Fruit peel directly affects the interaction between plants and the external environment and determines the fruit harvesting period and their ability to resist biotic or abiotic stress (Riglet et al., 2021). However, fruit cracking alters the mechanical properties and integrity of the peel, resulting in visual damage, decreased quality, significantly increased risk of pathogen and parasite infestation, shortened shelf life, and increased susceptibility to decay, thereby severely affecting the commercial value of fruits (Knoche and Lang, 2017; Jiang et al., 2019). As a common disorder, fruit cracking occurs in various horticultural crops, including apple (Opara, 1996), tomato (Emmons and Scott, 1997), pomegranate (Davarpanah et al., 2016), litchi (Wang et al., 2019), sweet cherry (Schumann and Knoche, 2020), and jujube (Liu et al., 2023). Both internal and external factors are involved in fruit cracking. Internal factors primarily include genetics and the intrinsic characteristics of the fruit, such as fruit size, shape, growth rate, moisture content, fruit peel properties, and fruit cracking-related gene expression (Khadivi-Khub, 2015; Yang et al., 2016). On the other hand, external factors primarily include growth environmental conditions, including temperature, light, and precipitation, and cultivation management practices, including irrigation, shading, and application of minerals and growth regulators (Dominguez et al., 2012). Moreover, cracking is associated with the loss of fruit hardness and cell wall integrity (Moctezuma et al., 2003; Cao et al., 2012).

Expansins (EXP) and Xyloglucan endotransglycosylase (XET) involved in cell wall relaxation are closely related to fruit softening and fruit cracking (Hayama et al., 2006; Asha et al., 2007). EXP, a nonenzymatic protein found in the cell wall of plants, plays a vital role in cell expansion. It breaks the hydrogen bonds between cellulose microfibrils and hemicelluloses, consequently regulating the relaxation degree between cell wall components and increasing cell wall flexibility (Feng et al., 2019). EXP was first identified in experiments on the “acid growth” of the cell wall (McQueen-Mason et al., 1992; Li et al., 1993). EXP is a large conserved gene family, with a relative molecular weight (MW) of approximately 26 kDa. It is divided into four subfamilies according to differences in gene sequences and structural composition: α-EXP (EXPA), β-EXP (EXPB), EXP-like A (EXLA), and EXP-like B (EXLB) (Kende et al., 2004). Advances in genome sequencing have resulted in the identification of EXP genes in numerous species, including Arabidopsis thaliana (Sampedro et al., 2006), rice (Sampedro et al., 2006), grape (Dal Santo et al., 2013), apple (Zhang et al., 2014a), corn (Zhang et al., 2014b), soybean (Zhu et al., 2014), tobacco (Ding et al., 2016), tomato (Lu et al., 2016), and cucumber (Liu et al., 2022a).

EXP plays an essential role in the development and growth of plants, including seed germination (Yan et al., 2014), root elongation (Noh et al., 2013), fruit ripening (Jiang et al., 2019), salt tolerance (Lv et al., 2013), and drought resistance (Chen et al., 2019). During seed softening, LeEXP4 expression increases in tomato, and this gene may participate in seed coat softening and cell wall relaxation, thereby promoting seed germination (Chen et al., 2001). After silencing LeEXP1, the hardness of the tomato decreased, with the promotion of hemicellulose decomposition (Brummell et al., 1999). In pear fruits, the ethylene inhibitor 1-methylcyclopropene inhibited fruit softening and suppressed PcEXP2, LeEXP3, and LeEXP5 expression, whereas an ethylene promoter induced their expression, indicating that these EXP proteins respond to ethylene induction and participate in fruit softening (Hiwasa et al., 2003). The analysis of the relationship between MdEXPA3 expression in the pericarp and mesocarp of ‘Fuji’ apple and fruit cracking during fruit growth revealed that inducing MdEXPA3 accumulation in the outer pericarp decreases the fruit’s sensitivity to cracking (Kasai et al., 2008). Xue et al. (2020) have reported that EXP proteins regulate water-induced tomato fruit cracking. The expression of EXPA-like genes is significantly higher in the fruit peels of the crack-resistant jujube variety ‘Wanzao 3’ than in the crack-susceptible jujube variety ‘Lifu Gongzao’ (Xin et al., 2021). Furthermore, simultaneously inhibiting polygalacturonase (PG) and EXP gene expression in tomato can decrease PG and EXP protein and water-soluble pectin contents in the fruit peel and increase cell wall thickness and hardness, thereby decreasing fruit cracking rate (Jiang et al., 2019). Therefore, EXP may play a vital role in fruit cracking regulation.

Netted melon (Cucumis melo L. var. reticulatus Naud.) is a type of thick-peeled melon belonging to the family Cucurbitaceae. The term ‘netted melon’ is used owing to the presence of net-like structures on the surface of its mature fruit (Sun et al., 2013). The netting is uniform and aesthetically pleasing, with sweet and juicy flesh, high sugar content, and a rich aroma. Furthermore, netted melon is resistant to storage and transportation, making it popular among consumers (Shi et al., 2015). At present, the cultivation area of netted melon exceeds 460,900 hectares annually in China (Chang et al., 2019). Netting degree is an important quality trait of netted melon. However, during netted melon production, adverse issues such as the uneven size of the fruit surface netting and uneven netting formation may occur. Under unfavorable environmental conditions, fruit cracking can occur at the site of netting formation, significantly affecting its commercial appeal. Many studies have reported that netting formation in netted melons is closely associated with environmental factors, including plant hormones, water, temperature, and light (Chen, 2010; Wang et al., 2011; Li et al., 2020). However, studies on the molecular mechanisms underlying the cracking resistance of netted melon are scarce. Herein, based on the genome data of melons, EXP genes associated with crack resistance were identified, and their features were characterized. Furthermore, using the transcriptome data of our laboratory, the EXP gene expression patterns in the mature fruit peels of crack-resistant and crack-susceptible varieties were elucidated. Our study findings provide important implications for exploring crack resistance-related genes in the fruit peels of netted melon and deciphering their molecular mechanisms.

Fruit cracking during growth and development is a challenging issue in fruit production. Previous studies have investigated fruit cracking from the perspectives of anatomical structure, fruit shape, and growth characteristics. For example, in the crack-susceptible tomato variety, the maximum pressure on the fruit peel is near the calyx (Peet, 1992). Larger cherry tomatoes are more prone to cracking than smaller ones (Sekse, 1987; Sekse, 2008). Additionally, there is a positive correlation between the cracking rate of grapefruit and its shape index (longitudinal diameter–transverse diameter ratio), and crack-susceptible grape varieties have larger transverse and longitudinal diameters (Zhang et al., 2020). Regarding growth characteristics, rapid fruit growth in tomatoes promotes cracking (Khadivi-Khub, 2015). Furthermore, the absolute growth rate of the transverse diameter is higher than that of the longitudinal diameter in crack-susceptible tomato varieties, whereas the absolute growth rates of the two diameters are extremely similar in crack-resistant varieties (Yang et al., 2016). However, the molecular mechanisms underlying the crack resistance of fruit peels remain unclear. Transcriptome sequencing of the resistant and susceptible-cracking watermelon peel revealed that several genes play roles in watermelon cracking resistance (Jiang et al., 2019). Transcriptome sequencing of the cracking susceptible and the cracking tolerant sweet cherry cultivars revealed significant changes in gene expression related to the biosynthesis of expansins, aquaporins, abscisic acid, ethylene, etc. (Michailidis et al., 2021). These results will contribute to the understanding and development of the molecular basis of fruit cracking and plant breeding.

In plant cells, the cell wall is an essential and unique structure that regulates cell shape and size, provides mechanical support and rigidity to cells, and functions as the first line of defense against external environmental stimuli (Liu et al., 2018). The decomposition, modification and composition of cell wall can affect the performance of pericarp and is an important factor in determining the sensitivity of fruit cracking (Bruggenwirth and Knoche, 2017; Yang et al., 2016; Jiang et al., 2019). Therefore, enzymes and proteins that regulate cell wall metabolism, such as polygalacturonase (PG), β-galactosidase (β-Gal), and expansin (EXP), are particularly important in the resistance to fruit cracking (Yu et al., 2020). EXPs can induce cell wall loosening and is crucial for regulating the cracking process of fruit peels (Wang et al., 2006; Kasai et al., 2008; Liu et al., 2022b).

Previous studies have elucidated the EXP gene family in many plants, with the systematic analysis of the EXP gene family in various plants, including Arabidopsis (model plant) (Sampedro et al., 2006), crops such as rice (Sampedro et al., 2006), vegetable crops such as tomato (Jiang et al., 2019) and pepper (Liu et al., 2022b), and fruit crops such as apple (Kasai et al., 2008), litchi (Li et al., 2014), and pomegranate (Xu et al., 2023). Subsequent studies have revealed a close relationship between EXPs and fruit cracking. Fruit cracking significantly hampers fruit appearance, facilitates pathogen invasion, and results in significant economic losses (Wang et al., 2021). During ripening, netted melon is prone to fruit cracking (Hu et al., 2021b). Therefore, there is an urgent and vital need to elucidate the role of EXP proteins in netted melon. Herein, 33 EXP genes were identified in the genome of netted melon, similar to the number identified in pumpkin (33) (Gao et al., 2020) and watermelon (30) (Liu et al., 2022a). Thirty-five EXP genes have been identified in the model plant Arabidopsis, whereas 56 EXP genes have been identified in rice, a monocot plant (Sampedro et al., 2006). This suggests the different replication methods of the EXP gene family in monocots and dicots, which is possibly associated with plant evolution (Cosgrove et al., 2002). Phylogenetic analysis revealed that the EXP gene family in plants can be divided into four subgroups: EXPA, EXPB, EXLA, and EXLB ( Figure 1 ), which are present in the EXP gene families of different plants (Sampedro et al., 2006; Liu et al., 2022a; Xu et al., 2023). A comparison of the number of members among the four subgroups within the EXP gene families in the five different plant species revealed that the EXPA subgroup comprises more members. Furthermore, the EXPB subfamily in monocot rice has significantly more members than the other four dicot plants ( Figure 1 ; Supplementary Table S2 ). This finding further suggests evolutionary differences in the gene replication and amplification of EXP genes between monocots and dicots, with monocots having more gene expansion and retention during gene replication (Zhu et al., 2014). Moreover, evolutionary tree analysis revealed that in the two largest subgroups, i.e., EXPA and EXPB, the major branches generally contain EXP genes from different species, whereas in some small branches, only EXP genes from the same species are present. These findings indicates that EXP genes undergo amplification before species differentiation. Similar results have been observed in tobacco (Ding et al., 2016) and pomegranate (Xu et al., 2023). Conserved protein motif analysis demonstrated that the CmEXP proteins in the same subgroup exhibit similar conserved elements. Gene structure analysis revealed that there are 1–4 introns in CmEXP genes, and this finding is in line with that of other plants (Dal Santo et al., 2013; Xu et al., 2023). These structural features suggest that the CmEXP genes in the same subgroup share similar structural characteristics and have been subjected to gene duplication events during evolution.

Gene duplication events are essential for genome rearrangement and amplification (Vision et al., 2000). The analysis of the duplication events in the gene family revealed that 13 gene pairs were duplicated in the CmEXP gene family ( Figure 3B ). This indicates that species-specific gene duplication has strongly affected this family’s evolution. Ka/Ks analysis revealed that the duplicated genes have undergone strong purification selection pressure ( Supplementary Table S3 ), indicating the highly conserved evolutionary pattern of CmEXP genes. The estimated duplication time for paralogous genes indicates that all paralogs, except for CmEXPA15/CmEXPA19, are ancient and range from 337 to 955 million years ago ( Supplementary Table S3 ). Homology analysis of the EXP gene families in different species revealed that CmEXP genes have 50 homologous genes in cucumber and melon, 44 homologous genes in Arabidopsis, and only 9 homologous genes in rice ( Figure 4 ). These results suggest evolutionary variation between monocots and dicots in terms of EXP genes, with higher homology in closely related species (melon and pumpkin, and melon and watermelon).

CREs help genes respond to growth, development, or environmental adaptation. Previous research has demonstrated that the upstream promoter region of CmEXP genes contains elements involved in plant growth, abiotic stress, and hormone induction, and this finding is consistent with that observed for other plant EXP gene promoters (Xu et al., 2023), indicating that the CREs are conserved in the EXP gene promoters across species. The CmEXP gene promoter contains multiple stress-related CREs ( Figure 4 ), suggesting that these genes participate in abiotic stress responses. Moreover, previous studies revealed that plant EXP genes can regulate abiotic stress. EaEXPA1 overexpression improves drought tolerance in transgenic sugarcane (Narayan et al., 2021). Furthermore, in transgenic Arabidopsis, the Osmanthus fragrans OfEXLA1 can improve salt and drought tolerance, which is regulated by the abscisic acid signaling pathway (Dong et al., 2023). When the wild peanut AdEXLB8 gene is heterogeneously expressed in tobacco, it leads to cell wall reorganization, enhancing both drought tolerance and resistance to Sclerotinia sclerotiorum and Meloidogyne incognita (Brasileiro et al., 2021). In addition, the promoter region contains multiple hormone-related CREs ( Figure 5B ), indicating that hormone signaling pathways regulate CmEXP gene functions. A recent study on jujube fruit cracking revealed that abscisic acid and MeJA treatment induce fruit cracking (Liu et al., 2023), suggesting that these two hormones negatively regulate jujube fruit cracking. The RNA-seq analysis of crack-resistant and crack-susceptible jujube varieties revealed that the most DEGs (including EXPA genes) are enriched in the cell wall synthesis pathway (Liu et al., 2023). This indicates the role of EXP genes in jujube fruit cracking.

To elucidate the role of CmEXP genes in the crack resistance of netted melon fruit, transcriptome sequencing on different fruit peel types was conducted. The CmEXP gene expression patterns were different between the non-cracked and cracked fruit peels of the crack-susceptible and crack-resistant netted melon varieties ( Figures 6C, D ). When comparing N17 with N25, 14 differentially expressed CmEXP genes were identified; in contrast, when comparing N25 and C25, 24 differentially expressed CmEXP genes were identified ( Figure 6E ). Fewer significant changes were observed in CmEXP gene expression in the non-cracked fruit peel of the different varieties; however, the CmEXP genes exhibited a more evident varying trend in the non-cracked and cracked fruit peels of the same variety. This indicates the importance of CmEXP genes in the crack resistance of netted melon. To identify the key CmEXP genes involved in crack resistance, the upregulated genes in both datasets were analyzed. Only one gene, i.e., CmEXPB1, was consistently upregulated ( Figure 6F ). Therefore, CmEXPB1 may play a vital role in the crack resistance of netted melon, and transgenic or gene silencing techniques can be used to validate its function (Liu et al., 2022b). Transcription factors are essential for regulating gene function (Donovan and Larson, 2022). Previous studies have demonstrated that various transcription factors regulate EXP gene expression. The banana transcription factor MaERF11 can inhibit MaEXP2, MaEXP7, and MaEXP8 gene expression, thereby regulating banana ripening (Han et al., 2016). Furthermore, the transcription factor MaBSD1 regulates MaEXP1 and MaEXP2 expression in response to banana ripening (Ba et al., 2014). ZmNAC11 and ZmNAC29, two NAC transcription factors in maize, can activate ZmEXPB15 expression, thereby improving grain size and weight by regulating nuclear elimination (Sun et al., 2022). To elucidate the transcriptional regulatory mechanism of CmEXPB1, we predicted its interacting transcription factors and identified 56 transcription factors that may regulate CmEXPB1 expression positively or negatively ( Figure 8A ). Nevertheless, additional research is warranted to validate the functions of these transcription factors.

Herein, 33 CmEXP genes were identified. Phylogenetic tree analysis of CmEXPs, CsEXPs, ClEXPs, AtEXPs, and OsEXPs revealed that CmEXP proteins can be categorized into four subfamilies: EXPA (20 members), EXPB (8 members), EXLA (3 members), and EXLB (2 members). The motifs and gene structures of the members of these subfamilies are highly conserved. CRE analysis in the promoter region suggests that CmEXP genes respond to development and stress. Furthermore, using transcriptome data, the CmEXP gene expression patterns in the non-cracked and cracked fruit peels of netted melon were analyzed, identifying 14 upregulated genes. Among them, CmEXPB1 was consistently upregulated in both datasets, indicating its potential role in the crack resistance of netted melon. Transcription factor prediction led to the identification of 21 and 35 positive and negative regulators, respectively, potentially involved in the regulation of CmEXPB1 expression. The above-mentioned findings enrich our understanding of the CmEXP gene family and suggest that CmEXPB1 is a candidate gene involved in regulating the crack resistance of netted melon.

The datasets presented in this study can be found in online repositories. The raw sequencing reads can be retrieved from National Center for Biotechnology Information (NCBI) database with the accession number PRJNA1026294.

YH: Data curation, Formal analysis, Investigation, Methodology, Project administration, Resources, Software, Writing – original draft. YL: Formal analysis, Investigation, Methodology, Software, Writing – original draft. BZ: Investigation. WH: Formal analysis. JC: Investigation. FW: Formal analysis. YC: Investigation. MW: Funding acquisition, Resources, Supervision, Writing – review & editing. HL: Supervision, Validation, Writing – review & editing. YZ: Conceptualization, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Software, Supervision, Validation, Visualization, Writing – original draft, Writing – review & editing.