Many plant species protect themselves against herbivores through mechanical or chemical so-called inducible defences (ID). These are regulated via a hormonal cascade which may be under epigenetic control and in which jasmonic acid (JA) plays a prominent role.

In this study, we indirectly tested the role of DNA methylation in the production of ID and the synthesis of hormones involved in the ID signalling cascade. Using different intensities of 5-azacytidine application, we aimed to produce plants of Trifolium repens with different levels of DNA methylation alteration. We then elicited the plants together with controls, i.e. plants with natural DNA methylation status, with JA and then indirectly recorded ID production in herbivore-choice trials in which the leaves of plants with different DNA methylation statuses were provided to caterpillars of a generalist herbivore, Spodoptera littoralis.

We also analysed the balance of several key defence hormones such as jasmonates, abscisic acid (ABA), indole-3-acetic acid (IAA) and salicylic acid in the plants. We found that the Spodoptera littoralis preferred demethylated plants over non-demethylated controls. Demethylation also reduced production of JA, ABA and IAA. We conclude that DNA methylation modulates expression of ID likely via regulation of signalling hormones involved in the establishment of defence.

Cafeteria data collected as an area eaten by caterpillars. Endogenous levels of jasmonates, IAA, ABA and SA were determined in plant tissue (20 mg of fresh weight) according to the method described by Floková et al. (2014). These phytohormones were extracted using an aqueous solution of methanol (10% MeOH/H₂O, v/v). A cocktail of stable isotope-labelled standards was added as follows: 5 pmol of [¹³C₆]IAA, 10 pmol of [²H₆]JA, [²H₂]JA-Ile, and [²H₆]ABA, 20 pmol of [²H₄]SA and [²H₅]OPDA (all from Olchemim Ltd, Czech Republic) per sample to validate the LC-MS method. The extracts were purified using Oasis HLB columns (30 mg/1 ml, Waters) and targeted analytes were eluted using 80% MeOH. Eluent containing all compounds was gently evaporated to dryness under a stream of nitrogen. Neutral and acidic phytohormones were determined using ultra-high-performance liquid chromatography-electrospray tandem mass spectrometry (an Acquity UPLC I-Class System coupled to a Xevo TQ-S MS, all from Waters).