Fig. 2: SYP72 maintains cellular integrity during pollen rehydration.

a, b syp72 mutation results in loss of plasma membrane integrity after rehydration. a Propidium iodide (PI) and fluorescein diacetate (FDA) double staining to assess plasma membrane integrity of mature pollen from the WT, the syp72-1 mutant, and the gSYP72 complementation line. Scale bars, 100 µm. b Frequency of FDA-positive pollen from the WT and three syp72 T-DNA insertion lines. Data represent the mean ± SD from four independent assays, with 500 pollen grains scored each time (n = 2,000). c Representative DIC images showing ruptured syp72 pollen during hydration (n = 60 biologically independent pollen grains). Scale bars, 10 µm. d, e DIC observation (d) and PI-FDA staining (e) assess plasma membrane integrity of mature pollen in the WT pistil during hydration. Scale bars, 50 µm. DIC observation of ruptured syp72 pollen grains in the pistil was performed three times, with three pistils analysed in each experiment (n = 9). f Frequency of FDA-positive pollen from the WT and the syp72-1 mutant in the WT pistil during hydration. Data represent the mean ± SD from four independent assays. (n = 658 pollen grains for WT and 528 for syp72-1). g Frequency of FDA-positive pollen from the WT and the syp72-1 mutant during a rehydration time course in distilled water. Data represent the mean ± SD from four independent assays, with 400 pollen grains scored each time. h, i Polyethylene glycol (PEG) treatment increases the frequency of FDA-positive pollen from the syp72-1 mutant during rehydration. h Representative images of PI-FDA double-stained syp72-1 pollen after PEG treatment. Scale bars, 100 µm. i Frequency of FDA-positive pollen from the syp72-1 mutant after PEG treatment. Data represent the mean ± SD from three independent assays, with 400 pollen grains scored each time. j, k PI-FDA double staining assessment of plasma membrane integrity in nondehiscent tricellular pollen grains dissected from anthers. j Representative images of PI-FDA-stained nondehiscent tricellular pollen. Scale bars, 100 µm. k Frequency of FDA-positive nondehiscent tricellular pollen grains from WT and syp72-1 plants. Data represent the mean ± SD from four independent assays, with 400 pollen grains scored each time. pc papilla cell. Arrows indicate ruptured pollen. Two-tailed Student’s t test was used for statistical analysis (*P < 0.05; ****P < 0.0001).