Atypical epigenetic mark in an atypical location: cytosine methylation at asymmetric (CNN) sites within the body of a non-repetitive tomato gene

We describe the existence of CNN-methylated epialleles that span Asr1, a non-transposon, protein-coding gene from tomato plants that lacks an orthologous counterpart in Arabidopsis. In addition, to test the hypothesis of a link between epigenetics modifications and the adaptation of crop plants to abiotic stress, we exhaustively explored the cytosine methylation status in leaf Asr1 DNA, a model gene in our system, resulting from water-deficit stress conditions imposed on tomato plants. We found that drought conditions brought about removal of methyl marks at approximately 75 of the 110 asymmetric (CNN) sites analysed, concomitantly with a decrease of the repressive H3K27me3 epigenetic mark and a large induction of expression at the RNA level. When pinpointing those sites, we observed that demethylation occurred mostly in the intronic region.These results demonstrate a novel genomic distribution of CNN methylation, namely in the transcribed region of a protein-coding, non-repetitive gene, and the changes in those epigenetic marks that are caused by water stress. These findings may represent a general mechanism for the acquisition of new epialleles in somatic cells, which are pivotal for regulating gene expression in plants.Epigenetics refers to mitotically and meiotically heritable variation in gene regulation and function that cannot be accounted for by changes in DNA sequence but rather results from enzyme-mediated chemical modifications to DNA and its associated chromatin proteins [1]. Over the last decade, epigenetic research has focused mainly on mammals, whereas plants have received less attention, although there is a fair amount of information on certain plant models such as Arabidopsis [2,3], rice [4] and maize [5].Whereas methylation in animal genomes occurs mostly in regulatory regions, methylation in Arabidopsis is found in transcribed sequences, not only at canonical CG sites but also at CNG (N denotes A, C or T) and CNN (asymmetric) sites. The latter sit