The role of DNA methylation in transgenerational stress resistance in a clonal plant

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Dwindling intraspecific diversity and global environmental change are threatening ecosystem functioning and human society. Assessing whether species may acquire stress resistance across generations in the absence of genetic change has thus never been more urgent. DNA methylation is hypothesized to facilitate rapid adaptation, particularly in clonal plants. Experimental evidence for this controversial hypothesis is however scarce. In my previous work, I found that the duckweed Spirodela polyrhiza – a clonally reproducing aquatic angiosperm – can acquire resistance to copper excess across generations in the absence of genetic change. Based on these results and the state of the art, I propose to use S. polyrhiza to address three fundamental questions on the role of DNA methylation in transgenerational stress resistance in clonal plants: 1) How common and stable are spontaneous and stress-induced methylome variants? 2) Do stress-induced methylome variants mediate transgenerational resistance? 3) Can populations adapt via selection on methylome variants? To answer these questions, we will first assess the accumulation of spontaneous methylome changes across generations in S. polyrhiza and investigate whether two contrasting stresses – copper excess and herbivory of the aphid Rhopalosiphum nymphaeae – induce methylome variants that are passed on to offspring using whole-genome bisulfite sequencing. Next, we will assess whether S. polyrhiza exhibits transgenerational resistance not only to copper excess but also to aphid herbivory and identify traits and genes that are associated with transgenerational resistance and stress-induced methylome variants using targeted metabolite analysis, transcriptomics and whole-genome bisulfite sequencing. We will manipulate the identified genes and the DNA methylation machinery with CRISPR-Cas9 and chemical inhibition to infer causation between stress-induced methylome variants and transgenerational resistance. Finally, we will assess whether S. polyrhiza adapts via selection on methylome variants by manipulating both the methylome and the efficacy of selection: a wild type and a knockout mutant deficient in the DNA methylation machinery will be grown for 80 generations under control, copper excess and aphid herbivory in minimally small (evolving through drift) and large (evolution with selection) populations. Assessing variation in resistance between small and large populations and between wild type and mutant under chemical inhibition of the methylome will allow us to infer whether selection on methylome variants facilitates rapid adaptation. Together, these experiments will provide novel insights into the sources of methylome variation and its contribution to adaptation, thereby testing a controversial hypothesis in evolutionary ecology. Considering the importance of clonal plants in natural and agricultural ecosystems and the rapid pace of global change, the results will be relevant to diverse biological fields.