Genome-Wide Identiﬁcation and Molecular Evolution of the Magnesium Transporter (MGT) Gene Family in Citrullus lanatus and Cucumis sativus

: Magnesium transporters (MGTs) play a prominent role in the absorption, transporta-tion, and storage of magnesium in plant cells. In the present study, MGT gene family members were identiﬁed and characterized into two species of Cucurbitaceae, including Cucumis sativus and Citrullus lanatus . Totals of 20 and 19 MGT genes were recognized in Citrullus lanatus and Cucumis sativus , respectively. According to their physicochemical properties, the members of each sub-class of MGTs in the species of Cucurbitaceae showed the close relationship. Proteins from NIPA class were identiﬁed as hydrophilic proteins with high stability. Based on phylogenetic analysis, MGT family members were classiﬁed into three groups, and NIPAs showed more diversity. Moreover, duplication events were not identiﬁed between the MGT genes in C. lanatus and C. sativus . According to pocket analysis, residues such as L, V, S, I, and A were frequently observed in the binding sites of MGT proteins in both studied species. The prediction of post-translation modiﬁcations revealed that MSR2 proteins have higher phosphorylation potentials than other sub-classes of MGT in both studied plants. The expression proﬁle of MGTs showed that MGTs are more expressed in root tissues. In addition, MGTs showed differential expression in response to abiotic/biotic stresses as well as hormone application and NIPAs were more induced in response to stimuli in watermelon. The results of this study, as the primary work of MGT gene family, can be used in programs related to Cucurbitaceae breeding. proteins in both species with nine helices, while two helices in CorAs and between two and three helices in MSR2s were predicted. Both C- and N-terminal of the transmembrane structure of CorA proteins were observed in the cytoplasm, while C- and N-terminal of some MSR2 proteins were predicted to locate in extracellular. In addition, a signal peptide was expected in the N-terminal of MSR2 proteins. Overall, the results indicate that MGT proteins have an almost conserved transmembrane structure in both plants.


Introduction
Magnesium (Mg) is a vital element for living cells involved in many critical cellular activities [1][2][3]. For instance, Mg as a cofactor is essential for the activity of many enzymes (>300 enzymes) such as kinase, polymerase, and H + -ATPase [4][5][6]. In addition, Mg, as the key atom of chlorophyll, affects the photosynthesis rate and plant growth [7]. Magnesium transporters (MGTs) are present in plants for Mg uptake, translocation, and cell storage. Based on sequence structure, MGT proteins have been classified into three groups, including MRS2, CorA, and NIPA [8]. The CorA protein, as a member of MGTs, was firstly identified in bacteria, Salmonella typhimurium [9], and in plants for the first time; MGT proteins were studied in the model plant, Arabidopsis [10]. The MRS2 and CorA proteins are recognized by a tripeptide conserved region, GMN (Glycine-Methionine-Asparagine), and two or three transmembrane (TM) domains in their C-terminal ends [11], while NIPAs contain several TMs in their structures [3,8]. However, our knowledge of the NIPA class is limited. Due to the important role of Mg in plants, members of the MGT gene family have been identified and studied in different plants such as Arabidopsis [12], Triticum turgidum and Camelina sativa [8], Pyrus communis [13], Table 1. Summary of MGTs properties in C. lanatus, C. sativus, and C. melo. Full details are provided in Table S1.

Attributes
C

Phylogenetic Analysis of the MGT Family
To better understand the evolutionary relationships of the MGT family, a phylogenetic tree was constructed for 85 members of this family in five different species, including Arabidopsis thaliana, Oryza sativa, Citrullus lanatus, Cucumis sativus, and Cucumis melo ( Figure 1). According to the phylogenetic tree, MGT family members can be classified into three groups, including groups I, II, and III. Subclass NIPA proteins showed more diversity, and they were divided into two groups. Moreover, subclasses CorA and MSR2 were more closely related and were put together in group III.

Evolutionary Process in MGT Genes in Citrullus lanatus and Cucumis sativus
Duplication events were not identified between the MGT genes in C. lanatus and C. sativus. To investigate the duplication events between MGTs in C. lanatus and C. sativus, the Ks, Ka, and Ka/Ks for all gene pairs were calculated ( Figure 2). The Ks value of MGTs in C. lanatus was detected between 1.8 and 2.8 (Figure 2a), and, in C. sativus, it was observed between 1.8 and 2.6 ( Figure 2b). Moreover, the frequency of Ka/Ks of MGTs in C. lanatus was more observed between 0.5 and 0.6 ( Figure 2c), while, in C. sativus, Ka/Ks ratio was frequently detected, ranging from 0.4 to 0.5 ( Figure 2d). It seems that a similar evolutionary process has occurred in C. lanatus and C. sativus for the MGT gene family, and changes (mutations and duplication events) in members of this gene family may have occurred before the derivation of these two species.

Protein Structure of MGTs in C. lanatus and C. sativus
The three-dimensional structures of the MGT proteins, along with their binding sites in the two plants, C. lanatus and C. sativus, was predicted ( Figure 3). Results showed that the members of each subclass, NIPA, CorA, and MSR2, have an almost similar structure in both studied plants. Moreover, various amino acids were predicted as ligand-binding residues in the MGT structures ( Figure 4; Table S2). L, V, S, I, and A were also observed in the binding sites of MGT proteins in C. lanatus and C. sativus, indicating that these residues in molecular function of MGTs.

Evolutionary Process in MGT Genes in Citrullus lanatus and Cucumis sativus
Duplication events were not identified between the MGT genes in C. lanatus and C. sativus. To investigate the duplication events between MGTs in C. lanatus and C. sativus, the Ks, Ka, and Ka/Ks for all gene pairs were calculated ( Figure 2). The Ks value of MGTs in C. lanatus was detected between 1.8 and 2.8 (Figure 2a), and, in C. sativus, it was observed between 1.8 and 2.6 ( Figure 2b). Moreover, the frequency of Ka/Ks of MGTs in C. lanatus was more observed between 0.5 and 0.6 ( Figure 2c), while, in C. sativus, Ka/Ks ratio was frequently detected, ranging from 0.4 to 0.5 (Figure 2d). It seems that a similar evolutionary process has occurred in C. lanatus and C. sativus for the MGT gene family, and changes (mutations and duplication events) in members of this gene family may have occurred before the derivation of these two species.

Transmembrane Structure of MGTs
The transmembrane structures of three subclasses of MGTs were predicted in C. lanatus and C. sativus ( Figure 5). The highest numbers of transmembrane helices were observed in NIPA proteins in both species with nine helices, while two helices in CorAs and between two and three helices in MSR2s were predicted. Both C-and N-terminal of the transmembrane structure of CorA proteins were observed in the cytoplasm, while C-and N-terminal of some MSR2 proteins were predicted to locate in extracellular. In addition, a signal peptide was expected in the N-terminal of MSR2 proteins. Overall, the results indicate that MGT proteins have an almost conserved transmembrane structure in both plants.

Protein Structure of MGTs in C. lanatus and C. sativus
The three-dimensional structures of the MGT proteins, along with their binding sit in the two plants, C. lanatus and C. sativus, was predicted ( Figure 3). Results showed th the members of each subclass, NIPA, CorA, and MSR2, have an almost similar structu in both studied plants. Moreover, various amino acids were predicted as ligand-bindi residues in the MGT structures ( Figure 4; Table S2). L, V, S, I, and A were also observ in the binding sites of MGT proteins in C. lanatus and C. sativus, indicating that these re idues in molecular function of MGTs.

Prediction of the Phosphorylation Site in MGT Proteins
The phosphorylation sites into MGTs of C. lanatus and C. sativus were predicted based on three amino acids, including serine (S), threonine (T), and tyrosine (Y) ( Figure 6). The predicted phosphorylation sites in MGTs of C. lanatus varied from 4 to 18 sites and from 3 to 19 in C. sativus. MSR2 proteins showed higher phosphorylation potential than NIPAs in both studied plants. Moreover, Cla97C07G128920, as a MSR2 protein in C. lanatus, and Csa_1G453990, as a CorA protein, were identified as the MGT proteins with a high phosphorylation potential.  Table S2.

Transmembrane Structure of MGTs
The transmembrane structures of three subclasses of MGTs were predicted in C. lanatus and C. sativus ( Figure 5). The highest numbers of transmembrane helices were observed in NIPA proteins in both species with nine helices, while two helices in CorAs and between two and three helices in MSR2s were predicted. Both C-and N-terminal of the transmembrane structure of CorA proteins were observed in the cytoplasm, while C-and N-terminal of some MSR2 proteins were predicted to locate in extracellular. In addition, a signal peptide was expected in the N-terminal of MSR2 proteins. Overall, the results indicate that MGT proteins have an almost conserved transmembrane structure in both plants.

Prediction of the Phosphorylation Site in MGT Proteins
The phosphorylation sites into MGTs of C. lanatus and C. sativus were predicted based on three amino acids, including serine (S), threonine (T), and tyrosine (Y) ( Figure  6). The predicted phosphorylation sites in MGTs of C. lanatus varied from 4 to 18 sites and from 3 to 19 in C. sativus. MSR2 proteins showed higher phosphorylation potential than NIPAs in both studied plants. Moreover, Cla97C07G128920, as a MSR2 protein in C. lanatus, and Csa_1G453990, as a CorA protein, were identified as the MGT proteins with a high phosphorylation potential.

Co-Expression Network of MGTs in Cucumber
To understand the potential interaction of the MGT gene family members with other genes, as well as the involved pathways, an interaction network of MGT proteins was drawn in cucumber ( Figure 7). The results show that NIPAs interacted highly with the protein phosphatase C (PP2C). In addition, some interactions were observed between NIPA and auxin-induced proteins. Moreover, MSR2 proteins showed strong interactions with acetyltransferase and CaCA antiporter proteins. Gene ontology (GO) analysis based on interaction network revealed molecular function terms including 8-amino-7-oxononanoate synthase activity, chlorophyllide a oxygenase activity, and transmembrane transporter activity were significantly involved in the MGT-interaction network. Moreover, biological processes, such as divalent metal ion transporter process, localization process, and cellular process, were linked with the MGT-interaction network.

Co-Expression Network of MGTs in Cucumber
To understand the potential interaction of the MGT gene family members with other genes, as well as the involved pathways, an interaction network of MGT proteins was drawn in cucumber ( Figure 7). The results show that NIPAs interacted highly with the protein phosphatase C (PP2C). In addition, some interactions were observed between NIPA and auxin-induced proteins. Moreover, MSR2 proteins showed strong interactions with acetyltransferase and CaCA antiporter proteins. Gene ontology (GO) analysis based on interaction network revealed molecular function terms including 8-amino-7-oxononanoate synthase activity, chlorophyllide a oxygenase activity, and transmembrane transporter activity were significantly involved in the MGT-interaction network. Moreover, biological processes, such as divalent metal ion transporter process, localization process, and cellular process, were linked with the MGT-interaction network.

Upstream Analysis of MGT Genes
The upstream region, 1500 bp, of the MGT genes C. lanatus and C. sativus was analyzed as the promoter region to identify cis-regulatory elements. All identified cis-elements were grouped according to their functions, including light-responsive elements (REs), hormone REs, stress REs, growth REs, and MYB binding site ( Figure 8). The cis-regulatory elements related to stress REs were more observed in the promoter region of MGTs in C. lanatus (Figure 8a). In contrast, hormone REs were more observed in the upstream region of MGTs from C. sativus (Figure 8b). Moreover, abscisic acid (ABA) REs were frequently identified in the promoter region of MGTs, more in the cucumber MGTs, while auxin REs and methyl jasmonate (MeJA) REs were more frequently observed in the promoter region of MGTs from watermelon (Figure 8b,e). Stress REs were classified into four groups, including anaerobic, biotic, drought, and low-temperature stress (Figure 8c,f). Moreover, the regulatory elements of anaerobic were recognized with high frequency in the promoter region of MGT genes. Moreover, low-temperature REs were observed most often in the upstream region of MGTs from watermelon (Figure 8c), while regulatory elements that were responsive to drought stress were more frequently observed in promoter region of cucumber MGTs (Figure 8f).

Upstream Analysis of MGT Genes
The upstream region, 1500 bp, of the MGT genes C. lanatus and C. sativus was analyzed as the promoter region to identify cis-regulatory elements. All identified cis-elements were grouped according to their functions, including light-responsive elements (REs), hormone REs, stress REs, growth REs, and MYB binding site ( Figure 8). The cisregulatory elements related to stress REs were more observed in the promoter region of MGTs in C. lanatus (Figure 8a). In contrast, hormone REs were more observed in the upstream region of MGTs from C. sativus (Figure 8b). Moreover, abscisic acid (ABA) REs were frequently identified in the promoter region of MGTs, more in the cucumber MGTs, while auxin REs and methyl jasmonate (MeJA) REs were more frequently observed in the promoter region of MGTs from watermelon (Figure 8b,e). Stress REs were classified into four groups, including anaerobic, biotic, drought, and low-temperature stress ( Figure  8c,f). Moreover, the regulatory elements of anaerobic were recognized with high frequency in the promoter region of MGT genes. Moreover, low-temperature REs were observed most often in the upstream region of MGTs from watermelon (Figure 8c), while regulatory elements that were responsive to drought stress were more frequently observed in promoter region of cucumber MGTs (Figure 8f).

Expression Profile of MGTs in C. lanatus and C. sativus
The expression patterns of MGT genes in different tissues of C. lanatus and C. sativus were provided based on the RNA-Seq datasets ( Figure 9). The MGTs in both studied plants showed low expressions in seeds, while high expression levels of MGTs were observed in root tissues. Three MSR2 genes, including Csa_2G225310, Csa_2G19910, and Csa_7G070800, and three NIPAs, Csa_7G291140, Csa_5G586580, and Csa_3G251940 showed high expression in shoot tissues of cucumber (Figure 9a). Moreover, an MSR2 gene, Cla97C02G033950, showed substantial expression levels in all studied tissues in watermelon (Figure 9b), suggesting the important role of this gene during watermelon growth and expansion. The expression levels of MGTs were also investigated according to available RNA-seq datasets related to biotic and abiotic stresses and hormone treatments ( Figure 10). The expression levels of a NIPA, Csa_3G129750, and an MSR2 gene, Csa_1G138280, of cucumber were upregulated in response to NaCl stress (Figure 10a). Moreover, in response to the nematode, the expression pattern of Csa_2G225310, as an MSR2 gene, was upregulated in cucumber, and Csa_4G646200, as a NIPA gene, showed an upregulation one day after infection (dai) by Pseudoperonospora cubensis (PC) (Figure 10a). In the watermelon, two NIPA genes, including Cla97C05G083800 and Cla97C03G063010, were upregulated in response to drought stress and under mosaic virus stress, two NIPAs, Cla97C05G083800 and Cla97C02G031120, also showed an upregulation (Figure 10b). Moreover, two NIPAs genes from watermelon were more induced in response to melatonin treatment and under low nitrogen content in the leaf (Figure 10b). It seems that NIPAs are more induced in response to stimuli in watermelon. Agronomy 2022, 12, x FOR PEER REVIEW 11 of 18  Table S3.

Expression Profile of MGTs in C. lanatus and C. sativus
The expression patterns of MGT genes in different tissues of C. lanatus and C. sativus were provided based on the RNA-Seq datasets (Figure 9). The MGTs in both studied plants showed low expressions in seeds, while high expression levels of MGTs were observed in root tissues. Three MSR2 genes, including Csa_2G225310, Csa_2G19910, and Csa_7G070800, and three NIPAs, Csa_7G291140, Csa_5G586580, and Csa_3G251940 showed high expression in shoot tissues of cucumber ( Figure 9a). Moreover, an MSR2 gene, Cla97C02G033950, showed substantial expression levels in all studied tissues in watermelon (Figure 9b), suggesting the important role of this gene during watermelon growth and expansion. The expression levels of MGTs were also investigated according to available RNA-seq datasets related to biotic and abiotic stresses and hormone treatments ( Figure 10). The expression levels of a NIPA, Csa_3G129750, and an MSR2 gene, Csa_1G138280, of cucumber were upregulated in response to NaCl stress (Figure 10a). Moreover, in response to the nematode, the expression pattern of Csa_2G225310, as an MSR2 gene, was upregulated in cucumber, and Csa_4G646200, as a NIPA gene, showed an upregulation one day after infection (dai) by Pseudoperonospora cubensis (PC) ( Figure  10a). In the watermelon, two NIPA genes, including Cla97C05G083800 and Cla97C03G063010, were upregulated in response to drought stress and under mosaic virus stress, two NIPAs, Cla97C05G083800 and Cla97C02G031120, also showed an upregulation (Figure 10b). Moreover, two NIPAs genes from watermelon were more induced in response to melatonin treatment and under low nitrogen content in the leaf (Figure 10b). It seems that NIPAs are more induced in response to stimuli in watermelon. The cis-regulatory elements were classified in hormone-responsive elements (REs), light REs, stress REs, growth REs, and MYB binding site in C. lanatus (a) and C. sativus (d). The percentage of cis-regulatory elements related to hormone REs in C. lanatus (b) and C. sativus (e), and stress REs in C. lanatus (c) and C. sativus (f). More details are provided in Table S3.

Discussion
Magnesium (Mg), in addition to being a basic element for plant growth as an essential cofactor, is also involved in the activity of enzymes and in metabolic and photosynthetic processes [29]. Magnesium transporters (MGTs) are fundamental in transmitting and maintaining Mg concentrations in various organelles and cell tissues. In the current study, as the first report, 20, 19, and 20 MGT genes were identified and characterized in three species of Cucurbitaceae, including C. lanatus, C. sativus, and C. melo, respectively. This number of genes is less than the number in Gossypium hirsutum (41 MGTs) [3], Camelina sativa (62 MGTs) [8], Triticum turgidum (41 MGTs) [8], and Brassica napus (36 MGTs) [14]; however, the number of identified genes is greater than that reported in the Theobroma cacao (18 MGTs) [3], Corchorus capsularis (16 MGTs) [3], Pyrus bretschneideri (16 MGTs) [13], Zea mays (12 MGTs) [15], Poncirus trifoliata (8 MGTs) [16], and Fagaria vesca (12 MGTs) [17]. The evolutionary events such as duplication and polyploidization have increased the number of MGTs in some plant species [30,31]. However, we did not find any duplication between the MGT family members in C. lanatus and C. sativus. We hypothesize that the duplication events probably did not occur after the derivation of these two species. Moreover, based on the structure of genes and physicochemical characteristics, an almost conserved state was observed between the studied species, further strengthening this hypothesis. Three subclasses of MGTs, including MSR2, CorA, and NIPA, were identified and compared, and the NIPAs showed a significant difference from the other two classes. For instance, the NIPAs were predicted as stable proteins. In addition, based on GRAVY as a solubility index [31,32], the NIPAs were predicted as hydrophilic proteins. There is limited information about the function of NIPA class in plants, and, due to the different structures, it is necessary to conduct more studies in the field of their functional analysis. Moreover, based on the analysis of gene structure, members of the MGT family had variations in the number of exons, especially in the NIPAs. The exon number can increase the diversity of the coding protein of a gene by affecting the post-transcriptional processes, such as alternative splicing [33,34]. Those with fewer exons can activate rapidly in response to stress, and these genes play a stronger role in the process of adapting to adverse environmental conditions [35]. Evolutionary events such as duplication can affect the structure of genes. Our results reveal that the Ka/Ks ratios of all MGTs in both studied species, C. lanatus and C. sativus, were less than 1.0 indicating that purifying the (negative) selection was the most important force for motiving the evolution of MGTs. Moreover, it was stated that most MGTs emerged before the disclosure of angiosperms [18]. According to the phylogenetic analysis, MGT gene family members can be classified into three main groups; more diversity was observed among the orthologous NIPAs than CorA and MSR2. It seems that the evolutionary processes in the NIPAs were different from the other two groups, CorA and MSR2.
The prediction of the 3D structure of the MGT proteins showed that each subclass had an almost conserved structure in C. lanatus and C. sativus; however, differences in their binding sites were observed, indicating a parallel evolutionary trend in MGTs of both species. Moreover, leucine, valine, serine, isoleucine, and alanine were frequently predicted in pocket sites, suggesting that these residues were more related to the possible interactions of MGTs [36,37]. The interaction network of the MGTs in C. sativus showed that NIPAs interact more with PP2C and auxin-induced proteins. PP2C is a phosphatases involved in ABA signaling, and it was reported previously that Mg could induce Mg-dependent phosphatases PP2C heterodimer in response to heat stress [38]. In addition, hormone response elements were observed in the upstream regions of the MGT genes in both plants, indicating an interaction between Mg and phytohormones. Moreover, interactions between MSR2s and acetyltransferase and CaCA antiporter proteins were observed, and further studies are needed on how they interact in cucumber cells. The phosphorylation process is one of the key post-translational modifications that significantly affect the activity and stability of target proteins and also affect the regulation of cellular signaling pathways in response to adverse conditions [31,39,40]. Results reveal that MSR2 sub-class members have more potential sites for phosphorylation than NIPAs in both studied plant species, C. lanatus and C. sativus, suggesting that MSR2s have more potential to interact with kinases and other signaling components. Based on the expression profile, MGTs were expressed in various tissues in C. lanatus and C. sativus, indicating that they are involved in different biological processes. However, most MGT genes were expressed in the root tissues, indicating that MGTs are more involved in the uptake of Mg in the root and then the distribution of mg in other tissues. Moreover, MGTs showed various expression patterns in response to biotic and abiotic stresses, as well as hormone application. Interestingly, the results disclose that NIPA members are more frequently induced in response to stimuli in watermelon than MSR2s and CorAs. For instance, Cla97C116215350 as a NIPA showed an upregulation in response to cold stress, melatonin application, and low nitrogen in leaf. Regarding the phylogenetic tree, two MGT genes of Arabidopsis, AT4G09640 and AT1G34470, which were involved in antiviral defense [41], showed a closed relationship with Cla97C116215350. Findings suggest that this sub-class of MGTs may be an appropriate target group for further molecular breeding to release the watermelon-resistant lines. The specialized expression of MGT genes can also be related to their promoter region. These genes appear to be more heavily influenced and induced by the pathways dependent on the phytohormones such as ABA, auxin and MeJA. Moreover, it was revealed that the cell-signal transduction associated with hormone concentrations is induced in response to Mg toxicity/deficiency [42,43]. Moreover, previous studies disclosed that MGTs interact with Ca 2+ sensors to induce the downstream signals correlated with plants' reaction to adverse environmental conditions [44].

Identification and Characterization of MGT Genes in C. lanatus, and C. sativus
To identify all sequences related to the MGT family, the MGT proteins of Arabidopsis thaliana were used as queries in the BLAST program in Ensembl Plants [45] against the genomes of C. lanatus and C. sativus. In addition, the orthologue of MGTs was identified in Cucumis melo, and Oryza sativa was identified in the same way. The non-redundant sequences of MGTs were checked using CDD search [46] and Pfam database [47] to validate the presence of MGT domains. To predict the physicochemical properties, including the instability index, GRAVY, isoelectric points (pI), and molecular weight (MW) of MGTs, the ProtParam tool was applied [48].

Phylogenetic Analysis of MGTs
The amino acid sequences of MGTs from C. lanatus, C. sativus, C. melo, Arabidopsis thaliana (as a model plant from dicots), and Oryza sativa (as a model plant from monocots) were used to construct a phylogenetic tree. In the first step, all sequences were aligned using a multiple alignment tool, Clustal-Omega [49]. Then, the output of the Clustal-Omega was submitted to the IQ-TREE webserver [50] to estimate the phylogenetic relationships of MGTs using the Maximum likelihood (ML) method under 1000 bootstrap replicates. In the final step, the phylogenetic tree of MGT proteins was prepared by the interactive tree of life (iTOL version 5) tool [51].

Prediction of Ka and Ks
To recognize the duplicated genes, the cDNA sequences of MGT genes in C. lanatus and C. sativus were processed by the ClustalX v.21 program [52]. According to the identity matrix, the gene pairs with more than 90% identity were screened as a duplicated gene pairs [53]. In the present study, to understand mutations that affected protein sequencing during the evolutionary process, synonymous substitution (K S ) and nonsynonymous substitution (K a ) were investigated for all paired genes of MGT family in C. lanatus and C. sativus. Ks, Ka, and Ka/Ks were calculated using TBtools software [54].

Transmembrane Structure and Pocket Site Analysis of MGTs
To predict the 3D structure and transmembrane structure of MGTs in C. lanatus and C. sativus, the amino acid sequences were submitted to the Phyre2 server [55], and the predicted models with the highest similarity were selected. The pocket sites of each MGT were identified using the Phyre investigator tool of the Phyre2 server.

Prediction of Phosphorylation Sites into MGTs
The phosphorylation sites of each MGT protein in C. lanatus and C. sativus were predicted based on three amino acids, including serine (S), tyrosine (Y), and threonine (T), using the NetPhos 3.1 Server [56]. To predict the sites to a high percentage of confidence, the score was adjusted to scores of more than 0.90.

Protein-Protein Interaction Network
To construct the protein-protein interaction network between MGTs in C. sativus, the sequences of all MGTs were submitted to the STRING v11.5 database [57]. A maximum number of interactors was adjusted to no more than 5 interactors for the first shell and no more than 20 interactors for the second shell. Finally, the interaction networks were illustrated using Cytoscape v3.8.2 [58].

Promoter Analysis of MGT Genes
To identify the known cis-regulatory elements related to the response of hormones and stresses as well as those involved in growth, the upstream region (1500 bp before the start codon) of each MGT gene in C. lanatus and C. sativus was screened by the PlantCARE tool [59]. Finally, cis-regulatory elements were grouped according to their functions.

Gene Expression Profile of MGT Genes
To extract the expression patterns of MGT genes in C. lanatus and C. sativus, the available RNA-seq data from CuGenDBv1 (http://cucurbitgenomics.org/) (accessed on 1 August 2022) were used. Three RNA-seq datasets of different tissues of C. sativus, in-cluding PRJNA80169 (leaf, stem, root, tendril, ovary, female, and male), PRJNA319011 (seed), and PRJNA263870 (phloem of fruit) were analyzed to extract the expression levels of MGTs. Moreover, the RNA-seq data of C. lanatus related to root tissue (PRJNA209092), 34 days after pollination (DAP) in fruit tissues (PRJNA221197), fruit flesh and fruit rind (SRP012849), seed (PRJNA319011), and phloem and vascular tissues (SRP012853) were used to find out the expression profile of MGTs. In addition to understanding the responses of MGTs to biotic/abiotic stresses and the exogenous application of hormones/elicitors, the RNA-seq datasets of C. sativus related to cold stress after 2 h and 12 h (PRJNA438923), NaCl (PRJNA437579), silica (PRJEB7612), GA at 12 h (PRJNA376073), nematode infection (PRJNA419665), powdery mildew infection (PRJNA321023), and one and two days after infection with Pseudoperonospora cubensis (PRJNA285071) and the RNA-seq datasets of C. lanatus, including PRJNA326331 (osmotic stress), PRJNA454040 (drought stress), PR-JNA389184 (mosaic virus), PRJNA328189 (cold stress and melatonin application), and PRJNA422970 (low nitrogen (N) stress in leaf and root) were used and analyzed. The expression data of MGTs were extracted based on FPKM values. The expression profiles of the MGTs were illustrated in heatmaps based on log2 transformed method of FPKM + 1 for expression in tissues and the log2 fold change in response to stresses and hormone applications using TBtools software.

Conclusions
In the present study, MGT gene family members were identified and analyzed in two candidate species of Cucurbitaceae, C. sativus and C. lanatus, in a first report. The results reveal that a similar evolutionary process for the MGT gene family members has probably occurred in C. lanatus and C. sativus, and duplication events between MGTs may have occurred before the derivation of these two species. The NIPAs class showed great structural diversity and different expression patterns from the MSR2 and CorA groups that should be considered more in future studies.