Functional Analyses of PtROS1-RNAi in Poplars and Evaluation of Its Effect on DNA Methylation

DNA methylation occurs mostly at the C5 position of dinucleotide symmetric CpG sites in genomic DNA. A balance is maintained in the plant genome between DNA methylation mediated by RNA-directed DNA methylation (RdDM) and DNA demethylation mediated by the DEMETER (DME) protein family and REPRESSOR OF SILENCING (ROS1). We used double-stranded RNA (dsRNA) silencing to suppress ROS1 protein expression in 'Nanlin895' (Populus deltoides x Populus euramericana 'Nanlin895'). Leaves of WT and transformant poplars revealed more symmetric methylation on CpG sites than roots and stems. In addition, leaves of transformant poplars revealed more methylated CpG sites in both 5.8S rDNA and histone H3 compared to WT types via 0, 50 and 100 mM NaCl treatments. In asymmetric methylation sites, transformant poplars exhibited more methylated CpHpG and CpHpH contexts than WT poplars. On the other hand, hypermethylation induced by PtROS1-RNAi construct resulted in pleiotropic phenotypic changes in transgenic poplars. The percentage of wavy leaves was increased maximum by similar to 45% in transgenic poplars. Also, the number of leaves was increased by similar to 200 number in transformants. Furthermore, shooting (%) and rooting (%) was decreased in transgenic poplars versus WT.