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Comparative Study
. 2002 Jan;14(1):101-18.
doi: 10.1105/tpc.010346.

The Arabidopsis SPIKE1 Gene Is Required for Normal Cell Shape Control and Tissue Development

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Free PMC article
Comparative Study

The Arabidopsis SPIKE1 Gene Is Required for Normal Cell Shape Control and Tissue Development

Jin-Long Qiu et al. Plant Cell. .
Free PMC article

Abstract

Regulated growth and cell shape control are fundamentally important to the function of plant cells, tissues, and organs. The signal transduction cascades that control localized growth and cell shape, however, are not known. To better understand the relationship between cytoskeletal organization, organelle positioning, and regulated vesicle transport, we conducted a forward genetic screen to identify genes that regulate cytoskeletal organization in plants. Because of the distinct requirements for microtubules and actin filaments during leaf trichome development, a trichome-based morphology screen is an efficient approach to identify genes that affect cytoplasmic organization. The seedling lethal spike1 mutant was identified based on trichome, cotyledon, and leaf-shape defects. The predicted SPIKE1 protein shares amino acid identity with a large family of adapter proteins present in humans, flies, and worms that integrate extracellular signals with cytoskeletal reorganization. Both the trichome phenotype and immunolocalization data suggest that SPIKE1 also is involved in cytoskeletal reorganization. The assembly of laterally clustered foci of microtubules and polarized growth are early events in cotyledon development, and both processes are misregulated in spike1 epidermal cells.

Figures

Figure 1.
Figure 1.
Arabidopsis Wild-Type and spk1-1 Seedling Phenotypes Grown under Different Conditions. (A) One-week-old seedlings grown on soil at 100% humidity. The wild type is at left, spk1-1 mutant at right. (B) Wild-type seedling at 9 DAG on agar-based medium. (C) spk1-1 seedling at 9 DAG on agar-based medium. (D) Scanning electron microscopy of wild-type leaf trichomes. (E) Scanning electron microscopy of spk1-1 leaf trichomes with aborted branches. (F) Scanning electron microscopy of spk1-1 leaf trichomes with two branches. (G) Leaf epidermal pavement cells of the wild type. (H) Leaf epidermal pavement cells of spk1-1. Bar in (A) = 0.5 mm; bars in (B) and (C) = 1 mm; bars in (D) to (F) = 100 μm; bars in (G) and (H) = 10 μm.
Figure 2.
Figure 2.
Cell and Organ Shape Defects during spk1-1 Cotyledon Development. (A) to (D) Scanning electron microscopy of the adaxial surface of wild-type cotyledons. (A) Wild-type cotyledon at 2 DAG. (B) Higher magnification of wild-type cotyledon epidermal cells at 2 DAG. (C) Wild-type cotyledon at 5 DAG. (D) Higher magnification of wild-type cotyledon epidermal cells at 5 DAG. (E) to (H) Scanning electron microscopy of the adaxial surface of spk1-1 cotyledons. (E) spk1-1 cotyledon at 2 DAG. (F) Higher magnification of spk1-1 cotyledon epidermal cells at 2 DAG. (G) spk1-1 cotyledon at 5 DAG. (H) Higher magnification of spk1-1 cotyledon epidermal cells at 5 DAG. Bars in (A), (C), (E), and (G) = 100 μm; bars in (B), (D), (F), and (H) = 10 μm.
Figure 3.
Figure 3.
Growth Rates of Wild-Type and spk1-1 Cotyledons. Maximum cotyledon lengths and widths were measured from 1 to 12 DAG. Closed squares, wild-type cotyledon length; open squares, spk1-1 cotyledon length; closed triangles, wild-type cotyledon width; open triangles, spk1-1 cotyledon width. Error bars indicate ±sd × 1.96 to reflect a 95% confidence interval for significance.
Figure 4.
Figure 4.
Structure of the SPK1 Gene and Nature of spk1 Mutant Alleles. (A) Exon and intron organization of the SPK1 gene. The positions and lengths of exons and introns are indicated by closed rectangles and lines, respectively. The positions of T-DNA inserts associated with spk1-1 and spk1-2 are shown above the gene diagram. (B) Altered expression of the SPK1 gene in spk1-1 plants. RT-PCR analysis shows SPK1 mRNA accumulation in spk1-1 and the wild type. The locations of the upstream (SPK1 RT2) and downstream (SPK1 RT1) primer pairs are labeled. Gene-specific primers for the ACT7 gene were used as a control. (+), with reverse transcriptase (RT); (−), without reverse transcriptase.
Figure 5.
Figure 5.
Expression of Double-Stranded SPK1 RNA Causes a spk1 Phenotype. (A) Wild-type plants transformed with the empty binary vector. (B) Wild-type plants transformed with DSCK. DSCK expresses double-stranded RNA corresponding to the 27th intron of SPK1. (C) Wild-type plants transformed with DS1. DS1 corresponds to 1089 bp of the SPK1 coding sequence. (D) Wild-type plants transformed with DS2. DS2 corresponds to 677 bp of the SPK1 coding sequence. Insets in each panel show scanning electron micrographs of the adaxial cotyledon epidermal cells of similarly staged plants. Bars = 1 mm; inset bars = 100 μm.
Figure 6.
Figure 6.
Scheme of the SPK1 Coding Sequence. The MOD1, MOD2, and CDMS domains are labeled to scale according to their positions in the predicted protein.
Figure 7.
Figure 7.
Alignment of the CDMS Domain of SPK1 with Homo sapiens DOCK180, Caenorhabditis elegans CED-5, Drosophila melanogaster MYOBLAST CITY (MBC), Saccharomyces cerevisiae Ylr422wp, and Dictyostelium discoideum DocA. Amino acid residues in black indicate identity, and those in gray indicate conserved substitutions. Residues that are identical or similar in four of seven sequences are highlighted. The numerical amino acid scale for each sequence is labeled at right.
Figure 8.
Figure 8.
Immunolocalization and Confocal Micrographs of the Cytoskeleton in Developing Wild-Type and spk1-1 Cotyledon Epidermal Cells. Epidermal cells were double labeled with antibodies directed against α-tubulin (green channel) or actin (red channel). Each image is a maximum projection of the upper half of the cells. (A), (C), and (E) Wild-type epidermal cells. (A) Cuboidal wild-type epidermal cell at 1 DAG. (B) Cuboidal spk1-1 epidermal cell at 1 DAG. (C) A wild-type epidermal cell at 2 DAG that has initiated lobe formation. The inset shows a single optical section through a plane of the transvacuolar cytoplasm. (B), (D), and (F) spk1-1 epidermal cells. (D) spk1-1 epidermal cell at 2 DAG. (E) A rapidly growing wild-type epidermal cell at 5 DAG. (F) An elongated spk1-1 epidermal cell at 5 DAG. White arrows, regions containing cortical microtubules that are in close proximity to the plasma membrane; black arrows, regions of the cell containing actin filaments that are in close proximity to the plasma membrane. Bars = 10 μm.

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