CmSN Regulates Fruit Skin Netting Formation in Melon

Abstract

Melon (Cucumis melo) includes more than ten botanical groups, many of which feature netting ornamentation on the surface of mature fruit. Ripe melons display a netted skin that signifies their ripeness and readiness for consumption. Previously, we identified SKIN NETTING (CmSN), which encodes an EamA-like transporter family protein, as the candidate gene controlling fruit skin netting formation in melon, while its biological functions remain unclear. In this study, we demonstrated that the expression of the CmSN gene was considerably lower in netted melons compared to smooth-skinned melons, indicating a negative correlation between CmSN expression and netting formation. Subsequently, we employed transient overexpression and virus-induced gene silencing (VIGS) experiments to explore the role of CmSN gene during fruit development. Overexpression of the CmSN gene inhibited netting development, whereas silencing it promoted netting formation. Using heterologous transformation in tomato, we further confirmed the effect of the CmSN gene on rind texture and toughness, as these tomatoes exhibited rougher and tougher skins. Analysis with near-isogenic lines (NILs) revealed that CmSN gene-bearing fruits (NIL_CmSN) possessed significantly harder rinds than the control smooth-skinned variety HB42, underscoring the role of CmSN in enhancing rind protection. Together, our research offers essential insights into the netting formation and genetic improvement of melon fruits.

1. Introduction

Melon (Cucumis melo) exhibits a rich diversity in morphology, especially in fruit characteristics. Pitrat classified melons into two main subspecies, subsp. melo and subsp. agrestis, along with 15 botanical varieties under them [1]. Several groups, such as cantalupensis, reticulatus, and ameri, have netting ornamentation on the surface of mature fruit [2]. The cantaloupe derives its name from its intricate net-like pattern, appealing to consumers worldwide. As a premium melon, it features a sturdy rind, thick pulp, rich nutrients, and excellent storability, but it is most esteemed for its aroma, juicy flavor, and unique skin pattern [3,4]. Its high market value has earned it the title of “noble of melons”.

The netting on the melon rind develops during the mid to late stages of fruit ripening [5,6,7], a result of the suberization process where cells fill surface cracks to form a thick cuticle. This structure aids in moisture retention, provides mechanical support, and protects against damage and pathogens [8]. Prominent netting marks the maturity and quality of the fruits, influencing the cantaloupe’s market value. Understanding the genetics and breeding techniques for netting formation in melon is essential for producers.

The formation of netting is a pivotal agronomic trait in melon breeding; however, limited research on its formation mechanism and genetic regulation has hindered progress in enhancing this trait through molecular breeding, thus restricting improvements in melon quality and market competitiveness. In our previous study, the SKIN NETTING (CmSN) gene was identified as a crucial regulator of netting formation in melon [9], offering significant insights into the molecular mechanisms of netting formation.

The annotation of the melon genome identifies the protein encoded by CmSN as a member of the EamA transporter family. Its homologous gene in Arabidopsis thaliana, WAT1 (AT1G75500), is primarily expressed in vascular tissues, such as the xylem. It facilitates auxin transport to regulate the formation of secondary cell walls in fibers, contributing to lignin accumulation and the development of lignified cells [10]. These walls, composed of cellulose, xylan, and lignin, are deposited during advanced stages of cell development [11]. In mature melons, the netted structure of the peel is abundant in lignified cell walls [3,11]. The analogous histological characteristics between Arabidopsis and melon, such as highly lignified cell walls, imply a potential involvement of WAT1. Netted melons, compared to those with smooth skins, possess a thicker cuticle layer, which may enhance peel firmness during rapid expansion and consequently increase the risk of cracking [3]. This netting pattern is perceived as a response to injury or callus formation, appearing when surface tension exceeds a specific threshold during fruit expansion [11]. In Arabidopsis, the expression of the EamA transporter gene notably decreases during callus formation, suggesting its possible role in inhibiting this process [12].

In this study, virus-induced gene silencing (VIGS) and transient overexpression experiments were conducted on various melon genotypes to elucidate the role of the CmSN gene in regulating netting formation. Subsequently, the function of the CmSN gene was studied via heterologous transformation in tomato. Furthermore, near-isogenic lines (NILs) were developed to verify the specific role of CmSN in muskmelons. Notably, our study revealed the molecular function of the CmSN gene in muskmelon netting formation, facilitating future research into the genetic regulation of muskmelon rind characteristics.

2. Materials and Methods

2.1. Plant Material and Growth Conditions

A total of eight melon inbred lines (H581, Cm4, Cm46, and M68 with netted fruit skins and H906, HNA05-D, 992H, and HB42 with smooth fruit skins) were used in the present study. All of them were cultivated at the Scientific Education Farm of Henan Agricultural University in 2023. Field management followed standard agricultural procedures. Six accessions (H581, Cm4, Cm46, H906, HNA05-D, and 992H) were utilized for transient transformation, while the other two were used for the development of near-isogenic lines.

Eight days after pollination, similarly sized and mature fruits of the six accessions were chosen for transient transformation. The middle side of each fruit was injected with the target vector, while the opposite side received an empty vector. Samples were collected at 3, 6, 9, and 15 days post-injection, respectively, and fruits without infection were used as controls. Each injection experiment includes three to five melon plants. All the samples were photographed, and the peel was frozen in liquid nitrogen and stored at −80 °C.

To establish near-isogenic lines (NILs), M68, the donor of the CmSN allele with netted fruit skins, was initially crossed with HB42, the recipient with smooth fruit skins. This was followed by a backcross with HB42, resulting in the BC₁F₁ generation. Through phenotypic selection and continuous backcrossing, the BC₂F₁ generation was obtained. Self-pollination of the BC₂F₁ plants resulted in BC₂F₂ progeny, among which the most likely homozygous plant for CmSN identified by genotyping was designated NIL_CmSN. The Melon Whole Genome Liquid Phase Chip is a 5K liquid-phase gene chip specifically designed for targeted capture sequencing of melons. This system comprises individually packaged 5K probe mixtures along with hybridization capture reagents. These probes, composed of double-stranded DNA, are developed from selected SNP loci. These loci were identified through the alignment of re-sequencing data from 52 diverse melon genotypes against the reference genome DHL92 V3.6.1 using BWA-mem (v0.7.17), with subsequent SNP calling performed by GATK V4.1 and additional filtering. From 112K polymorphic SNP loci, 4860 loci were chosen based on uniform distribution criteria. Through detailed analysis, 281 core functional loci were identified, and 148 SNPs were selected for probe synthesis. Ultimately, a total of 5008 SNP loci were integrated into the melon 5K liquid phase gene chip, significantly enhancing the efficiency and reducing the cycle time of melon breeding processes. The proportion of recurrent parent genome (PRPG) [13] in the target individuals was calculated by gene chip-based genotyping, with the NIL_CmSN of the highest PRPG of 98%.

Wild-type (WT) tomato cv. Ailsa Craig and three homozygous transgenic lines (CmSN-OE2, CmSN-OE8, and CmSN-OE10) were grown under long-day conditions with 16 h of light and 8 h of darkness, at 25 ± 2 °C and 75% relative humidity.

2.2. Transient Transformation of Melon Fruits

The complete coding sequence (CDS) of CmSN from H581 RNA was cloned into the plant overexpression vector pCAMBIA1305.4, with the vector named OE-CmSN. Following the method described by Li et al. [14], uniformly sized fruits from melon varieties H581, Cm4, and Cm46, 8–10 days after pollination, were selected for transient genetic transformation experiments. Samples were collected on the 3rd, 6th, and 9th days after injection, with each fruit serving as a biological replicate, and 3–7 replicates were obtained per time point. Fruit skin samples near the injection site were collected with a peeler, quickly frozen with liquid nitrogen, and stored at −80 °C for subsequent analysis. Primer information is listed in Table S1. In the Supplementary Table S1, lowercase letters represent the vector’s junction sequence and uppercase letters specify the gene-specific primers.

2.3. Virus-Induced Gene Silencing (VIGS) in Melon

The VIGS experiment followed the protocol from Li et al. [14], using the vectors pTRSV1 and pTRSV2. The CmSN fragment was amplified from H581 cDNA with primers CmSN-TRSV2-F and CmSN-TRSV2-R, then cloned into pTRSV2, and transformed into Agrobacterium tumefaciens strain GV3101, creating strain TRSV-CmSN. After 8–10 days of pollination, fruits with similar sizes from melon varieties H906, HNA05-D, and 992H were selected for transient transformation. Sampling was carried out on the 9th and 15th days post-injection, with each fruit being 1 replicate and 3–7 biological replicates collected per time point. All fruit skin samples were frozen with liquid nitrogen and stored at −80 °C. The primers are listed in Table S1.

2.4. Tomato Transformation and Phenotype Observation

To elucidate the function of the CmSN gene further, we heterologously overexpressed it in tomato. Firstly, cDNA synthesized from H581 RNA was utilized as the template, and specific primers were used for PCR amplification of the target gene. The amplified fragments were then inserted into the pHELLSGATE8 overexpression vector, and the verified constructs were transformed into Agrobacterium tumefaciens strain GV3101. These transformed Agrobacterium were subsequently used to infect tomato cotyledon explants. The explants were cultured to induce callus formation, shoot development, and root formation. Positive seedlings overexpressing CmSN were identified using selection markers [15]. Peels from WT and transgenic lines were segmented and placed in 2 mL tubes, fixed with 3% glutaraldehyde, and sealed to prevent dehydration. These samples were subsequently processed by Wuhan Servicebio Technology Co., Ltd. (Wuhan, China). for dehydration, drying, and gold sputtering, then imaged with a Regulus 8100 SEM (scanning electron microscope) (Hitachi, Japan). Lignin content was measured using a kit from Suzhou Comin Biotechnology Co., Ltd (Suzhou, China). Hardness measurement was according to Zhang et al. [16]. Primers sequence information is listed in Table S1.

2.5. Total RNA Extraction and RT-qPCR Analysis

Total RNA was extracted using an RNA extraction kit (Huayueyang, Beijing, China). Complementary DNA (cDNA) was synthesized from the total RNA using the HiFiScript cDNA Synthesis Kit (Vazyme, Nanjing, China). The SYBR Green PCR Master Mix (Vazyme, Nanjing, China) was used for qPCR with the Applied Biosystems StepOne™ Real-Time PCR System (BIO-RAD Thermal Cycler, Foster City, CA, USA). The actin gene of melon was employed as the internal control in all qPCR reactions [17]. Each sample was run with three biological and three technical replicates. The primers are listed in Table S1.

2.6. Statistical Analysis

All experiments proceeded independently and were repeated in triplicate to ensure reproducibility. Data analysis was executed using IBM SPSS statistics 22.0 software (International Business Machines Corp, New York, NY, USA) with analysis of variance procedures. Significant differences among the means were assessed using Student’s t-test at the p < 0.05 and p < 0.01 levels. The reported values represent the mean ± standard deviation.

3. Results

3.1. Measurement of CmSN Gene Expression in Different Rind Types

In this study, we first investigated CmSN gene expression in various melon inbred lines to determine its role in netting formation. Six melon inbred lines with different rind types were selected: three netted melons (H581, Cm4, and Cm46) and three smooth-skinned melons (H906, 992H, and HNA05-D) (Figure 1). The results showed that significantly lower CmSN expression was detected in netted melons compared to smooth-skinned ones, indicating a negative correlation between CmSN expression and netting formation (Figure 2). These results suggested that CmSN might play a crucial role in the netting formation process.

3.2. Overexpression of CmSN Inhibits Netting Formation in Melon Fruit Skin

To explore the role of the CmSN gene in the netting formation of melon fruits, we overexpressed the CmSN gene in three melon varieties with netted fruits (H581, Cm4, and Cm46). The pCAMBIA1305.4 empty vector was used as a negative control. We observed the formation of a reticulation structure in an area of approximately 2.5 cm² around the injection point of fruit surface. Starting three days post-injection, a significant reduction in netting formation was observed in the CmSN-overexpression fruits compared to the control, with the netting appearing shriveled from days six to nine (Figure 3A–C). In addition, RT-qPCR confirmed the increased CmSN expression in the fruit skin of the overexpressing melons (Figure 3D–F). These findings suggested that overexpression of the CmSN gene inhibited normal netting development in melon fruits.

3.3. Silencing CmSN Enhances Netting Formation in Melon Fruit Skin

To further investigate the role of the CmSN gene in fruit skin netting formation, three melon varieties with smooth-skinned fruits (H906, 992H, and HNA05-D) were selected due to their high CmSN gene expression in fruit skins. Using Agrobacterium-mediated transient transformation, the CmSN gene silencing vector was introduced into melon fruits, with the empty vector serving as a control. From 9 to 15 days post-injection, we observed the formation of cork-like tissue near the injection sites on the fruit skin, but without interconnected netting structure (Figure 4A–C). In addition, RT-qPCR confirmed a significant reduction in CmSN expression levels at the injection sites, validating successful gene silencing (Figure 4D–F). These results demonstrated that silencing the CmSN gene caused suberization in melon fruit skins but was insufficient for complete netting formation.

3.4. Heterologous Transformation of CmSN in Tomato Leads to Skin Roughness

To further investigate the function of CmSN, we established the CmSN overexpression lines in tomato (Figures S1–S3). To confirm the success of gene overexpression, we assessed the expression levels of the CmSN gene in genetically modified plants. Confirmed positive strains of sterile seedlings were cultivated for a month, after which RNA was extracted from the mature fruit peel for analysis. The results revealed that the expression levels of the CmSN gene were increased in several overexpression lines compared to the WT, with OE-CmSN-5 showing the lowest increase (20-fold) and OE-CmSN-8 showing the highest increase (3023-fold) (Figure 5). These findings highlighted significant gene expression variation among different lines and demonstrated the effectiveness of genetic modification in achieving gene overexpression.

To explore the effect of CmSN gene overexpression on tomato fruit peel, we utilized scanning electron microscopy (SEM) to examine the ultrastructure of fruit skin in transgenic tomatoes (OE-CmSN). We selected three transgenic lines (OE-CmSN-2, OE-CmSN-8, and OE-CmSN-9) for subsequent analysis due to their significantly upregulated CmSN expression. Phenotypic analysis revealed that transgenic tomatoes had rougher skin surfaces compared to WT tomatoes, along with enhanced toughness and elasticity. SEM observations indicated that transgenic lines lacked the microcracks which were found in WT fruit skin, exhibiting improved surface integrity (Figure 6). Moreover, higher lignin content was observed in the fruit skin of transgenic lines, contributing to thicker cell walls and corroborating the gene expression levels (Figure 7). These findings underscored the role of CmSN gene overexpression in modifying tomato fruit skin structure by increasing roughness, toughness, and elasticity.

3.5. NIL_CmSN Exhibits Netted Skin Phenotype During Fruit Development Process

In a prior study, we successfully cloned the CmSN gene from a segregating population derived from the crossing of the melon varieties H581 and H906 and developed a molecular marker co-segregating with CmSN [7]. To validate the functional role of the CmSN gene and examine the effects of genetic background on netting formation, we introduced the CmSN allele from the netted melon inbred line M68 into the smooth-skinned melon inbred line HB42 through backcrossing, with the developed marker as foreground marker. Sanger sequencing confirmed that the CmSN gene sequence of M68 matched that of H581, verifying M68 as a valid donor parent (Figure 8A). Through backcrossing and molecular marker-assisted selection, we successfully obtained the NIL_CmSN plant, with a PRPG of 98% (Figure S4). Mature fruits of this plant were covered in netted skin. Obviously, the netted skin phenotype had been successfully introduced into HB42 (Figure 8B). Given the significant commercial importance of melon fruits, we assessed the commercial potential of NIL_CmSN, and the principal trait evaluated was fruit rind hardness. Our results showed that NIL_CmSN fruits exhibited greater rind hardness compared to HB42, confirming the role of the CmSN gene in enhancing the mechanical protection of the rind (Figure 8C).

4. Discussion

The formation of netting in melon fruits results from a complex physiological and biochemical process. Researchers have identified rind cracking and tissue suberization during fruit development as essential factors in netting formation [5,6,7]. Initially, the rind hardens while the flesh continues to grow, creating pressure that leads to rind cracking. These cracks facilitate the proliferation and suberization of epidermal cells, forming netting [3]. At present, studies on the netting trait of melon fruits primarily address physiological aspects, and direct genetic evidence for fruit skin netting regulation remains elusive. Previously, we identified CmSN as a candidate gene controlling fruit skin netting formation in melon [9]. CmSHN1 (MELO3C010341), which was predicted from a QTL region, was identified as a primary candidate gene associated with netting density [18], but without function identification. This gene is classified as a WIN1-like ethylene response transcription factor, and its Arabidopsis counterpart, SHN1/WIN1, plays a role in cuticle and wax metabolism [19]. The homologous gene CsSHN1 has been implicated in cucumber fruit netting intensity through positional cloning [20]. The intricate nature of a netted epidermis, governed by mechanical, physicochemical, and metabolic factors [3], indicates the potential involvement of multiple genes in regulating this trait across melon varieties. The distinct roles of the CmSHN1 and CmSN genes in netting formation necessitate further molecular research. Further research using various molecular techniques is needed to clarify the specific roles of these genes in pattern formation. In this study, we utilized VIGS and transient expression techniques to silence and overexpress the CmSN gene in melon fruits with varying rind types, underscoring its critical role in netting formation. The results indicated that smooth-skinned melons with silenced CmSN gene exhibited significantly lower expression levels compared to controls, whereas overexpressed netted melons showed higher expression levels and noticeable rind changes. Despite these changes being localized and influenced by strain activity and temperature, the significant differences in gene expression support the involvement of CmSN in fruit skin netting formation. Our study provides insights into the genetic mechanisms of melon rind morphology and highlights the potential of CmSN as a genetic marker or breeding target to develop melons with desirable rind characteristics.

Given the complexities of the genetic transformation of melon and the extended duration required to observe its fruit phenotype, we employed tomato as a model plant to explore the function of the CmSN gene in this study. While the CmSN gene negatively regulates netting formation in melons, its overexpression in tomatoes enhanced peel toughness and lignin content and reduced fruit cracking, suggesting a similar regulatory function in tomatoes. Fruit firmness is a complex attribute determined by diverse characteristics of fruit cells, including their thickness, size, and arrangement [21,22,23]. In tomatoes, the exocarp cuticle, comprising cutin and wax, is vital for fruit hardness. During fruit ripening, changes in these components influence cuticle accumulation, thereby affecting peel strength and fruit storage [23]. Variations in cuticle biosynthesis genes generally result in a thinner cuticle, diminishing peel toughness and hardness [23,24]. In the present study, we observed that the peels of OE-CmSN transgenic tomatoes were consistently rougher and significantly tougher than WT, possibly due to defects in cuticle formation. The reduced fruit cracking suggests increased cuticle thickness. OE-CmSN transgenic tomatoes revealed notable differences in fruit surface tissue from the WT, which might affect the fruit texture. The precise causes of these differences remain unclear and warrant further investigation. Overall, these findings underscore the importance of the CmSN gene in fruit skin development, particularly in cuticle formation and texture maintenance.

NILs represent alternative tools for investigating gene function, especially for plant species without a stable genetic transformation system. The key to applying NIL is that it must have a high background recovery rate to ensure the consistency of the genetic background with the donor material, aside from the target gene, as much as possible [25,26,27,28,29,30,31]. We successfully developed appropriate near-isogenic lines (NIL), achieving a 98% background recovery rate through chip detection. Phenotypic analysis revealed no significant variations in traits other than those associated with netting. This strongly affirms the functional significance of the target gene CmSN in netting traits and underscores the considerable potential of chip detection technology in evaluating genetic backgrounds with restricted backcross generations. This approach assured uniform expression of the CmSN gene, enabling accurate functional assessment. The commercial value of melon is heavily influenced by fruit quality, especially rind firmness. Our research focused on NIL_CmSN fruits and demonstrated that the fruits of NIL_CmSN exhibited superior rind firmness compared to HB42, enhancing mechanical protection and resistance during transport and storage. This is likely due to the accumulation of lignin in the fruit peel cells, which enhances the rigidity of the cells. Consequently, the CmSN gene is instrumental in bolstering melon rind firmness. The results of the experiment indicate that the CmSN gene significantly influences the netting pattern in melons while not affecting other fruit traits. This positions the CmSN gene as a promising candidate for molecular-level enhancement of melon netting traits. In modern agricultural breeding, the use of genetic engineering to introduce targeted traits is highly valuable. Investigating the function of the CmSN gene provides new insights into developing uniquely phenotyped melon varieties, thereby increasing their market competitiveness. A deeper understanding of the regulation and role of the CmSN gene in netting formation could lead to significant advancements in melon breeding.

5. Conclusions

This study examined the role of the CmSN gene in the development of reticulated patterns on melon rinds, underlining its importance for breeding and genetic enhancement. The research demonstrated that CmSN gene expression was negatively correlated with rind reticulation formation, where netted melons exhibited lower CmSN expression compared to smooth-skinned melons. Utilizing virus-induced gene silencing (VIGS) and transient overexpression methods, we found that overexpressing CmSN hindered netting formation in melon fruits, while silencing it enhanced reticulation formation. Furthermore, the heterologous overexpression of CmSN in tomato confirmed its influence on rind structure and resilience, with these transgenic tomatoes presenting enhanced toughness and roughness. Evaluation of near-isogenic lines (NILs) revealed that NIL_CmSN fruits exhibited significantly tougher rinds than the control variety HB42, substantiating the contribution of CmSN to improving mechanical defense. In conclusion, our study offers novel insights into the genetic mechanisms underlying melon rind reticulation formation and establishes a foundation for future advancements in melon quality breeding.