Genome-Wide Identification of the SUN Gene Family in Melon (Cucumis melo) and Functional Characterization of Two CmSUN Genes in Regulating Fruit Shape Variation

Melon (Cucumis melo) is an important economic crop cultivated worldwide. A unique SUN gene family plays a crucial role in regulating plant growth and fruit development, but many SUN family genes and their function have not been well-characterized in melon. In the present study, we performed genome-wide identification and bioinformatics analysis and identified 24 CmSUN family genes that contain integrated and conserved IQ67 domain in the melon genome. Transcriptome data analysis and qRT-PCR results showed that most CmSUNs are specifically enriched in melon reproductive organs, such as young flowers and ovaries. Through genetic transformation in melons, we found that overexpression of CmSUN23-24 and CmSUN25-26-27c led to an increased fruit shape index, suggesting that they act as essential regulators in melon fruit shape variation. Subcellular localization revealed that the CmSUN23-24 protein is located in the cytoplasmic membrane. A direct interaction between CmSUN23-24 and a Calmodulin protein CmCaM5 was found by yeast two-hybrid assay, which indicated their participation in the calcium signal transduction pathway in regulating plant growth. These findings revealed the molecular characteristics, expression profile, and functional pattern of the CmSUN genes, and may provide the theoretical basis for the genetic improvement of melon fruit breeding.


Introduction
Melon (Cucumis melo L.) is a globally cultivated horticulture crop, bearing sweet and pleasant fruit with high nutritional value [1][2][3]. The long-term domestication process and wide distribution have produced multiple melon cultivars with diversified fruit traits, especially the fruit shape and size [4]. Fruit shape index (FSI) refers to the ratio of the fruit's longitudinal diameter to the transverse diameter, and is an important agronomic trait that affects melon consumption [5]. Melon FSI can vary between round, oval, and long, and has always been used as a fruit quality screening standard to classify different market groups and satisfy consumer preferences [6]. In melon, the molecular mechanism underlying the outstanding diversity of FSI needs to be further studied [3]. The establishment of fruit shape is mediated by cell differentiation and cell expansion, which are caused by multiple endogenous gene activity [7]. Considerable research on the tomato has found that the SUN gene is one of the pivotal regulators in fruit shape regulation [8,9]. Overexpression of SlSUN/SlIQD12leads to long slender fruits with an increased number of vertical cells and a reduced number of horizontal cells, and SlSUNs regulate the expression of genes involved in cell division, cell wall development, and the auxin pathway [10,11].
SUN genes encode the IQD protein, a plant-specific calmodulin-binding protein that consists of 67 amino acid residues, containing three copies of the IQ motif and separated in precise spacing by short sequences of 11 and 15 amino acid residues. IQ67 domain

CmSUN Family Gene Identification and Sequence Analyses
A total of 24 CmSUN members were identified by searching the Hidden Markov model PF00612 of the IQ motifs and blasting probe sequences in the melon protein database, including the 21 CmSUNs consistent with the previous study [30,34]. In the CmSUN family, CmSUN9-10a is the shortest protein consisting of 261 amino acids (aa), and the longest protein, CmSUN32b, is 845 aa in length. Physical and chemical analyses showed that the molecular weights of the corresponding proteins range from 29.9 to 93.4 k Dalton (kD), and the theoretical isoelectric points range from 5.59 (CmSUN32b) to 10.87 (CmSUN13-14a) ( Table 1). The average theoretical isoelectric point of 24 CmSUN proteins was 9.94, indicating that they contain many basic amino acids.

Expression Profiling of CmSUN Genes
The heat map of CmSUN expression was generated according to the transcriptomic analyses in different melon tissues ( Figure 5A). The significant differences in relative expression in the ten different tissues are indicated by salient letter notation. The relatively high transcripts of the CmSUNs accumulated in melon stem, ovary, and flower. In the late development stages of the fruit, such as the growing stage (18 days after pollination, DAP), ripening stage (36 DAP), climacteric stage (determined according to breathalyzer), and post-climacteric stage (48 h after climacteric stage), the CmSUNs displayed low expression levels. We further verified the expression patterns of CmSUN genes by performing qRT-PCR in different melon tissues. The qRT-PCR results were consistent with the transcriptome analyses ( Figure 5B). CmSUN25-26-27a has the highest expression in female flowers, and may have an active role in regulating gynoecium development; CmSUN13-14a and CmSUN25-26-27c are highly expressed in male flowers; CmSUN1-2b, CmSUN3, CmSUN17-18a, CmSUN23-24, and CmSUN30-31 showed the highest expression in the ovary compared to the other tissues, suggesting their potential role in fruit development regulation. . Phylogenetic tree analyses of SUN proteins from Arabidopsis, Solanum Lycopersicon, Cucumis sativus, and Cucumis melo. The blue represents AtSUNs, the green represents CsSUNs, the yellow represents CmSUNs, and the red represents SlSUNs. 'I~V' indicates different subfamily of SUN members in four species. The asterisk indicates the functionally studied CmSUN genes.

Expression Profiling of CmSUN Genes
The heat map of CmSUN expression was generated according to the transcriptomic analyses in different melon tissues ( Figure 5A). The significant differences in relative expression in the ten different tissues are indicated by salient letter notation. The relatively high transcripts of the CmSUNs accumulated in melon stem, ovary, and flower. In the late development stages of the fruit, such as the growing stage (18 days after pollination, DAP), ripening stage (36 DAP), climacteric stage (determined according to breathalyzer), and postclimacteric stage (48 h after climacteric stage), the CmSUNs displayed low expression levels. We further verified the expression patterns of CmSUN genes by performing qRT-PCR in different melon tissues. The qRT-PCR results were consistent with the transcriptome analyses ( Figure 5B). CmSUN25-26-27a has the highest expression in female flowers, and may have an active role in regulating gynoecium development; CmSUN13-14a and CmSUN25-26-27c are highly expressed in male flowers; CmSUN1-2b, CmSUN3, CmSUN17-18a, CmSUN23-24, and CmSUN30-31 showed the highest expression in the ovary compared to the other tissues, suggesting their potential role in fruit development regulation.

Overexpression of CmSUN23-24 and CmSUN25-26-27c Resulted in Melon Fruit Shape Variation
CmSUN23-24 and CmSUN25-26-27c were presumed as candidate genes for melon fruit shape QTL [37]. To verify the role of CmSUN23-24 and CmSUN25-26-27c in fruit regulation, we facilitated the transformation of the genes driven by the 35S promoter into the melon. We obtained six CmSUN25-26-27c-overexpressed transgenic lines, and two representative phenotypic lines were chosen for further characterization. Compared to the wild type plants, the vertical diameter of the CmSUN25-26-27c-Oe mature fruits was significantly increased, while the horizontal diameter showed no difference ( Figure 6A-C). Thus, the melon fruit shape index is more enlarged in the transgenic lines than in WT ( Figure 6D). In CmSUN25-26-27c-Oe lines, the fruit shape index was about 1.23 (Oe-L1, n > 20) and 1.21 (Oe-L2, n > 20), and the average FSI 1.22 was 11.9% larger than that in WT (1.09, n > 100). We measured the vertical and horizontal diameter of the transgenic fruits at DAP 1~7, which was the crucial time for determining the melon fruit shape. The data showed a similar fruit enlargement curve in the WT and transgenic lines ( Figure 7A-C). CmSUN25-26-27c-Oe lines had a higher FSI at early fruit elongation stages than WT, particularly in the fruits at 1 DAP and 3 DAP ( Figure 7A-C). Then we examined CmSUN25-26-27c expression in the ovaries at 1 DAP and found twofold elevated expression in the overexpression lines ( Figure 7D). In the ovaries at 3 DAP, CmSUN25-26-27c expression was increased by about 7.2 and 3.3 times in Oe-L1 and Oe-L2 lines compared to the WT, respectively ( Figure 7E). A scatterplot analysis was performed to verify the relevant between FSI and CmSUN25-26-27c expression ( Figure 7F). The correlation coefficient indicates the significant relevance (R 2 = 0.76) between gene expression and FSI ( Figure 7F). In the transcriptomic analyses, CmSUN25-26-27c transcripts were also highly accumulated

Overexpression of CmSUN23-24 and CmSUN25-26-27c Resulted in Melon Fruit Shape Variation
CmSUN23-24 and CmSUN25-26-27c were presumed as candidate genes for melon fruit shape QTL [37]. To verify the role of CmSUN23-24 and CmSUN25-26-27c in fruit regulation, we facilitated the transformation of the genes driven by the 35S promoter into the melon. We obtained six CmSUN25-26-27c-overexpressed transgenic lines, and two representative phenotypic lines were chosen for further characterization. Compared to the wild type plants, the vertical diameter of the CmSUN25-26-27c-Oe mature fruits was significantly increased, while the horizontal diameter showed no difference ( Figure 6A-C). Thus, the melon fruit shape index is more enlarged in the transgenic lines than in WT ( Figure 6D). In CmSUN25-26-27c-Oe lines, the fruit shape index was about 1.23 (Oe-L1, n > 20) and 1.21 (Oe-L2, n > 20), and the average FSI 1.22 was 11.9% larger than that in WT (1.09, n > 100). We measured the vertical and horizontal diameter of the transgenic fruits at DAP 1~7, which was the crucial time for determining the melon fruit shape. The data showed a similar fruit enlargement curve in the WT and transgenic lines ( Figure 7A-C). CmSUN25-26-27c-Oe lines had a higher FSI at early fruit elongation stages than WT, particularly in the fruits at 1 DAP and 3 DAP ( Figure 7A-C). Then we examined CmSUN25-26-27c expression in the ovaries at 1 DAP and found twofold elevated expression in the overexpression lines ( Figure 7D). In the ovaries at 3 DAP, CmSUN25-26-27c expression was increased by about 7.2 and 3.3 times in Oe-L1 and Oe-L2 lines compared to the WT, respectively ( Figure 7E). A scatterplot analysis was performed to verify the relevant between FSI and CmSUN25-26-27c expression ( Figure 7F). The correlation coefficient indicates the significant relevance (R 2 = 0.76) between gene expression and FSI ( Figure 7F). In the transcriptomic analyses, CmSUN25-26-27c transcripts were also highly accumulated in the male flower ( Figure 7G). The phenotypic analyses suggested that the CmSUN25-26-27c gene positively regulated melon fruit elongation and functions in the early developmental phase.
in the male flower ( Figure 7G). The phenotypic analyses suggested that the CmSUN25-26-27c gene positively regulated melon fruit elongation and functions in the early developmental phase.      In CmSUN23-24-Oe lines, the altered vertical diameter and unchanged horizontal diameter resulted in the increased fruit shape index ( Figure 8A-D). The average FSI (1.18, n > 40) of the mature fruits in CmSUN23-24-Oe lines was 8.3% larger than in WT (1.09, n > 100) ( Figure 8D). CmSUN23-24-Oe lines also had increased vertical diameter and FSI at early developmental stages ( Figure 9A-C). CmSUN23-24 expression was dramatically increased at 1 DAP and 3 DAP in ovaries in the severe line ( Figure 9D,E). The CmSUN23-24 expression showed a weak relevance (R 2 = 0.31) with the FSI (Figure 9F). The transcriptomic analyses in different melon tissues showed that CmSUN23-24 was highly accumulated in female flowers and ovary ( Figure 9G). The phenotype of CmSUN23-24-Oe lines is similar to CmSUN25-26-27c-Oe lines, indicating their redundant function in mediating melon fruit size development.
In CmSUN23-24-Oe lines, the altered vertical diameter and unchanged horizontal diameter resulted in the increased fruit shape index ( Figure 8A-D). The average FSI (1.18, n > 40) of the mature fruits in CmSUN23-24-Oe lines was 8.3% larger than in WT (1.09, n > 100) ( Figure 8D). CmSUN23-24-Oe lines also had increased vertical diameter and FSI at early developmental stages ( Figure 9A-C). CmSUN23-24 expression was dramatically increased at 1 DAP and 3 DAP in ovaries in the severe line ( Figure 9D,E). The CmSUN23-24 expression showed a weak relevance (R 2 = 0.31) with the FSI ( Figure 9F). The transcriptomic analyses in different melon tissues showed that CmSUN23-24 was highly accumulated in female flowers and ovary ( Figure 9G). The phenotype of CmSUN23-24-Oe lines is similar to CmSUN25-26-27c-Oe lines, indicating their redundant function in mediating melon fruit size development.

Yeast Two-Hybrid and Subcellular Localization
The transcriptional activation activity of CmSUN23-24 and CmSUN25-26-27c proteins were examined. We found that CmSUN23-24 and CmSUN25-26-27c did not contain transcriptional activation activity ( Figure 10A). CmCaM5 and CmCmL11 are homologous proteins of AtCaM1, which can interact with several IQD proteins. The Y2H result showed

Yeast Two-Hybrid and Subcellular Localization
The transcriptional activation activity of CmSUN23-24 and CmSUN25-26-27c proteins were examined. We found that CmSUN23-24 and CmSUN25-26-27c did not contain transcriptional activation activity ( Figure 10A). CmCaM5 and CmCmL11 are homologous proteins of AtCaM1, which can interact with several IQD proteins. The Y2H result showed that CmSUN23-24 had a direct interaction with CmCaM5 protein ( Figure 10B). However, there was no interaction between CmSUN25-26-27c, CmCaM5 and CmCmL11. We performed subcellular localization in tobacco leaves and found that CmSUN23-24 was localized in the cell membrane ( Figure 10C).

Yeast Two-Hybrid and Subcellular Localization
The transcriptional activation activity of CmSUN23-24 and CmSUN25-26-27c proteins were examined. We found that CmSUN23-24 and CmSUN25-26-27c did not contain transcriptional activation activity ( Figure 10A). CmCaM5 and CmCmL11 are homologous proteins of AtCaM1, which can interact with several IQD proteins. The Y2H result showed that CmSUN23-24 had a direct interaction with CmCaM5 protein ( Figure 10B). However, there was no interaction between CmSUN25-26-27c, CmCaM5 and CmCmL11. We performed subcellular localization in tobacco leaves and found that CmSUN23-24 was localized in the cell membrane ( Figure 10C).

Discussion
SUN family genes participate in plant disease defense [13], stress resistance, plant development [28,29,36], cell arrangement, and cytoskeleton regulation [8,9,24,25]. Arabidopsis [12] and Solanum lycopersicum [27] have nearly 30 members, and Soybean has 67 more members [28]. In previous studies, Monforte identified 24 CmSUNs in melon by blasting the protein sequences of the known tomato SUN genes into the melon genome [34]; Pan mentioned 21 proteins containing both IQ67 and DUF4005 domains and identified CmSUNs by their homology with the Arabidopsis genes [30]. The integrity of the IQ67 domain is crucial in binding with the Ca 2+ receptors Calmodulin/Calmodulin-like (CaM/CmL) [39]. However, some SUN members were identified incompletely due to the low conservation of the IQ67 domain in sequence and quantity during evolution. In this study, we considered the protein containing an integrated and typical conserved IQ67 domain with three IQ motifs precisely separated by 11 and 15 amino acid residues to be the CmSUN genes, following the same criteria as the SUN family gene identification in

Discussion
SUN family genes participate in plant disease defense [13], stress resistance, plant development [28,29,36], cell arrangement, and cytoskeleton regulation [8,9,24,25]. Arabidopsis [12] and Solanum lycopersicum [27] have nearly 30 members, and Soybean has 67 more members [28]. In previous studies, Monforte identified 24 CmSUNs in melon by blasting the protein sequences of the known tomato SUN genes into the melon genome [34]; Pan mentioned 21 proteins containing both IQ67 and DUF4005 domains and identified Cm-SUNs by their homology with the Arabidopsis genes [30]. The integrity of the IQ67 domain is crucial in binding with the Ca 2+ receptors Calmodulin/Calmodulin-like (CaM/CmL) [39]. However, some SUN members were identified incompletely due to the low conservation of the IQ67 domain in sequence and quantity during evolution. In this study, we considered the protein containing an integrated and typical conserved IQ67 domain with three IQ motifs precisely separated by 11 and 15 amino acid residues to be the CmSUN genes, following the same criteria as the SUN family gene identification in Arabidopsis and Oryza sativa [12]. We characterized 24 CmSUN members, including 21 consensus genes with the previous work. Moreover, we found two other protein families, Myosin (7 members) and CaMTA (2 members), and several proteins that contained IQ motifs. It is noteworthy that nine genes of the Myosin and CaMTA family contain 1-3 IQ motifs, but possess unique amino acid spacing patterns. The two gene families have long amino acid sequences (744-1523 aa), high molecular weights (84.7-172.8 kD), and average low isoelectric points (7.6). CmSUNs are relatively short in length, have a high average isoelectric point (9.9), and contain a typical conserved IQ67 domain and a conserved exon-intron structure. During the evolution, the first IQ motif is the most conservative in the sequences, and the second and third IQ are more distinctive [12]. In our preliminary results, MELO3C005949.2.1 and MELO3C005636.2.1, which contain two IQ motifs and the specific intervals, were also annotated as IQD proteins. However, considering the absence of the first IQ motif and low sequence conservation, we did not nominate the two genes as CmSUN members. Taken together, our solid work on identifying CmSUN family members in melon provided a theoretical foundation for further characterization of their function and molecular mechanism.
Expression analyses in melon tissues showed that CmSUN transcripts were mainly accumulated in the lateral organs and the young fruits. Most members tended to be highly expressed in flower and ovary, and gradually disappeared in the fruit at the late developmental stage. In cucumber, CsSUN30-31 had a high expression level in various tissues, which may have been involved in the many aspects of plant growth. The expression of CsSUN, CsSUN17-18b, and other three SUN genes was correlated with cucumber fruit development. CsSUN23-24 and CsSUN25-26-27c expression were significantly higher in long fruit than short fruit, while CsSUN21a and the other two genes had much higher expression in short fruit [38], meaning they had the opposite role in regulating fruit elongation. In tomato, SlSUN1 and SlSUN28 showed high expression in growing fruits; SlSUN5, SlSUN11, SlSUN12, SlSUN21, SlSUN22, and SlSUN27 were expressed actively in vegetative growth [27]. The expression pattern of SlSUNs in tomato was similar to CmSUNs in different melon tissues, which may imply their conservative function in horticulture plant development.
In the functional analyses, we revealed that the overexpression of CmSUN23-24 and CmSUN25-26-27c resulted in elongated fruits and increased fruit shape index, suggesting that they had an important role in melon fruit shape regulation. In cucumber, CsSUN (CsSUN25-26-27a) is a positive regulator in fruit elongation, and the phenotype has a consistency with its expression in fruits of different lengths [38]. In watermelon, ClSUN25-26-27a is a pivotal factor in regulating the round or elongated fruit shape [32,33]. In melon, CmSUN25-26-27c is homologous to CsSUN25-26-27a, CsSUN25-26-27c, and ClSUN25-26-27a. Overexpression of CmSUN23-24 and CmSUN25-26-27c altered the fruit shape at the early ovary developmental stage. The increased expression level, enlarged fruit shape index, and significant correlation coefficient in melon transgenic fruits demonstrated that CmSUN23-24 and CmSUN25-26-27c mediate fruit shape index variation.
SUN family genes usually act as downstream targets in the calcium signal transduction pathway, which contributes to various physiological activities in plant development [40][41][42][43]. Most CmSUNs are rich in basic amino acids and hydrophobic amino acids and provide advantages for them to bind with the Ca 2+ receptors Calmodulin/Calmodulinlike (CaM/CmL) [23,35]. Through performing the yeast two-hybrid assay, we found that CmSUN23-24 can directly bind to CmCaM5. CmSUN23-24 localized in the cell membrane, which may be regulated by the CaM and Ca 2+ binding complex in Ca 2+ signal transduction, and then participated in melon plant development. In Arabidopsis, IQD1 localized in microtubules and interacted with KLCR1 (kinesin light chain-related) protein or CaM2 to recruit them for cell division regulation [39]. AtIQD5 and AtIQD18 can also regulate the cell size and cell shape by protein interaction with microtubulin and then affecting the microtubule dynamics [24,25]. The combination between IQD and microtubulin was mediated by a DUF4005 domain [26]. CmSUN23-24 and CmSUN25-26-27c both contained the DUF4005 domain. Therefore, we speculated that CmSUN23-24 positively regulates melon fruit elongation through the CmCaM5-dependent Ca 2+ signaling transduction pathway and may have a positive regulation in microtubules. Although CmSUN25-26-27c has the potential CaM binding sites, there was no interaction between CmSUN25-26-27c and CmCaM5, implying that there may other CmCaMs/CmLs working with CmSUN25-26-27c and then participating in the calcium signal and microtubule regulation.

Identification of SUN Gene Family Members
The melon protein sequence file (CM3.6.1_pep) was downloaded from the CuGenDB (http://cucurbitgenomics.org/, accessed on 5 January 2022) online website [44], which refers to the melon genome reported by Garcia-Mas [45]. The Hidden Markov model file (PF00612) of the IQ motif was downloaded from the Pfam (http://pfam.xfam.org/, accessed on 5 January 2022) database [46]. The IQD protein sequences of Arabidopsis, Solanum lycopersicum and Cucumis sativus was downloaded from previous study [12]. We searched the melon IQD proteins in HMM (http://www.hmmer.org/, accessed on 6 January 2022) software [47] using the Hidden Markov model (PF00612) and blasted the melon protein data using IQD sequences from Arabidopsis, tomato and cucumber as the probe in CuGenDB. Redundant sequences were removed and the remaining sequences were aligned through the online websites of Pfam (http://pfam.xfam.org/, accessed on 9 January 2022) and Smart (http://smart.embl-heidelberg.de/, accessed on 9 January 2022) [48] to verify the existence of the IQ motifs. The integrity of IQ67 conserved domain was demonstrated by multi-sequence alignment in MEGA7 software [49]. The protein sequences with three IQ motifs separated by 11 and 15 amino acid residues were identified as CmSUNs.

Plant Materials and Growth Conditions
The melon (Cucumis melo cv. Hetao) inbred lines were cultivated in Dengkou county of Inner Mongolia region and used in this study. All plant materials were provided by Hasi lab at Inner Mongolia University, Hohhot, China. The melon seedlings grew in an artificial climate chamber under the conditions of 60% humidity, 16 h light (25 • C), and 8 h dark (18 • C). The female fruits at anthesis day were self-pollinated. The female flower, male flower, ovary, fruits from four different developmental stages, roots, stems, and leaves were collected for expression analyses. Samples from the young lateral root, the second or third section of stem, the 3 to 5 true leaves, the unpollinated female flower, and male flowers at anthesis, and the fruit mesocarp were frozen in liquid nitrogen and stored at −80 • C. The tissues were sampled from different plants at same growth phase.

Expression Analysis and Quantitative Real-Time PCR
Different tissues from melon were used for the total RNA extraction. Raw reads for RNA-Seq data were quoted from our previous work [56]. The expression level of CmSUNs in the above ten periods was obtained from statistical data analyses. The heat map was drawn by using TBtools software [57], and parameters were set as default.
The relative expression level of CmSUN23-24 and CmSUN25-26-27c in the transgenic melon were verified by qRT-PCR. Primers were designed by Primer 5. The primer information was listed in Supplemental Table S1. RNA was reversed to cDNA by using the PrimeScript™ RT Reagent Kit and gDNA Eraser Kit (Takara Bio, Shiga, Japan). SYBR ® Premix Ex Taq™ II (Takara Bio, Shiga, Japan) and 96-well Chromo4 Real-Time PCR System were used for qRT-PCR and 2 −∆∆CT method was utilized for expression level analyses. Three biological replicates and three technical replicates were performed for each gene.

Gene Cloning and Plant Transformation
The whole-length coding sequence of CmSUN23-24 (MELO3C006884) and CmSUN25-26-27c (MELO3C013004) were amplified by gene-specific primers. The primers are listed in Supplemental Table S1. The restriction sites XbaI and BamHI were used for constructing the overexpression vector. The target gene was recombined into the overexpression vector pCAMBIA-1305 driven by 35S promoter. To obtain transgenic plants, the recombinant plasmids were diluted to 100 ng/µL with 2 × SSC solution (sodium citrate buffer, pH = 7.0) and ddH 2 O. After 7 h in artificial pollination, 10 µL plasmid solution was injected into the melon fruits as described previously [58]. Fruits of the T 1 generation transgenic plants were sampled and detected by PCR with a pair of specific primers designed on pCAMBIA-1305 vector.

Subcellular Localization
The full-length coding sequence of CmSUN23-24 without the termination codon was jointed to the vector pCAMBIA-1300 at the upstream of GFP and transformed into agrobacterium GV3101. The primers are listed in Supplemental Table S1. The lower epidermis of tobacco leaves was infected for subcellular localization experiments. A confocal laserscanning microscope was used for observing the fluorescence signals under 488-nm wave length excitation.

Conclusions
We identified 24 SUN family genes in melon, and performed a comprehensive analysis. Twenty-four CmSUN members all contained typical IQ67 motifs precisely separated by 11 and 15 amino acids. Most CmSUNs transcripts were accumulated in the melon stem, ovary, and flower. The biological function of CmSUN23-24 and CmSUN25-26-27c in regulating melon fruit shape was revealed by the gene transformation analysis. Protein interaction between CmSUN23-24 and CmCaM5 suggested their participation in the Ca 2+ signaling transduction pathway. Future studies on the function and molecular mechanism of CmSUNs would be valuable for uncovering the gene regulation in fruit development in melon.