HYPERMETHYLATION OF TOBACCO HETEROCHROMATIC LOCI IN RESPONSE TO OSMOTIC-STRESS

Plants have to cope with a number of environmental stresses which may potentially induce genetic and epigenetic changes and thus contribute to genome variability. In the present study we inspected the DNA methy lation status of two heterochromatic loci (defined with repetitive DNA sequences HRS60 and GRS) in a tobacco cell culture exposed to osmotic stress. Investigations were performed on a TBY-2 cell suspension cult ure, and the stress was elicited with NaCl or D-mannitol. Using the re striction enzymes MspI/IHpaII and MboI/Sau3AI in combination with Sout hern hydridization we observed a reversible hypermethylation of the ex ternal cytosine at the CpCpG trinucleotides in cells grown under mild osmotic stress equal to a NaCl concentration of 10 g/l. There were no changes in the methylation of the internal cytosine as the CpG dinucle otides within the CCGG motifs (HpaII sites) appeared to be fully methy lated in tobacco DNA repetitive sequences under normal physiological c onditions. The data suggest epigenetic changes in the plant genome bas ed on de novo methylation of DNA in response to environmental stress.