Cytosine methylation of plastid genome in higher plants. Fact or artefact?

DNA methylation of chloroplast genome has been studied in a large variety o f angiosperm species using restriction enzyme analysis of three genomic loc i (totally encompassing about 10% of chloroplast genome) and bisulfite geno mic sequencing of tobacco ribulose bisphosphate carboxylase/oxygenase (larg e subunit) gene (rbcL). Except for CCWGG (W = A or T) sites that were parti ally refractory to the cleavage with methylation sensitive EcoRII in all lo ci. no cytosine methylation was found at the CCGG (dMspI/HpaII) and several other restriction sites tested. However, EcoRII was unable to completely d igest an unmethylated CCWGG site in the cloned rbcL gene on plasmid. Furthe r a bisulfite genomic sequencing performed on EcoRII-restricted DNA failed to show any 5-methylcytosine either within or outside inspected EcoRII site s along the 3 ' end of rbcL coding region. In conclusion our results do not support evidence for methylated cytosine residues in plant chloroplast gen omes and we suggest that results obtained with EcoRII should be interpreted with great care especially when small differences in methylation levels ar e analysed. (C) 2001 Elsevier Science Ireland Ltd. All rights reserved.