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First published online July 21, 2003
doi: 10.1242/10.1242/dev.00619

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SCD1 is required for cell cytokinesis and polarized cell expansion in Arabidopsis thaliana

¹ Department of Biochemistry, University of Wisconsin-Madison, 433 Babcock Drive, Madison, WI 53706, USA
² Department of Plant Biology, Ohio State University, 1735 Neil Avenue, Columbus, OH 43210, USA
³ Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, CO 80309, USA

View larger version (86K): [in a new window]   Fig. 1. Wild-type and cytokinesis-defective guard cells. (A-C) Stoma-specific expression of KAT1-GUS, a marker for guard cell identity (Nakamura et al., 1995), in the cotyledons of 5-day-old seedlings germinated in the absence (A) or presence (B,C) of 5 mM caffeine, an inhibitor of cell plate membrane consolidation. scd1-1 mutant guard cells phenocopy the effects of caffeine. (D-F) Light microscopy of Toluidine Blue-stained scd1-1 leaf epidermis from plants grown at 22°C. (G,H) KAT1-GUS was expressed in cytokinesis-defective scd1-1 guard cells. Hanging pores are indicated by arrows. A torus and oblate type guard cell are shown in F and H, respectively. Scale bars: 10 µm.

View larger version (36K): [in a new window]   Fig. 5. Identification and organization of SCD1 and scd1 alleles. (A) The scd1-1 mutation was mapped to a 65 kb region on BAC F27J15, containing 18 predicted open reading frames. Vertical lines show marker positions with an identifier number below each line and a box indicating the number of recombinants detected by each marker. (B) The intron/exon map of the SCD1 gene (At1g49040) in the forward orientation. Positions of the scd1-1 point mutation and the two T-DNA insertion alleles, scd1-2 and scd1-3 are indicated. Arrows denote the location and orientation of primers P1 through P7 described in Table 1 in the Materials and Methods section. TL, T-DNA left Border. (C) The domain structure of SCD1, with the position of the S131F mutation indicated by an asterisk. The tripartite DENN domain starts at the N terminus of SCD1 and extends for 430 amino acids (Levivier et al., 2001), the eight WD-40 repeats span a 500 amino acid region extending to the C terminus. (D) Location of the scd1-1 (S131F) mutation in an alignment of the DENN sub-motif `F' as identified by hydrophobic cluster analysis (Levivier et al., 2001). The alignment compares the sequence of 6 characterized DENN proteins from various organisms that interact with components of vesicle trafficking and MAPK signaling pathways. Residues shaded in black are identical throughout most of the 43 known and hypothetical proteins identified to date containing DENN domains that are present in the GenBank database. Those shaded in gray are conservative substitutions.

View larger version (79K): [in a new window]   Fig. 2. Defective stomata are binucleate. Cotyledons of 13-day-old wild-type (A) and scd1-1 seedlings (B,C) that express the nuclear (green) GFP N7 marker and (D) cotyledons from 4-day-old seedlings expressing plasma membrane GFP 29-1 marker (Cutler et al., 2000). Multinucleate scd1-1 epidermal cells with wall stubs (arrow) are shown in C and D. Two serial confocal optical sections were merged to create C. Cell walls were stained with propidium iodide (red) and chloroplasts are autofluorescent (blue). (D) The membrane in the cell wall stubs of cytokinesis-defective scd1-1 epidermal cells was contiguous with the plasma membrane. Scale bars: 10 µm.

View larger version (69K): [in a new window]   Fig. 4. The scd1-1 mutant is temperature-sensitive. (A) scd1-1 plants grown under continuous illumination at 22oC (left) and 16°C (right). (B) Aborted scd1-1 flower buds from plants grown at 22°C. (C) The fertile inflorescences of plants grown at 16°C. (D,E) Toluidine Blue stained leaf epidermal peels from plants grown at 22°C (D) show many defective guard cells whereas plants grown at 16°C (E) show normal guard cells and an increased number of meristemoids. (F) Temperature-shift experiment. scd1-1 flower buds that were developing during the 16°C incubation period (bracket) developed into fertile flowers, which produced siliques filled with scd1-1 seed. Upon return to restrictive conditions (22°C), flower bud development aborted once again. (G) Three-month-old scd1-3 mutant grown in soil at 18°C. Scale bars: 1 cm (A); 0.5 cm (B,C); 10 µm (D,E); 0.25 cm (G).

View larger version (87K): [in a new window]   Fig. 3. scd1 mutants display a dwarfed phenotype. (A) scd1-1, scd1-2 and scd1-3 mutants grown on MS-Suc under continuous light for 8 days are smaller, have shorter roots, and have darker green leaves than those of wild type. (B) Mutant shoots are smaller and darker green than wild type. (C) Comparison of wild type, scd1-1 and scd1-2 root cell size. Cell length was plotted as a function of the distance from the root tip. Each point represents an average of 20-50 cell measurements of a mixture of epidermal and cortical cells. (DH) Root hair development in wild type and scd1 mutant seedlings grown vertically on the surface of MS-Suc medium. (D) Stereomicroscope image of surface views of a wild type (WT) and a scd1-2 root. (E-H) Phase contrast images of (E-G) scd1/2 and (H) wild-type roots. scd1 mutants usually form small blebs (D,E,F) at the basal end of trichoblasts, which do not extend. (E) Enlargement of boxed area in D. Arrows in G indicate branched root hairs. Scale bars: 1 cm (A); 0.25 cm (B); 100 µm (D-H).

View larger version (79K): [in a new window]   Fig. 8. Effects of vesicle and cytoskeletal inhibitors on wild type and scd1 root growth and cell morphogenesis. CLSM optical sections of wild type (wt), scd1-1 and scd1-2 roots grown in (A) the absence and (B-E) the presence of caffeine (B), brefeldin A (C), latrunculin B (D) and propyzamide (E). The graph associated with each image represents the extent of root growth (y-axis; mm) over a 4- to 5-day period in the absence and presence of the inhibitors at the indicated concentrations (x-axis). The asterisk denotes the inhibitor concentration used in the underlying root section. 5-15 seedlings were measured for each treatment and genotype. Scale bars: 50 µm.

View larger version (36K): [in a new window]   Fig. 6. RT-PCR analysis of SCD1 expression in wild-type and mutant tissue. RT-PCR analysis of SCD1 mRNA expression in total RNA isolated from wild-type roots, rosette leaves, cauline leaves, immature flower buds, open flowers, scd1-1 leaf tissue, and actively dividing suspension-cultured cells (T87). A reaction (-) containing no DNA template was included as a negative control. RT-PCR amplified ubiquitin was used as a loading control. std: 1.0 and 0.5 kb markers.

View larger version (141K): [in a new window]   Fig. 7. scd1::T-DNA mutants showed defects in leaf trichome and epidermal pavement cell expansion. (A) wild type and (B) scd1-2 cotyledon epidermal cells stained with propidium iodide. The white arrowhead indicates a potential recently divided guard mother cell. (C) Wild-type and (D-G) scd1-2 leaf trichomes on emerging leaves. (E) An enlarged view of expanding unbranched scd1-2 trichome initials. (F,G) Older leaves of scd1-2 seedlings showing mechanically unstable trichomes and small gaps between pavement cells (arrowheads). Cell debris is often observed in craters surrounded by the subtending socket cells of broken trichomes. (H,I) Transmission electron micrographs of cells from very young leaves; (H) a wild-type telophase cell and (I) a cytokinesis-defective scd1-2 cell. C, chloroplast; CP, cell plate; CWS, cell wall stub; PCW, parental cell wall; G, Golgi; M, mitochondrion; N, nucleus; V, secretory vesicles. Scale bars: 10 µm (A,B); 50 µm (C-G); 1 µm (H,I).