Table 1.
PCR-based markers for molecular mapping.
Table 2.
Frequency of selected embryo-defective phenotypes.
Fig 1.
Range of cytokinesis-defective embryo phenotypes.
Nomarski images of whole mount cleared immature seed; embryos at the heart stage of development are shown on the left and endosperm nuclei (arrowhead) on the right: (a) wild type; (b) kn mutant derived from line 4–43 (see text); enlarged cell caught in mitosis is boxed and shown magnified; (c) kn keu double mutant; three nuclei of normal size (stars) are visible; (d) phenotype produced by a weak kiesel allele; (e) double mutant phenotype produced by line 4–43; (f) mutant arrested at globular stage with no apparent cell division defects derived from line 4–43. Scale bar equals 50 μm.
Table 3.
Loci mutating to kn-like phenotypes.
Fig 2.
Morphological and ultra-structural characteristics of tio and rsw mutants.
Left: Seedling morphology. Images of wild type (a) tio-12 (d) and rsw-lph (e) seedlings; root hairs were contrasted with methylene blue; scale bar equals 1 mm. Center: Anatomy of embryos. Confocal micrographs of wild type (b), tio-12 (e), and rsw-lph (h) embryos at the heart stage of development stained with Alexafluor 488 hydrazide; scale bar equals 30 μm. Right: Ultra-structure of embryonic cells. Transmission electron micrographs showing cells of wild type (c), tio-10 (f) and rsw-lph (i) embryos; the arrowheads point to cell wall stubs, and the stars in (c) and (f) mark the nucleus; the side of panels equals 10 μm.
Fig 3.
Molecular cloning of OPN/TIO and LPH/RSW7.
(a) Top: Genetic and physical map of the OPN/TIO region on the lower arm of chromosome 1. Numbers below the line indicate the number of recombination events between opn/tio mutations and the corresponding molecular markers (with italics referring to recombinants on the centromeric side, and the total number of meiotic events analyzed listed on the far right); two BAC clones (black bars) spanning the OPN/TIO transcription unit (arrow) are shown below the map. Bottom: Domain structure of the TIO protein. The N-terminus consists of a FU-type kinase domain (gray bar, with the ATP binding pocket in black); the C-terminus contains four repeat motifs, the first two of which share significant similarity with Armadillo and HEAT repeats (white boxes labeled “AH”), while the second two show borderline similarity to Armadillo repeats (“A”; [35]); the tio-12 allele (mutation 12–15) harbors a glycine to aspartic acid substitution in the ATP-binding pocket (listed in the dashed box, invariant positions of the consensus sequence marked with a star); the PROSITE consensus (motif no. P00107) of this sequence signature is: [LIV]-G-{P}-G-{P}-[FYWMGSTNH]-[SGA]-{PW}-[LIVCAT]-{PD}-x-[GSTACLIVMFY]-x(5,18)-[LIVMFYWCSTAR]-[AIVP]-[LIVMFAGCKR]-K; the tio-10 allele (mutation OX10) harbors a cystein to tyrosine substitution in a portion of OPN without significant similarity to known motifs. (b) Top: Genetic and physical map of the LPH/RSW7 region on the lower arm of chromosome 2, organized as in (a). Bottom: Domain structure of the predicted RSW7 protein. The N-terminal catalytic core (grey bar) and the neck domain (black bar) show strong similarity to members of the kinesin-5 family; the insertion sites of the two T-DNA alleles, rsw7-118 and rsw7-92, are marked with diamonds; the rsw7-lph allele harbors a glycine to arginine substitution in a conserved portion of the catalytic core; an alignment of this portion with human Eg5/KIF11 and E. nidulans BimC (GenBank accession nos. P52732, P17120) is shown in the dashed box, with stars representing invariant and colons conserved positions; folding of this domain is shown below the alignment and inferred from the crystal structure of rat brain kinesin [67] (L12/MT2: loop 12, microtubule binding domain 2; α5: alpha helix 5, L13: loop 13, β8: beta sheet 8).
Fig 4.
An allelic series of the RSW7 gene.
(a–d): kn-like phenotype of rsw7-lph/rsw7-1 embryos. Nomarski images of whole mount cleared immature seed containing wild type (a,c) and trans-heterozygous embryos (b,d) at the early heart (a,b) and torpedo stage (c,d); arrows point to examples of enlarged cells with polyploid nuclei. (e–h): Aberrant divisions in the root meristem of rsw7-1 and rsw7-lph/rsw7-118 seedlings. Nomarski images of wild type (e), and rsw7-lph/rsw7-118 trans-heterozygotes (f), grown at 25°C; and rsw7-1 grown at 16°C (permissive temperature) (g), and 25°C (non-permissive temperature) (h); examples of abnormally large cell within the meristem, presumably resulting from a failure of mitosis, are boxed in (g,h) and shown magnified (top); stars indicate the boundary between meristem and elongation zone, as marked by the appearance of highly vacuolated rectangular cells. Scale bars equal 50 μm.
Fig 5.
Effect of the rsw7-lph mutation on cellularization of the male gametophyte and endosperm.
(a): Pollen produced by rsw7-lph/+ plants; scanning electron micrograph showing a mixture of normal (star) and shriveled or collapsed grains; scale bar equals 10 μm. (b–e): Cell division defects of rsw-lph male gametophytes. DAPI staining of normal pollen (b) reveals two small, brightly staining sperm cell nuclei and the large, less brightly staining nucleus of the vegetative cell; pollen of rsw7-lph/+ plants is frequently mono-nucleate (c), bi-nucleate (d), or collapsed with no detectable DNA (e); scale bar in (e) equals 10 μm. (f, g): Absence of a cellularized endosperm in rsw-lph seed. Confocal micrographs of Schiff-stained seed show the presence of cell walls in the endosperm of seed containing wild type embryos (f) but not in seed containing mutant embryos (g); scale bar in (g) equals 50 μm.