Methylome Changes During In Vitro Regeneration Of Populus trichocarpa

Cytosine DNA methylation (5mC) is an epigenetic modification involved in gene expression regulation. DNA methylation has been implicated in the observed phenotypic variation among plants clonally propagated in in vitro culture systems. We investigated the in vitro methylome of the model tree species Populus trichocarpa, comparing internode explants, dedifferentiated calli, and internodes from regenerated plants, using methylated DNA immunoprecipitation followed by high-throughput sequencing. Using an exploratory false discovery rate (Q<0.1), we found that more than half (56%) of the methylated portion of the genome was differentially methylated among in vitro tissues. Methylation of four categories of transposons increased through dedifferentiation and redifferentiation. Gene body methylation was more frequent in callus than in either explants or regenerated internodes. Fifty percent of differentially-methylated genes underwent transient methylation, i.e. becoming methylated in calli, then demethylated in regenerants, and these genes mapped to large euchromatic regions on at least half of poplar’s nineteen chromosomes. Enriched Gene Ontology categories among transiently-methylated genes included stress response, cellular signaling, and protein catabolism. Comparisons of mC data with expression microarray data showed that expression of body-methylated genes was statistically lower than that of unmethylated or promoter-methylated genes, except in callus tissue, where expression of body-methylated genes was higher. Expression of promoter-methylated genes did not differ significantly from that of unmethylated genes in any tissue. Genes methylated at both promoters and gene bodies had lower expression than unmethylated or promoter-methylated genes. Overall, DNA methylation varied in a highly context-specific manner during in vitro regeneration.