<i>Atwapl1-1wapl2</i> plants exhibit reduced fertility.

<p>(A) Thirty five day-old wild-type and <i>Atwapl1-1wapl2</i> plants. (B) Alexander staining of wild-type, <i>Atwapl1-1</i>, <i>Atwapl2</i>, and <i>Atwapl1-1wapl2</i> pollen. Green pollen is nonviable. Size Bars = 10 µm. (C) Seed setting in siliques of wild type, <i>Atwapl1-2wapl2</i> and <i>Atwapl1-1wapl2</i> plants.</p

Female gametophyte development is altered in <i>Atwapl1-1wapl2</i> plants.

<p>Cleared ovules of wild-type (A–C) and <i>Atwapl1-1wapl2</i> (D–F) plants are shown at the Megaspore Mother Cell stage (A, D), wild type FG2 (B, E) and wild-type FG7 stages (C, F), CN: Central nucleus, EC: egg cell, SC: synergid cell. Female gametophytes were found to arrest at FG1 (E) and FG2 (F) in <i>Atwapl1-1wapl2</i> plants. Images shown for <i>Atwapl1-1wapl2</i> represent the most common phenotypes observed. Arrows indicate arrested nuclei. Size bar = 10 µM.</p

Inactivation of <i>AtWAPL</i> rescues <i>Atctf7</i> mutants.

<p>(A) Thirty day-old wild-type (left), <i>Atctf7</i> homozygous (middle) and <i>Atwapl1-1wapl2ctf7</i> triple homozygous (right) plants. (B) Alexander staining of anthers showing pollen viability in <i>AtCtf7^+/−</i>, <i>Atctf7</i>, <i>Atwapl1-1wapl2</i>, <i>Atwapl1-1wapl2Ctf7^+/−</i> and <i>Atwapl1-1wapl2ctf7</i> plants. Viable pollen stain red, while nonviable pollen stain green. Size Bar = 10 µm (C) Seed set in <i>Atwapl1-1wapl2Ctf7^+/−</i> and <i>Atwapl1-1wapl2ctf7</i> plants is lower than that observed in <i>Atwapl1-1wapl2</i> plants. Images shown represent the most common phenotypes observed.</p

Embryonic patterning is defective in the seeds of <i>Atwapl1-1wapl2</i> plants.

<p>Fertilized ovules of wild type (A–D) and <i>Atwapl1-1wapl2</i> (E–H) plants were cleared in Hoyers solution and viewed using DIC microscopy. Abnormal division planes were observed early in development, including in two (E) and four celled embryos (F). Asynchronous/abnormal cell division and growth was observed (F, G) with defects becoming more pronounced at the dermatogen (G) and globular stages (H). Images shown for <i>Atwapl1-1wapl2</i> represent the most common abnormal phenotypes observed at each stage. Size bar = 10 µm.</p

<i>Arabidopsis thaliana</i> WAPL Is Essential for the Prophase Removal of Cohesin during Meiosis

<div><p>Sister chromatid cohesion, which is mediated by the cohesin complex, is essential for the proper segregation of chromosomes in mitosis and meiosis. The establishment of stable sister chromatid cohesion occurs during DNA replication and involves acetylation of the complex by the acetyltransferase CTF7. In higher eukaryotes, the majority of cohesin complexes are removed from chromosomes during prophase. Studies in fly and human have shown that this process involves the WAPL mediated opening of the cohesin ring at the junction between the SMC3 ATPase domain and the N-terminal domain of cohesin's α-kleisin subunit. We report here the isolation and detailed characterization of <i>WAPL</i> in <i>Arabidopsis thaliana</i>. We show that Arabidopsis contains two <i>WAPL</i> genes, which share overlapping functions. Plants in which both <i>WAPL</i> genes contain T-DNA insertions show relatively normal growth and development but exhibit a significant reduction in male and female fertility. The removal of cohesin from chromosomes during meiotic prophase is blocked in <i>Atwapl</i> mutants resulting in chromosome bridges, broken chromosomes and uneven chromosome segregation. In contrast, while subtle mitotic alterations are observed in some somatic cells, cohesin complexes appear to be removed normally. Finally, we show that mutations in <i>AtWAPL</i> suppress the lethality associated with inactivation of <i>AtCTF7</i>. Taken together our results demonstrate that WAPL plays a critical role in meiosis and raises the possibility that mechanisms involved in the prophase removal of cohesin may vary between mitosis and meiosis in plants.</p></div

<i>Atwapl1-1wapl2</i> plants exhibit defects during male meiosis.

<p>DAPI stained chromosomes from male meiocytes of wild type (A–D, I–L, Q–S) and <i>Atwapl1-1wapl2</i> plants (E–H, M–P, U–W) are shown at leptotene (A, E), zygotene (B, F), pachytene (C, G), diplotene (D, H), diakinesis (I, M), metaphase I (J, N), anaphase 1 (K, O), telophase I (L, P), metaphase II (Q, U), telophase II (R, V) and tetrad stage (S, W). Alexander stained tetrads/polyads are shown in (T, X). Images shown for <i>Atwapl1-1wapl2</i> represent the most common phenotypes observed at each stage. Arrows in C & G denote chromocenters. Arrow in P denotes a lagging chromosome. Size bar = 5 µm.</p

<i>Atwapl1-1wapl2</i> plants show defects in mitosis.

<p>Root tips of wild type (A–C) and <i>Atwapl1-1wapl2</i> plants (D–F) were squashed and stained with DAPI. In wild type root tips the replicated chromosomes condense and align on the metaphase plate (A) followed by the even segregation of ten chromosomes to each pole during anaphase (B) and telophase (C). Most <i>Atwapl1-1wapl2</i> root tip cells appeared normal; however 20% of the cells contained metaphase chromosomes that appeared sticky (D). Uneven segregation of chromosomes, chromosome bridges, stretched chromosomes and chromosome fragments were subsequently observed at anaphase and telophase (E, F). Arrows denote a lagging chromosome and chromosome bridge in E and F, respectively. Size Bar = 10 µm.</p

<i>Atwapl1-1wapl2</i> meiocytes exhibit nonspecific association of centromeres.

<p>FISH was conducted using a 180(A–D, I–L) and <i>Atwapl1-1wapl2</i> (E–H, M–P) plants. DAPI-stained chromosomes are shown in red, centromere FISH signals in green. Ten signals are observed at interphase I cells of both lines (A, E). Five signals are typically observed during zygotene (B), pachytene (C), and diplotene (D) in wild type meiocytes. Clusters of centromere signals are typically observed in <i>Atwapl1-1wapl2</i> meiocytes during prophase I (F, G, H). In wild type five pairs of chromosomes are observed at metaphase I (I) that separate into two groups of five signals at anaphase I (J); two groups of five pairs of signals are observed at metaphase II (K) followed four groups of five signals at telophase II (L). Ten to twenty signals that show aberrant segregation are observed from anaphase I onward in <i>Atwapl1-1wapl2</i> meiocytes (M–P). Images shown for <i>Atwapl1-1wapl2</i> represent the most common phenotypes observed at each stage. Size bar = 10 µm.</p

<i>AtWAPL1</i> and <i>AtWAPL2</i> show similar expression patterns.

<p>(A) Relative transcript levels for <i>AtWAPL1</i> and <i>AtWAPL2</i> in different wild type tissues are shown. (B) <i>AtWAPL</i> transcript levels in bud tissue from wild type, <i>Atwapl1-1wapl2</i> and <i>Atwapl1-2wapl2</i> plants. Results are shown as means ± SD (<i>n</i> = 3). Asterisks represent significant differences between mutant and wild type levels (*<i>P</i><0.0001, **<i>P</i><0.001; Student's <i>t</i>-test).</p

<i>Atwapl1-1wapl2</i> meiocytes exhibit alterations in homologous chromosome pairing.

<p>FISH was conducted on male meiocytes from wild type (A–D) and <i>Atwapl1-1wapl2</i> (E–L) plants using a telomere repeat probe that binds to a region proximal to the centromere of chromosome 1. Two signals are observed at early leptotene (A, E, I). One signal reflecting synapsed chromosomes is observed at late zygotene and pachytene in wild type and some <i>Atwapl1-1wapl2</i> meiocytes (B, C F, G), while two signals are observed in others (J, K). Two closely spaced signals are typically observed at diplotene in wild type and many <i>Atwapl1-1wapl2</i> meiocytes (D, H) with two widely separated signals in others (L). Images shown for <i>Atwapl1-1wapl2</i> represent the most common phenotypes observed at each stage. Size bar = 10 µm.</p