Abstract
An unprecedented number of studies have explored hormone levels in plants; however, only a small fraction includes comprehensive metabolite analyses spanning multiple hormone classes. Here, we aim to establish a unique and detailed resource integrating the absolute concentrations of diverse hormone classes and their metabolites in tomato floral organs and early fruit tissues across developmental stages. We quantified 58 hormone metabolites from six chemical classes in whole flower buds, individual floral organs at five developmental stages, mature pollen, and early fruit tissues up to 15 days after anthesis. Hormone profiling was complemented by matched transcriptomic and shotgun proteomic analyses. This integrated dataset revealed distinct spatial and temporal hormone signatures, including a gradual decline in active auxin levels-especially in stamens-contrasting with the accumulation of oxidized and conjugated auxin forms toward anthesis. Multi-omics analyses identified three GRETCHEN HAGEN 3(GH3) genes (GH3-2, GH3-7, and GH3-15) likely involved in auxin inactivation within reproductive organs. In vitro enzyme assays and transient overexpression in Nicotiana benthamiana confirmed their capacity to conjugate indole-3-acetic acid (IAA) to various amino acids. CRISPR/Cas9-generated single, double, and triple gh3 mutants showed increased levels of free IAA in mature stamens. Proteomic profiling of gh3-2 stamens revealed upregulation of stress-related proteins under normal conditions, whereas under heat stress, gh3-2 stamens exhibited fewer proteomic changes than the wild type. Moreover, pollen from gh3-2 and gh3-7 mutants maintained higher viability after prolonged heat stress. This study offers the most comprehensive hormone-focused multi-omics resource for tomato reproductive development to date. It provides a detailed map of hormone distribution across floral and early fruit tissues, and demonstrates its utility by uncovering a stamen-specific auxin conjugation mechanism that contributes to pollen thermotolerance.