Experimental DNA demethylation associates with changes in phenotype and gene expression of tree seedlings

Epigenetic modifications, such as DNA methylation where methyl groups are added to cytosine base pairs, have the potential to impact phenotypic variation and gene expression, and could influence the ability of plants to respond to changing environmental conditions. One approach to determining the association between DNA methylation, phenotypic variation, and gene expression, is through the application of chemical demethylation agents, such as 5-Azacytidine, which inhibit DNA methylation and lead to a partial reduction in DNA methylation across the genome. In this study, we treated 5-month-old seedlings of the tree species of Quercus lobata with foliar application of 5-Azacytidine to test whether a reduction in genome-wide methylation would cause differential gene expression and change phenotypic development. First, we demonstrate that foliar application of 5-azayctidine lead to a 3-6% absolute reduction and 6.1-43% relative reductions in genome-wide methylation across CG, CHG, and CHH sequence contexts, with CHH showing the strongest relative reduction. We also show that the demethylation treatment had minimal impact on the soil microbiome. Seedlings treated with 5-Azacytidine showed a substantial reduction in new growth, which was less than half that of control seedlings, and but no significant differences in fluctuating asymmetry (i.e., deviations from bilateral symmetry) of leaves compared to control seedlings. Finally, treated seedlings showed differential expression of a total of 23 genes relative to control seedlings, the majority of which were upregulated. Overall, this study provides evidence that DNA methylation is involved in gene expression and phenotypic variation in seedlings.