Trimethylamine-N-oxide depletes urea in a peptide solvation shell.

Trimethylamine-N-oxide (TMAO) and urea are metabolites that are used by some marine animals to maintain their cell volume in a saline environment. Urea is a well-known denaturant, and TMAO is a protective osmolyte that counteracts urea-induced protein denaturation. TMAO also has a general protein-protective effect, for example, it counters pressure-induced protein denaturation in deep-sea fish. These opposing effects on protein stability have been linked to the spatial relationship of TMAO, urea, and protein molecules. It is generally accepted that urea-induced denaturation proceeds through the accumulation of urea at the protein surface and their subsequent interaction. In contrast, it has been suggested that TMAO's protein-stabilizing effects stem from its exclusion from the protein surface, and its ability to deplete urea from protein surfaces; however, these spatial relationships are uncertain. We used neutron diffraction, coupled with structural refinement modeling, to study the spatial associations of TMAO and urea with the tripeptide derivative glycine-proline-glycinamide in aqueous urea, aqueous TMAO, and aqueous urea-TMAO (in the mole ratio 1:2 TMAO:urea). We found that TMAO depleted urea from the peptide's surface and that while TMAO was not excluded from the tripeptide's surface, strong atomic interactions between the peptide and TMAO were limited to hydrogen bond donating peptide groups. We found that the repartition of urea, by TMAO, was associated with preferential TMAO-urea bonding and enhanced urea-water hydrogen bonding, thereby anchoring urea in the bulk solution and depleting urea from the peptide surface.

Metabolic engineering of high-salinity-induced biosynthesis of γ-aminobutyric acid improves salt-stress tolerance in a glutamic acid-overproducing mutant of an ectoine-deficient Halomonas elongata.

A moderately halophilic eubacterium, Halomonas elongata, has been used as cell factory to produce fine chemical 1,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid (ectoine), which functions as a major osmolyte protecting the cells from high-salinity stress. To explore the possibility of using H. elongata to biosynthesize other valuable osmolytes, an ectoine-deficient salt-sensitive H. elongata deletion mutant strain KA1 (ΔectABC), which only grows well in minimal medium containing up to 3% NaCl, was subjected to an adaptive mutagenesis screening in search of mutants with restored salt tolerance. Consequently, we obtained a mutant, which tolerates 6% NaCl in minimal medium by overproducing L-glutamic acid (Glu). However, this Glu-overproducing (GOP) strain has a lower tolerance level than the wild-type H. elongata, possibly because the acidity of Glu interferes with the pH homeostasis of the cell and hinders its own cellular accumulation. Enzymatic decarboxylation of Glu to γ-aminobutyric acid (GABA) by a Glu decarboxylase (GAD) could restore cellular pH homeostasis; therefore, we introduced an engineered salt-inducible HopgadBmut gene, which encodes a wide pH-range GAD mutant, into the genome of the H. elongata GOP strain. We found that the resulting H. elongata GOP-Gad strain exhibits higher salt tolerance than the GOP strain by accumulating high concentration of GABA as an osmolyte in the cell (176.94 µmol/g cell dry weight in minimal medium containing 7% NaCl). With H. elongata OUT30018 genetic background, H. elongata GOP-Gad strain can utilize biomass-derived carbon and nitrogen compounds as its sole carbon and nitrogen sources, making it a good candidate for the development of GABA-producing cell factories.IMPORTANCEWhile the wild-type moderately halophilic H. elongata can synthesize ectoine as a high-value osmolyte via the aspartic acid metabolic pathway, a mutant H. elongata GOP strain identified in this work opens doors for the biosynthesis of alternative valuable osmolytes via glutamic acid metabolic pathway. Further metabolic engineering to install a GAD system into the H. elongata GOP strain successfully created a H. elongata GOP-Gad strain, which acquired higher tolerance to salt stress by accumulating GABA as a major osmolyte. With the ability to assimilate biomass-derived carbon and nitrogen sources and thrive in high-salinity environment, the H. elongata GOP-Gad strain can be used in the development of sustainable GABA-producing cell factories.

Induction of tomato plant biochemical immune responses by the synthesized zinc oxide nanoparticles against wilt-induced Fusarium oxysporum.

The current study used zinc oxide nanoparticles (ZnO-NPs) to protect the tomato plant against Fusarium wilt. Gamma rays were used to synthesize ZnO-NPs, and the designed ZnO-NPs were characterized using high-resolution transmission electron microscopy (HRTEM), scanning electron microscope (SEM), dynamic light scattering (DLS), energy-dispersive X-ray spectroscopy (EDX), and ultraviolet-visible (UV-Vis.) spectroscopy. We found that the 20 kGy dose is the most effective for ZnO-NPs synthesis, with the highest O.D. = 1.65 (diluted 3 times) at 400 nm. The scale of ZnO-NPs ranged from 10.45 to 75.25 nm with an average diameter of 40.20 nm. The results showed that the designed ZnO-NPs showed promising activity as a potent inducer of plant physiological immunity against Fusarium wilt disease. Likewise, ZnO-NPs significantly reduced the wilt disease symptoms incidence by 28.57% and high protection by 67.99% against F. oxysporum. Additionally, infected tomato plants treated with ZnO-NPs show improved shoot length (44.71%), root length (40.0%), number of leaves (60.0 %), chlorophyll a (36.93%), chlorophyll b (16.46%), and carotenoids (21.87%) versus infected plants. Notably, in the treatment of tomato seedlings, the beneficial effects of ZnO-NPs extended to increase not only in osmolyte contents but also total phenol contents in comparison with control plants. In conclusion, the designed ZnO-NPs can control Fusarium wilt disease and improve and develop biochemical compounds responsible for defense against fusarial infection.

Strategies for the synthesis of the osmolyte glucosylglycerate and its precursor glycerate.

Glycosidic osmolytes are widespread natural compounds that protect microorganisms and their macromolecules from the deleterious effects of various environmental stresses. Their protective properties have attracted considerable interest for industrial applications, especially as active ingredients in cosmetics and healthcare products. In that regard, the osmolyte glucosylglycerate is somewhat overlooked. Glucosylglycerate is typically accumulated by certain organisms when they are exposed to high salinity and nitrogen starvation, and its potent stabilizing effects have been demonstrated in vitro. However, the applications of this osmolyte have not been thoroughly explored due to the lack of a cost-efficient production process. Here, we present an overview of the progress that has been made in developing promising strategies for the synthesis of glucosylglycerate and its precursor glycerate, and discuss the remaining challenges. KEY POINTS: • Bacterial milking could be explored for fermentative production of glucosylglycerate • Glycoside phosphorylases of GH13_18 represent attractive alternatives for biocatalytic production • Conversion of glycerol with alditol oxidase is a promising strategy for generating the precursor glycerate.

High-yield synthesis of 2-O-α-D-glucosyl-D-glycerate by a bifunctional glycoside phosphorylase.

Osmolytes are produced by various microorganisms as a defense mechanism to protect cells and macromolecules from damage caused by external stresses in harsh environments. Due to their useful stabilizing properties, these molecules are applied as active ingredients in a wide range of cosmetics and healthcare products. The metabolic pathways and biocatalytic syntheses of glycosidic osmolytes such as 2-O-α-D-glucosyl-D-glycerate often involve the action of a glycoside phosphorylase. Here, we report the discovery of a glucosylglycerate phosphorylase from carbohydrate-active enzyme family GH13 that is also active on sucrose, which contrasts the strict specificity of known glucosylglycerate phosphorylases that can only use α-D-glucose 1-phosphate as glycosyl donor in transglycosylation reactions. The novel enzyme can be distinguished from other phosphorylases from the same family by the presence of an atypical conserved sequence motif at specificity-determining positions in the active site. The promiscuity of the sucrose-active glucosylglycerate phosphorylase can be exploited for the high-yielding and rapid synthesis of 2-O-α-D-glucosyl-D-glycerate from sucrose and D-glycerate. KEY POINTS: • A Xylanimonas protaetiae glycoside phosphorylase can use both d-glycerate and fructose as glucosyl acceptor with high catalytic efficiency • Biocatalytic synthesis of the osmolyte 2-O-α-d-glucosyl-d-glycerate • Positions in the active site of GH13 phosphorylases act as convenient specificity fingerprints.

Expression of an engineered salt-inducible proline biosynthetic operon in a Glutamic acid Over-Producing mutant, Halomonas elongata GOP, confers increased proline yield due to enhanced growth under high-salinity conditions.

L-Proline (Pro) is an essential amino acid additive in livestock and aquaculture feeds. Previously, we created a Pro overproducing Halomonas elongata HN6 by introducing an engineered salt-inducible Pro biosynthetic mCherry-proBm1AC operon and deleting a putA gene that encoded a Pro catabolic enzyme in the genome of H. elongata OUT30018. Here, we report a generation of a novel Pro overproducing H. elongata HN10 strain with improved salt tolerance and higher Pro yield by expressing the mCherry-proBm1AC operon and deleting the putA gene in the genome of a spontaneous mutant H. elongata GOP, which overproduces glutamic acid (Glu) that is a precursor for Pro biosynthesis. The optimal salt concentration for growth of H. elongata HN10 was found to be 7% to 8% w/v NaCl, and the average Pro yield of 166 mg/L was achieved when H. elongata HN10 was cultivated in M63 minimal medium containing 4% w/v glucose and 8% w/v NaCl.

Revisiting blood pressure and body fluid status.

Homeostasis of body fluid is a key component for maintaining health. An imbalance of body sodium and water causes various pathological states, such as dehydration, volume overload, hypertension, cardiovascular and renal diseases, and metabolic disorders. Conventional concepts regarding physiology and pathophysiology of body sodium and water balance have been established by several assumptions. These assumptions are that the kidneys are the master regulator of body sodium and water content, and that sodium moves inside the body in parallel with water. However, recent clinical and basic studies have proposed alternative concepts. These concepts are that body sodium and water balance are regulated by various organs and multiple factors, such as physical activity and the environment, and that sodium accumulates locally in tissues independently of the blood status and/or water. Various concerns remain unclear, and the regulatory mechanism of body sodium, fluid, and blood pressure needs to be readdressed. In the present review article, we discuss novel concepts regarding the regulation of body sodium, water, and blood pressure with a particular focus on the systemic water conservation system and fluid loss-triggered elevation in blood pressure.

Exogenous glucosylglycerol and proline extend the chronological lifespan of Rhodosporidium toruloides.

Glucosylglycerol (GG) is an osmolyte found in a few bacteria (e.g., cyanobacteria) and plants grown in harsh environments. GG protects microbes and plants from salinity and desiccation stress. In the industry, GG is synthesized from a combination of ADP-glucose and glycerol-3-phosphate in a condensation reaction catalyzed by glucosylglycerol phosphate synthase. Proline, on the other hand, is an amino acid-based osmolyte that plays a key role in cellular reprograming. It functions as a protectant and a scavenger of reactive oxygen species. Studies on lifespan extension have focused on the use of Saccharomyces cerevisiae. Rhodosporidium toruloides, also known as Rhodotorula toruloides, is a basidiomycetous oleaginous yeast known to accumulate lipids to more than 70% of its dry cell weight. The oleaginous red yeast (R. toruloides) has not been intensely studied in the lifespan domain. We designed this work to investigate how GG and proline promote the longevity of this red yeast strain. The results obtained in our study confirmed that these molecules increased R. toruloides' viability, survival percentage, and lifespan upon supplementation. GG exerts the most promising effects at a relatively high concentration (100 mM), while proline functions best at a low level (2 mM). Elucidation of the processes underlying these favorable responses revealed that GG promotes the yeast chronological lifespan (CLS) through increased catalase activity, modulation of the culture medium pH, a rise in ATP, and an increase in reactive oxygen species (ROS) accumulation (mitohormesis). It is critical to understand the mechanisms of these geroprotector molecules, particularly GG, and the proclivity of its lifespan application; this will aid in offering clarity on its potential application in aging research.

Wheat ABA Receptor TaPYL5 Constitutes a Signaling Module with Its Downstream Partners TaPP2C53/TaSnRK2.1/TaABI1 to Modulate Plant Drought Response.

Abscisic acid receptors (ABR) play crucial roles in transducing the ABA signaling initiated by osmotic stresses, which has a significant impact on plant acclimation to drought by modulating stress-related defensive physiological processes. We characterized TaPYL5, a member of the ABR family in wheat (Triticum aestivum), as a mediator of drought stress adaptation in plants. The signals derived from the fusion of TaPYL5-GFP suggest that the TaPYL5 protein was directed to various subcellular locations, namely stomata, plasma membrane, and nucleus. Drought stress significantly upregulated the TaPYL5 transcripts in roots and leaves. The biological roles of ABA and drought responsive cis-elements, specifically ABRE and recognition sites MYB, in mediating gene transcription under drought conditions were confirmed by histochemical GUS staining analysis for plants harbouring a truncated TaPYL5 promoter. Yeast two-hybrid and BiFC assays indicated that TaPYL5 interacted with TaPP2C53, a clade A member of phosphatase (PP2C), and the latter with TaSnRK2.1, a kinase member of the SnRK2 family, implying the formation of an ABA core signaling module TaPYL5/TaPP2C53/TaSnRK2.1. TaABI1, an ABA responsive transcription factor, proved to be a component of the ABA signaling pathway, as evidenced by its interaction with TaSnRK2.1. Transgene analysis of TaPYL5 and its module partners, as well as TaABI1, revealed that they have an effect on plant drought responses. TaPYL5 and TaSnRK2.1 positively regulated plant drought acclimation, whereas TaPP2C53 and TaABI1 negatively regulated it. This coincided with the osmotic stress-related physiology shown in their transgenic lines, such as stomata movement, osmolytes biosynthesis, and antioxidant enzyme function. TaPYL5 significantly altered the transcription of numerous genes involved in biological processes related to drought defense. Our findings suggest that TaPYL5 is one of the most important regulators in plant drought tolerance and a valuable target for engineering drought-tolerant cultivars in wheat.

Balancing the Equation: A Natural History of Trimethylamine and Trimethylamine-N-oxide.

Trimethylamine (TMA) and its N-oxide (TMAO) are ubiquitous in prokaryote and eukaryote organisms as well as in the environment, reflecting their fundamental importance in evolutionary biology, and their diverse biochemical functions. Both metabolites have multiple biological roles including cell-signaling. Much attention has focused on the significance of serum and urinary TMAO in cardiovascular disease risk, yet this is only one of the many facets of a deeper TMA-TMAO partnership that reflects the significance of these metabolites in multiple biological processes spanning animals, plants, bacteria, and fungi. We report on analytical methods for measuring TMA and TMAO and attempt to critically synthesize and map the global functions of TMA and TMAO in a systems biology framework.

Effect of three water-regimes on morpho-physiological, biochemical and yield responses of local and foreign olive cultivars under field conditions.

BACKGROUND: Drought stress is among the most serious threats jeopardizing the economic yield of crop plants in Iran. In particular, in response to withholding irrigation, the reduction in performance and quality of a precious plant such as the olive tree is remarkable. Therefore, the selection of cultivars that are resistant or tolerant to drought has been recognized as one of the most effective long-term strategies for sustainably alleviating the adverse effects of this stress. In this view, our study evaluated the response of 8 olive cultivars including 4 elite native cultivars (Zard Aliabad, Roughani, Dezful, and Shengeh) and 4 foreign cultivars (Manzanilla, Sevillana, Konservolia, and Mission) to water shortage in the Dallaho Olive Research station of Sarpole-Zahab in Kermanshah province in 2020. Olive trees underwent 3 levels of irrigation treatment including 100% full irrigation (control), 75%, and 50% deficit irrigation. RESULTS: Based on the results, 50% deficit irrigation decreased both growth and pomological traits, but determined the highest dry matter percentage. As the severity of drought stress increased, with an accumulation of sodium and malondialdehyde, an incremental increase in osmolytes was observed, as well as an enhancement of the activity of antioxidant enzymes (peroxidase and catalase). In contrast, full irrigation led to an increase in photosynthetic pigments, calcium, and potassium. Dezful and Konservolia cultivars revealed a significantly higher growth rate, correlated in the former to higher levels of chlorophyll, compatible compounds, total phenolic content, relative water content, potassium to sodium ratio, catalase, and peroxidase activities compared with other cultivars. Konservolia showed the best yield parameters under 75% and 100% irrigation regimes, correlated to higher chlorophyll, potassium, and total phenolic content (in particular at 75% ET). CONCLUSIONS: Generally, the selection of more resilient or tolerant cultivars to sustain water scarcity stress is a widely operative solution to extend rainfed orchards in semi-arid environments. Our study showed that Dezful and Konservolia had the best adaptive mechanisms to cope with the detrimental effects of drought stress.

Glutamate Enhances Osmoadaptation of Anammox Bacteria under High Salinity: Genomic Analysis and Experimental Evidence.

An osmoprotectant that alleviates the bacterial osmotic stress can improve the bioreactor treatment of saline wastewater. However, proposed candidates are expensive, and osmoprotectants of anammox bacteria and their ecophysiological roles are not fully understood. In this study, a comparative analysis of 34 high-quality public metagenome-assembled genomes from anammox bacteria revealed two distinct groups of osmoadaptation. Candidatus Scalindua and Kuenenia share a close phylogenomic relation and osmoadaptation gene profile and have pathways for glutamate transport and metabolisms for enhanced osmoadaptation. The batch assay results demonstrated that the reduced Ca. Kuenenia activity in saline conditions was substantially alleviated with the addition and subsequent synergistic effects of potassium and glutamate. The operational test of two reactors demonstrated that the reduced anammox performance under brine conditions rapidly recovered by 35.7-43.1% as a result of glutamate treatment. The Ca. Kuenenia 16S rRNA and hydrazine gene expressions were upregulated significantly (p < 0.05), and the abundance increased by approximately 19.9%, with a decrease in dominant heterotrophs. These data demonstrated the effectiveness of glutamate in alleviating the osmotic stress of Ca. Kuenenia. This study provides genomic insight into group-specific osmoadaptation of anammox bacteria and can facilitate the precision management of anammox reactors under high salinity.

The role of the glycerol transporter channel Fps1p in cellular proteostasis during enhanced proteotoxic stress.

In response to osmotic shock, the components of high-osmolarity glycerol (HOG) pathway regulate the level of intracellular glycerol in yeast and ensure cell survival. Glycerol is a compatible solute and a stabiliser of proteins. Its role in maintaining proteostasis is less explored. We show that mild stress in the form of dietary restriction leads to increased glycerol level which increases cell viability. However, dietary restriction coupled with protein aggregation decreases intracellular glycerol level and attenuates cell viability. The transcript level of FPS1, the glycerol transporter channel, remains unchanged. However, its activity is altered under enhanced proteotoxic stress. Our results provide evidence for a probable role of the Fps1p channel in the cellular proteostasis network. KEY POINTS: • Dietary restriction led to increased accumulation of glycerol in Fps1-deleted yeast cells. • This led to lower protein aggregation in these cells. • Increased production of glycerol under dietary restriction was not linked to increased level of Fps1.

Exploring the Therapeutic Potential of Ectoine in Duchenne Muscular Dystrophy: Comparison with Taurine, a Supplement with Known Beneficial Effects in the mdx Mouse.

Duchenne Muscular Dystrophy (DMD) is a debilitating muscle disorder that condemns patients to year-long dependency on glucocorticoids. Chronic glucocorticoid use elicits many unfavourable side-effects without offering satisfying clinical improvement, thus, the search for alternative treatments to alleviate muscle inflammation persists. Taurine, an osmolyte with anti-inflammatory effects, mitigated pathological features in the mdx mouse model for DMD but interfered with murine development. In this study, ectoine is evaluated as an alternative for taurine in vitro in CCL-136 cells and in vivo in the mdx mouse. Pre-treating CCL-136 cells with 0.1 mM taurine and 0.1 mM ectoine prior to exposure with 300 U/mL IFN-γ and 20 ng/mL IL-1ß partially attenuated cell death, whilst 100 mM taurine reduced MHC-I protein levels. In vivo, histopathological features of the tibialis anterior in mdx mice were mitigated by ectoine, but not by taurine. Osmolyte treatment significantly reduced mRNA levels of inflammatory disease biomarkers, respectively, CCL2 and SPP1 in ectoine-treated mdx mice, and CCL2, HSPA1A, TNF-α and IL-1ß in taurine-treated mdx mice. Functional performance was not improved by osmolyte treatment. Furthermore, ectoine-treated mdx mice exhibited reduced body weight. Our results confirmed beneficial effects of taurine in mdx mice and, for the first time, demonstrated similar and differential effects of ectoine.

Upregulation of genes encoding plastidic isoforms of antioxidant enzymes and osmolyte synthesis impart tissue tolerance to salinity stress in bread wheat.

Wheat genotype Kharchia is a donor for salt tolerance in wheat breeding programs worldwide; however, the tolerance mechanism in Kharchia is yet to be deciphered completely. To avoid spending energy on accumulating organic osmolytes and to conserve resources for maintaining growth, plants deploy sodium (Na+) ions to maintain turgor. The enhanced ability to tolerate excess ion accumulation and ion toxicity is designated as tissue tolerance. In this study, salt-tolerant wheat genotype (Kharchia 65) and sensitive cultivars (HD2687, HD2009, WL711) were exposed to vegetative stage salinity stress (for four weeks). Kharchia 65 showed better tissue tolerance to salinity than the other genotypes based on different physiological parameters. Gene expression and abundance of chloroplast localized antioxidant enzymes and compatible osmolyte synthesis were upregulated by salinity in Kharchia 65. In Kharchia 65, the higher abundance of NADPH Oxidase (RBOH) transcripts and localization of reactive oxygen species (ROS) suggested an apoplastic ROS burst. Expression of calcium signaling genes of SOS pathway, MAPK6, bZIP6 and NAC4 were also upregulated by salinity in Kharchia 65. Considering that Kharchia local is the donor of salt tolerance trait in Kharchia 65, the publically available Kharchia local transcriptome data were analyzed. Our results and the in-silico transcriptome analysis also confirmed that higher basal levels and the stress-induced rise in the expression of plastidic isoforms of antioxidant enzymes and osmolyte biosynthesis genes provide tissue tolerance in Kharchia 65. Thus, in salinity tolerant genotype Kharchia 65, ROS burst mediated triggering of calcium signaling improves Na+ exclusion and tissue tolerance to Na+. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-022-01237-w.

Identification of the main glutamine and glutamate transporters in Staphylococcus aureus and their impact on c-di-AMP production.

A Staphylococcus aureus strain deleted for the c-di-AMP cyclase gene dacA is unable to survive in rich medium unless it acquires compensatory mutations. Previously identified mutations were in opuD, encoding the main glycine-betaine transporter, and alsT, encoding a predicted amino acid transporter. Here, we show that inactivation of OpuD restores the cell size of a dacA mutant to near wild-type (WT) size, while inactivation of AlsT does not. AlsT was identified as an efficient glutamine transporter, indicating that preventing glutamine uptake in rich medium rescues the growth of the S. aureus dacA mutant. In addition, GltS was identified as a glutamate transporter. By performing growth curves with WT, alsT and gltS mutant strains in defined medium supplemented with ammonium, glutamine or glutamate, we revealed that ammonium and glutamine, but not glutamate promote the growth of S. aureus. This suggests that besides ammonium also glutamine can serve as a nitrogen source under these conditions. Ammonium and uptake of glutamine via AlsT and hence likely a higher intracellular glutamine concentration inhibited c-di-AMP production, while glutamate uptake had no effect. These findings provide, besides the previously reported link between potassium and osmolyte uptake, a connection between nitrogen metabolism and c-di-AMP signalling in S. aureus.

An ICln homolog contributes to osmotic and low-nitrate tolerance by enhancing nitrate accumulation in Arabidopsis.

Nitrate (NO3- ) is a source of plant nutrients and osmolytes, but its delivery machineries under osmotic and low-nutrient stress remain largely unknown. Here, we report that AtICln, an Arabidopsis homolog of the nucleotide-sensitive chloride-conductance regulatory protein family (ICln), is involved in response to osmotic and low-NO3- stress. The gene AtICln, encoding plasma membrane-anchored proteins, was upregulated by various osmotic stresses, and its disruption impaired plant tolerance to osmotic stress. Compared with the wild type, the aticln mutant retained lower anions, particularly NO3- , and its growth retardation was not rescued by NO3- supply under osmotic stress. Interestingly, this mutant also displayed growth defects under low-NO3 stress, which were accompanied by decreases in NO3- accumulation, suggesting that AtICln may facilitate the NO3- accumulation under NO3- deficiency. Moreover, the low-NO3- hypersensitive phenotype of aticln mutant was overridden by the overexpression of NRT1.1, an important NO3- transporter in Arabidopsis low-NO3- responses. Further genetic analysis in the plants with altered activity of AtICln and NRT1.1 indicated that AtICln and NRT1.1 play a compensatory role in maintaining NO3- homeostasis under low-NO3- environments. These results suggest that AtICln is involved in cellular NO3- accumulation and thus determines osmotic adjustment and low-NO3- tolerance in plants.

Addressing the challenge of cold stress resilience with the synergistic effect of Rhizobium inoculation and exogenous melatonin application in Medicago truncatula.

Cold stress is an adverse environmental condition that limits the growth and yield of leguminous plants. Thus, discovering an effective way of ameliorating cold-mediated damage is important for sustainable legume production. In this study, the combined use of Rhizobium inoculation (RI) and melatonin (MT) pretreatment was investigated in Medicago truncatula plants under cold stress. Eight-week-old seedlings were divided into eight groups: (i) CK (no stress, noninoculated, no MT), (ii) RI (Rhizobium inoculated), (iii) MT (75 µM melatonin), (iv) RI+MT, (v) CS (cold stress at 4 °C without Rhizobium inoculation and melatonin), (vi) CS+RI, (vii) CS+MT, and (viii) CS+RI+MT. Plants were exposed to cold stress for 24 hrs. Cold stress decreased photosynthetic pigments and increased oxidative stress. Pretreatment with RI and MT alone or combined significantly improved root activity and plant biomass production under cold stress. Interestingly, chlorophyll contents increased by 242.81% and MDA levels dramatically decreased by 34.22% in the CS+RI+MT treatment compared to the CS treatment. Moreover, RI+MT pretreatment improved the antioxidative ability by increasing the activities of peroxidase (POD; 8.45%), superoxide dismutase (SOD; 50.36%), catalase (CAT; 140.26%), and ascorbate peroxidase (APX; 42.63%) over CS treated plants. Additionally, increased osmolyte accumulation, nutrient uptake, and nitrate reductase activity due to the combined use of RI and MT helped the plants counteract cold-mediated damage by strengthening the nonenzymatic antioxidant system. These data indicate that pretreatment with a combined application of RI and MT can attenuate cold damage by enhancing the antioxidation ability of legumes.

Cartap hydrochloride induced stress response in Anabaena variabilis ARM 441.

Cartap hydrochloride is a moderately hazardous nereistoxin analogue insecticide that is predominantly applied in paddy fields of India, at a recommended dose of 10 µg ml-1 to kill chewing and sucking insect pests of rice crop. Toxicity of cartap hydrochloride was studied on non-target free-living nitrogen fixing cyanobacterium Anabaena variabilis ARM 441 commonly used as algal biofertilizer in rice cultivation. Anabaena sp. could tolerate commercial grade insecticide up to 30 µg ml-1. However, at the recommended dose of 10 µg ml-1, it caused reduction in algal growth, total nitrogen and heterocyst frequency by 47.28, 24.29 and 17.72% respectively, as well as photosynthetic pigments under pure culture conditions. Scanning electron micrographs revealed cell rupture and breakage in filaments due to cartap exposure with the formation of akinetes. Cartap hydrochloride induced stress, since level of superoxide dismutase, peroxidase and catalase were increased by 108.57, 187.5 and 117% respectively. Generation of superoxide radicals and hydrogen peroxide were also increased by 152.48 and 34% respectively. Lipid peroxidation was increased by 31.03%, whereas there was decline in ascorbate content by 48.45%, however the glutathione content was increased by 128.57%. Increase in osmolytes such as proline from 8.6 to 32.8% and sucrose from 61.22 to 90.13% indicates their possible role in overcoming cartap induced oxidative stress and can be helpful in assessing its detrimental effect on Anabaena variabilis ARM 441, since cyanobacterial biofertilizers are purposely used in paddy fields as nitrogen contributors.

Combined Effects of Methylated Cytosine and Molecular Crowding on the Thermodynamic Stability of DNA Duplexes.

Methylated cytosine within CpG dinucleotides is a key factor for epigenetic gene regulation. It has been revealed that methylated cytosine decreases DNA backbone flexibility and increases the thermal stability of DNA. Although the molecular environment is an important factor for the structure, thermodynamics, and function of biomolecules, there are few reports on the effects of methylated cytosine under a cell-mimicking molecular environment. Here, we systematically investigated the effects of methylated cytosine on the thermodynamics of DNA duplexes under molecular crowding conditions, which is a critical difference between the molecular environment in cells and test tubes. Thermodynamic parameters quantitatively demonstrated that the methylation effect and molecular crowding effect on DNA duplexes are independent and additive, in which the degree of the stabilization is the sum of the methylation effect and molecular crowding effect. Furthermore, the effects of methylation and molecular crowding correlate with the hydration states of DNA duplexes. The stabilization effect of methylation was due to the favorable enthalpic contribution, suggesting that direct interactions of the methyl group with adjacent bases and adjacent methyl groups play a role in determining the flexibility and thermodynamics of DNA duplexes. These results are useful to predict the properties of DNA duplexes with methylation in cell-mimicking conditions.