Extended Data Fig. 3: Ovular NO status in wild type and NO-deficient mutants.
From: FERONIA controls pectin- and nitric oxide-mediated male–female interaction
a, Comparison of two methods of ovule NO quantification. Ovules from pollinated (16 h after pollination) and unpollinated wild-type pistils at the same developmental stage were examined. Images were acquired with identical conditions and a sampling of ovules is shown. In the direct scoring method, ovules were scored as positive (+) when signal at the filiform apparatus (arrow, filiform apparatus) was notably higher than in the synergid cell (right histogram). In the filiform-apparatus/synergid-cell signal-intensity-ratio method, average signal intensity of identical areas at the filiform apparatus and in the synergid cell (as background) was determined by ImageJ. A filiform apparatus synergid cell ratio of ≥1.2 was scored as + (left dot plot). The two methods gave comparable conclusions. Data are average ± s.d. n, number of ovules. P values were obtained by two-tailed t-tests. b, Full ovule images for Fig. 2b. The ejected pollen cytoplasm (red) marked the synergid cell, providing a clear spatial definition for NO located at the filiform apparatus (arrow). c, Comparison of filiform apparatus:synergid cell NO signal intensity in pollinated wild-type and fer-4 ovules (left dot plot) and direct scoring (right histogram). Results were comparable. Data (left dot plot) are average ± s.d. n, number of ovules. d, NO in wild-type, nia1 nia2 and noa1 mutant ovules. The results correlated reduced filiform-apparatus NO in these pollen-tube-penetrated ovules with elevated multiple pollen tube entrance (Fig. 2e). Data are average ± s.d. n, number of pistils. Numbers in plot denote the number of ovules examined. P values were obtained by two-tailed t-tests. Data are representative of three independent experiments. Scale bars, 50 μm. Box plots: centre line, median; box limits, lower and upper quartiles; dots, individual data points; whiskers, highest and lowest data points.
