Spraying N-(2-chloro-4-pyridyl)-N′-phenylurea (CPPU), an exogenous cytokinin (CK) growth regulator, is the conventional method for inducing fruit set during melon (Cucumis melo L.) production; however, the mechanism by which CPPU induces fruit set is unclear. Through histological and morphological observations, fruit size was comparable between CPPU-induced fruits and normal pollinated fruits because CPPU-induced fruits had higher cell density but smaller cell size compared with normal pollinated fruits. CPPU promotes the accumulation of gibberellin (GA) and auxin and decreases the level of abscisic acid (ABA) during fruit set. Moreover, application of the GA inhibitor paclobutrazol (PAC) partially inhibits CPPU-induced fruit set. Transcriptome analysis revealed that CPPU-induced fruit set specifically induced the GA-related pathway, in which the key synthase encoding gibberellin 20-oxidase 1 (CmGA20ox1) was specifically upregulated. Further study indicated that the two-component response regulator 2 (CmRR2) of the cytokinin signaling pathway, which is highly expressed at fruit setting, positively regulates the expression of CmGA20ox1. Collectively, our study determined that CPPU-induced melon fruit set is dependent on GA biosynthesis, providing a theoretical basis for the creation of parthenocarpic melon germplasm.

Cucurbits (Cucurbitaceae) include major horticultural crops with high nutritional and economic value that also serve as model plants for studying plant development and crop improvement. Conventional breeding methods have made important contributions to the production of cucurbit crops but have led to a breeding bottleneck because of the narrow genetic bases and low variation rates of these crops. With the development of molecular techniques, innovations in germplasm development through transgenesis and gene editing have led to breakthroughs in horticultural crop breeding. Although the development of genetic transformation and gene editing techniques for cucurbit crops has lagged behind that for other major crops, great progress has been made in recent years. Here, we summarize recent advances in improving the genetic transformation efficiency of cucurbit crops, including the screening of germplasm and the application of physical treatments, morphogenic genes, and selection markers. In addition, we review the application of gene editing technology to cucurbit crops, including CRISPR (clustered regularly interspaced short palindromic repeat)/Cas9 (CRISPR-associated nuclease 9)-mediated gene knockout and base editing. This work provides a reference for improving genetic transformation efficiency and gene editing technology for cucurbit crops.

A number of cis-regulatory elements (CREs) conserved during evolution have been found to be responsible for phenotypic novelty and variation. Cucurbit crops such as cucumber (Cucumis sativus), watermelon (Citrullus lanatus), melon (Cucumis melo) and squash (Cucurbita maxima) develop fruits from an inferior ovary and share some similar biological processes during fruit development. Whether conserved regulatory sequences play critical roles in fruit development of cucurbit crops remains to be explored. In six well-studied cucurbit species, we identified 392,438 conserved non-coding sequences (CNSs), including 82,756 that are specific to cucurbits, by comparative genomics. Genome-wide profiling of accessible chromatin regions (ACRs) and gene expression patterns mapped 20,865–43,204 ACRs and their potential target genes for two fruit tissues at two key developmental stages in six cucurbits. Integrated analysis of CNSs and ACRs revealed 4,431 syntenic orthologous CNSs, including 1,687 cucurbit-specific CNSs that overlap with ACRs that are present in all six cucurbit crops and that may regulate the expression of 757 adjacent orthologous genes. CRISPR mutations targeting two CNSs present in the 1,687 cucurbit-specific sequences resulted in substantially altered fruit shape and gene expression patterns of adjacent NAC1 (NAM, ATAF1/2 and CUC2) and EXT-like (EXTENSIN-like) genes, validating the regulatory roles of these CNSs in fruit development. These results not only provide a number of target CREs for cucurbit crop improvement, but also provide insight into the roles of CREs in plant biology and during evolution.

Melon (Cucumis melo L.) is a globally important fruit crop, but progress in molecular breeding has been hampered by limited functional dissection of genes associated with agronomic traits. Therefore, we developed a comprehensive genome resource based on the C. melo ssp. agrestis accession 13C. This resource includes a complete telomere-to-telomere genome assembly, including accurate quantification of 45S rDNA copy number in melon, a transcriptome atlas from 31 tissue samples, a phenotypically diverse EMS-induced mutant library and a stable transformation system. By sequencing 1,125 M2 families, we identified about 660,000 variants, which cover 97.33% of the annotated gene space. Leveraging these integrated resources, we identified and functionally characterized several key genes, including CLAVATA3 INSENSITIVE RECEPTOR KINASES 2 (CmCIK2), which regulates carpel number; PARA-AMINOBENZOIC ACID SYNTHASE (CmACDS), a central regulator of folate biosynthesis; and a mutant allele of the known gynoecious gene WIP DOMAIN PROTEIN 1 (CmWIP1). In addition, we discovered a specific EMS-induced variant in the fruit ripening regulator CmNAC-NOR, and further validated its function by generating targeted mutants. The CmNAC-NOR mutants exhibited delayed fruit ripening, thus providing a valuable resource for improving ripening traits in agrestis accessions. To facilitate broader utilization, we developed the Melon Information Resource, available at https://zhanglab.qau.edu.cn/melon/index.php, an integrated platform housing 13C comprehensive genome resources and associated convenient analysis tools. This unified and accession-specific resource offers unprecedented opportunities to accelerate gene discovery and trait improvement in melon through functional genomics and molecular breeding.

Cucurbitacin B (CuB) is the primary compound responsible for bitterness in melon (Cucumis melo L.) and negatively affects fruit quality and consumer acceptance. Although CuB biosynthesis is regulated by multiple transcription factors, the regulatory mechanisms underlying CuB biosynthesis across different tissues remain unclear. Here, we generated two independent knockout lines (Cmbt-18 and Cmbt-19) of the basic helix-loop-helix (bHLH) transcription factor gene CmBt (Bitter fruit) using CRISPR-Cas9. Loss of CmBt reduces CuB levels in fruits and roots and produces non-bitter leaves, indicating that CmBt predominantly regulates CuB accumulation in leaves. By contrast, its paralog CmBr (Bitter root) predominantly controls CuB accumulation in fruits and roots. These results redefine CmBt as a leaf-predominant regulator and CmBr as a fruit/root-predominant regulator of CuB biosynthesis, revising previous findings. Furthermore, we demonstrate that CmBt physically interacts with CmBr. The CmbtCmbr double mutants exhibit a more severe reduction in CuB content in fruits, leaves, and roots, compared with either single mutant, confirming a synergistic interaction between CmBt and CmBr. Reverse transcription quantitative PCR (RT-qPCR) and tobacco dual-luciferase assays further show that CmBt and CmBr synergistically activate the CuB biosynthetic gene CmBi. Additionally, the CmbtCmbr double mutant completely prevents CPPU-induced fruit bitterness. Our findings reveal that CmBt and CmBr exert tissue-specific, partially redundant, and synergistic roles in activating CmBi and promoting CuB biosynthesis. These results identify the CmBt-CmBr locus as a promising target for breeding non-bitter melon cultivars.