Genomic organization and expression profiles of nitrogen assimilation genes in Glycine max.

Background: Glutamine synthetase (GS), glutamate synthase (GOGAT), and nitrate reductase (NR) are key enzymes involved in nitrogen assimilation and metabolism in plants. However, the systematic analysis of these gene families lacked reports in soybean (Glycine max (L.) Merr.), one of the most important crops worldwide. Methods: In this study, we performed genome-wide identification and characterization of GS, GOGAT, and NR genes in soybean under abiotic and nitrogen stress conditions. Results: We identified a total of 10 GS genes, six GOGAT genes, and four NR genes in the soybean genome. Phylogenetic analysis revealed the presence of multiple isoforms for each gene family, indicating their functional diversification. The distribution of these genes on soybean chromosomes was uneven, with segmental duplication events contributing to their expansion. Within the nitrogen assimilation genes (NAGs) group, there was uniformity in the exon-intron structure and the presence of conserved motifs in NAGs. Furthermore, analysis of cis-elements in NAG promoters indicated complex regulation of their expression. RT-qPCR analysis of seven soybean NAGs under various abiotic stresses, including nitrogen deficiency, drought-nitrogen, and salinity, revealed distinct regulatory patterns. Most NAGs exhibited up-regulation under nitrogen stress, while diverse expression patterns were observed under salt and drought-nitrogen stress, indicating their crucial role in nitrogen assimilation and abiotic stress tolerance. These findings offer valuable insights into the genomic organization and expression profiles of GS, GOGAT, and NR genes in soybean under nitrogen and abiotic stress conditions. The results have potential applications in the development of stress-resistant soybean varieties through genetic engineering and breeding.

The Role of Glutamine Synthetase (GS) and Glutamate Synthase (GOGAT) in the Improvement of Nitrogen Use Efficiency in Cereals.

Cereals are the most broadly produced crops and represent the primary source of food worldwide. Nitrogen (N) is a critical mineral nutrient for plant growth and high yield, and the quality of cereal crops greatly depends on a suitable N supply. In the last decades, a massive use of N fertilizers has been achieved in the desire to have high yields of cereal crops, leading to damaging effects for the environment, ecosystems, and human health. To ensure agricultural sustainability and the required food source, many attempts have been made towards developing cereal crops with a more effective nitrogen use efficiency (NUE). NUE depends on N uptake, utilization, and lastly, combining the capability to assimilate N into carbon skeletons and remobilize the N assimilated. The glutamine synthetase (GS)/glutamate synthase (GOGAT) cycle represents a crucial metabolic step of N assimilation, regulating crop yield. In this review, the physiological and genetic studies on GS and GOGAT of the main cereal crops will be examined, giving emphasis on their implications in NUE.

Genome-wide identification of glutamate synthase gene family and expression patterns analysis in response to carbon and nitrogen treatment in Populus.

Glutamate synthase (GOGAT) is a key enzyme in glutamine synthetase (GS)/GOGAT cycle and at the hub of carbon and nitrogen metabolism, catalyzing the formation of glutamate from α-oxoglutarate and glutamine. In this study, members of GOGAT family in Populus trichocarpa were identified and analyzed by bioinformatics. The four PtGOGATs were divided into two subgroups: subgroup A (Fd-GOGAT1 and Fd-GOGAT2) and subgroup B (NADH-GOGAT1 and NADH-GOGAT2). Many important elements have been identified in the promoters of different PtGOGATs, including hormone- and light-responsive elements. Meanwhile, the transcript levels of PxGOGATs were affected by light and diurnal cycle. Quantitative real-time PCR showed PxFd-GOGATs and PxNADH-GOGATs were mainly expressed in leaves and roots in Populus × xiaohei T. S. Hwang et Liang, respectively. Under elevated CO2, PxGOGATs were suppressed in all tissues except the stem. And PxFd-GOGATs and PxNADH-GOGATs were strongly induced by nitrogen in leaves and roots, respectively. In addition, PxGOGATs were stimulated significantly in roots in response to NH4+and glutamine directly. Our results provide new insights about GOGATs in poplar and their expression patterns under exogenous substances, to lay molecular basis for studying gene function and provide a reference for exploring putative roles of GOGATs in carbon-nitrogen balance.

Nitrogen starvation induces genome-wide activation of transposable elements in Arabidopsis.

Nitrogen (N) availability is a major limiting factor for plant growth and agricultural productivity. Although the gene regulation network in response to N starvation has been extensively studied, it remains unknown whether N starvation has an impact on the activity of transposable elements (TEs). Here, we report that TEs can be transcriptionally activated in Arabidopsis under N starvation conditions. Through genetic screening of idm1-14 suppressors, we cloned GLU1, which encodes a glutamate synthase that catalyzes the synthesis of glutamate in the primary N assimilation pathway. We found that glutamate synthase 1 (GLU1) and its functional homologs GLU2 and glutamate transport 1 (GLT1) are redundantly required for TE silencing, suggesting that N metabolism can regulate TE activity. Transcriptome and methylome analyses revealed that N starvation results in genome-wide TE activation without inducing obvious alteration of DNA methylation. Genetic analysis indicated that N starvation-induced TE activation is also independent of other well-established epigenetic mechanisms, including histone methylation and heterochromatin decondensation. Our results provide new insights into the regulation of TE activity under stressful environments in planta.

A counter-enzyme complex regulates glutamate metabolism in Bacillus subtilis.

Multi-enzyme assemblies composed of metabolic enzymes catalyzing sequential reactions are being increasingly studied. Here, we report the discovery of a 1.6 megadalton multi-enzyme complex from Bacillus subtilis composed of two enzymes catalyzing opposite ('counter-enzymes') rather than sequential reactions: glutamate synthase (GltAB) and glutamate dehydrogenase (GudB), which make and break glutamate, respectively. In vivo and in vitro studies show that the primary role of complex formation is to inhibit the activity of GudB. Using cryo-electron microscopy, we elucidated the structure of the complex and the molecular basis of inhibition of GudB by GltAB. The complex exhibits unusual oscillatory progress curves and is necessary for both planktonic growth, in glutamate-limiting conditions, and for biofilm growth, in glutamate-rich media. The regulation of a key metabolic enzyme by complexing with its counter enzyme may thus enable cell growth under fluctuating glutamate concentrations.

A glutamate synthase mutant of Bradyrhizobium sp. strain ORS285 is unable to induce nodules on Nod factor-independent Aeschynomene species.

The Bradyrhizobium sp. strain ORS285 is able to establish a nitrogen-fixing symbiosis with both Nod factor (NF) dependent and NF-independent Aeschynomene species. Here, we have studied the growth characteristics and symbiotic interaction of a glutamate synthase (GOGAT; gltD::Tn5) mutant of Bradyrhizobium ORS285. We show that the ORS285 gltD::Tn5 mutant is unable to use ammonium, nitrate and many amino acids as nitrogen source for growth and is unable to fix nitrogen under free-living conditions. Moreover, on several nitrogen sources, the growth rate of the gltB::Tn5 mutant was faster and/or the production of the carotenoid spirilloxanthin was much higher as compared to the wild-type strain. The absence of GOGAT activity has a drastic impact on the symbiotic interaction with NF-independent Aeschynomene species. With these species, inoculation with the ORS285 gltD::Tn5 mutant does not result in the formation of nodules. In contrast, the ORS285 gltD::Tn5 mutant is capable to induce nodules on NF-dependent Aeschynomene species, but these nodules were ineffective for nitrogen fixation. Interestingly, in NF-dependent and NF-independent Aeschynomene species inoculation with the ORS285 gltD::Tn5 mutant results in browning of the plant tissue at the site of the infection suggesting that the mutant bacteria induce plant defence responses.

New Insights into the Transcriptional Regulation of Genes Involved in the Nitrogen Use Efficiency under Potassium Chlorate in Rice (Oryza sativa L.).

Potassium chlorate (KClO3) has been widely used to evaluate the divergence in nitrogen use efficiency (NUE) between indica and japonica rice subspecies. This study investigated the transcriptional regulation of major genes involved in the NUE in rice treated with KClO3, which acts as an inhibitor of the reducing activity of nitrate reductase (NR) in higher plants. A set of two KClO3 sensitive nitrate reductase (NR) and two nitrate transporter (NRT) introgression rice lines (BC2F7), carrying the indica alleles of NR or NRT, derived from a cross between Saeilmi (japonica, P1) and Milyang23 (indica, P2), were exposed to KClO3 at the seedling stage. The phenotypic responses were recorded 7 days after treatment, and samples for gene expression, physiological, and biochemical analyses were collected at 0 h (control) and 3 h after KClO3 application. The results revealed that Saeilmi (P1, japonica) and Milyang23 (P2, indica) showed distinctive phenotypic responses. In addition, the expression of OsNR2 was differentially regulated between the roots, stem, and leaf tissues, and between introgression lines. When expressed in the roots, OsNR2 was downregulated in all introgression lines. However, in the stem and leaves, OsNR2 was upregulated in the NR introgression lines, but downregulation in the NRT introgression lines. In the same way, the expression patterns of OsNIA1 and OsNIA2 in the roots, stem, and leaves indicated a differential transcriptional regulation by KClO3, with OsNIA2 prevailing over OsNIA1 in the roots. Under the same conditions, the activity of NR was inhibited in the roots and differentially regulated in the stem and leaf tissues. Furthermore, the transcriptional divergence of OsAMT1.3 and OsAMT2.3, OsGLU1 and OsGLU2, between NR and NRT, coupled with the NR activity pattern in the roots, would indicate the prevalence of nitrate (NO3¯) transport over ammonium (NH4+) transport. Moreover, the induction of catalase (CAT) and polyphenol oxidase (PPO) enzyme activities in Saeilmi (P1, KClO3 resistant), and the decrease in Milyang23 (P2, KClO3 sensitive), coupled with the malondialdehyde (MDA) content, indicated the extent of the oxidative stress, and the induction of the adaptive response mechanism, tending to maintain a balanced reduction-oxidation state in response to KClO3. The changes in the chloroplast pigments and proline content propose these compounds as emerging biomarkers for assessing the overall plant health status. These results suggest that the inhibitory potential of KClO3 on the reduction activity of the nitrate reductase (NR), as well as that of the genes encoding the nitrate and ammonium transporters, and glutamate synthase are tissue-specific, which may differentially affect the transport and assimilation of nitrate or ammonium in rice.

Hashimoto's Thyroiditis Induces Hippocampus-Dependent Cognitive Alterations by Impairing Astrocytes in Euthyroid Mice.

Background: Although studies have reported an increased risk for cognitive disorders in Hashimoto's thyroiditis (HT) patients, even in the euthyroid state, the mechanisms involved remain unclear. The hippocampus is a classic brain region associated with cognitive function, among which the formation of long-term potentiation (LTP) in the Schaffer collateral-CA1 pathway plays an important role in the process of learning and memory. Therefore, this study established a euthyroid HT model in mice and investigated whether and how HT itself has the ability to trigger LTP alterations accompanied by learning and memory abnormality. Methods: An experimental euthyroid HT model was established in NOD mice through immunization with porcine thyroglobulin (Tg). Morris water maze was measured to determine mice spatial learning and memory. We investigated the effect of HT on synaptic transmission and high-frequency stimulation-induced LTP in the Schaffer collateral-CA1 synapse of mice hippocampus in vivo. Then, animals were sacrificed for thyroid-related parameter measure as well as detection of cellular and molecular events associated with the induction of LTP. Results: HT mice showed intrathyroidal lymphocyte infiltration and rising serum thyroid autoantibody levels accompanied by normal thyroid function. The HT mice had poorer performance in Morris water maze than controls. These alterations were mirrored by abnormalities in synaptic plasticity in the Schaffer collateral-CA1 synapses of the hippocampus in vivo. The integrity of the synaptic structure is the premise for the production of LTP. As detected by transmission electron microscopy, the ultrastructure of synapse and astrocyte in the hippocampus were impaired in euthyroid HT mice. Additionally, Western blot and real-time polymerase chain reaction analyses confirmed that in HT mice, GS, GLAST, and GLT-1, key elements in glutamate-glutamine circulation located in astrocyte, were downregulated, accompanied by elevated levels of glutamate in the hippocampus, which impaired the material basis for LTP induction. NMDR2B expression in the hippocampus was also downregulated. Conclusion: HT can induce damage of LTP in the hippocampal Schaffer collateral-CA1 pathway in the euthyroid state, and this can be attributed, at least partly, to astrocytes impairment, which may underlie the deleterious effects of HT itself on hippocampal-dependent learning and memory function.

New species of Tulasnella associated with Australian terrestrial orchids in the Cryptostylidinae and Drakaeinae.

Many orchids have an obligate relationship with Tulasnella mycorrhizal fungi for seed germination and support into adulthood. Despite the importance of Tulasnella as mycorrhizal partners, many species remain undescribed. Here, we use multiple sequence locus phylogenetic analyses to delimit and describe six new Tulasnella species associated with Australian terrestrial orchids from the subtribes Cryptostylidinae and Drakaeinae. Five of the new species, Tulasnella australiensis, T. occidentalis, T. punctata, T. densa, and T. concentrica, all associate with Cryptostylis (Cryptostylidinae), whereas T. rosea associates with Spiculaea ciliata (Drakaeinae). Isolates representing T. australiensis were previously also reported in association with Arthrochilus (Drakaeinae). All newly described Tulasnella species were delimited by phylogenetic analyses of four loci (nuc rDNA internal transcribed spacer region ITS1-5.8S-ITS2 [ITS], C14436 [ATP synthase], C4102 [glutamate synthase], and mt 16S rDNA [mtLSU]). The pairwise sequence divergence between species for the ITS region ranged from 5.6% to 25.2%, and the maximum sequence divergence within the newly described species ranged from 1.64% to 4.97%. There was a gap in the distribution of within- and between-species pairwise divergences in the region of 4-6%, with only one within-species value of 4.97% (for two T. australiensis isolates) and one between-species value of 5.6% (involving an isolate of T. occidentalis) falling within this region. Based on fluorescence staining, all six new Tulasnella species are binucleate and have septate, cylindrical hyphae. There was some subtle variation in culture morphology, but colony diameter as measured on 3MN+vitamin medium after 6 wk of growth did not differ among species. However, T. australiensis grew significantly (P < 0.02) slower than others on ½ FIM and » potato dextrose agar (PDA) media. Formal description of these Tulasnella species contributes significantly to documentation of Tulasnella diversity and provides names and delimitations to underpin further research on the fungi and their relationships with orchids.

Glutamate synthase 1 is involved in iron-deficiency response and long-distance transportation in Arabidopsis.

Iron is an essential microelement for plant growth. After uptake from the soil, iron is chelated by ligands and translocated from roots to shoots for subsequent utilization. However, the number of ligands involved in iron chelation is unclear. In this study, we identified and demonstrated that GLU1, which encodes a ferredoxin-dependent glutamate synthase, was involved in iron homeostasis. First, the expression of GLU1 was strongly induced by iron deficiency condition. Second, lesion of GLU1 results in reduced transcription of many iron-deficiency-responsive genes in roots and shoots. The mutant plants revealed a decreased iron concentration in the shoots, and displayed severe leaf chlorosis under the condition of Fe limitation, compared to wild-type. Third, the product of GLU1, glutamate, could chelate iron in vivo and promote iron transportation. Last, we also found that supplementation of glutamate in the medium can alleviate cadmium toxicity in plants. Overall, our results provide evidence that GLU1 is involved in iron homeostasis through affecting glutamate synthesis under iron deficiency conditions in Arabidopsis.

Role of Glutamate Synthase in Biofilm Formation by Bacillus subtilis.

Bacillus subtilis forms robust biofilms in the presence of large amounts of carbon sources, such as glycerol. However, little is known about the importance of the metabolic systems, or the relationship between metabolic systems and regulatory systems, involved in biofilm formation. Glutamate synthase, encoded by gltAB, is an enzyme that converts 2-ketoglutarate (a tricarboxylic acid [TCA] cycle intermediate) and glutamine into glutamate, which is a general amino group donor in metabolism. Here, we show that a ΔgltA mutant exhibited early arrest of biofilm formation in complex medium containing glycerol. This phenotype was not due to glutamate auxotrophy. Consistent with its biofilm formation phenotype, the ΔgltA mutant exhibited an early decrease in expression of the epsA and tapA operons, which are responsible for production of biofilm matrix polymers. This resulted from decreased activity of their regulator, Spo0A, as evidenced by reduced expression of other Spo0A-regulated genes in the ΔgltA mutant. The ΔgltA mutation prevented biofilm formation only in the presence of large amounts of glycerol. Moreover, limited expression of citrate synthase (but not other TCA enzymes) restored biofilm-forming ability to the ΔgltA mutant. These results indicate that the ΔgltA mutant accumulates an inhibitory intermediate (citrate) in the TCA cycle in the presence of large amounts of glycerol. The ΔgltA mutant formed biofilms when excess iron was added to the medium. Taken together, the data suggest that accumulation of citrate ions by the ΔgltA mutant causes iron shortage due to chelation, which prevents activation of Spo0A and causes defective biofilm formation.IMPORTANCEBacillus subtilis, a model organism for bacterial biofilm formation, forms robust biofilms in a medium-dependent manner. Although the regulatory network that controls biofilm formation has been well studied, the importance of the underlying metabolic systems remains to be elucidated. The present study demonstrates that a metabolic disorder in a well-conserved metabolic system causes accumulation of an inhibitory metabolic intermediate that prevents activation of the system that regulates biofilm formation. These findings increase our understanding of the coordination between cellular metabolic status and the regulatory networks governing biofilm formation.

Symbiont population control by host-symbiont metabolic interaction in Symbiodiniaceae-cnidarian associations.

In cnidarian-Symbiodiniaceae symbioses, algal endosymbiont population control within the host is needed to sustain a symbiotic relationship. However, the molecular mechanisms that underlie such population control are unclear. Here we show that a cnidarian host uses nitrogen limitation as a primary mechanism to control endosymbiont populations. Nitrogen acquisition and assimilation transcripts become elevated in symbiotic Breviolum minutum algae as they reach high-densities within the sea anemone host Exaiptasia pallida. These same transcripts increase in free-living algae deprived of nitrogen. Symbiotic algae also have an elevated carbon-to-nitrogen ratio and shift metabolism towards scavenging nitrogen from purines relative to free-living algae. Exaiptasia glutamine synthetase and glutamate synthase transcripts concomitantly increase with the algal endosymbiont population, suggesting an increased ability of the host to assimilate ammonium. These results suggest algal growth and replication in hospite is controlled by access to nitrogen, which becomes limiting for the algae as their population within the host increases.

Metabolomics of Escherichia coli Treated with the Antimicrobial Carbon Monoxide-Releasing Molecule CORM-3 Reveals Tricarboxylic Acid Cycle as Major Target.

In the last decade, carbon monoxide-releasing molecules (CORMs) have been shown to act against several pathogens and to be promising antimicrobials. However, the understanding of the mode of action and reactivity of these compounds on bacterial cells is still deficient. In this work, we used a metabolomics approach to probe the toxicity of the ruthenium(II) complex Ru(CO)3Cl(glycinate) (CORM-3) on Escherichia coli By resorting to 1H nuclear magnetic resonance, mass spectrometry, and enzymatic activities, we show that CORM-3-treated E. coli accumulates larger amounts of glycolytic intermediates, independently of the oxygen growth conditions. The work provides several evidences that CORM-3 inhibits glutamate synthesis and the iron-sulfur enzymes of the tricarboxylic acid (TCA) cycle and that the glycolysis pathway is triggered in order to establish an energy and redox homeostasis balance. Accordingly, supplementation of the growth medium with fumarate, α-ketoglutarate, glutamate, and amino acids cancels the toxicity of CORM-3. Importantly, inhibition of the iron-sulfur enzymes glutamate synthase, aconitase, and fumarase is only observed for compounds that liberate carbon monoxide. Altogether, this work reveals that the antimicrobial action of CORM-3 results from intracellular glutamate deficiency and inhibition of nitrogen and TCA cycles.

Genetic analyses of nitrogen assimilation enzymes in Brassica juncea (L.) Czern & Coss.

Nitrogen (N) is a critical input for plant growth and development. A better understanding of N uptake and utilization is important to develop plant breeding strategies for improving nitrogen use efficiency (NUE). With that objective in mind, we assayed a SNP-genotyped association panel comprising 92 inbred lines for the activities of nitrate reductase (NR), nitrite reductase (NIR), glutamine synthetase (GS) and glutamate synthase (GOGAT). All these enzymes are associated with N assimilation. The experiments were carried out at two levels of N application: no added N (N0) and agrnomically recommened dose (100 kg/ha) of N application (N100). Genome wide association studies (GWAS) helped to identify several marker-trait associations (MTAs), involving chromosomes A01, A06, A08, B02, B04, B05 and B08. These explained high phenotypic variation (up to 32%). Annotation of the genomic region(s) in or around significant SNPs allowed prediction of genes encoding high affinity nitrate transporters, glutamine synthetase 1.3, myb-like transcription factor family protein, bidirectional amino acid transporter 1, auxin signaling F-box 3 and oxidoreductases. This is the first attempt to use GWAS for identification of enzyme QTLs to explain variation for nitrogen assimilation enzymes in Brassica juncea.

Genetic diversity, infection prevalence, and possible transmission routes of Bartonella spp. in vampire bats.

Bartonella spp. are globally distributed bacteria that cause endocarditis in humans and domestic animals. Recent work has suggested bats as zoonotic reservoirs of some human Bartonella infections; however, the ecological and spatiotemporal patterns of infection in bats remain largely unknown. Here we studied the genetic diversity, prevalence of infection across seasons and years, individual risk factors, and possible transmission routes of Bartonella in populations of common vampire bats (Desmodus rotundus) in Peru and Belize, for which high infection prevalence has previously been reported. Phylogenetic analysis of the gltA gene for a subset of PCR-positive blood samples revealed sequences that were related to Bartonella described from vampire bats from Mexico, other Neotropical bat species, and streblid bat flies. Sequences associated with vampire bats clustered significantly by country but commonly spanned Central and South America, implying limited spatial structure. Stable and nonzero Bartonella prevalence between years supported endemic transmission in all sites. The odds of Bartonella infection for individual bats was unrelated to the intensity of bat flies ectoparasitism, but nearly all infected bats were infested, which precluded conclusive assessment of support for vector-borne transmission. While metagenomic sequencing found no strong evidence of Bartonella DNA in pooled bat saliva and fecal samples, we detected PCR positivity in individual saliva and feces, suggesting the potential for bacterial transmission through both direct contact (i.e., biting) and environmental (i.e., fecal) exposures. Further investigating the relative contributions of direct contact, environmental, and vector-borne transmission for bat Bartonella is an important next step to predict infection dynamics within bats and the risks of human and livestock exposures.

Structural polymorphism of the Escherichia coli poly-α-L-glutamate synthetase RimK.

Bacterial RimK is an enzyme that catalyzes the polyglutamylation of the C-terminus of ribosomal protein S6 and the synthesis of poly-α-L-glutamate peptides using L-glutamic acid. In the present study, the crystal structure of the Escherichia coli RimK protein complexed with the ATP analogue AMP-PNP was determined at 2.05 Šresolution. Two different conformations of RimK, closed and open forms, were observed in the crystals. The structural polymorphism revealed in this study provided important information to understand the mechanism by which RimK catalyzes the synthesis of poly-α-L-glutamate peptides and the polyglutamylation of ribosomal protein S6.

Surface Display of Bacterial Laccase CotA on Escherichia coli Cells and its Application in Industrial Dye Decolorization.

Laccase CotA from Bacillus subtilis 168 was successfully displayed on the membrane of Escherichia coli cells using poly-γ-glutamate synthetase A protein (PgsA) from B. subtilis as an anchoring matrix. Further analyses demonstrated that the fusion protein PgsA/CotA efficiently translocates to the cell surface of E. coli with an enzymatic activity of 65 U/108 cells. Surface-displayed CotA was shown to possess improved enzymatic properties compared with those of the wild-type CotA, including higher thermal stability (above 90% activity at 70 °C and nearly 40% activity at 90 °C after 5-h incubation) and stronger inhibitor tolerance (approximately 80 and 65% activity when incubated with 200 and 400 mM NaCl, respectively). Furthermore, the whole-cell system was demonstrated to have high enzymatic activity against anthraquinone dye, Acid Blue 62, triphenylmethane dye, Malachite Green, and azo dye, Methyl Orange with the decolorization percentages of 91, 45, and 75%, after 5-h incubation, respectively.

Molecular characterization of a novel algal glutamine synthetase (GS) and an algal glutamate synthase (GOGAT) from the colorful outer mantle of the giant clam, Tridacna squamosa, and the putative GS-GOGAT cycle in its symbiotic zooxanthellae.

Giant clams harbor symbiotic zooxanthellae (Symbiodinium), which are nitrogen-deficient, mainly in the fleshy and colorful outer mantle. This study aimed to sequence and characterize the algal Glutamine Synthetase (GS) and Glutamate Synthase (GLT), which constitute the glutamate synthase cycle (or GS-GOGAT cycle, whereby GOGAT is the protein acronym of GLT) of nitrogen assimilation, from the outer mantle of the fluted giant clam, Tridacna squamosa. We had identified a novel GS-like cDNA coding sequence of 2325 bp, and named it as T. squamosa Symbiodinium GS1 (TSSGS1). The deduced TSSGS1 sequence had 774 amino acids with a molecular mass of 85 kDa, and displayed the characteristics of GS1 and Nucleotide Diphosphate Kinase. The cDNA coding sequence of the algal GLT, named as T. squamosa Symbiodinium GLT (TSSGLT), comprised 6399 bp, encoding a protein of 2133 amino acids and 232.4 kDa. The zooxanthellal origin of TSSGS1 and TSSGOGAT was confirmed by sequence comparison and phylogenetic analyses. Indeed, TSSGS1 and TSSGOGAT were expressed predominately in the outer mantle, which contained the majority of the zooxanthellae. Immunofluorescence microscopy confirmed the expression of TSSGS1 and TSSGOGAT in the cytoplasm and the plastids, respectively, of the zooxanthellae in the outer mantle. It can be concluded that the symbiotic zooxanthellae of T. squamosa possesses a glutamate synthase (TSSGS1-TSSGOGAT) cycle that can assimilate endogenous ammonia produced by the host clam into glutamate, which can act as a substrate for amino acid syntheses. Thus, our results provide insights into why intact giant clam-zooxanthellae associations do not excrete ammonia under normal circumstances.

Glutamate synthases from conifers: gene structure and phylogenetic studies.

BACKGROUND: Plants synthesize glutamate from ammonium by the combined activity of the enzymes glutamine synthetase (GS) and glutamate synthase (GOGAT) through the glutamate synthase cycle. In plants, there are two forms of glutamate synthases that differ in their electron donors, NADH-GOGAT (EC 1.4.1.14) and Fd-GOGAT (EC 1.4.7.1), which have differential roles either in primary ammonia assimilation or in the reassimilation of ammonium from different catabolic processes. Glutamate synthases are complex iron-sulfur flavoproteins containing functional domains involved in the control and coordination of their catalytic activities in annual plants. In conifers, partial cDNA sequences for GOGATs have been isolated and used for gene expression studies. However, knowledge of the gene structure and of phylogenetic relationships with other plant enzymes is quite scant. RESULTS: Technological advances in conifer megagenomes sequencing have made it possible to obtain full-length cDNA sequences encoding Fd- and NADH-GOGAT from maritime pine, as well as BAC clones containing sequences for NADH-GOGAT and Fd-GOGAT genes. In the current study, we studied the genomic organization of pine GOGAT genes, the size of their exons/introns, copy numbers in the pine genome and relationships with other plant genes. Phylogenetic analysis was performed, and the degree of preservation and dissimilarity of key domains for the catalytic activities of these enzymes in different taxa were determined. CONCLUSIONS: Fd- and NADH-GOGAT are encoded by single-copy genes in the maritime pine genome. The Fd-GOGAT gene is extremely large spanning more than 330 kb and the presence of very long introns highlights the important contribution of LTR retrotransposons to the gene size in conifers. In contrast, the structure of the NADH-GOGAT gene is similar to the orthologous genes in angiosperms. Our phylogenetic analysis indicates that these two genes had different origins during plant evolution. The results provide new insights into the structure and molecular evolution of these essential genes.

Glutamate synthase MoGlt1-mediated glutamate homeostasis is important for autophagy, virulence and conidiation in the rice blast fungus.

Glutamate homeostasis plays a vital role in central nitrogen metabolism and coordinates several key metabolic functions. However, its function in fungal pathogenesis and development has not been investigated in detail. In this study, we identified and characterized a glutamate synthase gene MoGLT1 in the rice blast fungus Magnaporthe oryzae that was important to glutamate homeostasis. MoGLT1 was constitutively expressed, but showed the highest expression level in appressoria. Deletion of MoGLT1 resulted in a significant reduction in conidiation and virulence. The ΔMoglt1 mutants were defective in appressorial penetration and the differentiation and spread of invasive hyphae in penetrated plant cells. The addition of exogenous glutamic acid partially rescued the defects of the ΔMoglt1 mutants in conidiation and plant infection. Assays for MoAtg8 expression and localization showed that the ΔMoglt1 mutants were defective in autophagy. The ΔMoglt1 mutants were delayed in the mobilization of glycogens and lipid bodies from conidia to developing appressoria. Taken together, our results show that glutamate synthase MoGlt1-mediated glutamate homeostasis is important for pathogenesis and development in the rice blast fungus, possibly via the regulation of autophagy.