Papaya (Carica papaya L.) is a widely cultivated tropical and subtropical fruit crop valued for its rich nutritional content, diverse food industry applications, and the medicinal use of papain. However, bitterness in papaya fruit, particularly in fibrous strands, negatively affects fruit quality and consumer acceptance; therefore, the development of papaya cultivars with stable and desirable quality is of great importance. To identify the bitter compounds in papaya fruit fibrous strands and elucidate the molecular mechanisms underlying their biosynthesis, we performed transcriptomic and metabolomic analyses of fibrous strands from two papaya cultivars at three developmental stages. We identified carpaine, dehydrocarpaine II, and their derivative alkaloids. Furthermore, we identified two key regulatory genes, CpNAC82 and CpHD-Zip ANT2, associated with alkaloid biosynthesis. Finally, using single-nucleus RNA sequencing technology, we constructed a comprehensive gene expression atlas of papaya fibrous strands and stems, successfully identifying multiple cell types, including epidermal cells, guard cells, parenchyma cells, and phloem cells. Epidermal and phloem cells serve as the primary sites of alkaloid metabolism in papaya. These findings provide new insights into the molecular mechanisms of bitterness in papaya’s fibrous strands and yield genomic resources for improving fruit quality in papaya.

Papaya is a trioecious species, but a unified framework for comparing floral sex differentiation has been lacking. We combined stereomicroscopy, paraffin sectioning, and scanning electron microscopy to establish a continuous 12-stage developmental system from floral primordium initiation to anthesis. Male, female, and hermaphroditic flowers followed a common developmental trajectory during Stages 1–4 and diverged at Stage 5. Male flowers formed fertile stamens and a reduced pistillode, female flowers developed a complete gynoecium without stamens, and hermaphroditic flowers displayed normal, aborted, or transitional pistils and carpellodic stamens. RNA sequencing and weighted gene co-expression network analysis of morphologically defined tissues identified gene modules associated with normal pistil development, distinct modes of pistil abortion, and stamen carpellody. These modules were enriched in chromatin and RNA regulation, protein homeostasis, floral-organ identity, hormone and receptor-kinase signaling, pollen-wall formation, and stress-responsive pathways, yielding 30 representative candidate genes. This developmental framework integrates developmental morphology with transcriptional programs underlying reproductive-organ fate and plasticity in papaya.