Fruit growth and development are generally initiated following successful pollination and fertilization. Seedless chestnut rose (Rosa sterilis), an elite promising fruit tree for both edible and medicinal purposes due to the extremely high vitamin C and superior quality, exhibits a naturally parthenocarpic character, however the underlying mechanism has been still unclear so far. Currently, gibberellins (GAs) were justified as the key hormone for parthenocarpy induction in seedless chestnut rose by endogenous hormone analysis and exogenous plant growth regulator (PGR) application. In total, 43 members of the GA oxidase gene family (RsGAoxs) were systematically identified and characterized based on genome-wide analysis of seedless chestnut rose. On the basis of transcriptomic analysis, overexpression experiments in tomato, as well as virus-induced gene silencing (VIGS) assay in seedless chestnut rose, RsGA3ox9 was substantially justified to be involved in the parthenocarpic fruitsetting of this species. Transcription factors RsMYB3, RsMYB8, and RsMYB73 were proven to positively regulate the expression of RsGA3ox9. Further, yeast two-hybrid (Y2H) and luciferase complementation assay illuminated that RsMYB8 and RsMYB73 may interact, leading to upregulating RsGA3ox9. Thereby, RsGA3ox9 substantially regulates parthenocarpy of seedless chestnut rose, and RsMYB8-RsMYB73 complex promotes parthenocarpic fruitsetting by upregulating RsGA3ox9, which may facilitate the seedless fruit breeding in chestnut rose (Rosa roxburghii Tratt.), as well as provide novel insights for better understanding the mechanism underlying the parthenocarpic fruitsetting in fruit species.

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