NADH-GOGAT Gene Research Articles

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.

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.

Structural Analysis of the Wheat Genes Encoding NADH-Dependent Glutamine-2-oxoglutarate Amidotransferases and Correlation with Grain Protein Content

BackgroundNitrogen uptake and the efficient absorption and metabolism of nitrogen are essential elements in attempts to breed improved cereal cultivars for grain or silage production. One of the enzymes related to nitrogen metabolism is glutamine-2-oxoglutarate amidotransferase (GOGAT). Together with glutamine synthetase (GS), GOGAT maintains the flow of nitrogen from NH4 + into glutamine and glutamate, which are then used for several aminotransferase reactions during amino acid synthesis.ResultsThe aim of the present work was to identify and analyse the structure of wheat NADH-GOGAT genomic sequences, and study the expression in two durum wheat cultivars characterized by low and high kernel protein content. The genomic sequences of the three homoeologous A, B and D NADH-GOGAT genes were obtained for hexaploid Triticum aestivum and the tetraploid A and B genes of Triticum turgidum ssp. durum. Analysis of the gene sequences indicates that all wheat NADH-GOGAT genes are composed of 22 exons and 21 introns. The three hexaploid wheat homoeologous genes have high conservation of sequence except intron 13 which shows differences in both length and sequence. A comparative analysis of sequences among di- and mono-cotyledonous plants shows both regions of high conservation and of divergence. qRT-PCR performed with the two durum wheat cvs Svevo and Ciccio (characterized by high and low protein content, respectively) indicates different expression levels of the two NADH-GOGAT-3A and NADH-GOGAT-3B genes.ConclusionThe three hexaploid wheat homoeologous NADH-GOGAT gene sequences are highly conserved – consistent with the key metabolic role of this gene. However, the dicot and monocot amino acid sequences show distinctive patterns, particularly in the transit peptide, the exon 16–17 junction, and the C-terminus. The lack of conservation in the transit peptide may indicate subcellular differences between the two plant divisions - while the sequence conservation within enzyme functional domains remains high. Higher expression levels of NADH-GOGAT are associated with higher grain protein content in two durum wheats.

Cell type distinct accumulations of mRNA and protein for NADH-dependent glutamate synthase in rice roots in response to the supply of NH 4+

Transient and NH 4 +-inducible accumulation of the mRNA for NADH-dependent glutamate synthase (NADH-GOGAT; EC 1.4.1.14) in the roots of rice seedlings was analyzed in situ to identify the cell types responsible for the induction. The mRNA was detected specifically in sclerenchyma cells (the third cell-layer from the root surface), and the maximal accumulation was seen at 3–6 h following the supply of NH 4 + ions. Expression of the NADH-GOGAT gene in sclerenchyma cells was also confirmed using transgenic rice plants expressing GUS reporter gene under the control of rice NADH-GOGAT promoter. On the other hand, clear signals for the NADH-GOGAT protein were detected in epidermial cells and exodermal cells (the first and second cell layers from the root surface) at 12 h, following the supply of NH 4 + ions. The distinct localization of mRNA and protein for NADH-GOGAT suggests that either the mRNA or the translated protein in the sclerenchyma cells is migrated to the root surface. In contrast to NADH-GOGAT protein, Fd-GOGAT (EC 1.4.7.1) protein was detected in sclerenchyma cells, cortex cells, and stele in the rice roots. The distinct localization of the two GOGAT species indicates that they have different roles in the nitrogen metabolism in rice roots.

Genetic manipulation and quantitative-trait loci mapping for nitrogen recycling in rice.

Immunocytological studies in this laboratory have suggested that NADH-dependent glutamate synthase (NADH-GOGAT; EC 1.4.1.14) in developing organs of rice (Oryza sativa L. cv. Sasanishiki) is involved in the utilization of glutamine remobilized from senescing organs through the phloem. Because most of the indica cultivars contained less NADH-GOGAT in their sink organs than japonica cultivars, over-expression of NADH-GOGAT gene from japonica rice was investigated using Kasalath, an indica cultivar. Several T0 transgenic Kasalath lines over-producing NADH-GOGAT under the control of a NADH-GOGAT promoter of Sasanishiki, a japonica rice, showed an increase in grain weight (80% as a maximum), indicating that NADH-GOGAT is indeed a key step for nitrogen utilization and grain filling in rice. A genetic approach using 98 backcross-inbred lines (BC(1)F(6)) developed between Nipponbare (a japonica rice) and Kasalath were employed to detect putative quantitative trait loci (QTLs) associated with the contents of cytosolic glutamine synthetase (GS1; EC 6.3.1.2), which is probably involved in the export of nitrogen from senescing organs and those of NADH-GOGAT. Immunoblotting analyses showed transgressive segregations toward lower or greater contents of these enzyme proteins in these BC(1)F(6). Seven chromosomal QTL regions were detected for GS1 protein content and six for NADH-GOGAT. Some of these QTLs were located in QTL regions for various biochemical and agronomic traits affected by nitrogen recycling. The relationships between the genetic variability of complex agronomic traits and traits for these two enzymes are discussed.

Transgenic tobacco plants that overexpress alfalfa NADH-glutamate synthase have higher carbon and nitrogen content.

This work reports the characterization of transgenic tobacco (Nicotiana tabacum L.) plants that constitutively overexpress NADH-GOGAT. Three independent transformants, designated GOS10, GOS13 and GOS19 (for GOGAT sense), with stable integration of the chimeric alfalfa NADH-GOGAT gene fused to the CaMV 35S promoter were studied. The transgene was stably integrated and inherited by the progeny. In these GOS lines, the expression of NADH-GOGAT mRNA and protein was detected at low levels in roots and leaves, while the expression of the host tobacco NADH-GOGAT gene was nearly undetectable. The roots of GOS lines showed an elevated (15-40%) enzyme activity as compared to control plants. When GOS plants were grown under greenhouse conditions and fed with either nitrate or ammonium as the sole nitrogen source, they showed higher total carbon and nitrogen content in shoots and increased shoot dry weight when plants were entering into the flowering stage, as compared to control plants. The observed phenotype of GOS plants was interpreted as reflecting a higher capacity to assimilate nitrogen due to a higher NADH-GOGAT activity.

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.

Okadaic Acid Mimics Nitrogen-Stimulated Transcription of the NADH-Glutamate Synthase Gene in Rice Cell Cultures.

Okadaic acid (OKA), a potent and specific inhibitor of protein serine/threonine phosphatases 1 and 2A, induced the accumulation of NADH-glutamate synthase (GOGAT) mRNA within 4 h in rice (Oryza sativa L.) cell cultures. In contrast to the transient accumulation of NADH-GOGAT mRNA by NH(4)(+), OKA caused a continuous accumulation for at least 24 h. The induction of NADH-GOGAT mRNA by OKA was not inhibited in the presence of methionine sulfoximine, which inhibited the NH(4)(+)-induced accumulation of mRNA. These results suggest that the OKA-sensitive protein phosphatase is involved in the regulation of NADH-GOGAT gene expression and probably plays a role in the signal transduction pathway downstream from NH(4)(+), although a signal transduction pathway other than that of nitrogen sensing could be responsible. Nuclear run-on assays demonstrated that the accumulation of NADH-GOGAT mRNA induced by the supply of either NH(4)(+) or OKA was mainly regulated at the transcription level. OKA effects were synergistic to the NH(4)(+)-induced expression of the NADH-GOGAT gene. In the presence of K-252a, a protein kinase inhibitor, the accumulation of NADH-GOGAT mRNA induced by either NH(4)(+) or OKA was reduced. The possible roles of protein phosphatases in the regulation of NADH-GOGAT gene expression are discussed.

Organization and structure of NADH-dependent glutamate synthase gene from rice plants

Genomic clones for NADH-dependent glutamate synthase (NADH-GOGAT; EC 1.4.1.14) were obtained from a genomic library of rice ( Oryza sativa L. cv. Sasanishki). A genomic clone (λOS42, 14 kb) covered an entire structural gene and a 3.7 kb 5′-upstream region from the first methionine. Another clone (λOS23, 14 kb) contained a 2.8 kb 3′-downstream region from the stop codon. A 7047 bp long clone (λOSR51) consisting of full length cDNA for NADH-GOGAT was isolated from a cDNA library prepared using mRNA from roots of rice seedlings treated with 1 mM NH 4Cl for 12 h. The presumed transcribed region (11.7 kb) consisted of 23 exons separated by 22 introns. Rice NADH-GOGAT is synthesized as a 2166 amino acid protein with a molecular mass of 236.7 kDa that includes a 99 amino acid presequence. DNA gel blot analysis suggested that NADH-GOGAT occurred as a single gene in rice. Primer extension experiments map the transcription start of NADH-GOGAT to identical positions. The 3.7 kb 5′-upstream region was able to transiently express a reporter gene in cultured rice cells. Putative motifs related to the regulation of NADH-GOGAT gene expression were looked for within the 5′-upstream region by database.

Inducible Accumulation of mRNA for NADH-Dependent Glutamate Synthase in Rice Roots in Response to Ammonium Ions

In developing young tissues of rice plants, NADHdependent glutamate synthase (NADH-GOGAT; EC 1.4.1.14) has been suggested to play an important role in the utilization of glutamine, which is transported from senescing tissues and roots (Hayakawa et al. 1994). Because NADH-GOGAT protein is located in specific cells in both the leaf blade and grain of rice (Hayakawa et al. 1994) and because its protein content and activity change dramatically during the early stage of the seed ripening (Hayakawa et al. 1993), the expression of NADH-GOGAT gene might be regulated as a cell specificand age specific manner. We recently showed that the mRNA, protein, and activity of NADH-GOGAT in roots of rice seedlings increased more than 10-fold within a day of the start of a supply of nitrogen (Yamaya et al. 1995). This increase was specific to NADH-GOGAT in the roots; little change was observed in the activity or protein content of Fd-GOGAT (EC 1.4.7.1) or GS (EC 6.3.1.2). A supply of NH^ was most effective on the apparent inducible increase in NADH-GOGAT, but adding NOJ, glutamine, or asparagine also caused rapid increases to a lesser extent in both rice roots (Yamaya et al. 1995) and rice cell cultures (Watanabe et al. 1996). In this study, we further characterized the expression of mRNA for NADH-GOGAT in rice roots in more detail, because our previous work with roots was mainly directed toward studying the response of activity and protein content for

Alfalfa NADH‐dependent glutamate synthase: structure of the gene and importance in symbiotic N2 fixation

Glutamate synthase (GOGAT), a key enzyme in ammonia (NH+4) assimilation, occurs as two forms in plants: a ferredoxin-dependent form (Fd-GOGAT) and an NADH-dependent form (NADH-GOGAT). These enzymes are encoded by distinct genes as evidenced by their cDNA and deduced amino acid sequences. This paper reports the isolation and characterization of a NADH-GOGAT gene from alfalfa (Medicago sativa L.), the first GOGAT gene to be isolated from a eukaryote. RNase protection and primer extension experiments map the transcription start site of NADH-GOGAT to nearly identical positions. The transcribed region of this gene, 12,214 bp, is comprised of 22 exons separated by 21 introns. The 2.7 kbp region 5' from the translation initiation site confers nodule-specific reporter gene activity when used in a chimeric beta-glucuronidase (GUS) construct and transformed into Lotus corniculatus and Medicago sativa. Both infected and uninfected cells display GUS activity. The abundance of NADH-GOGAT transcripts increases substantially in developing nodules of plants infected with effective rhizobia. However, this increase is not observed when nodules are induced by a variety of ineffective rhizobial strains. Thus, unlike many other plant genes involved in root nodule NH+4 assimilation, high levels of NADH-GOGAT expression are strictly associated with effective nodules indicating that NADH-GOGAT plays a central role in the functioning of effective root nodules. An alfalfa Fd-GOGAT PCR product showing greater than 85% identity to maize Fd-GOGAT was isolated and used to investigate the contribution of this enzyme to NH+4 assimilation in nodules. Fd-GOGAT mRNA was abundant in leaves and cotyledons but was not detected in alfalfa root nodules. Fd-GOGAT in alfalfa does not appear to play a significant role in symbiotic N2 fixation.

Molecular characterization of NADH-dependent glutamate synthase from alfalfa nodules.

Alfalfa NADH-dependent glutamate synthase (NADH-GOGAT), together with glutamine synthetase, plays a central role in the assimilation of symbiotically fixed nitrogen into amino acids in root nodules. Antibodies previously raised against purified NADH-GOGAT were employed to screen a cDNA library prepared using RNA isolated from nodules of 20-day-old alfalfa plants. A 7.2-kb cDNA clone was obtained that contained the entire protein coding region of NADH-GOGAT. Analysis of this cDNA and determination of the amino-terminal amino acids of the purified protein revealed that NADH-GOGAT is synthesized as a 2194-amino acid protein that includes a 101-amino acid presequence. The deduced amino acid sequence shares significant identity with maize ferredoxin-dependent GOGAT, and with both large and small subunits of Escherichia coli NADPH-GOGAT. DNA gel blot analysis of alfalfa genomic DNA suggests the presence of a single NADH-GOGAT gene or a small gene family. The expression of NADH-GOGAT mRNA, enzyme protein, and enzyme activity was developmentally regulated in root nodules. A dramatic increase in gene expression occurred coincidentally with the onset of nitrogen fixation in the bacteroid, and was absent in both ineffective plants that were nodulated with effective Rhizobium meliloti and effective plants that had been nodulated with ineffective R. meliloti strains. Maximum NADH-GOGAT expression, therefore, appears to require an effective, nitrogen-fixing symbiosis.