Transcriptomic analysis reveals the molecular basis of photoperiod-regulated sex differentiation in tropical pumpkins (Cucurbita moschata Duch.)

Abstract

Background: Photoperiod, or the length of the day, has a significant impact on the flowering and sex differentiation of photoperiod-sensitive crops. The "miben" pumpkin (the main type of Cucurbita moschata Duch.) is well-known for its high yield and strong disease resistance. However, its cultivation has been limited due to its sensitivity to photoperiod. This sensitivity imposes challenges on its widespread cultivation and may result in suboptimal yields in regions with specific daylength conditions. As a consequence, efforts are being made to explore potential strategies or breeding techniques to enhance its adaptability to a broader range of photoperiods, thus unlocking its full cultivation potential and further promoting its valuable traits in agriculture.

Results: This study aimed to identify photoperiod-insensitive germplasm exhibiting no difference in sex differentiation under different day-length conditions. The investigation involved a phenotypic analysis of photoperiod-sensitive (PPS) and photoperiod-insensitive (PPIS) pumpkin materials exposed to different day lengths, including long days (LDs) and short days (SDs). The results revealed that female flower differentiation was significantly inhibited in PPS_LD, while no differences were observed in the other three groups (PPS_SD, PPIS_LD, and PPIS_SD). Transcriptome analysis was carried out for these four groups to explore the main-effect genes of sex differentiation responsive to photoperiod. The main-effect gene subclusters were identified based on the principal component and hierarchical cluster analyses. Further, functional annotations and enrichment analysis revealed significant upregulation of photoreceptors (CmCRY1, F-box/kelch-repeat protein), circadian rhythm-related genes (CmGI, CmPRR9, etc.), and CONSTANS (CO) in PPS_LD. Conversely, a significant downregulation was observed in most Nuclear Factor Y (NF-Y) transcription factors. Regarding the gibberellic acid (GA) signal transduction pathway, positive regulators of GA signaling (CmSCL3, CmSCL13, and so forth) displayed higher expression levels, while the negative regulators of GA signaling, CmGAI, exhibited lower expression levels in PPS_LD. Notably, this effect was not observed in the synthetic pathway genes. Furthermore, genes associated with ethylene synthesis and signal transduction (CmACO3, CmACO1, CmERF118, CmERF118-like1,2, CmWIN1-like, and CmRAP2-7-like) showed significant downregulation.

Conclusions: This study offered a crucial theoretical and genetic basis for understanding how photoperiod influences the mechanism of female flower differentiation in pumpkins.

Keywords: Cucurbita moschata; Ethylene biosynthetic and ethylene response pathways; Gibberellin signaling pathway; Photoperiod; Photoperiod-mediated flowering processes; Sex differentiation.

Fig. 1

Flowering difference in PPIS versus PPS plants growing under moderate LD and SD conditions: (A) PPIS_LD, (B) PPS_LD, (C) PPIS_SD, (D) PPS_SD. Red arrows indicate first female flower bud

Fig. 2

Pearson correlation analysis and principal component analysis were conducted on 12 samples. (A) Pearson correlation illustrating the gene expression relationships among the 12 samples. (B) Principal component analysis plot displaying the clustering of RNA sequencing data for all sample types

Fig. 3

Gene expression patterns were obtained by hierarchical clustering analysis. Differentially expressed genes (DEGs) among four groups were categorized into six clusters depending on their expressions. Levels of gene expression are represented along the y-axis as log2(ratio), and four groups were represented along the x-axis as PPIS_LD, PPIS_SD, PPS_SD, and PPS_LD

Fig. 4

Gene ontology enrichment analysis of the genes of subclusters (1, 3, and 5) based on the biological process, molecular function, and cellular component

Fig. 5

DEGs from subclusters (1, 3, and 5) are associated with the photoperiodic flowering pathway, GA signaling pathway, and ethylene biosynthetic and ethylene response pathways

Fig. 6

Comparative analysis of the expression of GA20ox as gibberellin (GA) biosynthetic genes and GA2ox as the GA-deactivating enzyme, and the content of four kinds of GA in PPS and PPIS seedlings under LD and SD treatments. The GA levels are normalized to ng^.g^–1 F.W. n.d., not detected; n.q., not quantified. In all cases, the data are represented as means ± SD (n = 3). Values followed by the same letter were not significantly different (P > 0.05). This determination was made by a one-way analysis of variance followed by a post hoc Tukey’s HSD (Honestly Significant Difference) test

Fig. 7

Validation of gene expression patterns by real-time quantitative PCR

Fig. 8

Schematic diagram of the molecular basis of photoperiod-regulated sex differentiation in pumpkin