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First published online 4 August 2004
doi: 10.1242/dev.01307

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Interchangeable functions of Arabidopsis PIROGI and the human WAVE complex subunit SRA1 during leaf epidermal development

¹ Agronomy Department, Purdue University, Lilly Hall, 915 West State Street, West Lafayette, IN 47907-2054, USA
² Purdue Motility Group, Purdue University, Lilly Hall, 915 West State Street, West Lafayette, IN 47907-2054, USA

View larger version (190K): [in a new window]   Fig. 1. Epidermal phenotypes of pir trichomes, cotyledon pavement cells and hypocotyls. (A) SEM of developing trichomes on the upper surface of developing wild-type leaves. (B) SEM of developing trichomes on the upper surface of pir-3 leaves. (C) SEM of upper surface of 12 DAG (days after germination) wild-type cotyledon pavement cells. (D) SEM of upper surface of 12 DAG pir-3 cotyledon pavement cells. (E) Hypocotyl of dark-grown wild-type seedlings. (F) Hypocotyl of dark-grown pir-3 seedlings (boxed area is enlarged in the inset, which shows a higher magnification view of the non-adherant cell). (G) Wild-type plant (15 DAG). (H) pir-3 plant (15 DAG). Numbers in A and B are positioned in the lower right region of a trichome to indicate the developmental stage. White arrows indicate the gaps between adjacent pavement cells and black arrows indicate stomatal pores in C,D. Scale bars: 100 µm in A-F; 1 cm in G,H.

View larger version (57K): [in a new window]   Fig. 2. PIR encodes a homolog of human SRA1. Physical map of the PIR gene, mutant alleles and rescue of the mutant phenotype by overexpression. (A) The region of chromosome 5 that contains the PIR locus. Boxes indicate the position of molecular markers and the oval represents the PIR-containing BAC (T4C15). The measured recombination frequency for several markers in the mapping interval is given. (B) Physical structure of the PIR locus. The position of exons and introns are indicated by black rectangles and lines, respectively. The 5'-UTR and 3'-UTR are indicated by white rectangles. The location and nature of the pir-1, pir-2, pir-3, pir-5 and pir-T1 mutations are labeled. The location of the AT-AC intron is labeled. The 5'-UTR and 3'UTR sequences were derived from RIKEN clone RAFL04-10-J11 (GenBank Accession Numbers AV82143 and AV781909, respectively). (C) Overexpression of PIR rescues pir-3 trichome phenotype. (Left) Col mature trichome; (middle) pir-3 mature trichome; (right) mature trichome on a pir-3 plant that harbors a PIR overexpression construct.

View larger version (80K): [in a new window]   Fig. 3. Expression patterns of PIR, ATNAP125 and ARP2/3 subunit genes in major organs. Lane 1, no reverse transcriptase (-RT) in 5 DAG seedling; lane 2, plus (+) RT in 5 DAG seedling; lane 3, 5 DAG root (+RT); lane 4, 5 DAG hypocotyl (+RT); lane 5, 5 DAG cotyledon (+RT); lane 6, 15 DAG leaf (+RT); lane 7, stem (+RT); lane 8, inflorescence (+RT); lane 9, flower (+RT). No RT controls were conducted on all RNA samples, but the data are shown only for the seedling RNA sample.

View larger version (59K): [in a new window]   Fig. 4. Arabidopsis PIR and human SRA1 have interchangeable in vivo functions and interactions with ATNAP125. (A) Direct interaction of recombinant PIR and ATNAP125 in a GST-pull-down assay. (Top) Lanes 1-5, fractions probed with anti-ATNAP antibody; (bottom) same fractions probed with control antibody anti-AT3G. Lane 1, 5% of total binding reaction; lane 2, GST-PIR bead bound pellet fraction; lane 3, GST-PIR bead unbound; lane 4, GST bead bound; lane 5, GST bead unbound. (B)PIR and human SRA1 interact with ATNAP125 in a yeast two-hybrid assay. The bait and prey plasmids that were co-transformed into yeast are defined to the left of the corresponding yeast patches and ß-galactosidase assay results. pDS32PIR, full-length PIR; pDS32HSPIR, full-length human SRA1; pDS22ATNAP, full-length ATNAP125; pDS22HSNAP, full-length human NAP125. (C) Overexpression of human SRA1 can suppress pir-3 trichome distortion. Left to right: Col mature trichome; pir-3 mature trichome; mature trichome on a Col plant that harbors a human SRA1 overexpression construct; mature trichome on a pir-3 plant that harbors a human SRA1 overexpression construct. Scale bars: 100 µm.

View larger version (39K): [in a new window]   Fig. 5. Arabidopsis PIR and human SRA1 have similar RHO-family GTPase binding activities. (A) PIR and human SRA1 bind interchangeably to plant and animal RHO-family GTPases in the yeast two-hybrid assay. The bait and prey plasmids co-transformed into yeast are listed to the left of the corresponding patches and ß-galactosidase assay results. (B) PIR shows nucleotide-selectivity for interaction with ROP2 in yeast two-hybrid system. The bait and prey plasmids co-transformed into yeast are described to the left as in A.

View larger version (63K): [in a new window]   Fig. 6. Localization and quantification of actin filaments in whole mounted wild-type (Col), dis2 and pir stage 4 trichomes. (A-C) Wild-type. (D-F) dis2-1. (G-I) pir-3. (A,D,G) Maximum projection of whole-cell F-actin signals in stage 4 trichomes. (B,E,H) Maximum projection of signals from cortical actin filaments (within 2.5 µm of the plasma membrane) in the same branches of A, D and G, respectively. (C,F,I) Maximum projection of signals from actin filaments in the core branch cytoplasm. (J) Mean ratios±s.d. of core cytoplasmic to total cytoplasmic actin filament signals in stage 3/4 and stage 4/5 branches from 5-10 branches. Broken line indicates the lower limit of the 95% confidence level of the mean ratio for wild-type stage 4/5 trichomes. Scale bars: 10 µm.

View larger version (39K): [in a new window]   Fig. 7. Organization of actin filaments in wild-type (Col), dis2 and pir-3 early stage 5 trichomes. Trichomes belonging to this stage were identified by a pointed tip morphology, the absence of papillae, a cell height of at least 40 µm, and a branch length between 30 and 75 µm. Each image is a projection of planes that correspond to the core branch cytoplasm. (A) Wild-type. (B) dis2-1. (C,D) pir-3. Scale bars: 10 µm.