Figure 4.
Loss of function of ADF7 and ADF10 has differential effects on the actin cytoskeleton at the apex and subapex of pollen tubes. (A) Images of actin filaments stained with Alexa-Fluor-488-phalloidin in pollen tubes of WT, adf7, and adf10. Full and half Z projection images are shown in the left panel, and the distances from the tube tip are indicated on the upper left side. The corresponding optical slices are shown in the right panel. Regions covered by yellow shaded boxes and between two colored dotted lines indicate pollen tube tips. The green arrows indicate disorganized actin filaments in adf10 pollen tubes. Colored triangles at pollen tube tips indicate the accumulation of actin filaments close to the plasma membrane. Bar = 5 μm. (B) Transverse sections at the indicated distance from tube tips are shown in yellow shaded boxes in A. Blue arrows indicate actin filaments within the inner region of adf7 pollen tube. (C) Schematic diagram of the method for measuring the relative fluorescence intensity of actin filament staining at the tip of pollen tubes. The left panel shows that a line can be considered as being composed of many points. For a thin line, each point corresponds to a pixel. To measure the fluorescence intensity of actin filaments stained with Alexa-488-phalloidin at the pollen tube tip, a 3-μm wide line that fully covers the pollen tube tip region (0–3 μm from the tube tip) was drawn as shown in the right panel. The fluorescence intensity of each point with the width of one pixel was measured using the “Plot Profile” plugin in ImageJ. The intensity at each point was subsequently divided by the average fluorescence intensity within this 3-μm wide line to yield the relative fluorescence intensity of actin filament staining at each point. (D) Quantification of the relative fluorescence intensity of actin filament staining within the yellow shaded boxes shown in A at pollen tube tips. A 3-µm thick line was drawn, covering the yellow boxes shown in A, and the fluorescence intensity of each point on this line was divided by the average fluorescence intensity within the band to yield the relative fluorescence intensity (the method for measuring the relative fluorescence intensity of actin filament staining see the schematic diagram shown in C), which was plotted versus the distance across the pollen tube. The abscissa “0” indicates the extreme tip of the pollen tube. At least 35 pollen tubes were measured for each genotype. Data are presented as mean ± SD. (E) Plot of the fluorescence intensity of actin filament staining at different positions across the width of pollen tubes. (F) Time-lapse images of actin filaments decorated by Lifeact-eGFP in growing WT, adf7, and adf10 pollen tubes. Yellow dotted lines indicate the base of the apical actin structure and green arrows indicate disorganized actin filaments. Enlarged kymograph analyses of apical actin filaments during pollen tube growth are shown in the right panels. The ribbon corresponds to the base of the apical actin structure. Bars = 5 μm in all images. (G) Quantification of the distance from the tube tip to the base of the actin fringe in pollen tubes of WT, adf7, and adf10 via kymograph analysis. The red line indicates the average length of apical actin filaments. *P < 0.05 (Student’s t test), **P < 0.01 (Student’s t test). (H) Quantification of filament angles in apical and subapical regions of pollen tubes. A schematic diagram of angle measurement is shown on the left. The midline was set as a reference line in the pollen tube. The angles were measured between filaments and the midline towards the growth direction of pollen tubes. For filaments spanning the reference midline, the angles on both sides were included. ns, no statistical difference, **P < 0.01 (Student’s t test). (I) Schematic depiction of the role of ADF7 and ADF10 in regulating actin dynamics via coordination with the pH gradient in pollen tubes. Within apical and subapical regions of a WT pollen tube, actin filaments are mainly polymerized from the plasma membrane by the membrane-anchored class I formins and are arrayed into a distinct “apical actin structure” (Xu and Huang, 2020). ADF7 and ADF10 are two major actin-depolymerizing factors that promote the turnover of actin filaments but exhibit differential roles in response to the cytosolic pH gradient in pollen tubes. Specifically, ADF7 functions better at low pH and plays an important role in enhancing the turnover of actin filaments originating from the plasma membrane. This process is crucial for the generation of an apical region with fewer actin filaments. ADF10 functions better at high pH and plays a prominent role in enhancing the turnover and facilitating the organization of actin filaments at the alkaline subapex. The concerted action of ADF7 and ADF10 thus shapes the formation of the unique “apical actin structure” in the pollen tube.

Loss of function of ADF7 and ADF10 has differential effects on the actin cytoskeleton at the apex and subapex of pollen tubes. (A) Images of actin filaments stained with Alexa-Fluor-488-phalloidin in pollen tubes of WT, adf7, and adf10. Full and half Z projection images are shown in the left panel, and the distances from the tube tip are indicated on the upper left side. The corresponding optical slices are shown in the right panel. Regions covered by yellow shaded boxes and between two colored dotted lines indicate pollen tube tips. The green arrows indicate disorganized actin filaments in adf10 pollen tubes. Colored triangles at pollen tube tips indicate the accumulation of actin filaments close to the plasma membrane. Bar = 5 μm. (B) Transverse sections at the indicated distance from tube tips are shown in yellow shaded boxes in A. Blue arrows indicate actin filaments within the inner region of adf7 pollen tube. (C) Schematic diagram of the method for measuring the relative fluorescence intensity of actin filament staining at the tip of pollen tubes. The left panel shows that a line can be considered as being composed of many points. For a thin line, each point corresponds to a pixel. To measure the fluorescence intensity of actin filaments stained with Alexa-488-phalloidin at the pollen tube tip, a 3-μm wide line that fully covers the pollen tube tip region (0–3 μm from the tube tip) was drawn as shown in the right panel. The fluorescence intensity of each point with the width of one pixel was measured using the “Plot Profile” plugin in ImageJ. The intensity at each point was subsequently divided by the average fluorescence intensity within this 3-μm wide line to yield the relative fluorescence intensity of actin filament staining at each point. (D) Quantification of the relative fluorescence intensity of actin filament staining within the yellow shaded boxes shown in A at pollen tube tips. A 3-µm thick line was drawn, covering the yellow boxes shown in A, and the fluorescence intensity of each point on this line was divided by the average fluorescence intensity within the band to yield the relative fluorescence intensity (the method for measuring the relative fluorescence intensity of actin filament staining see the schematic diagram shown in C), which was plotted versus the distance across the pollen tube. The abscissa “0” indicates the extreme tip of the pollen tube. At least 35 pollen tubes were measured for each genotype. Data are presented as mean ± SD. (E) Plot of the fluorescence intensity of actin filament staining at different positions across the width of pollen tubes. (F) Time-lapse images of actin filaments decorated by Lifeact-eGFP in growing WT, adf7, and adf10 pollen tubes. Yellow dotted lines indicate the base of the apical actin structure and green arrows indicate disorganized actin filaments. Enlarged kymograph analyses of apical actin filaments during pollen tube growth are shown in the right panels. The ribbon corresponds to the base of the apical actin structure. Bars = 5 μm in all images. (G) Quantification of the distance from the tube tip to the base of the actin fringe in pollen tubes of WT, adf7, and adf10 via kymograph analysis. The red line indicates the average length of apical actin filaments. *P < 0.05 (Student’s t test), **P < 0.01 (Student’s t test). (H) Quantification of filament angles in apical and subapical regions of pollen tubes. A schematic diagram of angle measurement is shown on the left. The midline was set as a reference line in the pollen tube. The angles were measured between filaments and the midline towards the growth direction of pollen tubes. For filaments spanning the reference midline, the angles on both sides were included. ns, no statistical difference, **P < 0.01 (Student’s t test). (I) Schematic depiction of the role of ADF7 and ADF10 in regulating actin dynamics via coordination with the pH gradient in pollen tubes. Within apical and subapical regions of a WT pollen tube, actin filaments are mainly polymerized from the plasma membrane by the membrane-anchored class I formins and are arrayed into a distinct “apical actin structure” (Xu and Huang, 2020). ADF7 and ADF10 are two major actin-depolymerizing factors that promote the turnover of actin filaments but exhibit differential roles in response to the cytosolic pH gradient in pollen tubes. Specifically, ADF7 functions better at low pH and plays an important role in enhancing the turnover of actin filaments originating from the plasma membrane. This process is crucial for the generation of an apical region with fewer actin filaments. ADF10 functions better at high pH and plays a prominent role in enhancing the turnover and facilitating the organization of actin filaments at the alkaline subapex. The concerted action of ADF7 and ADF10 thus shapes the formation of the unique “apical actin structure” in the pollen tube.

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