Variation in DNA methylation and response to short-term herbivory in Thlaspi arvense

Abstract

Plant metabolic pathways and gene networks involved in the response to herbivory are well-established, but the impact of epigenetic factors as modulators of those responses is less understood. Here, we use the demethylating agent 5-azacytidine to uncover the role of DNA cytosine methylation on phenotypic responses after short-term herbivory in Thlaspi arvense plants that came from two European populations with contrasting flowering phenotypes expected to differ in the response to experimental demethylation. The experimental design followed a 2 × 3 factorial design, that was replicated for each flowering-type. First, half the seeds were submerged in a water solution of 5-azacytidine and the other half only in water, as controls. Then, we assigned control and demethylated plants to three herbivory categories (i) insect herbivory, (ii) artificial herbivory, and (iii) undamaged plants. The effects of the demethylation and herbivory treatments were assessed by quantifying genome-wide global DNA cytosine methylation, concentration of leaf glucosinolates, final stem biomass, fruit and seed production, and seed size. For most of the plant traits analysed, individuals from the two flowering-types responded differently. In late-flowering plants, global DNA methylation did not differ between control and demethylated plants but it was significantly reduced by herbivory. Conversely, in early-flowering plants, demethylation at seed stage was still evident in leaf DNA of reproductive individuals whereas herbivory did not affect their global DNA methylation. In late-flowering plants, artificial herbivory imposed a stronger reduction than insect herbivory in global DNA methylation and final stem biomass, and induced higher concentration of aliphatic glucosinolates. In early-flowering plants, the effects of herbivory were non-significant for the same traits. Finally, the effect of herbivory on reproductive parameters varied with the level of demethylation and the plant flowering-type. Although further investigations with more populations and families are required to confirm our results, they suggest that the genetic background of experimental plants and timing of damage can affect the response to herbivory and point towards multifaceted genetic-epigenetic interactions in determining herbivory-induced phenotypic plasticity.