Fig. 2

Time course of transcriptional silencing of P35S::GFP. β-estradiol was applied at time 0 to three BY-2 lines (8, 19, and 35). The establishment of TGS of P35S::GFP was monitored in the selected timepoints at the level of a GFP fluorescence (flow cytometry of about 10 thousand cells per sample); b transcription of the GFP and the P35S promoter hairpin (RT qPCR); c proportion of methylated cytosines in the target region (means of about 10 clones per sample after bisulfite conversion); d proportion of promoter-specific siRNAs (of about 50 mil reads per sample); e correlation between the level of GFP silencing (i.e., relative decrease in GFP expression) and cytosine methylation (relative to the maximal attainable level) and siRNA level (relative to the maximal attainable value; only for line 8). Error bars in (b) and (c) indicate standard errors, and asterixis in (c) indicate a significant difference (Mann–Whitney U test, p < 0.05) for lines 8 and 19