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Files in this Data Supplement:
Fig. S1. Comparison of root hairs. Columbia wild type (A) versus brk1-1 mutants (B). Root hairs were somewhat variable in length in both genotypes, but there was no apparent difference in the distribution of lengths, or in the thickness or frequency, of brk1-1 root hairs compared with wild type. Scale bar: 250 mm. arp2, arp3, arpc5, nap1 and sra1 mutant root hairs were also examined and found to be indistinguishable from wild type (not shown). Seeds were sown on plates containing 13 Murashige & Skoog (MS) salts, 0.05% MES and 1% agarose. Plants were grown for ~1 week on vertically oriented plates at 20-22°C in a 16 hour light/8 hour dark cycle. Seedlings were mounted on slides in water under a coverslip, and root hairs were imaged under bright-field conditions on a Nikon E600 microscope with a 43 objective. Images were captured using NIH ImageJ version 1.32j software with a DAGE MTI CCD72 camera coupled to a Scion LG-3 framegrabber.
Fig. S2. Effects of brk1 and ARP2/3 complex subunit mutations on rosette leaf epidermal pavement cell shapes. (A-F) Adaxial surfaces of fully expanded fifth rosette leaves from soil-grown plants. Two representative cells of intermediate size are outlined to highlight their shapes. (A) Columbia wild type; (B) brk1-1 mutant; (C) arp2 mutant; (D) arpc5 mutant; (E) arp2;brk1-1 double mutant; (F) arpc5;brk1-2 double mutant. Scale bar: 50 mm. (G) Form factors are plotted for each genotype analyzed (error bars illustrate standard deviations). t-tests show that at the P<0.01 confidence level, form factors for wild-type cells are significantly higher than those for all other genotypes. In addition, form factors for brk1 cells are significantly lower than those for both arp2 and arpc5 single mutants, but are not significantly different from those for brk1;arp2 or brk1;arpc5 double mutants. To visualize pavement cell shapes, fifth rosette leaves were fixed and cleared with chloral hydrate as described previously (Hamada et al., 2000). Specimens were mounted in chloral hydrate clearing solution, and visualized with DIC optics using a Nikon E600 microscope with a 203 objective. Images were captured using NIH ImageJ version 1.32j software with a DAGE MTI CCD72 camera coupled to a Scion LG-3 framegrabber. For each leaf analyzed, a set of images was collected spanning the entire width of the leaf along a straight line perpendicular to the tip-base axis approximately halfway from base to tip, but excluding the extreme edges and the midrib region, where cell shapes are atypical compared to the rest of the leaf. Using ImageJ, outlines were traced for every pavement cell in every image, and form factors calculated as described in Materials and methods for cotyledons.
Fig. S3. F-actin organization in expanding rosette leaf pavement cells transiently expressing GFP-ABD2. Intact plants grown for ~2 weeks on MS-agar plates were bombarded with a plasmid driving GFP-ABD2 expression from the 35S promoter (Wang et al., 2004). After 24 hours, expanding rosette leaves 3 and 4 were excised and mounted for confocal microscopy. Images were collected for cells with surface areas from 300-1600 mm2 (approximately corresponding to stage II cells of Fu et al., 2002). (A-C) Columbia wild type; (D-F) brk1-1 mutant. Arrowheads indicate areas of the cell cortex occupied by the highest density of F-actin; arrows indicate areas occupied by the lowest density of F-actin. Scale bar:10 mm. (G) Quantitative analysis of cortical F-actin distribution in expanding pavement cells of wild type (n=26) and brk1-1 mutant (n=26). Bright actin, upper half of the fluorescence intensity range; dim actin, bottom 25% of range; intermediate actin, 25-50% of range. Error bars shown are s.e.m.
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