GOGAT Genes Research Articles

Glutamine Synthetase and Glutamate Synthase Family Perform Diverse Physiological Functions in Exogenous Hormones and Abiotic Stress Responses in Pyrus betulifolia Bunge (P.be)

The unscientific application of nitrogen (N) fertilizer not only increases the economic input of pear growers but also leads to environmental pollution. Improving plant N use efficiency (NUE) is the most effective economical method to solve the above problems. The absorption and utilization of N by plants is a complicated process. Glutamine synthetase (GS) and glutamate synthase (GOGAT) are crucial for synthesizing glutamate from ammonium in plants. However, their gene family in pears has not been documented. This study identified 29 genes belonging to the GS and GOGAT family in the genomes of Pyrus betulaefolia (P.be, 10 genes), Pyrus pyrifolia (P.py, 9 genes), and Pyrus bretschneideri (P.br, 10 genes). These genes were classified into two GS subgroups (GS1 and GS2) and two GOGAT subgroups (Fd–GOGAT and NADH–GOGAT). The similar exon–intron structures and conserved motifs within each cluster suggest the evolutionary conservation of these genes. Meanwhile, segmental duplication has driven the expansion and evolution of the GS and GOGAT gene families in pear. The tissue–specific expression dynamics of PbeGS and PbeGOGAT genes suggest significant roles in pear growth and development. Cis–acting elements of the GS and GOGAT gene promoters are crucial for plant development, hormonal responses, and stress reactions. Furthermore, qRT–PCR analysis indicated that PbeGSs and PbeGOGATs showed differential expression under exogenous hormones (GA3, IAA, SA, ABA) and abiotic stress (NO3− and salt stress). In which, the expression of PbeGS2.2 was up–regulated under hormone treatment and down–regulated under salt stress. Furthermore, physiological experiments demonstrated that GA3 and IAA promoted GS, Fd–GOGAT, and NADH–GOGAT enzyme activities, as well as the N content. Correlation analysis revealed a significant positive relationship between PbeGS1.1, PbeGS2.2, PbeNADH–GOGATs, and the N content. Therefore, PbeGS1.1, PbeGS2.2, and PbeNADH–GOGATs could be key candidate genes for improving NUE under plant hormone and abiotic stress response. To the best of our knowledge, our study provides valuable biological information about the GS and GOGAT family in the pear for the first time and establishes a foundation for molecular breeding aimed at developing high NUE pear rootstocks.

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.

Identification of Wheat Glutamate Synthetase Gene Family and Expression Analysis under Nitrogen Stress.

Nitrogen (N), as the main component of biological macromolecules, maintains the basic process of plant growth and development. GOGAT, as a key enzyme in the N assimilation process, catalyzes α-ketoglutaric acid and glutamine to form glutamate. In this study, six GOGAT genes in wheat (Triticum aestivum L.) were identified and classified into two subfamilies, Fd-GOGAT (TaGOGAT2s) and NADH-GOGAT (TaGOGAT3s), according to the type of electron donor. Subcellular localization prediction showed that TaGOGAT3-D was localized in mitochondria and that the other five TaGOGATs were localized in chloroplasts. Via the analysis of promoter elements, many binding sites related to growth and development, hormone regulation and plant stress resistance regulations were found on the TaGOGAT promoters. The tissue-specificity expression analysis showed that TaGOGAT2s were mainly expressed in wheat leaves and flag leaves, while TaGOGAT3s were highly expressed in roots and leaves. The expression level of TaGOGATs and the enzyme activity of TaGOGAT3s in the leaves and roots of wheat seedlings were influenced by the treatment of N deficiency. This study conducted a systematic analysis of wheat GOGAT genes, providing a theoretical basis not only for the functional analysis of TaGOGATs, but also for the study of wheat nitrogen use efficiency (NUE).

Integrative analysis of transcriptome and metabolome reveals the mechanism of foliar application of Bacillus amyloliquefaciens to improve summer tea quality (Camellia sinensis)

Bacillus amyloliquefaciens is a promising microbial agent for quality improvement in crops; however, the effects of B. amyloliquefaciens biofertilizers on tea leaf metabolites are relatively unknown. Herein, a combination of metabolome profiling and transcriptome analysis was employed to investigate the effects of foliar spraying with B. amyloliquefaciens biofertilizers on tea leaf quality. The tea polyphenol to amino acid ratio (TP/AA), catechin, and caffeine levels decreased, but theanine level increased in tea leaves after foliar spraying with B. amyloliquefaciens. The differentially accumulated metabolites included flavonoids, phenolic acids, organic acids, amino acids, and carbohydrates. The decrease in catechin was correlated with the catechin/flavonoid biosynthesis pathway. The AMPD gene was highly associated with caffeine content, while the GOGAT gene was associated with theanine accumulation. Foliar spraying with B. amyloliquefaciens biofertilizers may improve summer tea quality. Our findings provide a basis for the application of B. amyloliquefaciens biofertilizers in tea plants and new insights on summer tea leaf resource utilization.

Interspecies interaction for nitrogen use efficiency via up-regulated glutamine and glutamate synthase under wheat-faba bean intercropping

• Intercropping drove higher grain N and total N accumulation, NTE, and NUE. • Interspecies interaction upregulated GS/GOGAT activity and gene expression in leaves. • GS/GOGAT stimulated by intercropping linked with higher straw and total N accumulation. • Upregulated GS/GOGAT after wheat heading stages contributed to higher NTE and NUE. Glutamine synthetase (GS) and the glutamate synthase (GOGAT) cycle are closely related to plant nitrogen (N) uptake, translocation, and remobilization, but little attention has been paid to their role in intercropping systems. A two-factor field trial involving two planting patterns (mono-cropped wheat [MW] and wheat intercropped with faba bean) and four N levels (N0, N1, N2, and N3 with respective application rates of 0, 90, 180, and 270 kg ha −1 for wheat) were studied. Nitrogen accumulation and allocation were determined, and the GS and GOGAT activities and their gene expression in the leaves were analyzed. The results showed that wheat total aboveground N accumulation and grain N accumulation were increased when wheat was intercropped with faba bean. Intercropped wheat (IW) presented higher N translocation efficiency (NTE) than MW (except for the N3 level during 2018–2019) during the reproductive growth stage. Thus, the grain yield (except for the N3 level), N harvest index (NHI), and apparent N recovery rate (ANR) were higher in IW than in MW. In addition, total GS, Fd-GOGAT, and NADH-GOGAT activities in the wheat leaves were increased by 27 %–42 %, 17 %–44 %, and 15 %–25 %, respectively, under the N0, N1, and N2 levels from the stem elongation to filling stages when wheat was intercropped with faba bean. On average, GS1, GS2, Fd-GOGAT, and NADH-GOGAT gene expression in the IW flag leaves was 1.4–4.3 times, 1.2–3.6 times, 1.2–2.2 times, and 1.4–6.7 times higher, respectively, than that in MW. Both GS and GOGAT activity and gene expression were tightly related to straw N accumulation and NTE rather than grain N accumulation based on simple correlation analysis. In addition, wheat total N accumulation at maturity was more sensitive to GS and GOGAT activity and gene expression in the flag leaves during the heading, flowering, and filling stages, and ANR only linked with GS and GOGAT activity at the filling stage. Hence, it could be concluded that up-regulated GS and GOGAT activity and gene expression induced by intercropping during the reproductive growth stages contributed to higher NTE and NUE in intercropping. These findings suggest that interspecies interactions modulated GS and GOGAT, which play a crucial role in the intercropping advantage and provide insight into the mechanism of enhanced NUE under intercropping.

Transcriptome Analysis of Two Near-Isogenic Lines with Different NUE under Normal Nitrogen Conditions in Wheat.

Simple SummaryHigh nitrogen use efficiency (NUE) in wheat (Triticum aestivum L.) is the key to ensure high yield and reduce pollution. Understanding the physiological and molecular changes that regulate NUE is important for the breeding of high-NUE wheat varieties. Carbon and nitrogen metabolism are the basic metabolic pathways in plants. It becomes important to reveal the underlying molecular mechanisms related to carbon and nitrogen metabolism, which may be helpful to improve NUE. In this paper, two wheat near-isogenic lines (NILs) with contrasting NUE were performed RNA-Sequencing (RNA-Seq) to identify candidate genes associated with carbon/nitrogen metabolism under normal nitrogen conditions. Our research may provide new insights into the comprehensive understanding of the molecular mechanism underlying NUE.Nitrogen (N) is an essential nutrient element for crop productivity. Unfortunately, the nitrogen use efficiency (NUE) of crop plants gradually decreases with the increase of the N application rate. Nevertheless, little has been known about the molecular mechanisms of differences in NUE among genotypes of wheat. In this study, we used RNA-Sequencing (RNA-Seq) to compare the transcriptome profiling of flag leaves at the stage of anthesis in wheat NILs (1Y, high-NUE, and 1W, low-NUE) under normal nitrogen conditions (300 kg N ha−1, corresponding to 1.6 g N pot−1). We identified 7023 DEGs (4738 upregulated and 2285 downregulated) in the comparison between lines 1Y and 1W. The responses of 1Y and 1W to normal N differed in the transcriptional regulatory mechanisms. Several genes belonging to the GS and GOGAT gene families were upregulated in 1Y compared with 1W, and the enhanced carbon metabolism might lead 1Y to produce more C skeletons, metabolic energy, and reductants for nitrogen metabolism. A subset of transcription factors (TFs) family members, such as ERF, WRKY, NAC, and MYB, were also identified. Collectively, these identified candidate genes provided new information for a further understanding of the genotypic difference in NUE.

Dynamic transcriptome analysis of root nitrate starvation and re-supply provides insights into nitrogen metabolism in pear (Pyrus bretschneideri)

Pear (Pyrus bretschneideri) is a popular fruit worldwide, but the irrational utilization of nitrogen as a fertilizer not only greatly affects the fruit’ quality, but also wastes resources and results in serious environmental pollution. To better understand the molecular mechanism in pear responsible for the regulation of nitrate transport and assimilation, RNA-seq was performed on samples collected in response to nitrate treatments. Here, 10,273 differentially expressed genes were obtained and annotated into 49 GO terms, 45 clusters having co-expression trends that involved 18 KEGG-defined significantly overrepresented pathways. The KEGG pathways revealed that 15 unigenes, including one NRT gene, two NR genes, one NiR gene, two GDH genes, six GS genes and three GOGAT genes, were related to nitrogen metabolism and significantly differentially expressed in response to nitrate starvation and a nitrate re-supply treatment. Furthermore, 449 transcription factors belonging to 35 different families were identified during the nitrate treatments. The expression patterns of 14 randomly selected differentially expressed genes were validated by qRT-PCR. This study provides valuable resources for investigating the genetics of the nitrogen metabolic pathways and improving nitrogen utilization efficiency in pear.

Glutamate synthases from conifers: gene structure and phylogenetic studies

BackgroundPlants 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.ResultsTechnological 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.ConclusionsFd- 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.

Allelic Variants of Glutamine Synthetase and Glutamate Synthase Genes in a Collection of Durum Wheat and Association with Grain Protein Content

Wheat is one of the most important crops grown worldwide. Despite the fact that it accounts for only 5% of the global wheat production, durum wheat (Triticum turgidum L. subsp. durum) is a commercially important tetraploid wheat species, which originated and diversified in the Mediterranean basin. In this work, the candidate gene approach has been applied in a collection of durum wheat genotypes; allelic variants of genes glutamine synthetase (GS2) and glutamate synthase (GOGAT) were screened and correlated with grain protein content (GPC). Natural populations and collections of germplasms are quite suitable for this approach, as molecular polymorphisms close to a locus with evident phenotypic effects may be closely associated with their character, providing a better physical resolution than genetic mapping using ad hoc constituted populations. A number of allelic variants were detected both for GS2 and GOGAT genes, and regression analysis demonstrated that some variations are positively and significantly related to the GPC effect. Additionally, these genes map into homoeologous chromosome groups 2 and 3, where several authors have localized important quantitative trait loci (QTLs) for GPC. The information outlined in this work could be useful in breeding and marker-assisted selection programs.

Molecular analyses of tomato GS, GOGAT and GDH gene families and their response to abiotic stresses

Glutamine synthetase (GS), glutamate synthase (GOGAT) and glutamate dehydrogenase (GDH) are closely related enzymes in plant nitrogen metabolism and potential targets for improving nitrogen use efficiency. However, little research has focused on the enzyme-encoding genes in tomato. Here, a comprehensive study of these genes was conducted. Six GS genes, two GOGAT genes and five GDH genes were identified in tomato. Bioinformatics and gene expression analyses suggested that these genes evolved species-specific regulatory properties and biological functions in tomato. SlNADH-GOGAT, SlGS1.1 and SlNAD-GDHB1 were abundantly expressed in roots, SlGS1.1 can be induced by nitrogen deprivation, and SlGS1.2, SlGS1.3, SlNADH-GOGAT and SlNAD-GDHB1 can be induced by the re-supply of nitrogen after 5 days of deprivation, they may play key roles in primary nitrogen assimilation. SlFd-GOGAT, SlGS1.1 and SlNAD-GDHA1-A2 were also highly expressed in fruits, indicating their important roles in fruit development and ripening. Tomato GS, GOGAT and GDH may be involved in stress responsiveness, since most of these genes modified their expression levels under drought, cold or heat stress treatment. We believe these findings will assist in the exploration of the genes’ biological functions and regulatory mechanisms, as well as the studies to improve nitrogen use efficiency, stress resistance and fruit quality in tomato.

Differential regulation of GS-GOGAT gene expression by plant growth regulators in Arabidopsis seedlings

Primary and secondary ammonium assimilation is catalyzed by the glutamine synthetase-glutamate synthase (GS-GOGAT) pathway in plants. The Arabidopsis genome contains five cytosolic GS1 genes (GLN1;1 - GLN1;5), one nuclear gene for chloroplastic GS2 isoform (GLN2), two Fd-GOGAT genes (GLU1 and GLU2) and a GLT1 gene coding for NADH-GOGAT. Even though the regulation of GS and GOGAT isoforms has been extensively studied in response to various environmental and metabolic cues in many plant species, little is known about the effects of phytohormones on their regulation. The objective of this study was to investigate the impact of representative plant growth regulators, kinetin (KIN), abscisic acid (ABA), gibberellic acid (GA3) and 2,4-dichlorophenoxyacetic acid (2,4-D), on the expression of A. thaliana GS and GOGAT genes. The obtained results indicate that GS and GOGAT genes are differentially regulated by growth regulators in shoots and roots. KIN and 2,4-D repressed GS and GOGAT expression in roots, with little effect on transcript levels in shoots. KIN affected all tested genes; 2,4-D was apparently more selective and less potent. ABA induced the expression of GLN1;1 and GLU2 in whole seedlings, while GA3 enhanced the expression of all tested genes in shoots, except GLU2. The observed expression patterns are discussed in relation to physiological roles of investigated plant growth regulators and N-assimilating enzymes.

Effects of NaCl stress on nitrogen metabolism of cucumber seedlings

To investigate the effects of NaCl stress on plant growth, nitrogen assimilation, proline and soluble protein contents, and gene expression of enzymes of nitrogen metabolism, a hydroponic experiment using cucumber (Cucumis sativus L.) seedlings was performed. The seedlings were grown in nutrient solution supplemented with 0 or 84 mM NaCl for up to 9 days. Plant biomass, especially root biomass, was significantly decreased under NaCl stress. Salinity significantly increased ammonium content, but decreased nitrate and soluble protein contents in leaves and roots. Salt stress caused a significant increase in proline content, which peaked on day 3 of NaCl treatment. Moreover, salt stress significantly decreased activities of nitrate reductase (NR), glutamine synthetase (GS), glutamate synthase (GOGAT), and glutamate dehydrogenase (GDH) in the roots and leaves on the 3rd, 6th, and 9th day of NaCl treatment. A semiquantitative RT-PCR approach showed that changes in NR, GS and GOGAT gene expression were consistent with the salt-induced changes in enzyme activities. These results suggest that salt stress-induced growth inhibition in cucumber seedlings may involve disruption of nitrogen absorption and decreased activities of enzymes associated with nitrogen assimilation.

Effects of NaCl Stress on Nitrogen Metabolism of Cucumber Seedlings

To investigate the effects of NaCl stress on plant growth, nitrogen assimilation, proline and sol uble protein contents, and gene expression of enzymes of nitrogen metabolism, a hydroponic experiment using cucumber (Cucumis sativus L.) seedlings was performed. The seedlings were grown in nutrient solu tion supplemented with 0 or 84 mM NaCl for up to 9 days. Plant biomass, especially root biomass, was sig nificantly decreased under NaCl stress. Salinity significantly increased ammonium content, but decreased nitrate and soluble protein contents in leaves and roots. Salt stress caused a significant increase in proline content, which peaked on day 3 of NaCl treatment. Moreover, salt stress significantly decreased activities of nitrate reductase (NR), glutamine synthetase (GS), glutamate synthase (GOGAT), and glutamate dehydrogenase (GDH) in the roots and leaves on the 3rd, 6th, and 9th day of NaCl treatment. A semi quantitative RT PCR approach showed that changes in NR, GS and GOGAT gene expression were consis tent with the salt induced changes in enzyme activities. These results suggest that salt stress induced growth inhibition in cucumber seedlings may involve disruption of nitrogen absorption and decreased activities of enzymes associated with nitrogen assimilation.

Transcriptional regulation of host [formula omitted] transporters and GS/GOGAT pathway in arbuscular mycorrhizal rice roots

Arbuscular mycorrhizal (AM) fungi play a key role in the nutrition of many land plants. AM roots have two pathways for nutrient uptake, directly through the root epidermis and root hairs and via AM fungal hyphae into root cortical cells, where arbuscules or hyphal coils provide symbiotic interfaces. Recent studies demonstrated that the AM symbiosis modifies the expression of plant transporter genes and that NH4+ is the main form of N transported in the symbiosis. The aim of the present work was to get insights into the mycorrhizal N uptake pathway in Oryza sativa by analysing the expression of genes encoding ammonium transporters (AMTs), glutamine synthase (GS) and glutamate synthase (GOGAT) in roots colonized by the AM fungus Rhizophagus irregularis and grown under two N regimes. We found that the AM symbiosis down-regulated OsAMT1;1 and OsAMT1;3 expression at low-N, but not at high-N conditions, and induced, independently of the N status of the plant, a strong up-regulation of OsAMT3;1 expression. The AM-inducible NH4+ transporter OsAMT3;1 belongs to the family 2 of plant AMTs and is phylogenetically related to the AM-inducible AMTs of other plant species. Moreover, for the first time we provide evidence of the specific induction of a GOGAT gene upon colonization with an AM fungus. These data suggest that OsAMT3;1 is likely involved in the mycorrhizal N uptake pathway in rice roots and that OsGOGAT2 plays a role in the assimilation of the NH4+ supplied via the OsAMT3;1 AM-inducible transporter.

Control of glutamate homeostasis in Bacillus subtilis: a complex interplay between ammonium assimilation, glutamate biosynthesis and degradation

Glutamate, the major amino group donor in anabolism, is synthesized by the combined action of the glutamine synthetase (GS) and the glutamate synthase (GOGAT) in Bacillus subtilis. The glutamate dehydrogenase (GDH) exclusively degrades glutamate. GS and GDH are both trigger enzymes, active in nitrogen metabolism and in controlling gene expression. Feedback-inhibited GS (FBI-GS) controls DNA-binding activities of two transcription factors, the repressor GlnR and TnrA, the global regulator of nitrogen metabolism. FBI-GS binds to and activates GlnR. This protein complex inhibits GS formation and thus glutamine synthesis. Moreover, FBI-GS inhibits DNA-binding activity of TnrA. Glutamate biosynthesis, the reaction linking carbon with nitrogen metabolism, is controlled by GDH. Together with glutamate GDH inhibits GltC, the transcription factor that activates expression of the GOGAT genes. Thus, GS and GDH control glutamine and glutamate synthesis, respectively, depending on the nitrogen status of the cell. B. subtilis lacking a functional GDH show a severe growth defect. Interestingly, the growth defect is suppressed by the rapid activation of an inactive GDH. Thus, maintenance of glutamate homeostasis is crucial for cellular vitality. This review covers the recent work on the complex control of glutamine and glutamate metabolism in the Gram-positive model organism B. subtilis.

Suppression of glutamate synthase genes significantly affects carbon and nitrogen metabolism in rice (Oryza sativa L.)

Rice (Oryza sativa) glutamate synthase (GOGAT, EC 1.4.1.14) enzymes have been proposed to have great potential for improving nitrogen use efficiency, but their functions in vivo and their effects on carbon and nitrogen metabolism have not been systematically explored. In this research, we analyzed transcriptional profiles of rice GOGAT genes using a genome-wide microarray database, and investigated the effects of suppression of glutamate synthase genes on carbon and nitrogen metabolism using GOGAT co-suppressed rice plants. Transcriptional profiles showed that rice GOGAT genes were expressed differently in various tissues and organs, which suggested that they have different roles in vivo. Compared with the wild-type, tiller number, total shoot dry weight, and yield of GOGAT co-suppressed plants were significantly decreased. Physiological and biochemical studies showed that the contents of nitrate, several kinds of free amino acids, chlorophyll, sugars, sugar phosphates, and pyridine nucleotides were significantly decreased in leaves of GOGAT co-suppressed plants, but the contents of free ammonium, 2-oxoglutarate, and isocitrate in leaves were increased. We conclude that GOGATs play essential roles in carbon and nitrogen metabolism, and that they are indispensable for efficient nitrogen assimilation in rice.

Influence of nitrogen on the expression of TaDof1 transcription factor in wheat and its relationship with photo synthetic and ammonium assimilating efficiency

Nitrogen is a crucial macronutrient needed in the greatest amount of all mineral elements required by plants. Development of crop varieties with high nitrogen use efficiency (NUE) is imperative for sustainable agriculture. Understanding how plant genes respond to different nitrogen conditions is essential for formulating approaches, for manipulating genes, for improving NUE. In the present study we analyzed the activity of three different enzymes involved in nitrogen assimilation viz., GS, GOGAT and GDH along with physiological parameters like chlorophyll variable yield (Fv/Fmax), photosynthesis rate and total chlorophyll content at four different growth stages of wheat plant development under different nitrogen treatments. For this study two different wheat varieties UP-2644 and Raj-4097 having high and low protein content, respectively in the grains were chosen. Gene expression profile of a Dof transcription factor (TaDof1 of wheat) was also included in the study to assess its role in nitrogen metabolism. Densitometry analysis at S(2) and S(3) stage of wheat spikes of both the wheat varieties grown at different nitrogen treatments showed that TaDof1 expression was up-regulated in low nitrogen treatment. In S(3) stage, in high protein content wheat variety UP-2644, TaDof1 expression was elevated in low and normal nitrogen treatment as compared to high nitrogen treatment. The gene expression profile of Dof 1 was found to coincide with the enzyme activities of GS, GOGAT at the S(3) stage. The activities of these enzymes were prolonged in the high protein content variety. Since, Dof transcription factor(s) have been previously reported to control the expression of genes involved nitrogen assimilation i.e., GS and GOGAT and may be the elevated expression of Dof 1 at the grain filling stage over expresses the GS and GOGAT genes thereby prolonging their activities.

Expression analysis of the glutamine synthetase and glutamate synthase gene families in young rice ( Oryza sativa) seedlings

Glutamine synthetase (GS) and glutamate synthase (GOGAT) serve for primary assimilation of N in higher plants. When NH 4 + is the major inorganic N source, rice plants must assimilate NH 4 + quickly in roots through the GS/GOGAT cycle to ameliorate the toxic effect of excess NH 4 +. The sequence of the rice genome is almost complete, facilitating the identification of the GS and GOGAT gene families in this species. Thus, we investigated the different members of GS and GOGAT genes, and analyzed the pattern of expression of each gene in young rice seedlings by quantitative real-time PCR, revealing a distinct expression pattern for these genes. Os Gln1;1 and Os Gln1;2 mainly functions in roots, and Os Gln2 and Os Glu1 are preferentially expressed in leaves. However, transcriptions of Os Gln1;1, Os Gln1;2, Os Gln2 and Os Glu1 in leaves are all increased by increased N level while those in roots not influenced or even decreased. Os Glt1 and Os Glt2 are expressed primarily in roots when N is limiting, but in leaves when non-limiting. Transcription of Os Glt1 is decreased, but Os Glt2 is increased by increased N level in roots and leaves. When rice roots are exposed to NO 3 − and NH 4 + for 2 h after N-starvation, transcriptions of Os Gln1;1, Os Glt1, Os Glt2 and Os Glu1 are all repressed by NO 3 − and NH 4 +. Os Gln1;2 expression shows significant up-regulation by NH 4 + and down-regulation by NO 3 − while Os Gln2 down-regulation by NH 4 + and up-regulation by NO 3 −.

Pathways for glutamate biosynthesis in the yeast Kluyveromyces lactis.

Purified glutamate synthase (GOGAT) from Kluyveromyces lactis was characterized as a high-molecular-mass polypeptide, a distinction shared with previously described GOGATs from other eukaryotic micro-organisms. Using degenerate deoxyoligonucleotides, designed from conserved regions of the alfalfa, maize and Escherichia coli GOGAT genes, a 300 bp PCR fragment from the K. lactis GOGAT gene KIGLT1 was obtained. This fragment was used to construct null GOGAT mutants of K. lactis by gene replacement. These mutants showed no growth defect phenotype and were able to grow on ammonium as sole nitrogen source. Double mutants obtained from a cross between a previously described KIGDH1 mutant and the K. lactis null GOGAT strain were full glutamate auxotrophs. These results indicate that glutamate biosynthesis in K. lactis is afforded through the combined action of KIGDH1 and KIGLT1 products.

Analysis of a promoter for the NADH - glutamate synthase gene in rice (Oryza sativa): cell type-specific expression in developing organs of transgenic rice plants

This paper originates from a presentation at the International Conference on Assimilate Transport and Partitioning, Newcastle, NSW, August 1999 The entire 3.7 kbp 5´-upstream region (–2840 to +886) from the translational start codon of NADH–glutamate synthase (NADH–GOGAT, EC 1.4.1.14) gene from rice (Oryza sativa L.) or the region sequentially deleted from the 5´-end was fused with the β−glucuronidase (GUS) reporter gene. The chimeric gene was introduced into calli derived from rice scutellum via Agrobacterium tumefaciens-mediated transformation and tissue-specific GUS activity determined in T0 generations. When the entire region was fused, GUS activity was detected in vascular bundles of the developing leaf blade and in dorsal and lateral vascular bundles of developing grains. This corresponds with our previous immunodetection of NADH–GOGAT protein (Hayakawa et al., Planta 193, 455–460, 1994). A series of deletion experiments showed that a 149-nucleotide region between –142 and +7 was essential for promoter activity in the NADH–GOGAT gene.