The carotenoid cleavage dioxygenase (CCD) gene family in plants comprises two subfamilies: CCD and 9-cis-epoxycarotenoid dioxygenase (NCED). Genes in the NCED subfamily are mainly involved in plant responses to abiotic stresses such as salt, low temperature, and drought. Members of the NCED subfamily are the most important rate-limiting enzymes in the biosynthesis of abscisic acid (ABA). In the present study, genome-wide analysis was performed to identify CCD gene members in six Cucurbitaceae species, including watermelon (Citrullus lanatus), melon (Cucumis melo), cucumber (C.sativus), pumpkin (Cucurbita moschata), bottle gourd (Lagenaria siceraria), and wax gourd (Benincasa hispida). A total of 10, 9, 9, 13, 8, 8 CCD genes were identified in the six species, respectively, and these genes were unevenly distributed in different chromosomes. Phylogenetic analysis showed that CCD genes of the six species clustered into two subfamilies: CCD and NCED, with five and three independent clades, respectively. The number of exons ranged from 1 to 15, and the number of motifs were set to 15 at most. The cis-acting elements analysis showed that a lot of the cis-acting elements were implicated in stress and hormone response. Melon seedlings were treated with salt, low temperature, drought, and ABA, and then tissue-specific analysis of CCDs expression were performed on the root, stem, upper leaf, middle leaf, female flower, male flower, and tendril of melon. The results showed that genes in CCD family exhibited various expression patterns. Different CCD genes of melon showed different degrees of response to abiotic stress. This study presents a comprehensive analysis of CCD gene family in six species of Cucurbitaceae, providing a strong foundation for future studies on specific genes in this family.

Seed germination is a critical stage and the first step in the plant’s life cycle. H₂O₂ and Ca²⁺ act as important signal molecules in regulating plant growth and development and in providing defense against numerous stresses; however, their crosstalk in modulating seed germination remains largely unaddressed. In the current study, we report that H₂O₂ and Ca²⁺ counteracted abscisic acid (ABA) to induce seed germination in melon and Arabidopsis by modulating ABA and gibberellic acid (GA₃) balance. H₂O₂ treatment induced a Ca²⁺ influx in melon seeds accompanied by the upregulation of cyclic nucleotide-gated ion channel(CNGC) 20, which encodes a plasma membrane Ca²⁺-permeable channel. However, the inhibition of cytoplasmic free Ca²⁺ elevation in the melon seeds and Arabidopsis mutant atcngc20 compromised H₂O₂-induced germination under ABA stress. CaCl₂ induced H₂O₂ accumulation accompanied by the upregulation of respiratory burst oxidase homologue(RBOH) D and RBOHF in melon seeds with ABA pretreatment. However, inhibition of H₂O₂ accumulation in the melon seeds and Arabidopsis mutant atrbohd and atrbohf abolished CaCl₂-induced germination under ABA stress. The current study reveals a novel mechanism in which H₂O₂ and Ca²⁺ signaling crosstalk offsets ABA to induce seed germination. H₂O₂ induces Ca²⁺ influx, which in turn increases H₂O₂ accumulation, thus forming a reciprocal positive-regulatory loop to maintain a balance between ABA and GA₃ and promote seed germination under ABA stress.

High salinity can severely inhibit the growth and development of watermelon (Citrullus lanatus L.). WRKY proteins are believed to mediate the adaptation of plants to abiotic stresses. Here, we identified the ClWRKY61 gene, which positively regulates the tolerance of watermelon to salt stress. Knockout of the ClWRKY61 reduced salt tolerance, while overexpression of the ClWRKY61 enhanced salt tolerance in watermelon according to phenotypic and physiological analyses. Yeast two-hybrid assays revealed that ClWRKY61 interacts with the ClLEA55 protein, and this interaction was further confirmed by luciferase complementation imaging, transient bimolecular fluorescence complementation, and GST pull-down assays. Knockout of the ClLEA55 resulted in lower salt tolerance compared to the wild-type plants. RNA-seq analysis indicated 421 up-regulated and 133 down-regulated genes in the ClWRKY61 knockout line under salt stress, containing 293 differentially expressed genes with W-box in their promoters. Thirteen genes encoding phytoene synthase, MYB transcription factor, sucrose synthase, alpha/beta-hydrolases superfamily protein, glutathione reductase, sugar transporter, LEA protein, WRKY transcription factor, ERF transcription factor, alpha-glucan water dikinase, and calcium-dependent protein kinase showed transcriptional changes in ClWRKY61 knockout line, ClWRKY61 overexpression line, and ClLEA55 knockout line under salt stress. These results provide an opportunity to mediate the regulation of salt stress in watermelon with WRKY proteins.

Watermelon is a crucial horticultural crop worldwide but its genetic base has become extremely narrow owing to long-term cultivation. Induced mutagenesis can create a range of variations with distinctive agricultural characteristics. To broaden the genetic diversity of watermelon, we established a mutagenesis library containing over 4000 M₁ seeds from an inbred line ‘M08’, which was irradiated by 350 Gy of ⁶⁰Co γ-rays for 3 h. The rates of germination, emergence, and survival of the M₁ seeds were reduced by 5.88%, 18.66%, and 41.96%, respectively. After phenotypic screening, 20 and 10 types of morphological changes were observed in the M₁ and M₂ generations, with approximately 10.57% and 14.17% mutation frequencies, respectively. Six mutants with desirable horticultural alterations were selected for additional presentation, including the leaf color mutant C₁-NO.1, the yellow peel mutant C₁-NO.2, the pericarp thickening mutant C₁-NO.3, the pericarp thinning mutant C₁-NO.4, the seedless mutant C₁-NO.5, and the C₂-No.1 mutant with normal female flowers and malformed male flowers. Moreover, the three mutants M₁-3, M₂-1, and M₁-5 were identified from our EMS-induced M₂ library, exhibiting the fusiform fruit, the dark green peel, and the yellow leaves, respectively. Compared to the wild type (WT), the photosynthetic pigments and parameters were negatively impacted in the yellow-leaf mutant M₁-5. For example, the total chlorophyll was 1.22 and 2.22 mg/g in the young and mature leaves of M₁-5, respectively, which were significantly lower than those in the WT (2.58 and 2.90 mg/g, respectively). Notably, some mutagenesis phenotypes could be stably inherited, including traits such as yellow leaf color, fusiform fruit shape, and thickening and thinning pericarp. Taken together, these results indicate that these two mutant libraries serve as essential resources to discover new phenotypic germplasms, thereby facilitating the genetic breeding and functional gene exploration in watermelon.

Sexual differentiation is an important developmental phenomenon in cucurbits that directly affects fruit yield. The natural existence of multiple flower types in melon offers an inclusive structure for studying the molecular basis of sexual differentiation. The current study aimed to identify and characterize the molecular network involved in sex determination and female development in melon. Male and female pools separated by the F₂ segregated generation were used for sequencing. The comparative multi-omics data revealed 551 DAPs and 594 DEGs involved in multiple pathways of melon growth and development, and based on functional annotation and enrichment analysis, we summarized four biological process modules, including ethylene biosynthesis, flower organ development, plant hormone signaling, and ubiquitinated protein metabolism, that are related to female development. Furthermore, the detailed analysis of the female developmental regulatory pathway model of ethylene biosynthesis, signal transduction, and target gene regulation identified some important candidates that might have a crucial role in female development. Two CMTs ((cytosine-5)-methyltransferase), one AdoHS (adenosylhomocysteinase), four ACSs (1-aminocyclopropane-1-carboxylic acid synthase), three ACOs (ACC oxidase), two ARFs (auxin response factor), four ARPs (auxin-responsive protein), and six ERFs (Ethylene responsive factor) were identified based on various female developmental regulatory models. Our data offer new and valuable insights into female development and hold the potential to offer a deeper comprehension of sex differentiation mechanisms in melon.

Flesh color is a critical commodity trait of melon, directly influencing consumer preference and the fruit's market value. This study investigates the crucial role of carotenoids in determining melon flesh coloration by examining color transitions and carotenoid profiles during fruit development in 'TianYu' (white flesh) and 'TianJu' (orange flesh) melons. Research has demonstrated that the color change in melon flesh results from carotenoid accumulation, with β-carotene being the primary compound responsible for the formation of orange flesh. Transcriptome analysis revealed that 2,459 and 947 differentially expressed genes (DEGs) were involved in melon pulp color change and orange pulp formation, respectively. Among these DEGs, the study identified and analyzed those related to carotenoid synthesis and metabolism. The results indicated that genes encoding key enzymes for pyruvate synthesis, phytoene synthase (PSY), phytoene desaturase (PDS), and lycopene β-cyclase (LCYB) play significant roles in fruit pulp color change and carotenoid accumulation. Notably, the genes encoding PDS (MELO3C017772.2) and LCYB (MELO3C020744.2) were found to be potentially critical genes for β-carotene synthesis and orange flesh formation. Moreover, functional enrichment analysis of DEGs emphasized the importance of transcriptional regulation and transcription factors such as MYB, WRKY, ERF, and bHLH, whose differential expression during color change suggests their potential to regulate carotenoid pathway genes. This study not only elucidates the molecular mechanisms underlying melon flesh color variation but also reveals the potential regulatory roles of key genes and transcription factors, providing valuable insights for enhancing melon quality, informing breeding strategies, and exploring potential genetic engineering approaches for color manipulation.