| Summary: | Natural herbicides that are based on allelopathy of compounds, can offer effective alternatives to chemical herbicides towards sustainable agricultural practices. Nerolidol, a sesquiterpenoid alcohol synthesized by many plant families, was shown to be the most effective allelopathic compound in a preliminary screening performed with several other sesquiterpenoids. In the present study, <i>Arabidopsis thaliana</i> seedlings were treated for 14 d with various cis-nerolidol concentrations (0, 50, 100, 200, 400, and 800 µM) to investigate its effects on root growth and morphology. To probe the underlying changes in root metabolome, we conducted untargeted gas chromatography mass spectrometry (GC-MS) based metabolomics to find out the specificity or multi-target action of this sesquiterpenoid alcohol. Oxidative stress (measured as levels of H<sub>2</sub>O<sub>2</sub> and malondialdehyde (MDA) by-product) and antioxidant enzyme activities, i.e., superoxide dismutase (SOD) and catalase (CAT) were also evaluated in the roots. Nerolidol showed an IC<sub>50</sub> (120 µM), which can be considered low for natural products. Nerolidol caused alterations in root morphology, brought changes in auxin balance, induced changes in sugar, amino acid, and carboxylic acid profiles, and increased the levels of H<sub>2</sub>O<sub>2</sub> and MDA in root tissues in a dose-dependent manner. Several metabolomic-scale changes induced by nerolidol support the multi-target action of nerolidol, which is a positive feature for a botanical herbicide. Though it warrants further mechanistic investigation, nerolidol is a promising compound for developing a new natural herbicide.
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