Osmotic Stress-related Genes Research Articles

Sugarcane ScDREB2B-1 Confers Drought Stress Tolerance in Transgenic Nicotiana benthamiana by Regulating the ABA Signal, ROS Level and Stress-Related Gene Expression

The dehydration-responsive element-binding protein (DREB) is a subgroup member of the AP2/ERF family and actively participates in the response of plants to abiotic stress. Although DREB genes have been studied in a variety of plant species, there are few reports of DREB genes in sugarcane (Saccharum spp.). In this study, a novel full-length cDNA sequence of the ScDREB2B-1 gene was cloned from the Saccharum hybrid ROC22, whose encoding protein contained only one AP2-conserved domain and was clustered into the DREB (A-2) subgroup. The diverse promoter elements in the ScDREB2B-1 gene and the accumulated transcripts of its homologous gene (SsAP2/ERF-107) in S. spontaneum under drought stress suggest that the ScDREB2B-1 gene may play a role in drought response. In addition, reverse transcription quantitative PCR analysis showed that the expression level of the ScDREB2B-1 gene was upregulated in the root and leaf of ROC22 under polyethylene glycol, sodium chloride and abscisic acid (ABA) treatments. The yeast two-hybrid experiment demonstrated that ScDREB2B-1 had transcriptional self-activation activity. Compared with wild-type plants, the overexpression of the ScDREB2B-1 gene improved the drought tolerance of the transgenic Nicotiana benthamiana by activating the ABA pathway to enhance the expression of the ABA-responsive gene (NbNCED) and ABA content, regulate the intracellular reactive oxygen species (ROS) level (enhance the transcripts of ROS synthase-related gene NbRbohB and the activities of catalase, peroxidase and superoxide dismutase) and increase the relative water content, proline content and expression level of osmotic stress-related genes (NbERD and NbLEA). Collectively, our data indicate that ScDREB2B-1 is a stress-inducible and ABA-responsive transcription factor gene that responds to drought stress by regulating ABA signaling, ROS levels and stress-related gene expression. This study contributes to a better understanding of the biological function of ScDREB2B-1, which could serve as a foundation for future resistance breeding in sugarcane.

The rice phosphoinositide-specific phospholipase C3 is involved in responses to osmotic stresses via modulating ROS homeostasis

Four members of phosphoinositide-specific phospholipase C (PI-PLC) are predicted in rice genome. Although the involvement of OsPLC1 and OsPLC4 in the responses of rice to salt and drought stresses has been documented, the role of OsPLC3 in which, yet, is elusive. Here, we report that OsPLC3 was ubiquitously expressed in various tissues during the development of rice. The expression of YFP-tagged OsPLC3 was observed at the plasma membrane (PM), cytoplasm and nucleus of rice protoplasts, onion epidermal cells and tobacco leaves. The catalytic activity of OsPLC3 was measured using the thin-layer chromatography (TLC) method. The inhibition of OsPLC3 expression was detected in the treatments of NaCl and mannitol. Overexpression (OE) of OsPLC3 produced plants showing more sensitive to osmotic stresses when they were compared to the wild-type (HJ) and osplc3 mutants, the phenomena such as decreased plant fresh weight and increased water loss rate (WLR) were observed. Under the treatment of NaCl or mannitol, expressions of a subset osmotic stress-related genes were altered, in both OE and osplc3 mutant lines. In addition, the expressions and the enzyme activities of reactive oxygen species (ROS) scavengers were significantly decreased in OE lines, leading to over-accumulation of ROS together with less osmotic adjustment substances including proline, soluble sugars and soluble proteins in OE plants which caused the growth inhibition. Thus, our results suggested that, via modulating ROS homeostasis, OsPLC3 is involved in responses to the osmotic stress in rice.

Initial Transcriptomic Response and Adaption of Listeria monocytogenes to Desiccation on Food Grade Stainless Steel.

The foodborne pathogen Listeria monocytogenes survives exposure to a variety of stresses including desiccation in the food industry. Strand-specific RNA sequencing was applied to analyze changes in the transcriptomes of two strains of L. monocytogenes (Lm 568 and Lm 08-5578) during desiccation [15°C, 43% relative humidity (RH)] on food grade stainless steel surfaces over 48 h to simulate a weekend with no food production. Both strains showed similar survival during desiccation with a 1.8–2 Log CFU/cm2 reduction after 48 h. Analysis of differentially expressed (DE) genes (>twofold, adjusted p-value <0.05) revealed that the initial response to desiccation was established after 6 h and remained constant with few new genes being DE after 12, 24, and 48 h. A core of 81 up- and 73 down-regulated DE genes were identified as a shared, strain independent response to desiccation. Among common upregulated genes were energy and oxidative stress related genes e.g., qoxABCD (cytochrome aa3) pdhABC (pyruvate dehydrogenase complex) and mntABCH (manganese transporter). Common downregulated genes related to anaerobic growth, proteolysis and the two component systems lmo1172/lmo1173 and cheA/cheY, which are involved in cold growth and flagellin production, respectively. Both strains upregulated additional genes involved in combatting oxidative stress and reactive oxygen species (ROS), including sod (superoxide dismutase), kat (catalase), tpx (thiol peroxidase) and several thioredoxins including trxAB, lmo2390 and lmo2830. Osmotic stress related genes were also upregulated in both strains, including gbuABC (glycine betaine transporter) and several chaperones clpC, cspA, and groE. Significant strain differences were also detected with the food outbreak strain Lm 08-5578 differentially expressing 1.9 × more genes (726) compared to Lm 568 (410). Unique to Lm 08-5578 was a significant upregulation of the expression of the alternative transcription factor σB and its regulon. A number of long antisense transcripts (lasRNA) were upregulated during desiccation including anti0605, anti0936, anti1846, and anti0777, with the latter controlling flagellum biosynthesis and possibly the downregulation of motility genes observed in both strains. This exploration of the transcriptomes of desiccated L. monocytogenes provides further understanding of how this bacterium encounters and survives the stress faced when exposed to dry conditions in the food industry.

A Glycine max sodium/hydrogen exchanger enhances salt tolerance through maintaining higher Na+ efflux rate and K+/Na+ ratio in Arabidopsis

BackgroundSoybean (Glycine max (L.)) is one the most important oil-yielding cash crops. However, the soybean production has been seriously restricted by salinization. It is therefore crucial to identify salt tolerance-related genes and reveal molecular mechanisms underlying salt tolerance in soybean crops. A better understanding of how plants resist salt stress provides insights in improving existing soybean varieties as well as cultivating novel salt tolerant varieties. In this study, the biological function of GmNHX1, a NHX-like gene, and the molecular basis underlying GmNHX1-mediated salt stress resistance have been revealed.ResultsWe found that the transcription level of GmNHX1 was up-regulated under salt stress condition in soybean, reaching its peak at 24 h after salt treatment. By employing the virus-induced gene silencing technique (VIGS), we also found that soybean plants became more susceptible to salt stress after silencing GmNHX1 than wild-type and more silenced plants wilted than wild-type under salt treatment. Furthermore, Arabidopsis thaliana expressing GmNHX1 grew taller and generated more rosette leaves under salt stress condition compared to wild-type. Exogenous expression of GmNHX1 resulted in an increase of Na+ transportation to leaves along with a reduction of Na+ absorption in roots, and the consequent maintenance of a high K+/Na+ ratio under salt stress condition. GmNHX1-GFP-transformed onion bulb endothelium cells showed fluorescent pattern in which GFP fluorescence signals enriched in vacuolar membranes. Using the non-invasive micro-test technique (NMT), we found that the Na+ efflux rate of both wild-type and transformed plants after salt treatment were significantly higher than that of before salt treatment. Additionally, the Na+ efflux rate of transformed plants after salt treatment were significantly higher than that of wild-type. Meanwhile, the transcription levels of three osmotic stress-related genes, SKOR, SOS1 and AKT1 were all up-regulated in GmNHX1-expressing plants under salt stress condition.ConclusionVacuolar membrane-localized GmNHX1 enhances plant salt tolerance through maintaining a high K+/Na+ ratio along with inducing the expression of SKOR, SOS1 and AKT1. Our findings provide molecular insights on the roles of GmNHX1 and similar sodium/hydrogen exchangers in regulating salt tolerance.

Genome-Wide Identification of Cassava Serine/Arginine-Rich Proteins: Insights into Alternative Splicing of Pre-mRNAs and Response to Abiotic Stress.

Serine/arginine-rich (SR) proteins have an essential role in the splicing of pre-messenger RNA (pre-mRNA) in eukaryote. Pre-mRNA with introns can be alternatively spliced to generate multiple transcripts, thereby increasing adaptation to the external stress conditions in planta. However, pre-mRNA of SR proteins can also be alternatively spliced in different plant tissues and in response to diverse stress treatments, indicating that SR proteins might be involved in regulating plant development and adaptation to environmental changes. We identified and named 18 SR proteins in cassava and systematically studied their splicing and transcriptional changes under tissue-specific and abiotic stress conditions. Fifteen out of 18 SR genes showed alternative splicing in the tissues. 45 transcripts were found from 18 SR genes under normal conditions, whereas 55 transcripts were identified, and 21 transcripts were alternate spliced in some SR genes under salt stress, suggesting that SR proteins might participate in the plant adaptation to salt stress. We then found that overexpression of MeSR34 in Arabidopsis enhanced the tolerance to salt stress through maintaining reactive oxygen species homeostasis and increasing the expression of calcineurin B-like proteins (CBL)-CBL-interacting protein kinases and osmotic stress-related genes. Therefore, our findings highlight the critical role of cassava SR proteins as regulators of RNA splicing and salt tolerance in planta.

CaDHN5, a Dehydrin Gene from Pepper, Plays an Important Role in Salt and Osmotic Stress Responses.

Dehydrins (DHNs), as a sub-family of group two late embryogenesis-abundant (LEA) proteins, have attracted considerable interest owing to their functions in enhancing abiotic stress tolerance in plants. Our previous study showed that the expression of CaDHN5 (a dehydrin gene from pepper) is strongly induced by salt and osmotic stresses, but its function was not clear. To understand the function of CaDHN5 in the abiotic stress responses, we produced pepper (Capsicum annuum L.) plants, in which CaDHN5 expression was down-regulated using VIGS (Virus-induced Gene Silencing), and transgenic Arabidopsis plants overexpressing CaDHN5. We found that knock-down of CaDHN5 suppressed the expression of manganese superoxide dismutase (MnSOD) and peroxidase (POD) genes. These changes caused more reactive oxygen species accumulation in the VIGS lines than control pepper plants under stress conditions. CaDHN5-overexpressing plants exhibited enhanced tolerance to salt and osmotic stresses as compared to the wild type and also showed increased expression of salt and osmotic stress-related genes. Interestingly, our results showed that many salt-related genes were upregulated in our transgenic Arabidopsis lines under salt or osmotic stress. Taken together, our results suggest that CaDHN5 functions as a positive regulator in the salt and osmotic stress signaling pathways.

ThPP1 gene, encodes an inorganic pyrophosphatase in Thellungiella halophila, enhanced the tolerance of the transgenic rice to alkali stress.

An inorganic pyrophosphorylase gene, ThPP1 , modulated the accumulations of phosphate and osmolytes by up-regulating the differentially expression genes, thus enhancing the tolerance of the transgenic rice to alkali stress (AS). Inorganic pyrophosphorylase is essential in catalyzing the hydrolysis of pyrophosphate to inorganic phosphate during plant growth. Here, we report the changes of physiological osmolytes and differentially expression genes in the transgenic rice overexpressing a soluble inorganic pyrophosphatase gene ThPP1 of Thellungiella halophila in response to AS. Analyses showed that the ThPP1 gene was a PPase family I member which is located to the cytoplasm. Data showed that the transgenic lines revealed an enhanced tolerance to AS compared to the wild type, and effectively increased the accumulations of inorganic phosphate and organic small molecules starch, sucrose, proline and chlorophyll, and maintained the balance of osmotic potential by modulating the ratio of Na+/K+ in plant cells. Under AS, total 379 of differentially expression genes were up-regulated in the leaves of the transgenic line compared with control, and the enhanced tolerance of the transgenic rice to the AS seemed to be associated with the up-regulations of the osmotic stress-related genes such as the L-type lectin-domain containing receptor kinase (L-type LecRK), the cation/H+ antiporter gene and the vacuolar cation/proton exchanger 1 gene (CAX1), which conferred the involvements in the biosynthesis and metabolic pathways. Protein interaction showed that the ThPP1 protein specifically interacted with a 16# target partner of the photosystem II light-harvesting-Chl-binding protein. This study suggested that the ThPP1 gene plays an important regulatory role in conferring the tolerance of the transgenic rice to AS, and is an effective candidate in molecular breeding for crop cultivation of the alkali tolerance.

Transcriptional and translational regulation by RNA thermometers, riboswitches and the sRNA DsrA in Escherichia coli O157:H7 Sakai under combined cold and osmotic stress adaptation.

The enteric pathogen Escherichia coli O157:H7 Sakai (EHEC) is able to grow at lower temperatures compared to commensal E.coli Growth at environmental conditions displays complex challenges different to those in a host. EHEC was grown at 37°C and at 14°C with 4% NaCl, a combination of cold and osmotic stress as present in the food chain. Comparison of RNAseq and RIBOseq data provided a snap shot of ongoing transcription and translation, differentiating transcriptional and post-transcriptional gene regulation, respectively. Indeed, cold and osmotic stress related genes are simultaneously regulated at both levels, but translational regulation clearly dominates. Special emphasis was given to genes regulated by RNA secondary structures in their 5'UTRs, such as RNA thermometers and riboswitches, or genes controlled by small RNAs encoded in trans The results reveal large differences in gene expression between short-time shock compared to adaptation in combined cold and osmotic stress. Whereas the majority of cold shock proteins, such as CspA, are translationally downregulated after adaptation, many osmotic stress genes are still significantly upregulated mainly translationally, but several also transcriptionally.

Differential gene expression profiling of Listeria monocytogenes in Cacciatore and Felino salami to reveal potential stress resistance biomarkers

The current study reports a) the in situ transcriptional profiles of Listeria monocytogenes in response to fermented sausage stress and b) an approach in which in situ RT-qPCR data have been combined with advanced statistical techniques to discover potential stress resistance or cell viability biomarkers. Gene expression profiling of the pathogen has been investigated using RT-qPCR to understand how L. monocytogenes responds to the conditions encountered during the fermentation and ripening of sausages. A cocktail of five L. monocytogenes strains was inoculated into the batter of Cacciatore and Felino sausages. The RT-qPCR data showed that the acidic and osmotic stress-related genes were up-regulated. The transcripts of the lmo0669 gene increased during the fermentation and ripening of Cacciatore, whereas gbuA and lmo1421 were up-regulated during the ripening of Felino and Cacciatore, respectively. sigB expression was induced in both sausages throughout the whole process. Finally, the virulence-related gene prfA was down-regulated during the fermentation of Cacciatore. The multivariate gene expression profiling analysis suggested that sigB and lmo1421 or sigB and gbuA could be used as different types of stress resistance biomarkers to track, for example, stress resistance or cell viability in fermented sausages with short (Cacciatore) or long (Felino) maturation times, respectively.

Rewiring global regulator cAMP receptor protein (CRP) to improve E. coli tolerance towards low pH

Bioprocesses such as production of organic acids or acid hydrolysis of bioresources during biofuel production often suffer limitations due to microbial sensitivity under acidic conditions. Approaches for improving the acid tolerance of these microbes have mainly focused on using metabolic engineering tools. Here, we tried to improve strain acidic tolerance from its transcription level, i.e. we adopted error-prone PCR method to engineer global regulator cAMP receptor protein (CRP) of Escherichia coli to improve its performance at low pH. The best mutant AcM1 was identified from random mutagenesis libraries based on its growth performance. AcM1 almost doubled (0.113h−1) the growth rate of the control (0.062h−1) at pH 4.24. It also demonstrated better thermotolerance than the control at 48°C, whose growth was completely inhibited at this temperature. Quantitative real time reverse transcription PCR results revealed a stress response overlap among low pH stress-, oxidative stress- and osmotic stress-related genes. The chief enzyme responsible for cell acid tolerance, glutamate decarboxylase, demonstrated over twofold activity in AcM1 compared to the control. Differential binding properties of AcM1 mutant CRP with Class-I, II, and III CRP-dependent promoters suggested that modifications to native CRP may lead to transcription profile changes. Hence, we believe that transcriptional engineering of global regulator CRP can provide a new strain engineering alternative for E. coli.

RhNAC3, a stress‐associated NAC transcription factor, has a role in dehydration tolerance through regulating osmotic stress‐related genes in rose petals

Petal cell expansion depends on cell wall metabolism, changes in cell turgor pressure and restructuring of the cytoskeleton, and recovery ability of petal cell expansion is defined as an indicator of dehydration tolerance in flowers. We previously reported that RhNAC2, a development-related NAC domain transcription factor, confers dehydration tolerance through regulating cell wall-related genes in rose petals. Here, we identify RhNAC3, a novel rose SNAC gene, and its expression in petals induced by dehydration, wounding, exogenous ethylene and abscisic acid (ABA). Expression studies in Arabidopsis protoplasts and yeast show that RhNAC3 has transactivation activity along its full length and in the carboxyl-terminal domain. Silencing RhNAC3 in rose petals by virus-induced gene silencing (VIGS) significantly decreased the cell expansion of rose petals under rehydration conditions. In total, 24 of 27 osmotic stress-related genes were down-regulated in RhNAC3-silenced rose petals, while only 4 of 22 cell expansion-related genes were down-regulated. Overexpression of RhNAC3 in Arabidopsis gave improved drought tolerance, with lower water loss of leaves in transgenic plants. Arabidopsis ATH1 microarray analysis showed that RhNAC3 regulated the expression of stress-responsive genes in overexpressing lines, and further analysis revealed that most of the RhNAC3-up-regulated genes were involved in the response to osmotic stress. Comparative analysis revealed that different transcription regulation existed between RhNAC3 and RhNAC2. Taken together, these data indicate that RhNAC3, as a positive regulator, confers dehydration tolerance of rose petals mainly through regulating osmotic adjustment-associated genes.

Pyrosequencing-Based Analysis of the Bacterial Community in Korean Traditional Seafood, Ojingeo Jeotgal

Jeotgal fermentation is dependent upon a diverse microbial community, although a detailed understanding of its microbial composition is limited to a relatively small number of jeotgal. Pyrosequencing-based bacterial community analysis was performed in fermented squid, ojingeo jeotgal. Leuconostoc was identified as the predominant bacterial genus, with Bacillus and Staphylococcus also accounting for a large proportion of the bacterial community. Phylogenetic analysis with 16S rRNA genes of Leuconostoc type species indicated that L. citreum- and L. holzapfelii-like strains could be the major Leuconostoc strains in jeotgal. High concentrations of NaCl were thought to be an important factor determining the makeup of the bacterial community in the fermented squid; however, a genomic survey with osmotic stress-related genes suggests the existence of more complex factors selecting the dominant bacterial species in fermented squid.

Genetic Determinants forn-Butanol Tolerance in Evolved Escherichia coli Mutants: Cross Adaptation and Antagonistic Pleiotropy betweenn-Butanol and Other Stressors

Cross-tolerance and antagonistic pleiotropy have been observed between different complex phenotypes in microbial systems. These relationships between adaptive landscapes are important for the design of industrially relevant strains, which are generally subjected to multiple stressors. In our previous work, we evolved Escherichia coli for enhanced tolerance to the biofuel n-butanol and discovered a molecular mechanism of n-butanol tolerance that also conferred tolerance to the cationic antimicrobial peptide polymyxin B in one specific lineage (green fluorescent protein [GFP] labeled) in the evolved population. In this work, we aim to identify additional mechanisms of n-butanol tolerance in an independent lineage (yellow fluorescent protein [YFP] labeled) from the same evolved population and to further explore potential cross-tolerance and antagonistic pleiotropy between n-butanol tolerance and other industrially relevant stressors. Analysis of the transcriptome data of the YFP-labeled mutants allowed us to discover additional membrane-related and osmotic stress-related genes that confer n-butanol tolerance in E. coli. Interestingly, the n-butanol resistance mechanisms conferred by the membrane-related genes appear to be specific to n-butanol and are in many cases antagonistic with isobutanol and ethanol. Furthermore, the YFP-labeled mutants showed cross-tolerance between n-butanol and osmotic stress, while the GFP-labeled mutants showed antagonistic pleiotropy between n-butanol and osmotic stress tolerance.

Degradation of NFAT5, a Transcriptional Regulator of Osmotic Stress-related Genes, Is a Critical Event for Doxorubicin-induced Cytotoxicity in Cardiac Myocytes

Nuclear factor-activated T cell 5 (NFAT5), a novel member of the NFAT family of proteins, was originally identified as a transcriptional factor responsible for adaptation to hyperosmotic stress. Though NFAT5 is ubiquitously expressed, the biological functions of NFAT5 remain to be clarified, especially in the tissues that are not exposed to hypertonicity, including hearts. In the present study, we focused on the cardioprotective roles of NFAT5 against the cardiotoxic anti-tumor agent doxorubicin (Dox). In cultured cardiomyocytes, transcripts of the hypertonicity-inducible genes, such as taurine transporter (TauT) and sodium/myo-inositol transporter, were down-regulated by Dox. Interestingly, NFAT5 protein, but not mRNA, was decreased in cardiomyocytes exposed to Dox. Treatment of proteasome inhibitors, MG-132 or proteasome-specific inhibitor 1, prevented the Dox-mediated decrease of NFAT5 protein. Further, ubiquitin-conjugated NFAT5 was not detected in cultured cardiomyocytes treated with MG-132 and/or Dox, as assessed by immunoprecipitation assay, suggesting Dox-induced degradation through ubiquitin-independent proteasome pathway. Importantly, inhibition of NFAT5 with overexpression of dominant-negative NFAT5 decreased cell viability and increased creatine kinase leakage into culture medium. Consistently, small interfering RNA targeting NFAT5 gene enhanced myocyte death. These findings suggest that Dox promoted the degradation of NFAT5 protein, reducing cell viability in cardiomyocytes. This is the first demonstration that NFAT5 is a positive regulator of cardiomyocyte survival.

Expression of osmotic stress-related genes in tissues of normal and hyposmotic rats.

TonEBP is a transcription factor that, when activated by hypertonicity, increases transcription of genes, including those involved in organic osmolyte accumulation. Surprisingly, it is expressed in virtually all tissues, including many never normally exposed to hypertonicity. We measured TonEBP mRNA (real-time PCR) and protein (Western blot analysis) in tissues of control (plasma osmolality 294 +/- 1 mosmol/kgH2O) and hyposmotic (dDAVP infusion plus water loading for 3 days, 241 +/- 2 mosmol/kgH2O) rats to test whether the ubiquitous expression of TonEBP mRNA is osmotically regulated around the normal plasma osmolality. TonEBP protein is reduced by hyposmolality in thymus and liver, but not in brain, and is not detected in heart and skeletal muscle. TonEBP mRNA decreases in brain and liver but is unchanged in other tissues. There are no general changes in mRNA of TonEBP-mediated genes: aldose reductase (AR) does not change in any tissue, betaine transporter (BGT1) decreases only in liver, taurine transporter (TauT) only in brain and thymus, and inositol transporter (SMIT) only in skeletal muscle and liver. Heat shock protein (Hsp)70-1 and Hsp70-2 mRNA increase greatly in most tissues, which cannot be attributed to decreased TonEBP activity. The conclusions are as follows: 1) TonEBP protein or mRNA expression is reduced by hyposmolality in thymus, liver, and brain. 2) TonEBP protein and mRNA expression are differentially regulated in some tissues. 3) Although AR, SMIT, BGT1, and TauT are regulated by TonEBP in renal medullary cells, other sources of regulation may predominate in other tissues. 4) TonEBP abundance and activity are regulated by factors other than tonicity in some tissues.