death or activity loss. Although the involvement of the plasma membrane is strongly suspected, the mechanism remains unclear. Here, the integrity and functionality of the yeast plasma membrane at different levels of dehydration and rehydration during an osmotic treatment were assessed using various fluorescent dyes. Flow cytometry and confocal microscopy of cells stained with oxonol, propidium iodide, and lucifer yellow were used to study changes in membrane polarization, permeabilization, and endocytosis, respectively. Cell volume contraction, reversible depolarization, permeabilization, and endovesicle formation were successively observed with increasing levels of osmotic pressure during dehydration. The maximum survival rate was also detected at a specific rehydration level, of 20 MPa, above which cells were strongly permeabilized. Thus, we show that the two steps of an osmotic treatment, dehydration and rehydration, are both involved in the induction of cell death. Permeabilization of the plasma membranes is the critical event related to cell death. It may result from lipidic phase transitions in the membrane and from variations in the area-to-volume ratio during the osmotic treatment.     

Sip18 hydrophilin prevents yeast cell death during desiccation stress

Aims: For this study, we performed a genetic screen of S. cerevisiae’s deletion library for mutants sensitive to dehydration stress, with which we aimed to discover cell dehydration–tolerant genes. Methods and Results: We used a yeast gene deletion set (YGDS) of 4850 viable mutant haploid strains to perform a genome‐wide screen for the identification of desiccation stress modifiers. SIP18 is among the genes identified as essential for cells surviving to drying/rehydration process. Deletion of SIP18 promotes the accumulation of reactive oxygen species and enhances apoptotic and necrotic cell death in response to dehydration/rehydration process. Conclusions: SIP18p acts as an inhibitor of apoptosis in yeast under dehydration stress, as suggested by its antioxidative capacity through the ROS accumulation reduction after an H₂O₂ attack. Significance and Impact of the Study: To our knowledge, this is the first systematic screen for the identification of putative genes essential to overcoming cell dehydration process. A broad range of identified genes could help to understand why some strains of high biotechnological interest cannot cope with the drying and rehydration treatments. Dehydration sensitivity makes these strains not suitable to be commercialized by yeast manufactures.     

State of adenosine phosphates during dehydration of yeast

Summary The effect of cultivation and dehydration conditions on the adenosine phosphate content of yeast cells has been studied. Irrespective of the cultivation conditions the total pool of adenosine phosphates was found to increase, mainly due to accumulation of ATP, during the exponential phase of cell growth and to decrease during transition of the culture into the stationary phase. Changes in the intracellular content of adenosine phosphates were parallel with changes in the respiratory activity of yeast cells cultivated under batch conditions. Yeast cells harvested at the exponential growth phase were sensitive to dehydration, losing a notable amount of adenosine phosphates as well as respiratory capacity during drying, leading to a massive dying-off of the cells. Yeast at the stationary phase was resistant to drying, and, during this process, accumulated ATP by mitochondrial oxidation of endogenous carbohydrates. The accumulated ATP was used by the dried yeast cells as an energy source in the first minutes of reactivation. On the basis of our results we recommend that the ATP content of dried yeast cells should be used as an indicator of their capacity to recover their viability by reactivation.     

Effect of xylitol and trehalose on dry resistance of yeasts

The effects of dehydration/rehydration on two strains of Saccharomyces cerevisiae: S600, a metabolically engineered xylose-utilising strain, and H158, the non-xylose-utilising host strain; and on the naturally xylose-utilising yeast Pachysolen tannophilus CBS 4044, were compared after glucose and xylose utilisation respectively. The yeast strains differed in their ability to excrete and accumulate intracellular xylitol. A high intracellular xylitol content before and after dehydration coincided with a higher viability after a dehydration/rehydration cycle. The intracellular trehalose content increased during dehydration in all three yeast strains, but this did not correspond to enhanced cell viability after dehydration/rehydration. The results are discussed in relation to the ability of xylitol and trehalose to structure water. Received: 9 July 1996 / Received revision: 29 October 1996 / Accepted: 2 November 1996     

Phase transitions and permeability changes in dry membranes during rehydration

Dry phospholipid bilayers are known to undergo transient changes in permeability during rehydration. In this review, we present evidence from which we suggest that this permeability change is due to a gel to liquid-crystaline phase transition accompanying rehydration. If the transition is avoided, as in lipids that remain in gel phase whether dry or rehydrated, the problem of leakage during rehydration is obviated, at least in part. Further, the evidence that the transition temperature for dry bilayers can be depressed by certain sugars is discussed. Finally, we show that these principles can be extended to intact cells. Using pollen grains as a model, we have measured the transition temperature for membrane phospholipids and show that the transition is correlated with physiological measurements including permeability changes and subsequent germination. From theT _m values taken from pollen grains at different water contents, we have constructed a phase diagram for the intact pollen that has high predictive value for physiological properties.     

Weak red light plays an important role in awakening the photosynthetic machinery following desiccation in the subaerial cyanobacterium Nostoc flagelliforme

The subaerial cyanobacterium Nostoc flagelliforme can survive for years in the desiccated state and light exposure may stimulate photosynthetic recovery during rehydration. However, the influence of light quality on photosynthetic recovery and the underlying mechanism remain unresolved. Exposure of field collected N. flagelliforme to light intensity ≥2 μmol photons m⁻² s⁻¹ showed that the speed of photosystem II (PSII) recovery was in the following order: red > green > blue ≈ violet light. Decreasing the light intensity showed that weak red light stimulated PSII recovery during rehydration. The chlorophyll fluorescence transient and oxygen evolution activity indicated that the oxygen evolution complex (OEC) was the activated site triggered by weak red light. The damaged D1 protein accumulated in the thylakoid membrane during dehydration and is degraded and resynthesized during dark rehydration. PsbO interaction with the thylakoid membrane was induced by weak red light. Thus, weak red light plays an important role in triggering OEC photoactivation and the formation of functional PSII during rehydration. In its arid habitats, weak red light could stimulate the awakening of dormant N. flagelliforme after absorbing water from nighttime dew or rain to maximize growth during the early daylight hours of the dry season.     

Glycerolipid analysis during desiccation and recovery of the resurrection plant Xerophyta humilis (Bak) Dur and Schinz

Xerophyta humilis is a poikilochlorophyllous monocot resurrection plant used as a model to study vegetative desiccation tolerance. Dehydration imposes tension and ultimate loss of integrity of membranes in desiccation sensitive species. We investigated the predominant molecular species of glycerolipids present in root and leaf tissues, using multiple reaction monitoring mass spectrometry, and then analysed changes therein during dehydration and subsequent rehydration of whole plants. The presence of fatty acids with long carbon chains and with odd numbers of carbons were detected and confirmed by gas chromatography. Dehydration of both leaves and roots resulted in an increase in species containing polyunsaturated fatty acids and a decrease in disaturated species. Upon rehydration, lipid saturation was reversed, with this being initiated immediately upon watering in roots but only 12–24 hr later in leaves. Relative levels of species with short‐chained odd‐numbered saturated fatty acids decreased during dehydration and increased during rehydration, whereas the reverse trend was observed for long‐chained fatty acids. X. humilis has a unique lipid composition, this report being one of the few to demonstrate the presence of odd‐numbered fatty acids in plant phosphoglycerolipids.     

Cryopreservation of wheat (Triticum aestivum L.) egg cells by vitrification

A procedure has been developed for the cryopreservation of wheat female gametes. The procedure involves loading the cells with 25% concentrated vitrification solution consisting of 30% glycerol, 10% sucrose, 120 mM ascorbic acid (AA) and 5% propylene glycol (PG), dehydration in 80% concentrated vitrification solution, droplet vitrification and storage in liquid nitrogen, unloading and rehydration of the cells by gradual addition of isolation solution. Supplementation with AA significantly increased the proportion of viable egg cells after de- and rehydration. During the early phase of rehydration AA reduced the probability of membrane damage caused by rapid water uptake. Maintaining the temperature of the cells at 0°C during the de- and rehydration processes increased cell survival. Microscopic examination of the semi-thin sections of untreated and viable cryopreserved cells revealed that the vitrification process might cause changes in cell structure.     

Changes in the content of individual lipid classes of a lichen <Emphasis Type="Italic">Peltigera aphthosa</Emphasis> during dehydration and subsequent rehydration

A lichen Peltigera aphthosa (L.) Willd. was subjected to a short-term (7 days) or a long-term (180 and 540 days) dehydration followed by rehydration. Then the composition and content of lipids, as well as the rate of their metabolism (the rate of sodium 2-¹⁴C-acetate incorporation) were investigated. The long-term dehydration resulted in a dramatic decrease in the content (per dry wt) of major extrachloroplastic phospholipids, mainly phosphatidylcholines and phosphatidylethanolamines. The rehydration of lichen thalli after a short-term and long-term dehydration also resulted in an enhanced breakdown of these lipid molecules; however, it was accompanied by their rather intense in vivo synthesis, which was decreased after long-term dehydration. In contrast to phospholipids, the betaine lipids, diacylglyceroltrimethylhomoserines (DGTSs), were involved in metabolic processes to a far lesser extent. In the course of rehydration, their content was virtually unchanged and decreased only after 540 days of dehydration. The rate of incorporation of sodium 2-¹⁴C-acetate into the DGTS molecules was moderate and did not change even after long-term dehydration. Glycolipids were characterized by a fair tolerance to hydrolytic processes and by an increase in the rate of their synthesis after 540 days of the lichen dehydration. Responses of neutral lipids to dehydration turned out to be different. The long-term dehydration (for 540 days) was accompanied by a decrease in the contents of free sterols and sterol esters, whereas the contents of di- and triacylglycerols remained unchanged. Rehydration resulted in a decrease in diacylglycerol and sterol ester contents. All neutral lipids were characterized by a dramatic decrease in the rate of de novo synthesis after long-term dehydration. It was suggested that the tolerance of lichen to long-term dehydration was appreciably determined by the tolerance of its phycobiont, in this case, a green alga Coccomyxa sp.; the bulk of its lipids was characterized by a minimum rate of breakdown and, at the same time, by a stable synthesis.Translated from Fiziologiya Rastenii, Vol. 52, No. 1, 2005, pp. 43–50.Original Russian Text Copyright © 2005 by Kotlova, Sinyutina.     

Germination and ion leakage are linked with phase transitions: of membrane lipids during imbibition of Typha latifolia pollen

In previous studies on the causes of imbibitional leakage in dry polien we have presented data which suggest that the leakage is due to a gel to liquid crystalline phase transition in membrane phospholipids during the rehydration event. In the present study we greatly extend and confirm those results. A supplemented phase diagram for the hydration dependent transition temperature of membrane phospholipids in pollen is presented. In pollen containing > 0.05 g H₂O g⁻¹ dry weight at the time of imbibition, this phase diagram for the phospholipids precisely predicts the conditions for rehydration under which germination is maximal and leakage is minimal. However, in extremely dry pollen, containing < 0.05 g H₂O g⁻¹ dry weight the predictive value of the phase diagram for phospholipids in the pollen is not in agreement with data for germination and leakage. Thus, an alternative explanation must be sought for leakage in these circumstances. We examined the available evidence and suggest here that a modified form of the non-bilayer phase hypothesis proposed by Simon (1974) may apply in the specialized case of extremely dry cells.     

Proteolysis upon dehydration of yeasts saccharomyces cerevisiae

The effect of dehydration on proteolysis and activity of proteases A, B and C in the cells of baker's yeast Saccharomyces cerevisiae was investigated. It can be concluded, that under investigated conditions of yeast Saccharomyces cerevisiae drying a decrease of proteases activity takes place. In cells a limited proteolysis takes place which is indicated by an increase in amino nitrogen content and a decrease of tryptophane synthase activity. Adding the protease inhibitor to yeast suspension prevents decrease of tryptophane synthase activity upon dehydration.     

Photosynthetic Performance and Water Use Efficiency of the Fern Cheilanthes Persica

Morphological, anatomical and physiological frond traits of Cheilanthes persica (Bory) Mett. ex Kuhn were studied to analyze its adaptive strategy. Mean frond life span is about 340 d. Mature fronds are characterized by 91 g m-2 areal dry mass (ADM) and 217 g m-2 succulence. The reduction of frond water content in July (dehydration phase) caused a 51 % decrease in frond surface area (SA). Fronds were dry in August (desiccation phase); nevertheless, in September they showed an increased SA (rehydration phase). Chlorophyll (Chl) a/b ratio, above 3, and the well developed palisade parenchyma (two layers, total thickness of 103.9 µm) are typical for sun leaves. Chl and carotenoid contents and net photosynthetic rate (PN) increased during frond development until the highest values in April-May (maturity phase). When mean air temperature reached 31.3 °C, stomatal conductance (gs) decreased by 34 % and PN by 33 %. The high pigment contents can dissipate the excess of radiant energy, particularly under unfavourable conditions, when PN is low. Rather high PN was found during the rehydration stage. The pronounced decline of mesophyll activity during the declining phase was confirmed by the lowest PN.