Quantification of PRF1 expression in prf1 mutants of Arabidopsis A, Diagram of PRF1 gene and T-DNA mutant alleles. Triangles, T-DNA insertions; black lines, introns; blue bars, exons; gray bars, nontranslated regions. Primers used for in B are labeled with arrows and sequences are given in Supplemental Table S1. B, Relative expression levels of PRF1 were examined by analysis of 5-d-old dark-grown seedlings from homozygous prf1 mutants (prf1-2, prf1-3) and wild-type (WT) siblings, all marked with GFP-ABD2 reporter. All data were normalized to GAPD transcript levels. Values represent mean ± SE from three biological repeats. C, Representative immunoblot of PRF1 and actin protein levels in 5-d-old dark-grown seedlings of prf1 mutants (prf1-2, prf1-3) and wild-type siblings, all marked with GFP-ABD2. Proteins were detected with anti-AtPRF1 or JLA-20 monoclonal antibodies, respectively. As a loading control, membranes were reprobed with PEP-carboxylase antibody. D, Quantitative analysis of PRF1 and actin protein levels measured by densitometry of the profilin or actin band from each genotype and then calculating the fold change compared to wild-type siblings. All data represent mean ± se from five biological repeats.

Perturbed actin architecture in prf1 mutants enhances hypocotyl growth and epidermal cell length in Arabidopsis. A, Representative images of 5-d-old dark-grown seedlings of wild type and prf1 mutants. Bar, 1 cm. B and C, Etiolated hypocotyls from homozygous prf1-2 (B) and prf1-3 (C) mutants were significantly longer than wild-type siblings over a developmental time course. Values represent mean ± SE, n ≥ 50 seedlings per genotype, *P < 0.05, **P < 0.005, Student’s t test. D, Epidermal cell lengths were measured from apical and basal regions of 5-d-old etiolated hypocotyls. The average length of epidermal cells from prf1-2 and prf1-3 mutants was significantly longer than that of wild-type siblings. Values represent mean ± se, n ≥ 50 cells from at least 10 hypocotyls per genotype, ***P < 0.001, Student’s t test. E, Representative micrographs of epidermal cells from the apical and basal regions of 5-d-old etiolated hypocotyls. Actin filament arrays in prf1-2 mutant cells appeared to be more sparse and less bundled compared to wild-type cells. Bar, 10 μm. F and H, Quantitative analysis of the percentage of occupancy (density) of actin filament arrays in epidermal cells from the basal region of hypocotyls. The density of actin arrays in prf1-2 (F) and prf1-3 (H) mutants was significantly decreased compared to wild-type siblings. Values represent mean ± se, n ≥ 300 cells from 10 hypocotyls per genotype, **P < 0.005, Student’s t test. G and I, Quantitative analysis of the extent of filament bundling (skewness) in epidermal cells from the basal region of hypocotyls. The same images used for F and H were analyzed for skewness. Compared to wild type, actin filament arrays in prf1-2 (G) and prf1-3 (I) mutants were significantly less bundled.

PRF1 is required for normal cortical actin organization in epidermal cells of the root elongation zone A, Representative images of 7-d-old light-grown roots of wild type and prf1 mutants. Bar, 1 cm. B and C, Root lengths were measured from light-grown seedlings over a developmental time course. Root lengths of prf1-2 and prf1-3 were significantly longer compared to wild-type siblings. Values represent mean ± SE, n ≥ 50 roots per genotype, ***P < 0.001, **P < 0.01 *P < 0.05, ^NDP > 0.05, Student’s t test. D, Epidermal cell lengths were measured from the elongation zone of 7-d-old light-grown roots. The cell length of prf1-1 and prf1-2 mutants was significantly longer compared to wild-type siblings. Values represent mean ± SE, n ≥ 200 cells from at least 15 roots, ***P < 0.001, Student’s t test. E, Representative micrographs of epidermal cells from the elongation zone of 7-d-old light-grown roots. Actin filament arrays in prf1-2 mutant cells appeared to be less dense compared to wild-type cells. Bar, 10 μm. F and H, Quantitative analysis of the percentage of occupancy (density) of actin filament arrays in epidermal cells from the elongation zone of roots. The density of actin arrays in prf1-2 (F) and prf1-3 (H) mutants was significantly decreased compared to wild type. Values represent mean ± SE, n ≥ 236 cells from 15 roots per genotype, **P < 0.005, ***P < 0.001, Student’s t test. G and I, Quantitative analysis of the extent of filament bundling (skewness) in epidermal cells from the elongation zone of roots. The same images used for F and H were analyzed for skewness. The extent of bundling of actin filament arrays in prf1-2 (G) and prf1-3 (I) mutants was not significantly different compared to wild type ND P ≥ 0.05.

The frequency of filament nucleation events is reduced in prf1 mutants. A, Three classes of actin filament origin were observed in epidermal cells of 5-d-old dark-grown hypocotyls. Actin filaments initiated from the end of an existing actin filament (top row), the side of an actin filament or bundle (middle row), and de novo in the cytoplasm. Green dots, New growing actin filament; red dots, preexisting actin filament. Bar, 5 μm. B, The overall frequency of actin nucleation was defined as the total number of filament origins per filament per minute in a 400 μm² region of interest. Compared to wild type, total nucleation frequency in prf1-2 and prf1-3 was significantly decreased. Values represent mean ± SE, n ≥ 380 events per genotype, ***P < 0.001, Student’s t test. C, The frequency of actin nucleation types was analyzed using the method indicated in B. Compared to wild type, nucleation frequency from filament sides and ends in prf1-2 and prf1-3 were significantly decreased, whereas de novo nucleation frequency was significantly increased compared to wild type. Values represent mean ± SE, n ≥ 380 events per genotype or treatment, ***P < 0.001, **P < 0.005, *P < 0.05, Student’s t test.

The proportion of filament ends with different elongation rates changes in prf1 mutants. A, A representative actin filament from a wild-type (top) epidermal cell (green dots) elongated rapidly (1.61 μm/s) and was subsequently disassembled by numerous severing events (red arrows). A representative actin filament from a prf1-2 mutant (bottom) epidermal cell elongated more slowly (0.74 μm/s) and persisted for a longer period before severing events occurred (red arrows). Bars, 5 μm. B, Box plots show the average filament elongation rate in epidermal cells from 5-d-old etiolated hypocotyls of prf1-2 and prf1-3. The box spans first and third quartiles. The line inside the box shows median value. Bars on the top and bottom represent the maximum and minimum value. The red circles represent mean values for single filament elongation rate. Compared to wild type, the filament elongation rates were significantly slower in prf1-2 and prf1-3. n ≥ 100 single actin filaments per genotype, ***P < 0.001, Student’s t test. C, Filament elongation rates were categorized into three populations: more than 2 μm/s, between 1 and 2 μm/s, and less than 1 μm/s, as described previously (). Compared to wild type, the percentage of filament elongation rates > 2 μm/s decreased in prf1-2 and prf1-3, whereas the percentage of filament elongation rates < 1 μm/s increased. n ≥ 100 single actin filaments per genotype or treatment, ***P < 0.001, χ² test.

Nucleation frequency and rate of actin filament elongation are inhibited by SMIFH2. A, Nucleation frequency was measured by counting the total nucleation events per filament in a 400 μm² over a 100 s time period after SMIFH2 treatment. Total nucleation frequency was inhibited by SMIFH2 treatment in a dose-dependent manner. Significant decrease of nucleation frequency appeared at concentrations of SMIFH2 greater than 25 μm. n > 300 events from 30 cells of 15 hypocotyls per treatment, Student’s t test, ***P < 0.001. B, Nucleation frequency by origins was analyzed in the same way as described for nucleation frequency in A. Compared to mock treatment, nucleation frequency of filaments originating from the side of a filament or bundle decreased significantly after SMIFH2 treatment. However, nucleation frequency of filaments originating de novo or from ends changed slightly. n > 300 events from 30 cells of 15 hypocotyls per treatment, Student’s t test, ***P < 0.001, **P < 0.01, *P < 0.05, ND P ≥ 0.05. C, Box plots show the average filament elongation rate in epidermal cells from 5-d-old etiolated hypocotyls without or with a 5 min, 25 μm SMIFH2 treatment. The box spans first and third quartiles. The line inside the box shows median value. Bars on the top and bottom represent the maximum and minimum values. The red circles represent mean values for single filament elongation rate. Compared to the mock, the filament elongation rates were significantly slower with SMIFH2 treatment. n ≥ 100 single actin filaments per treatment, **P < 0.005, Student’s t test. D, The proportion of single actin filament elongation rates was altered with SMIFH2 treatment. Filament elongation rates were categorized into three populations: more than 2 μm/s, between 1 and 2 μm/s, and less than 1 μm/s, as described previously (). Compared to mock treatment, the percentage of filament elongation rates > 2 μm/s decreased with SMIFH2 treatment, whereas the percentage of filament elongation rates < 1 μm/s increased. n ≥ 100 single actin filaments per treatment, *P < 0.05, χ² test.