Search

BackgroundSalicornia neei is a halophyte plant that has been proposed for use in the phytoremediation of the saline wastewater generated by land-based aquaculture, which usually contains elevated concentrations of ammonium resulting from protein metabolism. To identify the molecular mechanisms related to ammonium response, we analyzed the transcriptome of S. neei in response to growth in saline water containing 3 mM ammonium and the Michaelis–Menten ammonium removal biokinetics.ResultsThe RNA sequencing generated a total of 14,680,108 paired-end reads from the control and stressed conditions. De novo assembly using the CLC Genomic Workbench produced 86,020 transcripts and a reference transcriptome with an N50 of 683 bp.A total of 45,327 genes were annotated, representing 51.2% of the contig predicted from de novo assembly. As regards DEGs, a total of 9,140 genes were differentially expressed in response to ammonium in saline water; of these, 7,396 could be annotated against functional databases. The upregulated genes were mainly involved in cell wall biosynthesis, transmembrane transport and antiporter activities, including biological KEGG pathways linked to the biosynthesis of secondary metabolites, plant hormone signal transduction, autophagy, and nitrogen metabolism. In addiction, a set of 72 genes was directly involved in ammonium metabolism, including glutamine synthetase 1 (GLN1), glutamate synthase 1 (GLT1), and ferredoxin-dependent glutamate synthase chloroplastic (Fd-GOGAT). Finally, we observed that the ammonium uptake rate increased with increasing ammonium concentrations, and tended toward saturation in the range of 3 to 4 mM.ConclusionOur results support the hypothesis that an ammonium detoxification system mediated by glutamine and glutamate synthase was activated in S. neei when exposed to ammonium and saline water. The present transcriptome profiling method could be useful when investigating the response of halophyte plants to saline wastewater from land-based aquaculture.

Objective SCN8A epileptic encephalopathy is caused predominantly by de novo gain-of-function mutations in the voltage-gated-sodium channel Nav 1.6. The disorder is characterized by early onset of seizures and developmental delay. Most patients with SCN8A epileptic encephalopathy are refractory to current anti-seizure medications. Previous studies determining the mechanisms of this disease have focused on neuronal dysfunction as Nav 1.6 is expressed by neurons and plays a critical role in controlling neuronal excitability. However, glial dysfunction has been implicated in epilepsy and alterations in glial physiology could contribute to the pathology of SCN8A encephalopathy. In the current study, we examined alterations in astrocyte and microglia physiology in the development of seizures in a mouse model of SCN8A epileptic encephalopathy. Methods Using immunohistochemistry we assessed microglia and astrocyte reactivity before and after the onset of spontaneous seizures. Expression of glutamine synthetase, Nav 1.6 and Kir 4.1 channel currents were assessed in astrocytes in wildtype (WT) mice and mice carrying the N1768D SCN8A mutation (D/+). Results Astrocytes in spontaneously seizing D/+ mice become reactive and increase expression of glial fibrillary acidic protein (GFAP), a marker of astrocyte reactivity. These same astrocytes exhibited reduced barium-sensitive Kir 4.1 currents compared to age-matched WT mice and decreased expression of glutamine synthetase. These alterations were only observed in spontaneously seizing mice and not before the onset of seizures. In contrast, microglial morphology remained unchanged before and after the onset of seizures. Significance Astrocytes, but not microglia, become reactive only after the onset of spontaneous seizures in a mouse model of SCN8A encephalopathy. Reactive astrocytes have reduced Kir 4.1-mediated currents, which would impair their ability to buffer potassium. Reduced expression of glutamine synthetase would modulate the availability of neurotransmitters to excitatory and inhibitory neurons. These deficits in potassium and glutamate handling by astrocytes could exacerbate seizures in SCN8A epileptic encephalopathy. Targeting astrocytes may provide a new therapeutic approach to seizure suppression.

The accumulation of excess glutamate in the synapse leads to excitotoxicity, which is the underlying reason of neuronal death in intracranial tumors. We identified the expression levels of glutamate dehydrogenase, glutamine synthetase and sirtuin 4 in U87 cell line and various intracranial tumors. mRNA expressions of glutamate dehydrogenase (GDH), glutamine synthetase (GS) and sirtuin 4 (SIRT4) were analyzed in various intracranial tumors using qPCR. GDH, GS and SIRT4 protein expressions were analyzed in glioblastoma (U87) and glial (IHA-immortalized human astrocytes) cell lines via western blotting. The protein expressions of SIRT4 and GS were shown to be elevated and GDH protein expression was reduced in U87 cells in comparison to IHA cells. All types of intracranial tumors displayed lower GS mRNA expressions compared to controls. SIRT4 mRNA expressions were also shown to be lower in all the tumors and grades, although not significantly. GDH mRNA expression was found to be similar in all groups. The molecular mechanisms of glutamate metabolism and excitotoxicity should be discovered to develop therapies against intracranial tumors.

Glutamine synthetase (GS) is a decameric enzyme that plays a key role in nitrogen metabolism. Acetylation of the N-terminal degron (N-degron) of GS is essential for ubiquitylation and subsequent GS degradation. The full-length GS structure showed that the N-degron is buried inside the GS decamer and is inaccessible to the acetyltransferase. The structure of N-degron-truncated GS reported here reveals that the N-degron is not essential for GS decamer formation. It is also shown that the N-degron can be exposed to a solvent region through a series of conformational adjustments upon ligand binding. In summary, this study elucidated the dynamic movement of the N-degron and the possible effect of glutamine in enhancing the acetylation process.

Bungarus multicinctus is one of the top ten venomous snakes in China, and its bite causes acute and severe diseases, but its pathophysiology remains poorly elucidated. Thus, an animal model of Bungarus multicinctus bite was established by intramuscular injection of 30μg/kg of Bungarus multicinctus venom, and then the serum metabolites were subsequently screened, identified and validated by ultra-performance liquid chromatography-quadrupole-time of flight-mass spectrometry (UPLC-Q-TOF-MS) methods to explore the potential biomakers and possible metabolic pathways. Untargeted metabolomics analysis showed that 36 and 38 endogenous metabolites levels changed in ESI+ and ESI-, respectively, KEGG pathway analysis showed that 5 metabolic pathways, including mineral absorption, central carbon metabolism in cancer, protein digestion and absorption, aminoacyl-tRNA biosynthesis and ABC transporters might be closely related to Bungarus multicinctus bite. Targeted metabolomics analysis showed that there were significant differences in serum D-proline, L-leucine and L-glutamine after Bungarus multicinctus bite (P < 0.05). In addition, receiver operating characteristic (ROC) analysis showed that the diagnostic efficiency of L-Glutamine was superior to other potential biomarkers and the AUC value was 0.944. Moreover, we found evidence for differences in the pathophysiology of glutamine between Bungarus multicinctus bite group and normal group, specifically with the content of glutamine synthetase (GS) and glutaminase (GLS). Taken together, the current study has successfully established an animal model of Bungarus multicinctus bite, and further identified the links between the metabolic perturbations and the pathophysiology and the potential diagnostic biomakers of Bungarus multicinctus bite, which provided valuable insights for studying the mechanism of Bungarus multicinctus bite.

Nowadays, among several abiotic stresses, salt, especially NaCl and UV-B are of major concern. They lead to deleterious effects on plant growth and ultimately affect crop productivity. The present study was planned to find out some ameliorative solution against these stresses. Here, the modulatory action of two oxylipins, namely, methyl jasmonate (MeJA) and 12-Oxo-phytodienoic acid (OPDA) on growth, photosynthetic performance, nitrate/ammonia assimilating enzymes, and nutritive values of Luffa Mill. seedlings grown under NaCl (20 and 40 mM) and/or enhanced UV-B stresses (ambient: 8.2 kJ m−2 d−1 + additional: 2.2 kJ m−2 s−1) were analyzed. Both the stresses when given alone, negatively affected the fresh mass, root/shoot ratio, leaf area, photosynthetic pigments content, photosynthetic oxygen yield and, chlorophyll a fluorescence kinetic parameter. This decline was further aggravated upon combined exposure to the stressors. However, supplementation of MeJA/OPDA effectively counteracted the negative impact on important growth-regulating processes. The activities of nitrate reductase (NR), nitrite reductase (NiR), glutamine synthetase (GS), and glutamate synthase (GOGAT) enzymes, as well as the contents of inorganic nitrogen, protein, and carbohydrate, were increased with the supplementation of MeJA/OPDA. The increase in the Na+ and Cl‾ contents due to NaCl or/and UV-B was depreciated by MeJA or OPDA. Ameliorating behaviour of MeJA or OPDA is correlated with improved photosynthetic activity and nitrogen metabolism. These findings, point out that supplementation of MeJA/OPDA, particularly OPDA more favourably regulated the growth-promoting activities, which can be linked with the mitigation of NaCl and UV-B stress.

Bioavailable nitrogen is the limiting nutrient for most agricultural food production. Associative diazotrophs can colonize crop roots and fix their own bioavailable nitrogen from the atmosphere. Wild-type (WT) associative diazotrophs, however, do not release fixed nitrogen in culture and are not known to directly transfer fixed nitrogen resources to plants. Efforts to engineer diazotrophs for plant nitrogen provision as an alternative to chemical fertilization have yielded several strains that transiently release ammonia. However, these strains suffer from selection pressure for nonproducers, which rapidly deplete ammonia accumulating in culture, likely limiting their potential for plant growth promotion (PGP). Here we report engineered Azospirillum brasilense strains with significantly extend ammonia production lifetimes of up to 32 days in culture. Our approach relies on multicopy genetic redundancy of a unidirectional adenylyltransferase (uAT) as a posttranslational mechanism to induce ammonia release via glutamine synthetase deactivation. Testing our multicopy stable strains with the model monocot Setaria viridis in hydroponic monoassociation reveals improvement in plant growth promotion compared to single copy strains. In contrast, inoculation of Zea mays in nitrogen-poor, nonsterile soil does not lead to increased PGP relative to WT, suggesting strain health, resource competition, or colonization capacity in soil may also be limiting factors. In this context, we show that while engineered strains fix more nitrogen per cell compared to WT strains, the expression strength of multiple uAT copies needs to be carefully balanced to maximize ammonia production rates and avoid excessive fitness defects caused by excessive glutamine synthetase shutdown.

Therapy-induced senescence (TIS) is a major cellular response to anticancer therapies. While induction of a persistent growth arrest would be a desirable outcome in cancer therapy, it has been shown that, unlike normal cells, cancer cells are able to evade the senescence cell cycle arrest and to resume proliferation, likely contributing to tumor relapse. Notably, cells that escape from TIS acquire a plastic, stem cell-like phenotype. The metabolic dependencies of cells that evade senescence have not been thoroughly studied. In this study, we show that glutamine depletion inhibits escape from TIS in all cell lines studied, and reduces the stem cell subpopulation. In line with a metabolic reliance on glutamine, escaped clones overexpress the glutamine transporter SLC1A5. We also demonstrate a central role of glutamine synthetase that mediates resistance to glutamine deprivation, conferring independence from exogenous glutamine. Finally, rescue experiments demonstrate that glutamine provides nitrogen for nucleotides biosynthesis in cells that escape from TIS, but also suggest a critical involvement of glutamine in other metabolic and non-metabolic pathways. On the whole, these results reveal a metabolic vulnerability of cancer stem cells that recover proliferation after exposure to anticancer therapies, which could be exploited to prevent tumor recurrence.

Thiocyanate (SCN−) is a nitrogen-containing pollutant, which can be involved in the nitrogen (N) cycle and interferes with plant growth. The current study highlights a new insight into the N (nitrate [NO3-] and ammonium [NH4+]) utilization ways in rice seedlings under SCN− exposure to clarify the interactive effect on uptake and assimilation between these N-containing chemicals. Phenotypically, relative growth rates (RGR) of NO3−-fed seedlings were significantly higher than NH4+-fed rice seedlings at the same SCN− concentration. Both N fertilizations have no significant influence on SCN− content and its assimilation in rice seedlings. However, significant accumulation of NO3− and NH4+ were detected in shoots prior to roots under SCN− stress. Enzymatic assay and mRNA analysis showed that the carbonyl sulfide (COS) pathway of SCN− degradation occurred in both roots and shoots of NO3−-fed seedlings but only evident in roots of NH4+-fed seedlings. Moreover, the effect of SCN− on the activity of nitrate reductase (NR), glutamine synthetase (GS), and glutamate synthase (GOGAT) was negligible in NO3−-fed seedlings, while GOGAT activity was significantly inhibited in shoots of NH4+-fed seedlings. Nitrogen use efficiency (NUE) estimation provided positive evidence in utilizing NO3− over NH4+ as the main N source to support rice seedling growth during detoxification of exogenous SCN−. Overall, SCN− pollution has unexpectedly changed the rice preference for N source which shifted from NH4+ to NO3−, suggesting that the interactions of SCN− with different N sources in terms of uptake and assimilation in rice plants should not be overlooked, especially at the plant N nutritional level.

The effects of glutamine dipeptide (GDP) on the growth performance, intestinal digestive enzyme activity, immune metabolism index, and gene expression of related factors of Penaeus vannamei were investigated in present study. Results showed that GDP obviously increased the weight gain rate, specific growth rate, and survival rate of P. vannamei but decreased the feed conversion ratio (FCR). The activities of amylase, protease, catalase (CAT), glutathione peroxidase (GSH-Px), and Na+–K+–ATPase in the intestine of P. vannamei fed with 0.5% and 1% GDP were significantly higher than those of the control group (P

Melatonin (MT) and salicylic acid (SA) are known to improve plant tolerance to environmental stresses; however, no reports have evaluated the effect of their combined treatment on plants under both normal and stressful conditions. For the first time, the present study aimed to investigate MT and SA’s potential role in regulating polyamine and nitrogen metabolism in wheat plants subjected to salty soils, and counterbalancing oxidative damage induced by salt stress. Moreover, this research aimed to elucidate a possible link between melatonin, salicylic acid, nitrogen and polyamine levels. Wheat (Triticum aestivum L. cv. Sids 14) plants were grown under non-saline or saline conditions (6.0 and 12.0 dS m–1) and were foliar sprayed with 70 µM MT and/or 75 mg l−1 SA. The injury impacts of salt stress on wheat growth and production were significantly alleviated by exogenous MT and/or SA treatments. This was evidenced by increasing polyamines content through accelerating the metabolic flow from the precursor amino acids arginine and methionine to polyamines, decreasing the polyamines degradation, and enhancing the polyamines biosynthesis. Furthermore, MT and/or SA are involved in promoting nitrogen metabolism through up-regulating the activities of N uptake and metabolism related enzymes (nitrate reductase, nitrite reductase, glutamine synthetase, glutamate synthase) as well as enhancing the content of nitrogen, nitrate and protein in salt-stressed plants. The mitigation was also detected in reduced lipid peroxidation, hydrogen peroxide content, carbonyl content, and protease activity in stressed treated plants. Notably, the best response was registered for the combined treatment of MT and SA. Therefore, the co-application of MT and SA is a promising way for ameliorating salt toxicity in sustainable agricultural systems.

Understanding the reproductive stage salinity stress tolerance is a key target for breeding stress tolerant rice genotypes. Nitrate and ammonium are equally important nitrogen forms utilized by rice. We evaluated nitrate and ammonium assimilation during reproductive stage in control and salinity (10dSm-1 using NaCl) stressed rice plants. Osmotic stress tolerant rice genotype Shabhagidhan (SD) and high yielding yet osmotic and salinity stress sensitive genotype Pusa sugandh-5 (PS5) were evaluated. Salinity stress was given to plants during panicle emergence and flag leaves was collected after 1d, 3d 5d, 7d, 9d,12d and 15d after anthesis. Reproductive stage salinity stress resulted in decrease of membrane stability, relative water content and osmotic potential of rice plants. Reproductive stage salinity stress decreased the expression of nitrate reductase (OsNIA), nitrite reductase (OsNiR), Glutamine synthetase (OsGLN1.1, OsGLN1.2, OsGLN2) and glutamate synthase/GOGAT (OsFd-GOGAT, OsNADH-GOGAT) in flag leaves. In response to stress, SD showed better stress tolerance than PS5 in terms of higher yield stability. Variety SD showed higher leaf nitrate and ammonium content and maintained comparatively higher nitrate and ammonia metabolism enzyme activity than PS5. Salinity stress upregulated the activity of glutamate dehydrogenase enzyme and indirectly contributed to the higher proline content and maintenance of favourable osmotic potential in SD. Expression of GS2 which has role in photo respiratory ammonia assimilation was upregulated by salinity stress in PS5 in comparison to SD. Rice genotype showing better induction of nitrogen assimilatory genes will be more tolerant to reproductive stage salinity stress.

Transgenic rice with Bacillus thuringiensis (Bt) genes has been successfully cultivated in recent years. The stable and sustainable expression of Bt protein is the guarantee of effective resistance within these strains against target pests. It is well known that the synthesis of Bt protein in Bt crops needs nitrogen (N). Therefore, how is the efficacy of transgenic Bt rice against target pests in response to N application? In our study, we found that enhanced nitrogen inputs increased soluble protein content (SPC) and glutamine synthetase (GS), but decreased amino acid content (AAC); it is evident that the protein synthesis was enhanced, and degradation was reduced with increasing nitrogen application. Thus, both increased protein synthesis and decreased protein degradation contributed to the increased Bt protein content (BTC) in transgenic Bt rice. Responses of the three Bt rice lines to N proportions and N sources were different. All these differences can be explained by the changes of nitrogen metabolism of different genotypes. The BTC in leaves was higher than that in other organs, and the fully expanded green leaf had higher BTC than the senescent leaf and tender leaf. Despite the difference of Bt protein expression, all transgenic rice lines showed 100% resistance to target pests under different N conditions. Our results indicated that the response of Bt protein expression in transgenic Bt rice with different genotypes to N fertilizer was inconsistent; moreover, there exist obvious spatial-temporal differences. Therefore, in order to guarantee the stability and sustainability resistance of Bt rice, it is necessary to adopt appropriate N management practice according to the expression pattern of Bt protein of transgenic Bt rice with different genotypes.

Paraquat (1,1'-dimethyl-4,4'-bipyridinium dichloride; PQ) is a widely used herbicide in Brazilian crops, despite its banishment in many other countries. The present study investigated the effects of repeated dose of PQ on glutamate system, energy metabolism and redox parameters in the hippocampus of prepubertal rats. Twenty-two-day-old rats received daily intraperitoneal injections of PQ (10 mg/Kg) during 5 consecutive days and the effects of the pesticide were assessed 24 h after the last injection. The PQ exposure provoked cytotoxicity associated to decreased cell viability and increased glutamate excitotoxicity, as demonstrated by decreased 14C-glutamate uptake and increased 45Ca2+ uptake. Downregulated glutamine synthetase (GS) activity, further supports disrupted glutamate metabolism compromising the glutamate-glutamine cycle. Downregulated 14C-2-Deoxy-D-glucose indicates energy failure and upregulated lactate dehydrogenase (LDH) suggests the relevance of lactate as energy fuel. Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) upregulation suggest Krebs cycle replenishment and piruvate production. In addition, PQ disturbed the redox status inducing lipid peroxidation, evaluated by increased TBARS and imbalanced antioxidant system. Downregulated glutathione reductase (GR), gamma-glutamyltransferase (GGT), glutathione-S-transferase (GST) and glucose-6-P-dehydrogenase (G6PD) activities together with upregulated superoxide dismutase (SOD) and catalase activities corroborate the oxidative imbalance. The mechanisms underlying PQ-induced neurotoxicity involves the modulation of GSK-3β, NF-κB and NMDA receptors. These neurochemical and oxidative events observed may contribute to neuroinflammation and neurotoxic effects of PQ on hippocampal cells.

Sulfur compounds are known to play crucial roles in plants facilitating tolerance to heavy metal stress. Previous studies have shown the protective role of exogenous cysteine (Cys) to improve chromium (Cr) tolerance. In the present study, we aimed to evaluate the capability of Cys (0.5 mM) to reduce chromium toxicity (100 μM) in rapeseed by physiological and proteomic analyses. Seedlings treated with Cr showed reduced growth and activities of antioxidant enzymes, and increased malondialdehyde (MDA) content. Exogenous application of Cys reduced Cr accumulation and MDA content, restored catalase (CAT) and peroxidase (POD) activities, and caused significant enhancements in seedling growth. 2-DE gel electrophoresis and mass spectrometry were performed to investigate changes in the root proteome in response to Cys and Cr. The Cr-induced down-regulations of glycine-rich RNA-binding protein, lactoylglutathione lyase, fructose-bisphosphate aldolase, and glutamine synthetase were restored with Cys. Additionally, Cys removed the Cr-induced up-regulation of some proteins including pathogenesis-related protein, major latex protein (MLP) like proteins, 22.0 kDa HSP, triosephosphate isomerase, glyceraldehyde-3-phosphate dehydrogenase, glutamine amidotransferase, cysteine synthase, and L-lactate dehydrogenase. Taken together, proteomic analysis revealed that exogenous Cys helps plants to overcome Cr stress by modulating energy metabolism, amino acid metabolism, and defense mechanism.

In the current study, the role of chlorophyll precursor, 5-aminolevulinic acid (ALA) and its effect on growth attributes, photosynthetic pigments, indices of chlorophyll a fluorescence, nitrogen metabolism, and contents of protein and carbohydrate in seedlings of pigeon pea varities BAHAR and KB-17 under the enhanced ultraviolet (UV-B; 2.2 kJ m−2d−1) radiations was investigated. Ultraviolet radiations (UV-B) adversely affected the fresh mass of shoot and root, shoot and root length, photosynthetic pigments content (Chl a + b and ratios of Chl a/b and Chl/Car), photosynthetic oxygen yield and chlorophyll a fluorescence parameters: i.e., Fv/Fm (Phi_P0), ψ0 (Psi_0), φE0 (Phi_E0) and performance index of PS II (PIABS) along with Fv/F0 while increased the energy flux parameters, i.e., ABS/RC, TR0/RC, ET0/RC, and DI0/RC along with F0/Fv and also consequently raised the dark respiratory rate in test seedlings; however, exogenous application with both the doses of ALA (low dose: ALA1; 25 µM and high dose: ALA2; 100 µM) through seed priming notably counteracted UV-B induced negative effects on growth attributes and other important growth-regulating processes. Additionally, inorganic nitrogen contents, i.e., nitrate (NO3−) and nitrite (NO2−) and the activities of nitrate assimilating enzymes, viz., nitrate reductase and nitrite reductase and ammonia assimilating enzymes, viz., glutamine synthetase and glutamate synthase (GOGAT) along with carbohydrate and protein contents were rigorously affected and declined under UV-B toxicity; while in present study, ammonium (NH4+) content and glutamate dehydrogenase (GDH) activity in both BAHAR and KB-17 varieties exhibited reverse trend. Furthermore, ALA priming induced significant alliveation against UV-B stress on these parameters except NH4+ content and GDH activity, which exhibited an apparent reduction under similar conditions. Our results suggest that ALA priming, particularly high dose (ALA2) more efficiently protects Cajanus cajan seedlings by attenuating UV-B toxicity in both the varieties (BAHAR and KB-17) by maintaining carbon and nitrogen status of seedlings through regulating photosynthesis and nitrogen metabolism.

Glutamine synthetase (GS) plays an important role in nitrogen (N) metabolism in cucumber. In this study, we cloned and sequenced the CsGS1 gene, and analyzed the expression patterns and subcellular localization of the GS1 protein in response to different N conditions in order to determine its role in low-nitrogen (LN) tolerance. CsGS1 was abundantly expressed in the leaves of the low N-requiring cultivar D0328, while the high N-requiring cultivar D0422 showed similar expression levels across different tissues including leaves, shoots and roots. Furthermore, the GS1 protein was primarily localized in the cytoplasm of plant cells. Both cultivars were then transformed with the CsGS1 coding sequence or antisense sequence via Agrobacterium tumefaciens in order to overexpress and silence GS1 expression, respectively. Overexpression of CsGS1 significantly improved LN tolerance and photosynthetic parameters, and increased chlorophyll b content, biomass, plant height, root length, N accumulation and GS activity under LN condition compared to the control. CsGS1 silencing on the other hand significantly reduced the above indices. Taken together, CsGS1 is crucial for maintaining N metabolism in cucumber plants during N deprivation, and is a promising target for generating novel transgenic breeds with increasing nitrogen utilization efficiency.

Because it is the precursor for various essential cellular components, the amino acid serine is indispensable for every living organism. In plants, serine is synthesized by two major pathways: photorespiration and the phosphorylated pathway of serine biosynthesis (PPSB). However, the importance of these pathways in providing serine for plant development is not fully understood. In this study, we examine the relative contributions of photorespiration and PPSB to providing serine for growth and metabolism in the C3 model plant Arabidopsis thaliana. Our analyses of cell proliferation and elongation reveal that PPSB-derived serine is indispensable for plant growth and its loss cannot be compensated by photorespiratory serine biosynthesis. Using isotope labeling, we show that PPSB-deficiency impairs the synthesis of proteins and purine nucleotides in plants. Furthermore, deficiency in PPSB-mediated serine biosynthesis leads to a strong accumulation of metabolites related to nitrogen metabolism. This result corroborates 15N-isotope labeling in which we observed an increased enrichment in labeled amino acids in PPSB-deficient plants. Expression studies indicate that elevated ammonium uptake and higher glutamine synthetase/glutamine oxoglutarate aminotransferase (GS/GOGAT) activity causes this phenotype. Metabolic analyses further show that elevated nitrogen assimilation and reduced amino acid turnover into proteins and nucleotides are the most likely driving forces for changes in respiratory metabolism and amino acid catabolism in PPSB-deficient plants. Accordingly, we conclude that even though photorespiration generates high amounts of serine in plants, PPSB-derived serine is more important for plant growth and its deficiency triggers the induction of nitrogen assimilation, most likely as an amino acid starvation response.

Backgrouond Grain chalkiness lowers the market value of rice. Alleviating grain chalkiness is the most challenging issue in many rice-producing areas of the world. Nitrogen (N) metabolism has received increasing attention as a result of its relationship with grain chalkiness, although little information is available on the mechanism of N-induced grain chalk. Results A highly chalky rice variety OM052 was used to explore the protein synthesis and its accumulation in the grain exposed to N topdressing (N+) at the panicle initiation stage and a control (N-). The results showed that chalky kernels were stimulated by the N+ treatment and more prone to occur on the top and primary rachis. The grain protein content was increased because of the increased average and maximum rates of protein accumulation during grain filling, which was related to the enhanced activities of glutamine synthetase, glutamate synthase, glutamic oxalo-acetic transaminase and glutamate pyruvate transaminase under the N+ treatment. The activities of these enzymes at 15 days after flowering (DAF) were notably positively correlated with grain chalky traits and protein content. Conclusion N topdressing regulates the synthesis and accumulation of the protein by affecting the key enzymes, especially at 15 DAF, which is attributed to grain chalkiness in rice. © 2021 Society of Chemical Industry.

Salinity is one of the well-known abiotic stresses resulting in loss of wheat yield. The identification of differentially induced genes and then becoming aware of their function in salt-stressed wheat is imperative to enhance salinity resistance. To determine salt-responsive genes, we evaluated the transcriptome derived from control and stressed wheat by cDNA-AFLP procedure. A total of 31 transcript-derived fragments (TDFs) were sequenced with success. Most of the genes recognized herein, via BLASTX search of TDFs versus GenBank database, belonged to several functional groups such as transport, protein degradation, transcription regulation, signal transduction, cell defense, energy, and metabolism. Real-time PCR revealed that four TDFs were down-regulated and 18 were up-regulated of 22 TDFs. The HAK, CBL3, serine/threonine PK, cysteine proteinase, LTP, glutamine synthetase, PCAP, DNA glycosylase, PEPC, sulfate permease, and FRA2 genes were specified responsible for salinity stress tolerance. Further research on the new salt-responsive TDFs identified in our experiment can present useful information helping the improvement of wheat tolerance to a high level of salt in the field. These findings altogether improved our understanding of cellular mechanisms involved in wheat response to soil salinization.

This study aimed to investigate the impact of the herbicide monuron on various aspects of photosynthesis and diazotrophy in the cyanobacterium Nostoc muscorum Meg 1 isolated from a rice field in Cherrapunji, Meghalaya, India. The consequences of monuron exposure on various photosynthetic pigments, functioning of PSII, RuBisCO, nitrogenase and glutamine synthetase enzyme activities, heterocyst frequency, and on production of proteins and carbohydrates in the cyanobacterium were studied in a range of monuron doses (20–100 ppm) to gauge the herbicide’s effect on CO2 and N2 fixation and on net biomass production. The total amount of D1 protein (the host for PSII complex), RuBisCO (the key enzyme for CO2 fixation), nitrogenase (the enzyme responsible for the reduction of atmospheric nitrogen to ammonia), and ammonia assimilating enzyme glutamine synthetase (GS) contents under western blot analysis indicated interruption of new protein synthesis and breakdown of their existing enzyme molecules when exposed to higher monuron concentrations. All parameters studied showed enhanced expression under low dose monuron treatment (20 ppm) indicating a hormetic effect in the exposed organism. The expression of monuron toxicity on various parameters of CO2 and N2 fixation in a dose-dependent manner was immediately visible when cultures were treated with higher doses (40–100 ppm). Scanning and transmission electron microscopic studies further uncovered several undesirable changes in the morphology and ultrastructure of the organism due to herbicide treatment that could be correlated to compromised CO2 and N2 fixation.

Previous studies have suggested that multiwalled carbon nanotubes (MWCNTs) promote plant growth; however, the mechanism is yet to be fully understood. In this study, the effects of MWCNTs (20, 100, and 500 mg/L) on the carbon (C) and nitrogen (N) metabolism in maize were studied to explore the molecular mechanism of the action of MWCNTs on plants. The results showed that 100 mg/L MWCNTs increased the shoot fresh and dry weight, root fresh weight, and seedling length while other doses showed no significant effects. Further studies showed that 100 mg/L MWCNTs increased the chlorophyll content, transpiration rate, stomatal conductance, and intercellular CO2 concentration, by 50.6%, 60.8%, 47.2%, and 32.1%, respectively. Activities of key enzymes including sucrose synthase (SS), sucrose phosphate synthase (SPS) and phosphoenolpyruvate carboxylase (PEPC) that are involved in the carbon metabolism, and nitrate reductase (NR), glutamine synthetase (GS), and glutamate synthetase (GOGAT) that are involved in N metabolism, were all upregulated by 100 mg/L MWCNTs, which contributed to the increase of the accumulation of carbohydrates (sugar and starch), soluble protein, and N in plants. These findings suggest that MWCNTs can improve plant growth by regulating the key enzymes involved in C and N metabolism thereby enhancing the carbohydrate production and the use of N and improving plant growth. This study provides significant insights into the molecular mechanism of the positive effects of MWCNTs on plants and provide a basis for the agricultural application of MWCNTs.

Heterosis refers to the improved agronomic performance of F1 hybrids relative to their parents. Although this phenomenon is widely employed to increase biomass, yield, and stress tolerance of plants, the underlying molecular mechanisms remain unclear. To dissect the metabolic fluctuations derived from genomic and/or environmental differences contributing to the improved biomass of F1 hybrids relative to their parents, we optimized the growth condition for Arabidopsis thaliana F1 hybrids and their parents. Modest but statistically significant increase in the biomass of F1 hybrids was observed. Plant samples grown under the optimized condition were also utilized for integrated omics analysis to capture specific changes in the F1 hybrids. Metabolite profiling of F1 hybrids and parent plants was performed using gas chromatography-mass spectrometry. Among the detected 237 metabolites, 2-oxoglutarate (2-OG) and malate levels were lower and the level of aspartate was higher in the F1 hybrids than in each parent. In addition, microarray analysis revealed that there were 44 up-regulated and 12 down-regulated genes with more than 1.5-fold changes in expression levels in the F1 hybrid compared to each parent. Gene ontology (GO) analyses indicated that genes up-regulated in the F1 hybrids were largely related to organic nitrogen (N) process. Quantitative PCR verified that glutamine synthetase 2 (AtGLN2) was upregulated in the F1 hybrids, while other genes encoding enzymes in the GS-GOGAT cycle showed no significant differences between the hybrid and parent lines. These results suggested the existence of metabolic regulation that coordinates biomass and N metabolism involving AtGLN2 in F1 hybrids.

There is an increasing interest in the use of N2-fixing bacteria for the sustainable biofertilization of crops. Genetically-optimized bacteria for ammonium release have an improved biofertilization capacity. Some of these strains also cross-feed ammonium into microalgae raising additional concerns on their sustainable use in agriculture due to the potential risk of producing a higher and longer-lasting eutrophication problem than synthetic N-fertilizers. Here we studied the dynamic algal cross-feeding properties of a genetically-modified Azotobacter vinelandii strain which can be tuned to over-accumulate different levels of glutamine synthetase (GS, EC 6.3.1.20) under the control of an exogenous inducer. After switching cells overaccumulating GS into a noninducing medium, they proliferated for several generations at the expense of the previously accumulated GS. Further dilution of GS by cell division slowed-down growth, promoted ammonium-excretion and cross-fed algae. The final bacterial population, and timing and magnitude of algal N-biofertlization was finely tuned in a deferred manner. This tuning was in accordance with the intensity of the previous induction of GS accumulation in the cells. This bacterial population behavior could be maintained up to about 15 bacterial cell generations, until faster-growing and nonammonium excreting cells arose at an apparent high frequency. Further improvements of this genetic engineering strategy might help to align efficiency of N-biofertilizers and safe use in an open environment. KEY POINTS: • Ammonium-excreting bacteria are potential eutrophication agents • GS-dependent deferred control of bacterial growth and ammonium release • Strong but transient ammonium cross-feeding of microalgae.

The study of plant responses to increases in atmospheric carbon dioxide (CO2) concentration is crucial to understand and to predict the effect of future global climate change on plant adaptation and evolution. Increasing amount of nitrogen (N) can promote the positive effect of CO2, while how N forms would modify the degree of CO2 effect is rarely studied. The aim of this study was to determine whether the amount and form of nitrogen (N) could mitigate the effects of elevated CO2 (eCO2) on enzyme activities related to carbon (C) and N metabolism, the C/N ratio, and growth of Phoebe bournei (Hemsl.) Y.C. Yang. One-year-old P. bournei seedlings were grown in an open-top air chamber under either an ambient CO2 (aCO2) (350 ± 70 μmol•mol−1) or an eCO2 (700 ± 10 μmol•mol−1) concentration and cultivated in soil treated with either moderate (0.8 g per seedling) or high applications (1.2 g per seedling) of nitrate or ammonium. In seedlings treated with a moderate level of nitrate, the activities of key enzymes involved in C and N metabolism (i.e., Rubisco, Rubisco activase and glutamine synthetase) were lower under eCO2 than under aCO2. By contrast, key enzyme activities (except GS) in seedlings treated with high nitrate or ammonium were not significantly different between aCO2 and eCO2 or higher under eCO2 than under aCO2. The C/N ratio of seedlings treated with moderate or high nitrate under eCO2was significantly changed compared with the seedlings grown under aCO2, whereas the C/N ratio of seedlings treated with ammonium was not significantly different between aCO2 and eCO2. Therefore, under eCO2, application of ammonium can be beneficial C and N metabolism and mitigate effects on the C/N ratio.

Pancreatic ductal adenocarcinoma (PDAC) has a poor 5-year survival rate and lacks effective therapeutics. Therefore, it is of paramount importance to identify new targets. Using multiplex data from patient tissue, three-dimensional coculturing in vitro assays, and orthotopic murine models, we identified Netrin G1 (NetG1) as a promoter of PDAC tumorigenesis. We found that NetG1+ cancer-associated fibroblasts (CAF) support PDAC survival, through a NetG1-mediated effect on glutamate/glutamine metabolism. Also, NetG1+ CAFs are intrinsically immunosuppressive and inhibit natural killer cell–mediated killing of tumor cells. These protumor functions are controlled by a signaling circuit downstream of NetG1, which is comprised of AKT/4E-BP1, p38/FRA1, vesicular glutamate transporter 1, and glutamine synthetase. Finally, blocking NetG1 with a neutralizing antibody stunts in vivo tumorigenesis, suggesting NetG1 as potential target in PDAC. Significance: This study demonstrates the feasibility of targeting a fibroblastic protein, NetG1, which can limit PDAC tumorigenesis in vivo by reverting the protumorigenic properties of CAFs. Moreover, inhibition of metabolic proteins in CAFs altered their immunosuppressive capacity, linking metabolism with immunomodulatory function. See related commentary by Sherman, p. 230. This article is highlighted in the In This Issue feature, p. 211

Increasing post-flowering nitrogen (N) uptake is likely to improve seed N accumulation, ultimately leading to greater seed yield and N use efficiency (NUE). A comprehensive study on contrasting N utilization efficiency (NUtE) winter oilseed rape genotypes was conducted for three years (2017–2020) to unravel the effect of post-flowering N uptake on seed N accumulation. Compared to the low NUtE genotype, the high NUtE genotype displayed respectively 31%, 28% and 70% greater root biomass, length and volume, along with 40%, 44%, 46% and 82% higher root nitrate reductase (NR), glutamine synthetase (GS), glutamate synthetase (GOGAT), and glutamate dehydrogenase (GDH) activities after flowering. These were accompanied by a significant increase (P < 0.05) in catalases (134%), peroxidase (45%), glutathione (45%) and ascorbate peroxidase (41%), leading to 27% higher seed N accumulation, 23% higher seed yield and 60% higher NUE. Stronger post-flowering N uptake potential for high NUtE genotype was a main contributor to the enhanced seed N accumulation, and ultimately increased seed yield and NUE.

Graphical abstract, Highlights • Aridanin, scopoletin, naringenin and ferulic acid were identified in methanol fruit extract of Tetrapleura tetraptera (TT) by HPLC-DAD. • TT demonstrated high antioxidant activity and radical scavenging ability in vitro. • TT maintained cellular redox status, viability and mitochondrial membrane potential. • Aridanin, from TT has a good binding affinity with glutamine synthetase in silico. • TT optimized glutamine synthetase so as to attenuate the severity of excitotoxicity., Oxidative stress and excitotoxicity are some of the pathophysiological abnormalities in hypoxia-induced brain injury. This study evaluated the intrinsic antioxidant property of methanol fruit extract of Tetrapleura tetraptera (TT), traditionally used for managing brain diseases such as cerebral infarction in West Africa, and its ability to protect primary astrocytes from anoxia-induced cell death. The effect of the phytochemicals present in TT on excitotoxicity was assessed in silico, through docking with human glutamate synthetase (hGS). Chromatographic and spectrophotometric analyses of TT were performed. Primary astrocytes derived from neural stem cells were treated with TT and its effect on astrocyte viability was assessed. TT-treated astrocytes were then subjected to anoxic insult and, cell viability and mitochondrial membrane potential were evaluated. Molecular docking of hGS with detected phytochemicals in TT (aridanin, naringenin, ferulic acid, and scopoletin) was performed and the number of interactions with the lead compounds, aridanin, analyzed. HPLC-DAD analysis of TT revealed the presence of various bioactive phytochemicals. TT demonstrated notable antioxidant and radical scavenging activities. TT also protected astrocytes from anoxic insult by restoring cell viability and preventing alteration to mitochondrial membrane integrity. Aridanin, naringenin, ferulic acid, and scopoletin demonstrated good binding affinities with hGS indicating that Tetrapleura tetraptera is a potential source of new plant-based bioactives relevant in the therapy of neurodegenerative diseases.

Efficacy of glufosinate ammonium is influenced by temperature. However, several studies show variable results. The response of ryegrass (Lolium spp.) to glufosinate ammonium activity at different t...

Introduction. Few published studies have examined the role of glia-neuron interactions in neurodegen-eration. In this regard, the search for new experimental models is important. L-alpha-aminoadipic acid (L-AA), being a structural analogue of glutamate with a selective toxic effect on astroglia, is of particular interest. However, morphological and neurochemical changes caused by L-AA still remain unclear. The aim of the study was to characterize immunomorphological changes of glia and neurons in the striata of rats after L-AA administration. Materials and methods. On days 3 and 12 after L-AA stereotaxic administration, we studied astrocytic pro-teins localization using immunofluorescence methods: GFAP, vimentin, glutamine synthetase, along with oligodendroglia (by cyclonucleotidphosphatase expression), microglia (IBA1 Ca-binding protein), neuronal nuclear protein NeuN, and the astroglia proliferative activity (based on Ki67 localization). Results. We detected astrocyte death and a decrease in glutamine synthetase immunoreactivity in the le-sioned area, but no changes in the microglia reaction and the L-AA effect on neurons and oligodendroglia. The astrocyte loss was replenished by proliferation and migration of newly formed immature astrocytes, and a glial scar formed on day 12 after the surgery. Conclusion. L-AA administration, which causes the death of striatum astrocytes in the injection area, can serve as a convenient model for studying reactive changes in astroglia and astrocytic dysfunction while revealing the pathogenetic patterns of neurodegenerative processes. Keywords: astrocytes, striatum, L-alpha-aminoadipic acid, gliosis, glial toxin

Glutamine synthetase (GS) and glutamate synthase (GOGAT) play a central role in plant nitrogen (N) metabolism. In order to study the effect of powdery mildew (Blumeria graminis f. sp. tritici, Bgt) on N metabolism, field experiments were carried out to evaluate GS and GOGAT activity, GS expression and grain protein content (GPC) in susceptible (Xi'nong 979) and resistant (Zhengmai 103) wheat cultivars under three treatments. The three treatments were no inoculation (CK), inoculated once with Bgt (MP) and inoculated nine times with Bgt (HP). For Xi'nong 979, the activities of GS and GOGAT in grains as well as GS activity in flag leaves increased at 10–15 days after anthesis (DAA), and decreased significantly at 15 or 20–30 DAA in HP and MP. However, GS activity in grains decreased from 20 DAA, which was later than that of flag leaves (15 DAA). At the same time, GS expression in grains was up-regulated at early stage, with GS1 at 10 DAA and GS2 at 15 DAA, followed by a continuous down-regulation. This result indicated that GS and GOGAT activity as well as GS expression were inhibited by powdery mildew, indicating that N metabolism in grains was inhibited at 20–30 DAA. The current study also found out that the yield of the susceptible cultivar decreased significantly, while its GPC increased obviously in HP. It was shown that the increase of GPC was not due to the enhancement of N metabolism, but due to the passive increase caused by yield reduction.

Soil bacteria from the genus Streptomyces, phylum Actinobacteria, feature a complex metabolism and diverse adaptations to environmental stress. These characteristics are consequences of variable nutrition availability in the soil and allow survival under changing nitrogen conditions. Streptomyces coelicolor is a model organism for Actinobacteria and is able to use nitrogen from a variety of sources including unusual compounds originating from the decomposition of dead plant and animal material, such as polyamines or monoamines (like ethanolamine). Assimilation of nitrogen from these sources in S. coelicolor remains largely unstudied. Using microbiological, biochemical and in silico approaches, it was recently possible to postulate polyamine and monoamine (ethanolamine) utilization pathways in S. coelicolor. Glutamine synthetase-like enzymes (GS-like) play a central role in these pathways. Extensive studies have revealed that these enzymes are able to detoxify polyamines or monoamines and allow the survival of S. coelicolor in soil containing an excess of these compounds. On the other hand, at low concentrations, polyamines and monoamines can be utilized as nitrogen and carbon sources. It has been demonstrated that the first step in poly-/monoamine assimilation is catalyzed by GlnA3 (a γ-glutamylpolyamine synthetase) and GlnA4 (a γ-glutamylethanolamide synthetase), respectively. First insights into the regulation of polyamine and ethanolamine metabolism have revealed that the expression of the glnA3 and the glnA4 gene are controlled on the transcriptional level.

Dopamine plays numerous physiological roles in plants. We explored its role in the regulation of growth, nutrient absorption, and response to nitrogen (N) deficiency in Malus hupehensis Rehd. Under low N condition, plant growth slowed, and the net photosynthetic rates, chlorophyll contents, and maximal quantum yield of PSII (Fv/Fm) decreased significantly. However, the application of 100 μmol L−1 exogenous dopamine significantly reduced the inhibition of low N stress on plant growth. In addition to modifying root system architecture under low N supply, exogenous dopamine also changed the uptake, transport, and distribution of N, P, and K. Furthermore, exogenous dopamine enhances the tolerance to low nitrogen stress by increasing the activity of enzymes (nitrate reductase, nitrite reductase, glutamic acid synthase and glutamine synthetase) involved in N metabolism. We also found that exogenous dopamine promoted the expression of ethylene signaling genes (ERF1, ERF2, EIL1, ERS2, ETR1, and EIN4) under low N stress. Therefore, we hypothesized that ethylene might be involved in dopamine response to low N stress in M. hupehensis. Our results suggest that exogenous dopamine can mitigate low N stress by regulating the absorption of mineral nutrients, possibly through the regulation of the ethylene signaling pathway.

Background. The aim of this study was to research the effects of glutamine synthetase (GS) and glutamate aspartate transporter (GLAST) in rat Müller cells and the effects of an adenosine A2AR antagonist (SCH 442416) on GS and GLAST in hypoxia both in vivo and in vitro. Methods. This study used RT-PCR and Western blotting to quantify the expressions of GS and GLAST under different hypoxic conditions as well as the expressions of GS and GLAST at different drug concentrations. A cell viability assay was used to assess drug toxicity. Results. mRNA and protein expression of GS and GLAST in hypoxia Group 24 h was significantly increased. mRNA and protein expressions of GS and GLAST both increased in Group 1 μM SCH 442416 compared with other groups. One micromolar SCH 442416 could upregulate GS and GLAST’s activity in hypoxia both in vivo and in vitro. Conclusions. Hypoxia activates GS and GLAST in rat retinal Müller cells in a short time in vitro. (2) A2AR antagonists upregulate the activity of GS and GLAST in hypoxia both in vivo and in vitro.

Optimizing the supply of biosynthetic precursors and cofactors is usually an effective metabolic strategy to improve the production of target compounds. Here, the combination of optimizing precursor synthesis and balancing cofactor metabolism was adopted to improve tryptophan production in Escherichia coli. First, glutamine synthesis was improved by expressing heterologous glutamine synthetase from Bacillus subtilis and Bacillus megaterium in the engineered Escherichia coli strain KW001, resulting in the best candidate strain TS-1. Then icd and gdhA were overexpressed in TS-1, which led to the accumulation of 1.060 g/L tryptophan. Subsequently, one more copy of prs was introduced on the chromosome to increase the flux of 5-phospho-α- d -ribose 1-diphosphate followed by the expression of mutated serA and thrA to increase the precursor supply of serine, resulting in the accumulation of 1.380 g/L tryptophan. Finally, to maintain cofactor balance, sthA and pntAB, encoding transhydrogenase, were overexpressed. With sufficient amounts of precursors and balanced cofactors, the engineered strain could produce 1.710 g/L tryptophan after 48 h of shake-flask fermentation, which was 2.76-times higher than the titer of the parent strain. Taken together, our results demonstrate that the combination of optimizing precursor supply and regulating cofactor metabolism is an effective approach for high-level production of tryptophan. Similar strategies could be applied to the production of other amino acids or related derivatives.

Pomacea canaliculata, known as an invasive freshwater snail, is also called a golden apple snail; its survival and expansion are greatly affected by temperature. In this study, high-throughput sequencing (RNA-seq) was used to perform comparative transcriptome analysis on the muscular tissue (G_M) of snails in Guangdong and Hunan. Differential gene screening was performed with FDR 1 as the threshold, and a total of 1,368 differential genes were obtained (671 genes showed upregulation in snails from Guangdong, and 697 genes displayed upregulation in snails from Hunan). Fifteen genes were identified as candidate genes for the cold hardiness of Pomacea canaliculata. Among them, three genes were involved in energy metabolism (glycogen synthase, 1; DGK, 1; G6PD, 1); seven genes were involved in homeostasis regulation (HSP70, 2; BIP, 1; GPX, 1; GSTO 1, G6PD, 1; caspase-9, 1); two genes were involved in amino acid metabolism (glutamine synthetase, 1; PDK, 1); and four genes were involved in membrane metabolism (inositol-3-phosphate synthase, 1; Na+/K+-ATPase, 1; calcium-binding protein, 2). This study presents the molecular mechanisms for the cold hardiness of Pomacea canaliculata, which could provide a scientific basis for the forecast and prevention of harm from Pomacea canaliculata.

Injudicious use of chemical fertilizers particularly nitrogen to crops has increased dramatically in the last half-century and, therefore, identifying crop varieties with improved nitrogen efficiency is urgent for sustainable development. In this study, a pot experiment was conducted to screen the intrinsic nitrogen efficiency of eleven cabbage genotypes on the basis of various morpho-physiological and biochemical parameters related to nitrogen metabolism. High genotypic variation was found in nitrogen metabolizing enzymes. A significant variation in nitrate reductase, nitrite reductase, glutamine synthetase and glutamate synthase activity was found between contrasting (low and high) nitrogen efficient genotypes in all the nitrogen treatments. The experimental data were subjected to Principal component analysis and Agglomerate hierarchical clustering. Based on the data, Early Golden Acre and Pusa Drumhead were screened as low and high nitrogen efficiency genotypes respectively. Our study identifies low and high nitrogen efficient genotypes in cabbage which can be used in breeding programs and could help in addressing the problem of nitrate pollution in the environment and also could lessen the economic burden of fertilizers on farmers.

Euryhaline tilapia (Oreochromis mossambicus) are fish that tolerate a wide salinity range from fresh water to > 3× seawater. Even though the physiological effector mechanisms of osmoregulation that maintain plasma homeostasis in fresh water and seawater fish are well known, the corresponding molecular mechanisms that control switching between hyper- (fresh water) and hypo-osmoregulation (seawater) remain mostly elusive. In this study we show that hyperosmotic induction of glutamine synthetase represents a prominent part of this switch. Proteomics analysis of the O. mossambicus OmB cell line revealed that glutamine synthetase is transcriptionally regulated by hyperosmolality. Therefore, the 5′ regulatory sequence of O. mossambicus glutamine synthetase was investigated. Using an enhancer trapping assay, we discovered a novel osmosensitive mechanism by which intron 1 positively mediates glutamine synthetase transcription. Intron 1 includes a single, functional copy of an osmoresponsive element, osmolality/salinity-responsive enhancer 1 (OSRE1). Unlike for conventional enhancers, the hyperosmotic induction of glutamine synthetase by intron 1 is position dependent. But irrespective of intron 1 position, OSRE1 deletion from intron 1 abolishes hyperosmotic enhancer activity. These findings indicate that proper intron 1 positioning and the presence of an OSRE1 in intron 1 are required for precise enhancement of hyperosmotic glutamine synthetase expression.

β-N-Methylamino- l -alanine (BMAA), a neurotoxin naturally produced by cyanobacteria, diatoms and dinoflagellates, constitutes a serious environmental and health threat especially during acute blooms, which are becoming more frequent. This neurotoxin is implicated in several neurodegenerative diseases (ND) in humans through contaminated water or food consumption. Even low doses of neurotoxic compounds (NCs) can have lasting effects later in life. In this sense, early stages of development constitute a period of high sensitivity to environmental influence, particularly for the central nervous system. To understand the mechanisms underlying the delayed effects of NCs, newly hatched larvae of the mangrove rivulus fish, Kryptolebias marmoratus, were exposed to two sub-lethal doses of BMAA (20 μg/L and 15 mg/L) for 14 days. This fish naturally produces isogenic lineages due to its self-fertilizing reproduction, which is unique case among vertebrates. It thus provides genetic characteristics that allow scientists to study organisms’ true reaction norm, minimizing genetic variability and focusing exclusively on the effects of the environment. Effect assessment was performed at different levels of biological organization to detect inconspicuous effects of BMAA, since this molecule displays long retention in organisms. BMAA effects on life history traits as well as behavioral traits such as boldness and aggressiveness were assessed more than 100 days after exposure. In addition, the relative expression of 7 potential BMAA target genes was studied, given their involvement in neurotransmission or their association with individual variation in boldness and aggressiveness. Selected genes code for reticulon 4 (RTN4), glutamate vesicular transporter 1 (Slc17a7), glutamine synthetase a (Glula), dopamine receptor D4 (DRD4), monoamine oxidase A (MAOA), calmodulin (CaM) and epedymine (Epd). Despite observing no effects of BMAA on growth, reproduction and behavioral traits, BMAA induced a significant increase of the expression of CaM and MAOA genes at 20 μg/L BMAA compared to the control group. A significant decrease of expression was observed between this lowest BMAA dose and 15 mg/L for DRD4, MAOA and CaM genes. Our results suggest disruption of glutamate turnover, intracellular dopamine depletion and activation of astrocyte protective mechanisms, indicating that BMAA might be excitotoxic. Our study revealed that BMAA can have long-lasting effects on the brain that are suspected to affect phenotypic traits with aging. Furthermore, it highlights the importance of studying delayed effects in ecotoxicological studies.

This study aims to investigate the utility of digital protocols for Ki-67 immunohistochemistry quantitative analysis (“hot spot” method) in the setting of well-differentiated hepatocellular neoplasms. Resection cases of typical hepatic adenomas (HAs) (n = 40), atypical HAs (n = 9), and well-differentiated hepatocellular carcinomas (WD HCCs) (n = 56) were selected. HAs were further classified by immunohistochemistry using antibodies against liver fatty acid binding protein, glutamine synthetase, B-catenin, hepatic serum amyloid A, and C-reactive protein. Ki-67 proliferative index by immunohistochemistry was evaluated in all cases by digital analysis using a modified neuroendocrine tumor “hot spot” protocol. The proliferative rate of HAs (typical, median 1.2% (range 0–7.4%) and atypical, median 1.0% (range 0.3–3%)) was significantly lower than that of WD HCCs (median 4.5%, range 0–49.8%) (P

Low sunlight intensity from mid-June to mid-July in Southern China is the primary environmental factor that affects maize grain development. A field trial was conducted in 2016–2017 to investigate the effects of post-silking shading (i.e., 30% and 50% light deprivation using black net covers; ambient light as control) on grain filling, abscisic acid (ABA), and indole-3-acetic acid (IAA) contents, starch and protein contents, and enzymatic activities involved in biosynthesis by using two maize varieties, namely, the shade-sensitive Zhengdan958 (ZD958) and the shade-resistant Suyu30 (SY30). Results showed that grain filling was negatively affected by shading. Under 30% and 50% shading conditions, grain dry weight decreased by14.6% and 29.6% for ZD958, respectively, and 10.8% and 15.1% for SY30, respectively. Shading improved protein content, but restricted starch deposition. The activities of starch and protein synthetic enzymes were downregulated by shading, and the effect was severe when the plants were subjected to greater light deprivation (from 30 to 50%). Shading decreased IAA content, but increased ABA content. Under shading conditions, SY30 had higher activities of glutamine synthetase, sucrose synthase, and soluble starch synthase than ZD958. Moreover, SY30 generally had a higher IAA content, but lower ABA content than ZD958. These properties favored starch accumulation and increased grain weight. In conclusion, post-silking shading decreased IAA content and weakened the activities of starch and protein syntheses, but increased ABA content. These conditions restricted starch deposition, thereby resulting in grain weight loss.

Background l-Alanyl-l-glutamine (AQ) is a functional dipeptide with high water solubility, good thermal stability and high bioavailability. It is widely used in clinical treatment, post-operative rehabilitation, sports health care and other fields. AQ is mainly produced via chemical synthesis which is complicated, time-consuming, labor-intensive, and have a low yield accompanied with the generation of by-products. It is therefore highly desirable to develop an efficient biotechnological process for the industrial production of AQ. Results A metabolically engineered E. coli strain for AQ production was developed by over-expressing l-amino acid α-ligase (BacD) from Bacillus subtilis, and inactivating the peptidases PepA, PepB, PepD, and PepN, as well as the dipeptide transport system Dpp. In order to use the more readily available substrate glutamic acid, a module for glutamine synthesis from glutamic acid was constructed by introducing glutamine synthetase (GlnA). Additionally, we knocked out glsA–glsB to block the first step in glutamine metabolism, and glnE–glnB involved in the ATP-dependent addition of AMP/UMP to a subunit of glutamine synthetase, which resulted in increased glutamine supply. Then the glutamine synthesis module was combined with the AQ synthesis module to develop the engineered strain that uses glutamic acid and alanine for AQ production. The expression of BacD and GlnA was further balanced to improve AQ production. Using the final engineered strain p15/AQ10 as a whole-cell biocatalyst, 71.7 mM AQ was produced with a productivity of 3.98 mM/h and conversion rate of 71.7%. Conclusion A metabolically engineered strain for AQ production was successfully developed via inactivation of peptidases, screening of BacD, introduction of glutamine synthesis module, and balancing the glutamine and AQ synthesis modules to improve the yield of AQ. This work provides a microbial cell factory for efficient production of AQ with industrial potential.

Global warming has reached an alarming situation, which led to a dangerous climatic condition. The irregular rainfalls and land degradation are the significant consequences of these climatic changes causing a decrease in crop productivity. The effect of drought and its tolerance mechanism, a comparative roots proteomic analysis of chickpea seedlings grown under hydroponic conditions for three weeks, performed at different time points using 2-Dimensional gel electrophoresis (2-DE). After PD-Quest analysis, 110 differentially expressed spots subjected to MALDI-TOF/TOF and 75 spots identified with a significant score. These identified proteins classified into eight categories based on their functional annotation. Proteins involved in carbon and energy metabolism comprised 23% of total identified proteins include mainly glyceraldehyde-3-phosphate dehydrogenase, malate dehydrogenase, transaldolase, and isocitrate dehydrogenase. Proteins related to stress response (heat-shock protein, CS domain protein, and chitinase 2-like) contributed 16% of total protein spots followed by 13% involved in protein metabolism (adenosine kinase 2, and protein disulfide isomerase). ROS metabolism contributed 13% (glutathione S-transferase, ascorbate peroxidase, and thioredoxin), and 9% for signal transduction (actin-101, and 14-3-3-like protein B). Five percent protein identified for secondary metabolism (cinnamoyl-CoA reductase-1 and chalcone-flavononeisomerase 2) and 7% for nitrogen (N) and amino acid metabolism (glutamine synthetase and homocysteine methyltransferase). The abundance of some proteins validated by using Western blotting and Real-Time-PCR. The detailed information for drought-responsive root protein(s) through comparative proteomics analysis can be utilized in the future for genetic improvement programs to develop drought-tolerant chickpea lines.

Glufosinate inhibits glutamine synthetase (GS), a key enzyme for amino acid metabolism and photorespiration. Protoporphyrinogen oxidase (PPO) inhibitors block chlorophyll biosynthesis and cause protoporphyrin accumulation, a highly photodynamic intermediate. Both herbicides ultimately lead to plant death by a massive accumulation of reactive oxygen species (ROS) through different mechanisms. We investigated a potential synergistic effect by the mixture of the two herbicide mechanisms of action (MoAs). The tank mix between a low rate of glufosinate (280 g ai ha−1) with an ultra-low dose of saflufenacil (1 g ha−1) provided enhanced herbicidal activity compared with the products applied individually on Palmer amaranth (Amaranthus palmeriS. Watson). The synergism between the two herbicides was also confirmed by isobole analysis and field trials. The herbicide combination provided high levels of efficacy when applied at low temperature and low humidity. Mechanistically, glufosinate caused a transient accumulation of glutamate, the building block for chlorophyll biosynthesis. Consequently, inhibition of both GS and PPO resulted in greater accumulation of protoporphyrin and ROS, forming the physiological basis for the synergism between glufosinate and PPO inhibitors. While the synergy between the two herbicide MoAs provided excellent efficacy on weeds, it caused low injury to PPO-resistant waterhemp [Amaranthus tuberculatus(Moq.) Sauer] and high injury to both glufosinate-resistant and glufosinate-susceptible soybean [Glycine max(L.) Merr.]. Glufosinate enhances the activity of PPO inhibitors through glutamate and protoporphyrin accumulation, leading to increased levels of ROS and lipid peroxidation. The synergism between the two herbicide MoAs can help to overcome environmental effects limiting the efficacy of glufosinate. Future research is needed to optimize the uses for this herbicidal composition across different cropping systems.

Bisphenol A (BPA) is one of the most widely used chemicals in plastic industry, which enters the human body through occupational and food contact. We studied the protein changes in rat cerebral cortex to evaluate the neurotoxicity of BPA. Twenty-four adult male rats were randomly selected and divided into four groups and each group respectively received 0, 0.5, 5 and 50 mg/kg of BPA for 4 weeks orally. To determine the oxidative status, reduced glutathione content and the level of malondialdehyde were measured in brain cortical tissue. The proteins of each sample extracted and separated on a two-dimensional acrylamide gel electrophoresis. From the obtained protein map, the 10 most altered protein spots were used for mass spectroscopy analysis. The lipid peroxidation in both doses of 0.5 and 5 mg/kg was significantly higher than the control group, but the glutathione content had no significant difference between the groups. Based on the results of the MS data analysis by the MASCOT database search engine, 10 proteins with altered intensity were identified as pyruvate kinase, alpha-enolase, aconitate hydratase, creatine kinase B-type, phosphatidylethanolamine-binding protein 1, 14-3-3 protein eta, guanine nucleotide-binding protein subunit beta-1, dihydropyrimidinase-related protein 2, glutamine synthetase and the neurofilament light polypeptide. There are several reports suggesting that the increase or decrease in the level and activity of these 10 proteins, similar to those observed in this study, is related to some neurological and psychosocial disorders including neurodegenerative diseases, schizophrenia, depression, epilepsy and some brain tumors.

Drought stress is an important crop yield limiting factor worldwide. Plant physiological responses to drought stress are driven by changes in gene expression. While drought-responsive genes (DRGs) have been identified in maize, regulation patterns of gene expression during progressive water deficits remain to be elucidated. In this study, we generated time-series transcriptomic data from the maize inbred line B73 under well-watered and drought conditions. Comparisons between the two conditions identified 8,626 DRGs and the stages (early, middle, and late drought) at which DRGs occurred. Different functional groups of genes were regulated at the three stages. Specifically, early and middle DRGs display higher copy number variation among diverse Zea mays lines, and they exhibited stronger associations with drought tolerance as compared to late DRGs. In addition, correlation of expression between small RNAs (sRNAs) and DRGs from the same samples identified 201 negatively sRNA/DRG correlated pairs, including genes showing high levels of association with drought tolerance, such as two glutamine synthetase genes, gln2 and gln6. The characterization of dynamic gene responses to progressive drought stresses indicates important adaptive roles of early and middle DRGs, as well as roles played by sRNAs in gene expression regulation upon drought stress.