Sequence‐specific hypomethylation of the tobacco genome induced with dihydroxypropyladenine, ethionine and 5‐azacytidine

Higher plant DNA is methylated at CG and CNG targets. In this study we have investigated the tobacco methylation system in tissue culture using the methylation inhibitors 5-azacytidine (5-azaC), dihydroxypropyladenine (DHPA) and ethionine (Ethi), and methylation-sensitive restriction endonucleases HpaII, MspI, HhaI, EcoRII, ScrFI, and Fnu4HI. Surprisingly, CAG/CTG sequences, contrary to CG doublets and CCG/CGG triplets, appeared to be refractory to the inhibitory effect of 5-azaC. Thus 5-azaC cannot be considered a general inhibitor of DNA methylation in tobacco cells. On the other hand, DHPA, the inhibitor of S-adenosylhomocysteine (SAH) hydrolase, and Ethi caused hypomethylation of both CAG/CTG and CCG/CGG triplets but not of the CG doublets. The sensitivity of triplet-specific methylation to the inhibition of SAH hydrolase suggests the possibility that plant-specific DNA methylation at CNG targets might be modulated by alterations of the SAH/S-Adenosylmethionine ratio in plant cells.