Figure 3: ROS induce pollen tube rupture in the female gametophytes.

(a) Pistil-feeding assay. Excised wild-type pistils maintained in a reservoir of feeding medium and a single wild-type pistil pollinated by GUS-expressing pollen highlighting ovule targeting and cytoplasmic discharge (blue dots) in the female gametophytes. Supplementary Figure 5a shows an aniline blue-stained pollinated pistil after growth in the pistil-feeding assay, also confirming that normal pollen tube growth and ovule targeting were not affected. When fer-4 pistils were maintained in the pistil-feeding assays, they retained pollen tube–ovule interaction defects, harbouring comparable levels of pollen tube overgrowth as seen in pistils pollinated in planta (Supplementary Fig. 5b), confirming that the FER-regulated process was not affected in these assays. (b–e) Pollen tube rupture and entrance property in ovules from mock-treated (control) wild-type pistils and those fed with DPI, tiron and Na-benzoate. DPI inhibition is commonly used as a reflection of NADPH oxidase-dependent ROS synthesis, tiron and Na-benzoate are commonly used ROS scavengers⁶⁷ (Supplementary Fig. 3). Arrowheads in (b) indicate overgrown single-pollen tubes. ROS levels were reduced to almost non-detectable level in these treated ovules (Supplementary Fig. 5c,d). CuCl₂, another ROS scavenger (Supplementary Fig. 3), also reduced ovular ROS and induced pollen tube overgrowth (Supplementary Fig. 5d,e). Data=average ±s.e.m. from at least triplicate samples (each with at least three pistils) representative of three independent experiments; n=total number of ovules examined. **P≤0.01; *P≤0.05 as determined by Student’s t-tests. Scale bars, 250 μm (a); 100 μm (b).