Figure 3.
ADF7 is more abundant than ADF10 globally, and the two ADFs are differentially localized within the apical and subapical regions of the pollen tube. (A) Detection of ADF7 and ADF10 in Arabidopsis mature pollen by Western-blot analysis. ADF in total protein from Arabidopsis mature pollen was detected with an anti-ADF7 antibody, which recognizes both ADF7 and ADF10. UGPase, detected with an anti-UGPase antibody, was used as the loading control. The experiment was repeated more than three times and a representative result is shown. (B) Measurement of the relative abundance of ADF proteins in total protein from WT, adf7, and adf10 mature pollen. The average gray values of bands in A were measured. The relative abundance of ADF7 and ADF10 was calculated by dividing the gray values of the band probed with anti-ADF7 antibody by that of the band probed with anti-UGPase antibody. The relative abundance is marked on the top of each column. (C) The estimated concentration of total ADF, ADF7, and ADF10 in the cytosol. The calculation is described in the Materials and methods. (D) Western blot of total protein from Arabidopsis mature pollen derived from the transgenic plants ADF7pro::ADF7-eGFP;adf7 and ADF10pro::ADF10-eGFP;adf10. The blot was probed with anti-UGPase and anti-GFP antibodies. The experiment was repeated more than three times and a representative result is shown. (E) Ratio of ADF7 versus ADF10 in pollen. The ratios of ADF7 versus ADF10 and ADF7-eGFP versus ADF10-eGFP are presented. (F and G) Distribution of ADF7-eGFP (F) and ADF10-eGFP (G) in the XY, YZ, and XZ directions of the pollen tube. Z projection and X projection images are shown in the left panel with the corresponding 3D surface plot of fluorescence pixel intensity. Transverse sections at the indicated distance from the tube tips are shown in the right panel corresponding with the 3D distribution of fluorescence pixel intensity. Warm and cold colors indicate high and low fluorescence intensity, respectively. Bars = 5 μm in all images. (H and I) Quantification of the concentration of ADF7-eGFP and ADF10-eGFP from the extreme tip to the base of the pollen tube. The average concentration of ADF7-eGFP and ADF10-eGFP in different regions (0–3 μm, 3–10 μm, and 10–30 μm from the tube tip) is marked above the curve. More than 60 pollen tubes were measured for each protein. Data are presented as mean ± SD. (J) Percentage of ADF7-eGFP and ADF10-eGFP in different regions of pollen tubes. (K) Schematic showing the distribution of protein concentration and the assumed actin-depolymerizing activity of ADF7 and ADF10 in the pollen tube. The amount and activity of ADF7 and ADF10 are shown on the left and right sides, respectively, of the same pollen tube. Deeper colors represent higher disassembling activity. The color coding for the pH gradient is shown underneath the model. Source data are available for this figure: SourceData F3.

ADF7 is more abundant than ADF10 globally, and the two ADFs are differentially localized within the apical and subapical regions of the pollen tube. (A) Detection of ADF7 and ADF10 in Arabidopsis mature pollen by Western-blot analysis. ADF in total protein from Arabidopsis mature pollen was detected with an anti-ADF7 antibody, which recognizes both ADF7 and ADF10. UGPase, detected with an anti-UGPase antibody, was used as the loading control. The experiment was repeated more than three times and a representative result is shown. (B) Measurement of the relative abundance of ADF proteins in total protein from WT, adf7, and adf10 mature pollen. The average gray values of bands in A were measured. The relative abundance of ADF7 and ADF10 was calculated by dividing the gray values of the band probed with anti-ADF7 antibody by that of the band probed with anti-UGPase antibody. The relative abundance is marked on the top of each column. (C) The estimated concentration of total ADF, ADF7, and ADF10 in the cytosol. The calculation is described in the Materials and methods. (D) Western blot of total protein from Arabidopsis mature pollen derived from the transgenic plants ADF7pro::ADF7-eGFP;adf7 and ADF10pro::ADF10-eGFP;adf10. The blot was probed with anti-UGPase and anti-GFP antibodies. The experiment was repeated more than three times and a representative result is shown. (E) Ratio of ADF7 versus ADF10 in pollen. The ratios of ADF7 versus ADF10 and ADF7-eGFP versus ADF10-eGFP are presented. (F and G) Distribution of ADF7-eGFP (F) and ADF10-eGFP (G) in the XY, YZ, and XZ directions of the pollen tube. Z projection and X projection images are shown in the left panel with the corresponding 3D surface plot of fluorescence pixel intensity. Transverse sections at the indicated distance from the tube tips are shown in the right panel corresponding with the 3D distribution of fluorescence pixel intensity. Warm and cold colors indicate high and low fluorescence intensity, respectively. Bars = 5 μm in all images. (H and I) Quantification of the concentration of ADF7-eGFP and ADF10-eGFP from the extreme tip to the base of the pollen tube. The average concentration of ADF7-eGFP and ADF10-eGFP in different regions (0–3 μm, 3–10 μm, and 10–30 μm from the tube tip) is marked above the curve. More than 60 pollen tubes were measured for each protein. Data are presented as mean ± SD. (J) Percentage of ADF7-eGFP and ADF10-eGFP in different regions of pollen tubes. (K) Schematic showing the distribution of protein concentration and the assumed actin-depolymerizing activity of ADF7 and ADF10 in the pollen tube. The amount and activity of ADF7 and ADF10 are shown on the left and right sides, respectively, of the same pollen tube. Deeper colors represent higher disassembling activity. The color coding for the pH gradient is shown underneath the model. Source data are available for this figure: SourceData F3.

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