CRP4 coding sequence is unmethylated in leaves when transcribed from 35S promoter to produce translatable mRNA. (A) Bisulfite sequencing to assess methylation in leaves of the CRP4 coding region in a CRP4-GFP fusion gene under the control of the constitutive 35S promoter (left) and the synergid cell-specific CRP4 promoter (right). The region shown corresponds to that indicated by the yellow bar in Figure 2. We also performed bisulfite sequencing on the GFP coding sequence in the CRP4 promoter-directed construct but observed no methylation (data not shown). (B) Western blot detection of the CRP-GFP fusion protein in seedlings from four independent transgenic lines transformed with a 35S promoter-CRP4-GFP fusion construct (lanes 1–4, top band, top arrow). The ‘+’ lane shows control GFP expression in a previously published transgenic line (top band, bottom arrow) [23, 24]. The lower band in these lanes probably corresponds to a degradation product that reacts with the GFP antibody. A CRP4-GFP fusion protein is not detected in transgenic plants containing a CRP4-GFP fusion gene under the control of the CRP4 promoter (lanes 5 and 6; results from two independent lines shown). Tubulin is shown as a constitutively expressed control.