Validated phytohormone analysis with paired untargeted workflow enables new insights into soil phytohormonome

Abstract

The contribution of soil chemistry to plant growth and resilience, including presence of phytohormones, is increasingly recognized, yet characterization is limited by chemical complexity of soil matrices, diversity and low-abundance of metabolites. To enable further discoveries, we developed and characterized performance of a liquid chromatography-mass spectrometry method with solid phase extraction, integrating targeted and untargeted hormonomic approaches for comprehensive soil phytohormone profiling. Method performance was evaluated for fifteen plant growth-regulating compounds and precursors, including abscisic, gibberellic, jasmonic, and salicylic acids, auxins, cytokinins, karrikins, melatonin, and tryptophan, showing strong linearity (R{superscript 2} = 0.989-0.999), sensitivity (limits of detection and quantification 0.1-50.2 and 1.4-167.3 pg on-column, respectively), and precision (1.3-9.6% intraday; 3.4-34.8% interday). Soil composition impacted recovery; however, for most phytohormones rates were within 20% of matrix-adjusted spiked value, showing robustness across sandy, peat-rich, and clay-rich textures and suitable for use. We used the method to quantify analytes in research-relevant, active soils. Integration of untargeted analysis expanded coverage to 250 additional putative phytohormones and related metabolites, revealing chemical signatures potentially associated with plant community composition. This approach provides a versatile framework for investigating belowground phytohormone dynamics and their roles in plant physiology, resilience, and soil-plant feedbacks.