Higher Glutamine Synthetase Activity Research Articles

Nitrogen Supply Mitigates Heat Stress on Photosynthesis of Maize (Zea mays L.) During Early Grain Filling by Improving Nitrogen Assimilation

ABSTRACTHigh temperature during early grain‐filling stage is one of the serious abiotic stresses limiting maize yield in the North China Plain. Nitrogen (N) fertiliser has an important role in promoting crop growth, especially under abiotic stresses. However, its contribution to alleviating heat stress (HS) inhibition on maize photosynthesis during early grain‐filling stage is still unclear. Experiments with three N rates (LN, low nitrogen; MN, medium nitrogen; HN, high nitrogen) and two temperature (HS, heat stress; CK, ambient temperature as control) regimes were conducted to examine the effects of increasing N supply on photosynthesis, N assimilation, antioxidant system, and hormones homeostasis of maize during early grain‐filling stage using two maize hybrids Xianyu335 (XY335, heat‐sensitive) and Zhengdan (ZD958, heat‐tolerant). HS negatively affected photosynthesis of both two hybrids, exhibited lower net photosynthetic rate, chlorophyll content and activities of Rubisco and phosphoenolpyruvate carboxylase (PEPC) compared with CK, and then decreased dry matter accumulation of maize, with a lesser extent for ZD958 than XY335. However, increasing N supply alleviated the adverse effects of HS on maize photosynthesis due to improved N assimilation capacity. Under HS condition, greater N content and higher activities of glutamine synthetase and glutamate synthase in maize ear leaf were found in treatment of HN compared with LN and MN. HN with higher N assimilation capacity directly increased the net photosynthetic rate due to improved chlorophyll content, activities of Rubisco and PEPC and antioxidant capacity. HS‐induced abscisic acid (ABA) accumulation was also repressed by HN, and then enhanced the stomatal conductance and transpiration rate to maintain higher photosynthetic capacity compared with LN and MN. Moreover, the positive effects of increasing N supply on maize photosynthesis under HS condition exhibited a larger extent for XY335 than ZD958. As a result of improved photosynthesis and N assimilation capacity by adequate N supply, maize accumulated more biomass under HS, especially for heat‐sensitive hybrid.

Nitrate Reductase and Glutamine Synthetase Enzyme Activities and Chlorophyll in Sorghum Leaves (Sorghum bicolor) in Response to Organic Fertilization

Sorghum is a plant that mainly requires chemical nitrogen fertilization. There are organic fertilizers that can provide nutrients to plants with great benefits to the soil, such as chicken manure. To determine the influence of organic fertilization on nitrate reductase (NR), glutamine synthetase (GS), and the amount of chlorophyll, sorghum plants were grown using the following four treatments: soil (T1), soil + chicken manure 100 kg ha−1 of nitrogen (N) (T2), soil + chicken manure 200 kg ha−1 N (T3), and soil + ammonium sulfate 100 kg ha−1 N (T4). Leaves were sampled in the vegetative stage (VS), the reproductive stage (RS), and the maturation stage (MS). The highest NR activity occurred in plants with T2 and T3 in the VS. The highest GS activity was in T3 and T4 in the RS. The amount of chlorophyll a was the same in all phenological stages. However, the amount of chlorophyll b was influenced by the type of fertilization at different phenological stages. Organic fertilizers (OF) produced the highest NR activity. On the other hand, GS activity was higher with chemical fertilization (T4), which was equal to the second dose of organic fertilization (T3). Finally, chlorophyll a and b were influenced by both types of fertilization, and was different from T1.

Gene expression profiling indicates a shift in ammonia assimilation capacity along the hepatic acinus induced by different forms of selenium in vitamin–mineral mixes fed to beef steers grazing on toxic endophyte-infected tall fescue

Recently, we demonstrated that supplementing (with 3 mg Se per day) the diets of growing beef steers grazing on Se-deficient toxic endophyte-infected tall fescue-based forage with either organic Se (OSe, SEL-PLEX) or a 1:1 blend (MIX) of OSe and inorganic Se (ISe, sodium selenite) in vitamin–mineral mixes, rather than inorganic forms of Se, ameliorated several classic serum symptoms of fescue toxicosis. Importantly, higher levels of hepatic glutamine synthetase activity were observed in MIX and OSe steers. Accordingly, transcriptome level and targeted mRNA expression analyses were conducted on the same liver tissue to determine if Se treatments affected other hepatic metabolic pathways, especially those that are responsible for supplying substrates of glutamine synthetase. The effect of ISe, OSe, and MIX treatments (n = 8/treatment) on the relative abundances of mRNA [determined using microarray and real-time reverse-transcription PCR (RT-PCR)] and protein (determined using Western blotting) in liver tissue was assessed by ANOVA. Fisher’s protected LSD procedure was used to separate treatment means, with significance being declared at p ≤ 0.05. Microarray analysis identified (p< 0.01, false discovery rate of< 33%) 573 annotated differentially expressed gene (DEG) transcripts. Canonical pathway analysis identified the DEGs that are central to glutamine and glutamate biosynthesis/degradation and proline biosynthesis. Targeted RT-PCR analyses found that MIX and OSe steers had lower periportal ammonia-assimilation and urea-synthesizing capacities (lower glutaminase 2, key ornithine cycle enzymes, and mitochondrial ornithine/citrulline exchanger mRNA) than ISe steers. In addition, MIX and OSe steers had a higher capacity for pericentral ammonia assimilation (higher glutamine synthetase activity) and a higher capacity for the production of glutamate in pericentral hepatocytes from α-ketoglutarate (higher levels of glutamine dehydrogenase, and decreased levels of arginase 2 and ornithine aminotransferase and mRNA). The form of supplemental Se also affected steers’ capacity for hepatic proline metabolism, with OSe steers having a higher capacity for proline synthesis and MIX steers having a higher capacity for pyroline-5-carboxylate synthesis. In conclusion, supplementing the diets of growing beef steers grazing on toxic endophyte-infected tall fescue with MIX and OSe in vitamin–mineral mixes, rather than with inorganic forms of Se, shifts hepatic ammonia assimilation from periportal urea production to pericentral glutamine production, thus potentially increasing whole-animal N efficiency by increasing the supply of hepatic-synthesized glutamine.

Inorganic Nitrogen Uptake Characteristics of Three Typical Bloom-Forming Algae in the East China Sea

Inorganic nitrogen (N) is an important element for eutrophication and harmful algal bloom (HAB) formation. However, the roles of inorganic N in HAB outbreaks are still unclear. Here, we compared the affinities and abilities for inorganic N uptake and assimilation among three typical bloom-forming algae in the East China Sea (ECS), Skeletonema costatum, Prorocentrum donghaiense and Alexandrium pacificum by investigating the uptake and enzymatic (nitrate reductase (NR) and glutamine synthetase (GS) kinetics for nitrate and ammonia. The Ks of nitrate and ammonium in S. costatum was lower than those in P. donghaiense and A. pacificum. The NR activity of S. costatum and P. donghaiense exhibited a positive relationship with the nitrate concentration, and NR activity of S. costatum was nearly 4-fold higher than that of P. donghaiense at high nitrate concentration. However, the NR activity of A. pacificum could not be detected. The GS activity of three species decreased with the increase of ammonium concentrations, and the highest GS activity was detected in A. pacificum. S. costatum presented the highest affinity for nitrate and ammonium, followed by P. donghaiense and A. pacificum. Moreover, P. donghaiense exhibited the highest affinity for intracellular ammonium. Our results characterized the differences in inorganic nitrogen uptake among the three typical bloom-forming algae, which may contribute to the formation of blooms in the coastal waters of the ECS.

Macrophage-derived glutamine boosts satellite cells and muscle regeneration

Muscle regeneration is sustained by infiltrating macrophages and consequent satellite cell (SC) activation1–4. Macrophages and SC communicate in different ways1–5 but their metabolic interplay was never investigated so far. Here, we found that muscle injuries and aging are characterized by intratissutal glutamine restriction. Low glutamine levels endow macrophages with the metabolic ability to secrete glutamine via enhanced glutamine synthetase (GS) activity at the expense of glutamate dehydrogenase-1 (GLUD1)-mediated glutamine oxidation. Glud1 knockout (KO) macrophages display constitutively high GS activity which prevents glutamine shortage. Import of macrophage-derived glutamine by SC through the glutamine-transporter SLC1A5 activates mTOR and promotes SC proliferation and differentiation. Consequently, macrophage-specific deletion or pharmacological inhibition of GLUD1 improves muscle regeneration and functional recovery in response to acute injury, ischemia, or aging. Conversely, SLC1A5 blockade in SC or GS inactivation in macrophages negatively affects SC functions and muscle regeneration. These results highlight a metabolic cross-talk between SC and macrophages whereby macrophage-derived glutamine sustains SC functions. Thus, GLUD1 targeting offers new therapeutic opportunities for the regeneration of injured or aged muscles.

Phenotypical evidence of effective amelioration of ammonium-inhibited plant (root) growth by exogenous low urea

Ammonium and nitrate are major soil inorganic-nitrogen sources for plant growth, but many species cultivated with even low millimolar NH4+ as a sole N form display a growth retardation. To date, critical biological components and applicable approaches involved in the effective enhancement of NH4+ tolerance remain to be thoroughly explored. Here, we report phenotypical traits of urea-dependent improvement of NH4+-suppressed plant/root growth. Urea at 0.1 mM was sufficient to remarkably stimulate NH4+ (3 mM)-fed cotton growth, showing a 2.5∼4-fold increase in shoot- and root-biomass and total root-length, 20 % higher GS activity, 18 % less NH4+-accumulation in roots, and a comparable plant total-N content compared to the control, implying a novel role for urea in cotton NH4+detoxification. A similar phenomenon was observed in tobacco and rice. Moreover, comparisons between twelve NH4+-grown Arabidopsis accessions revealed a great degree of natural variation in their root-growth response to low urea, with WAR and Blh-1 exhibiting the most significant increase in primary- and lateral-root length and numbers, and Sav-0 and Edi-0 being the most insensitive. Such phenotypical evidence suggests a common ability of plants to accommodate NH4+-stress by responding to exogenous urea, providing a novel aspect for further understanding the process of urea-dependent plant NH4+ tolerance.

Soybean tolerance to drought depends on the associated Bradyrhizobium strain.

We evaluated the effect of three different Bradyrhizobium strains inoculated in two soybean genotypes (R01-581F, drought-tolerant, and NA5858RR, drought-sensitive) submitted to drought in two trials conducted simultaneously under greenhouse. The strains (SEMIA 587, SEMIA 5019 (both B. elkanii), and SEMIA 5080 (B. diazoefficiens)) were inoculated individually in each genotype and then submitted to water restriction (or kept well-watered, control) between 45 and 62days after emergence. No deep changes in plant physiological variables were observed under the moderate water restriction imposed during the first 10days. Nevertheless, photosynthesis and transpiration decreased after the severe water restriction imposed for further 7days. Water restriction reduced growth (- 30%) and the number of nodules (- 47% and - 58% for R01-581F and NA5858RR, respectively) of both genotypes, with a negative effect on N-metabolism. The genotype R01-581F inoculated with SEMIA 5019 strain had higher photosynthetic rates compared with NA5858RR, regardless of the Bradyrhizobium strain. On average, R01-581F showed better performance under drought than NA5858RR, with higher number of nodules (51 vs. 38 nodules per plant, respectively) and less accumulation of ureides in petioles (15μmolg-1 vs. 34μmolg-1, respectively). Moreover, plants inoculated with SEMIA 5080 had higher glutamine synthetase activity under severe water restriction, especially in the drought-tolerant R01-518F, suggesting maintenance of N metabolism under drought. The Bradyrhizobium strain affects the host plant responses to drought in which the strain SEMIA 5080 improves the drought tolerance of R01-518F genotype.

Effects of excess ammoniacal nitrogen (NH4+-N) on pigments, photosynthetic rates, chloroplast ultrastructure, proteomics, formation of reactive oxygen species and enzymatic activity in submerged plant Hydrilla verticillata (L.f.) Royle

Although excess ammoniacal-nitrogen (NH4+-N) results in the disturbance of various important biochemical and physiological processes, a detailed study on the effects of NH4+-N stress on the photosynthesis and global changes in protein levels in submerged macrophytes is still lacking. Here, the changes of excess NH4+-N on physiological parameters in Hydrilla verticillata (L.f.) Royle, a submerged macrophyte were investigated, including the contents of photosynthetic pigments, soluble sugars, net photosynthesis and respiration, glutamine synthetase (GS) and glutamate synthase (GOGAT) activities, chloroplast ultrastructure, chloroplast reactive oxygen species (ROS) accumulation and protein levels. Our results showed that the net photosynthetic rate and pigment content reached maximum values when the plants were treated with 1 and 2 mg L-1 NH4+-N, respectively, and decreased at NH4+-N concentrations at 5, 10, 15 and 20 mg L-1. This decrease might be caused by ROS accumulation. Compared that in 0.02 mg L-1 NH4+-N as a control, ROS generation in chloroplasts significantly increased in the presence of more than 2 mg L-1 NH4+-N. Consistently, the damages caused by over-accumulated ROS were observed in chloroplast ultrastructure, showing a loose thylakoid membranes and swollen grana/stroma lamellae. Furthermore, through proteomic analysis, we identified 91 differentially expressed protein spots. Among them, six proteins involved in photosynthesis decreased in abundance in response to excess NH4+-N. Surprisingly, the abundance of all the identified proteins that were involved in nitrogen assimilation and amino acid metabolism tended to increase under excess NH4+-N compared with the control, suggestive of the imbalanced carbon and nitrogen (C-N) metabolisms. In support, activated GS and GOGAT cycle was observed, evidenced by higher activities of GS and GOGAT enzymes. To our knowledge, this work is the first description that excess NH4+-N results in chloroplast ultrastructural damages and the first proteomic evidence to support that excess NH4+-N can lead to a decline in photosynthesis and imbalance of C-N metabolism in submerged macrophytes.

Variations in Nitrogen Metabolism are Closely Linked with Nitrogen Uptake and Utilization Efficiency in Cotton Genotypes under Various Nitrogen Supplies.

Cotton production is highly sensitive to nitrogen (N) fertilization, whose excessive use is responsible for human and environmental problems. Lowering N supply together with the selection of N-efficient genotypes, more able to uptake, utilize, and remobilize the available N, could be a challenge to maintain high cotton production sustainably. The current study aimed to explore the intraspecific variation among four cotton genotypes in response to various N supplies, in order to identify the most distinct N-efficient genotypes and their nitrogen use efficiency (NUE)-related traits in hydroponic culture. On the basis of shoot dry matter, CCRI-69 and XLZ-30 were identified as N-efficient and N-inefficient genotypes, respectively, and these results were confirmed by their contrasting N metabolism, uptake (NUpE), and utilization efficiency (NUtE). Overall, our results indicated the key role of shoot glutamine synthetase (GS) and root total soluble protein in NUtE. Conversely, tissue N concentration and N-metabolizing enzymes were considered as the key traits in conferring high NUpE. The remobilization of N from the shoot to roots by high shoot GS activity may be a strategy to enhance root total soluble protein, which improves root growth for N uptake and NUE. In future, multi-omics studies will be employed to focus on the key genes and pathways involved in N metabolism and their role in improving NUE.

Growth, Nutrient Assimilation, and Carbohydrate Metabolism in Korean Pine (Pinus koraiensis) Seedlings in Response to Light Spectra

A need is growing to plant superior Korean pine (Pinus koraiensis Siebold & Zucc.) seedlings to cope with the degradation of secondary forests in Northeast Eurasia. The goal of this study was to detect the physiological effect on the quality of Korean pine seedlings exposed to a range of spectra. One-year-old seedlings (n = 6) were cultured in three light-emitting diode (LED) spectra (69‒77 μmol m−2 s−1) of 13.9% red (R) + 77.0% green (G) + 9.2% blue (B) (R1BG5), 26.2% R + 70.2% G + 3.5% B (R2BG3), and 42.3% R + 57.3% G + 0.4% B (R3BG1). The spectrum of high-pressure sodium (HPS) lamps (43.9% R + 54.7% G + 1.5 B) was taken as the reference. Results showed that LED-lighting resulted in shorter seedlings with a greater diameter, shoot biomass, assessed quality, and sturdiness compared to those under the HPS-lighting. The R3BG1 spectrum reduced the shoot nitrogen (N) deficiency induced by the HPS spectrum, while the R1BG5 treatment induced a steady-state uptake of N and phosphorus (P) in whole-plant organs. The R1BG5 spectrum also resulted in a higher soluble sugar concentration and higher activities of glutamine synthetase and acid phosphatase in needles compared to the control. Seedlings in the R2BG3 spectrum had the highest concentrations of chlorophyll and soluble protein in the leaves. Overall, the R-high LED-spectrum could stimulate biomass accumulation in shoot, but meanwhile resulted in a P deficiency. Hence, the LED lighting in the R1BG5 spectrum is recommended to promote the quality of Korean pine seedlings.

Theme 6 Tissue biomarkers

Background: Glutamate excitotoxicity is a longstanding hypothesis in the pathophysiology of ALS. Prior studies have demonstrated increased plasma glutamate levels in ALS patients, which suggest a systemic defect in glutamate metabolism (1). The most abundant amino acid consumed in our diet is glutamate. Studies in healthy human subjects have demonstrated efficient metabolism of dietary glutamate via metabolism by enzymes present in the liver, gut lumen and residential gut bacteria. There is increasing evidence that the gut microbiota has significant CNS effects and intestinal dysbiosis has been found in the ALS transgenic SOD1G93A mouse model (2). If intestinal dysbiosis altered the prevalence of glutamine synthetase (GS) producing bacteria, dietary glutamate homeostasis could be impaired, leading to elevated plasma glutamate levels.Objectives: This study examined the degree of fluctuation in plasma glutamate levels seen with consumption of a protein shake in a cohort of ALS patients and family members from a single ALS center.Methods: Twenty six patients (87% of total cohort followed in the ALS center) underwent measurement of plasma amino acid analysis prior to and 1 hour following consumption of a 75 gm protein shake. A subset of 16 patients went on to receive a probiotic with high GS activity and completed serial protein challenges and amino acid analysis during the study. Ten unaffected family members of ALS patients underwent a similar protein challenge. Glutamate Metabolism Dysfunction (GMD) was defined as >30 umol difference post-prandially compared to fasting, and graded as mild (>30-60), moderate (>60-90) or severe (>90).Results: At baseline, 65.4% (17/26) ALS patients screened were GMD positive, compared to 30% (3/10) of tested family members. The severity of GMD in ALS patients was 41% mild, 29% moderate, 29% severe with only mild severity identified in family members. In the six month treatment phase, 75% (6/8) of patients with stable or improving GMD status saw significant improvements in their ALSFRS-R rate of decline, while 71.4% (5/7) with worsening or remaining severe GMD status experienced worsening of their rate of progression.Discussion and conclusions: Although limited by small sample size, this study does represent an excellent sampling within a single ALS center and is the first of its kind to investigate whether impairment in dietary glutamate metabolism exists in ALS patients. If validated in a larger ALS population, detection of glutamate metabolism dysfunction (GMD) could represent a novel biomarker linked to a potential therapeutic target in ALS patients. Planned microbiome analysis will also help in validating this hypothesis.

Inoculation with five diazotrophs alters nitrogen metabolism during the initial growth of sugarcane varieties with contrasting responses to added nitrogen

We examined the influence of inoculation with five species/strains of diazotrophic bacteria on the modulation of two enzymes involved in the assimilation of N and on the soluble N fractions in the sugarcane varieties RB867515 (adapted for low fertility soils) and IACSP95-5000 (adapted for medium to high fertility soils) under high- (3 mM) and low (0.3 mM)-N conditions in hydroponic cultivation for 59 days. The sugarcane plants were produced in three steps to obtain the hydroponic cultivation: the supply of 3 mM N for 30 days (first harvest), N depletion for 72 h (second harvest), and cultivation in high- and low-N conditions over 26 days (final harvest). Inoculation was performed by immersion of the minisetts in a diluted solution of five diazotrophic bacteria. After the final harvest, plants were divided into roots and shoots to assess their dry weight and N, P, and K accumulation. The variety played an important role in the interaction with diazotrophs, each showing distinct behavior in the activity of their N-assimilation enzymes. The nitrate reductase activity (NRa—EC 1.7.1.1) was increased in var. RB867515 by 26% in the shoots and by 48% in the roots after 72 h under N depletion, while var. IACSP95-5000 showed a reduced enzymatic activity in the roots (by 62%) but not in the shoots. Under high-N conditions, the inoculated IACSP95-5000 plants showed 31% higher glutamine synthetase activity (GSa—EC 6.31.2) compared with 19% in RB867515. Under low-N conditions, the GSas were 21% and 16% higher in the inoculated RB867515 and IACSP95-5000 plants, respectively, compared with that of the control. The content of nitrogen in the form of nitrate (N-nitrate) confirmed these varietal differences, but the soluble sugar content did not. The varieties utilized N sources differently, and inoculation modified the activity of two N-assimilation enzymes as well as the biomass accumulation, with the highest improvement seen in the low fertility-adapted variety RB867515; it showed a greater response to inoculation compared with that of the high fertility-adapted variety IACSP95-5000, with an increase in biomass and nutrient accumulation (N, P, K), especially when cultivated under low-N conditions. This suggests that the best response to inoculation with diazotrophs will be achieved using low fertility-adapted sugarcane varieties under low-N conditions.

Effects of light quality on carbon-nitrogen metabolism, growth, and quality of Chinese chives

We examined the effect of white + red (WR), white + blue (WB), white + green (WG), white + purple (WP) on the carbon-nitrogen metabolism, growth and quality of Chinese chives, with ‘Pingjiu 2’ as the material and white light (W) as the control. The results showed that photosynthetic rate (Pn) in WR treatment was significantly higher than that in the control (CK) and that there was no difference among WB, WG, WP and CK. The activity of RuBPCase in all the four treatments was higher than that in CK. Compared with other treatments, WR showed the highest total sugar content, then WP, with WB and WG showing the lowest. Compared with CK, sucrose phosphate synthase (SPS) activity in WR was higher, and lower in the other three treatments, especially the lowest in WB. WB showed the highest activity of sucrose synthetase (SS) and amylase (AMS), WR displayed the lowest as well as the similar or lower in WG or WP compared with CK. These results implied that the increases of the proportion of red and purple light could improve carbon assimilation and transformation and thus accelerate sugar accumulation of Chinese chives. The content of total nitrogen, protein nitrogen, the activities of NR, GS and GOGAT of WB were the highest among all the treatments. The GDH activity of WB was the lowest and notably lower compared with CK. WR showed the lower contents of total nitrogen, protein nitrogen, the activities of NR and GS, and higher activities of GOGAT and GDH than those in CK, while the GDH activity of WR was the highest. WG showed lower total nitrogen, no-protein nitrogen content, and GDH activity, but higher protein nitrogen, NR and GOGAT activities than those in CK. Compared with the control, WP showed the similar results as WG, besides the higher GS activity, with most of these parameters in WP being better than in WG. These results indicated that blue, purple and green light could promote nitrogen metabolism, while red light might inhibit protein synthesis in Chinese chives. The growth of Chinese chives was much better under red and purple light, but worse under blue light, which led to thinner leaves and lower growth mass. Chinese chives of WP with the lowest crude fiber content showed the optimal comprehensive quality. Therefore, purple light exhibited the best effect on the growth of Chinese chives.

Oral administration of trace element magnesium significantly improving the cognition and locomotion in hepatic encephalopathy rats

The therapeutic effects of iron, zinc and magnesium trace elements, as well as rifaximin were investigated and compared in HE rats. In this study, HE rats were treated with either ferrous sulfate (HE-Fe, 30 mg/kg/day), zinc sulfate (HE-Zn, 30 mg/kg/day), magnesium sulfate (HE-Mg, 50 mg/kg/day) or rifaximin (HE-Rf, 50 mg/kg/day), which was mixed with water and administered orally for 61 days. The Morris water maze (MWM) and open-field tests were used to evaluate cognitive and locomotor function. The blood ammonia levels before and after administration of the glutamine challenge test, manganese concentration and glutamine synthetase (GS) activity were measured. Significantly longer MWM escape latencies, less locomotor activity, higher blood ammonia levels, higher brain manganese concentrations and higher GS activity were observed in HE rats. However, HE-Mg and HE-Rf rats had significantly shorter MWM escape latencies, increased locomotor activity, lower blood ammonia, lower brain manganese concentrations and lower GS activity. Partial improvements were observed in HE-Fe and HE-Zn rats. The results indicated that oral administration of magnesium can significantly improve the cognitive and locomotor functions in HE rats by reducing the brain manganese concentration and regulating GS activity.

Responses of glutamine synthetase activity and gene expression to nitrogen levels in winter wheat cultivars with different grain protein content

Glutamine synthetase (GS) plays a central role in plant nitrogen (N) metabolism, which improves crops grain protein content. A pot experiment in field condition was carried out to evaluate GS expression and activity, and grain protein content in high (Wanmai16) and low grain protein (Loumai24) wheat cultivars under two N levels (0.05 and 0.15 g N kg−1 soil). High nitrogen (HN) resulted in significant increases in GS1 and GS2 expression at 10 days after anthesis (DAA), and higher GS activity during the entire grain filling stage. HN also significantly increased yield, grain protein content and protein fraction (except for glutenin of Luomai24) in two wheat cultivars, which indicated that it increased grain yield and protein content by improving nitrogen metabolism. Wanmai16 showed higher grain protein content, gliadin and glutenin content, and had higher expression level of GS2 both in flag leaves and grains at early grain filling stage. However, Luomai24 had greater yield and higher expression level of GS1. The difference expression of GS2 and GS1 genes indicates they had various contributions to the accumulation of protein and starch in wheat grains, respectively. The results suggest that GS2 would be serving as a potential breeding target for improving wheat quality.

Glutamine synthetase in Durum Wheat: Genotypic Variation and Relationship with Grain Protein Content.

Grain protein content (GPC), is one of the most important trait in wheat and its characterized by a very complex genetic control. The identification of wheat varieties with high GPC (HGPC), as well as the characterization of central enzymes involved in these processes, are important for more sustainable agricultural practices. In this study, we focused on Glutamine synthetase (GS) as a candidate to study GPC in wheat. We analyzed GS expression and its enzymatic activity in different tissues and phenological stages in 10 durum wheat genotypes with different GPC. Although each genotype performed quite differently from the others, both because their genetic variability and their adaptability to specific environmental conditions, the highest GS activity and expression were found in genotypes with HGPC and vice versa the lowest ones in genotypes with low GPC (LGPC). Moreover, in genotypes contrasting in GPC bred at different nitrogen regimes (0, 60, 140 N Unit/ha) GS behaved differently in diverse organs. Nitrogen supplement increased GS expression and activity in roots of all genotypes, highlighting the key role of this enzyme in nitrogen assimilation and ammonium detoxification in roots. Otherwise, nitrogen treatments decreased GS expression and activity in the leaves of HGPC genotypes and did not affect GS in the leaves of LGPC genotypes. Finally, no changes in GS and soluble protein content occurred at the filling stage in the caryopses of all analyzed genotypes.

Influence of different host associations on glutamine synthetase activity and ammonium transporter in Santalumalbum L.

The present study was aimed at understanding the role of different hosts in ammonium transporter1;2 expressions and glutamine synthetase(GS) activity and their effects on the growth parameters in the sandal. Sandal plant associated with leguminous host expressed better growth parameters. GS activity of leguminous hosts alone and in host associated sandals was analyzed using GS transferase assay. Highest GS activity was expressed in Mimosa pudica-sandal association compared to other leguminous and non-leguminous host associations. The association of N2 fixing host with sandal enhanced C and N levels in order to maintain the C/N value. The role of ammonium transporters in N nutrition of sandal-host association was elucidated by cloning AMT1;2 from the leaves, haustoria and roots of host associated sandal and quantifying the relative expression by the [Formula: see text] method. SaAMT1;2 was strongly up-regulated in leaves, roots and haustoria of leguminous host associated sandal compared to non-leguminous host associations. The relative increase in SaAMT1;2 expressions and up-regulated GS activity positively affected the growth parameters in sandal when associated with leguminous hosts.

Limitations to the development of recombinant human embryonic kidney 293E cells using glutamine synthetase-mediated gene amplification: Methionine sulfoximine resistance

To investigate the feasibility of glutamine synthetase (GS)-mediated gene amplification in HEK293 cells for the high-level stable production of therapeutic proteins, HEK293E cells were transfected by the GS expression vector containing antibody genes and were selected at various methionine sulfoximine (MSX) concentrations in 96-well plates. For a comparison, CHOK1 cells were transfected by the same GS expression vector and selected at various MSX concentrations. Unlike CHOK1 cells, HEK293E cells producing high levels of antibodies were not selected at all. For HEK293E cells, the number of wells with the cell pool did not decrease with an increase in the concentration of MSX up to 500μM MSX. A q-RT-PCR analysis confirmed that the antibody genes in the HEK293E cells, unlike the CHOK1 cells, were not amplified after increasing the MSX concentration. It was found that the GS activity in HEK293E cells was much higher than that in CHOK1 cells (P<0.05). In a glutamine-free medium, the GS activity of HEK293E cells was approximately 4.8 times higher than that in CHOK1 cells. Accordingly, it is inferred that high GS activity of HEK293E cells results in elevated resistance to MSX and therefore hampers GS-mediated gene amplification by MSX. Thus, in order to apply the GS-mediated gene amplification system to HEK293 cells, the endogenous GS expression level in HEK293 cells needs to be minimized by knock-out or down-regulation methods.

Biochemical and physiological characterization for nitrogen use efficiency in aromatic rice genotypes

In a set of 78 aromatic rice genotypes, cluster analysis was performed for yield and its related traits in field under two nitrogen (N) conditions viz., application of N fertilizer (N100) and without application of N fertilizer (N0) during wet season, 2011 and dry season, 2012. Basmati370 and Ranbir basmati were selected as high nitrogen use efficiency (NUE) genotypes and Kolajoha-3 and Ratnasundari as low NUE genotypes for characterization in terms of biochemical, physiological and agronomical aspects of NUE. A total of 32 biochemical, physiological and agronomical characters were measured in the selected four genotypes, growing in field under two N levels i.e., N0 and N100 during wet season 2012. Five efficiency parameters were also studied to determine their NUE. GS activity increased under low N and the increase was more in two high NUE genotypes (41.3%) than that of two low NUE genotypes (5.43%). NR activity increased with application of N fertilizer and low NUE genotypes expressed higher NR activity (8.8% and 2.02% more in N0 and N100 respectively). Chlorophyll content recorded maximum (3.6mgg−1) in low NUE genotypes under N100 condition, where as the chlorophyll content was minimum (0.43mgg−1) in high NUE genotypes under N0 condition. Electron transport rate (ETR), quantum yield (ΦPSII) and Fv/Fm were not affected by N levels but there were significant variations in non-photochemical quenching (qN) (15% more in N0) and photochemical quenching (qP) (25% more in N0). Grain yield, total dry matter and N uptake by grain and straw were higher in high NUE genotypes. Higher GS activity, maintenance of sufficient chlorophyll fluorescence and chlorophyll content in case of high NUE genotypes support their higher grain yield and total dry matter content under low N conditions by efficient N uptake, and utilization of nitrogen.

Proteomic analysis provides new insights into the adaptive response of a dinoflagellate Prorocentrum donghaiense to changing ambient nitrogen.

Nitrogen (N) is the major nutrient limiting phytoplankton growth and productivity over large ocean areas. Dinoflagellates are important primary producers and major causative agents of harmful algal blooms in the ocean. However, very little is known about their adaptive response to changing ambient N. Here, we compared the protein profiles of a marine dinoflagellate Prorocentrum donghaiense grown in inorganic N-replete, N-deplete and N-resupplied conditions using 2-D fluorescence differential gel electrophoresis. The results showed that cell density, chlorophyll a and particulate organic N contents presented low levels in N-deplete cells, while particulate organic carbon content and glutamine synthetase (GS) activity maintained high levels. Comparison of the protein profiles of N-replete, N-deplete and N-resupplied cells indicated that proteins involved in photosynthesis, carbon fixation, protein and lipid synthesis were down-regulated, while proteins participating in N reallocation and transport activity were up-regulated in N-deplete cells. High expressions of GS and 60 kDa chaperonin as well as high GS activity in N-deplete cells indicated their central role in N stress adaptation. Overall, in contrast with other photosynthetic eukaryotic algae, P. donghaiense possessed a specific ability to regulate intracellular carbon and N metabolism in response to extreme ambient N deficiency.