(A) Frequency of seeds containing a synergid nucleus at different endosperm stages (n for 2nES = 67/72, 4nES = 143/158, and 8nES = 179/141 for wild type/ecs1 ecs2-1, respectively). The image shows the second synergid nucleus at the two-nucleate endosperm stage. (B) Quantitative analyses of single fertilizations 8-10 HAP in ecs1 ecs2 (n=90) and wild-type ovules (n=89) using HTR10-mRFP. Successful double fertilization (DF) is visualized by two dispersed sperm nuclei signals (arrow heads). In the middle and right panel, only one dispersed sperm cell nuclear signal (arrow heads) can be observed, while an additional sperm nuclear signal (arrows) is condensed suggesting the unfertilized state. The positions of the dispersed sperm signals correspond to the position of central cell (middle) and egg cell (right), respectively, and thus suggest different single fertilization patterns (CC-F and EC-F). (C–E) Analyses of seed development 3 days after dual pollination. (C) Diagram of the dual pollination experiment. (D) Successful double fertilization by a single pollen tube from either pRPS5A::H2B_tdTomato (left panel) or pRPS5A::H2B_GFP (middle panel). Heterofertilization involving the content of two genetically distinct pollen tubes is depicted in the right panel. Endosperm nuclear signal (arrow heads), embryo nuclear signal (arrows). (E) Percentage of heterofertilization events in wild type (n=276) and ecs1 ecs2-1 (n=284) ovules. (F) Fluorescence signal in 6-day-old progenies of ecs1ecs2-1 female plants upon paternal introduction of pRPS5A::H2B-tdTomato. WT: wild-type like seeds, showing paternal marker expression in both embryo and endosperm. Only EN (I): paternal marker expression only in endosperm. Only EM (II): paternal marker expression only in embryo. Dotted line; embryo, yellow arrowhead; sperm nuclei. (G) Percentage of 6-day-old progenies of ecs1ecs2-1 or wild-type female plants crossed with pRPS5A::H2B-tdTomato, showing paternal marker expression in different fertilized tissues. (H) Assessment of paternally introduced marker expression in endosperm (EN) and embryo (EM) of 6-day-old siliques in ecs1 ecs2-1 or wild type. The ecs1 ecs2-1 seeds were divided into a normally looking and an abnormal seed fraction and analyzed individually. EM+EN; expression in both embryo and endosperm. (G) and (H), n=349/332 for wild type/ecs1 ecs2-1, respectively. Brightness was manually enhanced with Adobe Photoshop in (A–C). Data in (A), (B), (G), and (H) indicate mean ± SD. Two-tailed Mann-Whitney comparison test between wild type and mutants, *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. Scale bars, 20 μm (A), 25 μm (B), 75 µm (D), 50 μm (F).