plants Article Phytotoxicity, Morphological, and Metabolic Effects of the Sesquiterpenoid Nerolidol on Arabidopsis thaliana Seedling Roots 1, 2, 3 3 Marco Landi y , Biswapriya Biswavas Misra y , Antonella Muto , Leonardo Bruno and Fabrizio Araniti 4,* 1 Department of Agriculture, Food and Environment, University of Pisa, 56126 Pisa, Italy; [email protected] 2 Independent Researcher, Pine 211, Raintree Park Dwaraka Krishna, Namburu AP-522508, India; [email protected] 3 Dipartimento di Biologia, Ecologia e Scienze della Terra (DiBEST), Università della Calabria, 87040 Arcavacata di Rende, CS, Italy; [email protected] (A.M.); [email protected] (L.B.) 4 Department AGRARIA, University Mediterranea of Reggio Calabria Località Feo di Vito, 89124 Reggio Calabria, RC, Italy * Correspondence: [email protected] Both authors equally contributed to the manuscript. y Received: 26 August 2020; Accepted: 10 October 2020; Published: 12 October 2020 Abstract: 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, Arabidopsis thaliana 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 H2O2 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 IC50 (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 H2O2 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. Keywords: phytotoxicity; herbicide; root morphology; sesquiterpene alcohol; metabolomics 1. Introduction Weeds are one of the major threats to global agroecosystems, as they affect both crop productivity and quality [1]. The use of synthetic herbicides that are easy to apply and are economically accessible to farmers, is one of the popular and effective methods of weed management [2]. Nevertheless, the excessive use of chemical herbicides has negatively influenced the ecological equilibrium and human health [3]. Moreover, it has been clearly demonstrated that the majority of the known herbicides Plants 2020, 9, 1347; doi:10.3390/plants9101347 www.mdpi.com/journal/plants Plants 2020, 9, 1347 2 of 19 target a single specific metabolic action site [4–6], which is the main factor resulting in a rapidly evolving resistance to these synthetic chemicals. Herbicides with new mechanisms of action are extremely needed to counter this rapidly increasing evolution of herbicide resistance [7]. In addition, the attention of the public to possible hazardous effects of chemical herbicide to human health is continuously increasing, and new research activities are actively moving toward the search of naturally-derived herbicides, based on the allelopathic properties of some natural compounds [8–10]. Nowadays, it is of utmost importance for the use of a combination of agronomic, physical, mechanical, and chemical strategies for weed control in an Integrated Weed Management System (IWMS) [11,12] framework. In particular, the prospect of using secondary plant metabolites as natural herbicides, or as the backbone for herbicide discovery programs is becoming an effective alternative to the classic synthetic herbicides [13]. Natural compounds can effectively inhibit weed performances, act simultaneously on specific, and in most cases, multiple targets [14–17]. This ability to alter plant metabolism at different biochemical checkpoints increase the effectiveness of these compounds, but at the same time, it renders the identification of their main and most effective modes of action challenging [18]. Moreover, the chemical structure of natural products is generally more complex than synthetic herbicides, which means that they would not be easily obtained by traditional synthetic approaches based on massive chemical syntheses with the production of countless compounds whose biological activity is totally unknown [19]. Nerolidol (C15H26O; MW, 222.37 Da; IUPAC: 3,7,11-Trimethyl-1,6,10-dodecatrien-3-ol; also known as peruviol and penetrol), is a naturally occurring sesquiterpene alcohol found in the essential oils of diverse plants and flowers [20], and contributes to the fragrance/essence of the plants. This compound is largely studied for its multi-faceted pharmacological and biological activities [20–22] as well. Other compounds belonging to the sesquiterpenoid classes have shown a strong herbicidal activity [14,23]. In addition, nerolidol has already been mentioned as an important component of Asteraceae essential oil, whose natural herbicide activity was demonstrated [24]. Further, in our pilot studies, we have found that nerolidol resulted as a promising candidate among other sesquiterpenes (unpublished data) for allelopathy. Many allelochemicals, as stress inducers in acceptor plants, cause morphological alterations, and reduce weed germination and vigor [17,23,25–27]. However, their effectiveness in germination and growth inhibition of the weeds is, in most cases, the result of more complex metabolic alteration induced in the affected plants [16,27]. It is, therefore, essential to understanding the biochemical mechanism of action of these naturally-derived compounds for a future application as either a whole molecule, as a co-formulant, or as a backbone for a derived herbicide. In the present study, we focused on the effects of nerolidol on root morphology and root metabolome of Arabidopsis thaliana (L.) Heynh. Columbia-0 seedlings were treated for 14 d with a range of concentrations of this sesquiterpene alcohol. We also probed the effects of this compound on auxin balance to understand the underlying mechanism(s) responsible for the nerolidol-induced root alterations and the associated strong growth inhibition to elucidate the single- or multi-target activity of this potential allelochemical herbicide. 2. Materials and Methods 2.1. Root Bioassays in Arabidopsis thaliana Col-0 Cis-Nerolidol (Sigma-Aldrich, Milan, Italy, Cat. No. 72180-25ML) was firstly dissolved in 0.1% EtOH (ethanol, v/v) and was then diluted in deionized ddH2O to reach the final concentrations of: 0, 50, 100, 200, 400 and 800 µM. The same amount of EtOH was added to the control treatments (0 µM nerolidol). Arabidopsis seed sterilization and synchronization of the germination process were carried out as previously described by Araniti et al. [28]. To evaluate the phytotoxic effect of nerolidol on root morphology, seeds of Arabidopsis were germinated on Petri dishes (100 150 mm) containing agar × Plants 2020, 9, 1347 3 of 19 medium (0.8% w/v), enriched with micro- and macronutrients (Murashige-Skoog basal salt mixture, Sigma-Aldrich, Milan, Italy, Cat. No. M5524-50L) and supplemented with 1% sucrose as carbon source. Then, petri plates were transferred to a growth chamber (21 2 C temperature and 75 mol m 2 s 1 ± ◦ · − − light intensity). Immediately after germination of five seedlings, for each replicate and treatment, seedlings were transferred on treated Plates prepared as previously described. After 14 d of treatments the whole root system was imaged by scanning (STD 1600, Régent Instruments Inc., Quebec, QC, Canada) and Primary Root Length (PRL), and Number of Lateral Roots (NLR) were measured using WinRhizo Pro system v. 2002a (Instruments Régent Inc., Quebec, QC, Canada), whereas Root Hair Density (RHD) and Root Hair Length (RHL) were analyzed using a stereo-microscope (Olympus SZX9, Italy) and the software Image Pro Plus v6 (Media Cybernetics, Rockville, MD, USA). 2.2. Quantification of Indole-3-Acetic Acid (IAA) Quantification of indole-3-acetic acid (IAA) quantification was carried out following the method proposed by Rawlinson et al. [29] with some modifications. Arabidopsis roots were cut with a sharp blade below the hypocotyl, and were immediately frozen in liquid nitrogen, ground to a fine powder and were aliquoted. Further, weighed amounts of the powder (100 mg) per treatment and replicate were poured into 2 mL microcentrifuge tubes for extraction. To the samples, 20 µL of a 20 mg/mL solution of 3-indolepropionic acid (IPA) was added as internal standard for quantification and normalization purposes. Successively, 200 µL of NaOH (1% w/v), 147 µL of methanol (MeOH) and 34 µL of pyridine were added and the samples were vortexed for 40 s. To the extracted samples 20 µL of methyl chloroformate were added and samples were again vortexed for 30 s (and this step was repeated twice). To this extract, 400 µL of chloroform was added, samples were shaken for