5% CG and 13. 1% CHG methylation, respectively. In comparison, the methylation rates of T3 stage seedlings were more than 5 fold increased with 84. 5% CG and 83. 3% CHG methylation. At the asymmetric sites, the CHH methylations levels were Verdinexor (KPT-335)? 9 fold increased from 2. 7% in T2 to 25. 3% in T3 seed lings. The clearly increased levels of 35S promoter methylation were consistent Inhibitors,Modulators,Libraries with the observed loss of gene expression in this generation. De novo cytosine methylation is only acquired during vegetative growth To trace methylation changes of the 35S promoter at different times during plant growth, Inhibitors,Modulators,Libraries we sequentially sampled leaf material 30, 45 and 60 days post germin ation. The three silencing affected lines showed in both generations much higher 35S promoter methylation rates compared to the control lines.

The line PNA 8. 6. 1 indi cated the lowest methylation levels and showed through out the sampling period over both generations mean rates of only 0. 9% in CG, 1. 3% in CHG and Inhibitors,Modulators,Libraries 0. 8% in CHH methylation. These extremely low methylation levels in dicate a complete DNA conversion during the bisulfite treatment and therefore a negligible false positive signal due to incomplete conversion. The second control was investigated until the T4 generation and showed in all three generations consistent low rates of 35S promoter methylation. These two stable expressing lines indicated no tendency for an increase in 35S promoter methylation after a generational change or during vegetative growth. In contrast, the unstable lines ICE 4. 4, PNA 1. 2 and PNA 10.

1 all showed increasing levels of 35S promoter methylation during growth. As a consequence the methylation levels deviated strongly between seedlings and flowering plants within the same generation. For instance, the CHG methylation levels of line PNA 1. 2 indicated Inhibitors,Modulators,Libraries only 13. 1% in seedlings but 90. 9% in flowering plants. This re sembles an absolute methylation increase during plant development of more than 77% within only 45 days. The most rapid cytosine methylation increase was observed between seedlings and rosette stage plants, where the CG and CHG levels changed within 15 days with a velocity of more than 3% per day. Although the ICE 4. 4 line started initially with higher methylation levels in seedling stage, it followed a similar trend and all three independent lines showed a similarly dramatic increase in methylation over time.

During the growth of T3 plants, the promoter methylation levels increased only slightly and reached a plateau like level at around 90% for CG and CHG sites and ca. 30% for CHH sites. Surprisingly, at the generational transition low differ ences could be observed between the T2 and T3 plants. The mean methylation levels of the Inhibitors,Modulators,Libraries T3 seedlings inhibitor MG132 were highly similar to the levels found in the flowering T2 plants.