Cytosolic Glutamine Synthetase Research Articles

The significance of the two cytosolic glutamine synthetase enzymes, GLN1;3 and GLN1;5, in the context of seed development and germination in Arabidopsis thaliana

Glutamine synthetase (GS), an initial enzyme in nitrogen (N) plant metabolism, exists as a group of isoenzymes found in both cytosolic (GS1) and plastids (GS2) and has gathered significant attention for enhancing N use efficiency and crop yield.This work focuses on the A. thaliana GLN1;3 and GLN1;5 genes, the two predicted most expressed genes in seeds, among the five isogenes encoding GS1 in this species. The expression patterns were studied using transgenic marker line plants and qPCR during seed development and germination. The observed patterns highlight distinct functions for the two genes and confirm GLN1;5 as the most highly expressed GS1 gene in seeds. The GLN1;5, expression, oriented towards hypocotyl and cotyledons, suggests a role in protein turnover during germination, while the radicle-oriented expression of GLN1;3 supports a function in early external N uptake.While the single mutants exhibited a normal phenotype, except for a decrease in seed parameters, the double gln1;3/gln1;5 mutant displayed a germination delay, substantial impairment in growth, nitrogen metabolism, and number and quality of the seeds, as well as a diminishing in flowering. Although seed and pollen-specific, GLN1;5 expression is upregulated in the meristems of the gln1;3 mutants, filling the lack of GLN1;3 and ensuring the normal functioning of the gln1;3 mutants.These findings validate earlier in silico data on the expression patterns of GLN1;3 and GL1;5 genes in seeds, explore their different functions, and underscore their essential role in plant growth, seed production, germination, and early stages of plant development.

Bestrophin-2 and glutamine synthetase form a complex for glutamate release.

Bestrophin-2 (Best2), a member of the bestrophin family of calcium-activated anion channels, is expressed in nonpigmented epithelium (NPE) of the ciliary body with critical involvement in aqueous humor homeostasis. Best2 knockout mice exhibit a reduced intraocular pressure, suggesting the pharmaceutical potential of Best2 as a target for lowering ocular hypertension. However, little is known about the physiological binding partners and/or regulators of Best2. In this study, we performed a proteomic pulldown of bovine ciliary body lysate using purified Best2 proteins as the bait, and identified glutamine synthetase (GS), which catalyzes the conversion of glutamate and ammonia into glutamine, as a Best2 binding partner. We assessed the functional consequences of the interaction and discovered a mutually inhibitory effect: GS decreases Best2-mediated chloride conduction, while Best2 reduces cytosolic GS activity by tethering GS to the cell membrane. Since glutamate is the substrate of GS and an anion, we investigated the ability of Best2 to conduct glutamate. Remarkably, Best2 exhibits a directional permeability to glutamate heavily favoring glutamate exit from the cell, and the binding of GS to Best2 sensitized Best2 to intracellular glutamate, which promotes opening of Best2 and thus relieves the inhibitory effect of GS. We further demonstrated the physiological role of Best2 in conducting chloride and glutamate and the influence of GS in NPE cells. Moreover, by single-particle cryo-EM, we solved the structure of the Best2-GS complex, illustrating the interacting residues and extension of the Best2 ion conducting pathway through the GS central cavity. Together, our results reveal a novel mechanism of glutamate release through Best2-GS.

Development and marker-trait relationships of functional markers for glutamine synthetase GS1 and GS2 homoeogenes in bread wheat

GS1 and GS2 genes encode, respectively, the main cytosolic and the plastidic isoforms of glutamine synthetase (GS). In the present study, the wheat GS1 and GS2 homoeogenes located in the A, B and D genome chromosomes have been sequenced in a group of 15 bread wheat varieties including landraces, old commercial varieties and modern cultivars. Phenotypic characterization by multi-environment field trials detected significant effects of specific GS homoeogenes on three of the seven agronomic and grain quality traits analyzed. Based on the gene sequence polymorphisms found, biallelic molecular markers that could facilitate marker-assisted breeding were developed for genes GS1A, GS2A and GS2D. The remaining genes encoding main wheat GS were excluded because of being monomorphic (GS1D) or too polymorphic (GS1B and GS2B) in the sequencing panel varieties. A collection of 187 Spanish bread wheat landraces was genotyped for these gene-based molecular markers. Data analyses conducted with phenotypic records reported for this germplasm collection in López-Fernández et al. (Plants-Basel 10: 620, 2021) have revealed the beneficial influence of some individual alleles on thousand-kernel weight (TKW), kernels per spike (KS) and grain protein content. Furthermore, genetic interactions between GS1A, a cytosolic GS isoform coding gene, and GS2A or GS2D, plastidic GS enzyme coding genes, were found to affect TKW and KS. The finding that some alleles at one locus may mask the effect of positive alleles at hypostatic GS loci should be kept in mind if gene pyramiding strategies are attempted for the improvement of N-use efficiency-related traits.

Bestrophin-2 and glutamine synthetase form a complex for glutamate release.

Bestrophin-2 (BEST2) is a member of the bestrophin family of calcium-activated anion channels that has a critical role in ocular physiology1-4. Here we uncover a directional permeability of BEST2 to glutamate that heavily favours glutamate exit, identify glutamine synthetase (GS) as a binding partner of BEST2 in the ciliary body of the eye, and solve the structure of the BEST2-GS complex. BEST2 reduces cytosolic GS activity by tethering GS to the cell membrane. GS extends the ion conducting pathway of BEST2 through its central cavity and inhibits BEST2 channel function in the absence of intracellular glutamate, but sensitizes BEST2 to intracellular glutamate, which promotes the opening of BEST2 and thus relieves the inhibitory effect of GS. We demonstrate the physiological role of BEST2 in conducting chloride and glutamate and the influence of GS in non-pigmented ciliary epithelial cells. Together, our results reveal a novel mechanism of glutamate release through BEST2-GS.

Effects of low nitrogen supply on nitrogen uptake, assimilation and remobilization in wild bermudagrass

The natural mechanism of underlying the low nitrogen (N) tolerance of wild bermudagrass (Cynodon dactylon (L.) Pers.) germplasm was important for reducing N fertilizer input to turf while also maintaining acceptable turf quality. The growth, N uptake, assimilation and remobilization of two wild bermudagrass accessions (C291, low N tolerant and C716, low N sensitive) were determined under low N (0.5 mM) and control N (5 mM) levels. C291 exhibited lower reduction in shoot and plant dry weight than C716. Furthermore, C291 presented a lower decrease in 15NO3− influx compared with C716, maintained its root dry weight and root surface and showed obviously enhanced CyNRT2.2 and CyNRT2.3 expression resulting in higher shoot NO3−-N content than the control. Moreover, in C291, nitrate reductase (NR) activity had no significant difference with control, and cytosolic glutamine synthetase (GS1) protein content, glutamate synthetase (GOGAT) activity and glutamate dehydrogenase (GDH) activity higher than control, result in the soluble protein and free amino acid contents in the shoots did not differ compared with that in the control under low N conditions. Overall, the low N tolerant wild bermudagrass accessions adopted a low N supply based on improved root N uptake ability to achieve more nitrate to kept shoot N assimilation, and meanwhile increased N remobilization in the shoots, thereby maintaining a better N status in bermudagrass. The findings may help elucidate the low N tolerance mechanisms in bermudagrass and therefore facilitate genetic improvement of N use efficiency aiming to promote low-input turfgrass management.

A revised view on the evolution of glutamine synthetase isoenzymes in plants.

SUMMARYGlutamine synthetase (GS) is a key enzyme responsible for the incorporation of inorganic nitrogen in the form of ammonium into the amino acid glutamine. In plants, two groups of functional GS enzymes are found: eubacterial GSIIb (GLN2) and eukaryotic GSIIe (GLN1/GS). Only GLN1/GS genes are found in vascular plants, which suggests that they are involved in the final adaptation of plants to terrestrial life. The present phylogenetic study reclassifies the different GS genes of seed plants into three clusters: GS1a, GS1b and GS2. The presence of genes encoding GS2 has been expanded to Cycadopsida gymnosperms, which suggests the origin of this gene in a common ancestor of Cycadopsida, Ginkgoopsida and angiosperms. GS1a genes have been identified in all gymnosperms, basal angiosperms and some Magnoliidae species. Previous studies in conifers and the gene expression profiles obtained in ginkgo and magnolia in the present work could explain the absence of GS1a in more recent angiosperm species (e.g. monocots and eudicots) as a result of the redundant roles of GS1a and GS2 in photosynthetic cells. Altogether, the results provide a better understanding of the evolution of plant GS isoenzymes and their physiological roles, which is valuable for improving crop nitrogen use efficiency and productivity. This new view of GS evolution in plants, including a new cytosolic GS group (GS1a), has important functional implications in the context of plant metabolism adaptation to global changes.

Cytosolic Glutamine Synthetase GS1;3 Is Involved in Rice Grain Ripening and Germination.

Ammonium is combined with glutamate to form glutamine. This reaction is catalyzed by glutamine synthetase (GS or GLN). Plants harbor several isoforms of cytosolic GS (GS1). Rice GS1;3 is highly expressed in seeds during grain filling and germination, suggesting a unique role in these processes. This study aimed to investigate the role of GS1;3 for rice growth and yield. Tos17 insertion lines for GS1;3 were isolated, and the nitrogen (N), amino acid, and ammonium contents of GS1;3 mutant grains were compared to wild-type grains. The spatiotemporal expression of GS1;3 and the growth and yield of rice plants were evaluated in hydroponic culture and the paddy field. Additionally, the stable isotope of N was used to trace the foliar N flux during grain filling. Results showed that the loss of GS1;3 retarded seed germination. Seeds of GS1;3 mutants accumulated glutamate but did not show a marked change in the level of phytohormones. The expression of GS1;3 was detected at the beginning of germination, with limited promoter activity in seeds. GS1;3 mutants showed a considerably decreased ripening ratio and decreased N efflux in the 12th leaf blade under N deficient conditions. The β-glucuronidase gene expression under control of the GS1;3 promoter was detected in the vascular tissue and aleurone cell layer of developing grains. These data suggest unique physiological roles of GS1;3 in the early stage of seed germination and grain filling under N deficient conditions in rice.

Impacts of environmental conditions, and allelic variation of cytosolic glutamine synthetase on maize hybrid kernel production

Cytosolic glutamine synthetase (GS1) is the enzyme mainly responsible of ammonium assimilation and reassimilation in maize leaves. The agronomic potential of GS1 in maize kernel production was investigated by examining the impact of an overexpression of the enzyme in the leaf cells. Transgenic hybrids exhibiting a three-fold increase in leaf GS activity were produced and characterized using plants grown in the field. Several independent hybrids overexpressing Gln1-3, a gene encoding cytosolic (GS1), in the leaf and bundle sheath mesophyll cells were grown over five years in different locations. On average, a 3.8% increase in kernel yield was obtained in the transgenic hybrids compared to controls. However, we observed that such an increase was simultaneously dependent upon both the environmental conditions and the transgenic event for a given field trial. Although variable from one environment to another, significant associations were also found between two GS1 genes (Gln1-3 and Gln1-4) polymorphic regions and kernel yield in different locations. We propose that the GS1 enzyme is a potential lead for producing high yielding maize hybrids using either genetic engineering or marker-assisted selection. However, for these hybrids, yield increases will be largely dependent upon the environmental conditions used to grow the plants.

Nitrogen remobilization and its importance in nitrogen use efficiency (NUE) of crops

Nitrogen (N) remobilization during grain filling from pre-anthesis N uptake and stored in different tissues of crop N use efficiency (NUE). N is remobilized from to sink (young leaves or grains) with the help of nitrate/amino acid transporters. Nearly 80% of grain N in cereals is derived from N remobilized from vegetative tissues. Remobilization of N within the plant takes place from older leaves to young leaves, leaves to grains, senescing organs to grains, from storage parts to grains. Enzymes involved in N remobilization include glutamine synthetase (GS), glutamate dehydrogenase (GDH), asparagine synthetase (AS) and proteases. Among them, cytosolic GS plays a key role during N remobilization in cereals. There are various senescence-associated genes (SAG) involved in N remobilization from older degrading leaves to younger leaves and grains. Autophagy (ATG) is an important mechanism involved in the degradation of stored N in the form of various proteins to amino acids, which are transported to long-distance in the form of glutamine and asparagine via phloem tissue. There is a complex network of genes, mechanisms, and factors associated with N remobilization, which needs to be considered for improving NUE of crops.

Corrigendum to: Ammonium metabolism in Selaginella bryopteris in response to dehydration-rehydration and characterisation of desiccation tolerant, thermostable, cytosolic glutamine synthetase from plant.

Water deficit (WD) has adverse effects on plant growth, and acclimation requires responses allowing primary metabolism to continue. Resurrection plants can serve as model system to gain insight into metabolic regulation during WD. We herein report the response of a resurrection lycophyte, Selaginella bryopteris, to dehydration-rehydration cycle with emphasis on ammonium metabolism. Dehydration of S. bryopteris fronds resulted in decrease of total protein and increase of free ammonium levels and the effect was reversed on rehydration. The proline content increased twice after 24 h of dehydration, which again recovered to background levels comparable to that at full turgor state. The specific activity of glutamine synthetase (GS) didn't change significantly till 6 h and then declined by 21% after 24 h of dehydration, whereas specific activities of glutamate synthase (GOGAT) and aminating glutamate dehydrogenase (GDH) were enhanced significantly during dehydration. The deaminating activity of GDH also increased during dehydration albeit at a slower rate. Immunoblot analysis indicated overexpression of GS and GDH polypeptides during dehydration and their levels declined on rehydration. The results suggested significant role of GDH along with GS/GOGAT in production of nitrogen-rich amino acids for desiccation tolerance. Unlike higher plants S. bryopteris expressed GS only in cytosol. The enzyme had pH and temperature optima of 5.5 and 60°C, respectively, and it retained 96% activity on preincubation at 60°C for 30 min indicating thermostability. Hence, like higher plants the cytosolic GS from S. bryopteris has a conserved role in stress tolerance.

Ammonium metabolism in Selaginella bryopteris in response to dehydration-rehydration and characterisation of desiccation tolerant, thermostable, cytosolic glutamine synthetase from plant.

Water deficit (WD) has adverse effects on plant growth, and acclimation requires responses allowing primary metabolism to continue. Resurrection plants can serve as model system to gain insight into metabolic regulation during WD. We herein report the response of a resurrection lycophyte, Selaginella bryopteris, to dehydration-rehydration cycle with emphasis on ammonium metabolism. Dehydration of S. bryopteris fronds resulted in decrease of total protein and increase of free ammonium levels and the effect was reversed on rehydration. The proline content increased twice after 24 h of dehydration, which again recovered to background levels comparable to that at full turgor state. The specific activity of glutamine synthetase (GS) didn't change significantly till 6 h and then declined by 21% after 24 h of dehydration, whereas specific activities of glutamate synthase (GOGAT) and aminating glutamate dehydrogenase (GDH) were enhanced significantly during dehydration. The deaminating activity of GDH also increased during dehydration albeit at a slower rate. Immunoblot analysis indicated overexpression of GS and GDH polypeptides during dehydration and their levels declined on rehydration. The results suggested significant role of GDH along with GS/GOGAT in production of nitrogen-rich amino acids for desiccation tolerance. Unlike higher plants S. bryopteris expressed GS only in cytosol. The enzyme had pH and temperature optima of 5.5 and 60°C, respectively, and it retained 96% activity on preincubation at 60°C for 30 min indicating thermostability. Hence, like higher plants the cytosolic GS from S. bryopteris has a conserved role in stress tolerance.

Nitrogen Assimilation in the Highly Salt- and Boron-Tolerant Ecotype Zea mays L. Amylacea.

The Lluta Valley in Northern Chile is an important agricultural area affected by both salinity and boron (B) toxicity. Zea mays L. amylacea, an ecotype arisen because of the seed selection practiced in this valley, shows a high tolerance to salt and B levels. In the present study the interaction between B and salt was studied after 20 days of treatment at low (100 mM) and high salinity (430 mM NaCl), assessing changes in nitrogen metabolites and in the activity of key nitrogen-assimilating enzymes. Under non-saline conditions, the presence of excessive B favored higher nitrate and ammonium mobilization to leaves, increasing nitrate reductase (NR) activity but not glutamine synthetase (GS). Thus, the increment of nitrogen use efficiency by B application would contribute partially to maintain the biomass production in this ecotype. Positive relationships between NR activity, nitrate, and stomatal conductance were observed in leaves. The increment of major amino acids alanine and serine would indicate a photoprotective role of photorespiration under low-salinity conditions, thus the inhibition of nitrogen assimilation pathway (NR and GS activities) occurred only at high salinity. The role of cytosolic GS regarding the proline accumulation is discussed.

Transcriptional patterns of Coffea arabica L. nitrate reductase, glutamine and asparagine synthetase genes are modulated under nitrogen suppression and coffee leaf rust.

This study evaluated the transcriptional profile of genes related to nitrogen (N) assimilation in coffee plants susceptible and resistant to rust fungi under N sufficiency and N suppression. For this purpose, we inoculated young coffee leaves with Hemileia vastatrix uredospores and collected them at 0, 12, 24 and 48 hours post-inoculation (HPI) to evaluate the relative expressions of genes encoding cytosolic glutamine synthetase (CaGS1), plastid glutamine synthetase (CaGS2), nitrate reductase (CaNR), and asparagine synthetase (CaAS). The genes exhibited distinct patterns of transcriptional modulation for the different genotypes and N nutritional regimes. The resistant genotype (I59) presented high levels of transcription in response to pathogen inoculation for CaNR and CaGS1 genes, evaluated under N sufficiency in the initial moments of infection (12 HPI). The gene CaGS1 also showed a peak at 48 HPI. The susceptible genotype (CV99) showed increased transcript rates of CaNR at 12 and 24 HPI in response to rust inoculation. The transcriptional patterns observed for CV99, under N suppression, were high levels for CaAS and CaGS2 at all post-inoculation times in response to coffee leaf rust disease. In addition, CaGS1 was up-regulated at 48 HPI for CV99. Cultivar I59 showed high transcript levels at 12 HPI for CaAS and peaks at 24 and 48 HPI for CaGS2 in inoculated samples. Consequently, total chlorophyl concentration was influenced by N suppression and by rust infection. Regarding enzyme activities in vitro for glutamine synthetase and CaNR, there was an increase in infected coffee leaves (I59) and under N sufficiency. Moreover, CV99 was modulated in both N nutritional regimes for GS activity in response to rust. Our results indicate that N transport genes trigger a differential modulation between genotypes through the action of rust disease.

Effect of Phosphinothricin on Transgenic Downy Birch (Betula pubescens Ehrh.) Containing bar or GS1 Genes

Weeds are a big problem in agriculture and forestry, and herbicides are the main tools to control them. Phosphinotricin (ammonium glufosinate, PPT) is one of the most effective non-selective herbicides, to which weeds hardly gain resistance, but the reasons for its effect and toxicity to plants are still unclear, and especially, it is little studied in trees, including transgenic ones. We studied the physiological responses of downy birch (Betula pubescens Ehrh.) containing the herbicide resistance bar gene or the cytosol glutamine synthetase GS1 gene (the target enzyme of the herbicide) to PPT-based Basta herbicide treatment in various doses under open-air conditions during two years. Birch saplings with the bar gene were resistant to a double field dose (10 L/ha), but the expression of the GS1 gene only slightly increased resistance compared to the control. Herbicide treatment increased the ammonium level in leaf tissue by 3–8 times, but this, apparently, was not the main cause of plant death. Among leaf pigments, chlorophyll B was the most resistant to PPT, and carotenoids were the most sensitive. Responses of birch trees with the GS1 gene (accumulation of ammonium, pigment content, and dehydration) during treatment with a low dose of herbicide were less pronounced than in control plants. One-year-old control and transgenic plants with the GS gene died after 2.5 L/ha treatment, and two-year-old plants lost foliage after such treatment but remained alive and developed buds four weeks after treatment. Herbicide treatment of plants with the bar gene did not cause significant deviations in height (first year) or the accumulation of aboveground biomass (second year). The obtained results improve our understanding of the effect of PPT on woody plants and can be used both to clarify mechanisms of herbicide action and in plantation forestry.

Nepetalactone-rich essential oil mitigates phosphinothricin-induced ammonium toxicity in Arabidopsis thaliana (L.) Heynh.

Active ingredient of the commercial herbicide BASTA (B), phosphinothricin, acts as an inhibitor of glutamine synthetase (GS), a key enzyme in ammonium assimilation. The treatment with BASTA leads to an elevation of ammonium levels in plants and further to various physiological alterations, ammonium toxicity and lethality. Results of the present study emphasize the complexity underlying control mechanisms that determine BASTA interaction with essential oil (EO) from Nepeta rtanjensis (NrEO), bioherbicide inducing oxidative stress in target plants. Simultaneous application of NrEO and BASTA, two agents showing differential mode of action, suspends BASTA-induced ammonium toxicity in Arabidopsis thaliana plants. This is achieved through maintaining GS activity, which sustains a sub-toxic and/or sub-lethal ammonium concentration in tissues. As revealed by the present study, regulation of GS activity, as influenced by BASTA and NrEO, occurs at transcriptional, posttranscriptional, and/or posttranslational levels. Two genes encoding cytosolic GS, GLN1;1 and GLN1;3, are highlighted as the main isozymes in Arabidopsis shoots contributing to NrEO-induced overcoming of BASTA-generated ammonium toxicity. The effects of NrEO might be ascribed to its major component nepetalactone, but the contribution of minor EO components should not be neglected. Although of fundamental significance, the results of the present study suggest possible low efficiency of BASTA in plantations of medicinal/aromatic plants such as Nepeta species. Furthermore, these results highlight the possibility of using NrEO as a bioherbicide in BASTA-treated crop fields to mitigate the effect of BASTA residues in contaminated soils.

Roles of Cytosolic Glutamine Synthetases in Arabidopsis Development and Stress Responses.

Glutamine (Gln) has as a central role in nitrogen (N) and carbon (C) metabolism. It is synthesized during assimilation of ammonium by cytosolic and plastidial glutamine synthetases (GS; EC 6.1.1.3). Arabidopsis thaliana has five cytosolic GS (GS1) encoding genes designated as GLN1;1-GLN1;5 and one plastidial GS (GS2) gene. In this report that concerns cytosolic GS, we show by analyzing single, double and triple mutants that single genes were dispensable for growth under laboratory conditions. However, loss of two or three GS1 isoforms impacted plant form, function and the capacity to tolerate abiotic stresses. The loss of GLN1;1, GLN1;2 and GLN1;3 resulted in a significant reduction of vegetative growth and seed size. In addition, we infer that GLN1;4 is essential for pollen viability but only in the absence of GLN1;1 and GLN1;3. Transcript profiling revealed that expression of GLN1;1, GLN1;2, GLN1;3 and GLN1;4 was repressed by salinity and cold stresses. Among all single gln1 mutants, growth of gln1;1 seedlings showed an enhanced sensitivity to the GS inhibitor phosphinothricin (PPT), as well as to cold and salinity treatments, suggesting a non-redundant role for GLN1;1. Furthermore, the increased sensitivity of gln1;1 mutants to methyl viologen was associated with an accelerated accumulation of reactive oxygen species (ROS) in the thylakoid of chloroplasts. Our data demonstrate, for the first time, an involvement of the cytosolic GS1 in modulating ROS homeostasis in chloroplasts. Collectively, the current study establishes a link between cytosolic Gln production and plant development, ROS production and stress tolerance.

Cisgenic overexpression of cytosolic glutamine synthetase improves nitrogen utilization efficiency in barley and prevents grain protein decline under elevated CO2.

SummaryCytosolic glutamine synthetase (GS1) plays a central role in nitrogen (N) metabolism. The importance of GS1 in N remobilization during reproductive growth has been reported in cereal species but attempts to improve N utilization efficiency (NUE) by overexpressing GS1 have yielded inconsistent results. Here, we demonstrate that transformation of barley (Hordeum vulgare L.) plants using a cisgenic strategy to express an extra copy of native HvGS1‐1 lead to increased HvGS1.1 expression and GS1 enzyme activity. GS1 overexpressing lines exhibited higher grain yields and NUE than wild‐type plants when grown under three different N supplies and two levels of atmospheric CO2. In contrast with the wild‐type, the grain protein concentration in the GS1 overexpressing lines did not decline when plants were exposed to elevated (800–900 μL/L) atmospheric CO2. We conclude that an increase in GS1 activity obtained through cisgenic overexpression of HvGS1‐1 can improve grain yield and NUE in barley. The extra capacity for N assimilation obtained by GS1 overexpression may also provide a means to prevent declining grain protein levels under elevated atmospheric CO2.

Three cytosolic glutamine synthetase isoforms localized in different-order veins act together for N remobilization and seed filling in Arabidopsis.

Glutamine synthetase (GS) is central for ammonium assimilation and consists of cytosolic (GS1) and chloroplastic (GS2) isoenzymes. During plant ageing, GS2 protein decreases due to chloroplast degradation, and GS1 activity increases to support glutamine biosynthesis and N remobilization from senescing leaves. The role of the different Arabidopsis GS1 isoforms in nitrogen remobilization was examined using 15N tracing experiments. Only the gln1;1-gln1;2-gln1;3 triple-mutation affecting the three GLN1;1, GLN1;2, and GLN1;3 genes significantly reduced N remobilization, total seed yield, individual seed weight, harvest index, and vegetative biomass. The triple-mutant accumulated a large amount of ammonium that could not be assimilated by GS1. Alternative ammonium assimilation through asparagine biosynthesis was increased and was related to higher ASN2 asparagine synthetase transcript levels. The GS2 transcript, protein, and activity levels were also increased to compensate for the lack of GS1-related glutamine biosynthesis. Localization of the different GLN1 genes showed that they were all expressed in the phloem companion cells but in veins of different order. Our results demonstrate that glutamine biosynthesis for N-remobilization occurs in veins of all orders (major and minor) in leaves, it is mainly catalysed by the three major GS1 isoforms (GLN1;1, GLN1;2, and GLN1;3), and it is alternatively supported by AS2 in the veins and GS2 in the mesophyll cells.

Cytoplasmic glutamine synthetase gene expression regulates larval development in Bactrocera dorsalis (Hendel).

In insects, glutamine synthetase (GS), a key enzyme in the synthesis of glutamine, has been reported to be associated with embryonic development, heat shock response, and fecundity regulation. However, little is known about the influence of GS on postembryonic development. In this study, we demonstrate that blocking the activity of GS in the oriental fruit fly (Bactrocera dorsalis) with use of a GS-specific inhibitor (L-methionine S-sulfoximine), led to a significant delay in larval development, pupal weight loss, and inhibition of pupation. We further identify cloned and characterized two GS genes (BdGS-c and BdGS-m) from B. dorsalis. The two GS genes identified in B. dorsalis were predicted to be located in the cytosol (BdGS-c) and mitochondria (BdGS-m), and homology analysis indicated that both genes were similar to homologs from other Dipterans, such as Drosophila melanogaster and Aedes aegypti. BdGS-c was highly expressed in the larval stages, suggesting that cytosolic GS plays a predominant role in larval development. Furthermore, RNA interference experiments against BdGS-c, to specifically decrease the expression of cytosolic GS, resulted in delay in larval development as well as pupal weight loss. This study presents the prominent role played by BdGS-c in regulating larval development and suggests that the observed effect could have been modulated through ecdysteroid synthesis, agreeing with the reduced expression of the halloween gene spook. Also, the direct effects of BdGS-c silencing on B. dorsalis, such as larval lethality, delayed pupation, and late emergence, can be further exploited as novel insecticide target in the context of pest management.

Ammonium uptake capacity and response of cytosolic glutamine synthetase 1;2 to ammonium supply are key factors for the adaptation of ammonium nutrition in Arabidopsis thaliana

ABSTRACTPlant requires nitrogen for the growth, and it use nitrate and ammonium from the environment. Plant suffers from the toxicity when excess ammonium is supplied as a sole nitrogen, although it could be a good nitrogen source for plant growth. We hypothesized that the different responses of ecotypes to ammonium nutrient could partly account for the adaptation of Arabidopsis to an ammonium environment. The purpose of this study is to understand the different responses of ecotypes in ammonium environment. The growth of Arabidopsis thaliana ecotypes, Columbia was compared to those of Arabidopsis thaliana ecotypes, Landsberg erecta in ammonium nutrient. The ratio of shoot dry weight to root dry weight was compared to evaluate the adaptation of two ecotypes. The shoot:root ratio of Landsberg was significantly higher than that of Columbia. T-DNA insertion in cytosolic glutamine synthetase 1;2, one of the essential ammonium assimilatory enzymes, led a decrease of shoot:root ratio. We also measured the isotope-labeled ammonium uptake and the expression levels of ammonium transporter genes, and also the expression of ammonium assimilatory genes, glutamine synthetase genes and glutamate synthase genes, in roots after ammonium re-supply using real-time polymerase chain reaction analysis. We found that (1) ammonium uptake of Landsberg erecta was higher than that of Columbia, when ammonium was supplied at higher concentration, and (2) cytosolic glutamine synthetase 1;2 was highly increased by ammonium supply in the root of Landsberg erecta. The present study suggested the importance of these two factors for adaptation of Arabidopsis to an ammonium-rich environment.