Cytosine methylation changes in the ice plant Ppc1 promoter during transition from C3 to Crassulacean acid metabolism

The induction of Crassulacean acid metabolism (CAM) in Mesembryanthemum crystallinum is characterized by an increase in the expression of CAM cycle-related genes. The expression of Ppc1, a CAM-specific member of the phosphoenolpyruvate carboxylase gene family, increases rapidly during transition from C3 carbon assimilation to CAM. We used bisulfite sequencing to detect the changes in the cytosine methylation of two GC-rich regions of Ppc1 in C3 and CAM leaves. In the 800 bp 5′-flanking sequence of Ppc1, four cytosines located at the promoter region were unmethylated in C3 leaves but methylated in CAM leaves and four out of five cytosines found in the 5′ untranslated region were methylated in C3 leaves yet demethylated in CAM leaves. Analysis of the sequence contexts of 9 cytosines revealed that 2 were GC, 2 were CTT, and 5 were C(A/T)G methylations. Within 72 h of salt treatment in 6-week-old plants, the increase in methylation of a CTG site at the TATA box of the Ppc1 promoter coincided with the increase in the expression of Ppc1. The sequence-specific and stress-responsive cytosine methylation of Ppc1 indicates that epigenetic regulation is involved in the activation of Ppc1 during CAM transition.