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doi: 10.1242/10.1242/dev.00542

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Mutations in Arabidopsis condensin genes disrupt embryogenesis, meristem organization and segregation of homologous chromosomes during meiosis

Department of Botany, Division of Life Sciences, University of Toronto, 1265 Military Trail, West Hill, Ontario M1C 1A4, Canada

View larger version (71K): [in a new window]   Fig. 1. Functional complementation of the yeast smc2-6 temperature sensitive mutant with the AtCAP-E1 cDNA. (A) Diagram of the position on the plates of the cell types in B and C. (B) Rescue of mutant cells transformed with AtCAP-E1 cDNA in the presence of galactose at 37°C. (C) smc2-6 mutant cells transformed with AtCAP-E1 cDNA failed to grow at 37°C in the absence of galactose. (D,E) DAPI-stained mutant and transformed cells, respectively, exhibited the cut phenotype in the absence of galactose at 37°C. (F,G) Wild-type and transformed cells, respectively, showed normal chromosome segregation in the presence of galactose at 37°C.

View larger version (74K): [in a new window]   Fig. 2. Expression analysis of AtCAP-E1 and AtCAP-E2. RT-PCR was performed on total RNA from roots (R), stems (S), leaves (L), buds (B), and siliques (Si). (A) DNA gel blot of RT-PCR product pool. (B) PCR products were digested with Ssp I (AtCAP-E1-specific site) before DNA gel blotting. The intensity of the 662 bp fragment represents the relative abundance of the AtCAP-E1 transcript. (C) PCR products were digested with XbaI (AtCAP-E2-specific site) before DNA gel blotting. The intensity of the 459 bp fragment represents the relative abundance of the AtCAP-E2 transcript. (D) Ethidium bromide-stained gel of 1 µg of total RNA used for reverse transcription showing equal amounts of high quality total RNA. (E-I) AtCAP-E1 is expressed in meristems and mitotically active tissues. An AtCAP-E1::GUS reporter construct was introduced into wild-type Arabidopsis plants and GUS activity was examined in the transgenic plants. (E) GUS activity at the apex of an 8-day-old seedling. The apical dome and emerging true leaves stain intensely as does the tip of the root. (F) GUS activity in the primary root apical meristem and in early lateral root primordia (G) emerging from the pericycle cells. (H) GUS activity in developing leaves parallels the basipetal pattern of cell differentiation. (I) intense GUS activity in young floral buds.

View larger version (98K): [in a new window]   Fig. 3. Seed and embryo defects. (A) Silique from a wild-type Columbia plant with normal green developing seeds filling the entire length of the silique. (B,C) Siliques from (B) an E1-/-E2+/+ plant and (C) an E1+/+E2-/- plant with developing seeds similar to those of the wild-type silique. (D) Silique from an E1+/-E2-/- plant with the green seeds containing the normal embryos and brown seeds harboring the aborted embryos. Note the significant number of empty spaces between developing seeds resulting from unfertilized ovules. (E) Differential interference contrast (DIC) microscopy of a normal sibling embryo at the torpedo stage. (F) DIC image of an E1+/-E2-/- embryo arrested at the globular stage. (G,H) DIC images showing patterning defects in the suspensor and embryo proper. Arrows point to suspensors.

View larger version (63K): [in a new window]   Fig. 4. SMC2 is required for proper meiotic chromosome segregation. Pollen mother cells at both metaphase I and anaphase I from wild-type (A,D) and E1+/-E2-/- plants (B,C,E,F) were stained with DAPI and examined by fluorescence microscopy. In wild type, discrete signals and proper segregation of chromosomes were observed, whereas in a population of the mutant pollen mother cells, either relatively normal (B,E) or diffuse signals and chromatin bridges were often observed (C,F).

View larger version (51K): [in a new window]   Fig. 5. Reduced AtCAP-E1 and AtCAP-E2 levels correlate with developmental defects. (A-C) RT-PCR analysis of SMC2 expression in wild type and three representative antisense lines, 7, 13 and 14. (A,B) RNA was prepared from 7-day-old seedlings and subjected to RT-PCR with the same primers that were used for gene expression RT-PCR. Note that expression is high in wild-type seedlings, but dramatically reduced in the three antisense lines. (B) CAPS analysis (see Fig. 2B and the Methods) of the total RT-PCR pool, illustrating that both genes are affected by the antisense strategy. (C) The same RNA samples used in A and B were subjected to RT-PCR with primers for an actin gene, which served as an internal control for mRNA abundance in each of the samples. (D-K) Developmental defects in the T2 generation of antisense plants. (D) A wild-type Arabidopsis seedling at 12 days after germination. (E) A 12-day old antisense seedling exhibiting the severe phenotype where the SAM has failed to initiate any true leaves (c, cotyledons). (F) An 18-day-old antisense plant, illustrating the enlarged and degenerating SAM (white arrow) and an emerging axillary bud (black arrow). (G) Elongated leaf-like structures (arrows) initiated by an enlarged 18-day-old SAM. (H) Stem bifurcation (arrow) associated with fasciation. (I) Altered phyllotaxy of cauline leaves. (J,K) Stem and floral fasciation observed in E1+/-E2-/- plants.

View larger version (17K): [in a new window]   Fig. 6. Plant growth analysis of wild type and three antisense lines. (A) Plant height at 25, 30 and 35 days post germination. Height of the tallest branch was measured for the antisense plants. Standard deviations are indicated for each sample by vertical lines (n=18 per line). (B) Comparison of time (days post-germination) to the floral transition in wild-type and antisense plants (n=18 per line). The emergence of floral buds was detected in individual plants using a dissecting microscope. (C) Comparison of the average root length in 7-day-old vertically grown wild-type and antisense seedlings (n=48 per line).

View larger version (85K): [in a new window]   Fig. 7. Cytological and molecular characterization of phenotypic defects in the shoot apical meristems of antisense plants. (A-D) DAPI-stained sections of wild-type (A,B) and antisense (C,D) SAMs. Note the tunica/corpus organization in a wild-type SAM (A) is disrupted in the antisense SAM (C). The apical dome is enlarged and the cells of the antisense SAM are larger. (B,D) Higher magnification of A and C. The chromatin appears partially condensed and stretched. (E,F) Toluidine Blue-stained sections of 8-day-old wild-type (E) and 18-day-old antisense (F) plants. The enlarged SAM in F illustrates large disorganized cells. (G) Scanning electron micrograph of an 18-day-old antisense plant showing the enlarged and irregularly shaped SAM (M). (H,I) SEM of a wild-type (H) and an antisense (I) meristem, illustrating well patterned and regularly shaped cells on an emerging leaf of wild type (H), but a disorganized pattern of irregularly shaped cells in the antisense line (I). Note that the SAM in l H is hidden by the leaf primordia, but has clearly emerged in the antisense plant (I). (J-L) Histone H4 expression in the SAM of a wild-type (J) and an antisense (K) plant as seen by RNA in situ hybridization. (J) High level expression of histone H4 is observed in the wild-type SAM, leaf primordia and developing young leaves. (K) Apex of an antisense plant showing a dramatic reduction in histone H4 expression in the enlarged SAM. (L) Higher magnification photograph of an antisense SAM, illustrating that few cells are histone H4 positive. Bar=50 µm. Scale bars: 50 µm (A,C); 10 µm (B,D); 150 µm (G); 60 µm (H,I); 50 µm (J); 100 µm (K); 50 µm (L).

View larger version (87K): [in a new window]   Fig. 8. Phenotypic analysis of 10-day old wild-type and antisense roots. (A,B) Wild-type (A) and antisense (B) roots stained with Aniline Blue. The cell elongation zone of wild-type roots has small, undifferentiated cells with no root hairs. In contrast, antisense roots exhibit disorganized, enlarged, and precociously differentiated cells with root hairs just above the RAM. (C,D) Median optical sections of wild-type (C) and antisense (D) roots stained with propidium iodide showing the cell elongation zone. Note the densely cytoplasmic, small cells with large centrally located nuclei in the wild type (C) compared with the enlarged, vacuolated (v) cells with asymmetrically located nuclei in the epidermal and cortical cell layers in the antisense roots (D). In the antisense root, cell differentiation is also evident from the presence of fully differentiated xylem vessels (D, arrow). (E-F) Median optical sections of wild-type (E) and antisense (F) roots stained with Aniline Blue. Cell organization in wild-type roots is radially symmetric and the quiescent center (arrow in E) is well defined. In antisense roots (F) enlarged epidermal (e) and cortical (c) cells occur in the cell elongation zone, and the quiescent center is disorganized (H). Bars: 250 µm (A,B); 10 µm (C); and 50 µm (D-F).

View larger version (82K): [in a new window]   Fig. 9. Immunoblotting of wild type, mutant and antisense lines. (A) Total protein extracts were prepared from 7-day-old seedlings and subjected to immunoblotting. Lane 1: wild-type plant; lanes 2-4, antisense lines; lane 5, E1-/-E2+/+ (titan 3) plants; lane 6, E1+/+E2-/- plants; lane 7, E1+/-E2-/- plants. (B) Coomassie Blue-stained gel of the protein extracts used in A to show equal loading.