Fig. 2.

DOGL4 imprinting requires RdDM-mediated methylation of the paternal promoter. (A) Paternal silencing of DOGL4 in the endosperm is dependent on RdDM pathway. RdDM pathway-defective mutants including nrpd1, nrpde1, dcl3, and rdr2 were respectively used as pollen donors to cross with dogl4-1 mutant, and the expression of DOGL4 in these derived endosperms were determined and compared. Data are means ± SD of three technical replicates. (B and C) Expression analysis of GUS reporter gene in embryo and endosperm of F₁ seeds from reciprocal crosses between WT and a 3.7-kb DOGL4 promoter-driven GUS transgenic line (L4) (A) and between WT and a transgenic line (L14) with 1.9-kb DOGL4 promoter (B). Data are means ± SD of three technical replicates. (D) Diagram of bisulfite sequencing on four segments covering 1.9-kb promoter of DOGL4. (E) Bisulfite sequencing of the four segments of DOGL4 promoter in 7–9 DAP endosperm of Col wild type and the result showed that DNA methylation of paternal allele in CG sequence context at fragment 3 was increased compared with that of maternal allele. (F) Bisulfite sequencing of fragment 3 in F₁ endosperms from reciprocal crosses between Col and C24 ecotypes. (G) Bisulfite sequencing of fragment 3 in F₁ endosperm of Col/nrpd1.