Multi-Omics Profiling of Female Ovary Development Identifies Key Defects in Seed Production of Autotetraploid Watermelon

The autotetraploid watermelon lines serves as female parents to produce triploid seedless watermelon and their seed production directly impacts seed yield and high costing for triploid watermelon cultivars. Studying the key factors involved in autotetraploid seed production is critical for the triploid seedless watermelon programs. In this study, histological examination, antioxidant enzyme profiling, transcriptomic, and metabolomic analyses were employed to identify key developmental defects within the female ovary of the autotetraploid watermelon line K5M 4x which resulted in reduced seed production relative to the diploid line. Scanning electron microscopy revealed that diploid ovaries exhibited rapid pollen germination, directional pollen tube growth, and efficient penetration into the transmitting tract following pollination. In contrast, autotetraploid ovaries displayed delayed pollen germination, reduced pollen tube density, and aberrant tube morphology. Furthermore, ovary tissue analysis demonstrated that autotetraploid ovules were predominantly underdeveloped, resulting in significantly reduced seed set compared to the diploid line. Antioxidant enzyme activities including CAT, SOD, PPO, and POD were consistently elevated in autotetraploid ovaries, revealing enhanced oxidative stress and compensatory mechanisms. Transcriptomic analysis identified 1,027 and 686 differentially expressed genes at 1 DAP and 2 DAP, respectively, with enriched pathways including phenylpropanoid biosynthesis, plant hormone signal transduction, protein processing in the endoplasmic reticulum, and brassinosteroid biosynthesis. Metabolomic profiling revealed significant enrichment of tryptophan, tyrosine, starch and sucrose, and cysteine and methionine metabolic pathways. Integrated transcriptome–metabolome analysis revealed stage-specific gene–metabolite networks regulating auxin signaling, antioxidant enzyme homeostasis, and carbohydrate flux as primary determinants of impaired ovule development and reduced reproductive output in autotetraploid watermelon. These findings indicate that autotetraploidy alters pollen–pistil interactions and early ovule development, ultimately reducing reproductive efficiency in watermelon.