Both <i>AtFH3</i> and <i>AtFH5</i> are involved in the regulation of polarized pollen tube growth.

(A) Physical structure of AtFH3 (At4g15200) and AtFH5 (At5g54650). Filled black boxes, lines and white boxes represent exons, introns and untranslated regions (UTRs), respectively. The T-DNA insertion alleles Salk_150350, CSHL_GT24923, Salk_044464 and Salk_152090 were designated as fh3-1, fh3-2, fh5-2 and fh5-3, respectively. Red arrows indicate the position of T-DNA insertions. The black arrows indicate the position of primers used to analyze the transcript levels in pollen from WT and formin loss-of-function mutants. (B-E) qRT-PCR analysis of the amount of AtFH3 (B, C) and AtFH5 (D, E) transcripts in pollen derived from fh3, fh5 and fh3 fh5 mutant plants. Since T-DNA was inserted in the last exon of AtFH3, we used two primer pairs to detect the transcript level of AtFH3 in fh3-1 and fh3-2 and to determine whether the truncated AtFH3 transcript is transcribed in fh3-1 and fh3-2 mutant plants. eIF4A was used as the internal control. Data are presented as mean ± SE. (F) Micrographs of pollen derived from WT, fh3-1, fh5-2 and fh3-1 fh5-2 plants after 2 hours of incubation on standard germination medium are presented. Bar = 100 μm. (G) Quantification of pollen germination percentage in WT and formin loss-of-function mutants. The left panel is the plot of pollen germination percentage at different time points for WT, fh3-1, fh5-2 and fh3-1 fh5-2, and the right panel is the plot of pollen germination percentage at different time points for WT, fh3-2, fh5-3 and fh3-2 fh5-3. Data are presentend as mean ± SD, statistical comparisons were performed using Student’s t-test, *P H) Quantification of pollen tube growth rates. The left panel is the plot of pollen tube growth rates for WT, fh3-1, fh5-2 and fh3-1 fh5-2, and the right panel is the plot of pollen tube growth rates for WT, fh3-2, fh5-3 and fh3-2 fh5-3. Data are presented as mean ± SE, statistical comparisons were performed using ANOVA Post-Tukey, **P I) Quantification of pollen tube width. The left panel is the plot of pollen tube width for WT, fh3-1, fh5-2 and fh3-1 fh5-2, and the right panel is the plot of pollen tube width for WT, fh3-2, fh5-3 and fh3-2 fh5-3. Data are presented as mean ± SE, statistical comparisons were performed using ANOVA Post-Tukey, *P J) Micrographs of pollen tubes derived from WT and formin mutant plants. The blue and red lines in the bottom image indicate the actual length and linear length of the pollen tube, respectively. Bar = 100 μm. (K) Quantification of pollen tube morphology. The actual length was divided by the linear length and the values were plotted according to genotype to evaluate the morphology of the grown pollen tubes. Data are presented as mean ± SE, statistical comparisons were performed using ANOVA Post-Tukey, ** P < 0.01. More than 500 pollen tubes were measured for each genotype. Bar = 100 μm.