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

物种内多样性的减少和全球环境变化正在威胁生态系统的功能和人类社会。因此，在没有遗传变化的情况下，评估物种是否可能获得世代的抗逆性变得前所未有的紧迫。DNA甲基化被认为是为了促进快速适应，特别是在克隆植物中。然而，支持这一有争议的假说的实验证据很少。在我之前的工作中，我发现浮萍多根螺旋藻-一种克隆繁殖的水生被子植物-可以在没有遗传变化的情况下，通过世代获得对铜过量的抵抗力。基于这些结果和现状，我建议利用多根链霉菌来解决DNA甲基化在克隆植物跨代抗逆性中的作用的三个基本问题：1)自发和胁迫诱导的甲基组变体有多常见和稳定？2)胁迫诱导的甲基组变体是否介导了跨代抗性？3)群体能否通过对甲基组变体的选择来适应？为了回答这些问题，我们将首先评估自发的甲基组变化在多根草中的世代积累，并调查两种不同的胁迫--铜过量和棉铃虫的食草性--是否会诱导甲基组变异，并通过全基因组亚硫酸氢盐测序将其遗传给后代。接下来，我们将利用靶向代谢物分析、转录组分析和全基因组测序，评估多根链霉菌是否不仅表现出对铜过量的跨代抗性，而且还表现出对蚜虫食草性的跨代抗性，并鉴定与跨代抗性和胁迫诱导的甲基组变异相关的性状和基因。我们将利用CRISPR-Cas9和化学抑制来操纵已识别的基因和DNA甲基化机制，以推断应激诱导的甲基组变异和跨代耐药性之间的因果关系。最后，我们将通过操纵甲基组和选择的有效性来评估多根链霉菌是否通过对甲基组变体的选择来适应：在控制下，一个野生型和一个DNA甲基化机制缺陷的敲除突变体将在控制下生长80代，在最小的小种群(通过漂移进化)和大种群(进化和选择)中铜过剩和食草性蚜虫。评估在甲基组化学抑制下小种群和大种群之间以及野生型和突变体之间的抗性差异将使我们能够推断对甲基组变异体的选择是否有助于快速适应。总之，这些实验将为甲基组变异的来源及其对适应的贡献提供新的见解，从而检验进化生态学中一个有争议的假说。考虑到克隆植物在自然和农业生态系统中的重要性以及全球变化的快速步伐，结果将与不同的生物领域相关。

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.