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First published online 15 February 2006
doi: 10.1242/dev.02280

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BRICK1/HSPC300 functions with SCAR and the ARP2/3 complex to regulate epidermal cell shape in Arabidopsis

Stevan Djakovic^*, Julia Dyachok, Michael Burke, Mary J. Frank and Laurie G. Smith

Section of Cell and Developmental Biology, Division of Biological Sciences, University of California San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0116, USA.

View larger version (62K): [in a new window]   Fig. 1. Sequence and expression of Arabidopsis BRICK1. (A) Alignment of maize and Arabidopsis BRICK1 protein sequences. Dark and light shading highlight identical and similar residues, respectively. The Arabidopsis BRK1 protein sequence is supported by the sequence of full-length cDNA clone RAFL04-19-H22 (GenBank Accession Numbers AV821991 and AV782595). Asterisks indicate the locations of premature stop codons in mutant alleles brk1-1 and brk1-2. (B) RT-PCR analysis illustrating expression of Arabidopsis BRK1 and ubiquitin in all tissues analyzed (FB, flower buds; RT, root tips; ES, expanding siliques; EC, expanding cotyledons; MC, mature cotyledons; EL, expanding leaves; ML, mature leaves).

View larger version (36K): [in a new window]   Fig. 2. Mutant and wild-type seedlings at 2 weeks post germination. (A) Columbia wild type; (B) arp2 (wurm) mutant; (C) arpc5 (crk) mutant; (D) brk1-1 mutant; (E) brk1-1;arp2 double mutant; (F) brk1-2;arpc5 double mutant.

View larger version (111K): [in a new window]   Fig. 3. Scanning electron micrographs showing the effects of brk1 and arp2 (wurm) mutations on epidermal trichome morphology. (A,E) Columbia wild type; (B,F) brk1-1 mutant; (C,G) arp2 mutant; (D,H) brk1-1;arp2 double mutant. Scale bars: in A-D, 500 µm; in E-H, 100 µm. (I) Quantitative analysis of trichome branch lengths; bars show s.e.m. (J) P values resulting from two-tailed Student's t-tests showing which branch length differences are statistically significant (bold).

View larger version (100K): [in a new window]   Fig. 4. Analysis of BRK1p::BRK1-T7/His transgenic plants. (A) Scanning electron micrograph showing aberrant (white arrowheads) and normal (black arrowhead) trichome morphologies observed in some BRK1p::BRK1-T7/His transgenic plants. Scale bar: 100 µm. (B) Northern blot comparing BRK1 RNA levels in four BRK1p::BRK1-T7 transgenic lines with abnormal trichomes with four non-transgenic Columbia samples (blot was probed with a full-length BRK1 cDNA, which does not distinguish between endogenous and transgene mRNAs). BRK1:ß-tubulin mRNA ratios were higher in all four transgenic samples than in the wild-type samples.

View larger version (142K): [in a new window]   Fig. 5. F-actin organization in expanding trichomes fixed and labeled with anti-actin antibody. (A-D) Columbia wild type; (E-G,K) arp2 mutant; (H-J,L) brk1-1. (A,E,H) Stage 3 trichomes; (B,F,I) Stage 4 trichomes; (C,G,J) Stage 5 trichomes. Arrowheads in D,K,L indicate the fine network of transversely aligned cortical F-actin seen in trichomes of all genotypes analyzed. Arrowheads in E,F,H,I show misaligned F-actin bundles. Scale bars: 10 µm.

View larger version (129K): [in a new window]   Fig. 6. Effects of mutations in SCAR and ARP2/3 complex subunits on cotyledon pavement cell shapes. (A) Columbia wild type; (B) arp2 (wurm) mutant; (C) arpc5 (crk) mutant; (D) sra1 (pir) mutant; (E) brk1-1 mutant; (F) arp2;brk1-1 double mutant; (G) arpc5;brk1-2 double mutant. Arrows and arrowheads indicate intercellular gaps adjacent to stomata and at other locations, respectively. Scale bar: 100 µm. (H) Form factors are shown for each genotype analyzed (n=3-8 plants and 63-317 cells). Error bars illustrate s.e.m. (where n=number of plants analyzed).

View larger version (69K): [in a new window]   Fig. 7. F-actin organization in expanding cotyledon pavement cells transiently expressing GFP-talin. (A-C) Columbia wild-type (surface area is 2220 µm2 for cell in A and 5660 µm2 for cell in C); (D-F) brk1-1 mutant. White arrowheads indicate local enrichments of cortical F-actin associated with apparently emerging lobes; gray arrowheads indicate enrichments that do not appear to be associated with emerging lobes. Scale bar: 13 µm. (G) Quantitative analysis of cortical F-actin distribution in wild type (n=22 cells) and brk1-1 mutant (n=36 cells). Bright actin, upper half of the fluorescence intensity range; intermediate actin, 25-50% of range; dim actin, bottom 25% of range. Error bars shown are s.e.m.

View larger version (19K): [in a new window]   Fig. 8. Loss of SCAR1 protein in scar1 and brk1 mutants. Western blots of anti-SCAR1 immunoprecipitates from shoot tips of 3-week-old seedlings probed with anti-SCAR1. Lane 1, wild type; lane 2, scar1 mutant; lane 3, brk1-1 mutant. Black arrowhead indicates SCAR1 protein detected in wild-type extract, which is undetectable in scar1 extracts and dramatically reduced in brk1-1 extracts (the smaller protein also recognized by this antibody serves as a loading control).