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. 2025 Jun 18;12(9):uhaf148. doi: 10.1093/hr/uhaf148

OVATE family gene CmOFP6-19b negatively regulates fruit size in melon (Cucumis melo L.)

Junling Chi 1, Haimei Yan 2, Wenjing Zhang 3, Dingfang Tian 4, Gen Che 5,✉, Agula Hasi 6,✉
PMCID: PMC12373640  PMID: 40861040

Abstract

OVATE family proteins (OFPs) constitute a class of transcription factors regulating various developmental processes in plants. Nevertheless, their precise regulatory functions in melon (Cucumis melo L.) fruit development remain elusive. In this study, we identified expression profiling of melon OFP genes and revealed the molecular function of CmOFP6-19b gene mediating fruit size variation. Quantitative analysis revealed predominant CmOFP expression in reproductive organs (female/male flowers and ovaries), with distinct differential expression patterns observed among paralogs. Through melon genetic transformation, we revealed that CmOFP6-19b gene functions as a negative regulator in fruit enlargement. Overexpression of the CmOFP6-19b gene resulted in reduced fruit size, while its downregulation led to increased fruit size. Bimolecular fluorescence complementation and yeast two-hybrid assays confirmed nuclear-localized physical interaction between CmOFP6-19b and CmKNOX16. Overexpression of CmKNOX16 in melon produced smaller fruits, phenocopying the CmOFP6-19b-Oe lines. Quantitative real-time PCR (RT-qPCR) analysis showed negative correlation between CmOFP6-19b/CmKNOX16 expression level and fruit size, with peak expression levels observed in a cultivar displaying minimal longitudinal diameter. The results of histological section and expression analysis suggest that CmOFP6-19b and CmKNOX16 may affect melon fruit size by regulating genes related to cell division and cell expansion. In conclusion, our findings systematically characterized the phylogenetic architecture and expression divergence of CmOFP genes, and elucidated the function and molecular mechanism of CmOFP6-19b-CmKNOX16 regulatory module in mediating melon fruit development, providing a theoretical foundation for melon breeding.

Introduction

Melon (Cucumis melo), which belongs to the Cucurbitaceae crops, is characterized by its sweet flavor and rich nutritional content, with significant economic value and global cultivation [1, 2]. In melon agronomic trait research, fruit size is a crucial factor affecting yield and quality. Generally, fruit size is quantitatively assessed by measuring the longitudinal and transverse diameters of the fruit. The genetic regulation underlying melon fruit size determination involves a complex network of 26 FS (Fruit Size) consensus QTLs (quantitative trait loci), which were identified in melon [3–5]. Among them, only CmOFP1a (CmFSI8/CmOFP13), CmACS7, and CmCLV3 were validated as the candidate genes for the FS locus [5, 6]. CmACS7 is not only involved in flower sex determination, but also contributes to the elongated morphology of the fruit by coordinating cell division and cell expansion [7, 8]. CmCLV3 was able to regulate the variation of melon carpel number and then affect fruit shape [9, 10]. Overexpression of CmCRC and CmSUN23-24 led to melon fruit elongation [11, 12]. The OVATE gene was colocalized with melon fruit morphology QTL and found to regulate fruit shape and size [13–15]. Despite QTL analyses and several gene characterization being conducted in melon, the genetic regulation mechanism of the key regulators remains largely unknown.

In model horticultural plant tomato, OVATE gene was identified as a pivotal regulator in fruit development [16]. OVATE family proteins (OFPs) constitute a unique class of plant-specific transcription factors that extensively participate in diverse plant growth and developmental processes [17]. OVATE was first isolated in tomato (Solanum lycopersicum), where a single nucleotide polymorphism led to a morphological transition in fruit shape from spherical to pyriform [16]. Currently, phylogenetic analyses revealed OFP orthologs in many plant species, including model plant (Arabidopsis, tomato) and commercial crops (cucumber, grape, mango, strawberry, and wax melon) [18–24]. Strong overexpression of SlOFP20 interrupted the normal pollination process and led to sterility, while mild overexpression of SlOFP20 reduced the fruit length and increased fruit width, producing flattened and smaller fruits [13, 25]. Ectopic overexpression of grape VvOFP4 gene in both tomato and tobacco led to alterations in cellular shape, subsequently influencing the morphogenesis of both nutrient and reproductive organs [21]. Ectopic expression of cucumber CsOFP12-16c gene in Arabidopsis reduced silique length with apical blunting. Conversely, the knockdown of CsOVATE resulted in longer fruit neck, suggesting that OFP genes negatively regulate cucumber longitudinal growth [20, 26]. Regulation of OFP gene expression alters plant organ shape and size, and different OFP genes may be functionally redundant within this process. In melon, CmOFP13, underlying CmFSI8 locus, controls fruit shape development. Ectopic expression of CmFSI8/CmOFP13 in Arabidopsis resulted in kidney-shaped leaves and shortened siliques [6]. In rice, OsOFP19 forms functional protein complexes with OSH1 and DLT1, regulating brassinosteroid-mediated growth regulation and signaling homeostasis [27]. In Arabidopsis, AtOFP5 interacts with KNAT3 and BLH1 to regulate embryo sac development [28]. While conserved functions of OFP family members have been characterized in model crops/plants, the specific regulatory networks governing melon fruit development await systematic investigation.

Here, we performed comprehensive analysis of sequence characteristic and expression profiling of the melon CmOFPs. Meanwhile, we found the function of CmOFP6-19b in melon fruit development and explored the negative role of CmOFP6-19b-CmKNOX16 module in regulating fruit size.

Results

Structural and evolutionary analysis of CmOFPs

Genome-wide characterization identified 18 OVATE-domain containing proteins (CmOFPs) in melon [5]. Notably, CmOFP6-19b (MELO3C009515) exhibits phylogenetic affinity with established fruit morphogenesis regulators (OsOFP8, OsOFP9), strongly suggesting conserved regulatory functions in fruit architecture determination (Fig. 1A). Comparative analysis showed that OFP genes in Cucurbitaceae were clustered in one phylogenetic clade (Fig. 1A). The molecular characteristics of CmOFPs such as gene ID, chromosome location, CDS and protein length (AA), molecular weight (MW), isoelectric point (PI), and predicted subcellular localization are listed in Table S1. Five evolutionarily conserved motifs (motif 1–5) in CmOFP proteins were identified in melon (Fig. S1). All CmOFPs contain OVATE domain, motif 1, and motif 2, indicating critical structural roles in maintaining OVATE domain architecture. Gene structure analysis demonstrated an intron-less architecture in most (17/18) CmOFP genes, with CmOFP8b being the sole exception containing two exons (Fig. S1), reflecting strong evolutionary conservation of exon–intron organization. Gene duplication is one of the primary processes in genetic evolution. Collinearity analysis identified four segmental duplication events involving 10 CmOFP paralogs (CmOFP1a, CmOFP1b, CmOFP8a, CmOFP8b, CmOFP6-19a, CmOFP6-19b, CmOFP13a, CmOFP13d, CmOFP12-16a, and CmOFP12-16b) (Fig. 1B), suggesting tandem duplication as a major driver of CmOFP family expansion. Cross-species synteny analysis revealed 23 orthologous gene pairs between C. melo and Cucumis sativus OFPs, versus 11 pairs with Arabidopsis thaliana (Fig. 1C), indicating stronger evolutionary conservation within Cucurbitaceae (Fig. 1C). To decipher transcriptional regulation mechanisms, we analyzed 2-kb promoter regions upstream of CmOFP genes, identifying 12 functional cis-regulatory elements. The hormone-responsive elements (ABRE, CGTCA-motif, GARE-motif, TCA-element, and AuxRR-core) and light-responsive elements (Box4, G-Box, and GT1-motif) were identified in the CmOFPs promoter region. Statistical analysis showed ABRE (ABA), CGTCA-motif (JA), and GARE-motif (GA) occurred in 61%, 55%, and 44% of CmOFP promoters, respectively, indicating strong hormonal regulation potential (Fig. S2).

Figure 1.

Figure 1

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Phylogenetic analysis and collinear analysis of OFPs. (A) Phylogenetic tree was reconstructed using CmOFP6-19b and functionally characterized OFP orthologs implicated in fruit morphogenesis across angiosperms. CmOFP6-19b clustered within a conserved cucurbit-specific clade. (B) Chromosomal mapping revealed the segmental duplication events among CmOFPs. In the inner circle, melon chromosomes are scaled to physical distances. The outer circle indicates the density of genes on the chromosome. Solid lines indicate segmental duplication of CmOFP gene pairs. (C) Genome collinear relationship of OFP paralogs in melon, cucumber, and Arabidopsis. Solid lines indicate the collinear OFP gene pairs between different genomes.

Expression profile of CmOFPs and subcellular localization of CmOFP6-19b

To study the transcription levels of CmOFP genes in melon, we used RT-qPCR to examine the relative expression of all 18 CmOFPs in 10 developmental tissues (root, stem, leaf, male flower, female flower, ovary, and fruit at four different stages) (Fig. 2A). The results revealed that most of CmOFP genes were highly expressed in the female flowers, while a few of them exhibited specific expression in roots, ovaries, or male flowers. Notably, almost all CmOFP genes showed low expression in fruits. The expression of CmOFP1a, CmOFP5a, CmOFP8a, CmOFP10, CmOFP12-16a, CmOFP12-16b, CmOFP13b, CmOFP14, and CmOVATE was higher in female flower, whereas CmOFP13a was highly expressed in male flower. During the fruit development, only four genes, CmOFP1a, CmOFP1b, CmOFP6-19b, and CmOFP13c, showed transcript accumulation. Among them, CmOFP6-19b displayed broad expression patterns with peak accumulation in female flower, followed by ovary, root, female flower, and G-stage fruits, suggesting pleiotropic regulatory functions during plant development (Fig. 2A). The above results suggested that CmOFP genes mainly participate in the development of melon reproductive organs. Based on its unique spatiotemporal expression profile, CmOFP6-19b was prioritized for functional characterization. CmOFP6-19b coding sequence without the termination codon was fused to the GFP fragment driven by the 35S promoter. We transformed 35S::CmOFP6-19b:GFP construct into Nicotiana benthamiana leaves via Agrobacterium infiltration and detected the fluorescence by confocal microscope. The nuclear enrichment of CmOFP6-19b-GFP overlapping DAPI-stained nuclei was detected (Fig. 2B), confirming that CmOFP6-19b was localized to the plant nucleus.

Figure 2.

Figure 2

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Expression profile of CmOFP genes in different tissues and subcellular localization of CmOFP6-19b in N. benthamiana leaves. (A) The heatmap was generated using TBtools based on RT-qPCR results. L, Rt, S, FF, MF, O, G, R, C, and P represent leaf, root, stem, female flower, male flower, ovary, fruit at growing stage, fruit at ripening stage, fruit at climacteric stage, and fruit at postclimacteric stage, respectively. Three biological replicates and three technical replicates were performed for each gene. (B) Subcellular localization of CmOFP6-19b was performed in N. benthamiana leaves. Empty vector transformation was used as negative control. Bar = 100 μm.

Overexpression of CmOFP6-19b significantly reduced fruit size in melon

To determine the function of CmOFP6-19b in melon, we overexpressed CmOFP6-19b gene in melon by genetic transformation. Three independent overexpression transgenic lines showed significantly elevated expression level of CmOFP6-19b gene and altered fruit morphology (Fig. 3A–C). Reduction of ovary size was observed in CmOFP6-19b-Oe lines, with longitudinal diameter reduced by 15.8% (Oe1: 13.1 ± 0.2 mm vs WT 15.6 ± 0.6 mm; P < 0.01) at 1 day before anthesis (DBA) (Fig. 3D). Quantitative analysis of mature fruits (38 DAP) also revealed consistent reduction in fruit size and fruit weight. The Oe lines had significantly reduced fruit longitudinal diameter, transverse diameter, and weight by 9.93%–13.39%, 10.54%–13.28%, and 30.85%–34.24%, compared with WT mature fruits (Fig. 3E–G), while fruit shape index (FSI = longitudinal/transverse diameter) remained unaltered in Oe1 and Oe2 lines compared to WT, except for Oe3 line (Fig. 3H). Next, we followed the fruit morphology of WT and CmOFP6-19b-Oe1 fruits from pollination to the ripening stage. The growth phase progression of WT and CmOFP6-19b-Oe1 fruits was generally similar (Fig. 3I). Both longitudinal and transverse diameters of both fruits showed a faster growth rate from 1 to 20 days after pollination (DAP), and the growth rate gradually leveled from 20 to 32 days and stabilized after 32 days. Interestingly, the longitudinal and transverse diameters of CmOFP6-19b-Oe1 fruits were always smaller than those of WT fruits throughout the growth cycle (Fig. 3I). The longitudinal sections of WT and CmOFP6-19b-Oe1 fruits at 10 DAP showed that the transgenic fruits had an increased cell number and decreased cell area compared to WT (Fig. 3J–L). Through expression analysis of cell division- and cell expansion-related genes, we found that the expression levels of cell cycle regulatory genes (CDKF, CycB1;1, and CycB1;2) and cell expansion-related genes (EXPA and XTH) were significantly suppressed in the transgenic plants compared to WT (Fig. 3M). These findings establish CmOFP6-19b as a negative regulator of melon fruit size, operating through cell division and expansion processes.

Figure 3.

Figure 3

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Overexpression of CmOFP6-19b inhibits melon fruit size. (A) Ovary phenotype of WT and CmOFP6-19b-Oe at the DBA. Bar = 1 cm. (B) Mature fruit phenotype of WT and CmOFP6-19b-Oe lines. Bar = 5 cm. (C) Expression level of CmOFP6-19b in WT and Oe lines. (D) Longitudinal diameter of ovary at 1 DBA in WT and CmOFP6-19b-Oe lines (n = 20). (E–H) Longitudinal diameter, transverse diameter, weight and fruit shape index of mature fruits at 38 DAP in WT, and Oe lines (n = 20). (I) Growth curves of WT and CmOFP6-19b-Oe1 fruits. (J) Longitudinal sections of WT and CmOFP6-19b-Oe1 fruits at 10 DAP. Bar = 200 μm. (K, L) Measurement of cell number and cell area displayed in Panel J. (M) Expression of cell division- and cell expansion-related genes in melon fruit. **P < 0.01 and *P < 0.05, Student’s t-test.

Knockdown of CmOFP6-19b led to increased melon fruit size

We introduced CmOFP6-19b into melon under 35S promoter and identified two independent RNAi lines by assessing the CmOFP6-19b gene expression level in plants (Fig. 4A and B). Phenotypic characterization revealed that CmOFP6-19b-RNAi lines produced significantly enlarged fruits (Fig. 4A and B). In particular, the longitudinal diameter and transverse diameter of RNAi lines were increased by 6.11%–6.69% and 5.10%–6.65% with respect to WT (Fig. 4C and D). Fruit weight analysis showed 14.9% ± 16.9% increase in RNAi lines (RNAi5: 1523.8 ± 139.0 g vs WT 1302.5 ± 141.8 g, P < 0.01) (Fig. 4E). There was no difference in FSI between the CmOFP6-19b-RNAi lines and WT fruits (Fig. 4F). Longitudinal sections of WT and CmOFP6-19b-RNAi5 fruits showed a reduction in cell number and an increase in cell area compared to WT (Fig. 4G–I). Transcriptional profiling revealed significant upregulation of cell cycle progression genes CmCycB1;1 and CmCycB1;2, along with cell wall remodeling genes CmEXPA7 and CmXTH1 in RNAi5 fruits, and unaffected expression of CmCDKF and CmEXPA6 (Fig. 4J). Natural variation analysis across six melon accessions revealed significant intercultivar divergence in longitudinal diameter (30.7–9.4 cm), with cultivar E5 displaying minimal dimension (9.4 ± 0.4 cm) (Fig. 4K and L). RT-qPCR quantification identified 2.86-fold higher CmOFP6-19b expression in E5 fruits compared to B6, inversely correlating with longitudinal diameter (r = −0.527, P < 0.05) (Fig. 4M). This result suggested that there is a negative correlation between the expression level of CmOFP6-19b gene and the longitudinal diameter in melons (Fig. 4N).

Figure 4.

Figure 4

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Downregulation of CmOFP6-19b resulted in reduced melon fruit size. Expression level of CmOFP6-19b in different melon cultivars. (A) Mature fruit phenotype of WT and CmOFP6-19b-RNAi lines. Bar = 5 cm. (B) Expression level of CmOFP6-19b in WT and RNAi lines (**P < 0.01, Student’s t-test). (C–F) Mature fruit longitudinal diameter, transverse diameter, weight and fruit shape index of WT, and RNAi lines, respectively (n = 20, **P < 0.01, Student’s t-test). (G) Longitudinal sections of WT and CmOFP6-19b-RNAi5 fruits at 10 DAP. Bar = 200 μm. (H, I) Cell number and cell area (n = 10) displayed in Panel G (**P < 0.01, Student’s t-test). (J) Expression of cell division- and cell expansion-related genes in CmOFP6-19b-RNAi5 fruit. (K) Fruit shape observation of different melon cultivars, B6, B7, E1, E6, B5, and E5. Bar = 10 cm. (L) Statistical analysis of fruit longitudinal diameter in different melon cultivars. (M) Expression level of CmOFP6-19b in different melon cultivars. Significance analysis was performed using one-way ANOVA. (N) Pearson correlation analysis showed negative correlation between the CmOFP6-19b expression level and the melon fruit longitudinal diameter.

CmOFP6-19b and CmKNOX16 have a direct protein interaction

Evolutionary conservation analysis revealed OFP-KNOX functional interaction in different species, with characterized interactions in Arabidopsis, rice, and cotton. In melon, we identified a total of 21 CmKNOX proteins through genome-wide identification and named CmKNOX1–CmKNOX21 with chromosomal distribution (Fig. S3, Table S2). STRING database prediction identified CmKNOX16 as the candidate interactor of CmOFP6-19b, validated by the spatiotemporal coexpression pattern in male flower and growing fruit (Fig. 5A). Yeast two-hybrid assays confirmed that CmOFP6-19b had no transcriptional autoactivation and CmOFP6-19b interacted with CmKNOX16 (Fig. 5B). In addition, the interaction was further verified by bimolecular fluorescence complementation in N. benthamiana leaves. CmOFP6-19b and CmKNOX16 coexpression in the tobacco epidermal cells exhibited strong fluorescence of nuclear colocalization (Fig. 5C).

Figure 5.

Figure 5

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Expression pattern of CmKNOX16 and interaction assays of CmOFP6-19b and CmKNOX16. (A) The expression pattern of CmKNOX16 in different melon tissues. Rt, S, L, FF, MF, O, and G represent root, stem, leaf, female flower, male flower, ovary, and growing fruit. (B) Yeast two-hybrid assay. Positive interaction was examined by SD/−Ade/−His/−Leu/−Trp/−X-α-gal. pGBKT7-53 and pGADT7-T were used as positive controls, pGBKT7-lam and pGADT7-T were used as negative controls. (C) Bimolecular fluorescence complementation assay. CmOFP6-19b-NE and CmKNOX16-CE were coexpressed in N. benthamiana leaves. Empty vectors coexpressed with corresponding recombinant vectors were used as negative controls. Bar = 100 μm.

Overexpression of CmKNOX16 inhibits melon fruit size

To characterize the gene function of CmKNOX16 in melon fruit development, CmKNOX16 overexpression vector was transformed into melon. Three independent CmKNOX16-Oe lines showed significantly smaller fruits compared to WT fruit (Fig. 6A and B). Compared with WT fruits, CmKNOX16-Oe fruit showed reduced fruit longitudinal/transverse diameter, decreased fruit weight, and invariant FSI (Fig. 6C–F). The expression level of CmKNOX16 gene in different melon cultivars showed peak expression in E5 with the smallest longitudinal diameter (Fig. 6G). Correlation analysis revealed that the expression level of the CmKNOX16 gene was negatively correlated with the melon fruit longitudinal diameter (Fig. 6H). In comparison with WT, the longitudinal sections of WT and CmKNOX16-Oe2 fruits had increased cell number and enlarged cell area (Fig. 4I–K), with altered expression of several cell division and cell expansion genes (Fig. 4L). The results indicated that CmKNOX16 negatively regulates fruit size in melon, which is similar to the involvement of CmOFP6-19b in melon fruit regulation. This functional congruence between CmKNOX16 and CmOFP6-19b suggests their cooperative action in a conserved regulatory module.

Figure 6.

Figure 6

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Overexpression of CmKNOX16 inhibits melon fruit size. Expression level of CmKNOX16 in different melon cultivars. (A) Mature fruit phenotype of WT and CmKNOX16-Oe lines. Bar = 5 cm. (B) Expression level of CmKNOX16 in WT and Oe lines (**P < 0.01, Student’s t-test). (C–F) Mature fruit longitudinal diameter, transverse diameter, weight and fruit shape index of WT, and Oe lines, respectively. (n = 20, **P < 0.01 and *P < 0.05, Student’s t-test). (G) Expression level of CmKNOX16 in different melon cultivars. Significance analysis was performed using one-way ANOVA. (H) Pearson correlation analysis showed negative correlation between longitudinal diameter and CmKNOX16 expression level. (I) Longitudinal sections of WT and CmKNOX16-Oe2 fruits at 10 DAP. Bar = 200 μm. (J, K) Cell number and cell area (n = 10) displayed in Panel I (**P < 0.01, Student’s t-test). (L) Expression of cell division- and cell expansion-related genes in CmKNOX16 Oe2 fruit (**P < 0.01, Student’s t-test).

Discussion

The OVATE gene family, encoding plant-specific transcription factors, plays a pivotal role in modulating plant growth and development. In melon, fruit morphology constitutes critical agronomic traits of commercial importance. While previous investigations have identified numerous quantitative trait loci (QTL) and candidate genes associated with melon fruit morphology [5, 29], the molecular mechanisms underlying these genetic determinants remain largely unexplored. Notably, the OVATE family proteins (OFPs), particularly those implicated in fruit shape regulation, have been systematically characterized and functionally validated across multiple plant species, demonstrating their essential roles in governing fruit morphogenesis [20, 21, 24, 30]. Our structural analysis revealed that most CmOFP genes exhibit intron-less architectures, with the exception of CmOFP8b (Fig. S1), a genomic organization pattern consistent with OFP homologs in cucumber [20]. This structural feature may confer evolutionary advantages, as intron-deficient genes are postulated to enhance environmental adaptability through rapid transcriptional responses [31, 32]. Spatial expression profiling demonstrated predominant CmOFP transcript accumulation in floral organs and developing ovaries (Fig. 2), mirroring expression patterns observed in cucumber orthologs [19, 20]. This conserved expression signature implies evolutionary maintenance of OFP-mediated regulatory mechanisms in cucurbit fruit development.

Melon fruit morphology is predominantly determined during pre-anthesis developmental stages through precise regulation of ovary development [33–35], a phenomenon corroborated in related cucurbits, including watermelon and cucumber [36, 37]. The temporal–spatial expression pattern of CmOFP6-19b, characterized by high transcript levels in female flowers, ovaries, and development fruits, strongly suggests its regulatory involvement in melon morphogenesis. Quantitative phenotypic analysis revealed significant reductions in longitudinal diameter, transverse diameter, and fruit weight in CmOFP6-19b-Oe transgenic lines compared to WT (Fig. 3). Conversely, the fruits of transgenic lines with CmOFP6-19b-RNAi showed an opposite trend (Fig. 4), indicating this gene as a negative regulator of fruit size development. This functional conservation extends to SlOFP20 in tomato, which similarly inhibits fruit expansion [13, 25]. Moreover, subsequent analysis showed that CmOFP6-19b gene expression was negatively correlated with the fruit longitudinal diameter in melon cultivars (Fig. 4), reinforcing its inhibitory regulatory role. Fruit development is fundamentally governed by the coordinated regulation of cell proliferation and expansion processes [38, 39]. Cell cycle regulatory gene (CDK) and Cycs genes play important roles in cell division [40], as well as Expansions (EXPA) [41, 42] and XTH, which mediate cell wall loosening and directional organ elongation [43, 44]. In the CmOFP6-19b-Oe1 fruit, the expression levels of both cell division- and cell expansion-related genes, were significantly repressed (Fig. 3). In contrast, the expression of these genes was upregulated in CmOFP6-19b-RNAi5 fruit (Fig. 4). Notably, these genes were also observed to be differentially expressed in CmKNOX16 overexpression fruit (Fig. 6). The findings suggest that CmOFP6-19b and CmKNOX16 may affect fruit size by regulating the cell division and cell expansion processes.

Many functional OFP proteins frequently mediate developmental processes through interactions with homeodomain transcription factors, particularly KNOX/BELL heterodimers. OsOFP2 coordinates seed shape and lignin synthesis through interaction with the BLH-KNOX complex in rice [45]. In Arabidopsis, AtOFP4 regulates secondary cell wall formation through interaction with KNAT7 [46]. Our protein interaction assays confirmed physical association between CmOFP6-19b and CmKNOX16 (Fig. 5), suggesting their cooperative regulation of melon fruit development. The interactions between OFP and KNOX proteins have been reported in various model plants including Arabidopsis [28, 46], tomato [25], and rice [27, 45], exhibiting remarkable functional plasticity across angiosperm lineages, as evidenced by its species-specific regulatory specialization. In Arabidopsis, these protein complexes orchestrate secondary wall biosynthesis through GA pathway modulation [28, 46], whereas OsOFP19-OSH1 in rice coordinates leaf morphogenesis via brassinosteroid crosstalk [27]. Our findings reveal that in melon, the CmOFP6-19b-CmKNOX16 complex converges on conserved cell division and cell expansion targets (Figs 3–6). It is similar to suppression of SlOFP20-KNOX1 in tomato fruit development [25], suggesting fleshy fruits may have conserved cis-regulatory adaptations in evolution. KNOX transcription factors are involved in plant growth and development by regulating the activity of meristematic tissues [47]. In tomato, KNOX gene TKA-II constrained fruit size variation through gibberellin-mediated processes [48]. In rice, KNOX gene HOS59 resulted in inhibited kernels of development [49]. In melon, phenotypic characterization of CmKNOX16-Oe lines revealed parallel reductions in fruit dimensions and weight, accompanied by similar negative correlations with longitudinal expansion (Fig. 6). These congruent functional profiles of CmOFP6-19b and CmKNOX16 implicate their synergistic action in melon fruit size regulation, potentially through modulation of cell expansion/division-related gene networks (Fig. 7). Notably, our findings establish CmOFP6-19b and CmKNOX16 as pleiotropic developmental modulators coordinating melon fruit morphogenesis, providing crucial molecular targets for marker-assisted selection strategies in cucurbit breeding programs. The phylogenetically conserved OFP-KNOX interaction module, now substantiated in melon through this investigation, constitutes an evolutionary innovation fundamental to plant organogenesis and environmental adaptation. Nevertheless, the key mechanistic aspects in their epistatic relationships within hormone signaling pathways and potential post-translational modifications influencing complex formation need to be clarified in further investigation.

Figure 7.

Figure 7

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A proposed model for the regulatory mechanism of CmOFP6-19b in controlling fruit size.

Conclusion

The study provides comprehensive bioinformatic characteristics of the CmOFPs gene family and functional validation of CmOFP6-19b as a negative regulator of melon fruit size. The demonstrated interaction between CmOFP6-19b and CmKNOX16 establishes a regulatory module, potentially operating through transcriptional control of cellular division or expansion mechanisms. These findings not only elucidate molecular determinants of melon fruit morphology but also offer valuable genetic targets for precise breeding of commercial melon cultivars.

Materials and methods

Plant materials and growth conditions

Hetao melon (C. melo cv. Hetao) inbred lines were cultivated under standard agriculture condition in Dengkou county, Inner Mongolia region. Six representative melon cultivars, E1, B7, B5, B6, E6, and E5, were selected for developmental analysis, with mature fruits harvested at physiological ripening stage (R-stage). Significant variations in longitudinal diameter were observed among cultivars (detailed measurements provided in Table S3). Hetao melon seedlings were cultivated in an artificial climate chamber under conditions of 25°C with a 16-h/18°C light period and an 8-h/18°C dark period at 60% relative humidity. Various plant tissues, including roots, stems, young leaves, ovaries at the anthesis day, female flowers, male flowers, and fruit at growing stage (growing [G], ripening [R], climacteric [C], and postclimacteric [P] stages), were collected. All samples were frozen in liquid nitrogen and stored at −80°C.

Gene structure and promoter analysis

Gene structure, conserved motifs, and domains analysis of CmOFP genes were performed using TBtools software. Then, 2000-bp upstream sequences of CmOFP genes were extracted from melon genome database, and cis-elements were analyzed using PlantCARE (https://bioinformatics.psb.ugent.be/webtools/plantcare/html/).

Phylogenetic tree construction and collinearity analysis

The phylogenetic tree was performed using MEGA11 software. The protein sequences of CmOFP6-19b homologs and functionally characterized OFPs in other plants were downloaded from NCBI (https://blast.ncbi.nlm.nih.gov/Blast) and listed in Table S4. We downloaded the genome files and GFF annotation files of melon, cucumber, and Arabidopsis from CuGenDB (http://cucurbitgenomics.org) and TAIR (http://www.arabidopsis.org/), respectively. MCScanX was used to identify tandem duplication events of CmOFP genes within the melon species and to analyze the syntenic relationship of homologs in Arabidopsis and cucumber.

Melon transformation and RT-qPCR

Full-length coding sequences of CmOFP6-19b and CmKNOX16 were cloned into the pBI1305 vector. A 495-bp DNA fragment of CmOFP6-19b was amplified, and both sense and antisense fragments were inserted into the pFGC1008 vector. To obtain transgenic plants, recombinant plasmids of overexpression vectors and RNAi construct were introduced into melon female flower by the ovary injection method [50–52]. The recombinant plasmids (pBI1305-CmOFP6-19b, pBI1305-CmKNOX16, pFGC1008-CmOFP6-19b) were suspended in 0.1× SSC (pH 7.0) and its concentration was adjusted to 100 ng/μl concentration and then stored at −20°C. 6–7 hours after the self-pollination, the petals of the female flowers were removed to expose the stigma. Ten-microliter diluted recombinant plasmid delivery was achieved using sterile microinjectors, with strict adherence to bubble-free aspiration. The injection needle penetrated 2/3 of the ovarian depth along the pollen tube pathway, followed by gradual solution administration. Twenty biological replicates per construct resulted in 50%–60% fruit set efficiency with injected specimens. The transformed seeds were collected from mature fruits and germinated under controlled conditions. At the five-leaf developmental stage, genomic DNA was extracted from seedling leaves for PCR-based transgene detection. Positive transgenic seedlings were transferred to a greenhouse for phenotypic evaluation, followed by artificial self-pollination to obtain homozygous progeny. Pulp tissues from mature fruits (38 DAP) of both transgenic lines and WT plants were used for RT-qPCR analysis of target gene expression levels. Seeds of the identified CmOFP6-19b and CmKNOX16 transgenic melon lines were propagated and cultivated under controlled conditions, during which morphological characteristics and phenotypic were recorded.

To explore the transcript levels of CmOFP genes in different melon tissues, we reverse-transcribed the extracted total RNA into cDNA. The synthesized cDNA used as a template for RT-qPCR analysis, with CmGAPDH used as internal reference gene. The results of RT-qPCR were calculated by 2–ΔΔCT method and visualized through heatmaps using TBtools. All RT-qPCR experiments for detecting gene expression levels (Figs 2–4 and 6) were performed with three biological and three technical replicates. The specific primers used for RT-qPCR used were listed in Table S5.

Subcellular localization

The coding sequences of CmOFP6-19b excluding the stop codon were cloned into pCAMBIA1300-GFP expression vector. The primers used are listed in Table S6. pCAMBIA1300-CmOFP6-19b-GFP recombinant vector was transformed into Agrobacterium GV3101 and then injected into the lower epidermis of 4-week-old leaves of N. benthamiana. The infiltrated plants were subsequently maintained at ambient temperature (25°C) for 60 hours. Following infiltration, leaf samples were harvested and stained with DAPI solution at a final concentration of 0.5 μg/ml, followed by incubation in darkness in 5 minutes. The leaf samples were rinsed twice with 0.9% saline solution to eliminate unbound DAPI dye, and then the leaves were prepared for microscope observation. Fluorescence signal was observed using a laser scanning confocal microscope with an excitation wavelength of 488 nm.

Yeast two-hybrid assay

Full-length coding sequences of CmOFP6-19b and CmKNOX16 were inserted into pGBKT7 and pGADT7 vectors, respectively. The primers were listed in Table S6. The following combinations, pGBKT7-CmOFP6-19b + pGADT7, pGADT7-CmKNOX16 + pGBKT7, and pGBKT7-CmOFP6-19b + pGADT7-CmKNOX16, were cotransformed into the yeast strain AH109. Single-yeast strains grown on the SD/−Trp/−Leu selection plates were individually transferred into PCR tubes with 10 μl of ddH2O for resuspension and subsequent dilution. Yeast suspensions were prepared at three different concentrations: 100, 10-1, and 10–2, respectively. Yeast suspensions at different concentrations were spotted onto the SD/−His/−Leu/−Trp solid selection medium supplemented with 4 mg/ml X-α-gal for the both activation detection and protein–protein interaction analysis. pGBKT7-53 + pGADT7-T was used as a positive control and pGBKT7-lam + pGADT7-T as a negative control.

Bimolecular fluorescence complementation assay

Full-length sequences of CmOFP6-19b and CmKNOX16 excluding the termination codon were inserted into pSPYNE-35S and pSPYCE-35S, respectively. The primers were listed in Table S6. The combinations of CmOFP6-19b-NE + pSPYCE, CmKNOX16-CE + pSPYNE, and CmOFP6-19b-NE + CmKNOX16-CE were cotransformed into Agrobacterium GV3101 and injected into the abaxial leaf epidermis of N. benthamiana for approximately 60 hours. The infected leaves were initially stained with 0.5 μg/ml DAPI solution, then rinsed with 0.9% saline solution before microscope observation. Fluorescence signals were detected using a laser confocal microscope with 488-nm excitation wavelength.

Paraffin sectioning

Transgenic melon fruits (CmOFP6-19b-Oe1, CmOFP6-19b-RNAi5, and CmKNOX16-Oe2) and WT controls were harvested at 10 DAP. Fresh pulp tissues from the equatorial region were immediately fixed in 3.7% formaldehyde-acetic acid solution under vacuum infiltration for 24 hours at 4°C. Following gradient ethanol dehydration (50%, 70%, 85%, 95%, and 100%) samples were embedded in paraffin wax. The samples were sectioned and observed under a fluorescence microscope. Cell number and cell area were measured using Image J software. Pairwise comparisons between transgenic and WT lines were performed using GraphPad Prism 9, with significance thresholds set at P < 0.05 (*).

Supplementary Material

Web_Material_uhaf148
web_material_uhaf148.zip (774.4KB, zip)

Acknowledgements

This work was supported by the Applied Technology Research and Development Foundation of Inner Mongolia Autonomous Region, China (2021PT0001), the National Natural Science Foundation of China (32460769, 32202513), Inner Mongolia Autonomous Region universities ‘Young Science and Technology Talent Support Project’ (NJYT24067), and the Inner Mongolia Autonomous Region Department of Education First-class Scientific Research Project, China (YLXKZX-ND-030).

Contributor Information

Junling Chi, Key Laboratory of Herbage & Endemic Crop Biology, Ministry of Education, School of Life Science, Inner Mongolia University, 235 Daxue West Street, Saihan District, Hohhot 010070, China.

Haimei Yan, Key Laboratory of Herbage & Endemic Crop Biology, Ministry of Education, School of Life Science, Inner Mongolia University, 235 Daxue West Street, Saihan District, Hohhot 010070, China.

Wenjing Zhang, Key Laboratory of Herbage & Endemic Crop Biology, Ministry of Education, School of Life Science, Inner Mongolia University, 235 Daxue West Street, Saihan District, Hohhot 010070, China.

Dingfang Tian, Key Laboratory of Herbage & Endemic Crop Biology, Ministry of Education, School of Life Science, Inner Mongolia University, 235 Daxue West Street, Saihan District, Hohhot 010070, China.

Gen Che, Key Laboratory of Herbage & Endemic Crop Biology, Ministry of Education, School of Life Science, Inner Mongolia University, 235 Daxue West Street, Saihan District, Hohhot 010070, China.

Agula Hasi, Key Laboratory of Herbage & Endemic Crop Biology, Ministry of Education, School of Life Science, Inner Mongolia University, 235 Daxue West Street, Saihan District, Hohhot 010070, China.

Author contributions

Agula Hasi and Gen Che designed the project. Junling Chi wrote this paper, performed the experiments, and prepared the figures. Haimei Yan and Wenjing Zhang analyzed the data. Dingfang Tian contributed plant materials and mapping tools. All authors have read the drafts of this paper.

Data availability

All experimental data are available in the main text and supplementary data. RNA-Seq data in this study can be found at the NCBI SRA (https://www.ncbi.nlm.nih.gov/sra); the BioProject accession numbers are PRJNA543288 and PRJNA803327.

Conflict of interest statement

The authors declare that they have no conflict of interest.

Supplementary data

Supplementary data is available at Horticulture Research online.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Web_Material_uhaf148
web_material_uhaf148.zip (774.4KB, zip)

Data Availability Statement

All experimental data are available in the main text and supplementary data. RNA-Seq data in this study can be found at the NCBI SRA (https://www.ncbi.nlm.nih.gov/sra); the BioProject accession numbers are PRJNA543288 and PRJNA803327.

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