Methylation levels at most of the 35S-eGFP+hp promoters increased over time (Figure 3B). However, some showed only a slight increase (e.g., P2, P5, P8, and P9) or a decrease (P1, P3, P4, P6, and P10, see Figure 1B,C). The decrease in P4 methylation coincided with the reduced GFP signal observed when protein was immunoblotting extracts made at 3 and 7 dpi with anti-eGFP (Figure 4A). This decrease in protein accumulation may be caused by a destabilization of polyproline II helices in protein rich regions of the eGFP+hp sequence (Dietz et al., 1996).
The methylation levels of individual promoters were separated into classes that did or did not contain a TATA box. In no case did methylation in the TATA-less promoters decrease over time (Figure 3C). This result was expected for P4 and P6 since these promoters do not contain a TATA box. P1, P3, and P5 contain a TATA box and showed a comparable progression of methylation levels to P2 on the TATA-less promoter in the 35S-eGFP+hp construct (Figure 3C) (Somers, 2003). The TATA-containing promoters P8 and P10 all showed a decrease in methylation over time, but P8 was the only class to show a decrease in protein accumulation at 3 or 7 dpi when protein was immunoblotting extracts (Figure 4A). This latter result is equivocal due to the anti-eGFP antibody’s specificity for the N-terminal part of the protein, and did not correspond to the C-terminal part (see Figure 1B,C). Co-expressed in the 35S-eGFP construct with 35S-eGFP+hp, P8 and P10 also exhibited decreased protein accumulation (Figure 4B). The P9 promoter did not contain a TATA box, but was still methylated. P9 showed a large increase in methylation at 3 dpi in the 35S-eGFP+hp construct; however, protein accumulation remained steady at 7 dpi (Figure 3C). d2c66b5586