Data from: Improved demethylation in ecological epigenetic experiments: testing a simple and harmless foliar demethylation application

1. Experimental demethylation of plant DNA enables testing for epigenetic effects in a simple and straightforward way without the use of expensive and laborious DNA sequencing. Plants are commonly demethylated during their germination with the application of agents such as 5-azacytidine (5-azaC). However, this approach can cause unwanted effects such as underdeveloped root systems and high mortality of treated plants, hindering a full comparison with untreated plants, and can be applied only on plant reproducing by seeds. Here we test a simple alternative method of plant demethylation, designed to overcome the shortcomings of the germinating method. 2. We compared a novel method of demethylating plants, based on periodical spraying of 5-azaC aqueous solution on established seedlings, with the previous method in which seeds were germinated directly in 5-azaC solution. We quantified the amount of methylated DNA and measured various aspects of plant performance. Also, we demonstrated its applicability in ecological epigenetic experiments, by testing transgenerational effects of plant-plant competition. 3. We found that the spray application had similar DNA-demethylating efficiency than the germination method, particularly in the earlier phases of plant development, but without unwanted effects. The spray application method did not reduce plant growth and performance compared to untreated plants, as opposed to the traditional method which showed reduced growth. Also, the spray application method equalized the epigenetically-modified plant features of seedlings coming from plants grown under competition and plants growing without competition, demonstrating its application in ecological epigenetic experiments. 4. We conclude that regular spraying of 5-azaC solution onto established seedlings surpassed the germination-in-solution method in terms of vigor and fitness of treated plants. This novel method could thus be better suited for experimental studies seeking valuable insights into ecological epigenetics. Furthermore, the spray method can be suitable for clonal species reproducing asexually, and, most importantly, it opens the possibility of community-level experimental demethylation of plants.

1. 对植物DNA进行实验性去甲基化，可在无需开展昂贵且耗时的DNA测序的前提下，以简便直接的方式测试表观遗传效应。科研人员通常会在植物萌发阶段施加5-氮杂胞苷（5-azacytidine，5-azaC）等试剂来实现去甲基化。但该方法会引发诸多不良影响，例如植株根系发育不全、处理后植物死亡率居高不下，这会阻碍与未处理植株开展全面对比，且仅适用于以种子繁殖的植物。为此，本研究测试了一种简易的植物去甲基化替代方案，旨在克服传统萌发法的缺陷。
2. 本研究将一种新型植物去甲基化方法——即定期向定植幼苗喷施5-氮杂胞苷水溶液——与传统方法（将种子直接置于5-氮杂胞苷溶液中萌发）进行了对比。研究团队对甲基化DNA的含量进行了定量分析，并测定了植株生长表现的多项指标。此外，通过测试植物间竞争的跨代效应，本研究验证了该方法在生态表观遗传学实验中的适用性。
3. 研究结果显示，喷施法与萌发法的DNA去甲基化效率相近，尤其在植物发育的早期阶段，且不会引发不良影响。与传统方法会抑制植株生长不同，喷施法并未降低与未处理植株相比的生长及表现水平。此外，喷施法能够使来自竞争环境下生长植株的幼苗，与无竞争环境下生长植株的幼苗在表观遗传修饰特征上趋于一致，进一步证明了其在生态表观遗传学实验中的应用价值。
4. 本研究得出结论：相较于溶液萌发法，定期向定植幼苗喷施5-氮杂胞苷溶液的方法在处理植株的活力与适应度方面更具优势。因此，这种新型方法更适合用于旨在深入探究生态表观遗传学的实验研究。此外，喷施法还适用于无性繁殖的克隆物种，尤为重要的是，它为开展植物群落水平的实验性去甲基化研究提供了可能。