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.

Alleviation of Hg-, Cr-, Cu-, and Zn-Induced Heavy Metals Stress by Exogenous Sodium Nitroprusside in Rice Plants.

The cultivation of rice is widespread worldwide, but its growth and productivity are hampered by heavy metals stress. However, sodium nitroprusside (SNP), a nitric oxide donor, has been found to be effective for imparting heavy metals stress tolerance to plants. Therefore, the current study evaluated the role of exogenously applied SNP in improving plant growth and development under Hg, Cr, Cu, and Zn stress. For this purpose, heavy metals stress was induced via the application of 1 mM mercury (Hg), chromium (Cr), copper (Cu), and zinc (Zn). To reverse the toxic effects of heavy metals stress, 0.1 mM SNP was administrated via the root zone. The results revealed that the said heavy metals significantly reduced the chlorophyll contents (SPAD), chlorophyll a and b, and protein contents. However, SNP treatment significantly reduced the toxic effects of the said heavy metals on chlorophyll (SPAD), chlorophyll a and b, and protein contents. In addition, the results also revealed that heavy metals significantly increased the production of superoxide anion (SOA), hydrogen peroxide (H2O2), malondialdehyde (MDA), and electrolyte leakage (EL). However, SNP administration significantly reduced the production of SOA, H2O2, MDA, and EL in response to the said heavy metals. Furthermore, to cope with the said heavy metals stress, SNP administration significantly enhanced the activities of superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), and polyphenol peroxidase (PPO). Furthermore, in response to the said heavy metals, SNP application also upregulated the transcript accumulation of OsPCS1, OsPCS2, OsMTP1, OsMTP5, OsMT-I-1a, and OsMT-I-1b. Therefore, SNP can be used as a regulator to improve the heavy metals tolerance of rice in heavy-metals-affected areas.

Exogenously Applied Sodium Nitroprusside Mitigates Lead Toxicity in Rice by Regulating Antioxidants and Metal Stress-Related Transcripts.

Sustainable agriculture is increasingly being put in danger by environmental contamination with dangerous heavy metals (HMs), especially lead (Pb). Plants have developed a sophisticated mechanism for nitric oxide (NO) production and signaling to regulate hazardous effects of abiotic factors, including HMs. In the current study, we investigated the role of exogenously applied sodium nitroprusside (SNP, a nitric oxide (NO) donor) in ameliorating the toxic effects of lead (Pb) on rice. For this purpose, plants were subjected to 1.2 mM Pb alone and in combination with 100 µM SNP. We found that under 1.2 mM Pb stress conditions, the accumulation of oxidative stress markers, including hydrogen peroxide (H2O2) (37%), superoxide anion (O2-) (28%), malondialdehyde (MDA) (33%), and electrolyte leakage (EL) (34%), was significantly reduced via the application of 100 µM SNP. On the other hand, under the said stress of Pb, the activity of the reactive oxygen species (ROS) scavengers such as polyphenol oxidase (PPO) (60%), peroxidase (POD) (28%), catalase (CAT) (26%), superoxide dismutase (SOD) (42%), and ascorbate peroxidase (APX) (58%) was significantly increased via the application of 100 µM SNP. In addition, the application of 100 µM SNP rescued agronomic traits such as plant height (24%), number of tillers per plant (40%), and visible green pigments (44%) when the plants were exposed to 1.2 mM Pb stress. Furthermore, after exposure to 1.2 mM Pb stress, the expression of the heavy-metal stress-related genes OsPCS1 (44%), OsPCS2 (74%), OsMTP1 (83%), OsMTP5 (53%), OsMT-I-1a (31%), and OsMT-I-1b (24%) was significantly enhanced via the application of 100 µM SNP. Overall, our research evaluates that exogenously applied 100 mM SNP protects rice plants from the oxidative damage brought on by 1.2 mM Pb stress by lowering oxidative stress markers, enhancing the antioxidant system and the transcript accumulation of HMs stress-related genes.

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.

Melatonin Ameliorates Thermotolerance in Soybean Seedling through Balancing Redox Homeostasis and Modulating Antioxidant Defense, Phytohormones and Polyamines Biosynthesis.

Global warming is impacting the growth and development of economically important but sensitive crops, such as soybean (Glycine max L.). Using pleiotropic signaling molecules, melatonin can relieve the negative effects of high temperature by enhancing plant growth and development as well as modulating the defense system against abiotic stresses. However, less is known about how melatonin regulates the phytohormones and polyamines during heat stress. Our results showed that high temperature significantly increased ROS and decreased photosynthesis efficiency in soybean plants. Conversely, pretreatment with melatonin increased plant growth and photosynthetic pigments (chl a and chl b) and reduced oxidative stress via scavenging hydrogen peroxide and superoxide and reducing the MDA and electrolyte leakage contents. The inherent stress defense responses were further strengthened by the enhanced activities of antioxidants and upregulation of the expression of ascorbate-glutathione cycle genes. Melatonin mitigates heat stress by increasing several biochemicals (phenolics, flavonoids, and proline), as well as the endogenous melatonin and polyamines (spermine, spermidine, and putrescine). Furthermore, the positive effects of melatonin treatment also correlated with a reduced abscisic acid content, down-regulation of the gmNCED3, and up-regulation of catabolic genes (CYP707A1 and CYP707A2) during heat stress. Contrarily, an increase in salicylic acid and up-regulated expression of the defense-related gene PAL2 were revealed. In addition, melatonin induced the expression of heat shock protein 90 (gmHsp90) and heat shock transcription factor (gmHsfA2), suggesting promotion of ROS detoxification via the hydrogen peroxide-mediated signaling pathway. In conclusion, exogenous melatonin improves the thermotolerance of soybean plants and enhances plant growth and development by activating antioxidant defense mechanisms, interacting with plant hormones, and reprogramming the biochemical metabolism.

Rhizospheric Bacillus spp. Rescues Plant Growth Under Salinity Stress via Regulating Gene Expression, Endogenous Hormones, and Antioxidant System of Oryza sativa L.

Salinity has drastically reduced crop yields and harmed the global agricultural industry. We isolated 55 bacterial strains from plants inhabiting the coastal sand dunes of Pohang, Korea. A screening bioassay showed that 14 of the bacterial isolates secreted indole-3-acetic acid (IAA), 12 isolates were capable of exopolysaccharide (EPS) production and phosphate solubilization, and 10 isolates secreted siderophores. Based on our preliminary screening, 11 bacterial isolates were tested for salinity tolerance on Luria-Bertani (LB) media supplemented with 0, 50, 100, and 150 mM of NaCl. Three bacterial isolates, ALT11, ALT12, and ALT30, had the best tolerance against elevated NaCl levels and were selected for further study. Inoculation of the selected bacterial isolates significantly enhanced rice growth attributes, viz., shoot length (22.8-42.2%), root length (28.18-59%), fresh biomass (44.7-66.41%), dry biomass (85-90%), chlorophyll content (18.30-36.15%), Chl a (29.02-60.87%), Chl b (30.86-64.51%), and carotenoid content (26.86-70%), under elevated salt stress of 70 and 140 mM. Furthermore, a decrease in the endogenous abscisic acid (ABA) content (27.9-23%) and endogenous salicylic acid (SA) levels (11.70-69.19%) was observed in inoculated plants. Antioxidant analysis revealed an increase in total protein (TP) levels (42.57-68.26%), whereas it revealed a decrease in polyphenol peroxidase (PPO) (24.63-34.57%), glutathione (GSH) (25.53-24.91%), SOA (13.88-18.67%), and LPO levels (15.96-26.06%) of bacterial-inoculated plants. Moreover, an increase in catalase (CAT) (26-33.04%), peroxidase (POD) (59.55-78%), superoxide dismutase (SOD) (13.58-27.77%), and ascorbic peroxidase (APX) (5.76-22.74%) activity was observed. Additionally, inductively coupled plasma mass spectrometry (ICP-MS) analysis showed a decline in Na+ content (24.11 and 30.60%) and an increase in K+ (23.14 and 15.45%) and Mg+ (2.82 and 18.74%) under elevated salt stress. OsNHX1 gene expression was downregulated (0.3 and 4.1-folds), whereas the gene expression of OsPIN1A, OsCATA, and OsAPX1 was upregulated by a 7-17-fold in bacterial-inoculated rice plants. It was concluded that the selected bacterial isolates, ALT11, ALT12, and ALT30, mitigated the adverse effects of salt stress on rice growth and can be used as climate smart agricultural tools in ecofriendly agricultural practices.

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.

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.

Exogenous application of nitric oxide donors regulates short-term flooding stress in soybean.

Short-term water submergence to soybean (Glycine max L.) create hypoxic conditions hindering plant growth and productivity. Nitric oxide (NO) is considered a stress-signalling and stress-evading molecule, however, little is known about its role during flooding stress. We elucidated the role of sodium nitroprusside (SNP) and S-nitroso L-cysteine (CySNO) as NO donor in modulation of flooding stress-related bio-chemicals and genetic determinants of associated nitrosative stress to Daewon and Pungsannamul soybean cultivars after 3 h and 6 h of flooding stress. The results showed that exogenous SNP and CysNO induced glutathione activity and reduced the resulting superoxide anion contents during short-term flooding in Pungsannamul soybean. The exo- SNP and CysNO triggered the endogenous S-nitrosothiols, and resulted in elevated abscisic acid (ABA) contents in both soybean cultivars overtime. To know the role of ABA and NO related genes in short-term flooding stress, the mRNA expression of S-nitrosoglutathione reductase (GSNOR1), NO overproducer1 (NOX1) and nitrate reductase (NR), Timing of CAB expression1 (TOC1), and ABA-receptor (ABAR) were assessed. The transcripts accumulation of GSNOR1, NOX1, and NR being responsible for NO homeostasis, were significantly high in response to early or later phases of flooding stress. ABAR and TOC1 showed a decrease in transcript accumulation in both soybean plants treated with exogenous SNP and CySNO. The exo- SNP and CySNO could impinge a variety of biochemical and transcriptional programs that can mitigate the negative effects of short-term flooding stress in soybean.

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.

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.

Overexpression of RsMYB1 Enhances Anthocyanin Accumulation and Heavy Metal Stress Tolerance in Transgenic Petunia.

The RsMYB1 transcription factor (TF) controls the regulation of anthocyanin in radishes (Raphanus sativus), and its overexpression in tobacco and petunias strongly enhances anthocyanin production. However, there are no data on the involvement of RsMYB1 in the mechanisms underlying abiotic stress tolerance, despite strong sequence similarity with other MYBs that confer such tolerance. In this study, we used the anthocyanin-enriched transgenic petunia lines PM6 and PM2, which overexpress RsMYB1. The tolerance of these lines to heavy metal stress was investigated by examining several physiological and biochemical factors, and the transcript levels of genes related to metal detoxification and antioxidant activity were quantified. Under normal conditions (control conditions), transgenic petunia plants (T2-PM6 and T2-PM2) expressing RsMYB1, as well as wild-type (WT) plants, were able to thrive by producing well-developed broad leaves and regular roots. In contrast, a reduction in plant growth was observed when these plants were exposed to heavy metals (CuSO4, ZnSO4, MnSO4, or K2Cr2O7). However, T2-PM6 and T2-PM2 were found to be more stress tolerant than the WT plants, as indicated by superior results in all analyzed parameters. In addition, RsMYB1 overexpression enhanced the expression of genes related to metal detoxification [glutathione S-transferase (GST) and phytochelatin synthase (PCS)] and antioxidant activity [superoxide dismutase (SOD), catalase (CAT), and peroxidase (POX)]. These results suggest that enhanced expression levels of the above genes can improve metal detoxification activities and antioxidant activity, which are the main components of defense mechanism included in abiotic stress tolerance of petunia. Our findings demonstrate that RsMYB1 has potential as a dual-function gene that can have an impact on the improvement of anthocyanin production and heavy metal stress tolerance in horticultural crops.

Exo-ethylene application mitigates waterlogging stress in soybean (Glycine max L.).

BACKGROUND: Waterlogging (WL) is a key factor hindering soybean crop productivity worldwide. Plants utilize various hormones to avoid various stress conditions, including WL stress; however, the physiological mechanisms are still not fully understood. RESULTS: To identify physiological mechanisms during WL stress, different phytohormones, such as ethephon (ETP; donor source of ethylene), abscisic acid, gibberellins, indole-3-acetic acid, kinetin, jasmonic acid, and salicylic acid were exogenously applied to soybean plants. Through this experiment, we confirmed the beneficial effects of ETP treatment. Thus, we selected ETP as a candidate hormone to mitigate WL. Further mechanistic investigation of the role of ETP in waterlogging tolerance was carried out. Results showed that ETP application mitigated WL stress, significantly improved the photosynthesis pigment, and increased the contents of endogenous GAs compared to those in untreated plants. The amino acid contents during WL stress were significantly activated by EPT treatments. The amino acid contents were significantly higher in the 100 µM ETP-treated soybean plants than in the control. ETP application induced adventitious root initiation, increased root surface area, and significantly increased the expressions of glutathione transferases and relative glutathione activity compared to those of non-ETP-treated plants. ETP-treated soybeans produced a higher up-regulation of protein content and glutathione S-transferase (GSTs) than did soybeans under the WL only treatment. CONCLUSIONS: In conclusion, the current results suggest that ETP application enabled various biochemical and transcriptional modulations. In particular, ETP application could stimulate the higher expression of GST3 and GST8. Thus, increased GST3 and GST8 induced 1) increased GSH activity, 2) decreased reactive oxygen species (ROS), 3) mitigation of cell damage in photosynthetic apparatus, and 4) improved phenotype consecutively.

Endophytic Microbial Consortia of Phytohormones-Producing Fungus Paecilomyces formosus LHL10 and Bacteria Sphingomonas sp. LK11 to Glycine max L. Regulates Physio-hormonal Changes to Attenuate Aluminum and Zinc Stresses.

The compatible microbial consortia containing fungal and bacterial symbionts acting synergistically are applied to improve plant growth and eco-physiological responses in extreme crop growth conditions. However, the interactive effects of phytohormones-producing endophytic fungal and bacterial symbionts plant growth and stress tolerance under heavy metal stress have been least known. In the current study, the phytohormones-producing endophytic Paecilomyces formosus LHL10 and Sphingomonas sp. LK11 revealed potent growth and tolerance during their initial screening against combined Al and Zn (2.5 mM each) stress. This was followed with their co-inoculation in the Al- and Zn-stressed Glycine max L. plants, showing significantly higher plant growth attributes (shoot/root length, fresh/dry weight, and chlorophyll content) than the plants solely inoculated with LHL10 or LK11 and the non-inoculated (control) plants under metal stresses. Interestingly, under metal stress, the consortia exhibited lower metal uptake and inhibited metal transport in roots. Metal-induced oxidative stresses were modulated in co-inoculated plants through reduced hydrogen peroxide, lipid peroxidation, and antioxidant enzymes (catalase and superoxide dismutase) in comparison to the non-inoculated plants. In addition, endophytic co-inoculation enhanced plant macronutrient uptake (P, K, S, and N) and modulated soil enzymatic activities under stress conditions. It significantly downregulated the expression of heavy metal ATPase genes GmHMA13, GmHMA18, GmHMA19, and GmPHA1 and upregulated the expression of an ariadne-like ubiquitin ligase gene GmARI1 under heavy metals stress. Furthermore, the endogenous phytohormonal contents of co-inoculated plants revealed significantly enhanced gibberellins and reduced abscisic acid and jasmonic acid contents, suggesting that this endophytic interaction mitigated the adverse effect of metal stresses in host plants. In conclusion, the co-inoculation of the endophytic fungus LHL10 and bacteria LK11 actively contributed to the tripartite mutualistic symbiosis in G. max under heavy metal stresses; this could be used an excellent strategy for sustainable agriculture in the heavy metal-contaminated fields.

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.

Overexpressing sweetpotato peroxidase gene swpa4 affects nitric oxide production by activating the expression of reactive oxygen species- and nitric oxide-related genes in tobacco.

Reactive oxygen species (ROS) and nitric oxide (NO) are key signaling molecules involved in various developmental and stress responses in plants. NO and ROS production, which is triggered by various stimuli, activates downstream signaling pathways to help plants cope with abiotic and biotic stresses. Recent evidence suggests that the interplay between NO and ROS signaling plays a critical role in regulating stress responses. However, the underlying molecular mechanism remains poorly understood. We previously reported that transgenic tobacco overexpressing the swpa4 peroxidase (POD) gene from sweetpotato exhibits increased tolerance to stress. Overexpression of swpa4 also induces the generation of H2O2 and activates the expression of various extracellular acidic pathogenesis-related (PR) genes. Here, we show that swpa4 positively regulates the expression of ROS- and NO-related genes in transgenic tobacco plants. Plants expressing swpa4 exhibited increased expression of ROS-related genes and increased ROS-related enzyme activity under normal conditions and H2O2 treatment, whereas the expression of NO associated 1 (NOA1) only increased under normal conditions. Moreover, plants overexpressing swpa4 showed increased NO levels under normal conditions and after treatment with the NO donor sodium nitroprusside (SNP). Interestingly, treatment with a POD inhibitor dramatically reduced NO levels in swpa4 transgenic plants. These findings suggest that swpa4 regulates H2O2 and NO homeostasis in plants under stress conditions, thereby establishing a possible molecular link between the NO and ROS signaling pathways.