1. Introduction
Glutamine synthetase (GS; EC 6.3.1.2, L-glutamate: ammonia ligase ADP-forming) is the key enzyme responsible for primary nitrogen (N) assimilation in higher plants [1,2]. Glutamine synthetase catalyzes the ATP-dependent addition of ammonium (NH4+) to the γ-carboxyl group of glutamate to produce glutamine and takes part in the GS–GOGAT cycle, which serves as the cornerstone of N metabolism [3]. The GS gene family has been studied in certain plants, including Arabidopsis [4], maize (Zea mays) [5,6], and Populus (Populus trichocarpa) [7]. However, the entire GS gene family has not been identified in any of the species of Cucurbitaceae. The sources of ammonium assimilated by GS include the fixation of atmospheric N, direct nitrate or ammonia uptake from the soil, photorespiration, phenylalanine-ammonia lyase-catalyzed phenylalanine deamination, and the release of ammonium during storage via protein mobilization and plant senescence. Hence, in the context of nitrogen assimilation, GS is considered a candidate gene for transgenic approaches to increasing nitrogen-use efficiency (NUE). GS also responds to various abiotic stresses, including salt, cold, and drought, which have adverse effects on crop production [3].
Oligomorphic isozymes composed of GS polypeptides encoded by multiple nuclear genes are located in the cytoplasm or chloroplasts and are expressed in the nonphotosynthetic and photosynthetic tissues of higher plants [8]. Researchers have reported that the decametric structure of the plant GS holoenzyme consists of two face-to-face cyclic pentamer subunits [9,10]. In vascular plants, there are two main isoforms of GS, classified as cytosolic GS (GS1) and chloroplastic GS (GS2) according to their size and subcellular localization [11]. The genomic analysis of multiple angiosperm species showed that GS1 genes belonged to a small, multigene family [1], whereas GS2 was encoded by one to two genes. The cytosolic GS1 isoform assimilates ammonium from the soil, and the remobilization of ammonia is released via protein degradation in senescing leaves, whereas the larger chloroplast-localized GS2 isoform is responsible for the reassimilation of ammonium released during photorespiration and nitrate reduction in plastids [12,13]. The different expression patterns of these genes regulate glutamine production both spatially and temporally. For example, in rice (Oryza sativa), there are three genes coding for cytosolic GS1 (OsGS1.1, OsGS1.2, and OsGS1.3) and one gene coding for the plastidic GS2 (OsGS2). OsGS1.1 exists globally but is expressed more in the shoots, while OsGS1.2 is expressed mostly in the root. OsGS1.3 is almost undetectable except for the spikelets, and OsGS2 is abundant in the leaves [14].
The differences between GS isoforms are increasingly being studied. The main cause of ammonium toxicity in Arabidopsis was found to be ammonium assimilation of GS2 rather than ammonium accumulation [15]. Isotopic-tracing experiments and genetic evidence indicated that three of the five GS1s work together to remobilize nitrogen and fill seeds in Arabidopsis [16]. The different location, gene expression and function of each isoform of GS is well proven in wheat grain (Triticum aestivum L.) [17]. In Cucurbitaceae crops, it has been reported that GS responded to root-zone low temperature [18], low nitrogen stress [19], and intercropping allelopathy [20]. However, the detailed type, number and mechanism of functional global GS isoforms of Cucurbitaceae are still unclear.
There are several economically important species in the Cucurbitaceae family [21], such as cucumber, melon, and watermelon. Pumpkins are often used as rootstocks or as scions to afford these Cucurbitaceae plants higher stress tolerance, better quality, and higher yield [22,23,24]. Except for nitrogen sources, NH4+ is cytotoxic [25]. Cucumber, as a species of the Cucurbitaceae family, is highly influenced by NH4+ compared with other plants belonging to the family. Previous studies showed that grafting cucumber (Cucumis sativus L./Cucurbita moschata), compared with the control (Cucumis sativus L./Cucumis sativus L.), was less toxic alone, with a decrease in GS. In this study, we identified seven CmoGS and four CsaGS (Cucumber Glutamine Synthetase) genes through a genome-wide analysis of pumpkin and cucumber with reference to studies of other five species. We predicted the structure, subcellular localization, phylogeny, and function of GS family genes using bioinformatics methods to provide new insights into glutamine metabolism in pumpkin.
2. Materials and Methods
2.1. Screening of GS Family Genes in Pumpkin and Cucumber
By setting up a local database, the genome of Cucurbita moschata var. Rifu and Cucumis sativus L. including all information (cds, pep, DNA, and gff3) was obtained from CuGenDB (
2.2. Phylogenetic Analysis of GS Proteins in Pumpkin and Cucumber
First, to perform multiple-sequence alignment analysis, the full-length GS protein sequences from some representative species, including Populus trichocarpa, Oryza sativa, Vitis vinifera, Arabidopsis thaliana, and Zea mays were analyzed using MEGA-X software (Center of Evolutionary Functional Genomics Biodesign Institute, Arizona State University, Tempe, AZ, USA) [28]. The sequences of GS family genes of Arabidopsis and poplar were obtained through TAIR (
2.3. Structure and 5′-Upstream Regions Regulatory-Elements Analysis of GS Family Genes in Pumpkin and Cucumber
The distributions of different regions of GS family genes on pumpkin and cucumber chromosomes were drawn from the gff3 file. The potential regulatory elements in the 2000 bp 5′-upstream regions of GS genes were identified by TBtools [30] with the default parameters. The information extraction and preliminary drawing of the regulatory-element analysis were also completed using TBtools.
2.4. Evolution Analysis of GS Family Genes in Pumpkin and Cucumber
MCScanX software (Plant Genome Mapping Laboratory, University of Georgia, Athens, GA, USA) [28] was used to analyze the segmental duplications so as to replenish BLAST results. The collinearity of CmoGS and CsaGS family genes were also visualized using TBtools.
2.5. Gene Ontology Enrichment
GO enrichment analysis was performed using OmicShare tools (Gene Denovo, Guangzhou, China).
2.6. Relative Gene Expression with mRNA Abundance of GS Family Genes
To identify the relative gene expression levels of GS family genes, the published transcriptomic data (PRJNA385310 of pumpkin, PRJNA312872 of cucumber, and RNA-seq data in CuGenDB) [31] was downloaded. The data of four tissues, including roots, stems, leaves, and fruits harvested 46 days after pollination in Cucurbita moschata var. Rifu, and four tissues, including roots, stems, young leaves, and flesh-3weak-fruits in 12-week-old cucumber (Cucumis sativus), were utilized in our study. The parameter of log2(RPKM) was used to represent the expression level of each gene.
2.7. Semi-Quantitative RT-PCR Assays with Cold-Treated Pumpkin Seedlings
Pumpkin (Cucurbita moschata var. Rifu ‘Qianglishi’) was used in this study. About 100 seeds were put into sterile water at 55 °C for 10 min and 25 °C for 4 h, then germinated in a dark room at 28 °C for 24 h. The germinated seeds were sown in 50-hole seedling trays with a substrate (peat: vermiculite: perlite, volume ratio 2:1:1) in a light chamber (relative humidity: 70%, 28 °C/18 °C day/night; 16 h/8 h light/dark; light intensity 190–600 μmol m−2 s−1). Pumpkin seedlings with second true leaf were placed at 4 °C in the previous chamber (relative humidity: 70%; 4° C/4 °C day/night; 16 h/8 h light/dark; light intensity: 190–600 μmol m−2 s−1). Samples were taken from roots and leaves at 0 h, 6 h, 12 h, 24 h, 2 d, 3 d, 6 d, and 9 d after the cold treatment. Each replicate includes 3–4 independent plants, and three independent biological replicates were performed.
Total RNA was isolated from different pumpkin tissues using RNA plant Plus Reagent (Huayueyang Biotech, Co., Beijing, China) following manufacturer’s instructions. A total of 2 µL of each RNA sample was quantified using a NanoDrop 2000 (Thermo Fisher Scientific, Waltham, MA, USA). cDNA was synthesized by using PrimeScriptTM RT reagent kit (Perfect Real Time, Takara Biomedical Technology Co., Ltd., Beijing, China). The internal control PCR was performed with 30 cycles by amplifying ACT7. Semi RT-PCR was conducted with the reaction system (2 × T5 Super PCR Mix (TSE005, TSING KE Co., Beijing, China), 1.5 µM Primer and 2 ng cDNA) in 35 cycles using the primers listed in Supplementary Table S1.
3. Results
3.1. Genome-Wide Characterization of GS Family Genes in Pumpkins and Cucumbers
Seven and four genes encoding GS protein domains were identified by screening the pumpkin and cucumber genome databases, respectively. We analyzed amino-acid length, protein molecular weight, theoretical isoelectric point (PI), grand average of hydropathicity (GRAVY), instability index, predicted subcellular localization, and amino-acid composition of each gene. The predicted GS family genes of pumpkin and cucumber were distributed on different chromosomes. Additionally, the GRAVY values of the GS proteins were uniformly negative, indicating that these proteins may be hydrophilic. The difference between pumpkin and cucumber was that there was only one chloroplast-localized GS2 in cucumber but two in pumpkin. Furthermore, there were three and five cytosol-localized GS1s in cucumber and pumpkin, respectively. The longest (CmoCh06G014450, 479aa) and shortest (CmoCh01G003900, 340aa) of GS can be found in pumpkin, while the estimated length of all three GS1s of cucumber was 356 amino acids (Supplementary Table S2).
3.2. Evolutionary Relationships of GS Family Genes
To find the phylogenetics and taxonomy of CmoGS and CsaGS members, a phylogenetic tree was reconstructed based on the alignments of 39 amino-acid sequences, including seven from pumpkins (Cucurbita moschata var. Rifu), four from cucumbers (Cucumis sativus L.), five from grapes (Vitis vinifera), four from rice (Oryza sativa), seven from poplar (Populus trichocarpa), six from maize (Zea mays), and six from Arabidopsis thaliana (Supplementary Table S3). According to the phylogenetic relationships, Cucurbitaceae GSs were distributed in three groups, called Groups 1–3. Cucurbitaceae GS1s were distributed in Group 1 and Group 3, while Group 2 contained only GS2s. According to the homology of pumpkin and cucumber GS, the Cucurbitaceae GSs can be divided into four subfamilies (Groups 1–4). Each subfamily contains one CsaGS and one or two CmoGSs (Figure 1). The six CmoGSs, in addition to CmoCh15G007570, occurred in pairs, which may be due to chromosome-doubling events in pumpkin’s evolutionary history and a subsequent period of allotetraploidy [15]. It can be understood that Cucurbitaceae GS1s have a closer phylogenetic relationship with the GS1s of Arabidopsis, poplar, and grape than the other two monocotyledonous plants (Figure 1). Interestingly, Group 1 and Group 3 contained only GSs of dicotyledonous plants, and GSs of dicotyledonous plants were more closely related in other clades (e.g., yellow boxes).
3.3. Structural Analysis of Cucurbitacea GS Family Genes
Based on structural analysis, we found that there are 11–13 introns in these pumpkin and cucumber GS family genes. Considering their short protein length (340–479 amino acids), the exons of these genes are fully divided by introns (Figure 2). For the GS subfamily with two CmoGSs, the structure of CsaGS in this subfamily is more similar to that of the CmoGS, which may imply the evolution sequence of CmoGS replications in one subfamily. In particular, the intron length of CmoCh08G004920 is the longest. Compared to genes on other branches, CmoCh14G008450 and CmoCh15G007570 have a similar intron–exon structure, indicating that these sequences may have experienced a replication event.
3.4. Regulatory Elements in Cucurbitacea GS Family Genes
We investigated the regulatory elements in the 5′-upstream regions for the purpose of gaining insight into the function of GS family genes in pumpkins and cucumbers. In the 5′-upstream regions of GS family genes, the putative regulatory elements are abundant and not conserved among these genes. Even if the genes are of the same branch, their putative regulatory elements differ markedly in number and type (Figure 3, Supplementary Table S4). For example, 15, 33, and 13 regulatory elements were identified in the two putative CmoGS2s and one CsaGS2, of which the auxin-responsiveness element is only present in CmoCh06G014450. Phytohormone regulatory elements (abscisicacid responsiveness, auxin responsiveness, gibberellin responsiveness, MeJA responsiveness, and salicylicacid responsiveness) are located unevenly in the upstream regions of all of Cucurbitacea GS family genes. There seem to be fewer phytohormone regulatory elements in the 5′-upstream regions of cucumber (20, with an average of 5.00 for every CsaGS) than in the 5′-upstream regions of pumpkin (55, with an average of 7.86 for every CmoGS). Certain regulatory elements (e.g., low-temperature responsiveness) only exist in the regulatory regions of one to two GS genes, indicating that some genes are stimulated by specific signals but not others. Putative regulatory elements involved in light responsiveness and abscisic acid responsiveness were identified in all GS family genes. None of the regulatory elements in our study are unique to a subfamily.
3.5. Distribution and Duplication of GS Family Genes in Pumpkin and Cucumber
Cucurbitaceae GS genes are not located on scaffolds or unanchored contigs and exhibit uneven distribution on the chromosomes (Figure 4). Two CmoGS genes are located on Cmo_Chr14, while the others are located on Cmo_Chr1, Cmo_Chr6, Cmo_Chr8, Cmo_Chr15, and Cmo_Chr17. Furthermore, two CsaGS genes are located on Csa_Chr3, while the others are located on Csa_Chr5 and Csa_Chr7. Each CmoGS contains one collinear gene in cucumber, with the same as CsaGS in pumpkin. All CsaGS genes, except for Csa5G410730, contain a pair of collinear CmoGS genes, which are located in two chromosomes with high homology.
3.6. Functional Annotations of GS Gene Members in Pumpkin and Cucumber
To explore the relevant functions of GS, the enrichment of the GO (gene ontology) terms was analyzed. According to the GO enrichment analysis, the seven CmoGSs and four CsaGSs were classified into three ontological categories: biological process, cellular component, and molecular function, with 14 functional terms (Figure 5, Supplementary Table S5). We predicted that the GS family genes of pumpkin and cucumber were involved in many plant, biological, and physiological processes, particularly that all genes were involved in the process of glutamine biosynthesis, cell-wall macromolecule biosynthesis, organonitrogen-compound biosynthesis, glutamate–ammonia ligase activity, and ATP binding.
3.7. Analysis of the Expression Patterns of CmoGS Family Genes
To further explore the function of the GS family genes, a heat map (Figure 6, Supplementary Table S6) was constructed based on the published BioProject RNA-seq data (accession: CmoGSs: PRJNA385310 and CsaGSs: PRJNA312872) in the cucurbit genome database: CuGenDB. As expected, the three members of the GS2 subfamily (Group 2) are highly expressed in green tissues and barely expressed in the roots. Compared with GS2s, GS1s have more diverse expression patterns in a variety of tissues. Unlike other CmoGS1 replications, CmoCh08G004920 is highly expressed in all tissues, especially in the stem, suggesting its crucial role in intercellular nitrogen transport. The expression of all CsaGSs can be detected in the fruit, and the highest expression of Csa7G420690 indicates that it may play an important role in the development of the fruit. CmoCh08G004920, which belongs to Group 1 with Csa7G420690, also shows higher expression in the fruit. In conclusion, the expression pattern of the GS2 subfamily (Group 2) (CmoCh06G014450, CmoCh14G017140, and Csa3G150160) was similar, but that of the GS1 subfamily was different. In addition, different from all GS1 genes with high expression levels in cucumber, only one of the two GS1 genes in Group 1 and Group 3 was more active in pumpkin.
It was previously reported that the expression of GS was induced by cold in tea plants [32]. In order to test the potential function of CmoGS in pumpkin rootstock in glutamine regulation under cold stress, we conducted RT-PCR experiments on CmoGS genes. Semi RT-PCR assays of the first true leaves and roots of pumpkin treated at 4 °C were used to detect the expression level of CmoGSs under cold stress. The first true leaves and roots of pumpkin seedlings at 6 h, 12 h, 24 h, 2 days, 3 days, 6 days, and 9 days after cold treatment and before treatment were selected as samples. The result indicate that except for CmoCh08G004920, the expression trend of CmoGSs was changed to varying degrees by cold induction, although some changes only occurred in the root or leaf, such as those in CmoCh01G003900, CmoCh06G014450, CmoCh14G008450, and CmoCh14G017140 (Figure 7). The RNA abundance of CmoGSs remained unchanged or increased slightly under short-term cold stress within 12 h. Under the long-term cold stress of 2–9 days, the RNA abundance of only CmoCh01G003900 and CmoCh14G008450 in the first true leaf increased. Two genes with low-temperature-response regulatory elements, CmoCh06G014450 and CmoCh14G008450, were assumed to respond to cold stress. In general, most CmoGSs showed changes in the RNA content at low temperatures, and the trend of changes was different for different sites and genes. Compared with short-term cold stress, CmoGSs showed more significant changes in RNA abundance under long-term cold stress.
4. Discussion
GS isoforms have been analyzed in many plants, such as soybeans (Glycine max) [33], potatoes (Solanum tuberosum L) [34], and tomatoes (Solanum lycopersicum cv. Micro-Tom) [35], laying an important foundation for analyzing functional GS isoforms. Nevertheless, GS family members have not been identified in Cucurbitaceae, and few functional studies have been conducted. Thus, the structure and function of GS family members were analyzed herein through bioinformatics. According to homology comparison with the GS family of Arabidopsis thaliana and a domain search, seven and four members of the pumpkin and cucumber GS family genes were identified, respectively. Phylogenetic trees were then constructed with GS amino-acid sequences from seven plants, including pumpkins and cucumbers, with the aim of exploring the evolutionary relationships of Cucurbitaceae GSs. Promoter region analysis showed that the number and arrangement of regulatory elements in GSs differed between family members. The comparison of the number and types in the regulatory elements of GS family genes suggests that each GS gene may be subject to complex regulation, indicating that the functions of these genes are nonredundant. Chromosome 14 of pumpkins has high homology with Chromosome 1 and Chromosome 6, and CmoGSs are distributed pairwise on these chromosomes. Similar situations also occur on Chromosome 8 and Chromosome 17 [15]. This may be related to a doubling of the whole genome of pumpkin, and the duplication was considered to be the main cause of gene-family expansion [36,37]. Therefore, to a certain extent, the uneven distribution of family genes on chromosomes is caused by duplication of fragments. [38].
GS was considered one of the oldest functional genes [39,40]. There are two GS isoforms (GS1 and GS2) in plants, which are considered to have diverged by duplication from a common ancestor [41]. This separation may have occurred later than the appearance of vascular plants but earlier than the divergence of gymnosperms/angiosperms [9]. The majority of plants reported to date have only one GS2. By comparing the number of amino acids in the candidate GS proteins of pumpkins and the phylogenetic analysis, we proved that there are two GS2 duplicates in pumpkins, as in grapes and Medicago truncatula [9,42]. In this study, GS proteins from seven plants, including pumpkins and cucumbers, were used to reconstruct a phylogenetic tree (Figure 1). These proteins were divided into three groups based on their homology. Interestingly, GS1 proteins have a tendency to be separated according to whether they belonged to monocotyledons or dicotyledons. This suggests that the evolution of GS did not stop after the appearance of angiosperms. Thus, our findings facilitate the study of differentiation between monocotyledons and dicotyledons.
In other plants, members of GS family show spatially and temporally specific and nonoverlapping functions [5,43]. A heat map constructed based on the reported RNA-seq data also confirmed the tissue-specific expression patterns of GS genes in pumpkin and cucumber (Figure 6). Combined with the similar tissue-specific expression pattern and collinearity, the close homology between the two replicates of CmoGS2 was demonstrated. Differential expression between two CmoGS2 genes may depend on developmental processes or the presence of specific signals, based on their differential promoters. This also explains the differences in CmoGS1 gene expression; even members of a sister group with close homology have significantly different transcript abundance in tissues. The heat map shows the differential contribution of the CmoGS1 gene members. The high expression of CmoCh08G004920 in the roots and stem suggests that it may be endowed with powerful primary nitrogen assimilation and nitrogen-transport capacity, suggesting pumpkin has evolved a spare glutamine-synthesis-regulation mechanism. In other plants, these functions involve several major GS1s [32,44], so we should focus on CmoCh08G004920 in future research. Transcripts of the CmoGS2s are clustered in the leaves, where ammonia is produced by photorespiration release and nitrate reduction. While we are making unremitting efforts to improve NUE, we also facing new challenges. Studies have shown that the increase in CO2 reduces the nitrate reduction of C3 plants, but the ammonium utilization rate does not decrease [2,8,14]. Therefore, the means of cooperation between GS isoforms may change.
5. Conclusions
Our present work, to the best of our knowledge, is the first time the structure and expression patterns of all candidate members of the pumpkin and cucumber GS family genes has been studied. The Cucurbitaceae GS family genes exhibit complex regulatory and functional possibilities. We found that seven duplicates of CmoGSs and four duplicates of CsaGSs can be organized into three groups based on their homology and multispecies phylogenetic relationships, with two groups for cytosolic GS1 and one for chloroplast GS2. Based on the published RNA-seq data, we performed an expression analysis of the GS family, indicating that the gene expression is spatially specific. In particular, we found tissue-specific expression of pumpkin GS1 (CmoCh08G004920) with relatively high expression of other GS1s, indicating a candidate gene to improve nitrogen-utilization efficiency (NUE) in pumpkins. In general, this study paves the way to deepen our understanding of the GS family genes for future research on the regulation and function of GS family genes in cucurbits crops. It provides confidence in dealing with food safety issues of cucurbits vegetables that may be caused by climate change.
Conception and design of experiments: X.L. (Xiaojun Li), L.G. and W.Z.; performance of experiments: X.L. (Xiaojun Li), W.L., X.Z., T.W., Z.L., X.L. (Xiaohong Lu), C.W. and M.L.; analysis of data: X.L. (Xiaojun Li) and C.X.; writing of manuscript: X.L. (Xiaojun Li) and W.Z. All authors have read and agreed to the published version of the manuscript.
This work was supported by the National Key Research and Development Program of China (2019YFD1000300 and 2018YFD1000800) of W.Z. and the Earmarked Fund for China Agriculture Research System (CAS-23) of L.G.
The reported data can be found in
The authors declare no conflict of interest.
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Enlarge this image.Figure 1. Phylogenetic tree of glutamine synthetase (GS) family genes in pumpkin, cucumber, grape, rice, poplar, maize, and Arabidopsis. The phylogenetic tree was constructed by the neighbor-joining method, with 1000 bootstrap repeats. The three classifications and four subfamilies are represented by different colors. Pt: Populus trichocarpa. Os: Oryza sativa. Vv: Vitis vinifera. At: Arabidopsis thaliana. Zm: Zea mays.
Enlarge this image.Figure 2. Structural analysis of CmoGS and CsaGS members. GSs in different subfamilies are indicated by different colors. The lower number axis shows the number of deoxynucleotides.
Enlarge this image.Figure 3. The regulatory regions of CmoGS and CsaGS gene members. This shows an area of 2000 bp upstream of ATG. ATG is at the position of “0” in the figure. GS gene members in different subfamilies are indicated by different colors on the left. The different color blocks on the right represent different regulatory elements and correspond to the color blocks on the line.
Enlarge this image.Figure 4. Chromosome localization of duplicated CmoGS and CsaGS gene members in pumpkin and cucumber. The red lines represent the colinear genes.
Enlarge this image.Figure 6. The expression profile of CmoGS and CsaGS family genes. The RNA-seq data are from BioProject (pumpkin: accession: PRJNA385310 and cucumber: accession: PRJNA312872).
Enlarge this image.Figure 7. Semi RT-PCR identification of CmoGS expression in responding to cold stress. The time for cold treatment is shown at the top of the figure (h: hour, d: day). Actin: CmoACT7. S: first true leaf of pumpkin. R: root of pumpkin.
Supplementary Materials
The following are available online at
References
1. Bernard, S.M.; Habash, D. The importance of cytosolic glutamine synthetase in nitrogen assimilation and recycling. New Phytol.; 2009; 182, pp. 608-620. [DOI: https://dx.doi.org/10.1111/j.1469-8137.2009.02823.x] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/19422547]
2. Miflin, B.J.; Habash, D.Z. The role of glutamine synthetase and glutamate dehydrogenase in nitrogen assimilation and possibilities for improvement in the nitrogen utilization of crops. J. Exp. Bot.; 2002; 53, pp. 979-987. [DOI: https://dx.doi.org/10.1093/jexbot/53.370.979] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/11912240]
3. James, D.; Borphukan, B.; Fartyal, D.; Achary, V.M.M.; Reddy, M.K. Transgenic manipulation of Glutamine Synthetase: A Target with Untapped Potential in Various Aspects of Crop Improvement. Biotechnologies of Crop Improvement; Springer: Berlin/Heidelberg, Germany, 2018; Volume 2, pp. 367-416.
4. Peterman, T.K.; Goodman, H.M. The glutamine synthetase gene family of Arabidopsis thaliana light-regulation and differential expression in leaves, roots and seeds. Mol. Genet. Genom.; 1991; 230, pp. 145-154. [DOI: https://dx.doi.org/10.1007/BF00290662] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/1684022]
5. Li, M.G.; Villemur, R.; Hussey, P.J.; Silflow, C.D.; Gantt, J.S.; Snustad, D.P. Differential expression of six glutamine synthetase genes in Zea mays. Plant Mol. Biol.; 1993; 23, pp. 401-407. [DOI: https://dx.doi.org/10.1007/BF00029015] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/8106013]
6. Sakakibara, H.; Kawabata, S.; Takahashi, H.; Hase, T.; Sugiyama, T. Molecular Cloning of the Family of Glutamine Synthetase Genes from Maize: Expression of Genes for Glutamine Synthetase and Ferredoxin-Dependent Glutamate Synthase in Photosynthetic and Non-Photosynthetic Tissues. Plant Cell Physiol.; 1992; 33, pp. 49-58. [DOI: https://dx.doi.org/10.1093/oxfordjournals.pcp.a078219]
7. Castro-Rodríguez, V.; García-Gutiérrez, A.; Canales, J.; Avila, C.; Kirby, E.G.; Cánovas, F.M. The glutamine synthetase gene family in Populus. BMC Plant Biol.; 2011; 11, 119. [DOI: https://dx.doi.org/10.1186/1471-2229-11-119]
8. Lam, H.-M.; Coschigano, K.T.; Oliveira, I.C.; Melo-Oliveira, R.; Coruzzi, G.M. The Molecular-Genetics of Nitrogen Assimilation into Amino Acids in Higher Plants. Annu. Rev. Plant Biol.; 1996; 47, pp. 569-593. [DOI: https://dx.doi.org/10.1146/annurev.arplant.47.1.569] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/15012301]
9. Seabra, A.R.; Carvalho, H.; Pereira, P.J.B. Crystallization and preliminary crystallographic characterization of glutamine synthetase fromMedicago truncatula. Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun.; 2009; 65, pp. 1309-1312. [DOI: https://dx.doi.org/10.1107/S1744309109047381]
10. Unno, H.; Uchida, T.; Sugawara, H.; Kurisu, G.; Sugiyama, T.; Yamaya, T.; Sakakibara, H.; Hase, T.; Kusunoki, M. Atomic Structure of Plant Glutamine Synthetase-A key enzyme for plant productivity. J. Biol. Chem.; 2006; 281, pp. 29287-29296. [DOI: https://dx.doi.org/10.1074/jbc.M601497200]
11. McNally, S.; Hirel, B.; Gadal, P.; Mann, A.; Stewart, G. Evidence for a specific isoform content related to their possible physiological role and their compartmentation within the leaf. Plant Physiol.; 1983; 72, pp. 22-25. [DOI: https://dx.doi.org/10.1104/pp.72.1.22]
12. Andrews, M. The partitioning of nitrate assimilation between root and shoot of higher plants. Plant Cell Environ.; 1986; 9, pp. 511-519. [DOI: https://dx.doi.org/10.1111/1365-3040.ep11616228]
13. Hirel, B.; Lea, P.J.; Lea, P.J.; Morot-Gaudry, J.-F. Ammonia Assimilation. Plant Nitrogen; Springer: Berlin/Heidelberg, Germany, 2001; pp. 79-99.
14. Sun, H.; Huang, Q.-M.; Su, J. Highly effective expression of glutamine synthetase genes GS1 and GS2 in transgenic rice plants increases nitrogen-deficiency tolerance. J. Plant Physiol. Mol. Boil.; 2005; 31, pp. 492-498.
15. Hachiya, T.; Inaba, J.; Wakazaki, M.; Sato, M.; Toyooka, K.; Miyagi, A.; Kawai-Yamada, M.; Sugiura, D.; Nakagawa, T.; Kiba, T. et al. Excessive ammonium assimilation by plastidic glutamine synthetase causes ammonium toxicity in Arabidopsis thaliana. Nat. Commun.; 2021; 12, pp. 1-10. [DOI: https://dx.doi.org/10.1038/s41467-021-25238-7]
16. Moison, M.; Marmagne, A.; Dinant, S.; Soulay, F.; Azzopardi, M.; Lothier, J.; Citerne, S.; Morin, H.; Legay, N.; Chardon, F. et al. Three cytosolic glutamine synthetase isoforms localized in different-order veins act together for N remobilization and seed filling in Arabidopsis. J. Exp. Bot.; 2018; 69, pp. 4379-4393. [DOI: https://dx.doi.org/10.1093/jxb/ery217]
17. Wei, Y.; Xiong, S.; Zhang, Z.; Meng, X.; Wang, L.; Zhang, X.; Yu, M.; Yu, H.; Wang, X.; Ma, X. Localization, Gene Expression, and Functions of Glutamine Synthetase Isozymes in Wheat Grain (Triticum aestivum L.). Front. Plant Sci.; 2021; 12, [DOI: https://dx.doi.org/10.3389/fpls.2021.580405]
18. Anwar, A.; Li, Y.; He, C.; Yu, X. 24-Epibrassinolide promotes NO3- and NH4+ ion flux rate and NRT1 gene expression in cucumber under suboptimal root zone temperature. BMC Plant Biol.; 2019; 19, pp. 1-5.
19. Xin, M.; Wang, L.; Liu, Y.; Feng, Z.; Zhou, X.; Qin, Z. Transcriptome profiling of cucumber genome expression in response to long-term low nitrogen stress. Acta Physiol. Plant.; 2017; 39, 130. [DOI: https://dx.doi.org/10.1007/s11738-017-2429-2]
20. Liu, H.; Gao, Y.; Gao, C.; Liu, S.; Zhang, J.; Chen, G.; Zhang, S.; Wu, F. Study of the physiological mechanism of delaying cucumber senescence by wheat intercropping pattern. J. Plant Physiol.; 2019; 234–235, pp. 154-166. [DOI: https://dx.doi.org/10.1016/j.jplph.2019.02.003]
21. Sun, H.; Wu, S.; Zhang, G.; Jiao, C.; Guo, S.; Ren, Y.; Zhang, J.; Zhang, H.; Gong, G.; Jia, Z. et al. Karyotype Stability and Unbiased Fractionation in the Paleo-Allotetraploid Cucurbita Genomes. Mol. Plant; 2017; 10, pp. 1293-1306. [DOI: https://dx.doi.org/10.1016/j.molp.2017.09.003]
22. Condurso, C.; Verzera, A.; Dima, G.; Tripodi, G.; Crinò, P.; Paratore, A.; Romano, D. Effects of different rootstocks on aroma volatile compounds and carotenoid content of melon fruits. Sci. Hortic.; 2012; 148, pp. 9-16. [DOI: https://dx.doi.org/10.1016/j.scienta.2012.09.015]
23. Pulgar, G.; Villora, G.; Moreno, D.; Romero, L. Improving the Mineral Nutrition in Grafted Watermelon Plants: Nitrogen Metabolism. Biol. Plant.; 2000; 43, pp. 607-609. [DOI: https://dx.doi.org/10.1023/A:1002856117053]
24. Xing, W.-W.; Li, L.; Gao, P.; Li, H.; Shao, Q.-S.; Shu, S.; Sun, J.; Guo, S.-R. Effects of grafting with pumpkin rootstock on carbohydrate metabolism in cucumber seedlings under Ca(NO3)2 stress. Plant Physiol. Biochem.; 2015; 87, pp. 124-132. [DOI: https://dx.doi.org/10.1016/j.plaphy.2014.12.011] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/25579659]
25. Britto, D.T.; Kronzucker, H. NH4+ toxicity in higher plants: A critical review. J. Plant Physiol.; 2002; 159, pp. 567-584. [DOI: https://dx.doi.org/10.1078/0176-1617-0774]
26. Bateman, A.; Birney, E.; Cerruti, L.; Durbin, R.; Etwille, L.; Eddy, S.R.; Griffiths-Jones, S.; Howe, K.L.; Marshall, M.; Sonnhammer, E.L. The Pfam protein families database. Nucleic Acids Res.; 2002; 30, pp. 276-280. [DOI: https://dx.doi.org/10.1093/nar/30.1.276]
27. Gasteiger, E.; Gattiker, A.; Hoogland, C.; Ivanyi, I.; Appel, R.D.; Bairoch, A. ExPASy: The proteomics server for in-depth protein knowledge and analysis. Nucleic Acids Res.; 2003; 31, pp. 3784-3788. [DOI: https://dx.doi.org/10.1093/nar/gkg563]
28. Kumar, S.; Stecher, G.; Li, M.; Knyaz, C.; Tamura, K. MEGA X: Molecular Evolutionary Genetics Analysis across Computing Platforms. Mol. Biol. Evol.; 2018; 35, pp. 1547-1549. [DOI: https://dx.doi.org/10.1093/molbev/msy096] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/29722887]
29. Tang, H.; Bowers, J.E.; Wang, X.; Ming, R.; Alam, M.; Paterson, A.H. Synteny and Collinearity in Plant Genomes. Science; 2008; 320, pp. 486-488. [DOI: https://dx.doi.org/10.1126/science.1153917]
30. Chen, C.J.; Chen, H.; Zhang, Y.; Thomas, H.R.; Frank, M.H.; He, Y.H.; Xia, R. TBtools: An Integrative Toolkit Developed for Interactive Analyses of Big Biological Data. Mol. Plant.; 2020; 13, pp. 1194-1202. [DOI: https://dx.doi.org/10.1016/j.molp.2020.06.009]
31. Wei, G.; Tian, P.; Zhang, F.; Qin, H.; Miao, H.; Chen, Q.; Hu, Z.; Cao, L.; Wang, M.; Gu, X. et al. Integrative Analyses of Nontargeted Volatile Profiling and Transcriptome Data Provide Molecular Insight into VOC Diversity in Cucumber Plants (Cucumis sativus). Plant Physiol.; 2016; 172, pp. 603-618. [DOI: https://dx.doi.org/10.1104/pp.16.01051]
32. Cheng, G.; You, X.; Wu, Y.; Zhang, J. Analysis on gene differential expression of cold-resistance cultivar ‘Ziyangyuanye’of Camellia sinensis after low temperature stress. J. Plant Res. Environ.; 2013; 22, pp. 38-43.
33. Miao, G.H.; Hirel, B.; Marsolier, M.C.; Ridge, R.W.; Verma, D.P.S. Ammonia-regulated expression of a soybean gene encoding cytosolic Glutamine-Synthetase in transgenic lotus-corniculatus. Plant Cell; 1991; 3, pp. 11-22. [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/1688099]
34. Teixeira, J.; Pereira, S. High salinity and drought act on an organ-dependent manner on potato glutamine synthetase expression and accumulation. Environ. Exp. Bot.; 2007; 60, pp. 121-126. [DOI: https://dx.doi.org/10.1016/j.envexpbot.2006.09.003]
35. Scarpeci, T.E.; Marro, M.L.; Bortolotti, S.; Boggio, S.B.; Valle, E.M. Plant nutritional status modulates glutamine synthetase levels in ripe tomatoes (Solanum lycopersicum cv. Micro-Tom). J. Plant Physiol.; 2007; 164, pp. 137-145. [DOI: https://dx.doi.org/10.1016/j.jplph.2006.01.003] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/16513209]
36. Kong, H.; Landherr, L.L.; Frohlich, M.W.; Leebens-Mack, J.; Ma, H.; De Pamphilis, C.W. Patterns of gene duplication in the plant SKP1 gene family in angiosperms: Evidence for multiple mechanisms of rapid gene birth. Plant J.; 2007; 50, pp. 873-885. [DOI: https://dx.doi.org/10.1111/j.1365-313X.2007.03097.x]
37. Nakano, T.; Suzuki, K.; Fujimura, T.; Shinshi, H. Genome-Wide Analysis of the ERF Gene Family in Arabidopsis and Rice. Plant Physiol.; 2006; 140, pp. 411-432. [DOI: https://dx.doi.org/10.1104/pp.105.073783]
38. Zhu, Y.; Wu, N.; Song, W.; Yin, G.; Qin, Y.; Yan, Y.; Hu, Y. Soybean (Glycine max) expansin gene superfamily origins: Segmental and tandem duplication events followed by divergent selection among subfamilies. BMC Plant Biol.; 2014; 14, 93. [DOI: https://dx.doi.org/10.1186/1471-2229-14-93]
39. Kumada, Y.; Benson, D.; Hillemann, D.; Hosted, T.; Rochefort, D.; Thompson, C.; Wohlleben, W.; Tateno, Y. Evolution of the glutamine synthetase gene, one of the oldest existing and functioning genes. Proc. Natl. Acad. Sci. USA; 1993; 90, pp. 3009-3013. [DOI: https://dx.doi.org/10.1073/pnas.90.7.3009]
40. Pesole, G.; Bozzetti, M.P.; Lanave, C.; Preparata, G.; Saccone, C. Glutamine synthetase gene evolution: A good molecular clock. Proc. Natl. Acad. Sci. USA; 1991; 88, pp. 522-526. [DOI: https://dx.doi.org/10.1073/pnas.88.2.522]
41. Tingey, S.V.; Tsai, F.Y.; Edwards, J.W.; Walker, E.; Coruzzi, G.M. Chloroplast and cytosolic glutamine synthetase are encoded by homologous nuclear genes which are differentially expressed in vivo. J. Biol. Chem.; 1988; 263, pp. 9651-9657. [DOI: https://dx.doi.org/10.1016/S0021-9258(19)81566-1]
42. Seabra, A.R.; Vieira, C.P.; Cullimore, J.V.; Carvalho, H.G. Medicago truncatula contains a second gene encoding a plastid located glutamine synthetase exclusively expressed in developing seeds. BMC Plant Biol.; 2010; 10, pp. 1-16. [DOI: https://dx.doi.org/10.1186/1471-2229-10-183]
43. Wang, X.; Wei, Y.; Shi, L.; Ma, X.; Theg, S.M. New isoforms and assembly of glutamine synthetase in the leaf of wheat (Triticum aestivum L.). J. Exp. Bot.; 2015; 66, pp. 6827-6834. [DOI: https://dx.doi.org/10.1093/jxb/erv388] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/26307137]
44. Thomsen, H.C.; Eriksson, D.; Møller, I.S.; Schjoerring, J.K. Cytosolic glutamine synthetase: A target for improvement of crop nitrogen use efficiency?. Trends Plant Sci.; 2014; 19, pp. 656-663. [DOI: https://dx.doi.org/10.1016/j.tplants.2014.06.002] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/25017701]
© 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/). Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.
Details
; Wang, Cuicui 1 ; Zhang, Xiaojing 1 ; Wang, Tao 1 ; Liu, Zixi 1 ; Gao, Lihong 1 ; Zhang, Wenna 1
1 Beijing Key Laboratory of Growth and Developmental Regulation for Protected Vegetable Crops, China Agricultural University, Beijing 100193, China;
2 College of Life Science and Technology, Honghe University, Mengzi 661100, China;