Epigenetic mechanisms are important for control of plant development, and may play a particularly important role in trees given their long life cycles and distinctive and stable tissue types. To help understand the role of epigenetics in tree development, we produced transgenic poplars with reduced activity of the DDM1 genes, whose activity are known to be critical for the maintenance of DNA methylation in plant genomes. DNA methylation is widely recognized as a major element of epigenetic variation, where its presence is usually associated with loss of gene expression. The DDM1 gene is necessary for the maintenance of DNA methylation in the extensive heterochromatic fractions of the genome.
We identified two highly similar DDM1 homologs in poplar (PtDDM1) and used RNAi to suppress both of their transcripts with a single construct. PtDDM- RNAi transgenic poplars showed a wide range of suppression efficiency, with the most strongly suppressed gene insertion events (lines) having a reduction in RNA expression of 70% based on combined stem and leaf in vitro materials and real time RT-PCR. Six transgenic lines were analyzed for their total cellular cytosine DNA methylation by HPLC (High Performance Liquid Chromatography). The DNA methylation percentages were generally correlated with PtDDM1 expression. A greenhouse study identified variation in growth rate associated with events, but these were not associated with PtDDM1 gene expression. There were also no visible differences in morphology of the transgenic lines. However, after dormancy, transgenic trees with strong PtDDM1 suppression that were growing out of doors in a covered "lathouse" showed a severe mottled leaf phenotype in all of its ramets, and two other transgenic events with strong PtDDM1 suppression showed similar but less severe symptoms in some of their ramets. A study of in vitro callogenesis of stem explants showed an inverse correlation between DDM1 expression and the percentage of rapidly growing callus; however, these results were not repeated with leaf explants or in a second experiment with a different experimental design. The second study showed a positive correlation between PtDDM1 expression and callus size. We speculated that the reduced methylation promoted tissue dedifferentiation, redifferentiation, and cell division, and that the mottled leaf phenotype was a result of DNA methylation change in the poplar genome. Our results suggest that transgenic demethylation may be a useful tool for promoting in vitro regeneration, but requires considerably more study of different target genes and suppression methods.