Severe Osmotic Stress Research Articles

Cellular, Physiological, and Biochemical Basis of Adaptive Response to Variable Osmotic Environments by the River Shad, Tenualosa ilisha

The river shad, hilsa (Tenualosa ilisha), is an anadromous fish that migrates from marine to freshwater for spawning. This transition/migration poses severe osmotic stress that hilsa needs to successfully minimize. The present study was conducted to evaluate the cellular (ultrastructure of gill and kidney), physiological (hemocyte counts, blood, and water osmolality), and biochemical (free amino acids, free fatty acids, blood glucose, and cortisol levels) parameters of hilsa collected from four different environmental salinity levels (0‰, 10‰, 20‰, and 30‰ salinity levels; using 10 fish samples from each salinity). Results indicate that increased amount of fatty/mucus cells in the gill and lower number of glomerular capsules in kidney were observed at low (0‰ and 10‰) salinities compared to high (20‰ and 30‰) salinities. Water and blood osmolality (total ionic content) showed declining trends with lowering salinities. Total number of blood cells was also found to vary significantly P < 0.05 among salinities. Salinity-specific blood cortisol and glucose levels of hilsa were observed as 0‰ > 10‰ > 30‰ > 20‰. Total essential and free amino acids of hilsa blood showed significantly declining trends P < 0.05 with salinity reductions. No significant differences were observed between 10‰, 20‰, and 30‰ salinities for different types of fatty acids, while hilsa collected from 0‰ showed significantly higher P < 0.05 levels of fatty acids compared to the remaining three salinities. Findings indicate that hilsa rapidly alters cellular, physiological, and biochemical traits for successful transition between different salinity habitats.

Regulation of reactive oxygen species and phytohormones in osmotic stress tolerance during seed germination in indica rice.

Climate change due to global warming is now affecting agricultural production worldwide. In rice, one of the most important crops, water limitation due to irregular rainfall in rainfed lowlands during crop growth limits yield. Dry direct-sowing has been proposed as a water-efficient approach to cope with water stress during rice growth, but poor seedling establishment due to drought during germination and emergence is a problem. Here, we germinated indica rice cultivars Rc348 (drought tolerant) and Rc10 (drought sensitive) under osmotic stress induced by PEG to elucidate mechanisms of germination under drought. Rc348 had higher germination rate and germination index under severe osmotic stress of -1.5 MPa, above those of Rc10. Rc348 showed up-regulated GA biosynthesis, down-regulated ABA catabolism, and up-regulated α-amylase gene expression in imbibed seeds under PEG treatment compared to that of Rc10. During germination, reactive oxygen species (ROS) play important roles in antagonism between gibberellic acid (GA) and abscisic acid (ABA). Embryo of Rc348 treated with PEG had significantly greater expression of NADPH oxidase genes and higher endogenous ROS levels, together with significantly increased endogenous GA1, GA4 and ABA contents compared to that of Rc10. In aleurone layers treated with exogenous GA, expression of α-amylase genes was higher in Rc348 than in Rc10, and expression of NADPH oxidase genes was enhanced with significantly higher ROS content in Rc348, suggesting higher sensitivity of GA to ROS production and starch degradation in aleurone cells of Rc348. These results suggest that the osmotic stress tolerance of Rc348 is due to enhancement of ROS production, GA biosynthesis, and GA sensitivity, resulting in a higher germination rate under osmotic stress.

Factors affecting cryopreservation-associated damages in sperm motility of cockerels (Gallus gallus domesticus)

ABSTRACT 1. Sperm are exposed to severe osmotic stress during cryopreservation, which results in impairment of fertilisation ability, including motility and viability, in poultry. Sperm osmotolerance is regulated by many extracellular factors and varies widely in birds, leading to uncertainty in the nature of the osmotic injury. 2. Tail bending is a primary response resulting from cell swelling from excessive osmotic stress. However, the underlying mechanism responsible for tail bending is largely unknown. This study examined the relationship between osmotic stress and post-thaw motility, with a particular focus on the role of Na+/K+ ATPase (NKA) in the tail bending response. 3. Cryopreserved sperm exhibited rapidly reduced motility when maintained at 37°C. The combination of temperature change and osmotic stress was a primary factor responsible for tail bending. This work tested a hypothesis known to be associated with post-thaw tail abnormality in other species and found that cold shock, that is not accompanied by an apoptotic response, may occur. Ouabain inhibition of Na+/K+ ATPase activity alleviated the tail bending response in fresh and post-thaw sperm. 4. These results demonstrated that the combination of temperature change and osmotic stress has a primary impact on the reduction of post-thaw motility, with a particular role in NKA activity, in the tail bending response of chicken sperm. These results provide a foundation for establishing cryopreservation methodology to ensure the optimal fertilisation potential of cryopreserved chicken sperm.

Thylakoid proteome variation of Eutrema salsugineum in response to drought and salinity combined stress

It is well known that plant responses to stress involve different events occurring at different places of the cell/leaf and at different time scales in relation with the plant development. In fact, the organelles proteomes include a wide range of proteins that could include a wide range of proteins showing a considerable change in cellular functions and metabolism process. On this basis, a comparative proteomics analysis and fluorescence induction measurements were performed to investigate the photosynthetic performance and the relative thylakoid proteome variation in Eutrema salsugineum cultivated under salt stress (200 mM NaCl), water deficit stress (PEG) and combined treatment (PEG + NaCl) as a hyperosmotic stress. The obtained results showed a significant decrease of plant growth under drought stress conditions, with the appearance of some toxicity symptoms, especially in plants subjected to combined treatment. Application of salt or water stress alone showed no apparent change in the chlorophyll a fluorescence transients, primary photochemistry (fluorescence kinetics of the O-J phase), the PQ pool state (J-I phase changes), (Fv/Fm) and (Fk/Fj) ratios. However, a considerable decrease of all these parameters was observed under severe osmotic stress (PEG + NaCl). The thylakoid proteome analysis revealed 58 proteins showing a significant variation in their abundance between treatments (up or down regulation). The combined treatment (PEG + NaCl) induced a decrease in the expression of the whole PSII core subunit (D1, D2, CP43, CP47, PsbE and PsbH), whereas the OEC subunits proteins remained constant. An increase in the amount of PsaD, PsaE, PsaF, PsaH, PsaK and PsaN was detected under drought stress (PEG5%). No significant change in the accumulation of Cyt b6 and Cyt f was observed. Some regulated proteins involved in cellular redox homeostasis were detected (glutamine synthetase, phosphoglycerate kinase, transketolase), and showed a significant decrease under the combined treatment. Some oxidative stress related proteins were significantly up-regulated under salt or drought stress and could play a crucial role in the PSI photoprotection and the control of ROS production level.

Influence of NaCl induced salt stress on the seedling growth of citrus species

Salinity is a commonly occurring abiotic stress and poses as a major impediment in citrus production as the crop has less tolerance ability to this stress. It causes severe osmotic stress restraining overall performance of the plant. One month old seedlings of Rangpur lime, Rough lemon and Indian wild orange were established in sand media and later exposed to stress for 48 hrs in a low cost polyhouse. The different levels of sodium chloride (0 mM, 75 mM and 100 mM) was used for simulating salt stress. The morpho-physiological parameters viz., shoot and root length, fresh and dry weight of shoot and root and Relative water content (RWC) were reduced on exposing to stress with severity being in 100 mM NaCl. On comparing citrus species, Rangpur lime performed better at both stress levels than Rough lemon and Indian wild orange exhibiting its tolerance towards salinity.

Functional Characterization and Evolutionary Analysis of Glycine-Betaine Biosynthesis Pathway in Red Seaweed Pyropia yezoensis.

The red seaweed Pyropia yezoensis is an ideal research model for dissecting the molecular mechanisms underlying its robust acclimation to abiotic stresses in intertidal zones. Glycine betaine (GB) was an important osmolyte in maintaining osmotic balance and stabilizing the quaternary structure of complex proteins under abiotic stresses (drought, salinity, etc.) in plants, animals, and bacteria. However, the existence and possible functions of GB in Pyropia remain elusive. In this study, we observed the rapid accumulation of GB in desiccated Pyropia blades, identifying its essential roles in protecting Pyropia cells against severe osmotic stress. Based on the available genomic and transcriptomic information of Pyropia, we computationally identified genes encoding the three key enzymes in the GB biosynthesis pathway: phosphoethanolamine N-methyltransferase (PEAMT), choline dehydrogenase (CDH), and betaine aldehyde dehydrogenase (BADH). Pyropia had an extraordinarily expanded gene copy number of CDH (up to seven) compared to other red algae. Phylogeny analysis revealed that in addition to the one conservative CDH in red algae, the other six might have originated from early gene duplication events. In dehydration stress, multiple CDH paralogs and PEAMT genes were coordinating up-regulated and shunted metabolic flux into GB biosynthesis. An elaborate molecular mechanism might be involved in the transcriptional regulation of these genes.

Comparison of changes in water status and photosynthetic parameters in wild type and abscisic acid-deficient sitiens mutant of tomato (Solanum lycopersicum cv. Rheinlands Ruhm) exposed to sublethal and lethal salt stress

Abscisic acid (ABA) regulates many salt stress-related processes of plants such as water balance, osmotic stress tolerance and photosynthesis. In this study we investigated the responses of wild type (WT) and the ABA-deficient sitiens mutant of tomato (Solanum lycopersicum cv. Rheinlands Ruhm) to sublethal and lethal salt stress elicited by 100 mM and 250 mM NaCl, respectively. Sitiens mutants displayed much higher decrease in water potential, stomatal conductance and net CO2 assimilation rate under high salinity, especially at lethal salt stress, than the WT. However, ABA deficiency in sitiens caused more severe osmotic stress and more moderate ionic stress, higher K+/Na+ ratio, in leaf tissues of plants exposed to salt stress. The higher salt concentration caused irreversible damage to Photosystem II (PSII) reaction centres, severe reduction in the linear photosynthetic electron transport rate and in the effective quantum yields of PSII and PSI in sitiens plants. The cyclic electron transport (CET) around PSI, which is an effective defence mechanism against the damage caused by photoinhibition in PSI, decreased in sitiens mutants, while WT plants were able to increase CET under salt stress. This suggests that the activation of CET needs active ABA synthesis and/or signalling. In spite of ABA deficiency, proline accumulation could alleviate the stress injury at sublethal salt stress in the mutants but its accumulation was not sufficient at lethal salt stress.

Protein Profiles Underlying the Effect of Plant Growth-Promoting Rhizobacteria on Canola under Osmotic Stress

Plant growth-promoting bacteria recover the negative effects of osmotic stress on canola. For comprehending the role of bacteria in canola under osmotic stress, Sarigol and Hyola308 as drought-sensitive and -tolerant cultivars, respectively, inoculated with Enterobacter sp. S16-3 as plant growth-promoting bacteria were treated with polyethylene glycol. Bacterial inoculation increased the root volume of Hyola308 under osmotic stress. Using a gel-free/label-free proteomic technique, 115 commonly changed proteins were identified in the root between inoculated Sarigol and Hyola308 under osmotic stress. In both cultivars with bacterial inoculation, proteins related to protein metabolism, cell organization, and signaling were increased in roots under osmotic stress. On the other hand, bacterial inoculation increased and decreased the abundance of malate dehydrogenase (EC 1.1.1.37) in roots of Hyola308 and Sarigol, respectively, under severe osmotic stress. Furthermore, in leaf, bacterial inoculation increased the abundance of tricarboxylic acid cycle-related proteins in Hyola308 under severe osmotic stress. These results suggest that bacterial inoculation might increase the tolerance to severe osmotic stress by modifying proteins related to energy metabolism in both leaf and root. Enhancement of energy metabolism elicited by bacterial inoculation might provide a connection between cell metabolism and root growth, which might cause regulated growth and increased tolerance in drought-tolerant canola under osmotic stress.

Microbial Diversity in a Hypersaline Sulfate Lake: A Terrestrial Analog of Ancient Mars.

Life can persist under severe osmotic stress and low water activity in hypersaline environments. On Mars, evidence for the past presence of saline bodies of water is prevalent and resulted in the widespread deposition of sulfate and chloride salts. Here we investigate Spotted Lake (British Columbia, Canada), a hypersaline lake with extreme (>3 M) levels of sulfate salts as an exemplar of the conditions thought to be associated with ancient Mars. We provide the first characterization of microbial structure in Spotted Lake sediments through metagenomic sequencing, and report a bacteria-dominated community with abundant Proteobacteria, Firmicutes, and Bacteroidetes, as well as diverse extremophiles. Microbial abundance and functional comparisons reveal similarities to Ace Lake, a meromictic Antarctic lake with anoxic and sulfidic bottom waters. Our analysis suggests that hypersaline-associated species occupy niches characterized foremost by differential abundance of Archaea, uncharacterized Bacteria, and Cyanobacteria. Potential biosignatures in this environment are discussed, specifically the likelihood of a strong sulfur isotopic fractionation record within the sediments due to the presence of sulfate reducing bacteria. With its high sulfate levels and seasonal freeze-thaw cycles, Spotted Lake is an analog for ancient paleolakes on Mars in which sulfate salt deposits may have offered periodically habitable environments, and could have concentrated and preserved organic materials or their biomarkers over geologic time.

Mitigating effects of acetylcholine supply on soybean seed germination under osmotic stress

Acetylcholine (Ach) is a common neurotransmitter in animals, also synthesized in plants, which can have an influence on plant response to stress, also acting as a signaling molecule between root and shoot. The objective of this study was to analyze the possible mitigating effects of exogenous application of Ach on soybean germination under different levels of osmotic potential. The experiments were conducted with soybean [Glycine max (L.) Merrill] genotype Intacta. The seeds were first treated with Ach solutions with the following concentrations: 0.0 (control); 0.5; 1. 0 and 2.0 mM. Then, the seeds were subjected to two water potentials, −0.5 and −1.0 MPa, reached by using mannitol solutions, for the induction of osmotic stress, and a control condition with distilled water. Thus, 12 treatments were established in a double factorial 4 × 3, with 4 levels of Ach and 3 osmotic potential treatments (0.0, −0.5 and −1.0 MPa) with four replicates per treatment. The results showed that the concentration of 1.0 mM Ach, without osmotic stress, presented higher values for total dry mass of the seedlings compared to the control treatment (without Ach supply). In the treatments conducted to test the effectiveness of Ach on the mitigation of severe osmotic stress effects (−1.0 MPa), results showed that the concentration of 0.5 mM Ach showed positive results for the following parameters; dry weight of shoot, root dry weight, total dry mass, which were significantly higher than treatment under 1.0 MPa.

Copper-induced metabolic variation of oysters overwhelmed by salinity effects

In estuarine environments, Cu (copper) contamination is simultaneously coupled with salinity variation. In this study, 1H NMR was applied to investigate the metabolic disturbance of estuarine oysters Crassostrea hongkongensis under both Cu and salinity stresses. Oysters were exposed to dissolved Cu (50 μg L−1) at different salinities (10, 15 and 25 psu) for six weeks, and the Cu accumulation in the oyster tissues was higher at lowered salinity. Based on the NMR-metabolomics results, disturbances induced by Cu and salinity was mainly related to osmotic regulation, energy metabolism and glycerophospholipid metabolism, as indicated by the alteration of important metabolic biomarkers such as alanine, citrate, glucose, glycogen, betaine, taurine, hypotaurine and homarine in the gills. At lower salinity, oysters accumulated higher energy related compounds (e.g., glucose and glycogen) and amino acids (e.g., aspartate, dimethylglycine and lysine), with the enhancement of ATP/ADP production and accumulation of oxidizable amino acids catabolized from protein breakdown. With Cu exposure, the synthesis from glycine to dimethylglycine was observed to cope with severe osmotic stress, together with the elevation of lysine and homarine. The effects induced by Cu were much similar for each salinity treatment, but the combination of Cu and salinity turned out to be consistent with the singular salinity effects. Therefore, salinity played a dominant role in affecting the metabolites of oysters when combined with Cu exposure. This study indicated that salinity should be taken into consideration in order to predict the Cu toxicity in estuarine organisms.

Osmotic Stress-Induced Defective Glial Proteostasis Contributes to Brain Demyelination after Hyponatremia Treatment.

Adequate protein folding is necessary for normal cell function and a tightly regulated process that requires proper intracellular ionic strength. In many cell types, imbalance between protein synthesis and degradation can induce endoplasmic reticulum (ER) stress, which if sustained, can in turn lead to cell death. In nematodes, osmotic stress induces massive protein aggregation coupled with unfolded protein response and ER stress. In clinical practice, patients sustaining rapid correction of chronic hyponatremia are at risk of osmotic demyelination syndrome. The intense osmotic stress sustained by brain cells is believed to be the major risk factor for demyelination resulting from astrocyte death, which leads to microglial activation, blood-brain barrier opening, and later, myelin damage. Here, using a rat model of osmotic demyelination, we showed that rapid correction of chronic hyponatremia induces severe alterations in proteostasis characterized by diffuse protein aggregation and ubiquitination. Abrupt correction of hyponatremia resulted in vigorous activation of both the unfolded protein response and ER stress accompanied by increased autophagic activity and apoptosis. Immunofluorescence revealed that most of these processes occurred in astrocytes within regions previously shown to be demyelinated in later stages of this syndrome. These results identify osmotic stress as a potent protein aggregation stimuli in mammalian brain and further suggest that osmotic demyelination might be a consequence of proteostasis failure on severe osmotic stress.

Efficacy of a New Ocular Surface Modulator in Restoring Epithelial Changes in an In Vitro Model of Dry Eye Syndrome

ABSTRACTPurpose: So far tear substitutes have demonstrated a limited role in restoring ocular surface damage in dry eye syndrome (DES). The aim of this study was to assess the efficacy of a new ocular surface modulator in an in vitro model of human corneal epithelium (HCE) damaged by severe osmotic stress mirroring the features of dry eye conditions.Methods: A reconstructed HCE model challenged by the introduction of sorbitol in the culture medium for 16 h was used to induce an inflammatory pathway and to impair the tight junctions integrity determining a severe modification of the superficial layer ultrastructure. At the end of the overnight stress period in the treated HCE series, 30 μl of the ocular surface modulator (T-LysYal, Sildeha, Switzerland) and of hyaluronic acid (HA) in the control HCE series were applied for 24 h. The following parameters were quantified: scanning electron microscopy (SEM), trans-epithelial electrical resistance (TEER), immunofluorescence analysis of integrin β1 (ITG-β1), mRNA expression of Cyclin D-1 (CCND1), and ITG-β1.Results: In the positive control after the osmotic stress the HCE surface damage was visible at the ultrastructural level with loss of cell–cell interconnections, intercellular matrix destruction, and TEER reduction. After 24 h of treatment with T-LysYal, HCE showed a significant improvement of the ultrastructural morphological organization and increased expression of ITG-β1 at the tissue level when compared to positive and control series. A significant increase of mRNA expression for ITG-β1 and CCND1 was shown in the HA-treated cells compared to T-LysYal. TEER measurement showed a significant reduction in all groups after 16 h without modifications after the treatment period.Conclusions: This study has shown the possibility of a new class of agents denominated ocular surface modulators to restore corneal cells damaged by dry eye conditions. Further in vivo studies are certainly necessary to confirm these results.

PEG-induced osmotic stress in Mentha x piperita L.: Structural features and metabolic responses

The present study investigated whether osmotic stress induced by the exposure of peppermint (Mentha x piperita L.) to moderate and severe stress for short periods of time changes the plant's physiological parameters, leaf anatomy and ultrastructure and essential oil. Plants were exposed to two levels of polyethyleneglycol (50 g L−1 and 100 g L−1 of PEG) in a hydroponic experiment. The plants exposed to 50 g L−1 maintained metabolic functions similar to those of the control group (0 g L−1) without changes in gas exchange or structural characteristics. The increase in antioxidant enzyme activity reduced the presence of free radicals and protected membranes, including chloroplasts and mitochondria. In contrast, the osmotic stress caused by 100 g L−1 of PEG inhibited leaf gas exchange, reduced the essential oil content and changed the oil composition, including a decrease in menthone and an increase in menthofuran. These plants also showed an increase in peroxidase activity, but this increase was not sufficient to decrease the lipid peroxidation level responsible for damaging the membranes of organelles. Morphological changes were correlated with the evaluated physiological features: plants exposed to 100 g L−1 of PEG showed areas with collapsed cells, increases in mesophyll thickness and the area of the intercellular space, cuticle shrinkage, morphological changes in plastids, and lysis of mitochondria. In summary, our results revealed that PEG-induced osmotic stress in M. x piperita depends on the intensity level of the osmotic stress applied; severe osmotic stress changed the structural characteristics, caused damage at the cellular level, and reduced the essential oil content and quality.

An Ethylene-responsive Factor BpERF11 Negatively Modulates Salt and Osmotic Tolerance in Betula platyphylla.

Ethylene responsive factors (ERFs) play important roles in the abiotic stress; however, only a few ERF genes from woody plants have been functionally characterized. In the present study, an ERF gene from Betula platyphylla (birch), BpERF11, was functionally characterized in response to abiotic stress. BpERF11 is a nuclear protein, which could specifically bind to GCC boxes and DRE motifs. BpERF11-overexpressing and BpERF11 RNA interference (RNAi) knockdown plants were generated for gain- and loss-of-function analysis. BpERF11 negatively regulates resistance to salt and severe osmotic stress, and the transgenic birch plants overexpressing BpERF11 shows increased electrolyte leakage and malondialdehyde (MDA) contents. BpERF11 inhibits the expression of an AtMYB61 homologous gene, resulting in increased stomatal aperture, which elevated the transpiration rate. Furthermore, BpERF11 downregulates the expression of P5CS, SOD and POD genes, but upregulates the expression of PRODH and P5CDH, which results in reduced proline levels and increased reactive oxygen species (ROS) accumulation. BpERF11 also significantly inhibits the expression of LEA and dehydrin genes that involve in abiotic stress tolerance. Therefore, BpERF11 serves as a transcription factor that negatively regulates salt and severe osmotic tolerance by modulating various physiological processes.

Effect of osmotic stress on osmolyte accumulation and ammonia assimilating enzymes in chickpea cultivars

An investigation was conducted to evaluate the effect of osmotic stress on four chickpea cultivars, viz., two drought tolerant (ICC 4958 and Vijay), and two drought susceptible (JG-216 and PG 1808) subjected to mild (−0.4 MPa) and severe (−1.2 MPa) osmotic stress induced by PEG-6000. The activity of ammonia assimilation enzymes, viz., nitrate reductase (NR) glutamine synthetase (GS), glutamate synthase (GOGAT) decreased, while activity of proline synthesizing enzyme Δ1 pyrroline-5-carboxlate synthetase (P5CS), and contents of proline, glycine-betaine and mannitol increased significantly in both drought tolerant and susceptible chickpea cultivars under mild and severe osmotic stress conditions. The extent of increase was higher in tolerant than susceptible cultivars. ICC-4958 recorded higher accumulation of osmolytes, viz., proline, glycine-betaine, mannitol and P5CS activity with less decrease in the activity of NR, GS, and GOGAT.

Oscillatory Electrical Response to Osmotic Stress in Soybean Plants

Oscillations in plants can occur at various levels. Their physiological processes evolve during time, presenting a variable dynamics1. Different oscillatory dynamics in different physiological processes in plants have been observed under different environmental conditions. Oscillatory dynamics allow to organisms flexibility in responses to environmental changes2. Recent studies have evidenced the role of electrical signaling in the physiological processes. At the whole-plant level, oscillations of electrical frequency-coded signals are believed to play an important role in communication between the roots and shoots3. The aim of this work was to test the hypothesis that a severe osmotic stress causes changes in the pattern of electrical signaling in soybean plants, allowing discrimination among signs under different water conditions. Two-week-old soybean seedlings were used for the experiments, carried out under constant environmental conditions and inside a Faraday cage. Electrical time series (n=30) were obtained with an electronic acquisition data system. On previous day of experiments, the plants were transferred to hydroponic cultivation and Needle electrodes were inserted into the stem, for acclimatization. Osmotic stress was applied by replacing the nutrient solution by mannitol solution at -2MPa. Power frequency analysis has confirmed differences in oscillatory characteristic of plant bioelectrical data. Both conditions showed a power spectrum following a power-law distribution, indicating a scale-free pattern of the dynamics. On the other hand, while in the time series scored before osmotic stimuli the range of frequencies from 0.15 to 16Hz followed the power-law trend, after osmotic stimuli this range was from 0.10 to 5Hz. Moreover, before stimuli it was observed a second set of frequencies ranging from 11 to 13Hz, which it was not present in the time series after stimuli. These results have indicates a decrease trend of complexity of the electrical signal to root-shoot communication of soybean seedlings under osmotic stress.

Excluded Volume Effects Inside the Living Cell

Proteins and nucleic acids fold and behave in a highly occupied matrix of macromolecules, the cellular environment. Although this environment is filled up to a volume of 40% with macromolecules, the effect of crowding on biochemical reactions as well as biophysical properties of proteins has been rarely considered. Therefore, studies on macromolecular crowding are mainly conducted using artificially created polymer based substances as mimics of the in cell environment. Still, a common concept how the excluded volume effect in the cell affects protein folding, function and structure is lacking. Understanding these properties will be important to unravel the underlying mechanism of protein misfolding and aggregation as well as the behavior of intrinsically disordered proteins in a living cell.We introduce a FRET based random coil polymer to characterize crowding both in vitro and inside a living cell. We find different compression of the polymer in artificial crowding substances compared to protein crowders such as BSA or oocyte lysate. Injection of the probe inside cells confirms this result and reveals a heterogeneous environment which, on average, shows comparable polymer conformations as in diluted buffer. The polymer conformation is used to quantify crowding differences in the cytosol and the nucleus as well as to identify crowding in cells influenced by different extrinsic conditions. Severe osmotic stress is used to probe compression of the polymer in the highly concentrated cytosolic environment.We conclude that the FRET labelled polymer provides a new approach to investigate and characterize the cellular solvation properties with high spatio-temporal resolution in a variety of systems and identify crowding differences due to the architecture of the cellular matrix. Therefore, it will help to understand how the cell, as the native environment in which proteins evolved, might modulate and tune biomolecule properties and functions.

Oxygen transfer efficiency and environmental osmolarity response to neutralizing agents on l-lactic acid production efficiency by Lactobacillus paracasei

The effects of Ca(OH)2, NH4OH and NaOH as neutralizing agents on the efficiency of l-lactic acid production by Lactobacillus paracasei were investigated in this study. Fermentation performance with Ca(OH)2 was superior to NH4OH and NaOH because it had the highest oxygen transfer rate (OTR) and lowest environmental osmotic pressure. Much smaller bubbles were generated using a calcium lactate solution compared with those generated using ammonium lactate and sodium lactate solutions, indicating that Ca(OH)2 had the highest OTR. Moreover, experiments demonstrated that ammonium lactate and sodium lactate caused more severe osmotic stress on cell growth than calcium lactate. In conclusion, the effects of neutralizing agents on l-lactic acid production efficiency could be ascribed to the contribution of lactates to OTR and environmental osmotic stress.

Photosystem I shows a higher tolerance to sorbitol‐induced osmotic stress than photosystem II in the intertidal macro‐algae Ulva prolifera (Chlorophyta)

The photosynthetic performance of the desiccation-tolerant, intertidal macro-algae Ulva prolifera was significantly affected by sorbitol-induced osmotic stress. Our results showed that photosynthetic activity decreased significantly with increases in sorbitol concentration. Although the partial activity of both photosystem I (PS I) and photosystem II (PS II) was able to recover after 30 min of rehydration, the activity of PS II decreased more rapidly than PS I. At 4 M sorbitol concentration, the activity of PS II was almost 0 while that of PS I was still at about one third of normal levels. Following prolonged treatment with 1 and 2 M sorbitol, the activity of PS I and PS II decreased slowly, suggesting that the effects of moderate concentrations of sorbitol on PS I and PS II were gradual. Interestingly, an increase in non-photochemical quenching occurred under these conditions in response to moderate osmotic stress, whereas it declined significantly under severe osmotic stress. These results suggest that photoprotection in U. prolifera could also be induced by moderate osmotic stress. In addition, the oxidation of PS I was significantly affected by osmotic stress. P700(+) in the thalli treated with high concentrations of sorbitol could still be reduced, as PS II was inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), but it could not be fully oxidized. This observation may be caused by the higher quantum yield of non-photochemical energy dissipation in PS I due to acceptor-side limitation (Y(NA)) during rehydration in seawater containing DCMU.