Cytosine methylation occurs in a CDC48 homologue and a MADS-box gene during adventitious shoot induction in Petunia leaf explants1

Abstract

The DNA methylase inhibitors, 5-azacytidine and 5-aza-2'-deoxycytidine inhibited adventitious shoot induction in Petunia leaf cultures. Cytosine methylation status at CCGG sites in shoot- and callus-inducing culture treatments was analysed by coupled restriction enzyme digestion (HpaII or MspI) and random amplification. Two differentially methylated genomic DNA bands from the PCR products were cloned (OPU9-1 and OPU9-2) and sequenced. The open reading frames contained in OPU9-1 and OPU9-2 showed similarity to CDC48 and MADS-box genes, respectively. Cytosine methylation was restored at CCGG sites when the leaf explants were transferred from medium containing the drugs to medium without the drugs, simultaneously recovering the ability to develop adventitious shoot buds. Furthermore, combined bisulphite treatment and restriction analysis revealed differential methylation of CGCG sites in the drug-treated and control cultures. These results demonstrate that cytosine methylation at CCGG and CGCG sites within a MADS-box gene and a CDC48 homologue, among others, shows strong positive correlation with adventitious shoot bud induction in Petunia leaf explants.