Volume 8, Issue 12, Pages 1694-1709 (December 2015) Profilin Regulates Apical Actin Polymerization to Control Polarized Pollen Tube Growth  Xiaonan Liu, Xiaolu Qu, Yuxiang Jiang, Ming Chang, Ruihui Zhang, Youjun Wu, Ying Fu, Shanjin Huang  Molecular Plant  Volume 8, Issue 12, Pages 1694-1709 (December 2015) DOI: 10.1016/j.molp.2015.09.013 Copyright © 2015 The Author Terms and Conditions

Figure 1 PRF4 and PRF5 Redundantly Regulate Polarized Pollen Tube Growth. (A) Physical structures of the PRF4 and PRF5 genes and the insertion of T-DNA alleles. Both genes contain three exons (black boxes) and two introns (gray boxes). prf4 (N375684) was inserted in the promoter region of PRF4, and prf5 (Salk_116290) was inserted in the second intron of PRF5. (B and C) prf4 and prf5 are knock-down alleles. (B) The expression of PRF4 and PRF5 was determined by semi-quantitative RT–PCR and quantitative real-time (qRT) PCR. The left panel shows semi-quantitative RT–PCR analysis of PRF4 and PRF5 transcript levels. eIF4A was used as the internal control. The middle and right panels show the qRT PCR analysis of PRF4 and PRF5 transcript levels. eIF4A was used as the internal control. (C) Western blot probed with anti-PRF5 serum. Anti-tubulin antibody was used as the control. Two micrograms of Arabidopsis pollen total extract was loaded per lane. The left right panels show the representative immunoblot of profilin level and the quantitative analysis of relative amount of profilin in pollen, respectively. The data represent mean ± SE, n = 3. *P < 0.05, **P < 0.01 by Student's t-test. (D) Profilin mutants have pollen germination percentage similar to that of WT. The germination percentage for WT and profilin mutants at 3 h was plotted. (E) The average length of pollen tubes is decreased in profilin mutants. The length of WT, prf4, prf5, and prf4 prf5 pollen tubes was determined after germinating for 2 h on standard germination medium, and the length distribution was plotted. (F) Pollen tube growth is slower in profilin mutants. WT, prf4, prf5, and prf4 prf5 pollen was germinated on standard germination medium. Micrographs were obtained when the average length of the pollen tubes reached about 300 μm (0 min) and then again after a further 40 min. Scale bar, 50 μm. (G) Loss of function of PRF4 or PRF5, or both, inhibits pollen tube growth. (H and I) Quantification of the morphological change in profilin loss-of-function pollen tubes. (H) The images in the panel on the left show that prf4 prf5 pollen tubes grow crookedly. Scale bar, 25 μm. The green and red lines indicate the actual length and linear length of the pollen tube, respectively. The actual length was divided by the linear length and plotted versus genotype to evaluate the growth behavior of the tubes, as shown in the panel on the right. (I) The width of pollen tubes is increased in prf4 prf5 mutants. Error bars in (D), (G), and (I) represent SE, error bars in (H) represent SD. Statistical analysis was performed with Student's t-test: *P < 0.05; **P < 0.01. Molecular Plant 2015 8, 1694-1709DOI: (10.1016/j.molp.2015.09.013) Copyright © 2015 The Author Terms and Conditions

Figure 2 The F-Actin Level Decreases and Actin Filaments Become Disorganized in Profilin Mutant Pollen Tubes. (A) Organization of actin filaments in WT, prf4, prf5, and prf4 prf5 pollen tubes. Actin filaments were revealed by staining with Alexa-488 phalloidin. Scale bar, 5 μm. Red boxed region corresponds to the subapex and apex. Figure 4C shows the enlarged Z-series stacks of actin filament organization in this region. (B) The F-actin level decreases in profilin mutant pollen tubes. The apical–subapical region refers to the region about 10 μm away from the tip. The shank region refers to the region about 20 μm away from the tip. Error bars represent SE. The statistical difference was determined by Student's t-test. *P < 0.05; **P < 0.01. (C) The angles between the actin filaments and pollen tube growth axis are larger in the subapical regions of profilin loss-of-function pollen tubes. Histograms of the angle distribution are shown for WT, prf4, prf5, and prf4 prf5 pollen tubes. More than 1000 actin filaments from 27 pollen tubes of each genotype were counted. (D) Average filament lengths are reduced in the subapical regions of profilin loss-of-function pollen tubes. Histograms of filament length distribution are shown for WT, prf4, prf5, and prf4 prf5 pollen tubes. More than 300 actin filaments from 27 pollen tubes of each genotype were counted. Molecular Plant 2015 8, 1694-1709DOI: (10.1016/j.molp.2015.09.013) Copyright © 2015 The Author Terms and Conditions

Figure 3 Tip-Directed Vesicle Transport Is Impaired and the Cell Wall Composition Is Altered in prf4 prf5 Pollen Tubes. (A and B) RabA4b-positive vesicles are diffusely distributed in prf4 prf5 pollen tube tips. (A) Representative images of WT and prf4 prf5 pollen tubes expressing Lat52:YFP-RabA4b. The average pixel fluorescence intensity in the red and blue boxed regions was determined for use in (B). Scale bar, 5 μm. (B) The accumulation of RabA4b-positive vesicles is decreased in prf4 prf5 pollen tubes. The average pixel fluorescence intensities in the red and blue boxed regions (shown in A) were determined, and the value in the red region was divided by the value in the blue region to yield a ratio to assess the extent of accumulation of RabA4b-positive vesicles. Error bars represent SE. (C and D) RabA4b-positive vesicles are less dynamic in prf4 prf5 pollen tubes. (C) Time course of recovery of YFP-RabA4b fluorescence after photobleaching. Red boxes indicate the photobleached regions. Scale bar, 5 μm. (D) The recovery rate of YFP-RabA4b fluorescence is reduced in prf4 prf5 pollen tubes. YFP-RabA4b fluorescence was plotted against recovery time. The data were obtained from more than 15 pollen tubes and averaged, which were subsequently fitted with the exponential function. Error bars represent SE. (E–H) Immunocytological characterization of WT and prf4 prf5 pollen tubes. Pollen tubes were immunostained with JIM7 (E) and JIM5 (G) antibodies to detect pectins with high and low degrees of esterification, respectively. Median optical sections of pollen tubes were captured and the relative labeling intensities along the meridional cell surface were quantified (F and H). The black curve represents the mean relative fluorescence of all analyzed tubes; gray areas represent the SD. Scale bar, 5 μm. Molecular Plant 2015 8, 1694-1709DOI: (10.1016/j.molp.2015.09.013) Copyright © 2015 The Author Terms and Conditions

Figure 4 Actin Polymerization at the Apical Membrane Is Defective in prf4 prf5 Pollen Tubes. (A) Actin filaments continuously grow out from the apical membrane during the growth of WT pollen tubes. The growing actin filaments (decorated with EGFP-lifeact) that originate from the apical membrane are highlighted in green. Scale bar, 10 μm. (B) The amount of actin filaments is sustainably maintained during the growth of WT pollen tubes. The upper panel indicates a pollen tube of interest. The two green lines mark the region containing bright actin filaments originating from the apical membrane. The yellow band indicates the region that was subjected to kymograph analysis. Scale bar, 10 μm. (C) Actin filaments within the region marked by green dashed lines are brighter than those further from the apex. (D) Actin filaments continuously emerge from the apical membrane of prf4 prf5 pollen tubes during growth. Growing actin filaments originating from the apical membrane are shown in green. Scale bar, 10 μm. (E) The tip region containing bright actin filaments is thinner than that in the WT pollen tube shown in (B). (F) Actin filaments within the region marked by green dashed lines are less bright than those further from the apex. (G) The bright actin filaments originating from the apical membrane in prf4 prf5 pollen tubes are thinner on average than those in WT pollen tubes. (H) Time-lapse images of actin filaments at the cortex within the apical region close to the tip in a WT pollen tube. The entire series is shown in Supplemental Movie 1. Scale bar, 5 μm. (I) Time-lapse images of actin filaments at the cortex within the apical region close to the tip in a prf4 prf5 pollen tube. The entire series is shown in Supplemental Movie 2. Scale bar, 5 μm. In (H) and (I), apical membrane-originated actin filaments are labeled with different-colored dots and their elongating ends are indicated with similar-colored arrows. Molecular Plant 2015 8, 1694-1709DOI: (10.1016/j.molp.2015.09.013) Copyright © 2015 The Author Terms and Conditions

Figure 5 PRF5Y6A Only Partially Rescues Actin Polymerization Phenotype. (A) Generation of recombinant PRF5Y6A. The mutant protein was run on an SDS–PAGE gel and visualized by Coomassie blue staining. (B) The G-actin-binding activities of PRF5Y6A and PRF5 are similar. PRF5 (left panel) and PRF5Y6A (right panel) inhibit spontaneous actin nucleation in a dose-dependent manner with similar activities. (C) The affinity of PRF5Y6A for poly-L-proline is decreased. PRF5, closed circles; PRF5Y6A, closed squares. The KD values for binding to PLP are 0.25 mM for PRF5 and 2.22 mM for PRF5Y6A. (D–G) PRF5Y6A partially rescues the actin organization defect within apical and subapical regions of the pollen tube. (D) PRF5 transcript levels in pgPRF5Y6A;prf4 prf5 are similar to those in WT, prf4 and pgPRF5;prf4 prf5. (E) PRF5 restores pollen tube growth to that of prf4, but PRF5Y6A cannot. (F) Micrograph of pollen tube actin filaments stained with Alexa-488 phalloidin. Red boxed region corresponds to the subapex and apex selected for the determination of actin filament level shown in (G). Scale bar, 5 μm. (G) PRF5Y6A only partially restores the amount of actin filaments within apical and subapical regions of prf4 prf5 pollen tubes to that of prf4 pollen tubes. Error bars in (E) and (G) represent SE. *P < 0.05, **P < 0.01 by Student's t-test. Molecular Plant 2015 8, 1694-1709DOI: (10.1016/j.molp.2015.09.013) Copyright © 2015 The Author Terms and Conditions

Figure 6 Both PRF4 and PRF5 Distribute Uniformly in Pollen Tubes. To determine the subcellular localization of PRF4 and PRF5, pollen tubes derived from EGFP-gPRF4;prf4 and EGFP-gPRF5;prf5 were observed under a confocal microscope. Simultaneous expression of mCherry in the same pollen tube was captured, aiming to determine whether profilin distributes uniformly in the pollen tube as reported previously (Grote et al., 1995; Mittermann et al., 1995; Vidali and Hepler, 1997). (A–D) PRF4 (A and B) and PRF5 (C and D) distribute uniformly in the pollen tube. The upper panels in (A) and (C) show the stack and ten longitudinal Z-series optical sections (at 0.5-μm intervals) of EGFP-PRF4 and mCherry (A) or EGFP-PRF5 and mCherry (C) in the pollen tube. The lower panels in (A) and (C) show the transverse sections of either EGFP-PRF4 and mCherry (A) or EGFP-PRF5 and mCherry (C) starting from the tip at 0.5-μm intervals. Scale bar, 5 μm. (B and D) Quantitative measurements of the fluorescence pixel intensity of EGFP and mCherry in pollen tubes. The average fluorescence pixel intensity of EGFP-PRF4 and mCherry (B) or EGFP-PRF5 and mCherry (D) was plotted against the distance from pollen tube tip. More than 15 pollen tubes were measured for EGFP-gPRF4;prf4 and EGFP-gPRF5;prf5 plants harboring Lat52:mCherry, and the shaded regions indicate SE. Molecular Plant 2015 8, 1694-1709DOI: (10.1016/j.molp.2015.09.013) Copyright © 2015 The Author Terms and Conditions

Figure 7 Schematic Diagram of the Polymerization of Actin at the Apical Membrane. Monomeric actin is predicted to be buffered by equimolar levels of profilin, so that actin mainly exists as profilin–actin complexes in the pollen tube. Actin filaments are generated at the apical membrane from profilin–actin complexes by apically localized formins. Molecular Plant 2015 8, 1694-1709DOI: (10.1016/j.molp.2015.09.013) Copyright © 2015 The Author Terms and Conditions