Knockdown of BRP4 expression arrests the mitotic cell-cycle progression in male gametophytes. (A, B) DAPI staining of tetrads, microspores, and bicellular and tricellular pollen from WT (A) and transgenic proBRP4:BRP4 RNAi L7 (B) plants. Note that the abnormal uninucleate pollen (red arrows) and bicellular-like pollen (green arrow) are still present at the bicellular and tricellular development stages in transgenic proBRP4:BRP4 RNAi plants. The yellow arrow indicates shrunken pollen. Bar, 20 µm. Tds, tetrads; MSp, microspores; BP, bicellular pollen; TP, tricellular pollen. (C) Quantification of different types of abnormally developed pollen from WT and transgenic proBRP4:BRP4 RNAi plants (L3 and L7). BP, bicellular pollen, TP, tricellular pollen. At the BP stage, about 1000 pollen grains were examined, and approximately 2500 pollen grains were examined at the TP stage. (D) Distribution of the DNA content of generative-cell nuclei at prophase from WT bicellular pollen (n=42) and generative-like cell nuclei at anthesis in proBRP4:BRP4 RNAi L3 (n=55) and L7 (n=50). The relative C-value of each nucleus was determined by the DAPI fluorescence value normalized against the mean fluorescence of WT nuclei (DNA=2C).