Drought stress poses significant environmental challenges to agricultural plants, especially tomatoes, by hindering their growth and reducing yields. Biostimulants like fulvic acids (FA) have emerged promising strategies for mitigating drought effects and enhancing water-use efficiency. However, the regulatory mechanisms of FA-induced drought tolerance are not yet fully understood. This study aimed to characterize FA-induced drought tolerance mechanism in tomatoes. Four-week-old plants were treated with FA at 240 mg per plant, and drought conditions were imposed by withholding 75% of the water supplied to well-watered plants. The plant growth performance and the physiological responses were evaluated. Leaf samples were collected at two stages: the early drought stage (3 days after treatment) and the later stage (7 days after treatment), for untargeted hormonomics and metabolomics analysis using ultra-high-performance liquid chromatography coupled with high-resolution mass spectrometry (UHPLC-HRMS). Under drought conditions, control plants exhibited significant stress symptoms, including reduced height and leaf wilting during the later phase. In contrast, FA-treated plants developed less drought symptoms and improved stomatal conductance. The hormonomics and metabolomics analysis identified 114 hormones and 243 metabolites in ESI . Using orthogonal partial least-squares discriminant analysis (OPLS-DA), we determined that 39 hormones and 162 metabolites (with a VIP score > 1.0) were significant discriminants among the different treatments. Under drought conditions, 2-hydroxy melatonin and abscisic acid (ABA) levels were significantly increased in FA-treated plants, along with higher concentrations of amino acids such as glycine and threonine. These findings suggest that fulvic acids modulate the phytohormones ABA and melatonin to induce drought tolerances, orchestrating a response that enhances drought tolerance by sustaining elevated levels of osmoprotective amino acids.
