Pan-Genome-Wide Identification and Transcriptome-Wide Analysis of ZIP Genes in Cucumber

: The ZRT/IRT-like proteins (ZIPs) play critical roles in the absorption, transport, and intra-cellular balance of metal ions essential for various physiological processes in plants. However, little is known about the pan-genomic characteristics and properties of ZIP genes in cucumber ( Cucumis sativus L.). In this study, we identified 10 CsZIP genes from the pan-genome of 13 C . sativus accessions. Among them, only CsZIP10 showed no variation in protein sequence length. We analyzed the gene structure, conserved domains, promoter cis -elements, and phylogenetic relationships of these 10 CsZIP genes derived from “9930”. Based on phylogenetic analysis, the CsZIP genes were classified into three branches. Amino acid sequence comparison revealed the presence of conserved histidine residues in the ZIP proteins. Analysis of promoter cis -elements showed that most promoters contained elements responsive to plant hormones. Expression profiling in different tissues showed that most CsZIP genes were expressed at relatively low levels in C . sativus leaves, stems, and tendrils, and CsZIP7 and CsZIP10 were specifically expressed in roots, indicating their potential involvement in the absorption and transport of metal ions. Transcriptomic data indicated that these 10 ZIP genes displayed responses to both downy mildew and powdery mildew, and CsZIP1 was significantly downregulated after both salt and heat treatments. In conclusion, this study deepens our understanding of the ZIP gene family and enhances our knowledge of the biological functions of CsZIP genes in C . sativus .


Introduction
The homeostasis of metal ions plays a crucial role in the growth and development of plants.Appropriate concentrations of metal ions are required by plants to sustain biochemical reactions and physiological processes within cells.However, an excess or deficiency of metal ions may lead to severe growth issues and physiological disruptions.Therefore, maintaining suitable concentrations of metal ions is vital for the survival of plants.The ZIP gene family performs a significant role in the metal ion homeostasis of plants.This family encodes the zinc ion transport proteins responsible for regulating the absorption and distribution of zinc as well as other metal ions.Through the activity of these proteins, plants can respond to changes in environmental metal ion levels and maintain a balanced state of metal ions within cells [1,2].Most ZIP proteins consist of 309-476 amino acid residues and possess eight putative transmembrane (TM) domains.They exhibit a common membrane topology, with the N-and C-terminal ends located extracytoplasmically [3].The cytoplasmic loop between TM3 and TM4 contains a histidine-rich domain (HRD), which functions as the metal binding domain involved in metal transport.Additionally, the amphipathic nature of TM5 and TM4 creates a cavity that allows for the passage of metal ions [2,3].
Currently, more than 300 members of the ZIP protein family have been identified from plants, and the function of some ZIP members has been identified.The ZIP gene, AtIRT1, was first identified in Arabidopsis thaliana.It encodes an iron transporter protein and is mainly expressed in the roots [4].The previous study has shown that AtIRT1 also exhibits specific expression in the companion cells of the cortex and plays a role in iron transport in aerial organs [5].AtIRT2 is another IRT gene that functions similarly to AtIRT1 in A. thaliana.AtIRT2 is located on the vacuole membrane and transports Fe to the vacuole to prevent it from being poisoned in the cytoplasm [6].AtZIP1 and AtZIP3 are mainly expressed in roots.AtZIP1 is a vacuolar transport protein that transports Zn/Mn from the root cells to the cytoplasm [7].AtZIP3 functions in the uptake of Zn and Fe in plant roots [2].
AtZIP2 promotes Mn/Zn uptake and transport in xylem parenchyma cells, thereby facilitating the transport of Mn/Zn towards the shoots [7].In contrast, AtZIP4 transports metal ions into plant cells or between tissues [2].In addition, some ZIP transporter proteins directly participate in the enrichment of Zn in edible parts [8,9].The expression levels of the ZIP gene family in plants exhibit dynamic changes.For example, under zinc deficiency conditions, the expression levels of some ZIP transport proteins increase.However, when zinc levels return to normal or increase, their expression levels decrease [10].
In rice (Oryza sativa L.), OsIRT1 and OsIRT2 are iron-transport proteins that directly absorb Fe 2+ and Zn 2+ into root cells [11][12][13][14].When O. sativa is exposed to iron deficiency conditions, the expression of OsIRT1 and OsIRT2 in the root system is upregulated [15].OsZIP1 is located in the plasma membrane.Under the condition of excessive metal ions, OsZIP1 plays a role as a transporter in O. sativa [16].Furthermore, studies have shown that the overexpression of OsZIP1 can increase the accumulation of metal ions in plant tissues [13,14].OsZIP4 and OsZIP5, as plasma membrane-localized Zn transporters, participate in Zn 2+ uptake in O. sativa [17,18].Overexpression of OsZIP4 and OsZIP5 can increase the content of Zn 2+ in plants [17,18].OsZIP8 is also a Zn transporter protein in O. sativa, participating in the uptake and distribution of Zn 2+ [19].Generally speaking, ZIP transport proteins are primarily involved in the absorption and transport of Zn and Fe.However, these proteins also play a role in the absorption of other metal ions, such as Cu and Mn [7].
Additionally, they can also participate in the transport and absorption of a variety of toxic metals, such as cadmium (Cd), in plants [20].For example, overexpression of OsIRT1 in O. sativa has been found to enhance its resistance to Cd [21].ZIP2 and ZIP3 have been demonstrated the involvement in the uptake and transport of Cd in cabbage, with an increase in their expression levels [22].In tomato (Solanum lycopersicum L.), the expression of SlZIP4 is positively correlated with content of Cd within a specific range [23].Additionally, ZIP genes also participate in the absorption and transport of Cd in mulberry and tobacco [24,25].
In recent botanical studies, one of the core issues of concern is the mechanism of plant tolerance to biotic and abiotic stresses.In barley, 3029 up-regulated genes and 3017 downregulated genes involved in drought tolerance were successfully identified [26].In Qingke (Hordeum vulgare L. var.nudum), drought induced the expression of HOVUSG1548800, HOVUSG2056400 and HOVUSG5062900 [27].In tea plant (Camellia sinensis L.), most ZIP genes are involved in the response of tea to abiotic stress.Under NaCl treatment, the expression of ZIP1, ZIP6, ZIP9, ZIP11, and ZIP12 genes decreased.Under cold stress, ZIP1, ZIP3, ZIP6, ZIP7, ZIP11, and ZIP12 genes were up-regulated.Under MeJA treatment, the transcription level of ZIP4 decreased [28].
In H. vulgare, HOVUSG6558000, HOVUSG5063500, HOVUSG5062800, and HOV-USG5062900 were identified as the main response genes to powdery mildew infection [27].In sweet potato (Ipomoea batatas L.), according to bioinformatics analysis and RT-qPCR analysis, IbNBS75, IbNBS219, and IbNBS256 were found to respond to stem nematode infection [29].In Arachis duranensis, the expression level of AdLTPs changed.Three AdLTPs are associated with resistance to nematode infection, and DOF and WRI1 transcription factors may regulate the response of AdLTP to nematode infection [30].
Due to relatively limited research on the ZIP gene family in both abiotic and biotic stress conditions, it is crucial to investigate the role of the ZIP gene family in non-biological and biological stress to enhance our understanding of its internal functionalities.
Previous studies have reported the pan-genomic analysis of the ZIP gene family in different plants including A. thaliana, peanut, and Populus trichocarpa.Arabidopsis had 15 ZIP members, peanut 30, and Populus trichocarpa 16 [31-33].However, there is no report on the CsZIP gene family yet in C. sativus.Therefore, it is necessary to analyze the CsZIP gene family.
C. sativus is a popular cash crop due to its unique flavor and refreshing crisp texture [34].Due to the crucial role of the CsZIP gene family in the absorption and transportation of metal ions, and in order to explore the function of ZIP gene family in abiotic and biotic stresses, it is imperative to analyze and identify the CsZIP gene family in C. sativus.The updated genome database of C. sativus provides a better resource for identifying and characterizing CsZIP genes.In this study, bioinformatics methods were used to study the sequence similarity, gene structure, and expression pattern of CsZIP gene family, aiming to deepen the understanding of CsZIP gene and improve the understanding of its biological function.

Identification of ZIP Genes in C. sativus
The analysis and identification of ZIP genes in C. sativus were conducted by utilizing the genome sequence data available on the Cucurbit Genomics Data website (http://cucurbitgenomics.org/organism/20 (accessed on 1 January 2023)).The hidden Markov model (HMM) profile files of the ZIP conserved domain (PF02535) were downloaded from the Pfam database (http://pfam.xfam.org/(accessed on 3 January 2023)).The ZIP gene was searched from the C. sativus genome database using HMMER 3.0, and the default parameter and cutoff value were 0.01.The Pfam tool (http://pfam.janelia.org(accessed on 5 January 2023)) and SMART (http://smart.embl-heidelberg.de/(accessed on 5 January 2023)) were employed for confirming the conserved domain of ZIP.The prediction of ZIP candidates was conducted through the ExPASy Proteomics Server as described by Artimo et al. in 2012.Subsequently, the subcellular localization of the protein was analyzed using the Cell-PLoc tool [35].

Phylogenetic Analysis and Multiple Sequence Alignment
The MEGA software (v7.0) was utilized to align the protein sequences of ZIP from A. thaliana, C. sativus, melon, and O. sativa using the ClustalW algorithm.Subsequently, the evolutionary relationship between ZIP proteins was analyzed using the neighborjoining method software, and the number of repetitions was 1000 times.The resulting phylogenetic trees underwent graphical representation and enhancement through Evolview (https://evolgenius.info//evolview-v2/#login (accessed on 3 February 2023)).

Gene Duplication Analysis and Genome Distribution
The CsZIP loci, obtained from the C. sativus genome database (http://cucurbitgenomics.org/organism/20 (accessed on 4 February 2023)), were visualized on chromosomes using MapChart software (MapChart 2.32) [39].From Orthomcl [40], the homology of ZIP gene between C. sativus and A. thaliana, Cucumis melo, and O. sativa were obtained.Circos shows the chromosomal location and collinearity of the ZIP gene [41].The comparison between all coding sequences (CDS) from Arabidopsis and C. sativus genomes was conducted using Clustal W. Subsequently, repeated pairs and homologous pairs were identified based on the alignment result.The Computational Biology Unit (CBU) Ka/Ks calculation tool was then utilized to compute the non-synonymous (Ka) and synonymous (Ks) nucleotide substitution rates.The divergence times of the duplicated genes were calculated using the formula T = Ks/2r Mya (millions of years), where r equals 1.5 × 10 −8 [42].

Analysis of Promoter Regions of CsZIP Genes
The 1.5-kb sequences upstream of the initiation codons (ATG) of CsZIP genes were obtained from the cucurbit genomics data website (http://cucurbitgenomics.org/organism/ 20 (accessed on 5 February 2023)), and analyzed for cis-elements in the promoter region using the online tool PlantCARE [43].

Plant Materials and Growth Conditions
In this study, the temporal and spatial expression patterns of ZIP genes were analyzed using the C. sativus inbred line "China long" 9930 (North China type).C. sativus were grown in greenhouses at Shandong Agricultural University in China, following standard water management and pest control practices.

RNA isolation and Real-Time PCR Analysis
To collect samples for expression analysis, the roots, stems, leaves, female and male flowers, ovaries, and tendrils of C. sativus plants were collected.These samples were immediately frozen in liquid nitrogen and stored at −80 • C for RNA isolation.Total RNA extraction was performed using TRIzol according to the manufacturer's instructions, followed by DNase I treatment to eliminate potential DNA contamination.Subsequently, cDNA synthesis was conducted using the RevertAid First Strand Synthesis Kit.Primer sequences for the selected genes were designed using Primer Premier 5.0 (Table S4), with the C. sativus actin gene serving as an internal control.qRT-PCR validation of the selected genes was carried out using the Ultra SYBR Green Mixture qPCR kit (CWBIO, Beijing, China) on an iCycler iQ TM real-time PCR detection system.The relative expression levels of the selected genes were determined using the 2 −∆∆Ct method [45].

Identification of CsZIP Genes from C. sativus Pan-Genome
With the release of a graph-based pan-genome [46], we could identify ZIP genes in C. sativus by analyzing 13 accession genomes.We obtained the HMM configuration profile files and extracted CsZIP proteins from the C. sativus genome database.The candidate genes were further analyzed using NCBI, Pfam, and SMART.As a result, a total of 10 CsZIP genes were identified in the 13 C. sativus accessions (Table 1).Of these accessions, only "Cuc80" and "PI183967" contain nine CsZIP genes (Table 2).To avoid confusion, we renamed these genes as CsZIP1-CsZIP10 based on their chromosomal order (Table 3).To further investigate the variation in CsZIP genes among different C. sativus accessions, we counted the length of the identified CsZIP proteins (Table 2).Among the 13 C. sativus accessions, only one gene, CsZIP10, had the same protein length across all accessions.The lengths of CsZIP1, CsZIP4, CsZIP6, and CsZIP8 differed in only one accession.CsZIP2, CsZIP3, and CsZIP7 exhibited variations in protein length in two accessions.CsZIP9 showed protein variations in three accessions, while CsZIP5 exhibited protein length differences in multiple accessions.All CsZIP protein sequences can be found in Dataset S2.

Characterization of CsZIP Genes from Chinese Long 9930
Subsequent analyses primarily focused on the genes identified in the '9930 ′ genome, as it encompassed all the 10 CsZIPs and is the first C. sativus genome sequenced and has been updated to the V3 version.The gene name, gene number, gene intron number, chromosome position, gene length, protein length, isoelectric point, relative molecular weight, and subcellular localization prediction of CsZIPs are presented in Table 3. Sequence analysis revealed that the length of CsZIP proteins ranged from 228 to 594 amino acids, with molecular weights ranging from 27.20 to 61.98 kDa.The longest and most complex gene structure was found in CsZIP6, while the shortest gene was CsZIP8.CsZIP4 had the highest relative molecular weight, whereas CsZIP3 the lowest.The isoelectric points of these proteins ranged from 5.58 to 8.53.Furthermore, subcellular localization prediction indicated that most CsZIP proteins were localized to the cell membrane, except CsZIP2 and CsZIP7, which were found in the chloroplasts (Table 3).

Analysis of Phylogenetic Relationship and Gene Structure and Protein Motif
To analyze the phylogenetic relationship of CsZIP proteins, we constructed the phylogenetic tree of these 10 proteins (Figure 1).Based on the phylogenetic tree, the CsZIP family exhibited division into three distinct clades.Specifically, CsZIP2, CsZIP5, and CsZIP10 were grouped within the first clade, CsZIP8 and CsZIP1 were positioned in the second clade, while the remaining CsZIPs were categorized under the third clade (Figure 1).The number of introns varied among CsZIP genes, ranging from 0 to 10: CsZIP6 has the most introns, and CsZIP8 has none (Figure 1).The structures of the CsZIP6, CsZIP3, and CsZIP4 genes were similar, suggesting they are more closely related (Figure 1).
Subsequent analyses primarily focused on the genes identified in the 9930′ genome, as it encompassed all the 10 CsZIPs and is the first C. sativus genome sequenced and has been updated to the V3 version.The gene name, gene number, gene intron number, chromosome position, gene length, protein length, isoelectric point, relative molecular weight, and subcellular localization prediction of CsZIPs are presented in Table 3. Sequence analysis revealed that the length of CsZIP proteins ranged from 228 to 594 amino acids, with molecular weights ranging from 27.20 to 61.98 kDa.The longest and most complex gene structure was found in CsZIP6, while the shortest gene was CsZIP8.CsZIP4 had the highest relative molecular weight, whereas CsZIP3 the lowest.The isoelectric points of these proteins ranged from 5.58 to 8.53.Furthermore, subcellular localization prediction indicated that most CsZIP proteins were localized to the cell membrane, except CsZIP2 and CsZIP7, which were found in the chloroplasts (Table 3).

Analysis of Phylogenetic Relationship and Gene Structure and Protein Motif
To analyze the phylogenetic relationship of CsZIP proteins, we constructed the phylogenetic tree of these 10 proteins (Figure 1).Based on the phylogenetic tree, the CsZIP family exhibited division into three distinct clades.Specifically, CsZIP2, CsZIP5, and CsZIP10 were grouped within the first clade, CsZIP8 and CsZIP1 were positioned in the second clade, while the remaining CsZIPs were categorized under the third clade (Figure 1).The number of introns varied among CsZIP genes, ranging from 0 to 10: CsZIP6 has the most introns, and CsZIP8 has none (Figure 1).The structures of the CsZIP6, CsZIP3, and CsZIP4 genes were similar, suggesting they are more closely related (Figure 1).We further analyzed the conserved motifs of CsZIP family and found that motif type and arrangement were very similar among the members of the first and second clades, in all of which only motif 9 was present, but diverse among the members of the third clade, and motif number ranged from one (CsZIP3 and CsZIP4) to seven (CsZIP7) (Figure 2).Motif 2 was present in all the CsZIPs except CsZIP3, indicating that motif 2 is highly conserved (Figure 2).CsZIP6, CsZIP3, and CsZIP4 are the CsZIP proteins that did not contain motif 1 (Figure 2).The variations in motifs among CsZIP proteins could potentially account for their functional diversity.The amino acid sequence for each motif is presented in the Figure S1.
Motif 2 was present in all the CsZIPs except CsZIP3, indicating that motif 2 is highly con served (Figure 2).CsZIP6, CsZIP3, and CsZIP4 are the CsZIP proteins that did not contai motif 1 (Figure 2).The variations in motifs among CsZIP proteins could potentially a count for their functional diversity.The amino acid sequence for each motif is presente in the Figure S1.

Multiple Sequence Alignment
To analyze the characteristics of ZIP protein homologous sequences in compariso to other species, we conducted a comparative study of ZIP proteins in Arabidopsis, Cucum melo, O. sativa, and C. sativus.The transmembrane domains IV-VII exhibited the highe degree of conservation (Figure 3).Previous studies have reported that transmembran domains IV and V are amphiphilic and play a crucial role in transport, a conclusion th has been further corroborated by subsequent research [47].In the mutant irt1, mutation in the conserved histidine residues or adjacent polar charged residues within transmem brane domains IV and V were found to abolish their transport function [48].Notably, th IV regions of the sequence alignment in Arabidopsis, Cucumis melo, O. sativa, and C. sativu showed the presence of conserved histidine residues (Figure 3).

Multiple Sequence Alignment
To analyze the characteristics of ZIP protein homologous sequences in comparison to other species, we conducted a comparative study of ZIP proteins in Arabidopsis, Cucumis melo, O. sativa, and C. sativus.The transmembrane domains IV-VII exhibited the highest degree of conservation (Figure 3).Previous studies have reported that transmembrane domains IV and V are amphiphilic and play a crucial role in transport, a conclusion that has been further corroborated by subsequent research [47].In the mutant irt1, mutations in the conserved histidine residues or adjacent polar charged residues within transmembrane domains IV and V were found to abolish their transport function [48].Notably, the IV regions of the sequence alignment in Arabidopsis, Cucumis melo, O. sativa, and C. sativus showed the presence of conserved histidine residues (Figure 3).

Phylogenetic Analysis among Different Species
We obtained a comprehensive understanding of the relative relationship of CsZIP proteins by constructing a phylogenetic tree using the amino acid sequences of ZIP proteins from Arabidopsis, C. sativus, Cucumis melo, and O. sativa (Figure 4).The phylogenetic analysis revealed three major clades: Group 1, 2, and 3.In Group 1, there were seven proteins from Arabidopsis, three from C. sativus, three from Cucumis melo, and eight from O. sativa.Group 2 consisted of six proteins from Arabidopsis, three from C. sativus, three from Cucumis melo, and three from O. sativa.Group 3 included five proteins from Arabidopsis, four from C. sativus, four from Cucumis melo, and five from O. sativa (Figure 4).It is worth noting that the number of ZIP proteins varied within each subgroup for Arabidopsis, C. sativus, Cucumis melo, and O. sativa.Specifically, half O. sativa ZIP proteins were grouped into Group 1, while the distribution among the three clades was relatively equal for the other species.Further investigation within the Group 3 revealed a close relationship between CsZIP6, CmZIP8, AtIAR1, and OsZIP14 (Figure 4).For reference, the amino acid sequences of the ZIP proteins can be found in Dataset S1.This analysis provides valuable insights into the interrelationships of ZIP proteins in different plant species and serves as a foundation for further exploration of their biological functions.

Phylogenetic Analysis among Different Species
We obtained a comprehensive understanding of the relative relationship of CsZIP proteins by constructing a phylogenetic tree using the amino acid sequences of ZIP pro teins from Arabidopsis, C. sativus, Cucumis melo, and O. sativa (Figure 4).The phylogeneti analysis revealed three major clades: Group 1, 2, and 3.In Group 1, there were seven pro teins from Arabidopsis, three from C. sativus, three from Cucumis melo, and eight from O sativa.Group 2 consisted of six proteins from Arabidopsis, three from C. sativus, three from Cucumis melo, and three from O. sativa.Group 3 included five proteins from Arabidopsis four from C. sativus, four from Cucumis melo, and five from O. sativa (Figure 4).It is worth other species.Further investigation within the Group 3 revealed a close relationship between CsZIP6, CmZIP8, AtIAR1, and OsZIP14 (Figure 4).For reference, the amino acid sequences of the ZIP proteins can be found in Dataset S1.This analysis provides valuable insights into the interrelationships of ZIP proteins in different plant species and serves as a foundation for further exploration of their biological functions.sativa.In MEGA 7.0, the neighbor-joining (NJ) method was used to construct a root-free amino acid sequence similarity tree, which was repeated 1000 times.

Chromosome Localization and Collinearity Analysis
The chromosomal distribution analysis of CsZIP genes in C. sativus revealed an uneven distribution pattern across seven chromosomes (Figure 5).Specifically, three ZIP genes (CsZIP3, 4, and 5) were located on chromosome 4, two on each of chromosome 6 and 7, respectively, and one on each of chromosome 1, 2, and 5, and none on chromosome 3 (Figure 5).sativa.In MEGA 7.0, the neighbor-joining (NJ) method was used to construct a root-free amino acid sequence similarity tree, which was repeated 1000 times.

Chromosome Localization and Collinearity Analysis
The chromosomal distribution analysis of CsZIP genes in C. sativus revealed an uneven distribution pattern across seven chromosomes (Figure 5).Specifically, three ZIP genes (CsZIP3, 4, and 5) were located on chromosome 4, two on each of chromosome 6 and 7, respectively, and one on each of chromosome 1, 2, and 5, and none on chromosome 3 (Figure 5).We further conducted a collinearity analysis of the ZIP gene family members between C. sativus and other plant species, including Arabidopsis, Cucumis melo, and O. sativa (Figure 6).Our results revealed that a total of eight genes were identified to be present in both C. sativus and Arabidopsis, suggesting a conserved relationship between these two species (Figure 6).In the case of C. sativus and Cucumis melo, all members of the ZIP gene family We further conducted a collinearity analysis of the ZIP gene family members between C. sativus and other plant species, including Arabidopsis, Cucumis melo, and O. sativa (Figure 6).Our results revealed that a total of eight genes were identified to be present in both C. sativus and Arabidopsis, suggesting a conserved relationship between these two species (Figure 6).In the case of C. sativus and Cucumis melo, all members of the ZIP gene family exhibited homologous alignment, indicating a high level of homology during the long differentiation process between these two closely related species (Figure 6).However, only two genes showed homology between C. sativus and O. sativa.We further conducted a collinearity analysis of the ZIP gene family members between C. sativus and other plant species, including Arabidopsis, Cucumis melo, and O. sativa (Figure 6).Our results revealed that a total of eight genes were identified to be present in both C. sativus and Arabidopsis, suggesting a conserved relationship between these two species (Figure 6).In the case of C. sativus and Cucumis melo, all members of the ZIP gene family exhibited homologous alignment, indicating a high level of homology during the long differentiation process between these two closely related species (Figure 6).However, only two genes showed homology between C. sativus and O. sativa.

Analysis of Cis-Acting Elements in Promoter Region of CsZIP Genes
The analysis of the 1500 bp sequence upstream of the ZIP gene in C. sativus revealed the presence of various cis-elements (Figure 7).Specifically, the promoter regions of all the genes in this family contained elements related to some of gibberellin, auxin, salicylic acid, jasmonic acid, low temperature, and drought stress.Most of the gene promoters contained hormone-related elements, indicating their potential involvement in hormone signaling pathways.Notably, gibberellin-related elements were found in the promoters of several family members, including CsZIP1, CsZIP2, CsZIP4, CsZIP6, CsZIP7, CsZIP8, and CsZIP10 (Figure 7).This suggests that these genes may play crucial roles in gibberellin-mediated

Analysis of Cis-Acting Elements in Promoter Region of CsZIP Genes
The analysis of the 1500 bp sequence upstream of the ZIP gene in C. sativus revealed the presence of various cis-elements (Figure 7).Specifically, the promoter regions of all the genes in this family contained elements related to some of gibberellin, auxin, salicylic acid, jasmonic acid, low temperature, and drought stress.Most of the gene promoters contained hormone-related elements, indicating their potential involvement in hormone signaling pathways.Notably, gibberellin-related elements were found in the promoters of several family members, including CsZIP1, CsZIP2, CsZIP4, CsZIP6, CsZIP7, CsZIP8, and CsZIP10 (Figure 7).This suggests that these genes may play crucial roles in gibberellin-mediated processes.Auxin response elements were predominantly enriched in CsZIP6, CsZIP8, and CsZIP10 gene promoters (Figure 7).This finding suggests that these specific genes may be involved in the regulation of plant growth and development through auxin signaling pathways.

Expression of CsZIP Genes in Different Tissues
This section was dedicated to exploring the functions of ZIP genes in C. sativus development by analyzing RNA-seq data derived from various tissues.The expression patterns of CsZIP gene family were examined using data from the Cucurbit Genomics Data website and NCBI SAR database, and heat maps were generated to visualize the expression levels across tissues.The results revealed that the expression of ZIP genes in C. sativus leaves, stems, and tendrils was relatively lower compared to other tissues (Figure 8a).In contrast, CsZIP7 and CsZIP10 showed the highest expression levels in roots, suggesting their possible involvement in regulating ion transport in the underground parts of C. sativus.CsZIP2, CsZIP5, and CsZIP9 were expressed in flowers, indicating they may play a role in the growth and development of flowers (Figure 8a).On the other hand, CsZIP1, CsZIP3, CsZIP4, CsZIP6, and CsZIP8 exhibited major expression in the ovary, especially CsZIP3, CsZIP4, and CsZIP8 displayed high expression levels in the unfertilized ovary (Figure 8a).These findings indicate that these five ZIP genes have regulatory functions in fruit development.

Expression of CsZIP Genes in Different Tissues
This section was dedicated to exploring the functions of ZIP genes in C. sativus development by analyzing RNA-seq data derived from various tissues.The expression patterns of CsZIP gene family were examined using data from the Cucurbit Genomics Data website and NCBI SAR database, and heat maps were generated to visualize the expression levels across tissues.The results revealed that the expression of ZIP genes in C. sativus leaves, stems, and tendrils was relatively lower compared to other tissues (Figure 8a).In contrast, CsZIP7 and CsZIP10 showed the highest expression levels in roots, suggesting their possible involvement in regulating ion transport in the underground parts of C. sativus.CsZIP2, CsZIP5, and CsZIP9 were expressed in flowers, indicating they may play a role in the growth and development of flowers (Figure 8a).On the other hand, CsZIP1, CsZIP3, CsZIP4, CsZIP6, and CsZIP8 exhibited major expression in the ovary, especially CsZIP3, CsZIP4, and CsZIP8 displayed high expression levels in the unfertilized ovary (Figure 8a).These findings indicate that these five ZIP genes have regulatory functions in fruit development.
tivus.CsZIP2, CsZIP5, and CsZIP9 were expressed in flowers, indicating they may play a role in the growth and development of flowers (Figure 8a).On the other hand, CsZIP1, CsZIP3, CsZIP4, CsZIP6, and CsZIP8 exhibited major expression in the ovary, especially CsZIP3, CsZIP4, and CsZIP8 displayed high expression levels in the unfertilized ovary (Figure 8a).These findings indicate that these five ZIP genes have regulatory functions in fruit development.To further validate the reliability of the RNA-seq results, qRT-PCR analysis of ZIP gene expression in different tissues was performed.Overall, the qRT-PCR results were consistent with the RNA-seq data, confirming the reliability of the datasets (Figure 8b).However, an inconsistency was observed in the expression of CsZIP4 between male flowers and tendrils, indicating some variations in gene expression in specific tissues (Figure 8b).Therefore, these genes are likely involved in ion transport, flower differentiation, and development, and potentially regulate fruit development.

Expression Profiles of CsZIP Genes under Abiotic and Biotic Stresses
Due to the lack of transcriptomic data on metal ion transport, and in order to enhance our understanding of the functional roles within the ZIP gene family and predict their potential applications in the future, we investigated the impact of various environmental stresses on the expression of CsZIP genes, and we assessed their comprehensive expression patterns in response to different stress conditions, including salt, downy mildew (DM, caused by Pseudoperonospora cubensis), powdery mildew (PM, caused by Podosphaera fusca), and heat, based on publicly available transcriptome data.This analysis enabled us to gain further insights into the potential roles of CsZIP genes in mediating plant response to diverse stresses.
Under salt stress, specific expression patterns of CsZIP genes were revealed through transcriptome data analysis.CsZIP4, CsZIP5, CsZIP6, and CsZIP8 genes were up-regulated in response to NaCl treatment; in contrast, CsZIP1, CsZIP3, and CsZIP10 genes showed down-regulation (Figure 9a).It is worth noting that Si treatment has been reported to enhance stress resistance and stimulate plant growth.Interestingly, when plants were solely subjected to Si treatment, most CsZIP genes exhibited up-regulation in their expression levels.However, the expression of the CsZIP1 gene was found to be down-regulated (Figure 9a).Additionally, we observed a similar down-regulation of CsZIP1 gene expression after exogenous salt and Si treatment.Therefore, it can be concluded that under salt stress conditions, most CsZIP genes tend to be up-regulated, while the CsZIP1 gene specifically demonstrates down-regulation in response to salt stress (Figure 9a).S2.
We also examined the response of CsZIP genes to heat stress (Figure 9b).At 3 h following exposure to high temperatures, the expression levels of CsZIP2, CsZIP3, CsZIP4, CsZIP5, CsZIP6, CsZIP7, CsZIP8, and CsZIP9 were up-regulated, whereas CsZIP1 was down-regulated, and similar patterns emerged at 6 h after heat stress, suggesting that these genes may play a crucial role in heat tolerance (Figure 9b).
To explore the role of CsZIPs in biotic stress resistance, we conducted an analysis of CsZIP expression using the RNA-Seq database.Following inoculation with powdery mildew (PM), we observed distinct gene expression patterns between the susceptible and resistant C. sativus lines (Figure 10a).After inoculation with PM, the expression of CsZIP3, CsZIP4, CsZIP7, and CsZIP9 was down-regulated in both the resistant and susceptible lines.In contrast, the expression of CsZIP5 was up-regulated (Figure 10a).S2.
We also examined the response of CsZIP genes to heat stress (Figure 9b).At 3 h following exposure to high temperatures, the expression levels of CsZIP2, CsZIP3, CsZIP4, CsZIP5, CsZIP6, CsZIP7, CsZIP8, and CsZIP9 were up-regulated, whereas CsZIP1 was down-regulated, and similar patterns emerged at 6 h after heat stress, suggesting that these genes may play a crucial role in heat tolerance (Figure 9b).
To explore the role of CsZIPs in biotic stress resistance, we conducted an analysis of CsZIP expression using the RNA-Seq database.Following inoculation with powdery mildew (PM), we observed distinct gene expression patterns between the susceptible and resistant C. sativus lines (Figure 10a).After inoculation with PM, the expression of CsZIP3, CsZIP4, CsZIP7, and CsZIP9 was down-regulated in both the resistant and susceptible lines.In contrast, the expression of CsZIP5 was up-regulated (Figure 10a).
These findings indicate that CsZIP genes are responsive to different environmental stresses and may play diverse roles in mediating plant responses to these stresses.Further research is needed to fully understand the specific functions of each CsZIP gene and their contributions to stress resistance in C. sativus.
To explore the role of CsZIPs in biotic stress resistance, we conducted an analysis of CsZIP expression using the RNA-Seq database.Following inoculation with powdery mildew (PM), we observed distinct gene expression patterns between the susceptible and resistant C. sativus lines (Figure 10a).After inoculation with PM, the expression of CsZIP3, CsZIP4, CsZIP7, and CsZIP9 was down-regulated in both the resistant and susceptible lines.In contrast, the expression of CsZIP5 was up-regulated (Figure 10a).S3.

Discussion
The ZIP (ZRT/IRT-like protein) gene family is involved in the absorption and transport of metal ions in plants, playing a crucial role in plant growth, development, and response to heavy metal stress [1].In this study, a comprehensive genome-wide identification of CsZIPs was performed, and a total of 10 genes were identified in C. sativus.We further investigated their gene structure, phylogenetic relationship, composition of cis-regulatory elements in promoter, chromosome localization, collinearity analysis, and expression patterns under different elemental stresses.These findings enhance our understanding of the ZIP gene family and provide bases for better elucidating the function and evolutionary relationship of CsZIPs in C. sativus.
Previous studies have reported the identification of the ZIP gene family in various plant species including A. thaliana, peanut, and Populus trichocarpa.Arabidopsis had 15 ZIP members, peanut 30, and P. trichocarpa 16 [31,47,49,50].However, in this study, only 10 CsZIP genes were identified (Figure 1).Of the 13 C. sativus germplasm resources analyzed, "Cuc80" and "PI183967" contained 9 CsZIP genes, whereas the remaining varieties harbored 10 ZIP genes (Table 2).Notably, only CsZIP10 exhibited a consistent amino acid sequence length, while the lengths of other CsZIPs varied among the different varieties (Table 2).
In the evolutionary process of plant genomes, the emergence of gene family members often accompanies gene duplication, leading to new functions.For example, most of the AhZIP genes experienced gene duplication events except AhIRT1.1/1.3,AhZIP7.2/7.8, and AhZIP3.5/3.6 [47].However, unfortunately, we did not detect any gene duplication events in the CsZIP genes in C. sativus.
The CsZIP gene promoters contain many cis-acting elements that respond to various hormones and stresses, including gibberellins, methyl jasmonate, abscisic acid, and low temperature.In S. lycopersicum, zinc deficiency tolerance is determined by auxin signaling [32].Furthermore, the response elements of three ZIP genes in sunflower are regulated by salicylic acid and methyl jasmonate under iron deficiency [33].These findings suggest that the expression of CsZIP genes can be induced by several plant hormones, such as salicylic acid, MeJA, gibberellins, and auxins.Methyl jasmonate acts as a crucial plant hormone in defense against biotic and abiotic stresses by triggering defense mechanisms and regulating growth [52].Auxin, on the other hand, controls plant development, with tryptophan being a key amino acid in the auxin synthesis pathway.As Zn participates in tryptophan synthesis, CsZIP genes might play a significant role in auxin biosynthesis [53].Being similar to auxin, gibberellins also regulate various plant growth and development processes, including seed germination and fruit development [54].Salicylic acid has been shown to greatly enhance plant stress resistance [55].Additionally, several cis-acting elements associated with biotic and abiotic stress responses have been identified in the promoter region of CsZIP genes (Figure 7).This suggests that the CsZIP gene family may adapt to different environmental stresses through hormone signals.
In general, the expression patterns of genes are correlated with their functions [51].The expression data of CsZIPs in different tissues provide valuable insights into the possible functions of the ZIP gene family.Previous studies have confirmed that ZIP genes are predominantly expressed in roots, regardless of the plant species, such as Arabidopsis, O. sativa, or barley, with many genes being induced under deficiencies of environmental zinc or other metallic elements [7,19,56,57].Existing research has indicated that within tomatoes, the SlZIP gene family comprises 15 members, with 13 expressing in the roots, exhibiting significantly high expression levels.Among these, 11 SlZIP genes are expressed in fruits, albeit with varying expression across different developmental stages, indicating the involvement of SlZIP genes in the accumulation of zinc and iron in S. lycopersicum fruits or their redistribution among different tissues [58].Similarly, CsZIP7 and CsZIP10 exhibit high expression in C. sativus roots, while CsZIP10 has higher expression levels in stems compared to other tissues (Figure 8).This suggests that CsZIP7 and CsZIP10 may play functional roles in these two tissues.Furthermore, it was observed that CsZIP2, CsZIP5, and CsZIP9 show higher expression levels in flowers, while CsZIP3, CsZIP4, and CsZIP8 exhibit the highest expression levels in fertilized ovaries.CsZIP1 and CsZIP6 are also expressed in ovaries (Figure 8).Based on this information, it can be inferred that these CsZIP genes may participate in the regulation of fruit development by regulating the accumulation of Zn and Fe or their redistribution among different tissues.
Previous studies have primarily focused on the regulatory roles of ZIP genes in plant metal ion uptake and transport.For instance, AtZIP3 is known to play a crucial role in facilitating the absorption of Zn and Fe from the soil into plant roots [8].On the other hand, AtZIP2 functions by mediating the uptake of Mn/Zn into the parenchyma cells of the xylem, thereby enabling the efficient transportation of Mn/Zn to the aboveground parts of plants [7].However, in this study, we found that CsZIP genes could respond to different environmental stresses.We investigated the expression of CsZIP genes under different environmental stresses, including powdery mildew, downy mildew, salt, and heat.The results showed differential expression of CsZIP genes in response to high temperature (Figure 9b), salt, silicon (Figure 9a), powdery mildew (Figure 10a), and downy mildew treatments (Figure 10a).These results provide valuable clues suggesting that CsZIP genes may have significant functions in multiple stress conditions.The functionality and regulatory mechanisms of the CsZIP gene family will require further validation in the future, particularly regarding their functional responses under different environmental stressors.Utilizing gene-editing technologies such as CRISPR-Cas9 to generate mutants will help explore the precise roles of CsZIP genes in plant growth, development, and stress responses.Further investigations into the regulatory mechanisms of the CsZIP gene family, encompassing transcriptional, translational, and post-transcriptional regulations, will provide a deeper understanding of these genes' functions across various biological processes.These potential future research directions will contribute to a more comprehensive elucidation of the significance of the CsZIP gene family in plants, thereby enhancing our understanding of regulatory mechanisms governing plant adaptability and stress resilience.

Conclusions
In this study, we performed a pan-genome-wide identification of the ZIP gene family in C. sativus.A total of 10 members were identified.Eleven of the thirteen accessions contained all the CsZIP genes.The ZIP gene family has three evolutionary branches and contains conserved histidine residues.The CsZIP gene promoters contained elements that responded to plant hormone signaling pathways, indicating that plant hormone signals may have an impact on CsZIP-mediated biotic and abiotic stresses.The expression patterns of CsZIPs in different tissues showed that the CsZIP gene family may be related to the growth and development of C. sativus.Finally, CsZIPs could response to different environmental stresses.In summary, this study helps to improve the current understanding of the CsZIP gene family and provides a basis for exploring their functions in C. sativus.

Figure 1 .
Figure 1.Phylogenetic tree and gene structure of ZIP family members in C. sativus.The phylogenetic tree was constructed using the neighbor-joining (NJ) method with 1000 bootstrap replicates, based on the alignment of the identified 10 ZIP proteins in C. sativus.The gene structures of the identified 10 ZIP genes in C. sativus were generated utilizing the Gene Structure Display Server v.2.0.In the structures, the green box represents the UTR, the yellow box represents the exon, and the black line represents the intron.

Figure 1 .
Figure 1.Phylogenetic tree and gene structure of ZIP family members in C. sativus.The phylogenetic tree was constructed using the neighbor-joining (NJ) method with 1000 bootstrap replicates, based on the alignment of the identified 10 ZIP proteins in C. sativus.The gene structures of the identified 10 ZIP genes in C. sativus were generated utilizing the Gene Structure Display Server v.2.0.In the structures, the green box represents the UTR, the yellow box represents the exon, and the black line represents the intron.

Figure 2 .
Figure 2. C. sativus ZIP proteins exhibit conserved motifs identified by MEME, highlighted wi colored boxes.

Figure 2 .
Figure 2. C. sativus ZIP proteins exhibit conserved motifs identified by MEME, highlighted with colored boxes.

Figure 3 .
Figure 3. Amino acid sequence alignment for ZIP proteins from Arabidopsis, C. sativus, Cucumis melo and O. sativa.The alignment of ZIP proteins was conducted via MAFFT v.5.3, employing defau settings.Black boxes indicate residues that are entirely conserved, while gray boxes highlight res dues with a high level of conservation.* represents the positions of the amino acids, which are 430 450, and 470, respectively.

Figure 3 .
Figure 3. Amino acid sequence alignment for ZIP proteins from Arabidopsis, C. sativus, Cucumis melo, and O. sativa.The alignment of ZIP proteins was conducted via MAFFT v.5.3, employing default settings.Black boxes indicate residues that are entirely conserved, while gray boxes highlight residues with a high level of conservation.* represents the positions of the amino acids, which are 430, 450, and 470, respectively.

Figure 4 .
Figure 4. Phylogenetic tree for the ZIP proteins from Arabidopsis, C. sativus, Cucumis melo, and O.sativa.In MEGA 7.0, the neighbor-joining (NJ) method was used to construct a root-free amino acid sequence similarity tree, which was repeated 1000 times.

Figure 4 .
Figure 4. Phylogenetic tree for the ZIP proteins from Arabidopsis, C. sativus, Cucumis melo, and O.sativa.In MEGA 7.0, the neighbor-joining (NJ) method was used to construct a root-free amino acid sequence similarity tree, which was repeated 1000 times.

Figure 6 .
Figure 6.Synteny analysis of ZIPs between C. sativus and other plant species (A.thaliana, Oryza sativa, and Cucumis melo): the collinear blocks are marked by gray lines, while the collinear gene pairs with ZIP genes are highlighted by red lines.

Figure 6 .
Figure 6.Synteny analysis of ZIPs between C. sativus and other plant species (A.thaliana, Oryza sativa, and Cucumis melo): the collinear blocks are marked by gray lines, while the collinear gene pairs with ZIP genes are highlighted by red lines.

Agriculture 2024 , 20 Figure 7 .
Figure 7. Predicted cis-elements in promoter regions of C. sativus ZIP genes.The promoter region was defined as a 1.5 kb sequence upstream of the translation initiation codon of the CsZIP gene.The cis-acting elements were identified utilizing the Plant CARE online tool.Various types of cis-acting elements are denoted by distinctively colored closed boxes.

Figure 7 .
Figure 7. Predicted cis-elements in promoter regions of C. sativus ZIP genes.The promoter region was defined as a 1.5 kb sequence upstream of the translation initiation codon of the CsZIP gene.The cis-acting elements were identified utilizing the Plant CARE online tool.Various types of cis-acting elements are denoted by distinctively colored closed boxes.

Figure 8 .
Figure 8. Temporal-spatial expression of C. sativus ZIP genes.(a) The heatmap showcases the expression patterns of CsZIP genes across nine distinct C. sativus tissues.The RNA-seq datasets, acquired via accession number PRJNA80169 from the Cucurbit Genomics Data website, were used.Colors on the scale signify Log 2 (FPKM) values, where blue and red represent low and high expression levels, respectively.Detailed FPKM values for CsZIP genes can be located in Table S1.(b) Validation of RNA-seq results for four CsZIP genes using qRT-PCR.The error bars represent the standard error of the mean (n = 3).FF: female flower; R: root; L: leaf; MF: male flower; O-fer: expanded fertilized ovary; S: stem; O: unexpanded ovary; O-unfer: expanded unfertilized ovary; T: tendril.

Figure 9 .
Figure 9. Expression of CsZIP genes in response to salt and hot stimuli.(a) Heatmap for displaying the expression profile of CsZIP genes in response to salt stresses.(b) The heatmap illustrates the expression patterns of CsZIP genes in response to high temperature (42 °C).RNA−seq datasets with accession numbers GSE151055 and GSE116265 were retrieved from the NCBI SAR database.The color scale is representative of Log2(FPKM) values, where green signifies low expression, red represents high expression, and black indicates no expression.The FPKM value of CsZIP genes under salt and hot treatments are listed in TableS2.

Figure 10 .
Figure 10.Expression analysis of CsZIPs under biotic stresses: The transcriptional levels of CsZIP

Figure 9 .
Figure 9. Expression of CsZIP genes in response to salt and hot stimuli.(a) Heatmap for displaying the expression profile of CsZIP genes in response to salt stresses.(b) The heatmap illustrates the expression patterns of CsZIP genes in response to high temperature (42 • C).RNA-seq datasets with accession numbers GSE151055 and GSE116265 were retrieved from the NCBI SAR database.The color scale is representative of Log 2 (FPKM) values, where green signifies low expression, red represents high expression, and black indicates no expression.The FPKM value of CsZIP genes under salt and hot treatments are listed in TableS2.

Figure 10 . 8 Figure 10 .
Figure 10.Expression analysis of CsZIPs under biotic stresses: The transcriptional levels of CsZIP genes after infection with powdery mildew (PM) for 48 h (a) and with downy mildew (DM) for 1−8 Figure 10.Expression analysis of CsZIPs under biotic stresses: The transcriptional levels of CsZIP genes after infection with powdery mildew (PM) for 48 h (a) and with downy mildew (DM) for 1-8 days post-inoculation (b) are shown on the heatmaps.The color scale shows increasing expression levels from green to red.ID, PM-inoculated susceptible C. sativus line D8 leaves; NID, non-inoculated D8 leaves; IS, PM-inoculated resistant C. sativus line SSL508-28 leaves; NIS, non-inoculated SSL508-28 leaves; CT, without inoculation; DPI, days post-inoculation.The FPKM value of CsZIP genes under powdery mildew and downy mildew are listed in TableS3.

Table 1 .
The origins of the different C. sativus accessions.

Table 2 .
The protein lengths of CsZIPs in different C. sativus accessions.