Strawberry (Fragaria × ananassa) is one of the most popular fruits consumed in the United States, valued for its taste, nutritional benefits, and economic importance. Achieving year-round production in the United States has become essential to meet the growing consumer demand for fresh, locally grown strawberries. Controlled environment agriculture (CEA), including greenhouse and vertical farm-based productions, hold great promise for year-round strawberry production. A key factor in achieving year-round strawberry production is understanding the regulations of flowering. While genetic aspects of flowering in strawberries have been widely studied, the role of hormonal regulation remains relatively understudied. This experiment aims to characterize the hormonal regulations of flowering in the perpetual strawberry cultivars (‘Albion’ and ‘Seascape’). We investigated the flowering time and pattern of two perpetual strawberry cultivars grown from bare-root transplants under greenhouse conditions. Additionally, we used an untargeted hormonomics approach using ultra-high performance liquid chromatography coupled with high-resolution mass spectrometry (UHPLC-HRMS) at three key developmental stages: 3-leaf, runnering, and flowering. The cultivar variations in runnering and flowering were observed. ‘Albion’ showed flowering approximately eight weeks post-transplantation, whereas ‘Seascape’ primarily exhibited vegetative growth, characterized by minimal flowering and significantly higher runner production than ‘Albion’. The hormonomics analysis identified 102 hormone-related compounds in ESI mode, spanning several hormonal pathways such as cytokinins (CK), tryptophan (Trp) derivatives, gibberellins (GA), melatonin, jasmonic acid (JA), salicylic acid (SA), abscisic acid (ABA), auxins (IAA), and brassinosteroids (BR). CK was the most abundant group (27%), followed by Trp derivatives (23%) in both cultivars. Partial least squares–discriminant analysis (PLS-DA) showed that among the hormones, CK, GA, and precursor of JA and IAA are primary discriminants between two cultivars and different developmental stages. In the seascape, the accumulations of CK (N6-benzyladenine-7-glucoside, thidiazuron) and GA (gibberellin A5) increased as the developmental stage progressed. Conversely, accumulation of cis-12-oxo-phytodienoic acid (OPOD), a precursor for the JA biosynthesis, increased in the ‘Albion’ as developmental stages progressed. This obtained data reveals a complex interaction of phytohormones involved in the regulation of strawberry flowering. CK and GA accumulation are favorable for vegetative growth, while JA may play a significant role in strawberry flowering. These findings improve our understanding of phytohormonal regulation of perpetual strawberry flowering and could lead to effective strategies for managing flowering time and sustainable year-round strawberry production in CEA.
