Hyperosmotic Stress Conditions Research Articles

Function of L-Pipecolic Acid as Compatible Solute in Corynebacterium glutamicum as Basis for Its Production Under Hyperosmolar Conditions.

Pipecolic acid or L-PA is a cyclic amino acid derived from L-lysine which has gained interest in the recent years within the pharmaceutical and chemical industries. L-PA can be produced efficiently using recombinant Corynebacterium glutamicum strains by expanding the natural L-lysine biosynthetic pathway. L-PA is a six-membered ring homolog of the five-membered ring amino acid L-proline, which serves as compatible solute in C. glutamicum.Here, we show that de novo synthesized or externally added L-PA partially is beneficial for growth under hyper-osmotic stress conditions. C. glutamicum cells accumulated L-PA under elevated osmotic pressure and released it after an osmotic down shock. In the absence of the mechanosensitive channel YggB intracellular L-PA concentrations increased and its release after osmotic down shock was slower. The proline permease ProP was identified as a candidate L-PA uptake system since RNAseq analysis revealed increased proP RNA levels upon L-PA production. Under hyper-osmotic conditions, a ΔproP strain showed similar growth behavior than the parent strain when L-proline was added externally. By contrast, the growth impairment of the ΔproP strain under hyper-osmotic conditions could not be alleviated by addition of L-PA unless proP was expressed from a plasmid. This is commensurate with the view that L-proline can be imported into the C. glutamicum cell by ProP and other transporters such as EctP and PutP, while ProP appears of major importance for L-PA uptake under hyper-osmotic stress conditions.

Alanine, proline and urea are major organic osmolytes in the snail Theodoxus fluviatilis under hyperosmotic stress.

Hyperosmotic stress may result in osmotic volume loss from the body to the environment in animals that cannot control the water permeability of their integument. Euryhaline animals (which have a wide tolerance range of environmental salinities) have generally evolved the ability to counteract cell volume shrinkage by accumulating inorganic and organic osmolytes within their cells to balance internal and external osmolalities. Molluscs use very different combinations of amino acids and amino acid derivatives to achieve this goal. Theodoxus fluviatilis is a neritid gastropod that is distributed not only in limnic habitats in Europe but also in brackish waters (e.g. along the shoreline of the Baltic Sea). Animals from brackish sites survive better in high salinities than animals from freshwater locations. The results of the present study indicate that these differences in salinity tolerance cannot be explained by differences in the general ability to accumulate amino acids as organic osmolytes. Although there may be differences in the metabolic pathways involved in osmolyte accumulation in foot muscle tissue, the two groups of animals accumulate amino acid mixtures equally well when stepwise acclimated to their respective maximum tolerable salinity for extended periods. Among these amino acids, alanine and proline, as well as the osmolyte urea, hold a special importance for cell volume preservation in T. fluviatilis under hyperosmotic stress. It is possible that the accumulation of various amino acids during hyperosmotic stress occurs via hydrolysis of storage proteins, while alanine and proline are probably newly synthesised under conditions of hyperosmotic stress in the animals.

Living without the cell wall.

This study found that when filamentous actinomycetes are exposed to osmotic stress they extrude previously undetected cell wall-deficient cells, termed S-cells, that enable actinomycetes to thrive under hyperosmotic stress conditions.

A novel uORF-based regulatory mechanism controls translation of the human MDM2 and eIF2D mRNAs during stress

Short upstream open reading frames (uORFs) are the most prevalent cis-acting regulatory elements in the mammalian transcriptome which can orchestrate mRNA translation. Apart from being “passive roadblocks” that decrease expression of the main coding regions, particular uORFs can serve as specific sensors for changing conditions, thus regulating translation in response to cell stress. Here we report a novel uORF-based regulatory mechanism that is employed under conditions of hyperosmotic stress by at least two human mRNAs, coding for translation reinitiation/recycling factor eIF2D and E3 ubiquitin ligase MDM2. This novel mode of translational control selectively downregulates their expression and requires as few as one uORF. Using a set of reporter mRNAs and fleeting mRNA transfection (FLERT) technique, we provide evidence that the phenomenon does not rely on delayed reinitiation, altered AUG recognition, ribosome stalling, mRNA destabilization or other known mechanisms. Instead, it is based on events taking place at uORF stop codon or immediately downstream. Functional aspects and implications of the novel regulatory mechanism to cell physiology are discussed.

Stress-driven increase in proline levels, and not proline levels themselves, correlates with the ability to withstand excess salt in a group of 17 Italian rice genotypes.

In most plant species, a rapid increase in free proline content occurs following exposure to hyperosmotic stress conditions. However, inconsistent results were reported concerning the role of such an increase on the plant response to water shortage or excess salt. Therefore, the possibility that proline accumulation may help the cell to withstand stress conditions, or that it simply represents a stress marker, is still a matter of debate. A possible relationship between proline accumulation and salt tolerance was investigated in a set of 17 Italian rice varieties. Rice seedlings were exposed to increasing salt concentrations during germination and early growth. The resulting levels of free proline were measured separately in shoots and roots and compared to those in untreated controls. Results were related to the corresponding ability of a given genotype to tolerate stress conditions. Neither absolute proline levels in untreated or in salt-stressed seedlings showed a straightforward relationship to the relative tolerance to salt, estimated as conductivity values able to reduce growth by 10 or 50%. Conversely, a highly significant correlation was found between the increase in proline levels in shoots and the ability to withstand stress. The results strengthen a recent hypothesis suggesting than an increase in proline metabolic rates, more than the resulting proline content, may help the cell to counteract the effects of abiotic stress conditions.

A molecular dynamics approach towards evaluating osmotic and thermal stress in the extracellular environment

Objective: A molecular dynamics approach to understanding fundamental mechanisms of combined thermal and osmotic stress induced by thermochemical ablation (TCA) is presented.Methods: Structural models of fibronectin and fibronectin bound to its integrin receptor provide idealized models for the effects of thermal and osmotic stress in the extracellular matrix. Fibronectin binding to integrin is known to facilitate cell survival. The extracellular environment produced by TCA at the lesion boundary was modelled at 37 °C and 43 °C with added sodium chloride (NaCl) concentrations (0, 40, 80, 160, and 320 mM). Atomistic simulations of solvated proteins were performed using the GROMOS96 force field and TIP3P water model. Computational results were compared with the results of viability studies of human hepatocellular carcinoma (HCC) cell lines HepG2 and Hep3B under matching thermal and osmotic experimental conditions.Results: Cell viability was inversely correlated with hyperthermal and hyperosmotic stresses. Added NaCl concentrations were correlated with a root mean square fluctuation increase of the fibronectin arginylglycylaspartic acid (RGD) binding domain. Computed interaction coefficients demonstrate preferential hydration of the protein model and are correlated with salt-induced strengthening of hydrophobic interactions. Under the combined hyperthermal and hyperosmotic stress conditions (43 °C and 320 mM added NaCl), the free energy change required for fibronectin binding to integrin was less favorable than that for binding under control conditions (37 °C and 0 mM added NaCl).Conclusion: Results quantify multiple measures of structural changes as a function of temperature increase and addition of NaCl to the solution. Correlations between cell viability and stability measures suggest that protein aggregates, non-functional proteins, and less favorable cell attachment conditions have a role in TCA-induced cell stress.

Nrf3 promotes UV-induced keratinocyte apoptosis through suppression of cell adhesion

The transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) is a key regulator of the cellular stress response, but the biological functions of the related Nrf3 protein are largely unknown. Here we demonstrate a novel pro-apoptotic function of Nrf3 in mouse and human keratinocytes. In response to UV irradiation, Nrf3-deficient keratinocytes were protected from apoptosis in vitro and in vivo. The protective function was also seen under oxidative or hyperosmotic stress conditions, but not when apoptosis was induced by disruption of cell–matrix interactions. Mechanistically, we show that Nrf3-deficient keratinocytes exhibit stronger cell–cell and cell-matrix adhesion, which correlates with higher cell surface integrin levels and enhanced activation of focal adhesion kinase. Nrf3-deficient cells also formed more and larger focal adhesions and exhibited a higher motility. These results suggest that the strong expression of Nrf3 in basal keratinocytes promotes their elimination in response to DNA damage-inducing agents, thereby preventing accumulation of mutated stem and transit amplifying cells in the epidermis.

The molecular mechanism and post-transcriptional regulation characteristic of Tetragenococcus halophilus acclimation to osmotic stress revealed by quantitative proteomics

Tetragenococcus halophilus is a moderate halophilic bacterium which was widely used in fermentation processes, growing in a broad range of salinity conditions, and can survive a saturated 26.47% w/w NaCl concentration. However, the mechanism of this outstanding ability to acclimate to extracellular osmotic stress still remains unknown. The current study firstly conducted a quantitative proteomic analysis to identify alterations of the cellular proteome under both hypo-osmotic and hyper-osmotic stress conditions. A total of 1405 proteins were identified and differentially accumulated proteins were analyzed, further functional annotations were performed using Gene Ontology and Kyoto Encyclopedia of Genes and Genomes. The results revealed that both hypo- and hyper-osmotic stresses have prominent impacts on the synthesis of proteins involving in multiple cellular functions. Further analyses of the differentially accumulated proteins suggested that the adaptation strategies T. halophilus applies to deal with hypo- and hyper-osmotic stress conditions may be distinct. Comparison of the differentially accumulated proteins in both transcriptomic and proteomic study indicated the existence of post-transcriptional modification during salinity adaptation of T. halophilus. The current study generated a proteomic atlas of differentially accumulated proteins under both hypo- and hyper-osmotic stress conditions, provided an overview of the molecular mechanism of osmotic acclimation of T. halophilus. SignificanceThe current study aimed to reveal how the moderately halophilic Tetragenococcus halophilus adapt to extracellular salinity stress, which is the first proteomic study analyzing the differences in proteome of Tetragenococcus halophilus between hypo- and hyper-osmotic stress to our knowledge. By analyzing the differences in the accumulating levels of the proteome via isobaric labeling-based quantitative proteomic study, we identified proteins with significantly different accumulation levels which may play important roles in the adaptation process to extracellular salinity stress. Examining the cellular functions of these proteins according to Gene Ontology and Kyoto Encyclopedia of Genes and Genomes, a draft view of how the bacterium act to acclimate to osmotic stress has been drawn. Further analysis revealing the differences between the transcriptome and proteome suggested that some proteins may undergo post-transcriptional regulation during acclimation process, which still remains unstudied and needs further investigations. The results of the current study can help researchers to gain insights and further reveal the halophilic mechanism of halophiles.

Hyperosmotic stress stimulates autophagy via polycystin-2.

Various intracellular mechanisms are activated in response to stress, leading to adaptation or death. Autophagy, an intracellular process that promotes lysosomal degradation of proteins, is an adaptive response to several types of stress. Osmotic stress occurs under both physiological and pathological conditions, provoking mechanical stress and activating various osmoadaptive mechanisms. Polycystin-2 (PC2), a membrane protein of the polycystin family, is a mechanical sensor capable of activating the cell signaling pathways required for cell adaptation and survival. Here we show that hyperosmotic stress provoked by treatment with hyperosmolar concentrations of sorbitol or mannitol induces autophagy in HeLa and HCT116 cell lines. In addition, we show that mTOR and AMPK, two stress sensor proteins involved modulating autophagy, are downregulated and upregulated, respectively, when cells are subjected to hyperosmotic stress. Finally, our findings show that PC2 is required to promote hyperosmotic stress-induced autophagy. Downregulation of PC2 prevents inhibition of hyperosmotic stress-induced mTOR pathway activation. In conclusion, our data provide new insight into the role of PC2 as a mechanosensor that modulates autophagy under hyperosmotic stress conditions.

Heterologous Expression of theCarrot Hsp17.7 gene Increased Growth, Cell Viability, and Protein Solubility in Transformed Yeast (Saccharomyces cerevisiae) under Heat, Cold, Acid, and Osmotic Stress Conditions.

In industrial fermentation of yeast (Saccharomyces cerevisiae), culture conditions are often modified from the optimal growth conditions of the cells to maintain large-scale cultures and/or to increase recombinant protein production. However, altered growth conditions can be stressful to yeast cells resulting in reduced cell growth and viability. In this study, a small heat shock protein gene from carrot (Daucus carota L.), Hsp17.7, was inserted into the yeast genome via homologous recombination to increase tolerance to stress conditions that can occur during industrial culture. A DNA construct, Translational elongation factor gene promoter-carrot Hsp17.7 gene-Phosphoribosyl-anthranilate isomerase gene (an auxotrophic marker), was generated by a series of PCRs and introduced into the chromosome IV of the yeast genome. Immunoblot analysis showed that carrot Hsp17.7 accumulated in the transformed yeast cell lines. Growth rates and cell viability of these cell lines were higher than control cell lines under heat, cold, acid, and hyperosmotic stress conditions. Soluble protein levels were higher in the transgenic cell lines than control cell lines under heat and cold conditions, suggesting the molecular chaperone function of the recombinant Hsp17.7. This study showed that a recombinant DNA construct containing a HSP gene from carrot was successfully expressed in yeast by homologous recombination and increased tolerances to abiotic stress conditions.

Environmental Stress and Aroma Production During Wine Fermentation

The sensory description of wine uses the widest range of descriptive terminology of all food products, reflecting the complex nature of a product whose character depends on the balance of hundreds of individual flavour-active compounds. There are many tools that can influence flavour profiles or wine styles, one of which is the choice of a specific yeast strain. Yeasts contribute to wine flavour by producing volatile metabolites with different flavour profiles. The impact of changing environmental conditions on the production of flavour compounds by yeast strains remains largely unexplored. This is the first study investigating the impact of two mild fermentation stresses, hyperosmotic and temperature stress, on aroma production in synthetic must by commercial Saccharomyces cerevisiae wine strains. Hyperosmotic stress was imposed by cultivation of the yeast for 21 days in the must containing either 0.3 or 0.5 M sorbitol. The transient temperature stresses were applied for 16 h at 8° or 37°C for either two or eight days after commencement of the fermentation. Greater glycerol and acetic acid levels were produced by most yeasts when only hyperosmotic stress was applied. Hyperosmotic and temperature stress conditions produced a limited number of significant changes to the profile of the esters, higher alcohols and volatile fatty acids. These changes differed significantly for each strain and stress treatment, suggesting that the fermentation conditions can significantly alter the aromatic profile of a wine, although these stress impacts cannot be predicted in general. The changes to the aromatic profile are specific to each individual wine yeast strain.

Alternative Oxidase Pathway Optimizes Photosynthesis During Osmotic and Temperature Stress by Regulating Cellular ROS, Malate Valve and Antioxidative Systems.

The present study reveals the importance of alternative oxidase (AOX) pathway in optimizing photosynthesis under osmotic and temperature stress conditions in the mesophyll protoplasts of Pisum sativum. The responses of photosynthesis and respiration were monitored at saturating light intensity of 1000 μmoles m–2 s–1 at 25°C under a range of sorbitol concentrations from 0.4 to 1.0 M to induce hyper-osmotic stress and by varying the temperature of the thermo-jacketed pre-incubation chamber from 25 to 10°C to impose sub-optimal temperature stress. Compared to controls (0.4 M sorbitol and 25°C), the mesophyll protoplasts showed remarkable decrease in NaHCO3-dependent O2 evolution (indicator of photosynthetic carbon assimilation), under both hyper-osmotic (1.0 M sorbitol) and sub-optimal temperature stress conditions (10°C), while the decrease in rates of respiratory O2 uptake were marginal. The capacity of AOX pathway increased significantly in parallel to increase in intracellular pyruvate and reactive oxygen species (ROS) levels under both hyper-osmotic stress and sub-optimal temperature stress under the background of saturating light. The ratio of redox couple (Malate/OAA) related to malate valve increased in contrast to the ratio of redox couple (GSH/GSSG) related to antioxidative system during hyper-osmotic stress. Further, the ratio of GSH/GSSG decreased in the presence of sub-optimal temperature, while the ratio of Malate/OAA showed no visible changes. Also, the redox ratios of pyridine nucleotides increased under hyper-osmotic (NADH/NAD) and sub-optimal temperature (NADPH/NADP) stresses, respectively. However, upon restriction of AOX pathway by using salicylhydroxamic acid (SHAM), the observed changes in NaHCO3-dependent O2 evolution, cellular ROS, redox ratios of Malate/OAA, NAD(P)H/NAD(P) and GSH/GSSG were further aggravated under stress conditions with concomitant modulations in NADP-MDH and antioxidant enzymes. Taken together, the results indicated the importance of AOX pathway in optimizing photosynthesis under both hyper-osmotic stress and sub-optimal temperatures. Regulation of ROS through redox couples related to malate valve and antioxidant system by AOX pathway to optimize photosynthesis under these stresses are discussed.

Stress Tolerance Profiling of a Collection of Extant Salt-Tolerant Rice Varieties and Transgenic Plants Overexpressing Abiotic Stress Tolerance Genes

Environmental stress tolerance is an important trait for crop improvement. In recent decades, numerous genes that confer tolerance to abiotic stress such as salinity were reported. However, the levels of salt tolerance differ greatly depending on growth conditions, and mechanisms underlying the complicated nature of stress tolerance are far from being fully understood. In this study, we investigated the profiles of stress tolerance of nine salt-tolerant rice varieties and transgenic rice lines carrying constitutively expressed genes that are potentially involved in salt tolerance, by evaluating their growth and viability under salt, heat, ionic and hyperosmotic stress conditions. Profiling of the extant varieties and selected chromosome segment substitution lines showed that salt tolerance in a greenhouse condition was more tightly correlated with ionic stress tolerance than osmotic stresses. In Nona Bokra, one of the most salt-tolerant varieties, the contribution of the previously identified sodium transporter HKT1;5 to salt tolerance was fairly limited. In addition, Nona Bokra exhibited high tolerance to all the stresses imposed. More surprisingly, comparative evaluation of 74 stress tolerance genes revealed that the most striking effect to enhance salt tolerance was conferred by overexpressing CYP94C2b, which promotes deactivation of jasmonate. In contrast, genes encoding ABA signaling factors conferred multiple stress tolerance. Genes conferring tolerance to both heat and hyperosmotic stresses were preferentially linked to functional categories related to heat shock proteins, scavenging of reactive oxygen species and Ca(2+) signaling. These comparative profiling data provide a new basis for understanding the ability of plants to grow under harsh environmental conditions.

Functional characterization and expression analysis of rice δ(1)-pyrroline-5-carboxylate dehydrogenase provide new insight into the regulation of proline and arginine catabolism.

While intracellular proline accumulation in response to various stress conditions has been investigated in great detail, the biochemistry and physiological relevance of proline degradation in plants is much less understood. Moreover, the second and last step in proline catabolism, the oxidation of δ1-pyrroline-5-carboxylic acid (P5C) to glutamate, is shared with arginine catabolism. Little information is available to date concerning the regulatory mechanisms coordinating these two pathways. Expression of the gene coding for P5C dehydrogenase was analyzed in rice by real-time PCR either following the exogenous supply of amino acids of the glutamate family, or under hyperosmotic stress conditions. The rice enzyme was heterologously expressed in E. coli, and the affinity-purified protein was thoroughly characterized with respect to structural and functional properties. A tetrameric oligomerization state was observed in size exclusion chromatography, which suggests a structure of the plant enzyme different from that shown for the bacterial P5C dehydrogenases structurally characterized to date. Kinetic analysis accounted for a preferential use of NAD+ as the electron acceptor. Cations were found to modulate enzyme activity, whereas anion effects were negligible. Several metal ions were inhibitory in the micromolar range. Interestingly, arginine also inhibited the enzyme at higher concentrations, with a mechanism of uncompetitive type with respect to P5C. This implies that millimolar levels of arginine would increase the affinity of P5C dehydrogenase toward its specific substrate. Results are discussed in view of the involvement of the enzyme in either proline or arginine catabolism.

Functional properties and structural characterization of rice δ(1)-pyrroline-5-carboxylate reductase.

The majority of plant species accumulate high intracellular levels of proline to cope with hyperosmotic stress conditions. Proline synthesis from glutamate is tightly regulated at both the transcriptional and the translational levels, yet little is known about the mechanisms for post-translational regulation of the enzymatic activities involved. The gene coding in rice (Oryza sativa L.) for δ1-pyrroline-5-carboxylate (P5C) reductase, the enzyme that catalyzes the second and final step in this pathway, was isolated and expressed in Escherichia coli. The structural and functional properties of the affinity-purified protein were characterized. As for most species, rice P5C reductase was able to use in vitro either NADH or NADPH as the electron donor. However, strikingly different effects of cations and anions were found depending on the pyridine nucleotide used, namely inhibition of NADH-dependent activity and stimulation of NADPH-dependent activity. Moreover, physiological concentrations of proline and NADP+ were strongly inhibitory for the NADH-dependent reaction, whereas the NADPH-dependent activity was mildly affected. Our results suggest that only NADPH may be used in vivo and that stress-dependent variations in ion homeostasis and NADPH/NADP+ ratio could modulate enzyme activity, being functional in promoting proline accumulation and potentially also adjusting NADPH consumption during the defense against hyperosmotic stress. The apparent molecular weight of the native protein observed in size exclusion chromatography indicated a high oligomerization state. We also report the first crystal structure of a plant P5C reductase at 3.40-Å resolution, showing a decameric quaternary assembly. Based on the structure, it was possible to identify dynamic structural differences among rice, human, and bacterial enzymes.

Identification of the correct form of the mis-annotated response regulator Rre1 from the cyanobacterium Synechocystis sp. PCC 6803.

Two-component systems have been extensively described in the control of gene expression in response to different environmental signals in the cyanobacterium Synechocystis sp. PCC 6803. The Hik34-Rre1 two-component system has been shown to regulate a set of genes under certain stress conditions. Some evidence indicates that another histidine kinase, probably Hik2, is acting upstream of Rre1 in the regulation of some genes in response to hyperosmotic and salt stress. In the present study, a mis-annotation of the Rre1 protein has been identified and the correct version has been functionally characterized in vitro. By using EMSA assays, we have demonstrated that phosphorylation of Rre1 by Hik2 increases the affinity of the response regulator for the adhA promoter region, a gene that has been demonstrated previously to be specifically regulated by the Hik34-Rre1 system. These results suggest that Hik2 might cooperate with Hik34 in the regulation of the adhA gene by transferring the phosphoryl group to Rre1 under salt and hyperosmotic stress conditions.

Stability of the osmoregulated promoter-derived proP mRNA is posttranscriptionally regulated by RNase III in Escherichia coli.

The enzymatic activity of Escherichia coli endo-RNase III determines the stability of a subgroup of mRNA species, including bdm, betT, and proU, whose protein products are associated with the cellular response to osmotic stress. Here, we report that the stability of proP mRNA, which encodes a transporter of osmoprotectants, is controlled by RNase III in response to osmotic stress. We observed that steady-state levels of proP mRNA and ProP protein are inversely correlated with cellular RNase III activity and, in turn, affect the proline uptake capacity of the cell. In vitro and in vivo analyses of proP mRNA revealed RNase III cleavage sites in a stem-loop within the 5' untranslated region present only in proP mRNA species synthesized from the osmoregulated P1 promoter. Introduction of nucleotide substitutions in the cleavage site identified inhibited the ribonucleolytic activity of RNase III on proP mRNA, increasing the steady-state levels and half-life of the mRNA. In addition, decreased RNase III activity coincided with a significant increase in both the half-life and abundance of proP mRNA under hyperosmotic stress conditions. Analysis of the RNA bound to RNase III via in vivo cross-linking and immunoprecipitation indicated that this phenomenon is related to the decreased RNA binding capacity of RNase III. Our findings suggest the existence of an RNase III-mediated osmoregulatory network that rapidly balances the expression levels of factors associated with the cellular response to osmotic stress in E. coli. Our results demonstrate that RNase III activity on proP mRNA degradation is downregulated in Escherichia coli cells under osmotic stress. In addition, we show that the downregulation of RNase III activity is associated with decreased RNA binding capacity of RNase III under hyperosmotic conditions. In particular, our findings demonstrate a link between osmotic stress and RNase III activity, underscoring the growing importance of posttranscriptional regulation in modulating rapid physiological adjustment to environmental changes.

Two tandem RNase III cleavage sites determine betT mRNA stability in response to osmotic stress in Escherichia coli.

While identifying genes regulated by ribonuclease III (RNase III) in Escherichia coli, we observed that steady-state levels of betT mRNA, which encodes a transporter mediating the influx of choline, are dependent on cellular concentrations of RNase III. In the present study, we also observed that steady-state levels of betT mRNA are dependent on RNase III activity upon exposure to osmotic stress, indicating the presence of cis-acting elements controlled by RNase III in betT mRNA. Primer extension analyses of betT mRNA revealed two tandem RNase III cleavage sites in its stem-loop region, which were biochemically confirmed via in vitro cleavage assays. Analyses of cleavage sites suggested the stochastic selection of cleavage sites by RNase III, and mutational analyses indicated that RNase III cleavage at either site individually is insufficient for efficient betT mRNA degradation. In addition, both the half-life and abundance of betT mRNA were significantly increased in association with decreased RNase III activity under hyper-osmotic stress conditions. Our findings demonstrate that betT mRNA stability is controlled by RNase III at the post-transcriptional level under conditions of osmotic stress.

Intracellular mobility and nuclear trafficking of the stress-activated kinase JNK1 are impeded by hyperosmotic stress

The c-Jun N-terminal kinases (JNKs) are a group of stress-activated protein kinases that regulate gene expression changes through specific phosphorylation of nuclear transcription factor substrates. To address the mechanisms underlying JNK nuclear entry, we employed a semi-intact cell system to demonstrate for the first time that JNK1 nuclear entry is dependent on the importin α2/β1 heterodimer and independent of importins α3, α4, β2, β3, 7 and 13. However, quantitative image analysis of JNK1 localization following exposure of cells to either arsenite or hyperosmotic stress did not indicate its nuclear accumulation. Extending our analyses to define the dynamics of nuclear trafficking of JNK1, we combined live cell imaging analyses with fluorescence recovery after photobleaching (FRAP) protocols. Subnuclear and subcytoplasmic bleaching protocols revealed the slowed movement of JNK1 in both regions in response to hyperosmotic stress. Strikingly, while movement into the nucleus of green fluorescent protein (GFP) or transport of a GFP-T-antigen fusion protein as estimated by initial rates and time to reach half-maximal recovery (t1/2) measures remained unaltered, hyperosmotic stress slowed the nuclear entry of GFP-JNK1. In contrast, arsenite exposure which did not alter the initial rates of nuclear accumulation of GFP, GFP-T-antigen or GFP-JNK1, decreased the t1/2 for nuclear accumulation of both GFP and GFP-JNK1. Thus, our results challenge the paradigm of increased nuclear localization of JNK broadly in response to all forms of stress-activation and are consistent with enhanced interactions of stress-activated JNK1 with scaffold and substrate proteins throughout the nucleus and the cytosol under conditions of hyperosmotic stress.

Rapid identification of potential drought tolerance genes from Solanum tuberosum by using a yeast functional screening method

Identification of major stress tolerance genes of a crop plant is important for the rapid development of its stress-tolerant cultivar. Here, we used a yeast functional screen method to identify potential drought-tolerance genes from a potato plant. A cDNA expression library was constructed from hyperosmotic stressed potato plants. The yeast transformants expressing different cDNAs were selected for their ability to survive in hyperosmotic stress conditions. The relative tolerances of the selected yeast transformants to multiple abiotic stresses were also studied. Specific potato cDNAs expressed in the tolerant yeast transformants were identified. Sixty-nine genes were found capable of enhancing hyperosmotic stress tolerance of yeast. Based on the relative tolerance data generated, 12 genes were selected, which could be most effective in imparting higher drought tolerance to potato with better survival in salt and high-temperature stresses. Orthologues of few genes identified here are previously known to increase osmotic stress tolerance of yeast and plants; however, specific studies are needed to confirm their role in the osmotic stress tolerance of potato.