The melon genome sequence DHL92 v4.0 was downloaded from the CuGenDB database (http://cucurbitgenomics.org/) to identify C3H genes (Garcia-Mas et al., 2012). Using the Hidden Markov Model (HMM) profile PF00642 (https://www.ebi.ac.uk/interpro/entry/pfam/#table), a genome-wide search was conducted on the melon genome protein data using the HMMER tool, retaining genes with an e-value of less than or equal to e⁻⁵ (Potter et al., 2018). Additionally, 50 Arabidopsis C3H protein sequences were downloaded from the UniProt database (https://www.uniprot.org/) (UniProt Consortium, 2021) and subjected to a BLAST search against the Arabidopsis C3H protein sequences with an e-value of less than or equal to e⁻⁵ (Ye et al., 2006). After removing redundant sequences, the remaining dataset could be used for further analysis. Ultimately, 38 C3H genes were identified in the melon genome, and their chromosomal distribution was mapped using the TBtools (V2.131) (Chen et al., 2020b). Following the same methodology, 35 and 38 C3H members were identified in cucumber and watermelon, respectively. Protein physicochemical properties were analyzed using ExPASy (http://web.expasy.org/protparam/) (Wilkins et al., 1999), and subcellular localization was predicted using WoLF PSORT II (https://www.genscript.com/wolf-psort.html?src=leftbar) (Horton et al., 2007). Finally, a phylogenetic tree of the melon C3H gene family was constructed following a previous study (Deng et al., 2023). This process involved using MEGA 11 software to perform a ClustalW multiple sequence alignment of the CmC3H and AtC3H sequences and constructing the phylogenetic tree with the Neighbor-Joining (NJ) method, with 1,000 bootstrap replications, using the Poisson correction model and the pairwise deletion option (Deng et al., 2023).

Conserved motifs were identified in the 38 CmC3H protein sequences using the MEME tool (http://meme-suite.org/tools/meme), specifying a maximum of 10 motifs with default parameters (Bailey et al., 2009). The positions and numbers of C3H domains within the CmC3H protein sequences were analyzed using the NCBI CDD online tool (https://www.ncbi.nlm.nih.gov/cdd/) (Marchler-Bauer et al., 2015). Exon and intron positions and numbers within the CmC3H genes were identified based on melon genome data. The phylogenetic tree, conserved motifs, domains, and gene structures of CmC3H family members were subsequently clustered and visualized through TBtools (V2.131).

Gene duplication, including segmental and tandem duplications, among the 38 CmC3H members was analyzed using the MCScanX tool (Wang et al., 2012). Constructing chromosome homology maps using SRplot online (https://www.bioinformatics.com.cn/?keywords=circos) Rcircos circle diagram tool (Tang et al., 2023a). Homologous C3H gene pairs between melon and Arabidopsis, rice, cucumber, and watermelon were identified. The Ka (nonsynonymous substitution rate), Ks (synonymous substitution rate), and Ka/Ks ratios of all duplicated genes were computed through the KaKs_Calculator tool(Version 3.0) (Zhang et al., 2006).

Promoter sequences (2,000 bp upstream) of the 38 CmC3H genes were extracted and annotated to identify cis-elements through the PlantCARE online tool (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) (Lescot et al., 2002). The positional distribution of cis-elements related to abiotic and biotic stresses, phytohormone responsiveness, and plant growth and development was mapped according to previous studies (Zhang et al., 2024).

The expression patterns of the CmC3H family members were analyzed by retrieving FPKM (Fragments Per Kilobase of exon model per Million mapped fragments) values from the melon transcriptome database (https://melonet-db.dna.affrc.go.jp/ap/top) across eleven distinct tissues: dry seeds, root, middle stem, upper stem, leaves, tendril, petal, stigma, ovary at 4 Days After Flowering (DAF4), fruit flesh at 50 Days After Flowering (DAF50), and fruit epicarp at 50 Days After Flowering (DAF50) (Yu et al., 2023). The expression pattern of the CmC3H family was visualized using TBtools (V2.131). In this figure, the expression levels are represented by color intensity, with darker colors corresponding to higher expression levels.

The protein sequences of CmC3H family members were uploaded to the STRING database (https://string-db.org/) for node comparison, with interactions among CmC3H members predicted based on Arabidopsis protein interaction data (Szklarczyk et al., 2023). The binding motif profile for the Arabidopsis C3H12 transcription factor (MA1756.2) was obtained from the JASPAR Plantae database (https://jaspar.elixir.no/search?q=&collection=CORE&tax_group=plants) (Castro-Mondragon et al., 2022). Subsequently, the 2,000 bp promoter sequences of all genes in the melon genome were extracted and analyzed using the motif FIMO tool (https://meme-suite.org/meme/) to detect genes potentially binding with the C3H transcription factor. Finally, target gene domain prediction was performed using the PFAM database (Mistry et al., 2021), and KEGG (Kyoto Encyclopedia of Genes and Genomes) and GO (Gene Ontology) enrichment analyses were conducted on the target genes using OmicShare Tools (https://www.omicshare.com/tools).

The seeds of the melon variety ‘Super Sweet White Sugar Jar’ were soaked, germinated, and then sown. The plants were grown in a greenhouse under a 16-hour light/8-hour dark photoperiod, with temperatures maintained at 28°C during the day and 18°C at night, until they reached the three-leaf stage. Fusarium oxysporum was isolated from wilted melon plants, and a spore suspension with a concentration of 1×10⁸ spores/mL was prepared. The plants were inoculated via root irrigation, applying 10 mL of spore suspension per plant. Root, middle stem, and first true leaf samples were collected at 0 hours (CK), 12 hours, 24 hours, and 48 hours post-inoculation.

Drought stress treatment was applied by placing the three-leaf stage melon plants in a 15% PEG6000 solution, and leaf samples were collected at 0 hours (CK), 12 hours, 24 hours, and 48 hours. For heavy metal lead stress treatment, Pb (NO₃)₂ was dissolved in distilled water and applied to the pots, bringing the Pb ion concentration in the soil to 2000 mg/kg. Leaf samples were collected at 0 hours (CK), 12 hours, 24 hours, and 48 hours. All experiments were conducted with three biological replicates, and the collected samples were immediately placed in liquid nitrogen and stored at -80°C for further analysis.

The expression levels of 10 CmC3H genes at four time points (0 hours, 12 hours, 24 hours, and 48 hours) under drought stress (leaves; three biological replicates), heavy metal lead stress (leaves; three biological replicates), and Fusarium wilt infection (roots, stems, and leaves; three biological replicates) were analyzed using quantitative real-time PCR (qRT-PCR).The qRT-PCR primers for the selected CmC3H genes were designed using Primer Premier 5 (Singh et al., 1998). Total RNA was extracted from all samples using the Spectrum™ Plant Total RNA Kit (Merck KGaA), with the quality and concentration assessed using a Nanodrop 2000. First-strand cDNA was synthesized using the SweScript All-in-One First Strand cDNA Synthesis kit (TRANS, G3337). The qPCR reactions were performed using a CFX96 Real-Time PCR Detection System (Bio-Rad, Hercules, CA) with 2×SYBR Green qPCR Master Mix (no ROX) (TRANS, G3320). The cycling conditions were set as follows: 95°C for 30 seconds of pre-denaturation, followed by 40 cycles of 95°C for 15 seconds and 60°C for 30 seconds. The LOC103490203 gene was used as the internal reference gene in melon, and the relative expression levels of the target genes were calculated using the 2^−ΔΔCT method (Livak and Schmittgen, 2001). Differences among treatments were assessed by the least significance difference using one-way analysis in SPSS 19.0 (SPSS China, Beijing, China).

The subcellular localization of the target gene was predicted using the WOLF PSORT website. The coding sequence of CmC3H21 was cloned into the pBI121-GFP vector to construct the pBI121-CmC3H21-GFP recombinant plasmid. Subsequently, the pBI121-CmC3H21-GFP plasmid and mCherry (a nuclear marker) were introduced into Agrobacterium tumefaciens strain GV3101. The Agrobacterium culture was activated and grown until the OD600 reached 1.5, followed by centrifugation to remove the supernatant. The pellet was resuspended in an infiltration buffer (10 mM MES, 10 mM MgCl₂, and 200 µM acetosyringone) and vortexed to adjust the OD600 to 1.0. The resulting suspension was infiltrated into the abaxial side of leaves from approximately five-week-old tobacco (Nicotiana benthamiana) seedlings. After infiltration, the plants were kept in the dark at 22°C for 48 hours. The infiltrated leaves were then excised and imaged using a confocal laser scanning microscope (TCS SP8, Leica) to visualize the subcellular localization of the fluorescently tagged proteins.

A total of 38 C3H genes were identified in the melon genome, and these are unevenly distributed across 12 chromosomes ( Figure 1 ). Specifically, the genes are distributed as follows: 5, 6, 2, 5, 1, 3, 4, 5, 1, 1, 3, and 2 on chromosomes Chr01 to Chr12, respectively. These genes were renamed CmC3H1 through CmC3H38 based on their chromosomal locations. The physicochemical properties of the CmC3H family members were further analyzed ( Supplementary Table S1 ) (“aa” denotes amino acids, and “Da” refers to Daltons). The analysis indicated that the number of amino acids in CmC3H members ranges from 202 aa (CmC3H1) to 1034 aa (CmC3H25), with molecular weights varying from 22128.07 Da (CmC3H1) to 114623.34 Da (CmC3H25). The Instability Index ranges from 28.7 (CmC3H16) to 82.26 (CmC3H29), while the Aliphatic Index varies from 36.53 (CmC3H31) to 78.96 (CmC3H16). All CmC3H members are hydrophilic proteins. The theoretical isoelectric points (pI) range from 4.92 to 9.65, with 17 members having a pI below 7 and 21 members having a pI above 7. Subcellular localization analysis revealed that three genes, CmC3H9, CmC3H20, and CmC3H31, lack a start codon, preventing the prediction of their subcellular localization. Among the remaining members, 31 are predicted to localize in the nucleus, 3 in the chloroplast, and 1 in the cytoplasm.

A Neighbor-Joining phylogenetic tree of C3H family members from A. thaliana, melon (C. melo), cucumber (C. sativus), and watermelon (C. lanatus) was constructed using MEGA 11. The resulting tree was categorized into four groups: Group I, Group II, Group III, and Group IV ( Figure 2 ; Supplementary Table S2 ).Groups I, II, III, and IV contained 42, 33, 26, and 60 members, respectively. The CmC3H genes were distributed across these groups, with 10, 9, 5, and 14 genes in Groups I to IV, respectively, accounting for 26.3%, 23.7%, 13.2%, and 36.8% of the total. The C3H family consisted of 38 members, while cucumber and watermelon had 35 and 38 C3H genes, respectively, indicating similar gene numbers among the three species. Clustering analysis revealed that C3H genes in melon, cucumber, and watermelon were highly conserved, suggesting strong evolutionary conservation among these closely related species. Analysis of motifs and domains in the CmC3H family revealed that members within the same subgroup shared similar motif types and domain structures. These findings support the phylogenetic subgroup classification based on evolutionary analysis and further validate the rationale behind subgroup categorization in the evolutionary tree (Liu et al., 2024).

A total of 10 motifs were annotated in the protein sequences of CmC3H family members. A Neighbor-Joining phylogenetic tree was further constructed specifically for CmC3H family members ( Figure 3A ). The analysis indicated that, unlike the broader phylogenetic distribution shown in Figure 2 , the CmC3H members within the same group were not strictly clustered within the same major branches. Additionally, significant differences in the types and numbers of conserved protein motifs were observed among CmC3H members across the four groups ( Figure 3B ). As illustrated in Figure 3B , the protein motifs in the 10 CmC3H members of Group I were primarily motifs 1, 2, 3, and 10. The 9 CmC3H members of Group II mainly contained motifs 1, 4, 6, and 7. The 5 CmC3H members in Group III predominantly featured motifs 5 and 6. The 14 CmC3H members in Group IV primarily exhibited motifs 1 and 4. Moreover, according to the protein domain annotation results, each CmC3H family member contained at least one C3H domain ( Figure 3C ). In Group III, four CmC3H members contained five C3H domains situated at different positions within their protein sequences, and one member contained six C3H domains. For the other members, most of the C3H domains were distributed across one to three locations.

The gene structure of CmC3H family members is highly diverse, with the number of exons ranging from 1 to 16 and the number of introns ranging from 0 to 15 ( Figure 3D ). Significant differences were observed in the number of exons and introns among CmC3H members across the four groups, and also within members of the same group. In Group I, eight members had fewer than five exons, and two members had more than ten exons. In Group II, eight members had fewer than ten exons, and one member had more than ten exons. In Group III, one member had six exons, three members had seven exons, and one member had twelve exons. In Group IV, all 14 members had fewer than ten exons. Overall, the diversity in protein motifs and gene structures suggests that CmC3H family members may have diverse functions in regulating melon growth and development.

A pair of segmental duplication genes was identified within the CmC3H family (CmC3H31/CmC3H34) ( Figure 4A ). Additionally, the C3H family members in melon were identified to have 20, 7, 39, and 38 homologous gene pairs with A. thaliana, rice, cucumber, and watermelon, respectively ( Figure 4B ; Supplementary Table S3 ). Furthermore, the Ka/Ks values for both segmental duplication gene pairs and homologous gene pairs were less than 1, except for 6 homologous gene pairs between melon and rice, for which Ka/Ks values were not calculable ( Supplementary Table S3 ). These results indicate that C3H genes in dicotyledonous plants are likely to be highly conserved, whereas the conservation between monocots and dicots may be lower. Notably, the C3H homologous gene pairs between melon and cucumber, as well as between melon and watermelon, primarily exhibit a 1:1 distribution, indicating a high level of conservation of C3H family members across these three species during evolution.

Potential protein-protein interactions among CmC3H family members were predicted using the Arabidopsis database in the STRING tool (https://cn.string-db.org/) ( Figures 5A, B ; Supplementary Table S6 ). Potential interactions were identified among 29 CmC3H members, connected by 76 edges, forming a complex interaction network. For example, CmC3H26 potentially interacts with CmC3H27, CmC3H30, and other members. Conversely, some members, such as CmC3H28, show potential interaction with only a single member, CmC3H36. Notably, CmC3H13 was predicted to be a core gene, interacting with 13 other members. GO enrichment analysis revealed that CmC3H family members are involved in various protein functions, including nucleus (GO:0005634), ribonucleoprotein complex (GO:1990904), DNA binding (GO:0003677), metal ion binding (GO:0046872), nucleic acid binding (GO:0003676), and RNA processing (GO:0006396).

Using the binding motif profile of the Arabidopsis C3H12 transcription factor (MA1756.2), 3,243 target genes were identified in the melon genome, all of which matched the TGTCAGA sequence type ( Figure 5C ; Supplementary Table S7 ). GO enrichment analysis of these target genes ( Figure 5D ) indicated that, in the biological process category, most target genes were enriched in proteolysis (GO:0006508), regulation of DNA-templated transcription (GO:0006355), and protein phosphorylation. Five protein functional categories in the biological process category, including proteolysis (GO:0006508), regulation of DNA-templated transcription (GO:0006355), protein phosphorylation (GO:0006468), transmembrane transport (GO:0055085), and carbohydrate metabolic process (GO:0005975), were enriched with more than 40 target genes, based on the number of target genes enriched in Gene Ontology (GO) terms ( Supplementary Table S8 ). In the cellular component category, the target genes were primarily enriched in the nucleus (GO:0005634), cytoplasm (GO:0005737), and membrane (GO:0016020). Overall, the potential interactions among CmC3H members suggest a coordinated regulatory network in melon, and the target gene analysis indicates that CmC3H genes may regulate a wide range of genes involved in various growth and developmental processes.

A total of 102 types of cis-elements were identified in the CmC3H family members. In addition to numerous light-responsive elements (such as G-box, MRE, and ATCT-motif), three major categories of cis-elements were classified: hormone regulation, stress response, and growth and development ( Figure 6A ; Supplementary Table S4 ). Hormone-regulatory cis-elements include ten types: TGA-element, TATC-box, TCA-element, ABRE, AuxRR-core, CGTCA-motif, TGACG-motif, GARE-motif, P-box, and TGA-box. Stress-responsive cis-elements include six types: TC-rich repeats, LTR, ARE, GC-motif, MBS, and WUN-motif. Growth and development-related cis-elements comprise nine types: MSA-like, circadian, RY-element, CAT-box, motif I, GCN4_motif, HD-Zip 1, AACA_motif, and CellCycle-1b.

Furthermore, the total number of hormone regulation, stress response, and growth and development-related cis-elements within the CmC3H family members was quantified. The CGTCA-motif (cis-element involved in MeJA-responsiveness), TGACG-motif (cis-element involved in MeJA-responsiveness), ARE (cis-element essential for anaerobic induction), and ABRE (cis-element involved in abscisic acid responsiveness) were particularly abundant, with 54, 54, 82, and 110 occurrences, respectively ( Figure 6B ). Overall, the presence of these cis-elements suggests that CmC3H family members may play a significant role in various physiological processes in melon, contributing to the normal growth and development of tissues under diverse conditions.

The FPKM values for 36 CmC3H family members were obtained across eleven melon tissues: dry seeds, root, middle stem, upside stem, leaves, tendril,petal, stigma, ovary at 4 Days After Flowering (DAF4), fruit flesh at 50 DaysAfter Flowering (DAF50), and fruit epicarp at 50 Days After Flowering (DAF50). A heatmap was generated to visualize their expression patterns ( Figure 7 ; Supplementary Table S5 ). The results indicate that the expression levels of 13 genes (CmC3H7, CmC3H9, CmC3H16, CmC3H18, CmC3H19, CmC3H22, CmC3H23, CmC3H25, CmC3H26, CmC3H30, CmC3H31, CmC3H34, and CmC3H37) show significant expression in dry seeds. The expression levels of seven genes (CmC3H8, CmC3H10, CmC3H11, CmC3H12, CmC3H17, CmC3H28, and CmC3H38) are significantly expressed in root. The expression levels of twelve genes (CmC3H3, CmC3H8, CmC3H11, CmC3H12, CmC3H13, CmC3H15, CmC3H17, CmC3H22, CmC3H28, CmC3H29, CmC3H32, and CmC3H38) are significantly expressed in middle stem. The expression levels of five genes (CmC3H1, CmC3H2, CmC3H9, CmC3H29, and CmC3H36) are significantly expressed in upside stem.Only CmC3H1 shows significant expression in leaves. The expression levels of three genes (CmC3H1, CmC3H4, and CmC3H32) are significantly expressed in tendril. The expression levels of three genes (CmC3H4, CmC3H14, and CmC3H27)are significantly expressed in petal. The expression levels of eight genes (CmC3H4, CmC3H13, CmC3H17, CmC3H21, CmC3H28, CmC3H32, CmC3H36, and CmC3H38) are significantly expressed in stigma. The expression levels of eight genes (CmC3H2, CmC3H5, CmC3H10, CmC3H14, CmC3H15, CmC3H22, CmC3H27, and CmC3H29) show significant expression in ovary at DAF4. The expression levels of five genes (CmC3H16, CmC3H24, CmC3H30, CmC3H33, and CmC3H35) are significantly expressed in fruit flesh at DAF50. The expression levels of five genes (CmC3H5, CmC3H13, CmC3H21, CmC3H24, and CmC3H34) are significantly expressed in fruit epicarp at DAF50. Notably, several genes, such as CmC3H4, CmC3H11, CmC3H17, and CmC3H29, are significantly expressed in multiple tissues. Overall, CmC3H family members exhibit distinct tissue-specific expression patterns in melon.

The expression levels of 10 CmC3H genes in melon leaves were measured over time under drought stress treatment ( Figure 8A ; Supplementary Table S9 ). The results showed that the expression levels of six genes (CmC3H4, CmC3H7, CmC3H13, CmC3H24, CmC3H33, and CmC3H38) exhibited significant changes at 48 hours, with expression levels ranging from 2 to 5 times those at 0 hours. Notably, the expression levels of CmC3H11 and CmC3H21 increased over time, particularly at 48 hours, where their expression levels were over 50 times higher than at 0 hours. In contrast, following lead stress treatment, the expression levels of the 10 CmC3H genes in melon leaves exhibited relatively minor changes over time, showing a general trend of decreasing expression, indicating that expression was suppressed ( Figure 8B ; Supplementary Table S9 ). Expression changes of the 10 CmC3H genes were further examined in the roots, stems, and leaves of melon under Fusarium wilt treatment ( Figure 8C ; Supplementary Table S9 ). The results showed that the expression levels of five genes (CmC3H4, CmC3H15, CmC3H33, CmC3H21, and CmC3H11) increased significantly in roots at 12 and 24 hours, particularly CmC3H4, whose expression increased by approximately 348 and 126 times at 12 and 24 hours, respectively, compared to 0 hours. In the stems, the expression levels of eight genes (CmC3H7, CmC3H35, CmC3H38, CmC3H33, CmC3H21, CmC3H11, CmC3H13, and CmC3H24) increased significantly at 24 hours, with CmC3H24 and CmC3H33 increasing approximately 669 and 153 times, respectively, compared to 0 hours. Additionally, CmC3H33 expression at 48 hours increased approximately 696 times compared to 0 hours. In the leaves, the expression levels of nine genes(CmC3H7, CmC3H35, CmC3H38, CmC3H15, CmC3H33, CmC3H21, CmC3H11, CmC3H13, and CmC3H24) significantly increased at 24 hours following Fusarium wilt treatment.

Based on the aforementioned results, we selected CmC3H21 for further subcellular localization experiments. According to bioinformatics predictions, the CmC3H21 protein is likely localized in the nucleus ( Supplementary Table S1 ). To validate this prediction, we employed the Agrobacterium infiltration method to introduce the protein fusion into tobacco leaves to observe the subcellular localization of CmC3H21. The results showed that CmC3H21-GFP was co-expressed with mCherry in the nucleus( Figure 9 ), indicating that CmC3H21 is expressed in the nucleus.