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).


Introduction
Nitrogen (N) plays a key role in the anabolic process of proteins, nucleic acids, chlorophyll, hormones, and other substances that participate in the construction of living organisms [1].Wheat, as one of the three major grain crops, currently covers about 200 million ha in the world, providing a lot of nutrition for people [2].The use of N fertilizer is very important for increasing wheat yield, but it also places huge burdens on the environment such as soil acidification and water eutrophication [3].It is estimated that around two-thirds of the N fertilizer used to grow cereals is wasted worldwide [4].Improving the nitrogen use efficiency (NUE) of wheat can reduce the use of fertilizer, and reduce the production costs of farmers [5].Therefore, it is of great significance to further study the mechanisms of N absorption, transport, and the metabolism of wheat, for alleviating human food needs and reducing environmental pollution.
Plant roots absorb inorganic N from the soil mainly in the form of nitrate (NO 3 − ) and ammonium (NH 4 + ).Ammonium is the main inorganic N source for plant growth and development [6].Plants take up ammonium in soil through ammonium transporter (AMT)-mediated transport pathway [7].Nitrate in soil is absorbed by plants through two transport systems, the high-affinity and low-affinity transport systems (HATS and LATS); the selection of a transport pathway depends on its concentration in the soil [8].Nitrate in plants can be reduced to form nitrite, and then ammonium is produced by the catalytic reaction of nitrite reductase [9,10].Ammonium in plants can participate in the GS-GOGAT cycle, and the generation of glutamine and glutamate can be used to join in the biosynthesis of other amino acid derivatives, and ultimately participate in the growth and metabolism of plants [11,12].
According to the classification of electron donor types, GOGAT can be classified into two categories: NADH-GOGAT and Fd-GOGAT, which use reduced ferredoxin (Fd) and nicotinamide adenine dinucleotide (NADH) as electron donors, respectively [13].Fd-GOGAT is a monomeric protein with a molecular weight ranging from 145 kDa to 180 kDa [14] and is highly expressed in photosynthetic tissues.Its activity can be significantly induced by light or exogenous sucrose [15,16].NADH-GOGAT is a protein with a molecular weight of about 200 kDa and can be detected in the non-photosynthetic tissue of plants [13,15].
Many studies have demonstrated the function of GOGAT in plants.In rice, the phenotypic characteristics of the OsFd-GOGAT mutant plants showed chlorosis under natural conditions, and slight premature aging under low light treatment [17].Overexpression of NADH-GOGAT (EC 1.4.1.14) in rice could increase the grain weight, and affect plant nitrogen-related utilization [18].However, in corn, the NADH-GOGAT gene has the opposite effect.Overexpression of NADH-GOGAT gene influences shoot biomass accumulation.However, there are no significant effects on grain yield.Overexpression of NADH-GOGAT increases glutamate-derived amino acid contents and results in decreased concentrations of glusose-6-P, arabinose and fructose-6-P, which ultimately affect the generation of the C skeleton required for GOGAT synthesis.Finally, the primary carbon and N metabolism balance of maize is affected [19].CRISPR/Cas9-mediated targeted mutagenesis of the Fd-GOGAT suppressor ARE1 (abnormal cytokinin response1 repressor1) [20], delayed senescence and increased wheat yield in the field [21].
GOGAT plays an important role in grain yield and quality by participating in key N metabolism processes.At present, many studies have been conducted on maize, rice, durum wheat, etc., but the related research on wheat is neither systematic nor in-depth.Therefore, in this study, bioinformatics was used to identify and analyze the members of the GOGAT gene family in bread wheat.The temporal and spatial expression patterns of TaGOGATs were analyzed by RT-qPCR in wheat seedlings under N deficiency treatment and different tissues of wheat under normal N concentrations at the filling stage.The enzyme activity in leaf and root tissues was analyzed under an N hunger treatment.Through the above analyses, a regulation model of wheat GOGAT under N stress was preliminarily studied.

Identification of TaGOGAT Gene Family Members
In this study, members of the wheat glutamate synthase gene family were identified by Hidden Markov Model (HMM) screening and BLAST comparison.Glutamate synthase gene family information files were downloaded from the Ensembl database (http://plants.ensembl.org/index.html(accessed on 12 January 2024)).The HMM file for the GOGAT conservative domain (PF01645) was obtained from the Pfam database (https://www.ebi.ac.uk/interpro (accessed on 13 March 2024)).The members of the TaGOGAT gene family were identified using HMMER 3.0 software.It has been reported that the GOGAT gene is highly conserved across species [22].The gene sequences of three GOGATs (AT5G53460, AT5G04140, AT2G41220) from Arabidopsis and four GOGATs (KAI9392052, KAI9380114, KAI9384106, PNS99918) from Populus were downloaded from the NCBI (National Center for Biotechnology) database (https://www.ncbi.nlm.nih.gov/(accessed on 13 March 2024)).The NCBI-BLAST method was used to search for the homologous proteins of GOGAT in wheat.The same proteins obtained by the two methods were taken as the preliminary identification results.Using the Batch CD-Web Search Tool (https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi(accessed on 9 March 2024)) and SMART website (https://smart.embl.de/(accessed on 9 March 2024)), six TaGOGAT gene family members were identified by removing the proteins that did not possess complete conserved domains.Information on the gene sequence, protein-coding sequence (CDS), amino acid sequence, gene position on chromosome, etc., was queried in the Ensembl database.The protein molecular weight, isoelectric point, hydrophobicity and instability coefficient were obtained through the ExPASy online analysis website (https://www.expasy.org/(accessed on 14 March 2024)).The WoLF PSORT II website (https://www.genscript.com/(accessed on 8 May 2024)) was used for subcellular localization prediction.The protein transmembrane structure was analyzed based on the TMHMM 2.0 website (https://www.healthtech.dtu.dk/ (accessed on 6 April 2024)).Chromosome location mapping was carried out by TBtools-II software v2.096.

Phylogenetic Tree Analysis of GOGAT and Ka/Ks Ratio Calculation
The amino acid sequences of TaGOGATs and its homologous genes in Arabidopsis and Populus were analyzed by MEGA 11.The optimal algorithm model was determined as WAG + G + I + F, the Bootstrap value was selected as 1000, and the maximum likelihood (ML) method was used for phylogenetic tree analysis.TBtools-II software plug-in (Simple Ka/Ks Calculator (NG)) was used to calculate the Ka/Ks ratio of the TaGOGATs gene, and Excel 2016 software was used to draw the scatter plot for the analysis of the selection pressure during the evolution process.

Conserved Motif Identification and Gene Structure Analysis
MEME 5.5.5 (https://meme-suite.org/meme/ (accessed on 27 March 2024)) was used to identify GOGAT conserved motifs in Arabidopsis, Populus and wheat (T.aestivum).The number of identified motifs was set to 10. Gene annotation files were downloaded from the Ensembl database.The Batch Web CD-Search Tool was used to obtain the information on the GOGAT protein conserved domain, and the gene structure map was drawn by TBtools-II software (Gene Structure View (Advanced)).

Collinearity Analysis of TaGOGATs Gene
The collinearity analysis plug-in (one Step MCScanX) of TBtools-II software was used to analyze the collinearity within species, based on the MCScanX algorithm kernel.

Prediction Analysis of Promoter Cis-Acting Elements
2 kb upstream sequence of GOGAT genes were downloaded from the Ensembl database, using the PlantCARE website (https://bioinformatics.psb.ugent.be/webtools/plantcare/html/ (accessed on 15 March 2024)) to predict the cis-acting elements of promoters.R language was used to draw heat maps to analyze the discrepancy in the type and number of cis-acting elements between different promoters.

TaGOGATs Amino Acid Sequence Alignment and Protein Tertiary Structure Analysis
The amino acid sequence alignment analysis of TaGOGATs was performed using the ESPript 3.0 website (https://espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi(accessed on 16 April 2024)).Using the SWISS-MODEL website (https://swissmodel.expasy.org/(accessed on 5 May 2024)), the protein tertiary structure was predicted, and the optimal homologous protein model was found based on the homologous alignment method.

Plant Material and Gene Expression Analysis
The roots, stems, leaves, flag leaves and seeds of Chinese spring wheat at the filling stage were selected as samples for tissue-specific gene expression analysis.Wheat seeds with full grains and a relatively consistent size were selected and placed on the germination tray, kept in the dark for 2 days at 25 • C to permit germination, and then cultured under 16 h of light and 8 h of darkness for 4 days.After removing endosperm, the wheat seedlings were transferred to a 96-well plastic box (10.5 × 7 × 4 cm) and hydroponic with a modified Hoagland nutrient solution (Macro-nutrition: 1 mM KH 2 PO 4 , 4 mM CaCl 2 , 2 mM MgSO 4 •7 H 2 O, 5 mM KCl, 8 mM NH 4 NO 3 ; Fe and Micro-nutrition was consistent with normal Hoagland solution, NH 4 NO 3 as the only N source) for 3 days; ddH 2 O was used every day to ensure a constant volume of culture solution, and N deficiency culture was performed when they reached 2 leaves and 1 heart.The seedlings with similar growth were selected, and the root tissue was washed with ddH 2 O and dried with filter paper.Wheat seedlings were transferred to the modified Hoagland nutrient solution (exclude NH 4 NO 3 ) for N starvation culture.Leaf and root tissues were collected after treatment for 0, 0.5, 1.0, 1.5, 2.0, 3.0, 6.0 and 24.0 h, respectively, as samples for TaGOGATs gene expression pattern analysis under N stress.All samples were frozen using liquid N after sampling, and stored at −80 • C. Each sample contained 4 biological replicates.Trizol reagent (Invitrogen, Waltham, MA, USA) was selected for total RNA extraction [23].The NanoDrop 2000 (Thermo Scientific, Waltham, MA, USA) was used to determine the RNA concentration of the samples.A total of 1 µg RNA was taken from each sample for reverse transcription, and the reverse transcription reagent was HiScript RT SuperMix (Vazyme, Nanjing, China).The generated cDNA was used as the template for the RT-qPCR reaction.Each reaction system consisted of 10 µL SYBR qPCR Master Mix, 0.4 µL of forward primer, 0.4 µL of reverse primer, 1 µg of cDNA, and, finally, the volume was supplemented to 20 µL using ddH 2 O.The gene expression was analyzed using the ABI 7500 Real-Time PCR instrument (Applied Biosystems, Waltham, MA, USA), the RT-qPCR reaction process includes: 1. holding stage: 95 The primers used for the reaction were displayed in Table S1, and TaActin (wheat β-Actin gene [24]) was used as the internal reference gene [25].The relative expression level of the target genes was calculated using the ∆∆CT method; the technique was repeated 3 times per sample.

Enzyme Activity Test
The samples were derived from leaf and root tissues in 2.7 and stored at −80 • C. The materials were fully ground using a mortar pre-cooled with liquid N, collected in a 2 mL EP tube, and NADH-GOGAT extract buffer was added (0.1 M phosphate buffer, pH = 7.5).The homogenate was centrifuged at 4 • C and 8000× g for 10 min.The supernatant was collected and the enzyme activity was determined using the NADH-GOGAT kit (GOGAT-2-Y) at 340 nm [26].The Eppendorf Centrifuge-5810R and BECKMAN COULTER DU-640 nucleic acid and protein analyzer were utilized to handle and measure the absorbance values of the samples.The technique was repeated 3 times for each assay and were incorporated into the enzyme activity detection kit purchased from Comin Biotechnology Co. Ltd., Suzhou, China (http://www.cominbio.com/index.html(accessed on 10 May 2024)).

Identification of TaGOGATs and Analysis of Gene Family Information
According to the Pfam code of the conserved domain (PF01645), two methods, HMM and BLAST, were used to query the members of TaGOGATs gene family.Six wheat glutamate synthase gene sequences were identified after screening.According to the chromosome positions and the type of electron donor, three of the genes annotated as Fd-GOGAT were named TaGOGAT2s (TaGOGAT2-A, TaGOGAT2-B, TaGOGAT2-D), and the other three NADH-GOGAT were named TaGOGAT3s (TaGOGAT3-A, TaGOGAT3-B, TaGOGAT3-D).
The physicochemical characteristics of TaGOGATs were analyzed.TaGOGATs with the same electron donor had similar characteristics.The molecular weight of the TaGOGAT2s was about 170,000 D, which was smaller than the 230,000 D of TaGOGAT3s.However, the molecular weight was reversed with the exon number, as TaGOGAT2s with a low molecular weight had more exons at 33 to 34 (Table 1).The instability coefficient analysis showed suggested that TaGOGAT3s had higher protein stability, while TaGOGAT2s was an unstable protein.Subcellular localization prediction showed that except for TaGOGAT3-D located in Genes 2024, 15, 827 5 of 17 the mitochondria, the other five TaGOGATs were all located in chloroplasts.The results of the six TaGOGATs in terms of the protein isoelectric point, protein hydrophobicity, and transmembrane structure prediction manifested few differences; the isoelectric point ranged from 6.22 to 6.36, and all were hydrophilic proteins without a transmembrane domain (Table S2).These results indicate that TaGOGATs may have various biological functions.

Chromosome Localization Analysis of TaGOGATs
Common wheat was diploid (AA) in its ancestry, and after two natural hybridization and chromosome-doubling events in its evolutionary history, hexaploid wheat (AABBDD) was formed [27].It contains 3 genomes (ABD), with a total of 21 chromosomes (Figure 1).Chromosome analysis showed that the six genes were located on the A, B and D genomes of chromosomes 2 and 3, respectively, and the relative positions of GOGAT genes with the same electron donor type were similar on chromosomes Chr 2A (TaGOGAT2-A), Chr 2B (TaGOGAT2-B), Chr 2D (TaGOGAT2-D), Chr 3A (TaGOGAT3-A), Chr 3B (TaGOGAT3-B) and Chr 3D (TaGOGAT3-D).The TaGOGATs gene family did not detect tandem and proximal duplication gene replication events.a low molecular weight had more exons at 33 to 34 (Table 1).The instability coe analysis showed suggested that TaGOGAT3s had higher protein stability, TaGOGAT2s was an unstable protein.Subcellular localization prediction showed cept for TaGOGAT3-D located in the mitochondria, the other five TaGOGATs wer cated in chloroplasts.The results of the six TaGOGATs in terms of the protein iso point, protein hydrophobicity, and transmembrane structure prediction manifest differences; the isoelectric point ranged from 6.22 to 6.36, and all were hydrophi teins without a transmembrane domain (Table S2).These results indicate that TaG may have various biological functions.

Chromosome Localization Analysis of TaGOGATs
Common wheat was diploid (AA) in its ancestry, and after two natural hybrid and chromosome-doubling events in its evolutionary history, hexaploid (AABBDD) was formed [27].It contains 3 genomes (ABD), with a total of 21 chrom (Figure 1).Chromosome analysis showed that the six genes were located on the A D genomes of chromosomes 2 and 3, respectively, and the relative positions of G genes with the same electron donor type were similar on chromosomes C (TaGOGAT2-A), Chr 2B (TaGOGAT2-B), Chr 2D (TaGOGAT2-D), Chr 3A (TaGOGA Chr 3B (TaGOGAT3-B) and Chr 3D (TaGOGAT3-D).The TaGOGATs gene family detect tandem and proximal duplication gene replication events.

Phylogenetic Tree Construction and Protein Conserved Motif Analysis of GOGAT
The phylogenetic tree of the wheat and the reported GOGATs (Fd-GOGATs and NADH-GOGATs) from Arabidopsis (Arabidopsis thaliana) and Populus (Populus trichocarpa) was constructed for the analysis of the phylogenetic relationships.The results showed that TaGOGAT3s were clustered in a clade with the NADH-GOGATs from the other two species.TaGOGAT2s were clustered to a separate branch, indicating that the interspecies relationship of Fd-GOGATs in wheat is far greater than that of NADH-GOGATs.Moreover, TaGOGAT2-A and TaGOGAT2-B, TaGOGAT3-A and TaGOGAT3-D were more closely related (Figure 2A).Amino acid conserved motifs analysis showed that each GOGAT had 10 complete conserved motifs in the same order (Figure 2B).Visual analysis of the gene structure showed that NADH-GOGATs had two domains, gltB and gltD, while Fd-GOGATs had only the gltB domain.TaGOGAT2-A and TaGOGAT2-B had 33 exons, and TaGOGAT2-D had 34 exons.TaGOGAT3-A, TaGOGAT3-B and TaGOGAT3-D only contained 23 exons (Figure 2C).Six GOGATs of wheat had complete conserved domains (Figure 2B,C).lationship of Fd-GOGATs in wheat is far greater than that of NADH-GOGATs.Moreover, TaGOGAT2-A and TaGOGAT2-B, TaGOGAT3-A and TaGOGAT3-D were more closely related (Figure 2A).Amino acid conserved motifs analysis showed that each GOGAT had 10 complete conserved motifs in the same order (Figure 2B).Visual analysis of the gene structure showed that NADH-GOGATs had two domains, gltB and gltD, while Fd-GOG-ATs had only the gltB domain.TaGOGAT2-A and TaGOGAT2-B had 33 exons, and TaGOGAT2-D had 34 exons.TaGOGAT3-A, TaGOGAT3-B and TaGOGAT3-D only contained 23 exons (Figure 2C).Six GOGATs of wheat had complete conserved domains (Figure 2B,C).

A B C
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Collinearity Analysis of TaGOGATs Genes
The Tbtools-II tool and MCScanX algorithm were used to explore the repetitive events of six TaGOGATs genes.The results showed that there are four collinear events in TaGOGATs, which are present on chromosomes 2B, 2D, 3A, 3B and 3D.TaGOGAT2-A

Collinearity Analysis of TaGOGATs Genes
The Tbtools-II tool and MCScanX algorithm were used to explore the repetitive events of six TaGOGATs genes.The results showed that there are four collinear events in TaGOGATs, which are present on chromosomes 2B, 2D, 3A, 3B and 3D.TaGOGAT2-A did not have gene collinearity, while TaGOGAT3-A, TaGOGAT3-B and TaGOGAT3-D had gene collinearity events with each other (Figure 3).Repetitive events could be classified into 5 types according to gene replication types, which were tandem repeats, proximal repeats, transposed repeats, dispersed repeats, and single copies [28].Both TaGOGAT2-B/D and TaGOGAT3s are transposed repeats, while TaGOGAT2-A is a dispersed repeat.

Ka/Ks Analysis of TaGOGATs
Six TaGOGAT genes were classified into two branches by phylogenetic tree constructing.In order to study the TaGOGATs gene evolution was affected by selection pressure or not, the ratio of non-synonymous replacement rate (Ka) to synonymous replacement rate (Ks) was calculated.The results showed that the six genes had significant differences in Ks values, ranging from 0.029552 to 0.066745 (Table 2).The Ka/Ks ratios were all much less than 1, indicating that the related genes undergo the purifying selection.In most cases, selection eliminated harmful mutations and kept the protein sequence stable (Figure 4).

Ka/Ks Analysis of TaGOGATs
Six TaGOGAT genes were classified into two branches by phylogenetic tree constructing.In order to study the TaGOGATs gene evolution was affected by selection pressure or not, the ratio of non-synonymous replacement rate (Ka) to synonymous replacement rate (Ks) was calculated.The results showed that the six genes had significant differences in Ks values, ranging from 0.029552 to 0.066745 (Table 2).The Ka/Ks ratios were all much less than 1, indicating that the related genes undergo the purifying selection.In most cases, selection eliminated harmful mutations and kept the protein sequence stable (Figure 4).

Analysis of Cis-Acting Elements of TaGOGATs Gene Promoter
The cis-acting element is a DNA sequence on the promoter of genes that influences the gene expression.The 2kb upstream region from the GOGATs translation start site in wheat, Arabidopsis and Populus was analyzed for promoter cis-acting elements.The results showed that there was no obvious difference among species, but there was a significant discrepancy between TaGOGAT2-A and TaGOGAT2-B/D.The distribution regularity of cisacting elements among TaGOGAT3-A, TaGOGAT3-B and TaGOGAT3-D is conservative, and the number of cis-acting elements in TaGOGAT3s is higher than that in TaGOGAT2s (Figure 5).
The TaGOGATs promoter region also contains several zein metabolic regulatory elements (O2site), which are involved in plant biosynthesis and developmental related biological pro-

Analysis of Cis-Acting Elements of TaGOGATs Gene Promoter
The cis-acting element is a DNA sequence on the promoter of genes that influences the gene expression.The 2kb upstream region from the GOGATs translation start site in wheat, Arabidopsis and Populus was analyzed for promoter cis-acting elements.The results showed that there was no obvious difference among species, but there was a significant discrepancy between TaGOGAT2-A and TaGOGAT2-B/D.The distribution regularity of cis-acting elements among TaGOGAT3-A, TaGOGAT3-B and TaGOGAT3-D is conservative, and the number of cis-acting elements in TaGOGAT3s is higher than that in TaGOGAT2s (Figure 5).

Multiple Amino Acid Sequences Analysis of TaGOGATs
The conserved sequences and functional regions of six TaGOGATs in wheat were analyzed.The results show that the six GOGAT proteins had high homology (Figure S1).The conserved binding regions have been reported in Populus GOGAT proteins [22], andcould also be found in TaGOGATs.A putative FMN-binding region (Figure S1B) and a putative [3Fe-4S] cluster-binding region were included in all proteins (Figure S1C).The putative NAD(P)H-binding regions was only contained in three NADH-GOGATs (Figure S1D).

Secondary and Tertiary Structure Characteristics Prediction of TaGOGAT Proteins
The tertiary structure model of TaGOGAT proteins was predicted by AFDB search and the AlphaFold v2 algorithm.The results showed that the optimal TaGOGAT2s Many cis-acting elements related to hormone response, stress response, and developmental response processes were found in the promoter of GOGAT genes.Hormone response elements include abscisic acid response element (ABRE), auxin response element (TGA-element), gibberellin response element (P-box/GARE-motif), jasmonic acid response element (TGACG-motif/CGTCA-motif) and salicylic acid response element (TCA-element).Stress response related elements containing hypoxia-induced response elements (ARE/GC-motif), low-temperature response elements (LTR), droughtinduced response elements (MYB binding site, MBS), light response dependent elements (AE-box/ACE/Gap-box/TCTmotif/TCCC-motif/GATA-motif/I-box/Sp1/Box4/G-Box).The TaGOGATs promoter region also contains several zein metabolic regulatory elements (O2-site), which are involved in plant biosynthesis and developmental related biological processes.The number of ABRE, G-box and sp1 in TaGOGAT3s promoters is obviously higher than that of other binding components, while the number of ARE binding components is higher in TaGOGAT2 promoters compared with other promoters (Figure 6).In conclusion, the prediction results of TaGOGATs cis-acting elements indicate that TaGOGAT may be involved in biological metabolic processes such as growth and development, hormone response, and the stress response of wheat.

Multiple Amino Acid Sequences Analysis of TaGOGATs
The conserved sequences and functional regions of six TaGOGATs in wheat were analyzed.The results show that the six GOGAT proteins had high homology (Figure S1).The conserved binding regions have been reported in Populus GOGAT proteins [22], andcould also be found in Ta GOGATs.A putative FMN-binding region (Figure S1B) and a putative [3Fe-4S] cluster-binding region were included in all proteins (Figure S1C).The

Multiple Amino Acid Sequences Analysis of TaGOGATs
The conserved sequences and functional regions of six TaGOGATs in wheat were analyzed.The results show that the six GOGAT proteins had high homology (Figure S1).The conserved binding regions have been reported in Populus GOGAT proteins [22], andcould also be found in TaGOGATs.A putative FMN-binding region (Figure S1B) and a putative [3Fe-4S] cluster-binding region were included in all proteins (Figure S1C).The putative NAD(P)H-binding regions was only contained in three NADH-GOGATs (Figure S1D).

Secondary and Tertiary Structure Characteristics Prediction of TaGOGAT Proteins
The tertiary structure model of TaGOGAT proteins was predicted by AFDB search and the AlphaFold v2 algorithm.The results showed that the optimal TaGOGAT2s template was Q69RJ0.1.A (Ferredoxin-dependent glutamate synthase).A Seq identity score greater than 90 indicated that the model had high homology with the comparison protein, and a global model quality estimate score (GMQE) greater than 0.9 indicated high data reliability (Figure 7A,B).Q9LV03.1.A (Glutamate synthase 1 [NADH]) was the optimal TaGOGAT3s template, with a Seq identity greater than 78 and a GMQE greater than 0.8 (Figure 7C,D).The PDB file of the optimal homology model for protein secondary structure analysis of TaGOGAT was downloaded on the ESPript 3.0 website.The results showed that the secondary structure characteristics of TaGOGAT2s were similar.This situation also existed in TaGOGAT3s (Table S3), which further verified the rationality of protein tertiary structure analysis at the level of the secondary structure.
template was Q69RJ0.1.A (Ferredoxin-dependent glutamate synthase).A Seq identity score greater than 90 indicated that the model had high homology with the comparison protein, and a global model quality estimate score (GMQE) greater than 0.9 indicated high data reliability (Figure 7A,B).Q9LV03.1.A (Glutamate synthase 1 [NADH]) was the optimal TaGOGAT3s template, with a Seq identity greater than 78 and a GMQE greater than 0.8 (Figure 7C,D).The PDB file of the optimal homology model for protein secondary structure analysis of TaGOGAT was downloaded on the ESPript 3.0 website.The results showed that the secondary structure characteristics of TaGOGAT2s were similar.This situation also existed in TaGOGAT3s (Table S3), which further verified the rationality of protein tertiary structure analysis at the level of the secondary structure.TaGOGAT3s protein optimum homologous protein template.In Figure A and C, the blue peptide indicates that the amino acid consistency is greater than 70%, the red peptide indicates that the amino acid consistency is less than 50%, and the yellow peptide indicates that the amino acid consistency is 60-70%

Expression Patterns Analysis of TaGOGATs in Different Tissues
To study the expression patterns of TaGOGATs in different tissues of wheat, RNA was extracted from the roots, stems, leaves, flag leaves and seeds of wheat at the filling stage, and the TaGOGATs expression level was analyzed by RT-qPCR.As TaGOGATs gene sequences located on the same chromosome are highly similar, two pairs of primers were designed to detect the gene expression levels of Fd-GOGATs located on chromosome 2 and NADH-GOGATs located on chromosome 3, respectively (Table S1).The results showed  (A,C), the blue peptide indicates that the amino acid consistency is greater than 70%, the red peptide indicates that the amino acid consistency is less than 50%, and the yellow peptide indicates that the amino acid consistency is 60-70%.

Expression Patterns Analysis of TaGOGATs in Different Tissues
To study the expression patterns of TaGOGATs in different tissues of wheat, RNA was extracted from the roots, stems, leaves, flag leaves and seeds of wheat at the filling stage, and the TaGOGATs expression level was analyzed by RT-qPCR.As TaGOGATs gene sequences located on the same chromosome are highly similar, two pairs of primers were designed to detect the gene expression levels of Fd-GOGATs located on chromosome 2 and NADH-GOGATs located on chromosome 3, respectively (Table S1).The results showed that TaGOGATs were expressed in different tissues of wheat.The TaGOGAT2s gene was mainly expressed in leaves and flag leaves, and was the lowest in seeds (Figure 8A).The TaGOGAT3s gene had the lowest gene expression level in seeds.The relative expression level of TaGOGAT3s in leaves was slightly higher than that in the root tissues, but it was not significant (Figure 8B).The results were similar to that in other species [22].

The Expression Analysis of TaGOGATs in Different Tissues of Wheat Seedling under N Deficiency Treatment
TaGOGAT is one of the key enzymes in the wheat N metabolism pathway.In this study, RT-qPCR technology was used to analyze the changes in TaGOGATs gene expression levels in the root and leaf tissues of wheat seedlings under different N deficiency treatment times.The results showed that the expression of TaGOGATs gene was affected by the N hungry condition.In wheat root tissues, the expression level of TaGOGAT2s was upregulated by N deficiency induction, and induced to the highest level at 0.5 h, then gradually decreased to the pre-treatment level (0 h) at 6 h (Figure 9A).The TaGOGAT3s gene expressions were downregulated, but there were no significant changes at 0.5 h (Figure 9B).In leaf tissues, the relative expression level of TaGOGAT2s slightly increased at 0.5 h, then decreased, and suddenly reached a higher level at 24 h (Figure 9C).For TaGOGAT3s, the relative expression level gradually increased to the highest level at 1.5 h, and then decreased little by little, but was still higher than that in the pre-treatment level (Figure 9D).Overall, TaGOGATs could participate in the regulatory response of wheat N stress.

The Expression Analysis of TaGOGATs in Different Tissues of Wheat Seedling under N Deficiency Treatment
TaGOGAT is one of the key enzymes in the wheat N metabolism pathway.In this study, RT-qPCR technology was used to analyze the changes in TaGOGATs gene expression levels in the root and leaf tissues of wheat seedlings under different N deficiency treatment times.The results showed that the expression of TaGOGATs gene was affected by the N hungry condition.In wheat root tissues, the expression level of TaGOGAT2s was upregulated by N deficiency induction, and induced to the highest level at 0.5 h, then gradually decreased to the pre-treatment level (0 h) at 6 h (Figure 9A).The TaGOGAT3s gene expressions were downregulated, but there were no significant changes at 0.5 h (Figure 9B).In leaf tissues, the relative expression level of TaGOGAT2s slightly increased at 0.5 h, then decreased, and suddenly reached a higher level at 24 h (Figure 9C).For TaGOGAT3s, the relative expression level gradually increased to the highest level at 1.5 h, and then decreased little by little, but was still higher than that in the pre-treatment level (Figure 9D).Overall, TaGOGATs could participate in the regulatory response of wheat N stress.

Analysis of TaGOGATs Enzyme Activity in Wheat Seedling Stage
The tissue-specific analysis of TaGOGATs showed that TaGOGAT3s can be expressed in different wheat tissue.The enzyme activity analysis showed that the enzyme activity in wheat root tissues was less affected by N stress, and suddenly decreased at 3 h, and then quickly recovered (Figure 10A).In leaf tissues, the change trend of TaGOGAT3s' enzyme activity declined at 0.5 h, followed by a slightly increase with the increase in treatment, and then decreased at 3 and 6 h, and finally increasing to a high level at 24 h (Figure 10B).

Analysis of TaGOGATs Enzyme Activity in Wheat Seedling Stage
The tissue-specific analysis of TaGOGATs showed that TaGOGAT3s can be expressed in different wheat tissue.The enzyme activity analysis showed that the enzyme activity in wheat root tissues was less affected by N stress, and suddenly decreased at 3 h, and then quickly recovered (Figure 10A).In leaf tissues, the change trend of TaGOGAT3s' enzyme activity declined at 0.5 h, followed by a slightly increase with the increase in treatment, and then decreased at 3 and 6 h, and finally increasing to a high level at 24 h (Figure 10B).

Analysis of TaGOGATs Enzyme Activity in Wheat Seedling Stage
The tissue-specific analysis of TaGOGATs showed that TaGOGAT3s can be expressed in different wheat tissue.The enzyme activity analysis showed that the enzyme activity in wheat root tissues was less affected by N stress, and suddenly decreased at 3 h, and then quickly recovered (Figure 10A).In leaf tissues, the change trend of TaGOGAT3s' enzyme activity declined at 0.5 h, followed by a slightly increase with the increase in treatment, and then decreased at 3 and 6 h, and finally increasing to a high level at 24 h (Figure 10B).

Discussion
Currently, the GOGAT gene family were identified [22,29] in Populus and Arabidopsis species.Two Fd-GOGAT (AT5G04140, AT2G41220) and one NADH-GOGAT (AT5G53460) were identified in Arabidopsis.The poplar GOGAT gene family contains two Fd-GOGAT (KAI9392052, KAI9380114) and two NADH-GOGAT (KAI9384106, PNS99918).In wheat, six GOGAT genes were distinguished, which were located on chromosome 2 and chromosome 3, respectively.(Figure 2A).Through gene structure analysis, it was found that GOGAT of the same electron donor type had a similar domain distribution pattern.TaGOGAT2s contains only one gltB-conserved domain, while TaGOGAT3s contains gltB-and gltD-conserved domains, indicating that the two types of GOGAT may have biological functional diversity due to their different protein spatial structures (Figure 2C).Similar structures were found in Arabidopsis and Populus GOGAT genes.The gltB gene encodes the α subunit of GOGAT, and gltD is involved in encoding the β subunit [30].Studies have shown that gltB and gltD are very important for maintaining GOGAT protein biological activity.gltB mutants of Escherichia coli are unable to utilize substances, such as proline and glycine, as the sole N source [31], and mutations of this gene in pea bacteroid RU2307 lead to an increase in the intracellular Gln: Glu ratio.Two amino acid uptake systems (Aap and Bra), both ABC-types, were inhibited [32].The gltD gene mutants also showed similar characteristics.The gltD::Tn5 mutant of Bradyrhizobium ORS285 could not use ammonium, nitrate, and many amino acids as N sources for its growth, and could not undergo the N fixation reaction under natural conditions [33].
These results suggest that the function of GOGAT is relatively conservative among species.By evolutionary tree analysis of 13 species, including prokaryotes and eukaryotes, Cao et al. found that only prokaryotes Fd-GOGAT, and then speculated that, in the evolutionary history of GOGAT, Fd-GOGAT appeared first, and the emergence of NADH-GOGAT resulted in functional differentiation [22].Four GOGATs have been identified in Populus, and subcellular localization predictions show that they are all located in chloroplasts.Two of these proteins depend on Fd as electron donors, and the other two proteins have NADH.Interestingly, subcellular localization prediction analysis showed that TaGOGAT3-D were located in mitochondria, and the remaining five TaGOGAT proteins were all in chloroplasts, which may indicate that they were involved in different biological reactions.The Ka/Ks ratio between wheat gene pairs was found to be much less than 1 through gene analysis, indicating that TaGOGATs were subjected to purification selection in the evolutionary process, which also indicated that TaGOGATs were highly conserved, consistent with the previous conclusions (Figure 4).Analysis of cis-acting elements of gene promoters showed that TaGOGATs contained many different types of acting elements (Figure 5).For example, TaGOGAT3s contained several gibberellin-responsive elements (P-box, GARE-motif), indicating that it may be involved in gibberellin response regulating (Figure 6).In order to analyze the effects of abscisic acid (ABA), gibberellanic acid (GA3) and other hormones on the expression levels of two Fd-GOGAT (GLU1 and GLU2) and one NADH-GOGAT(GLT1) in Arabidopsis, Arabidopsis seedlings treated with different hormone concentrations were analyzed.The results showed that, except for GLU2, GA3 enhanced the expression of other genes in shoots [34].This is consistent with our analysis.Through amino acid sequence comparison analysis, it was found that the amino acid sequence of TaGOGAT with the same electron donor type was highly consistent (Figure S1); the spatial structure analysis of the proteins also led to the same conclusion (Figure 7).The FMN-binding sites and [3Fe-4S] cluster-binding regions were contained in all six TaGOGAT proteins.TaGOGAT3s included other predicted NAD(P)H binding sites.These binding sites have also been reported in Populus GOGAT-related studies [22], and the difference in the protein domain reflects the difference of its biological reaction process.
The GS/GOGAT cycle participates in plant N assimilation.Six GOGAT genes were isolated in bread wheat by the partial barley sequence [35] and NADH-GOGAT rice genome sequence [36], which were located on wheat chromosomes 2 and 3, respectively.The expression levels of NADH-GOGAT, except for NADH-GOGAT-3D, in durum wheat were correlated with grain protein content [37].NADH-GOGAT, located on wheat chromosomes 3A and 3B, were identified as a major candidate for NUE by meta-QTL.NO 3 − and NH 4 + are the main forms of inorganic N absorbed by plants, and their ion concentration and morphology could influence the expression of plant GOGAT genes.NADH-GOGAT (OsGlt1 and OsGlt2) were mainly expressed in rice root tissues under N-restricted treatment, but in leaves under unrestricted conditions [38].The expression and activity of GOGAT and GS could be regulated by the ratio of ammonium-nitrogen, and the relationship between them showed a significant positive correlation [39].Both high and low concentrations of NH 4 + could inhibit the expression of GOGAT gene.High NH 4 + concentrations could produce toxicity and inhibit the N assimilation process [40].Many studies have shown that Fd-GOGAT is mainly expressed in photosynthetic tissues, while NADH-GOGAT is expressed in non-photosynthetic plastid tissues [41].The transcription levels of six GOGAT genes in wheat roots, stems, leaves, flag leaves and seeds were tested; it was shown that TaGOGAT2s were mainly expressed in leaves, while TaGOGAT3s were highly expressed in roots (Figure 8).GOGAT gene expression in wheat seedlings had been detected under N deficiency treatment.It was found that the expression level of TaGOGAT2s in root tissues increased first, induced by N deficiency, and then decreased, and that it was the highest at 0.5 h and decreased to the pre-treatment level at 24 h (Figure 9A).This pattern can also be observed in the regulation of TaGOGAT3s gene expression in leaf tissues, with the highest transcription level at 1.5 h followed by a gradual decrease thereafter (Figure 9C).
However, the transcription levels of TaGOGAT3s in wheat root tissues showed an overall downward trend (Figure 9B).It was reported that plant hormone abscisic acid (ABA) could enhance the bioactivity of GS and GOGAT, thereby reducing NH 4 + accumulation and cellular oxidative damage [42].TabZIP60 could bind to the ABRE elements on the TaNADH-GOGAT-3B promoter and negatively regulate its expression [43].ABA-responsive cis-acting elements (ABRE) were found in all gene promoters except TaGOGAT2-B (Figures 5  and 6).However, GOGAT was the highly tissue-specific gene.Thus, the down-regulated expression of TaGOGAT3s in root tissues under N deficiency conditions may be related to the recovery of NH 4 + concentrations in plants.Enzyme activity in TaGOGAT2s is highly correlated with light source and external sucrose induction [44].The effect of N starvation induction on enzyme activity in TaGOGAT3s in wheat leaf and root tissues were studied.Enzyme activity analysis showed that the enzyme activity in TaGOGAT3s in wheat leaf tissue was significantly negatively regulated (Figure 10B).However, in wheat root tissues, the change in enzyme activity level was less affected by N stress (Figure 10A).Balotf et al. found that gene expression and enzyme activity in GOGAT were inhibited in wheat leaf tissue after 7 days of cultivation under nitrogen-starved conditions [45].

Conclusions
As one of the basic elements of an organism, N is very important for the growth and metabolism of wheat.GOGAT, as a key enzyme in the process of plant N assimilation, affects wheat N absorption efficiency.In this study, we identified the TaGOGATs gene family through bioinformatics analysis and identified a total of six genes, which were located on chromosomes 2 and 3. TaGOGAT2s was mainly expressed in leaves and flag leaves, while TaGOGAT3s was mainly expressed in roots and leaves.the expression of the GOGATs gene could be induced in leaves and roots under the treatment of N deficiency.Enzyme activity in TaGOGAT3s in wheat leaf tissues was significantly affected by N deficiency stress, and the regulation mechanism of enzyme activity in leaf and root tissue may be different.This paper provides a systematic approach for TaGOGAT gene family analysis.The research results indicate that TaGOGAT may participate in regulating wheat NUE.

Supplementary Materials:
The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/genes15070827/s1,Table S1: RT-PCR Primer sequence; Table S2: Information of wheat glutamate synthase family members; Table S3: The number of the secondary structure type in TaGOGAT proteins.Figure S1: Amino acid sequence alignment of wheat glutamate synthase TaGOGATs.
Author Contributions: S.Z. and P.G. designed the experiments and revised the manuscript; S.L. and B.J. wrote the manuscript; J.W., P.Z. and F.D. prepared the plant materials and performed the experiment; F.Y. and C.M. performed the data analysis.All authors have read and agreed to the published version of the manuscript.

Figure 1 .
Figure 1.Location information of TaGOGATs on chromosomes.The six rectangles are the somes.The chromosome names are labeled in the left side of the chromosome, and the gen

Figure 1 .
Figure 1.Location information of TaGOGATs on chromosomes.The six rectangles are the chromosomes.The chromosome names are labeled in the left side of the chromosome, and the gene name are marked on the right side.Chromosome-length information is provided in the scale at the far left of the image.

18 Figure 3 .
Figure 3. Collinearity analysis of TaGOGATs gene.The red lines indicate collinear gene pairs.The graph shows the gene name, gene density information, GC skew information, gene gap distribution information and chromosome name from the outside to the inside.The legend on the right corresponds to the size of the gene densities.

Figure 3 .
Figure 3. Collinearity analysis of TaGOGATs gene.The red lines indicate collinear gene pairs.The graph shows the gene name, gene density information, GC skew information, gene gap distribution information and chromosome name from the outside to the inside.The legend on the right corresponds to the size of the gene densities.

Figure 4 .
Figure 4. Ka/Ks scatter plot of TaGOGATs gene.Each point represents the coordinate of a gene pair.

Figure 4 .
Figure 4. Ka/Ks scatter plot of TaGOGATs gene.Each point represents the coordinate of a gene pair.

Figure 5 .
Figure 5. Prediction of promoter cis-acting elements.The 2 kb upstream region from the GOGATs translation start site in wheat, Arabidopsis and Populus were analyzed.

Figure 6 .
Figure 6.Predictive analysis of promoter cis-acting elements.In the figure, the vertical coordinate indicates the gene name, and the horizontal coordinate indicates the binding element name.

Figure 5 .
Figure 5. Prediction of promoter cis-acting elements.The 2 kb upstream region from the GOGATs translation start site in wheat, Arabidopsis and Populus were analyzed.

18 Figure 5 .
Figure 5. Prediction of promoter cis-acting elements.The 2 kb upstream region from the GOGATs translation start site in wheat, Arabidopsis and Populus were analyzed.

Figure 6 .
Figure 6.Predictive analysis of promoter cis-acting elements.In the figure, the vertical coordinate indicates the gene name, and the horizontal coordinate indicates the binding element name.

Figure 6 .
Figure 6.Predictive analysis of promoter cis-acting elements.In the figure, the vertical coordinate indicates the gene name, and the horizontal coordinate indicates the binding element name.

Figure 7 .
Figure 7. Tertiary structure prediction map of wheat TaGOGAT protein.Based on the homology model-building method, the optimal spatial structure model of the homologous proteins was predicted: (A) TaGOGAT2s protein tertiary structure diagram; (B) TaGOGAT2s protein optimum homologous protein template; (C) TaGOGAT3s protein tertiary structure diagram; and (D)TaGOGAT3s protein optimum homologous protein template.In FigureA and C, the blue peptide indicates that the amino acid consistency is greater than 70%, the red peptide indicates that the amino acid consistency is less than 50%, and the yellow peptide indicates that the amino acid consistency is 60-70%

Figure 7 .
Figure 7. Tertiary structure prediction map of wheat TaGOGAT protein.Based on the homology model-building method, the optimal spatial structure model of the homologous proteins was predicted: (A) TaGOGAT2s protein tertiary structure diagram; (B) TaGOGAT2s protein optimum homologous protein template; (C) TaGOGAT3s protein tertiary structure diagram; and (D) TaGOGAT3s protein optimum homologous protein template.In(A,C), the blue peptide indicates that the amino acid consistency is greater than 70%, the red peptide indicates that the amino acid consistency is less than 50%, and the yellow peptide indicates that the amino acid consistency is 60-70%.

Figure 8 .
Figure 8. Analysis of GOGAT gene expression patterns in different tissues of wheat.RNA was extracted from wheat roots, stems, leaves, flag leaves, and seeds at the filling stage for RT-qPCR: (A) TaGOGAT2s; and (B) TaGOGAT3s.Sample significance was obtained based the on t-test method (*, 0.01 ≤ p < 0.05; **, p < 0.01).

Figure 9 .
Figure 9.The relative expression analysis of TaGOGAT genes: (A) the relative expression analysis of TaGOGAT2s in wheat root tissue; (B) the relative expression analysis of TaGOGAT3s in root tissue; (C) the relative expression analysis of TaGOGAT2s in leaf tissue; and (D) the relative expression analysis of TaGOGAT3s in leaf tissue.Sample significance was obtained based on t-test method (*, 0.01 ≤ p < 0.05; **, p < 0.01).

Figure 10 .
Figure 10.Activity analysis of TaGOGAT3s enzyme: (A) analysis of TaGOGAT3s enzyme activity in the root tissue of wheat seedlings; and (B) analysis of TaGOGAT3s enzyme activity in leaf tissue.Sample significance was obtained based on t-test method (**, p < 0.01).

Figure 9 .
Figure 9.The relative expression analysis of TaGOGAT genes: (A) the relative expression analysis of TaGOGAT2s in wheat root tissue; (B) the relative expression analysis of TaGOGAT3s in root tissue; (C) the relative expression analysis of TaGOGAT2s in leaf tissue; and (D) the relative expression analysis of TaGOGAT3s in leaf tissue.Sample significance was obtained based on t-test method (*, 0.01 ≤ p < 0.05; **, p < 0.01).

Genes 2024 , 18 Figure 9 .
Figure 9.The relative expression analysis of TaGOGAT genes: (A) the relative expression analysis of TaGOGAT2s in wheat root tissue; (B) the relative expression analysis of TaGOGAT3s in root tissue; (C) the relative expression analysis of TaGOGAT2s in leaf tissue; and (D) the relative expression analysis of TaGOGAT3s in leaf tissue.Sample significance was obtained based on t-test method (*, 0.01 ≤ p < 0.05; **, p < 0.01).

Figure 10 .
Figure 10.Activity analysis of TaGOGAT3s enzyme: (A) analysis of TaGOGAT3s enzyme activity in the root tissue of wheat seedlings; and (B) analysis of TaGOGAT3s enzyme activity in leaf tissue.Sample significance was obtained based on t-test method (**, p < 0.01).

Figure 10 .
Figure 10.Activity analysis of TaGOGAT3s enzyme: (A) analysis of TaGOGAT3s enzyme activity in the root tissue of wheat seedlings; and (B) analysis of TaGOGAT3s enzyme activity in leaf tissue.Sample significance was obtained based on t-test method (**, p < 0.01).

Table 1 .
The basic nucleic acid and protein information of TaGOGAT genes in wheat.

Table 1 .
The basic nucleic acid and protein information of TaGOGAT genes in wheat.

Table 2 .
Ka/Ks analysis of gene pairs.