Halotolerant Pseudomonas koreensis S4T10 mitigate salt and drought stress in Arabidopsis thaliana.

Salt and drought are documented among the most detrimental and persistent abiotic stresses for crop production. Here, we investigated the impact of Pseudomonas koreensis strain S4T10 on plant performance under salt and drought stress. Arabidopsis thaliana Col-0 wild type and atnced3 mutant plants were inoculated with P. koreensis or tap water and exposed to NaCl (100 mM) for five days and drought stress by withholding water for seven days. P. koreensis significantly enhanced plant biomass and photosynthetic pigments under salt and drought stress conditions. Moreover, P. koreensis activated the antioxidant defence by modulating glutathione (GSH), superoxide dismutase (SOD), peroxidase (POD), and polyphenol oxidase (PPO) activities to scavenge the reactive oxygen species produced due to the stress. In addition, the application of P. koreensis upregulated the expression of genes associated with antioxidant responses, such as AtCAT1, AtCAT3, and AtSOD. Similarly, genes linked to salt stress, such as AtSOS1, AtSOS2, AtSOS3, AtNHX1, and AtHKT1, were also upregulated, affirming the positive role of P. koreensis S4T10 in streamlining the cellular influx and efflux transport systems during salt stress. Likewise, the PGPB inoculation was observed to regulate the expression of drought-responsive genes AtDREB2A, AtDREB2B, and ABA-responsive genes AtAO3, AtABA3 indicating that S4T10 enhanced drought tolerance via modulation of the ABA pathway. The results of this study affirm that P. koreensis S4T10 could be further developed as a biofertilizer to mitigate salt and drought stress at the same time.

In Silico Characterisation of the Aedes aegypti Gustatory Receptors.

Aedes aegypti, also known as the dengue mosquito or the yellow fewer mosquito, is the vector of dengue, chikungunya, Zika, Mayaro and yellow fever viruses. The A. aegypti genome contains an array of gustatory receptor (GR) proteins that are related to the recognition of taste. In this study, we performed in silico molecular characterization of all 72 A. aegypti GRs reported in the latest version of A. aegypti genome AaegL5. Phylogenetic analysis classified the receptors into three major clads. Multiple GRs were found to encode multiple transcripts. Physicochemical attributes such as the aliphatic index, hydropathicity index and isoelectric point indicated that A. aegypti gustatory receptors are highly stable and are tailored to perform under a variety of cellular environments. Analysis for subcellular localization indicated that all the GRs are located either in the extracellular matrix or the plasma membrane. Results also indicated that the GRs are distributed mainly on chromosomes 2 and 3, which house 22 and 49 GRs, respectively, whereas chromosome 1 houses only one GR. NCBI-CDD analysis showed the presence of a highly conserved 7tm_7 chemosensory receptor protein superfamily that includes gustatory and odorant receptors from insect species Anopheles gambiae and Drosophila melanogaster. Further, three significantly enriched ungapped motifs in the protein sequence of all 72 A. aegypti gustatory receptors were found. High-quality 3D models for the tertiary structures were predicted with significantly higher confidence, along with ligand-binding residues. Prediction of S-nitrosylation sites indicated the presence of target cysteines in all the GRs with close proximity to the ligand-bindings sites within the 3D structure of the receptors. In addition, two highly conserved motifs inside the GR proteins were discovered that house a tyrosine (Y) and a cysteine (C) residue which may serve as targets for NO-mediated tyrosine nitration and S-nitrosylation, respectively. This study will help devise strategies for functional genomic studies of these important receptor molecules in A. aegypti and other mosquito species through in vitro and in vivo studies.

Enhanced Resistance of atnigr1 against Pseudomonas syringae pv. tomato Suggests Negative Regulation of Plant Basal Defense and Systemic Acquired Resistance by AtNIGR1 Encoding NAD(P)-Binding Rossmann-Fold in Arabidopsis thaliana.

Nitric oxide (NO) regulates several biological and physiological processes in plants. This study investigated the role of Arabidopsis thaliana Negative Immune and Growth Regulator 1 (AtNIGR1), encoding an NAD(P)-binding Rossmann-fold superfamily, in the growth and immunity of Arabidopsis thaliana. AtNIGR1 was pooled from the CySNO transcriptome as a NO-responsive gene. Seeds of the knockout (atnigr1) and overexpression plants were evaluated for their response to oxidative [(hydrogen peroxide (H2O2) and methyl viologen (MV)] or nitro-oxidative [(S-nitroso-L-cysteine (CySNO) and S-nitroso glutathione (GSNO)] stress. Results showed that the root and shoot growth of atnigr1 (KO) and AtNIGR1 (OE) exhibited differential phenotypic responses under oxidative and nitro-oxidative stress and normal growth conditions. To investigate the role of the target gene in plant immunity, the biotrophic bacterial pathogen Pseudomonas syringae pv. tomato DC3000 virulent (Pst DC3000 vir) was used to assess the basal defense, while the Pst DC3000 avirulent (avrB) strain was used to investigate R-gene-mediated resistance and systemic acquired resistance (SAR). Data revealed that AtNIGR1 negatively regulated basal defense, R-gene-mediated resistance, and SAR. Furthermore, the Arabidopsis eFP browser indicated that the expression of AtNIGR1 is detected in several plant organs, with the highest expression observed in germinating seeds. All results put together suggest that AtNIGR1 could be involved in plant growth, as well as basal defense and SAR, in response to bacterial pathogens in Arabidopsis.

Prognostic performance of the REDS score, SOFA score, NEWS2 score, and the red-flag, NICE high-risk, and SIRS criteria to predict survival at 180 days, in emergency department patients admitted with suspected sepsis - An observational cohort study.

Background: Patients admitted to hospital with sepsis are at persistent risk of poor outcome after discharge. Many tools are available to risk-stratify sepsis patients for in-hospital mortality. This study aimed to identify the best risk-stratification tool to prognosticate outcome 180 days after admission via the emergency department (ED) with suspected sepsis. Methods: A retrospective observational cohort study was performed of adult ED patients who were admitted after receiving intravenous antibiotics for the treatment of a suspected sepsis, between 1st March and 31st August 2019. The Risk-stratification of ED suspected Sepsis (REDS) score, SOFA score, Red-flag sepsis criteria met, NICE high-risk criteria met, the NEWS2 score and the SIRS criteria, were calculated for each patient. Death and survival at 180 days were noted. Patients were stratified in to high and low-risk groups as per accepted criteria for each risk-stratification tool. Kaplan-Meier curves were plotted for each tool and the log-rank test performed. The tools were compared using Cox-proportional hazard regression (CPHR). The tools were studied further in those without the following specified co-morbidities: Dementia, malignancy, Rockwood Frailty score of 6 or more, long-term oxygen therapy and previous do-not-resuscitate orders. Results: Of the 1,057 patients studied 146 (13.8%) died at hospital discharge and 284 were known to have died within 180 days. Overall survival proportion was 74.4% at 180 days and 8.6% of the population was censored before 180 days. Only the REDS and SOFA scores identified less than 50% of the population as high-risk. All tools except the SIRS criteria, prognosticated for outcome at 180 days; Log-rank tests between high and low-risk groups were: REDS score p < 0.0001, SOFA score p < 0.0001, Red-flag criteria p = 0.001, NICE high-risk criteria p = 0.0001, NEWS2 score p = 0.003 and SIRS criteria p = 0.98. On CPHR, the REDS [Hazard ratio (HR) 2.54 (1.92-3.35)] and SOFA [HR 1.58 (1.24-2.03)] scores out-performed the other risk-stratification tools. In patients without the specified co-morbidities, only the REDS score and the SOFA score risk-stratified for outcome at 180 days. Conclusion: In this study, all the risk-stratification tools studied were found to prognosticate for outcome at 180 days, except the SIRS criteria. The REDS and SOFA scores outperformed the other tools.

Phytohormonal Regulation Through Protein S-Nitrosylation Under Stress.

The liaison between Nitric oxide (NO) and phytohormones regulates a myriad of physiological processes at the cellular level. The interaction between NO and phytohormones is mainly influenced by NO-mediated post-translational modifications (PTMs) under basal as well as induced conditions. Protein S-nitrosylation is the most prominent and widely studied PTM among others. It is the selective but reversible redox-based covalent addition of a NO moiety to the sulfhydryl group of cysteine (Cys) molecule(s) on a target protein to form S-nitrosothiols. This process may involve either direct S-nitrosylation or indirect S-nitrosylation followed by transfer of NO group from one thiol to another (transnitrosylation). During S-nitrosylation, NO can directly target Cys residue (s) of key genes involved in hormone signaling thereby regulating their function. The phytohormones regulated by NO in this manner includes abscisic acid, auxin, gibberellic acid, cytokinin, ethylene, salicylic acid, jasmonic acid, brassinosteroid, and strigolactone during various metabolic and physiological conditions and environmental stress responses. S-nitrosylation of key proteins involved in the phytohormonal network occurs during their synthesis, degradation, or signaling roles depending upon the response required to maintain cellular homeostasis. This review presents the interaction between NO and phytohormones and the role of the canonical NO-mediated post-translational modification particularly, S-nitrosylation of key proteins involved in the phytohormonal networks under biotic and abiotic stresses.

The Prevalence of Potential Drug-Drug Interactions in CKD-A Retrospective Observational Study of Cerrahpasa Nephrology Unit.

Background and Objectives: Chronic kidney disease (CKD) is usually linked with polypharmacy and patients are invariably at risk of complex medication regimens. The present study was designed to estimate the potential drug-drug interactions (pDDIs) through the prescription patterns provided to patients of the Nephrology Transplant Unit of Cerrahpasa Medical Faculty patients. Materials and Methods: 96 patients were included in the study. pDDIs among every combination of the prescribed drug were analyzed using the Thomson Reuters Micromedex. Results: We found 149 pDDIs making 2.16 interactions per prescription with incidence rates of 69.7%. Approximately 4.1% of interactions were of significant severity, 75.1% moderate severity, and 20.8% were classified as minor pDDIs. The most frequent interactions were found between iron and aluminum, calcium or magnesium-containing products (21.37%), calcium channel blockers and beta-blockers (8.96%); and aspirin and aluminum, calcium, or magnesium-containing products (7.58%). We identified 42 drug pairs with probability of clinical significance. The most commonly reported clinical outcomes of the pDDIs were hypo- or hypertension (39.24%), decreased drug efficacy (24.05%), and arrhythmia (9.49%). Aluminum, calcium, or magnesium-containing drug products (33.10%) constituted the primary class of drugs involved in interactions. Conclusions: This study showed pharmacodynamics (49%), pharmacokinetics (42.94%) interactions, polypharmacy and gender as determinant of pDDIs. A comprehensive multicenter research is required to decrease the morbidity and ease the state burden.

60Co-γ Radiation Alters Developmental Stages of Zeugodacus cucurbitae (Diptera: Tephritidae) Through Apoptosis Pathways Gene Expression.

Radiation is considered as a promising insect pest control strategy for minimizing postharvest yield losses. Among various techniques, irradiation is a method of choice as it induces lethal biochemical or molecular changes that cause a downstream cascade of abrupt physiological abnormalities at the cellular level. In this study, we evaluated the effect of 60Co-γ radiation on various developmental stages of Zeugodacus cucurbitae Coquillett and subsequent carry-over effects on the progeny. For this purpose, we treated eggs with 30- and 50-Gy radiation doses of 60Co-γ. We found that radiation significantly affected cellular antioxidants, insect morphology, and gene expression profiles. Our results indicate that in response to various doses of irradiation reactive oxygen species, catalase, peroxidase, and superoxide dismutase activities were increased along with a significant increase in the malondialdehyde (MDA) content. We observed higher mortality rates during the pupal stage of the insects that hatched from irradiated eggs (50 Gy). Furthermore, the life span of the adults was reduced in response to 50 Gy radiation. The negative effects carried over to the next generation were marked by significantly lower fecundity in the F1 generation of the irradiation groups as compared to control. The radiation induced morphological abnormalities at the pupal, as well as the adult, stages. Furthermore, variations in the gene expression following irradiation are discussed. Taken together, our results signify the utility of 60Co-γ radiation for fruit fly postharvest management.

Perturbations in nitric oxide homeostasis promote Arabidopsis disease susceptibility towards Phytophthora parasitica.

Phytophthora species can infect hundreds of different plants, including many important crops, causing a number of agriculturally relevant diseases. A key feature of attempted pathogen infection is the rapid production of the redox active molecule nitric oxide (NO). However, the potential role(s) of NO in plant resistance against Phytophthora is relatively unexplored. Here we show that the level of NO accumulation is crucial for basal resistance in Arabidopsis against Phytophthora parasitica. Counterintuitively, both relatively low or relatively high NO accumulation leads to reduced resistance against P. parasitica. S-nitrosylation, the addition of a NO group to a protein cysteine thiol to form an S-nitrosothiol, is an important route for NO bioactivity and this process is regulated predominantly by S-nitrosoglutathione reductase 1 (GSNOR1). Loss-of-function mutations in GSNOR1 disable both salicylic acid accumulation and associated signalling, and also the production of reactive oxygen species, leading to susceptibility towards P. parasitica. Significantly, we also demonstrate that secreted proteins from P. parasitica can inhibit Arabidopsis GSNOR1 activity.

A Novel DUF569 Gene Is a Positive Regulator of the Drought Stress Response in Arabidopsis.

In the last two decades, global environmental change has increased abiotic stress on plants and severely affected crops. For example, drought stress is a serious abiotic stress that rapidly and substantially alters the morphological, physiological, and molecular responses of plants. In Arabidopsis, several drought-responsive genes have been identified; however, the underlying molecular mechanism of drought tolerance in plants remains largely unclear. Here, we report that the "domain of unknown function" novel gene DUF569 (AT1G69890) positively regulates drought stress in Arabidopsis. The Arabidopsis loss-of-function mutant atduf569 showed significant sensitivity to drought stress, i.e., severe wilting at the rosette-leaf stage after water was withheld for 3 days. Importantly, the mutant plant did not recover after rewatering, unlike wild-type (WT) plants. In addition, atduf569 plants showed significantly lower abscisic acid accumulation under optimal and drought-stress conditions, as well as significantly higher electrolyte leakage when compared with WT Col-0 plants. Spectrophotometric analyses also indicated a significantly lower accumulation of polyphenols, flavonoids, carotenoids, and chlorophylls in atduf569 mutant plants. Overall, our results suggest that novel DUF569 is a positive regulator of the response to drought in Arabidopsis.

Treatment variables associated with outcome in emergency department patients with suspected sepsis.

BACKGROUND: Early treatment is advocated in the management of patients with suspected sepsis in the emergency department (ED). We sought to understand the association between the ED treatments and outcome in patients admitted with suspected sepsis. The treatments studied were: (i) the time to antibiotics, where time zero is the time the patient was booked in which is also the triage time; (ii) the volume of intravenous fluid (IVF); (iii) mean arterial pressure (MAP) after 2000 ml of IVF and (iv) the final MAP in the ED. METHODS: We performed a retrospective analysis of the ED database of patients aged ≥ 18 year who met two SIRS criteria or one red flag sepsis criteria on arrival, received intravenous antibiotics for a suspected infection and admitted between 8th February 2016 and 31st August 2017. The primary outcome measure was all-cause in-hospital mortality. The four treatments stated above were controlled for severity of illness and subject to multivariate logistic regression and Cox proportional-hazard regression to identify independent predictors of mortality. RESULTS: Of the 2,066 patients studied 272 (13.2%) died in hospital. The median time to antibiotics was 48 (interquartile range 30-82) minutes. The time to antibiotics was an independent predictor of mortality only in those who developed refractory hypotension (RH); antibiotics administered more than 55 mins after arrival was associated with an odds ratio (OR) for mortality of 2.75 [95% confidence interval (CI) 1.22-6.14]. The number-needed-to-treat was 4. IVF > 2000 ml (95% CI > 500- > 2100), except in RH, and a MAP ≤ 66 mmHg after 2000 ml of IVF were also independent predictors of mortality. The OR for mortality of IVF > 2,000 ml in non-RH was 1.80 (95% CI 1.15-2.82); Number-needed-to-harm was 14. The OR for morality for a MAP ≤ 66 mmHg after 2000 ml of IVF was 3.42 (95% CI 2.10-5.57). A final MAP < 75 mmHg in the ED was associated with, but not an independent predictor of mortality. An initial systolic blood pressure of < 100 mmHg has a sensitivity of 63.3% and specificity of 88.4% for the development of RH. CONCLUSION: In this study, antibiotics were found to be time-critical in RH. Intravenous fluids > 2000 ml (except in RH) and a MAP ≤ 66 mmHg after 2000 ml of IVF were also independent predictors of mortality.

Functional Insight of Nitric-Oxide Induced DUF Genes in Arabidopsis thaliana.

Advances in next-generation sequencing technologies facilitate the study of plant molecular functions in detail and with precision. Plant genome and proteome databases are continually being updated with large transcriptomic or genomic datasets. With the ever-increasing amount of sequencing data, several thousands of genes or proteins in public databases remain uncharacterized, and their domain functions are largely unknown. Such proteins contain domains of unknown function (DUF). In the present study, we identified 231 upregulated and 206 downregulated DUF genes from the available RNA-Seq-based transcriptome profiling datasets of Arabidopsis leaves exposed to a nitric oxide donor, S-nitroso-L-cysteine (CysNO). In addition, we performed extensive in silico and biological experiments to determine the potential functions of AtDUF569 and to elucidate its role in plant growth, development, and defense. We validated the expression pattern of the most upregulated and the most downregulated DUF genes from the transcriptomic data. In addition, a loss-of AtDUF569 function mutant was evaluated for growth, development, and defense against biotic and abiotic stresses. According to the results of the study, AtDUF569 negatively regulates biotic stress responses and differentially regulates plant growth under nitro-oxidative stress conditions.

Spinal Cord Stimulator Paddle Lead Surgery Complicated by Cerebrospinal Fluid Leak and Fistula Formation.

Spinal cord stimulation (SCS) paddle leads placed via laminectomy procedures have become common as more data accumulates with regards to their clinical efficacy. In this paper, we describe a case of a 72-year-old male patient with failed back surgery syndrome (FBSS) who underwent a thoracic laminectomy for permanent paddle lead placement. He went on to develop a complication that resulted in a large cerebrospinal fluid leak with a cerebrospinal fluid fistula formation.

Differential expression of AtWAKL10 in response to nitric oxide suggests a putative role in biotic and abiotic stress responses.

Plant defense against pathogens and abiotic stresses is regulated differentially by communicating signal transduction pathways in which nitric oxide (NO) plays a key role. Here, we show the biological role of Arabidopsis thaliana wall-associated kinase (AtWAK) Like10 (AtWAKL10) that exhibits greater than a 100-fold change in transcript accumulation in response to the NO donor S-nitroso-L-cysteine (CysNO), identified from high throughput RNA-seq based transcriptome analysis. Loss of AtWAKL10 function showed a similar phenotype to wild type (WT) with, however, less branching. The growth of atwakl10 on media supplemented with oxidative or nitrosative stress resulted in differential results with improved growth following treatment with CysNO but reduced growth in response to S-nitrosoglutatione (GSNO) and methyl-viologen. Further, atwakl10 plants exhibited increased susceptibility to virulent Pseudomonas syringae pv tomato (Pst) DC3000 with a significant increase in pathogen growth and decrease in PR1 transcript accumulation compared to WT overtime. Similar results were found in response to Pst DC3000 avrB, resulting in increased cell death as shown by increased electrolyte leakage in atwakl10. Furthermore, atwakl10 also showed increased reactive oxygen species accumulation following Pst DC3000 avrB inoculation. Promoter analysis of AtWAKL10 showed transcription factor (TF) binding sites for biotic and abiotic stress-related TFs. Further investigation into the role of AtWAKL10 in abiotic stresses showed that following two weeks water-withholding drought condition most of the atwakl10 plants got wilted; however, the majority (60%) of these plants recovered following re-watering. In contrast, in response to salinity stress, atwakl10 showed reduced germination under 150 mM salt stress compared to WT, suggesting that NO-induced AtWAKL10 differentially regulates different abiotic stresses. Taken together, this study further elucidates the importance of NO-induced changes in gene expression and their role in plant biotic and abiotic stress tolerance.

Novel and conserved functions of S-nitrosoglutathione reductase in tomato.

Nitric oxide (NO) is emerging as a key signalling molecule in plants. The chief mechanism for the transfer of NO bioactivity is thought to be S-nitrosylation, the addition of an NO moiety to a protein cysteine thiol to form an S-nitrosothiol (SNO). The enzyme S-nitrosoglutathione reductase (GSNOR) indirectly controls the total levels of cellular S-nitrosylation, by depleting S-nitrosoglutathione (GSNO), the major cellular NO donor. Here we show that depletion of GSNOR function impacts tomato (Solanum lycopersicum. L) fruit development. Thus, reduction of GSNOR expression through RNAi modulated both fruit formation and yield, establishing a novel function for GSNOR. Further, depletion of S. lycopersicum GSNOR (SlGSNOR) additionally impacted a number of other developmental processes, including seed development, which also has not been previously linked with GSNOR activity. In contrast to Arabidopsis, depletion of GSNOR function did not influence root development. Further, reduction of GSNOR transcript abundance compromised plant immunity. Surprisingly, this was in contrast to previous data in Arabidopsis that reported that reducing Arabidopsis thaliana GSNOR (AtGSNOR) expression by antisense technology increased disease resistance. We also show that increased SlGSNOR expression enhanced pathogen protection, uncovering a potential strategy to enhance disease resistance in crop plants. Collectively, our findings reveal, at the genetic level, that some but not all GSNOR activities are conserved outside the Arabidopsis reference system. Thus, manipulating the extent of GSNOR expression may control important agricultural traits in tomato and possibly other crop plants.

Comprehensive Analyses of Nitric Oxide-Induced Plant Stem Cell-Related Genes in Arabidopsis thaliana.

Plant stem cells are pluripotent cells that have diverse applications in regenerative biology and medicine. However, their roles in plant growth and disease resistance are often overlooked. Using high-throughput RNA-seq data, we identified approximately 20 stem cell-related differentially expressed genes (DEGs) that were responsive to the nitric oxide (NO) donor S-nitrosocysteine (CySNO) after six hours of infiltration. Among these DEGs, the highest number of positive correlations (R ≥ 0.8) was observed for CLAVATA3/EMBRYO SURROUNDING REGION-RELATED (CLE) 12. Gene ontology (GO) terms for molecular function showed DEGs associated with signal transduction and receptor activity. A promoter study of these DEGs showed the presence of cis-acting elements that are involved in growth as well as the regulation of abiotic and biotic stress. Phylogenetic analysis of the Arabidopsis stem cell-related genes and their common orthologs in rice, soybean, poplar, and tomato suggested that most soybean stem cell-related genes were grouped with the Arabidopsis CLE type of stem cell genes, while the rice stem cell-related genes were grouped with the Arabidopsis receptor-like proteins. The functional genomic-based characterization of the role of stem cell DEGs showed that under control conditions, the clv1 mutant showed a similar phenotype to that of the wild-type (WT) plants; however, under CySNO-mediated nitrosative stress, clv1 showed increased shoot and root length compared to WT. Furthermore, the inoculation of clv1 with virulent Pst DC3000 showed a resistant phenotype with fewer pathogens growing at early time points. The qRT-PCR validation and correlation with the RNA-seq data showed a Pearson correlation coefficient of >0.8, indicating the significantly high reliability of the RNA-seq analysis.

Silver Nanoparticles on Nanoscale Silica Platelets (AgNP/NSP) and Nanoscale Silica Platelets (NSP) Inhibit the Development of Fusarium oxysporum f. sp. niveum.

Nanotechnology has attracted much attention recently because of its agricultural applications. In this study, we analyzed the ability of two potential nanomaterials (NMs), nanoscale silica platelets (NSP) and silver nanoparticles on nanoscale silica platelets (AgNP/NSP), to control Fusarium wilt [caused by Fusarium oxysporum f. sp. niveum (Fon)] disease in watermelon. Both AgNP/NSP and NSP significantly reduced Fon mycelial growth and spore viability. In addition, AgNP/NSP decreased the mycelium viability at concentrations of 150 and 200 ppm. Scanning and transmission electron microscopy showed significant morphological effects on Fon cells, such as increased roughness and interior hollowing after AgNP/NSP and NSP treatments. Further, fluorescence staining experiments showed that a concomitant increase in membrane permeability occurred after treatment with NMs. The biochemical effects of NM treatment included a significant reduction in secreted cellulase activity. Interestingly, the addition of cysteine as a reducing agent decreased effects of NSP on Fon spores, suggesting suppression of Fon spore development attributable to oxidative stress. Taken together, these results indicate that AgNP/NSP and NSP may potentially serve as nanofungicides for future control of Fusarium wilt and other fungal diseases.

Nitric oxide- induced AtAO3 differentially regulates plant defense and drought tolerance in Arabidopsis thaliana.

BACKGROUND: Exposure of plants to different environmental insults instigates significant changes in the cellular redox tone driven in part by promoting the production of reactive nitrogen species. The key player, nitric oxide (NO) is a small gaseous diatomic molecule, well-known for its signaling role during stress. In this study, we focused on abscisic acid (ABA) metabolism-related genes that showed differential expression in response to the NO donor S-nitroso-L-cysteine (CySNO) by conducting RNA-seq-based transcriptomic analysis. RESULTS: CySNO-induced ABA-related genes were identified and further characterized. Gene ontology terms for biological processes showed most of the genes were associated with protein phosphorylation. Promoter analysis suggested that several cis-regulatory elements were activated under biotic and/or abiotic stress conditions. The ABA biosynthetic gene AtAO3 was selected for validation using functional genomics. The loss of function mutant atao3 was found to differentially regulate oxidative and nitrosative stress. Further investigations for determining the role of AtAO3 in plant defense suggested a negative regulation of plant basal defense and R-gene-mediated resistance. The atao3 plants showed resistance to virulent Pseudomonas syringae pv. tomato strain DC3000 (Pst DC3000) with gradual increase in PR1 gene expression. Similarly, atao3 plants showed increased hypersensitive response (HR) when challenged with Pst DC3000 (avrB). The atgsnor1-3 and atsid2 mutants showed a susceptible phenotype with reduced PR1 transcript accumulation. Drought tolerance assay indicated that atao3 and atnced3 ABA-deficient mutants showed early wilting, followed by plant death. The study of stomatal structure showed that atao3 and atnced3 were unable to close stomata even at 7 days after drought stress. Further, they showed reduced ABA content and increased electrolyte leakage than the wild-type (WT) plants. The quantitative polymerase chain reaction analysis suggested that ABA biosynthesis genes were down-regulated, whereas expression of most of the drought-related genes were up-regulated in atao3 than in WT. CONCLUSIONS: AtAO3 negatively regulates pathogen-induced salicylic acid pathway, although it is required for drought tolerance, despite the fact that ABA production is not totally dependent on AtAO3, and that drought-related genes like DREB2 and ABI2 show response to drought irrespective of ABA content.

S-nitrosocysteine-responsive genes modulate diverse regulatory pathways in Oryza sativa: a transcriptome profiling study.

Rice (Oryza sativa L.) is a major food crop and also a well-established genetic model. Nitric oxide (NO) and its derivatives are important signalling molecules that actively participate in various signalling pathways in response to different stresses. In this study, we performed RNA-seq mediated transcriptomic analysis of rice after treatment with the nitric oxide donor, S-nitroso-L-cysteine (CySNO), generating an average of 37.5 and 41.5 million reads from control and treated leaf samples respectively. More than 95% of the reads were successfully mapped to the O. sativa reference genome yielding a total of 33539 differentially expressed genes (DEGs, P < 0.05). Further analyses identified 825 genes with at least 2-fold change in the expression following treatment with CySNO (P < 0.01). The DEGs identified were involved in diverse molecular functions such as catalytic activity, binding, transport, and receptor activity and were mostly located in the membrane, organelles such as nucleus, Golgi apparatus and mitochondria. DEGs also contained several genes that regulate responses to abiotic stresses such as drought, heat, cold and salt stress and biotic stresses. We also found significantly similar expression patterns of CySNO-responsive DEGs of rice with the CySNO-responsive DEGs of Arabidopsis in a previous study. Expression patterns of genes involved in key biological functions were verified using quantitative real time (qRT)-PCR. The findings of this study suggest that NO regulates the transcriptional control of genes involved in a wide variety of physiological functions in rice, and that NO-mediated transcriptional networks are highly conserved across the plant kingdom. This study provides useful information regarding the transcriptional response of plants to nitrosative stress.

Nitric oxide and S-nitrosoglutathione function additively during plant immunity.

Nitric oxide (NO) is emerging as a key regulator of diverse plant cellular processes. A major route for the transfer of NO bioactivity is S-nitrosylation, the addition of an NO moiety to a protein cysteine thiol forming an S-nitrosothiol (SNO). Total cellular levels of protein S-nitrosylation are controlled predominantly by S-nitrosoglutathione reductase 1 (GSNOR1) which turns over the natural NO donor, S-nitrosoglutathione (GSNO). In the absence of GSNOR1 function, GSNO accumulates, leading to dysregulation of total cellular S-nitrosylation. Here we show that endogenous NO accumulation in Arabidopsis, resulting from loss-of-function mutations in NO Overexpression 1 (NOX1), led to disabled Resistance (R) gene-mediated protection, basal resistance and defence against nonadapted pathogens. In nox1 plants both salicylic acid (SA) synthesis and signalling were suppressed, reducing SA-dependent defence gene expression. Significantly, expression of a GSNOR1 transgene complemented the SNO-dependent phenotypes of paraquat resistant 2-1 (par2-1) plants but not the NO-related characters of the nox1-1 line. Furthermore, atgsnor1-3 nox1-1 double mutants supported greater bacterial titres than either of the corresponding single mutants. Our findings imply that GSNO and NO, two pivotal redox signalling molecules, exhibit additive functions and, by extension, may have distinct or overlapping molecular targets during both immunity and development.

Occurrence and molecular characterization of Cucumber green mottle mosaic virus in cucurbit crops of KPK, Pakistan

Field survey of the cucurbit crops revealed a high incidence of Cucumber green mottle mosaic virus (CGMMV) in Khyber Pakhtunkhwa Province (KPK), Pakistan. Among the seven districts surveyed, average percent incidence of CGMMV was recorded up to 58.1% in district Nowshera, followed by 51.1% in district Charsada, 40.5% in district Swabi and 37.3% in district Mardan. In Swat and Dir districts average incidence CGMMV was recorded upto 31.2% and 29.4%, respectively. Among the different crops highest incidence in plain areas of KPK was recorded in bottle gourd (59.3%) followed by 56.3% in Squash, 54.5% in Pumpkin, 45.5% in Melon, 41.7% in Cucumber and 29.9% in Sponge gourd. In Northern hilly areas highest incidence of CGMMV (52.9%) was observed in pumpkin, followed by 49.6% in bottle gourd, 47.3% in squash, 45.1% in Melon 42.3% in cucumber and 41.6% in sponge gourd. Little variability was observed in the coat protein amino acid sequence identities of CGMMV Pakistan isolate, when compared with other reported isolates.