A Genome-Wide Identification and Comparative Analysis of the Heavy-Metal-Associated Gene Family in Cucurbitaceae Species and Their Role in Cucurbita pepo under Arsenic Stress

The heavy-metal-associated (HMA) proteins are a class of PB1-type ATPases related to the intracellular transport and detoxification of metals. However, due to a lack of information regarding the HMA gene family in the Cucurbitaceae family, a comprehensive genome-wide analysis of the HMA family was performed in ten Cucurbitaceae species: Citrullus amarus, Citrullus colocynthis, Citrullus lanatus, Citrullus mucosospermus, Cucumis melo, Cucumis sativus, Cucurbita maxima, Cucurbita moschata, Cucurbita pepo, and Legenaria siceraria. We identified 103 Cucurbit HMA proteins with various members, ranging from 8 (Legenaria siceraria) to 14 (Cucurbita pepo) across species. The phylogenetic and structural analysis confirmed that the Cucurbitaceae HMA protein family could be further classified into two major clades: Zn/Co/Cd/Pb and Cu/Ag. The GO-annotation-based subcellular localization analysis predicted that all HMA gene family members were localized on membranes. Moreover, the analysis of conserved motifs and gene structure (intron/exon) revealed the functional divergence between clades. The interspecies microsynteny analysis demonstrated that maximum orthologous genes were found between species of the Citrullus genera. Finally, nine candidate HMA genes were selected, and their expression analysis was carried out via qRT-PCR in root, leaf, flower, and fruit tissues of C. pepo under arsenic stress. The expression pattern of the CpeHMA genes showed a distinct pattern of expression in root and shoot tissues, with a remarkable expression of CpeHMA6 and CpeHMA3 genes from the Cu/Ag clade. Overall, this study provides insights into the functional analysis of the HMA gene family in Cucurbitaceae species and lays down the basic knowledge to explore the role and mechanism of the HMA gene family to cope with arsenic stress conditions.


Introduction
Cucurbits are one of the major crop families with high economic value and are widely cultivated worldwide.Four genera, Cucurbita (squash and pumpkins), Cucumis (cucumbers and melons), Citrullus (watermelons), and Lagenaria (bottle gourd), are among the ten most economically significant vegetable crops in the world, while numerous others have regional significance [1].Nevertheless, the global Cucurbit growth and yield are adversely influenced by environmental stresses such as droughts, salinity, and high concentrations of heavy metals and metalloids [2,3].Arsenic (As) is a non-essential metalloid ubiquitous in soil at low levels; however, agricultural soils are threatened by toxic contamination from anthropogenic activities,

Phylogenetic Analysis, Synteny Analysis, and Gene Duplication Events of the Cucurbit HMA Family
The amino acid sequences of the Cucurbit species and Arabidopsis were imported into MEGA 7 [39], and multiple sequence alignment was performed using ClustalW [40] with gap-open and gap-extension penalties of 10 and 0.1, respectively.Alignment was used to build a phylogenetic tree based on the neighbor-joining (NJ) method.After a bootstrap analysis with 1000 replicates, the tree was exported into Newick format to display it by using iTOL software (version 6.8.1, http://itol.embl.de/index.shtml,accessed on 15 October 2022).
A syntenic relationship analysis of the HMA gene family between species of Citrullus, Cucumis, and Cucurbita genera was carried out and visualized using TBTools with the E-value < 1× 10 −10 [38].Non-synonymous (Ka), synonymous substitution (Ks), and Ka/Ks ratios for duplicated gene pairs were calculated in the Ka/Ks calculation tool (http: //services.cbu.uib.no/tools/kaks,accessed on 15 May 2023) using the CDS of Cucurbits.The duplication date was estimated according to the following formula, Million Years Ago (MYA) = Ks/2λ, assuming a clock-like rate (λ) of 6.56 synonymous substitutions per 10 −9 years [41].

Motif Analysis and Promoter Cis-Element Identification
HMA proteins were subjected to the Multiple Em for Motif Elicitation (MEME) tool found in the MEME suite (https://meme-suite.org/meme/, accessed on 15 October 2022) to identify common and species-specific motifs in the Cucurbit proteins.The analysis was performed with a maximum number of motifs of ten and an optimum motif width from 6 to 50.To identify motif function, discovered MEME motifs were searched in the ExPASy-PROSITE database using the ScanProsite tool (https://prosite.expasy.org/scanprosite/,accessed on 15 October 2022).Furthermore, promoter sequences (2000 bp upstream) from Cucurbit HMA genes were submitted to The PlantCARE database (http://bioinformatics. psb.ugent.be/webtools/plantcare/html,accessed on 15 October 2022) to analyze potential cis-regulatory elements within promoter sequences of the Cucurbit HMA genes.

Gene Ontology (GO) Annotation of HMA Proteins
The functional annotation, including cellular component, molecular function, and biological process of HMA proteins, was performed using OmicsBox Software (https: //www.biobam.com/omicsbox/,accessed on 15 November 2022).The amino acid sequences of HMA proteins were imported into the OmicsBox program to execute three steps: (1) BLASTp against the NCBI non-redundant protein database, (2) mapping and retrieval of GO terms associated with the BLAST results, and (3) annotation of GO terms associated with each query to relate the sequences to known protein function.

Expression Pattern of the HMA Family in C. pepo under Different Cu Treatments
RNA-seq gene expression data of CpeHMA genes were retrieved from the NCBI GEO DataSets (Accession: GSE173716) from a previously published work by Marmiroli et al. [42].The fragments per kilobase transcripts per million mapped reads (FPKM) expression values for root, leaf, and pollen tissues from C. pepo exposed to copper oxide (CuO) nanoparticles, bulk CuO (100 mg kg −1 ), and CuSO 4 (320 mg kg −1 ) were used to generate a heatmap and compare the expression of CpHMA1-14 genes identified by using the tydir and ggplot2 packages (https://ggplot2.tidyverse.org/,accessed on 15 July 2023).

Expression Pattern with RT-qPCR of the HMA Family in Cucurbita pepo under As Treatment
C. pepo var.cylindrica "Golden" seeds were germinated directly in the soil.Seedlings were transferred to 0, 50, 100, and 200 µM As (V) soil treatments and irrigated with the same water concentration every other day.Roots, leaves, flowers, and fruit tissues were collected at the anthesis stage, as previously reported by Obrero et al. [43,44].Tissues were immediately frozen in liquid nitrogen and stored at −80 • C. Further, total RNA was isolated from roots, leaves, flowers, and fruits of C. pepo according to the E.Z.N.A. Plant RNA Kit (Omega Bio-Tek, Norcross, GA, USA) following manufacturer instructions.An equivalent concentration of total RNA of the different tissues was used to synthesize first-strand cDNA with the SuperScript™ IV First-Strand Synthesis System (Invitrogen, Waltham, MA, USA).Equal cDNA concentration of samples was used for the qRT-PCR analysis.A StepOnePlus Real-Time PCR system (Applied Biosystems, Foster City, CA, USA) was employed for the qRT-PCR analysis with a final reaction volume of 20 µL containing 1 µL of cDNA template, 2 µL of forward and reverse primer at 10 µM, 10 µL of SYBR Green PCR Master (ROX) (Roche, Shanghai, China), and 7 µL of nuclease-free water.Three replicates per treatment were run to compute the average Ct values that were further analyzed with the 2 −∆∆Ct method [45].The Actin gene was used as an endogenous control to normalize the relative gene expression.HMA primers for qRT-PCR were designed in Primer3Plus software (version 3.3.0)[46] and are listed in Table S1.

Identification of HMA Genes in Cucurbits
A total of 103 genes potentially encoding HMA proteins were identified and classified: 10 from C. amarus, 9 from C. colocynthis, 10 from C. lanatus, 9 from C. mucusospermus, 10 from C. melo, 9 from C. sativus, 12 from C. maxima, 12 from C. moschata, 14 from C. pepo, and 8 from L. siceraria (Table 1).Moreover, amino acid length, molecular weight, and isoelectric point of Cucurbit HMA proteins were deducted from their protein sequences and are listed in Table S2.The protein length of Cucurbit HMA proteins generally varied between 356 and 1251 amino acid residues.The molecular weight was determined to range from 37.8 KDa to 143.5 KDa, while the isoelectric point (pI) ranged from 4.96 pH to 9.23 pH.

Chromosomal Location and Gene Structure of HMA Genes
HMA genes in Cucurbits were found to be located in various chromosomes with a wide distribution, except for CpeHMA1 from C. pepo found on the unanchored scaffold.Nevertheless, not all Cucurbit species showed the presence of HMA genes in all of their chromosomes, as depicted in Figure 1.HMA genes were detected in chromosomes   Next, we utilized a biosequence structure tool to produce gene structure schematic diagrams and assess the exon/intron arrangement of coding and genome sequences in HMA genes across Cucurbit species.Our analysis of the gene structures indicated significant variation in intron positions, lengths, and numbers across all species studied.Nonetheless, members most closely related shared similar exon/intron structures either according to the number of introns or exon length.Specifically, the number of exons present in Citrullus species ranged from 3 to 25, ranged from 3 to 17 in Cucumis species, ranged from 5 to 19 in Cucurbita species, and ranged from 6 to 16 in L. siceraria species.The detailed gene structure of the Cucurbit HMA genes is in Figure 2a.Further, we also observed that the location of the domains in the HMA proteins follows the forward pattern reported in Arabidopsis [36], i.e., HMA proteins in the clade of Cu/Ag showed HMA, E1-E2_ATPase, and hydrolase domains, while HMA proteins in the Zn/Co/Cd/Pb exhibited only the E1-E2_ATPase and hydrolase domains (Figure 2b).Next, we utilized a biosequence structure tool to produce gene structure schematic diagrams and assess the exon/intron arrangement of coding and genome sequences in HMA genes across Cucurbit species.Our analysis of the gene structures indicated significant variation in intron positions, lengths, and numbers across all species studied.Nonetheless, members most closely related shared similar exon/intron structures either according to the number of introns or exon length.Specifically, the number of exons present in Citrullus species ranged from 3 to 25, ranged from 3 to 17 in Cucumis species, ranged from 5 to 19 in Cucurbita species, and ranged from 6 to 16 in L. siceraria species.The detailed gene structure of the Cucurbit HMA genes is in Figure 2a.Further, we also observed that the location of the domains in the HMA proteins follows the forward pattern reported in Arabidopsis [36], i.e., HMA proteins in the clade of Cu/Ag showed HMA, E1-E2_ATPase, and hydrolase domains, while HMA proteins in the Zn/Co/Cd/Pb exhibited only the E1-E2_ATPase and hydrolase domains (Figure 2b).

Phylogenetic Analysis, Synteny Analysis, and Gene Duplication Events of the Cucurbit HMA Gene Family
To examine the phylogenetic relationships among the Cucurbit HMA proteins, an unrooted phylogenetic tree was constructed from alignments of the 103 full-length HMA sequences (Figure 3).The HMA proteins were classified into two major clades, the Zn/Co/Cd/Pb and the Cu/Ag.While the number of HMA genes in Citrullus, Cucumis, and L. siceraria species was similar compared to Arabidopsis [36], Cucurbita species exhibited distinct, independent duplication events.Specifically, four paralog pairs (CmaHMA7-CmaHMA8, CmoHMA7-CmoHMA8, CpHMA1-CpHMA2, and CpeHMA9-CpeHMA10) were found.The HMA genes from Citrullus, Cucumis, and Cucurbita species were subjected to synteny and a gene duplication analysis to confirm further the results from the phylogenetic tree (Figure 4).The syntenic analysis among the HMA genes of the three genera revealed collinearity among species; in the Citrullus genera, specifically between C. amarus and C. lanatus with ten events, while in the Cucurbita genera, between C. maxima and C. moschata with 12 events where chromosome/position/HMA copy number are conserved.Conversely, although the Cucumis genera shows nine events, HMA genes are not posi- The HMA genes from Citrullus, Cucumis, and Cucurbita species were subjected to synteny and a gene duplication analysis to confirm further the results from the phylogenetic tree (Figure 4).The syntenic analysis among the HMA genes of the three genera revealed collinearity among species; in the Citrullus genera, specifically between C. amarus and C. lanatus with ten events, while in the Cucurbita genera, between C. maxima and C. moschata with 12 events where chromosome/position/HMA copy number are conserved.Conversely, although the Cucumis genera shows nine events, HMA genes are not positionally conserved.In addition, gene duplication events of the HMA gene family were found in all species, the Cucurbita genera being the highest.Interestingly, tandem duplication occurs in pairs labeled as CamHMA2-3, CamHMA4-5, CcoHMA2-3, ClaHMA3-4, CmuHMA3-4, CmeHMA9-10, CmaHMA1-2, CmaHMA7-8, CmoHMA1-2, CmoHMA7-8, CpeHMA4-5, and CpeHMA9-10 while in C. sativus and L. siceraria, no tandem duplications were found.The non-synonymous rate (Ka), the synonymous rate (Ks), the Ka/Ks, and the duplication date using the Ks values of these pairs are presented in Table 2.In general, the Ka/Ks of tandem and segmental duplicates is less than 1, indicating they were under purifying selection.Moreover, the Ks of Citrullus duplication pairs ranges from 0.27 to 0.47 with divergent times that range from 21 to 36 MYA; similarly, in Cucumis and L. siceraria, the Ks value ranges from 0.27 to 0.  In addition, gene duplication events of the HMA gene family were found in all species, the Cucurbita genera being the highest.Interestingly, tandem duplication occurs in pairs labeled as CamHMA2-3, CamHMA4-5, CcoHMA2-3, ClaHMA3-4, CmuHMA3-4, CmeHMA9-10, CmaHMA1-2, CmaHMA7-8, CmoHMA1-2, CmoHMA7-8, CpeHMA4-5, and CpeHMA9-10 while in C. sativus and L. siceraria, no tandem duplications were found.The non-synonymous rate (Ka), the synonymous rate (Ks), the Ka/Ks, and the duplication date using the Ks values of these pairs are presented in Table 2.In general, the Ka/Ks of tandem and segmental duplicates is less than 1, indicating they were under purifying selection.Moreover, the Ks of Citrullus duplication pairs ranges from 0.27 to 0.47 with divergent times that range from 21 to 36 MYA; similarly, in Cucumis and L. siceraria, the Ks value ranges from 0.27 to 0.44 and 0.33 to 0.44, and the divergent times from 20 to 33 MYA and 24 to 34 MYA, respectively.Nevertheless, Cucurbita duplication pairs' Ks values range from 0.01 to 0.53 and divergent times from 1.27 to 40.88, which means a spectrum range of both, synonymous mutation, as well as diversification time.Interestingly, the tandem duplicates CmaHMA7-8, CmoHMA7-8, and CpeHMA10-CpeHMA9 were speculated to diverge in recent time, <3 MYA ago, in contrast to the Citrullus, Cucumis, and L. siceraria duplicates that resulted >20 Mya.

Conserved Motif Analysis and Cis-Elements of HMA Proteins
We identified ten common conserved motifs in HMA proteins by analyzing motif composition with MEME motif analysis, as shown in Figure 5. Common motifs ranged in length from 29 to 50 aa.The position and number of motifs vary according to the substrate specificity of Cu/Ag and Zn/Cd/Co/Pb clades (Figure S1).Furthermore, a ScanProsite analysis indicated that most of the motifs in the Cucurbit HMA family were associated with common functions of ATPase, such as the E1-E2 ATPase phosphorylation site, which is integral to the ATPase function.Details of the ten conserved motifs in common and for each Cucurbit species are given in Table S3.
length from 29 to 50 aa.The position and number of motifs vary acc specificity of Cu/Ag and Zn/Cd/Co/Pb clades (Figure S1).Furtherm ysis indicated that most of the motifs in the Cucurbit HMA famil common functions of ATPase, such as the E1-E2 ATPase phospho integral to the ATPase function.Details of the ten conserved mot each Cucurbit species are given in Table S3.To identify common putative cis-elements that can influence curbit HMA genes, 2000-bp DNA sequences upstream of the sta HMA proteins for each species were analyzed using the PlantCAR 20 common elements identified associated with environmental str To identify common putative cis-elements that can influence the expression of Cucurbit HMA genes, 2000-bp DNA sequences upstream of the start codon (ATG) for the HMA proteins for each species were analyzed using the PlantCARE database.There were 20 common elements identified associated with environmental stresses and plant hormonal processes.Table S4 shows all the identified common regulatory elements for the Cucurbit HMA family.The cis-regulatory elements associated with stress responses and possibly involved in the activation against heavy metals were TC-rich repeats, engaged in defense and stress response, LTR, associated with low temperatures, TCA cis-elements, related to salicylic acid that has an attenuation against biotic and abiotic stresses, and associated with heavy metal toxicity.

GO Annotation of HMA Proteins
The GO analysis performed with Omics Box suggested the putative participation of HMA genes in multiple biological processes, molecular functions, and cellular components (Figure 6 and Table S5).For instance, all 103 HMA proteins identified were predicted to be found in the membrane as a cellular component.Likewise, all HMA proteins were associated with the ATP-hydrolysis and binding activity for molecular function, followed by ATPase-coupled cation transmembrane transport activity.HMA proteins were involved in copper, cadmium, zinc transport, homeostasis, and metal ion binding for biological processes.

GO Annotation of HMA Proteins
The GO analysis performed with Omics Box suggested the putative participation of HMA genes in multiple biological processes, molecular functions, and cellular components (Figure 6 and Table S5).For instance, all 103 HMA proteins identified were predicted to be found in the membrane as a cellular component.Likewise, all HMA proteins were associated with the ATP-hydrolysis and binding activity for molecular function, followed by ATPase-coupled cation transmembrane transport activity.HMA proteins were involved in copper, cadmium, zinc transport, homeostasis, and metal ion binding for biological processes.S5.

Gene Expression Pattern of HMA Genes in Tissues of Cucurbita Pepo under Cu and As Treatments
To gain insights into the expression patterns and functions of HMA genes in C. pepo, we analyzed the expression profiles of CpeHMA genes.We based our analysis on their FPKM values derived from RNA-seq data acquired during copper (Cu) treatments, a heavy metal known to be linked with the HMA gene family.This initial investigation was a foundation for comparing subsequent expression results under arsenic exposure and validating our assumptions regarding gene functionality (Table S6).A phylogenetically clustered heatmap was used to visualize each gene s specific gene expression patterns in root, leaf, and pollen tissue under CuO nanoparticles, bulk CuO, and bulk CuSO4 (Figure 7).Genes in clade Cu/Ag exhibited high expression levels, whereas genes in clade Zn/Cd/Co/Cu exhibited low expression levels.CpeHMA14, a member of the Cu/Ag clade, exhibited the highest expression among treatments and tissues, whereas CpeHMA12, a member of the Zn/Cd/Co/Pb clade, exhibited the lowest expression.The maximum fold-  S5.

Gene Expression Pattern of HMA Genes in Tissues of Cucurbita Pepo under Cu and as Treatments
To gain insights into the expression patterns and functions of HMA genes in C. pepo, we analyzed the expression profiles of CpeHMA genes.We based our analysis on their FPKM values derived from RNA-seq data acquired during copper (Cu) treatments, a heavy metal known to be linked with the HMA gene family.This initial investigation was a foundation for comparing subsequent expression results under arsenic exposure and validating our assumptions regarding gene functionality (Table S6).A phylogenetically clustered heatmap was used to visualize each gene's specific gene expression patterns in root, leaf, and pollen tissue under CuO nanoparticles, bulk CuO, and bulk CuSO 4 (Figure 7).Genes in clade Cu/Ag exhibited high expression levels, whereas genes in clade Zn/Cd/Co/Cu exhibited low expression levels.CpeHMA14, a member of the Cu/Ag clade, exhibited the highest expression among treatments and tissues, whereas CpeHMA12, a member of the Zn/Cd/Co/Pb clade, exhibited the lowest expression.The maximum fold-change expression levels of CpeHMA genes were detected in root and leaf tissues, while low or relatively no expression was found in pollen tissues.Members of the Cu/Ag clade, CpeHMA7 and CpeHMA8, were highly expressed under bulk CuO and CuSO 4 with a 21-fold and 14-fold change, respectively.On the other hand, the highest expressed gene for leaf tissue was also a member of the Cu/Ag family, CpeHMA6, with 19-and 5-fold alterations under bulk CuSO 4 and CuO, respectively.CpeHMA3 and CpeHMA8, members of the Cu/Ag clade, had the maximum expression levels in pollen under CuO NPs, with a five-and six-fold change, respectively.Based on gene homology and duplication events, we evaluated the expression pattern of nine C. pepo HMA genes (CpeHMA2, CpeHMA3, CpeHMA5, CpeHMA6, CpeHMA8, CpeHMA10, CpeHMA11, CpeHMA12, and CpeHMA14) in root, leaf, flower, and fruit tissues under 50 µM, 100 µM, and 200 µM As treatment through RT-qPCR.Notably, no downregulated genes were observed among all the evaluated HMA genes and tissues.However, genes belonging to the Cu/Ag clade exhibited significantly higher differential expression across all tissues (Figure 8).Interestingly, CpeHMA6 showed upregulation in all As treatments across all tissue types.CpeHMA3 displayed the highest expression in leaf tissue with a six-fold upregulation under the 50 µM treatment.In roots, CpeHMA6, CpeHMA3, CpeHMA11, CpeHMA2, and CpeHMA8 exhibited differential expression with fold changes of 27, 9, 4, and 3, respectively, in the 200 µM treatment.Moreover, the flower tissue exhibited the most remarkable fold-change pattern, as all the studied genes showed upregulation across all treatments.CpeHMA6 consistently exhibited significant expression with a 36-fold change under the 200 µM treatment, while CpeHMA2 and CpeHMA14 showed fold changes of >20 and >30, respectively, among different treatments.In contrast, the expression of the studied CpeHMA genes did not show significant differential expression in leaf and fruit tissues compared to root and flower tissues.The exception was CpeHMA6, which exhibited a notable three-fold upregulation under the 200 µM treatment in both leaf and fruit tissues.Based on gene homology and duplication events, we evaluated the expression pattern of nine C. pepo HMA genes (CpeHMA2, CpeHMA3, CpeHMA5, CpeHMA6, CpeHMA8, CpeHMA10, CpeHMA11, CpeHMA12, and CpeHMA14) in root, leaf, flower, and fruit tissues under 50 µM, 100 µM, and 200 µM As treatment through RT-qPCR.Notably, no downregulated genes were observed among all the evaluated HMA genes and tissues.However, genes belonging to the Cu/Ag clade exhibited significantly higher differential expression across all tissues (Figure 8).Interestingly, CpeHMA6 showed upregulation in all As treatments across all tissue types.CpeHMA3 displayed the highest expression in leaf tissue with a six-fold upregulation under the 50 µM treatment.In roots, CpeHMA6, CpeHMA3, CpeHMA11, CpeHMA2, and CpeHMA8 exhibited differential expression with fold changes of 27, 9, 4, and 3, respectively, in the 200 µM treatment.Moreover, the flower tissue exhibited the most remarkable fold-change pattern, as all the studied genes showed upregulation across all treatments.CpeHMA6 consistently exhibited significant expression with a 36-fold change under the 200 µM treatment, while CpeHMA2 and CpeHMA14 showed fold changes of >20 and >30, respectively, among different treatments.In contrast, the expression of the studied CpeHMA genes did not show significant differential expression in leaf and fruit tissues compared to root and flower tissues.The exception was CpeHMA6, which exhibited a notable three-fold upregulation under the 200 µM treatment in both leaf and fruit tissues.

Discussion
The HMA "heavy metal ATPase transporter" is a type of ATPase known as t type ATPase.It belongs to the P-type ATPase family, comprising ion pumps utiliz ergy from ATP hydrolysis to uptake, translocate, compartmentalize, and detoxify metal ions within plant cells [9,16].Although HMA members have been identifie analyzed in Arabidopsis [36] and several crops such as rice [18], soybeans [20], and P [19], a comprehensive identification and characterization of this gene family in the bits have not been performed.In this study, a total of 103 heavy metal ATPase ( proteins were identified across ten different Cucurbit species.The subsequent ph netic analysis of the HMA gene family revealed the division of HMA proteins in distinct subfamilies (Zn/Co/Cd/Pb P1B-ATPase and the Cu/Ag P1B-ATPase) ba their structural and functional characteristics as described in previous studies [15 Cucurbita plants, especially C. pepo with 14 members, exhibited a higher abunda HMA proteins, despite having a smaller genome size (271.4Mb for C. maxima, 26 for C. moschata, and 263 Mb for C. pepo) [47,48] in comparison to Cucumis (375 Mb melo) [49] and Citrullus (425 Mb for C. lanatus) [50], indicating that genome size m have a positive correlation with the number of HMA family members.The length quences and isoelectric points of proteins significantly varied, indicating a high de diversification among the HMA genes in Cucurbits.Moreover, the domain struc

Discussion
The HMA "heavy metal ATPase transporter" is a type of ATPase known as the P 1Btype ATPase.It belongs to the P-type ATPase family, comprising ion pumps utilizing energy from ATP hydrolysis to uptake, translocate, compartmentalize, and detoxify heavy metal ions within plant cells [9,16].Although HMA members have been identified and analyzed in Arabidopsis [36] and several crops such as rice [18], soybeans [20], and Populus [19], a comprehensive identification and characterization of this gene family in the Cucurbits have not been performed.In this study, a total of 103 heavy metal ATPase (HMA) proteins were identified across ten different Cucurbit species.The subsequent phylogenetic analysis of the HMA gene family revealed the division of HMA proteins into two distinct subfamilies (Zn/Co/Cd/Pb P1B-ATPase and the Cu/Ag P1B-ATPase) based on their structural and functional characteristics as described in previous studies [15,20,28].Cucurbita plants, especially C. pepo with 14 members, exhibited a higher abundance of HMA proteins, despite having a smaller genome size (271.4Mb for C. maxima, 269.9 Mb for C. moschata, and 263 Mb for C. pepo) [47,48] in comparison to Cucumis (375 Mb for C. melo) [49] and Citrullus (425 Mb for C. lanatus) [50], indicating that genome size may not have a positive correlation with the number of HMA family members.The length of sequences and isoelectric points of proteins significantly varied, indicating a high degree of diversification among the HMA genes in Cucurbits.Moreover, the domain structure of HMA genes from the significant clades was similar to the pattern shown in Arabidopsis [15].Nevertheless, it is important to mention that all HMA Cucurbit genes possess a hydrolase domain since several HMA genes in other species have a lack or disruption of this domain, such as MtHMA8 in Medicago and ZmHMA9 in Zea mays [21,28].This suggests that all Cucurbit HMA proteins may play an active role in metal transport due to ATP-hydrolysis-dependent mechanisms of energy required for transport [8,9].Additionally, protein localization is the fundamental concept for understanding interactions at the systems' level, and the function of transporters is inextricably linked to their subcellular localization [51].In Arabidopsis, AtHMA2 is expressed mainly in vascular tissues [52]; however, previous studies in both Arabidopsis and Oryza sativa have demonstrated that different HMAs exhibit diverse subcellular localizations [19,53].In Cucurbits, all HMA proteins were predicted to localize within the cell membrane.Membrane proteins play a vital role in regulating plant responses to heavy metal stress, as they facilitate the transport of metals across membranes, thereby contributing to metal homeostasis and detoxification processes [54].Expression of CsHMA3 and CsHMA4 was found to confer tolerance to Cd and Zn with metal efflux tolerance and accumulation of Cd and Pb through sequestration, proving to be part of the Zn/Co/Cd/Pb clade and suggesting its role in plant translocation from the plasma membrane and bioaccumulation of these metals into the vacuoles [34].However, experimental validation is needed to locate HMAs and understand their role in other Cucurbits.
Furthermore, an important feature of P 1B -ATPases is the presence of soluble metal binding domains (MBDs) that regulate transport activity [55].The conserved structure characteristics of two cysteines (CxxC) of the HMA domain give HMA genes the basic function of binding metal ions through thiol groups [56,57].P 1B -type ATPases are capable of driving the efflux out of cells of both essential transition metal ions (e.g., Zn 2+ , Cu + , and Co 2+ ) and toxic metal ions (e.g., Ag + , Cd 2+ , and Pb 2+ ), contributing to their homeostasis maintenance [15,32].Previous studies on members of the HMA gene family in Arabidopsis focused on heavy metal stress.Several genes, i.e., AtHMA4, AtHMA2, and AtHMA3, have been identified as Cd transporters involved in transporting Cd across the cell membrane and from the cytoplasm to the vacuole [10,11].However, the molecular basis of HMA metal ion specificity remains unclear [58].According to Smith et al. [8], HMA proteins appear to have functional roles in transporting manganese, iron, nickel, and other thiophilic heavy metals and metalloids such as arsenic.In plants, arsenic can easily enter through phosphate (P) transporters (arsenate) and aquaporin channels (arsenite), inhibiting plant growth and reducing crop yield [59][60][61].After entering the plant, arsenic can be sequestered in the form of As-cysteine-rich peptides such as phytochelatins and then translocated into vacuoles mainly by ABC transporter subfamily C (ABCC) [62][63][64].Nonetheless, different studies have reported alternative and independent arsenic transporters, such as the silicon transporters Ls1 and Ls2 that transport As (III) and the peptide transporter OsPTR7 associated with the translocation of methylated-As species in Oryza sativa [24,59,65,66].
Although HMA proteins have not been previously characterized in arsenic transport, transcriptomic analyses showed that P-type ATPase genes were upregulated in roots and shoots of the hyper-accumulator Pteris vitatta in response to arsenic, which implies the role of P-type ATPase in the translocation of this metalloid [35].Additionally, vacuolar proteomics showed that P-type ATPases were highly abundant compared to other metal transporters under arsenic stress.Likewise, previous studies have also reported the participation of non-elucidated transporters for arsenic in Pteris vitatta when treated with a mix of Ag-As due to the inhibition of the entrance, translocation, and the enhancement of As tolerance when Ag and As are supplemented simultaneously; it is known that Ag transport and homeostasis across plant tissues are mediated via HMA proteins from the Cu/Ag clade [67,68].However, metal homeostasis in plants must be regulated using several complex processes [54], and the collaboration of transporters in different tissues may play an important role in plant metal distribution [19].
In order to understand the role of HMAs, we analyzed the gene expression levels from C. pepo genes (CpeHMA) in roots, leaves, and pollen from C. pepo under Cu treatments [42], a well-recognized HMA-related metal from proteins in the Ag/Cu clade, and arsenic stress in roots, leaves, flowers, and fruit.Under either Cu or As, genes that belong to the Zn/Co/Cd/Pb clade, CpeHMA2, CpeHMA4, CpeHMA5, CpeHMA9, CpeHMA10, CpeHMA11, and CpeHMA12, exhibited low to no expression levels in all tissues, whereas genes in the Cu/Ag clade such as CpeHMA3, CpeHMA6, CpeHMA7, CpeHMA8, and CpeHMA14 in the Cu treatments and CpeHMA3, CpeHMA6, CpeHMA8, and CpeHMA14 in the As treatments were highly expressed.CpeHMA6 showed a high upregulation in leaf tissue under Cu treatment and in root and flower tissues under As stress.It was observed from the phylogenetic tree that CpHMA6 is orthologous to AtHMA8 from Arabidopsis, which is related to the Cu transport through the thylakoid membrane [69][70][71].Cu is an essential metal due to its function as an enzyme cofactor for a number of physiological processes [72].Nevertheless, As (V) can act as a P analog in the phosphorylation process that occurs in the thylakoid membrane, leading to the disruption of the ATP production process and thus threatening the energy homeostasis of the cell [59,73].
Moreover, the CpeHMA3 gene, which is classified within the Cu/Ag clade, exhibited significant upregulation in response to both Cu and As treatments.Notably, the Cu/Ag clade lacks annotated HMA domains, a distinctive feature of this particular clade.The absence of highly conserved regions, particularly those associated with similar functionality observed in other species, can have an impact on both the affinity of the protein for various ionic metals and its inherent characteristics, such as heavy metal binding properties [13].Moreover, CpeHMA8 is an orthologue of AtHMA5 and OsHMA5 in Arabidopsis and Oryza sativs, respectively, while CpeHMA14 is an orthologue of AtHMA7.AtHMA5 is located in the plasma membrane and is involved in the Cu translocation from roots to shoots or Cu detoxification of roots [74].OsHMA5 is involved in loading Cu to the xylem of the roots and other organs [33].Nevertheless, a study in Populus trichocarpa suggested that PtHMA5 may differ in function from AtHMA5 and OsHMA5, where it was found to have a significant role in Ag detoxification in addition to Cu detoxification [19].Highly expressed CpeHMA7 in root tissues under CuSO 4 is orthologous to AtHMA7, also known as RAN1.The AtHMA7 gene has been recognized as an ATP-dependent copper transporter that interacts with the ethylene receptor ETR1, which is primarily found in the endoplasmic reticulum regulating plant growth and development [75].Therefore, considering the high expression observed in CpeHMA3, CpeHMA6, CpeHMA7, CpeHMA8, and CpeHMA14 across the different tissues under Cu and As stress, it is plausible to hypothesize that HMA proteins may play a role in the transportation and tolerance mechanisms of arsenic in Cucurbita pepo, similar to their involvement in copper transport.However, further study and confirmation are required to elucidate the specific molecular pathways with which these CpeHMA genes respond to arsenic stress.
Moreover, through the examination of cis-regulatory elements in the HMA gene family, it has been observed that all genes harbor multiple cis-elements associated with abiotic stress, including those related to heavy metals [27,76].Other cis-elements identified were the ABRE, ARE elements, LTR, and TC-rich repeats, which are associated with various stress responses, such as abscisic acid stress, anaerobic induction, low-temperature stress, defense mechanisms, and oxidative stress, which may be involved in generating a response to arsenic-induced oxidative stress [26,28].Consequently, these findings suggest that the HMA genes in Cucurbits may be activated and potentially play a role in responding to other stress conditions.

Conclusions
Metal transporters play vital roles in distributing and transforming essential, nonessential, and even toxic metal ions in plants.This study comprehensively analyzed the HMA gene family in ten Cucurbit species.A total of 103 HMA genes from species of the Cucurbitaceous family were characterized and classified into two groups based on a phylogenetic analysis and their structural characteristics.According to their evolutionary metal association, the Cucurbit HMA genes had conserved or divergent gene structures, protein motif patterns, and cis-regulatory elements.The expression profiles of CpeHMA genes in various tissues/organs of C. pepo in response to both Cu and As stress indicate that the members of this gene family might be involved in transporting As metal ions lanatus, C. maxima, and C. moschata in Chr2 have three, four, and four members, respectively.Similarly, C. melo has the highest number of HMA proteins in Chr11 with three members, while C. pepo in Chr5 has 4. Furthermore, similar quantities of members in not only one chromosome of several species are present due to tandem duplicates; C. colocynthis and C. sativus have the highest amount in Chr2 and Chr4 with two and three members each, respectively, and C. mucosospermus has the same highest amount in Chr1 and Chr2 with two members each.

Figure 2 . 20 Figure 3 .
Figure 2. Gene structure and protein domains of the HMA family in Cucurbit species.(a) Exon/intron structures of HMA genes.Exons are represented as orange boxes, introns as strings, and promoter UTRs in black.(b) The protein domain structure pattern of HMA gene family, and different-colored rectangles represent different structural domains; the green rectangles represent E1-E2 ATPase; the yellow rectangle represents hydrolase; the pink rectangle represents HMA; the dark-green rectangle represents E1-E2 ATPase superfamily.The gene name is on the left side of each sequence, and the below scale indicates the length in kb and aa, respectively.Genes 2023, 14, x FOR PEER REVIEW 8 of 20

Figure 3 .
Figure 3. Phylogenetic relationship of the HMA gene family.Phylogenetic analysis of Cucurbits and Arabidopsis was carried out using the neighbor-joining method with 1000 bootstraps.Based on genetic and functional studies, the phylogeny was divided into two major clades, P1B-ATPases; zinc (Zn)/cobalt (Co)/cadmium (Cd)/lead (Pb) group is highlighted in red, and the copper (Cu)/silver (Ag) group is highlighted in green.
44 and 0.33 to 0.44, and the divergent times from 20 to 33 MYA and 24 to 34 MYA, respectively.Nevertheless, Cucurbita duplication pairs Ks values range from 0.01 to 0.53 and divergent times from 1.27 to 40.88, which means a spectrum range of both, synonymous mutation, as well as diversification time.Interestingly, the tandem duplicates CmaHMA7-8, CmoHMA7-8, and CpeHMA10-CpeHMA9 were speculated to diverge in recent time, <3 MYA ago, in contrast to the Citrullus, Cucumis, and L. siceraria duplicates that resulted >20 Mya.

Figure 5 .
Figure 5. Common conserved motifs of HMA proteins among Cucurbita colocynthis, C. lanatus, C. mucusospermus, C. melo, C. sativus, C. maxima, C siceraria.The overall height of the stack indicates the degree of sequence co residues suggests the relative frequency of each residue at that position.T are represented below each stack.

Figure 5 .
Figure 5. Common conserved motifs of HMA proteins among Cucurbitaceae plants: C. amarus, C. colocynthis, C. lanatus, C. mucusospermus, C. melo, C. sativus, C. maxima, C. moschata, C. pepo, and L. siceraria.The overall height of the stack indicates the degree of sequence conservation.The height of residues suggests the relative frequency of each residue at that position.Typed sequences of motifs are represented below each stack.

Figure 6 .
Figure 6.Gene ontology analysis results for Cucurbit species.Cellular Component (CC), Molecular Function (MF), and Biological Processes (BP) were identified with the OmicsBox program.Specific results for genes of each species are found in Supplementary TableS5.

Figure 6 .
Figure 6.Gene ontology analysis results for Cucurbit species.Cellular Component (CC), Molecular Function (MF), and Biological Processes (BP) were identified with the OmicsBox program.Specific results for genes of each species are found in Supplementary TableS5.

Genes 2023 ,
14,  x FOR PEER REVIEW 13 of 20 change expression levels of CpeHMA genes were detected in root and leaf tissues, while low or relatively no expression was found in pollen tissues.Members of the Cu/Ag clade, CpeHMA7 and CpeHMA8, were highly expressed under bulk CuO and CuSO4 with a 21fold and 14-fold change, respectively.On the other hand, the highest expressed gene for leaf tissue was also a member of the Cu/Ag family, CpeHMA6, with 19-and 5-fold alterations under bulk CuSO4 and CuO, respectively.CpeHMA3 and CpeHMA8, members of the Cu/Ag clade, had the maximum expression levels in pollen under CuO NPs, with a fiveand six-fold change, respectively.

Figure 7 .
Figure 7. Phylogenetic tree and heat map of gene expression levels of HMA genes in root, leaf, and pollen tissues of C. pepo exposed to CuO NPs, bulk CuO, and bulk CuSO4 (Accession: GSE173716).The bar to the right of the heat map represents normalized expression values in each treatment.

Figure 7 .
Figure 7. Phylogenetic tree and heat map of gene expression levels of HMA genes in root, leaf, and pollen tissues of C. pepo exposed to CuO NPs, bulk CuO, and bulk CuSO 4 (Accession: GSE173716).The bar to the right of the heat map represents normalized expression values in each treatment.

, 14 ,Figure 8 .
Figure 8. Expression pattern of HMA genes from C. pepo in different tissues under As stress.R expression levels of the CpHMA genes in (a) root, (b) leaf, (c) flower, and (d) fruit tissue of plants treated with 50, 100, and 200 µM of arsenic in soil were determined with qRT-PCR.T expression level for each HMA gene in the control plants with no As was normalized to 1 2^Δ ΔCT method suggests.The results represent the means of the biological replicates with thei ard deviation represented as error bars."*", "**", and "***" indicate genes statistically signi differentially expressed between the treatment and the control using a t-test at the level of p p ≤ 0.01, and p ≤ 0.001, respectively.

Figure 8 .
Figure 8. Expression pattern of HMA genes from C. pepo in different tissues under As stress.Relative expression levels of the CpHMA genes in (a) root, (b) leaf, (c) flower, and (d) fruit tissue of C. pepo plants treated with 50, 100, and 200 µM of arsenic in soil were determined with qRT-PCR.The gene expression level for each HMA gene in the control plants with no As was normalized to 1, as the 2ˆ∆ ∆CT method suggests.The results represent the means of the biological replicates with their standard deviation represented as error bars."*", "**", and "***" indicate genes statistically significantly differentially expressed between the treatment and the control using a t-test at the level of p ≤ 0.05, p ≤ 0.01, and p ≤ 0.001, respectively.

Table 1 .
Comparative analysis of HMA proteins between Cucurbitaceae and other plant species and their distribution in the Zn/Cd/Co/Pb and Cu/Ag major clades.

Table 2 .
Ka, Ks, Ka/Ks ratio, and divergent time of the duplicated HMA genes in Cucurbitaceous plants.