A 29‐bp Promoter Deletion in CsCEPR1 Underlies Increased Seed and Fruit Size During Cucumber Domestication

Domestication signifies the beginning of crop breeding and is essential for modern breeding efforts. Seeds play a crucial role in conveying both domestication and evolutionary information. Wild species generally produce small seeds with pronounced dormancy, enabling them to survive in challenging environments. In contrast, modern cultivars feature larger seeds with reduced dormancy, optimising them for agricultural production (Fuller et al. 2014; Wang et al. 2018). Cucumber (Cucumis sativus var. sativus) is an important vegetable, having been domesticated from its wild relative, C. sativus var. hardwickii. Cultivated cucumbers exhibit larger seeds with diminished dormancy compared to their wild ancestors, likely resulting from domestication and artificial selection (Ren et al. 2009). Although several quantitative trait loci (QTLs) or genes linked to the domestication of seed size have been identified, a few genes have been thoroughly characterised (Wang et al. 2018, 2020). Cloning and characterising such genes in cucumber will deepen our understanding of domestication and facilitate molecular breeding. We previously mapped the Seed Size 2. 1 (SS2. 1) QTL between the SSR05743 and SSR13131 markers on chromosome 2 utilising CG64 (also known as ‘PI183967’, a wild cucumber with small seeds and strong dormancy) and ‘931’ (a cultivated cucumber with large seeds and reduced dormancy) (Figure 1a and Table S1) (Wang et al. 2014). Among the genes, five genes showed non-synonymous variations and only one gene displayed differential expression in early seed development between two parental lines (Figure S1a and Table S1). Notably, CsaV3₂G030560 exhibiting higher expression in the wild cucumber was also subject to significant selection pressure on seed size during domestication, as demonstrated by the analysis of 115 resequenced accessions (Figure 1b). CsaV3₂G030560 encodes a receptor protein-tyrosine kinase that is homologous to CEPR1 in Arabidopsis (Figure S1b and Table S2). Consequently, we designated this gene as CsCEPR1. Previous studies have shown that CEPR1 plays a critical role in seed size regulation (Taleski et al. 2020; Xu et al. 2021). CsCEPR1 is primarily expressed in root, stem, and mature seed (Figure S2a and Table S3; Guan et al. 2024). We identified a 29-bp deletion in the promoter region of cultivated cucumbers with large seeds, contrasting with all wild cucumbers that possess small seeds (Figure S2b-d). The canonical W-box and CAAT-box motifs, which function as binding sites for WRKY transcription factors and serve as common cis-acting elements for regulating transcription efficiency, respectively, were present in wild cucumbers but absent in cultivated varieties (Figure 1c). Dual-luciferase reporter assays indicated that the promoter with the insertion exhibited significantly higher transcriptional activity (Figure 1d, e and Figure S3). We propose that the 29-bp insertion–deletion polymorphism (InDel) influences the expression level of CsCEPR1, thereby regulating seed size. We introduced the full coding sequence of CsCEPR1, driven by the 35S promoter, into the cultivated cucumber ‘CG104’ (with lower CsCEPR1 expression) to create overexpression (OE) transgenic plants (Figure S4a). RT-qPCR analysis confirmed the successful overexpression of CsCEPR1 (Figure S4b and Table S4). Phenotypic analysis indicated that seeds from CsCEPR1-OE plants displayed a smaller size and increased dormancy (Figure 1f, Figures S4c and S5a). Additionally, CsCEPR1-OE plants exhibited a significant reduction in fruit length (Figure 1g and Figure S5b). These observations suggest that CsCEPR1 negatively regulates both seed and fruit sizes. CEPR1 homologues serve as receptors for C-terminally encoded peptides (CEPs) (Taleski et al. 2020; Xu et al. 2021). Six CsCEPs were identified in cucumber (Table S3) (Liu et al. 2021). We explored the interactions between CsCEPR1 and CsCEPs, and the results demonstrated that CsCEPR1 interacts with CsCEP2, CsCEP3, CsCEP5, and CsCEP6, as evidenced by split-luciferase complementation (LCI) and yeast two-hybrid (Y2H) assays (Figure 1h, i). The subcellular localization assay showed that both the proteins of CsCEPR1CG64 and CsCEPR1931 were localised to the cytoplasm (Figure S6). These support the conclusion that CsCEPR1 functions as a receptor for CsCEPs. We conducted transcriptome analysis on early developmental seeds from both ‘CG104’ and CsCEPR1-OE. We identified 117 up-regulated and 347 down-regulated differentially expressed genes (DEGs) in CsCEPR1-OE. Gene Ontology (GO) enrichment analysis revealed a significant association with phytohormone-related pathways (Figure 1j). Among these DEGs, 15 auxin-related genes were significantly down-regulated in CsCEPR1-OE plants (Figure 1k and Table S5). Previous studies have indicated that auxin plays a crucial role in regulating fruit size in cucumber (Chen et al. 2024). Active indoleacetic acid (IAA) concentrations were significantly lower in the early developmental seeds of CsCEPR1-OE compared to those of ‘CG104’ (Figure 1l). CsCEPR1 showed a significantly increased expression level after the exogenous naphthaleneacetic acid (NAA) treatment, suggesting the negative feedback regulation between CsCEPR1 and auxin (Figure S7). Therefore, we propose that CsCEPR1 regulates cucumber seed size, potentially through the auxin signalling pathway. J. H. performed experiments, analysed data, and drafted the manuscript. J. G. contributed to data analyses and revised the manuscript. S. D. , J. G. , and H. M. helped analyse the data. S. Z. and J. S. designed the experiments, supervised the study, and revised the manuscript. All authors approved the final submitted manuscript. Raw data in this study are available in Supporting Information. The raw transcriptome data have been deposited in the Genome Sequence Archive (https: //ngdc. cncb. ac. cn/gsa/) of the National Genomics Data Center, part of the China National Center for Bioinformation, under accession number PRJCA043228. Figure S1: Identification of CsCEPR1 in cucumber. Figure S2: CsCEPR1 regulates seed size in cucumber accessions. Figure S3: Transient expression assay in Nicotiana benthamiana leaves shows that CsCEPR1Ref exhibits higher promoter activity than CsCEPR1Del. Figure S4: Overexpression of CsCEPR1 resulted in decreased seed germination. Figure S5: CsCEPR1 negatively influences seed and fruit sizes. Figure S6: Subcellular localization of CsCEPR1CG64 and CsCEPR1931 in Nicotiana benthamiana leaves. Figure S7: Negative feedback regulation between CsCEPR1 and auxin. Table S1: Expression levels and the sequence variations of candidate genes at SS2. 1 in ‘CG64’ and ‘931’ during early seed development are depicted in relation to Figure 1. Table S2: Details regarding the homologues of CEPR1 across various species are presented in relation to Figure S1. Table S3: The expression profiles (TPM) of CsCEPR1 and CsCEPs genes across various tissues, as derived from RNA-seq data. Table S4: The expression levels of CsCEPR1 in ‘CG104’ and CsCEPR1-OE plants associated with the data presented in Figure S4b. Table S5: Expression (FPKM) of DEGs associated with the auxin pathway in early developmental seeds of ‘CG104’ and CsCEPR1-OE as analysed by RNA sequencing, related to Figure 1o. Table S6: Sequences of primers used in this study. Table S7: Summary statistics of the RNA-seq data. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. 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