The Opposing Actions of Arabidopsis CHROMOSOME TRANSMISSION FIDELITY7 and WINGS APART-LIKE1 and 2 Differ in Mitotic and Meiotic Cells

Abstract

Sister chromatid cohesion, which is mediated by the cohesin complex, is essential for the proper segregation of chromosomes during mitosis and meiosis. Stable binding of cohesin with chromosomes is regulated in part by the opposing actions of CTF7 (CHROMOSOME TRANSMISSION FIDELITY7) and WAPL (WINGS APART-LIKE). In this study, we characterized the interaction between Arabidopsis thaliana CTF7 and WAPL by conducting a detailed analysis of wapl1-1 wapl2 ctf7 plants. ctf7 plants exhibit major defects in vegetative growth and development and are completely sterile. Inactivation of WAPL restores normal growth, mitosis, and some fertility to ctf7 plants. This shows that the CTF7/WAPL cohesin system is not essential for mitosis in vegetative cells and suggests that plants may contain a second mechanism to regulate mitotic cohesin. WAPL inactivation restores cohesin binding and suppresses ctf7-associated meiotic cohesion defects, demonstrating that WAPL and CTF7 function as antagonists to regulate meiotic sister chromatid cohesion. The ctf7 mutation only had a minor effect on wapl-associated defects in chromosome condensation and centromere association. These results demonstrate that WAPL has additional roles that are independent of its role in regulating chromatin-bound cohesin.

Figure 1.

Inactivation of WAPL Rescues Growth in ctf7 Plants. (A) Thirty day-old ctf7 homozygous plant. (B) Left to right: 30-d-old wild-type, ctf7, wapl1-1 wapl2 Ctf7^+/−, and wapl1-1 wapl2 ctf7 plants.

Figure 2.

wapl1-1 wapl2 ctf7 Plants Show Alterations in Cell Cycle Progression, Mitotic Chromosome Segregation, and DNA Repair. (A) Flow cytometry data on somatic cells from 9-d-old seedlings from wild-type, ctf7, wapl1-1 wapl2 ctf7, and wapl1-1 wapl2 plants. The percentage of cells with 2C, 4C, 8C, 16C, and “intermediate” DNA contents are shown. Data are shown as means ± sd (n = 10,000) from at least three biological samples and three technical replicates. Asterisks represent significant differences (*P < 0.5, **P < 0.01, and ***P < 0.001; Student’s t test) relative to the wild type. (B) Comet images of leaf nuclei from 9-d-old seedlings exposed to Mitomycin C analyzed before and after a 60-min recovery period. Purple and red areas correspond to areas of high DNA density, while blue, green, and yellow correspond to lower density tail-DNA. Bar = 10 µm. (C) Percentage of tail-DNA in cells before and immediately after treatment (0 min) and after a 60-min recovery. Asterisks represent significant differences (*P < 0.5; Student’s t test) relative to the wild type. (D) Representative images of leaf nuclei from 9-d-old wild-type, ctf7, wapl1-1 wapl2, and wapl1-1 wapl2 ctf7 plants stained for DNA with DAPI (left column) or anti-H₃K₉me₂ antibody (middle). Merged images are shown in the right column. (E) Relative levels of chromocenter condensation. Sharp, well-defined anti- H₃K₉me₂ labeling patterns were classified as condensed chromocenters. Decondensed chromocenters appeared hazy and less well defined. Severely decondensed chromocenters were loosely shaped and hazy in appearance. Data are shown as means ± sd (n = 100) from at least three biological samples and three technical replicates. Asterisks represent significant differences (*P < 0.5, **P < 0.01, and ***P < 0.001; Student’s t test) relative to the wild type. Bar = 10 µm.

Figure 3.

wapl1-1 wapl2 ctf7 and wapl1-1 wapl2 Exhibit Developmental Differences in DNA Repair Gene Transcript Levels. (A) One-week-old leaves. (B) Two-week-old leaves. Transcript levels of ATM, ATR, BRCA2B, DMC1, CDC45, BRCA1, RAD51, TOPOII-α, CYCLINB1.1, and PARP2 were measured in Arabidopsis leaves using RT-qPCR. Graphs show representative data from two independent experiments each performed in triplicate. Error bars show the sd. Asterisks represent significant differences (*P < 0.05 and **P < 0.005; Student’s t test) relative to the wild type.

Figure 4.

Pollen, Ovule, and Embryo Development Is Defective in wapl1-1 wapl2 ctf7 Plants. (A) Alexander staining showing alterations in tetrad formation in wapl1-1 wapl2 ctf7, monad (i), dyad (ii), triad (iii), abnormal tetrad (iv), polyads with five (v), and seven uneven (vi) microspores. Bar = 5 μm. (B) Relative distribution of different types of polyads in wapl1-1 wapl2 ctf7 plants. (C) Female gametophytes of the triple mutant wapl1-1 wapl2 ctf7 often abort early during development. Cleared ovules from stages FG0, FG1, FG2, and FG7 in wapl-1 wapl2 ctf7 are shown. (i) Ovule arrested at FG0, with no identifiable nuclei. (ii) FG1 arrested ovule with one nucleus. (iii) FG2 arrested ovule with two nuclei. (iv) Normal appearing ovule at FG7. Solid arrows indicate nuclei, while the dashed arrow denotes no trace of a nucleus. Bar = 5 μm. (D) Embryonic patterning is defective in wapl1-1 wap2 ctf7 fertilized ovules. Fertilized ovules of wild type (i and ii) and wapl1-1 wapl2 ctf7 (iii to v) plants were cleared in Hoyer’s solution and viewed using differential interference contrast microscopy. Bars = 10 μm.

Figure 5.

Arabidopsis wapl1-1 wapl2 ctf7 Plants Exhibit Defects during Male Meiosis. DAPI-stained chromosomes from male meiocytes of wild-type ([A] to [D] and [Q] to [T]), ctf7 ([E] to [H]), wapl1-1 wapl2 ([I] to [L]), and wapl1-1 wapl2 ctf7 ([M] to [P] and [U] to [X]) plants are shown at pachytene ([A], [E], [I], and [M]), diakinesis ([B], [F], [J], and [N]), metaphase I ([C], [G], [K], and [O]), anaphase I ([D], [H], [L], and [P]), telophase I ([Q] and [U]), metaphase II ([R] and [V]), anaphase II ([S] and [W]), and telophase I/tetrad ([T] and [X]). Bars = 5 μm.

Figure 6.

wapl1-1 wapl2 ctf7 Male Meiocytes Exhibit Nonspecific Association of Centromeres. FISH was conducted on male meiocytes from wild-type ([A] to [D] and [Q] to [T]), ctf7 ([E] to [H]), wapl1-1 wapl2 ([I] to [L]), and wapl1-1 wapl2 ctf7 ([M] to [P] and [U] to [X]) plants with a centromere probe. DAPI-stained chromosomes are shown in red, and the centromere signal is shown in green. Cells are shown at leptotene ([A], [E], [I], and [M]), zygotene ([B], [F], [J], and [N]), pachytene ([C], [G], [K], and [O]), diakinesis ([D], [H], [L], and [P]), metaphase I (Q), early anaphase I (U), anaphase I ([R] and [V]), anaphase II ([S] and [W]), and telophase II ([T] and [X]). Bar = 5 µm.

Figure 7.

Cohesin Establishment Is Recovered in wapl1-1 wapl2 ctf7 Meiocytes. Meiotic spreads of wild-type ([A] to [D]), ctf7 ([E] to [H]), wapl1-1 wapl2 ([I] to [L]), and wapl1-1 wapl2 ctf7 ([M] to [P]) plants were prepared and stained with anti-SYN1 antibody (green) and propidium iodide (red). Meiocytes in wild-type, wapl1-1 wapl2, and the wapl1-1 wapl2 ctf7 plants exhibited similar SYN1 staining patterns from leptotene to pachytene. The ctf7 mutant did not exhibit a clear SYN1 signal during leptotene (E), zygotene (F), pachytene (G), or diakinesis (H). In wild-type meiocytes, SYN1 is removed from the arms of chromosomes during diplotene and diakinesis (C); it was not detectable during pro-metaphase I (D). Strong SYN1 signals are detectable in wapl1-1 wapl2 during diakinesis, metaphase, and anaphase a ([J] to [L]). SYN1 signals resemble the wild type on wapl1-1 wapl2 ctf7 chromosomes during leptotene and pachytene ([M] and [N]). During diplotene, condensed diakinesis (O), and late diakinesis/metaphase, SYN1 signals remained strong. Little to no SYN1 signal is detected by metaphase/anaphase I (P). Bar = 5 μm.

Figure 8.

The Radial Microtubule System Is Disrupted in wapl1-1 wapl2 ctf7Plants. Spindles of male meiocytes from wild-type ([A] to [F]) and wapl1-1 wapl2 ctf7 ([G] to [L]) plants were stained using anti-β-tubulin antibody (green). DNA was counterstained with propidium iodide (red). Male meiocytes are shown at metaphase I ([A] and [G]), anaphase I ([B] and [H]), telophase I ([C] and [I]), metaphase II ([D] and [J]), anaphase II ([E] and [K]), and telophase II/tetrad stages ([F] and [L]). Bars = 10 μm.