Arabidopsis separase AESP is essential for embryo development and the release of cohesin during meiosis

Abstract

To investigate how and when sister chromatid cohesion is released from chromosomes in plants, we isolated the Arabidopsis thaliana homolog of separase (AESP) and investigated its role in somatic and meiotic cells. AESP is similar to separase proteins identified in other organisms but contains several additional structural motifs. The characterization of two Arabidopsis T-DNA insertion alleles for AESP demonstrated that it is an essential gene. Seeds homozygous for T-DNA insertions in AESP exhibited embryo arrest at the globular stage. The endosperm also exhibited a weak titan-like phenotype. Transgenic plants expressing AESP RNA interference (RNAi) from the meiosis-specific DMC1 promoter exhibited alterations in chromosome segregation during meiosis I and II that resulted in polyads containing from one to eight microspores. Consistent with its predicted role in the release of sister chromatid cohesion, immunolocalization studies showed that the removal of SYN1 from chromosome arms and the centromeres is inhibited in the RNAi mutants. However, the release of SYN1 during diplotene occurred normally, indicating that this process is independent of AESP. Therefore, our results demonstrate that AESP plays an essential role in embryo development and provide direct evidence that AESP is required for the removal of cohesin from meiotic chromosomes.

Figure 1.

AESP Gene and Protein Structures. (A) AESP gene structure. The positions of exons are shown as black boxes. The positions and directions of primers used in this study are shown as horizontal arrows. The positions of T-DNA insertions are shown as inverted triangles. The scale bar is shown at bottom. (B) Motifs in ESP1 proteins from Arabidopsis (a), human (b), mouse (c), C. elegans (d), Saccharomyces cerevisiae (e), and Drosophila (f).

Figure 2.

Amino Acid Sequence Alignment of the Peptidase C-50 Domain in ESP1 Proteins. The peptidase domains of human (Q14674), mouse (P60330), Arabidopsis (AY823256), S. cerevisiae (NP011612), C. elegans (AAK77200), and Drosophila (AAQ72557) separase proteins are shown aligned. Identical amino acids are shaded in black, and similar amino acids are shaded in gray. Missing residues are shown as dashes. ClustalW (http://www.ebi.ac.uk/clustalw) and BOXSHADE 3.33 (http://www.ch.embnet.org/software/BOX_form.html) were used to produce the alignment.

Figure 3.

AESP Transcript Levels in Wild-Type and RNAi Plants. (A) AESP transcript levels were analyzed in root (R), bud (B), stem (S), and leaf (L) using RT-PCR. The ACT8 gene was used as an internal control. (B) RT-PCR analysis of AESP mRNA levels in wild-type and transgenic plants containing an AESP-RNAi construct driven by the ATDMC1 promoter. Plants 1, 2, and 7 exhibited reduced fertility and AESP RNA levels. Plant 5 showed normal levels of AESP RNA and was fertile. The ACT8 gene was used as an internal control.

Figure 4.

AESP Is Required for Seed Development. Bright-field images of stained sections taken from embryos ([A] to [F]) and endosperm nuclei ([G] to [I]) in wild-type ([A] to [C] and [I]) and abnormal ([D] to [H]) seeds in the same siliques of heterozygous Aesp-2 plants. Mutant embryos were arrested at the globular stage ([D] to [F]) relative to wild-type embryos at the globular (A), heart (B), and curled cotyledon (C) stages. Some periphery (G) and chalazal (H) endosperm nuclei and nucleoli were enlarged relative to those of wild-type embryos (I). Bars = 20 μm.

Figure 5.

ATDMC1-AESP-RNAi Plants Exhibit Reduced Fertility. (A) to (G) Anthers and pollen development in wild-type and ATDMC1-AESP-RNAi plants. Wild-type flowers had elongated stamens (A) and mature pollen grains ([C], arrow), whereas ATDMC1-AESP-RNAi flowers had short stamens (B) and shrunken pollen grains (D). The siliques in RNAi plants were dramatically shorter (E) and generated fewer seeds (F) in contrast with wild-type siliques (G). (H) Relative number of microspores in ATDMC1-AESP-RNAi mutant polyads (n = 200 polyads).

Figure 6.

ATDMC1-AESP-RNAi Plants Are Defective in Meiosis I and Meiosis II. Meiotic spreads of wild-type ([A] to [G]) and ATDMC1-AESP-RNAi ([I] to [O], [Q], and [R]) plants were prepared with Carnoy's fixative and stained with 4′,6-diamidino-2-phenylindole (DAPI). Tetrads of wild-type plants (H) and polyads of ATDMC1-AESP-RNAi plants ([P], [S], and [T]) were stained with toluidine blue. No differences were detected in wild-type and mutant meiocytes during prophase I, including late pachytene ([A] and [I]) and diakinesis ([B] and [J]), and metaphase I ([C] and [K]). Homologous chromosomes segregated evenly during anaphase I (D) and telophase I (E) in wild-type meiocytes, whereas in mutant meiocytes, homologous chromosomes were tangled and stretched at the metaphase I-to-anaphase I transition (L), which resulted in chromosome bridges ([M], arrows) and stretched and fragmented chromosomes ([N], arrow) at anaphase I and telophase I. Wild-type meiocytes at meiotic metaphase II (F), telophase II (G), and tetrad stage (H) are shown. Most meiocytes in the RNAi lines displayed more than four groups of chromosomes at telophase II (O) and produced polyads with more than four microspores, which contained different amounts of genetic material (P). Bivalents did not segregate in some meiocytes at meiosis I (Q) and meiosis II (R) in RNAi plants, resulting in polyads with fewer than four microspores ([S] and [T]). Stages of the abnormal meiocytes are approximate and based on cell morphology and the stage of the surrounding tissue. Bars = 5 μm.

Figure 7.

SYN1 Removal Is Defective in ATDMC1-AESP-RNAi Plants. Meiotic spreads of wild-type ([A] to [E]) and ATDMC1-AESP-RNAi ([F] to [O]) plants were prepared and stained with anti-SYN1 antibody (green) and propidium iodide (red). Meiocytes in wild-type plants and the RNAi mutant exhibited similar SYN1 staining at pachytene ([A] and [F]), diakinesis ([B] and [G]), prometaphase I ([C] and [H]), and metaphase I ([D] and [I]). SYN1 labeling was not detected after anaphase I (E) in wild-type meiocytes. SYN1 was still detected on chromosome arms and at centromeric regions at anaphase I ([J] and [K]) and on intact bivalents at various stages of meiosis II in the meiocytes of RNAi plants ([L] to [O]). Bar = 5 μm.