Parallel gas chromatography and photoacoustic spectroscopy for quantifying ethylene responses to nine modulating compounds in Arabidopsis and wheat

Abstract Background Ethylene is a gaseous plant hormone controlling development, stress responses, and senescence, yet its quantitative measurement remains methodologically heterogeneous across plant research. Here, we present a unified experimental workflow that combines gas chromatography with flame ionization detection (GC–FID) and laser‑based photoacoustic spectroscopy (PAS) for the quantification of endogenous ethylene production in plants. Although both techniques are widely used, a direct experimental comparison using identical biological samples has not been reported previously. Results Using Arabidopsis seedlings and wheat leaf segments, we demonstrate that GC–FID and PAS values are offset by a system-dependent proportional factor while following highly consistent dose‑response trends across a broad range of ethylene‑modulating treatments. Across all compounds and concentrations, ethylene values measured by both methods were strongly correlated. GC–FID provided superior linearity at high ethylene outputs, whereas PAS offered high sensitivity for low‑to‑moderate ethylene levels and faster sample throughput. The workflow defines a consistent set of experimental parameters, including delayed treatment application, normalization of ethylene production to plant fresh weight (FW), and minimization of biomass‑related artefacts. Applying this approach, we systematically evaluated inhibitors and inducers of ethylene biosynthesis and signaling, including aminoethoxyvinylglycine (AVG), aminooxyacetic acid (AOA), 2‑aminoisobutyric acid (AIB), 2‑picolinic acid (2‑PA), 1-aminocyclopropane‑1‑carboxylic acid (ACC), silver thiosulfate (STS), thidiazuron (TDZ), ethephon (ETH), and mebendazole (MEB). The data reveal pronounced species‑specific sensitivities and clear toxicity thresholds for several compounds. Importantly, the parallel ethylene measurement workflow is compatible with downstream LC–MS/MS based phytohormone profiling. Correlation analysis of ethylene, its precursor ACC, multiple phytohormones, and FW is consistent with ACC availability as a significant determinant of ethylene production in Arabidopsis and wheat, while many apparent hormonal associations reflect indirect stress or growth effects rather than direct regulatory coupling. Conclusion This study provides a robust and extensible platform for ethylene quantification and interpretation across plant systems. The parallel GC–FID/PAS approach improves cross‑validation of measurements and enables direct integration of ethylene production into broader phytohormonal analyses, facilitating reliable comparison of ethylene‑related studies across laboratories and plant species.