Figure 1: FER mediates a ROS maximum at the filiform apparatus/synergid cell region.

(a) An ovule sketch showing a penetrating pollen tube. A, antipodal cells; FG, female gametohyte; CC, central cell; EC, egg cell; SC, synergid cell; FA, filiform apparatus; In, Oi, inner and outer integuments; M, micropyle; PT, pollen tube; VN, pollen tube vegetative nucleus; Sp, sperm cell. (b) Aniline blue-stained WT and fer-4 ovules (left) and quantification of pollen tube (PT)–ovule interaction defects (right). Arrowhead, a single, ruptured tube; arrows, multiple overgrown (reflecting non-rupture) tubes appearing as thick bundles. Data=average±s.e.m. from at least triplicate samples (each with at least three pistils) from a representative analysis; n=total number of ovules examined. **P≤0.01 by Student’s t-test. (c) H₂DCF-DA staining to show ovular ROS (left panel) and dose-dependent DPI inhibition (right panel). For comparative ROS analysis, a region of interest (ROI) with identical areas at the synergid cell/filiform apparatus region (Supplementary Fig. 2) from the ovules compared was quantified by ImageJ. Data in histogram=average±s.d. of ovules from at least three pistils (n=number of ovules examined) from a representative of three independent analyses. (d,e) Co-imaging of the discharged pollen tube cytoplasm (red, arrowheads) and ROS (green, arrows) in a wild-type ovule (d) and of pollen tube cytoplasm (red, arrowheads) and with FER-GFP (green, arrows) in a pFER::FER-GFP transformed ovule (e).