Skip to main content
NCBI home page
Wiley Open Access Collection logoLink to Wiley Open Access Collection
. 2019 Jul 24;224(1):229–241. doi: 10.1111/nph.16014

The Arabidopsis anaphase‐promoting complex/cyclosome subunit 8 is required for male meiosis

Rong‐Yan Xu 1,2,†, Jing Xu 1,†, Liudan Wang 1,†, Baixiao Niu 1,3, Gregory P Copenhaver 4,5, Hong Ma 1,6, Binglian Zheng 1, Yingxiang Wang 1,✉
PMCID: PMC6771777  PMID: 31230348

Summary

  • Faithful chromosome segregation is required for both mitotic and meiotic cell divisions and is regulated by multiple mechanisms including the anaphase‐promoting complex/cyclosome (APC/C), which is the largest known E3 ubiquitin‐ligase complex and has been implicated in regulating chromosome segregation in both mitosis and meiosis in animals. However, the role of the APC/C during plant meiosis remains largely unknown. Here, we show that Arabidopsis APC8 is required for male meiosis.

  • We used a combination of genetic analyses, cytology and immunolocalisation to define the function of AtAPC8 in male meiosis.

  • Meiocytes from apc8‐1 plants exhibit several meiotic defects including improper alignment of bivalents at metaphase I, unequal chromosome segregation during anaphase II, and subsequent formation of polyads. Immunolocalisation using an antitubulin antibody showed that APC8 is required for normal spindle morphology. We also observed mitotic defects in apc8‐1, including abnormal sister chromatid segregation and microtubule morphology.

  • Our results demonstrate that Arabidopsis APC/C is required for meiotic chromosome segregation and that APC/C‐mediated regulation of meiotic chromosome segregation is a conserved mechanism among eukaryotes.

Keywords: APC/C, Arabidopsis thaliana, chromosome segregation, meiosis, spindle assembly

Introduction

In meiosis, faithful chromosome segregation is crucial for ploidy stability during sexual life cycles (Zamariola et al., 2014b). Unlike mitosis, which has one round of sister chromatid segregation, meiosis has two rounds of segregation following a single round of premeiotic DNA replication. Meiosis I results in segregation of homologous chromosomes and meiosis II separates the sister chromatids. Several mechanisms ensure accurate segregation of chromosomes during meiosis, including the stepwise removal of cohesion from sister chromatid arms and centromeres (Rankin & Dawson, 2016; Bolanos‐Villegas et al., 2017). However, the mechanisms that regulate the attachment of spindle microtubules to kinetochores to ensure proper chromosome alignment and the onset of anaphase (Di Fiore et al., 2016; Ji et al., 2018) are not as well understood, particularly in plants including Arabidopsis.

The anaphase‐promoting complex/cyclosome (APC/C) is a multisubunit RING E3 ubiquitin ligase that regulates the progression of the eukaryotic cell cycle together with Cdc20 (its mitosis‐specific activator) or Cdh1 (its interphase‐specific activator) (Kashevsky et al., 2002; Pines, 2011; Sivakumar & Gorbsky, 2015; Severson et al., 2016). In plants, the APC/C consists of at least 11 core subunits, each of which is encoded by a single gene, except for APC3, which is encoded by two genes, APC3a/CDC27a and APC3b/CDC27b/HOBBIT (HBT). The Arabidopsis APC/C activator Cell Division Cycle 20.1 is required for bivalent alignment and chromosome segregation during meiosis (Niu et al., 2015), suggesting that the APC/C subunits may also have meiotic functions in plants. During meiotic maturation in mammalian oocytes, the APC/C is involved in virtually every stage (Homer, 2013) and disruption of either the APC/C or its substrates results in severe phenotypes. For example, deletion of the APC/C subunit APC2 gene causes major defects in fertility (McGuinness et al., 2009). In C. elegans, the APC/C gene mat‐3/APC8 has been defined by temperature‐sensitive embryonic lethal alleles, which lead to defects in meiosis and mitosis (Garbe et al., 2004). In Xenopus, the APC is required for the second meiotic anaphase (Peter et al., 2001). In addition, genetic inactivation of APC/C components causes lethality in many species (Yu et al., 1998; Yamashita et al., 1999; Cullen et al., 2000; Bentley et al., 2002; Garbe et al., 2004; Pal et al., 2007; Jin et al., 2010). In plants, the APC/C core complex and its activators have been reported to play important roles in growth and development (Heyman & De Veylder, 2012; Wang et al., 2013; Guo et al., 2016). Although meiotic functions of the known plant APC/C subunits have not been reported, several meiotic regulators associated with APC/C have been identified. THREE DIVISION MUTANT1/MALE STERILE 5 (TDM1/MS5) regulates meiotic exit and is a putative meiotic APC/C component based on its shared structural similarities with the APC/C subunit CDC16/Cut9/APC6 (Cromer et al., 2012; Cifuentes et al., 2016). The direct interaction between TDM1with the APC/C activator CDC20.1 and the APC/C core component CDC27b (HOBBIT) further supports the hypothesis that TDM1 acts via the activation of the APC/C and/or by modifying its specificity. In addition, TDM1 also interacts with TAM/CYCA1;2 (an A‐type cyclin) and OSD1 (an APC/C inhibitor). Both proteins are required to prevent meiosis termination after meiosis I (Bulankova et al., 2010; d'Erfurth et al., 2010; Cromer et al., 2012). PATRONUS1 (PANS1) is required for the protection of centromere cohesion at meiosis II and interacts directly with CDC27b and CDC20.1 (Cromer et al., 2013; Zamariola et al., 2014a; Singh et al., 2015), suggesting that PANS1 could be an APC/C regulator or target.

An allele of Arabidopsis APC8, apc8‐1, was previously identified in a genetic screen for enhancers and/or suppressors of weak allele of DCL1 (dcl1‐14), which is required for miRNA biogenesis. Analysis of apc8‐1 showed that APC8 acts as a dual integrator mediating both microRNA‐dependent cyclin B1 transcription and its degradation during postmeiotic male gametophyte development (Zheng et al., 2011). More recently, the soybean GmILPA1 gene encoding an APC8‐like protein, has been shown to have a role in the regulation of leaf petiole angle (Gao et al., 2017), suggesting multiple functions for APC8.

Here, we report that Arabidopsis APC8 is required for male meiosis. Analysis of chromosome dynamics in apc8‐1 meiocytes shows irregular alignment of bivalents at metaphase I, improper segregation of sister chromatids at anaphase II, and the subsequent formation of polyads. Furthermore, apc8‐1 exhibits abnormal microtubule organisation from metaphase I onward. These results provide evidence that the APC/C subunit plays an important role during meiotic chromosome segregation in plants.

Materials and Methods

Plant materials

All the Arabidopsis thaliana lines used in the study are in the ‘Columbia’ ecotype background. Wild‐type Col‐0 plants were used as controls. The apc8‐1 allele is a point mutation (c. 925G>A) resulting in the substitution of a conserved amino acid, D309N, adjacent to the TPR domain. Transgenic complementation of apc8‐1 with an APC8‐YFP fusion has been described previously (Zheng et al., 2011). PCR amplification using primers (Supporting Information Table S1) of a 251‐bp fragment including the SNP was used for genotyping. Digestion of the PCR products with ApoI followed by agarose gel analysis, yields bands of c. 190 bp and c. 60 bp using apc8‐1 DNA and 251 bp using wild‐type DNA (Zheng et al., 2011). The other apc T‐DNA insertion alleles apc1‐2/at5g05560 (Salk_152651), apc2‐2/at2g04660 (Salk_010621), apc4‐1/at4g21530 (Salk_024729), apc5‐2/at1g06590 (Salk_036491), and apc11/at3g05870 (Salk_019654) were obtained from the Arabidopsis Biological Resource Center. All plants were grown in a glasshouse with a 16 h : 8 h, day : night photoperiod at 20°C with 70% humidity; 1–5 mm radicles harvested from germinated seeds were used for mitotic analyses.

Real‐time PCR

Total RNA was extracted from inflorescences and 14‐d‐old seedlings using TRIzol reagent (Invitrogen). Each experiment included three biological replicates collected from independent plants and three technical replicates for each biological replicate. Reverse transcription was conducted using a Prime Script RT reagent kit with gDNA Eraser (cat. no. RR047A; TaKaRa), the products were then used as the template for quantitative PCR. Primers are listed in Table S1. PCR analysis was conducted using the Step One Plus Real‐Time PCR system (Applied Biosystems, Carlsbad, CA, USA) with iTaq Universal SYBR Green Supermix (cat. no. 72‐5124; Bio‐Rad). The cycle threshold (CT) value of the gene was normalised to CT of the internal control (actin2), and then the relative expression between samples was calculated using delta CT method (Schmittgen & Livak, 2008). The statistical significance of different gene expression levels was using the two‐tailed Student's t‐test.

Cytological analyses

Inflorescences of Arabidopsis wild‐type and mutant plants were collected and fixed in a modified Carnoy's fixative (three ethanol : one acetic acid) for a minimum of 3 h. Pollen viability was analysed using a modified Alexander staining method by Peterson et al. (2010). Tetrad‐staged meiocytes were collected and stained with carbol fuchsin as previously described (Wang et al., 2014). Images of pollen and tetrads were obtained using a Zeiss Axio Imager microscope and processed using Adobe Photoshop CS6. Anthers at meiotic stages were collected from fixed inflorescences and used for cytological analyses. Chromosome spreading and fluorescence in situ hybridisation (FISH) analyses were performed as described previously (Wang et al., 2014).

Immunolocalisation

Immunolocalisation of SYN1 and CENH3 were performed as described previously (Chelysheva et al., 2010), with modified antibody dilutions (below). For immunolocalisation of tubulin, inflorescences were fixed in 4 : 1 methanol and acetone mix. Selected materials were digested as previously described (Wang et al., 2014). Meiotic stage anthers were squashed and immobilised on glass slides coated with poly l‐Lys. After drying, the slides were incubated in phosphate‐buffered saline (PBS) with 1% Triton X‐100 for 1 h at room temperature, followed by blocking in PBS with 1.0 mM EDTA, 0.1% Tween‐20 for 1 h at room temperature. Samples were incubated at 4°C overnight with the primary antibodies. After rinsing in PBS buffer with 0.1% Tween‐20 three times, slides were incubated with secondary antibodies at 37°C for 2 h and washed three times in PBS. Immunolocalisation samples were then stained with antifade medium containing 2 mg ml−1 DAPI (Vector Laboratories, Burlingame, CA, USA). Observations were recorded using a Zeiss Axio Imager fluorescence microscope and processed using Adobe Photoshop CS6. The dilution of primary antibodies was 1 : 100 for CENH3 (ab72001; Abcam, Cambridge, MA, USA), 1 : 200 for SYN1 (Wang et al., 2012a), SMC3 (Wang et al., 2016) and ASY1 (Wang et al., 2012a), and 1 : 500 for α‐tubulin (Beyotime Biotech, Shanghai, China). All secondary antibodies labelled with fluorophore were diluted 1 : 1000.

Yeast two‐hybrid assay

The full‐length cDNA of AtAPC8, AtAPC10 and AtCDC20.1 were obtained from reverse transcription PCR (RT‐PCR) of mRNA extracted from inflorescences of Arabidopsis plants. The PCR product of AtAPC8 was cloned into the pEASY‐Blunt Cloning Vector (Transgene Biotech Co., Beijing, China; CB101‐01). The product of the APC8‐1 point mutation was amplified from apc8‐1 cDNA to produce a clone of the mutant allele designated APC8‐D309N. Primers are listed in Table S1 and Vazyme Super‐Fidelity DNA polymerase was used (Vazyme Biotech Co., Nanjing, China; P515‐01). The primers used to amplify AtAPC10 (NdeI/EcoRI) and AtCDC20.1 (NdeI/BamHI) included engineered restriction enzyme sites. The amplicons were digested with the corresponding enzymes and ligated into the pGADT7 vector using T4 DNA ligase (Thermo Scientific Biotech Co., Waltham, MA, USA; EL0014). PCR products of APC8 and APC8‐D309N were purified and ligated into the pGBKT7 vector using NOVOREC PLUS DNA recombinase (Novoprotein Biotech Co., Suzhou, China; NR001A). The recombinant plasmids were verified by DNA sequencing and transformed into Y187 yeast strain (pGADT7) and Y2H Gold yeast strain (pGBKT7), accordingly. Transformants were mated on YPDA (yeast, peptone, dextrose, adenine) medium for 48 h, and selected on double dropout medium without leucine and tryptophan (DDO) plates for 48 h as well as the quadruple dropout medium lacking leucine, tryptophan, histidine, and adenine, and with X‐α‐Gal (g) (QDO) for 6 d.

Structural modelling

The predicted structural model of APC8 was developed using the Phyre2 server (http://www.sbg.bio.ic.ac.uk/phyre2/html/page.cgi?xml:id=index) (Kelley et al., 2015) based on the structure of human CDC23 (PDB code: 4UI9) (Schreiber et al., 2011).

Results

Arabidopsis APC8 is required for meiosis

The Arabidopsis apc8‐1 point mutant is hypomorphic and has pleiotropic phenotypes, including abnormal development of the meristem, leaves and shoots, as well as reduced fertility with short siliques and mature pollen with no or single sperm‐like cells (Zheng et al., 2011), indicating that APC8 plays an important role in vegetative and reproductive development. Although the apc8‐1 mutant is defective in vegetative growth (Fig. 1a–c), its floral organs are generally similar to wild‐type organs (Fig. 1d–f). Consistent with the observed reduced fertility, apc8‐1 anthers have reduced pollen production compared with wild‐type (Fig. 1d–f). Furthermore, Alexander staining of anthers showed that apc8‐1 has fewer viable pollen grains (25 ± 6/anther, n = 30) (Fig. 1h) than in either wild‐type (525 ± 41/anther, n = 15) or the APC8‐YFP complementation line (498 ± 38/anther, n = 15) (Fig. 1g,i). At the tetrad meiocyte stage (n = 30 per genotype), the four microspores in wild‐type and APC8‐YFP were uniform in size (Fig. 1j,l), whereas the polyads in apc8‐1 had > 4 microspores of unequal size (Fig. 1k), further supporting a meiotic defect in apc8‐1. Finally, apc8‐1 had an average of 12.8 seeds per silique (n = 11) compared with wild‐type with an average of 51.6 (n = 13) (Fig. S1b; Student's t‐test, P < 0.01). To verify that the embryo lethal phenotype of the null apc8 alleles is not unusual, we obtained T‐DNA insertion alleles for five of the 11 other APC/C subunits (APC1, APC2, APC4, APC5 and APC11). PCR analysis of the respective alleles in segregating populations from each line failed to identify any homozygous mutant plants, supporting the essential role of most APC/C subunits. Consistent with this observation, the heterozygotes apc1‐2 +/− (33.3, n = 5), apc2‐2 +/− (30.4, n = 5), apc4‐1 +/− (35.2, n = 5), and apc11 +/− (25.6, n = 5), have fewer viable seeds in their siliques compared with wild‐type (51.6, n = 13) (Fig. S1). The segregation ratio of normal vs dead seeds for apc1‐2 +/−, apc2‐2 +/− and apc4‐1 +/− is consistent with a 3 : 1 ratio, but lower for apc11 +/−. Together, these data support the essential role of APC/C subunits in regulating the cell cycle and that APC8 is also required for meiosis.

Figure 1.

Figure 1

Open in a new tab

Phenotypes of wild‐type (WT), apc8‐1 and APC8‐complemented Arabidopsis. WT (a), apc8‐1 (b), and APC8‐complemented plants (c). Flowers of WT (d), apc8‐1 (e), and APC8‐complemented plants (f). Alexander staining of anthers to assay pollen viability in WT (g), apc8‐1 (h), and APC8‐complementated plants (i). In total, six independent plants were analysed. Viable and inviable pollen grains are stained in red and dark green, respectively. Tetrads stained with carbol fuchsin were prepared from WT (j), apc8‐1 (k), and APC8‐complementated plants (l), respectively. Arrowhead indicates micronucleus. Bars: (a–c) 5 cm; (d–f) 1 mm; (g–i) 100 µm; (j–l) 25 µm.

The apc8‐1 mutant is a point mutation (925G>A). To examine the expression of APC8 in this mutant, we performed qRT‐PCR analyses using seedlings and inflorescences from wild‐type and apc8‐1. The expression of APC8 showed no significant difference in apc8‐1 compared with wild‐type in leaf tissue (Fig. S2a), but was significantly higher in apc8‐1 inflorescences (P < 0.001) relative to wild‐type (Fig. S2a), suggesting that the point mutation in APC8 had a positive effect on its expression in inflorescences. Previous studies have demonstrated that AtTDM1 and AtOSD1 are an APC/C component and inhibitor, respectively (Cromer et al., 2012; Cifuentes et al., 2016), and AtPANS1 is an APC regulator (Cromer et al., 2013; Zamariola et al., 2014a; Singh et al., 2015). We analysed the expression of these three genes in inflorescences of wild‐type, apc8‐1, APC8‐complementated plants, and AtTDM1 and AtOSD1 expression showed a significant elevation in apc8‐1 (P < 0.01) and APC8‐YFP plants (P < 0.05) compared with wild‐type (Fig. S2b), while the AtPANS1 expression was unaltered among the samples examined (Fig. S2b). Taken together, these results suggested that APC/C and its components are aberrantly activated in the apc8‐1 mutant and this increased activity triggers the need for more AtOSD1 to facilitate proper inhibition.

AtAPC8 ensures proper bivalent alignment and subsequent accurate segregation of sister chromatids

To investigate the meiotic defects in apc8‐1 in detail, chromosome spreads from male meiocytes stained with 4′,6‐diamidino‐2‐phenylindole (DAPI) were assessed. Wild‐type and apc8‐1 chromosomes have similar thread‐like structures through pachytene (Fig. 2a) that condense into five bivalents at diakinesis (Fig. 2b). At metaphase I the five bivalents, pushed by the spindle, are aligned along the equator in wild‐type (Fig. 2c), whereas c. 36.7% (n = 30) of apc8‐1 meiocytes have disorderly in bivalent alignment (Fig. 2c), similar to the phenotype of the previously identified cdc20.1 mutant (Niu et al., 2015). Even though some bivalents are not aligned at the metaphase I plate, the homologues still successfully segregate at anaphase I (Fig. 2d). However, some lagging chromosomes are observed, indicating aberrant segregation dynamics. Nonetheless, at later stages, no obvious chromosome behaviour defects were observed from telophase I to prophase II (Fig. 2e,f). Similar to metaphase I, we also observed a misalignment of chromosomes at metaphase II plate in one or two poles in the apc8‐1 mutant in 40% (n = 30) of meiocytes (Fig. 2g). However, c. 85% (n = 30) of anaphase II meiocytes have uneven separation of sister chromosomes (Fig. 2h). As a consequence, c. 90% of apc8‐1 meiocytes (n = 35) form abnormal tetrads or polyads (Fig. 2j). As a control, the APC8‐complementated plants exhibited similar meiotic chromosome behaviours to that of wild‐type at all the observed stages (Fig. S3). Because apc8‐1 is a weak mutant allele of APC8, these analyses are likely to underestimate the role of APC8 in meiosis. Therefore, these data provide evidence that APC8 is required for proper meiotic chromosome alignment and segregation.

Figure 2.

Figure 2

Open in a new tab

Meiotic chromosome configurations of wild‐type (WT), apc8‐1 and APC8‐complemented Arabidopsis. In each panel, left column, WT; middle column, apc8‐1; right column, APC8‐complemented chromosome spreads. (a) pachytene; (b) diakinesis; (c) metaphase I; (d) anaphase I; (e) telophase I; (f) prophase II; (g) metaphase II; (h) anaphase II; (i) telophase II; and (j) tetrad stage. All pictures were taken with a ×100 objective using a fluorescence microscope (Zeiss Axio Imager). Bars, 5 μm.

The abnormal vegetative development in apc8‐1 indicates a possible defect in mitotic cell division. To test this possibility, we examined mitotic chromosome spreads from root tip cells harvested from wild‐type, apc8‐1 and APC8‐complementated plants. We observed several types of mitotic anomalies in apc8‐1 cells including 3.03% (n = 99) with abnormally aligned sister chromatids at metaphase (Fig. S4f), 11.9% (n = 42) with lagging chromosomes at anaphase (Fig. S4fg), and 12.5% (n = 24) at telophase (Fig. 4h). By contrast, no abnormal chromosome configurations were observed in mitotic cells from wild‐type or APC8‐complementated plants (Figs S4a–d, 4i–l). These data support the idea that the abnormal vegetative development observed in apc8‐1 is at least partially due to mitotic chromosome segregation defects.

Figure 4.

Figure 4

Open in a new tab

FISH analysis of meiotic chromosomes in wild‐type (WT) and apc8‐1 Arabidopsis using a centromere probe. Chromosomes spreads were hybridised with a 180‐bp repeat centromere DNA probe. Blue images show chromosomes stained with DAPI; red spots show DNA signals at centromeres. (a, b) pachytene, (c, d) diakinesis, (e, f) metaphase I, (g, h) anaphase I, (i, j) prophase II, (k, l) metaphase II, (m, n) telophase II, and tetrad stages (o, p), respectively. Arrowheads in row (d) show centromeres in a pair of lagging chromosomes in the apc8‐1 mutant. Arrows in (l, n) shows misaligned chromosomes at metaphase II in apc8‐1. All pictures were taken with a ×100 objective using a fluorescence microscope (Zeiss Axio Imager). Bars, 5 μm.

Cohesin complex and chromosome axis protein ASY1 appears to be unaffected in atapc8‐1

To ensure successful chromosome segregation, both mitosis and meiosis require proper assembly of cohesin complexes between the sister chromatids (Klein et al., 1999; Watanabe, 2004; Brooker & Berkowitz, 2014). The timely removal of cohesin complexes by separase depends on the degradation of cyclin B1 by the APC/C (Hellmuth et al., 2015; Sivakumar & Gorbsky, 2015). The misalignment of meiotic bivalents in apc8‐1 resembles the phenotype observed in cdc20.1 meiocytes (Niu et al., 2015) in which cohesin establishment is normal. We analysed the distribution of Arabidopsis SYN1 (Cai et al., 2003), a homologue of the yeast meiosis‐specific cohesin REC8 (Meiotic recombination‐deficient 8), in wild‐type and apc8‐1 meiotic chromosome spreads. SYN1in wild‐type pachytene meiocytes is distributed in a linear pattern along chromosomes (Fig. 3a, n = 17), consistent with its typical distribution characteristics (Chelysheva et al., 2010). SYN1 continues to colocalise with chromosomes through diakinesis (wild‐type, n = 18; apc8‐1, n = 23) and metaphase I (wild‐type, n = 15; apc8‐1, n = 30). No obvious differences are seen in apc8‐1 (Fig. 3a, n = 30) at any stage. This finding suggests that loading of SYN1 onto chromosomes is unaffected in apc8‐1, consistent with the cdc20.1 phenotype (Niu et al., 2015). To validate the lack of a cohesin defect in apc8‐1, we examined another cohesin component, SMC3 (Lam et al., 2005), and found that the SMC3 signal exhibited no obvious difference at zygotene, pachytene, or diakinesis (Fig. S5) between wild‐type and apc8‐1 (n = 21), confirming that cohesin establishment is normal in apc8‐1. However, due to the limited sensitivity of the technique and/or very low protein levels, it was not possible to examine SYN1 or SMC3 during meiosis II. Therefore, whether the removal of cohesin at anaphase I and/or II is regulated by APC8 remains an open question. We also examined the localisation of the chromosome axis component ASY1 (Armstrong et al., 2002) in apc8‐1 and found no difference compared with wild‐type (Fig. 3b; n = 30), suggesting that the early assembly of the chromosome axis appears to be normal.

Figure 3.

Figure 3

Open in a new tab

Immunolocalization of AtSYN1 and AtASY1 in wild‐type (WT) and apc8‐1 Arabidopsis. (a) SYN1 and (b) ASY1 were immunolocalised to chromosomes from zygotene to metaphase I in WT and apc8‐1. Grey images show DAPI‐stained chromosomes; red colour along the chromosomes indicates SYN1 and ASY1 signals. Thirty cells for each stage were observed. All pictures were taken with a ×100 objective using a fluorescence microscope (Zeiss Axio Imager). Bars, 5 μm.

AtAPC8 is required for the normal alignment of the centromere and the kinetochore protein AtCENH3 in meiosis

Given the normal establishment of cohesion in apc8‐1, the misalignment of bivalents at metaphase I may indicate a defect in orientating the chromosomes on the spindle. To test this hypothesis, we analysed chromosome spreads using FISH with a probe corresponding to the 180‐bp centromere repeat. Chromosome configurations were indistinguishable between wild‐type and apc8‐1 at diakinesis (Fig. 4a–d). At metaphase I, c. 95% (n = 30) of wild‐type meiocytes had five pairs of centromere signals, with each pair distributed to both sides of one bivalent (Fig. 4e). By contrast, c. 40% (n = 30) of apc8‐1 meiocytes had asymmetrically aligned centromere signals (Fig. 4f), this is consistent with the misalignment observed with DAPI‐stained chromosome spreads (Fig. 2c). At anaphase I, centromere signals in wild‐type and apc8‐1 (n = 30) formed two clusters, each with five centromeres (Fig. 4g–h), with no obvious differences. During meiosis I, in wild‐type the two sister chromatids are mono‐oriented and the sister kinetochores are fused and co‐migrate during segregation of homologous chromosomes (Sarangapani et al., 2014). By comparison, the misaligned bivalents in apc8‐1 meiocytes suggest that the kinetochore orientation on the spindle may be defective.

Interestingly, at prophase II, wild‐type meiocytes have five concentrated centromeres at both poles (Fig. 4i), but in apc8‐1 meiocytes (n = 30) the pairs of centromeric signals are slightly separated (Fig. 4j), suggesting a defect in the protection of centromeric cohesin complexes between sister chromatids in meiosis II. At metaphase II, wild‐type chromosomes are aligned (Fig. 4k), while apc8‐1 (n = 30) chromosomes are misalignment (Fig. 4l), providing additional evidence that APC8 is required for chromosome segregation in meiosis II. Finally, abnormal chromosome segregation in apc8‐1 (Fig. 4n), compared with wild‐type (Fig. 4m), leads to the production of dysfunctional tetrads with four nuclei containing various amounts of DNA (Fig. 4p), or to polyads. These results are consistent with APC8 playing a role in meiotic chromosome segregation and proper interaction of the spindle microtubules and sister kinetochores at metaphase I. To explore this further we examined the immunolocalisation of the kinetochore marker protein CENH3 (CENP‐A) and found that CENH3 signals are indistinguishable between wild‐type and apc8‐1 at pachytene and diakinesis (Fig. 5a–d). At metaphase I, we detected CENH3 signals at two sides of the well aligned bivalents in wild‐type (Fig. 5e). By contrast, in apc8‐1 the distribution of CENH3 appears asymmetric (Fig. 5f), and c. 40% of cells (n = 30) in have one separated bivalent, which corresponds to the misalignment of bivalents at metaphase I. Based on these observations, we hypothesise that APC8 plays an important role in the normal sister kinetochore mono‐orientation during meiosis.

Figure 5.

Figure 5

Open in a new tab

Immunolocalization of AtCENH3/CENP‐A in wild‐type (WT) and apc8‐1 Arabidopsis. CENH3 was immunolocalised to meiotic chromosomes during pachytene (a, b), diakinesis (c, d), and metaphase I (e, f) using an anti‐HTR12 (CENH3) antibody. Blue images show DAPI‐stained chromosomes; red spots are CENH3 signals. White dotted lines outline the CENH3 signal in a bivalent (the middle panels of e and f). Arrowhead in (f) marks the bivalent with an abnormal CENH3 signal. All pictures were taken with a ×100 objective using a fluorescence microscope (Zeiss Axio Imager). Bars, 5 μm.

Formation of normal spindle morphology during meiosis and mitosis requires AtAPC8

The spindle is required for chromosome movement and segregation during mitosis and meiosis (Nagasaka et al., 2011; Duro & Marston, 2015), and accurate chromosome segregation is accomplished by proper attachment of chromosomes to the spindle microtubules via the kinetochore (Chan et al., 2005; Severson et al., 2016). We hypothesise that the aberrant chromosome segregation in apc8‐1 is caused by defects in establishing a normal spindle. To test this we examined microtubule assembly. At diakinesis in both wild‐type and apc8‐1 we found no obvious differences in microtubule arrangement. In both cases the microtubules were organised in a perinuclear arrangement (Fig. 6a,b). At metaphase I, the microtubules in wild‐type were rearranged into a bipolar spindle with sharply focused poles (Fig. 6c), whereas the apc8‐1 spindles (n = 30 cells) appeared twisted or narrow (87%), or had misaligned chromosomes (7%) (Fig. 6d1,d2). During anaphase I, the wild‐type spindle lengthened along the polar axis, pulling the chromosomes to separate them (Fig. 6e) and, at metaphase II, the microtubules formed two smaller spindles (Fig. 6i). By contrast, in apc8‐1 the main spindle had two large unfocused poles and, along the side of the main spindle, 10% (n = 30) had an unusual small spindle‐like structure around a chromosome (Fig. 6f2). Subsequently, 10% (n = 30) had a multipolar structure that formed at telophase I (Fig. 6h2) and at metaphase II, and 30% of cells (n = 30) formed two spindles in addition to a mini‐spindle that harbored the misaligned chromosomes (Fig. 6j2). Finally, wild‐type formed tetrads with microtubules surrounding the four nuclei (Fig. 6k), whereas in apc8‐1 40% (n = 30) of meioses resulted in polyads with multipolar spindles (Fig. 6l2).

Figure 6.

Figure 6

Open in a new tab

Immunolocalization of microtubules in meiocytes in wild‐type (WT) and apc8‐1 Arabidopsis. Microtubule organisation from diakinesis to tetrad in WT (a, c, e, g, i and k), normal microtubule arrangement (b1, d1, f1, h1, j1 and l1), and abnormal microtubule arrangement (b2, d2, f2, h2, j2 and l2) in apc8‐1. Microtubules were immunolocalised with an antitubulin antibody. Chromosomes were stained with DAPI. All pictures were taken with a ×100 objective using a fluorescence microscope (Zeiss Axio Imager). Bars, 5 μm.

We also observed the morphology mitotic spindles and found that during prophase microtubules were in a perinuclear arrangement in both wild‐type and apc8‐1 (Fig. S6a,b). At metaphase, regular spindles were observed in wild‐type (Fig. S6c); however, in apc8‐1 abnormal spindles with misaligned chromosomes were observed and a minispindle‐like structure was found next to the abnormal main spindle (Fig. S6d, arrowhead). Subsequently, free chromosomes were observed in both anaphase and telophase in apc8‐1 (Fig. S6f,h). Taken together, these results indicated that APC8 is required for normal microtubule organisation and spindle assembly. Furthermore, the defective microtubule organisation and dysfunctional spindles observed during mitosis and meiosis are likely to be the primary cause of the metaphase I chromosome misalignment and aberrant segregation at meiosis II in apc8‐1.

The atapc8‐1 point mutation disrupts interaction with AtCDC20.1

CDC20.1, an APC/C activator, interacts with APC3s, APC8 and APC10 (Fülöp et al., 2005; Perez‐Perez et al., 2008; Kevei et al., 2011; Qiao et al., 2016). To investigate whether apc8‐1 has altered protein interactions, we used yeast two‐hybrid assays and found that APC8 interacts with APC10, CDC20.1 and itself, while APC8D309N still interacts with APC10 and itself, but not with CDC20.1. (Fig. S7a). APC8 self‐interaction is consistent with homodimer formation of the human homologue (Schreiber et al., 2011). Previous analyses demonstrated that the TPR domain of the APC/C of various subunits is required for protein−protein interaction (Kevei et al., 2011; Qiao et al., 2016). We modelled the structure of the full‐length APC8 using the SWISS‐MODEL (https://www.swissmodel.expasy.org) protein structure prediction tool (Waterhouse et al., 2018). Protein sequence alignment showed that Arabidopsis APC8 (61−562 aa) has c. 60% similarity to human CDC23 (47−547 aa) (Fig. S7b), which has four electron microscopy structures deposited in the Protein Structure Database (PDB code: 4UI9, 5A31, 5KHU and 5G04). We built the predicted APC8 model using the 4UI9 structure as template (Fig. S7c,d), and found that D309 in APC8 corresponds to D301 in CDC23 while, in the 4UI9 structure, D301 and Y364 are closed to each other. We therefore speculated that the amino acid substitution in APC8D309N may affect the interaction between D309 and Y372 (corresponding to Y364 in human CDC23), and interfere with the TPR domain's ability to facilitate protein−protein interaction.

Discussion

AtAPC/C is indispensable for normal development and meiosis in Arabidopsis

As an essential E3 ubiquitin protein ligase that mediates the mitotic transition, the APC/C is highly conserved among eukaryotes (Peters, 2002). Mutations in APC/C subunits are severely detrimental in vertebrates and plants, including Arabidopsis (Capron et al., 2003; Kwee & Sundaresan, 2003; McGuinness et al., 2009; Eloy et al., 2011; Wang et al., 2012b; Wang et al., 2013; Zhang et al., 2014). In Arabidopsis, functional characterisation of the APC/C subunits APC1, APC2, APC4, APC6, APC8, and APC13 has demonstrated their indispensability for female gametogenesis and embryogenesis (Capron et al., 2003; Kwee & Sundaresan, 2003; Eloy et al., 2011; Zheng et al., 2011; Wang et al., 2012b, 2013). Genetic screening for mutants with defects in zygote development identified APC11 and the null allele of this gene as embryonic lethal (Guo et al., 2016). The probable function of the APC/C during mitotic cell division is to regulate cyclin B degradation. However, whether the APC/C subunit is required for meiotic cell division has not been previously reported. Here, we used several approaches to analyse the meiotic phenotypes of a previously identified weak allele of apc8‐1 (Zheng et al., 2011) and found that, in addition to the previously reported phenotypes, the reduced fertility in apc8‐1 is, at least partially, caused by meiotic defects. We provide several lines of cytological evidence to support this conclusion. First, unlike wild‐type tetrads, the apc8‐1 meiocytes produced polyads, indicative of unequal meiotic chromosome segregation. Second, chromosome behaviour assessed by DAPI staining showed that misaligned chromosomes were observed at both mitosis and meiosis metaphase I in apc8‐1, further supporting a defect in chromosome segregation. Third, we also found that chromosome segregation defects in apc8‐1 were likely to be due to abnormal spindle orientation rather than the loading or removal of cohesin. Taken together, our results demonstrated that APC8 is essential for meiosis in plants.

A potential role for AtAPC8 in meiotic chromosome segregation

Based on our results and previous findings, we present a model to illustrate the role of the APC/C and associated factors in bivalent alignment during meiotic prophase I (Fig. 7). During wild‐type prometaphase I of meiosis, kinetochores can attach to microtubules (K‐MT) normally or erroneously, and erroneous attachments can be corrected by a complex system involving the mitotic checkpoint complex (MCC), Aurora kinase (AUR1) and/or the APC/C complex, as well as its associated factors OSD1 and TDM1. During attachment correction, the ability of CDC20.1 to activate APC/C is partially inhibited by MCC and/or OSD1 or both. Under these conditions CDC20.1 promotes AUR1 correction of erroneous K‐MT attachment. In wild‐type, once all correction is implemented, the spindle aligns chromosomes at the equatorial plate at metaphase I. In apc8‐1, APC8D309N does not interact with CDC20.1 and erroneous spindle attachments persist or their correction is delayed, leading to chromosome misalignment at metaphase I (Fig. 7). Previous studies demonstrated that OSD1 is an APC/C inhibitor, while TDM1 is present throughout meiosis and its activity must be inhibited during meiosis I (Cromer et al., 2012; Cifuentes et al., 2016). Our qRT‐PCR data show that the expression of both genes is elevated in apc8‐1 compared with wild‐type, indicating that there may be a feedback mechanism between APC/C and its regulators.

Figure 7.

Figure 7

Open in a new tab

A model showing the potential role of AtAPC8 in bivalent alignment during prometaphase I. In wild‐type, at meiotic prometaphase I, the initial attachment of kinetochores to microtubules (K‐MT) is likely asynchronous and stochastic. Erroneous attachment activates a robust SAC signal, in which a SAC effector−MCC complex inhibits APC/C activity via regulation of CDC20.1. The correction of erroneous K‐MT attachments is mediated by the Aurora kinase (AUR) promoted by CDC20.1. Once all corrections are implemented, the spindle aligns chromosomes at the equatorial plate. In apc8‐1, APC8D309N does not interact with CDC20.1, which comprises AUR ability to correct K‐MT attachments. As a consequence, the bivalents are not well aligned at metaphase I.

Although we observed c. 40% abnormal metaphase I alignment of bivalents in apc8‐1, this does not lead to uneven segregation of homologues at anaphase I. A possible explanation for this observation is that spindle assembly checkpoint (SAC) activity may be compromised in apc8‐1, thereby delaying meiotic progression. Furthermore, the meiosis I apc8‐1 defects cannot explain the c. 95% frequency of abnormal tetrads at telophase II. It is plausible that APC8 may have a role in chromosome segregation during meiosis II, but this requires further investigation.

Conservation and divergence of APC/C activity in meiosis among species

The APC/C is highly conserved with 11 subunits in both plants and animals. Analysis of APC/C subunit null alleles that show they are essential in all species tested ranging from fungi to mice (Peters, 2006), suggested a conserved function in the cell cycle. Because APC/C subunits are essential, much of our understanding of their role in chromosome segregation during meiosis has been derived from studies focusing on APC/C co‐activators or regulators (Cooper et al., 2000; Pesin & Orr‐Weaver, 2008; Kimata et al., 2011; Okaz et al., 2012; Holt et al., 2014). These regulators include yeast Ama1, a member of the Cdc20 protein family that directs meiosis I, but not meiosis II, APC/C function (Cooper et al., 2000; Okaz et al., 2012). In fission yeast, Mes1 interacts with two co‐activators, Slp1/Cdc20 and Fzr1/Mfr1, in a different manner to control the activity of the APC/C required for the meiosis I/meiosis II transition (Kimata et al., 2011). In mammals, the APC/CFZR1 meiosis‐specific activity regulates spermatogonial proliferation as well as early prophase I in both male and female germ cells (Holt et al., 2014). Although FZR is not a homologue of Ama1, it activates the APC/C in a similar manner when CDK1 activity is low (Peters, 2006). Additionally, APC/CFZR1 regulates a wide range of cell‐cycle events (Holt et al., 2014), suggesting a conservation of function utilising different proteins in different species. A similar conservation of function using different protein players has been recently described for the Arabidopsis SAC compared with animals and yeast (Komaki & Schnittger, 2017). The essential function of APC/C subunits in plants has limited our understanding of their function during chromosome segregation. Evidence suggests that Arabidopsis TDM1 is an APC/C component and is required for the termination of meiosis II (Cromer et al., 2012; Cifuentes et al., 2016). OSD1 promotes entry into the second meiotic division, likely to be through inhibition of APC/C activity (Cromer et al., 2012). Here we reported the role of APC8 in bivalent alignment based on the analysis of a weak mutant. Given the conservation of APC/C function across wide species boundaries, APC8 may play a similar role in other species, but this needs to be validated in future studies.

Author contributions

R‐YX performed immunolocalisation of microtubules, analyses of CENH3 and other assessments; JX performed real‐time PCR, yeast two‐hybrid assays; R‐YX, B‐XN, JX and L‐DW performed FISH, SYN1 analysis and chromosome spreading; B‐LZ provided the mutant line apc8‐1 and revised the manuscript; Y‐XW guided the study and helped in designing the experiments; R‐YX, JX, HM, GPC and Y‐XW contributed to data interpretation and writing the manuscript. R‐YX, JX and LW contributed equally to this work.

Supporting information

Please note: Wiley Blackwell are not responsible for the content or functionality of any Supporting Information supplied by the authors. Any queries (other than missing material) should be directed to the New Phytologist Central Office.

Fig. S1 Silique images and seed numbers in wild‐type, atapc mutants and the APC8‐complementated plants.

Fig. S2 Relative transcript levels of AtAPC8, AtOSD1, AtTDM1 and AtPANS1 in wild‐type (Col‐0), atapc8‐1 and APC8‐complementated plant.

Fig. S3 FISH analysis of meiotic chromosomes in APC8‐complementated plants using a centromere probe.

Fig. S4 Mitotic chromosome behaviours in wild‐type, atapc8‐1 and APC8‐complementated plants.

Fig. S5 Immunolocalisation of AtSMC3 in wild‐type and atapc8‐1.

Fig. S6 Immunolocalisation of microtubules during mitosis in wild‐type and atapc8‐1 mutant.

Fig. S7 Examination of protein−protein interaction by yeast two‐hybrid assay and modelling the APC8 protein structure.

Table S1 Primers used in this study.

Click here for additional data file. (1.4MB, pdf)

Acknowledgements

We greatly appreciate Jianhua Gan from the School of Life Sciences at Fudan University for assistance on the APC8 structure modelling. This work was supported by grants from the National Natural Science Foundation of China (31570314 and 31870293) the State Key Laboratory of Genetic Engineering, Fudan University and Rijk Zwaan. GPC is supported by a US National Science Foundation grant (IOS‐1844264). BN is supported by the Natural Science Foundation of Jiangsu Province (BK20160454).

References

  1. Armstrong SJ, Caryl AP, Jones GH, Franklin FC. 2002. Asy1, a protein required for meiotic chromosome synapsis, localizes to axis‐associated chromatin in Arabidopsis and Brassica. Journal of Cell Science 115: 3645–3655. [DOI] [PubMed] [Google Scholar]
  2. Bentley AM, Williams BC, Goldberg ML, Andres AJ. 2002. Phenotypic characterization of Drosophila ida mutants: defining the role of APC5 in cell cycle progression. Journal of Cell Science 115: 949–961. [DOI] [PubMed] [Google Scholar]
  3. Bolanos‐Villegas P, De K, Pradillo M, Liu D, Makaroff CA. 2017. In favor of establishment: regulation of chromatid cohesion in plants. Frontiers in Plant Science 8: 846. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Brooker AS, Berkowitz KM. 2014. The roles of cohesins in mitosis, meiosis, and human health and disease. Methods in Molecular Biology 1170: 229–266. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bulankova P, Riehs‐Kearnan N, Nowack MK, Schnittger A, Riha K. 2010. Meiotic progression in Arabidopsis is governed by complex regulatory interactions between SMG7, TDM1, and the meiosis I‐specific cyclin TAM. Plant Cell 22: 3791–3803. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cai X, Dong FG, Edelmann RE, Makaroff CA. 2003. The Arabidopsis SYN1 cohesin protein is required for sister chromatid arm cohesion and homologous chromosome pairing. Journal of Cell Science 116: 2999–3007. [DOI] [PubMed] [Google Scholar]
  7. Capron A, Serralbo O, Fülöp K, Frugier F, Parmentier Y, Dong A, Lecureuil A, Guerche P, Kondorosi E, Scheres B et al 2003. The Arabidopsis anaphase‐promoting complex or cyclosome: molecular and genetic characterization of the APC2 subunit. Plant Cell 15: 2370–2382. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Chan GK, Liu ST, Yen TJ. 2005. Kinetochore structure and function. Trends in Cell Biology 15: 589–598. [DOI] [PubMed] [Google Scholar]
  9. Chelysheva L, Grandont L, Vrielynck N, le Guin S, Mercier R, Grelon M. 2010. An easy protocol for studying chromatin and recombination protein dynamics during Arabidopsis thaliana meiosis: immunodetection of cohesins, histones and MLH1. Cytogenetic and Genome Research 129: 143–153. [DOI] [PubMed] [Google Scholar]
  10. Cifuentes M, Jolivet S, Cromer L, Harashima H, Bulankova P, Renne C, Crismani W, Nomura Y, Nakagami H, Sugimoto K et al 2016. TDM1 regulation determines the number of meiotic divisions. PLoS Genetics 12: e1005856. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Cooper KF, Mallory MJ, Egeland DB, Jarnik M, Strich R. 2000. Ama1p is a meiosis‐specific regulator of the anaphase promoting complex/cyclosome in yeast. Proceedings of the National Academy of Sciences, USA 97: 14548–14553. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Cromer L, Heyman J, Touati S, Harashima H, Araou E, Girard C, Horlow C, Wassmann K, Schnittger A, De Veylder L et al 2012. OSD1 promotes meiotic progression via APC/C inhibition and forms a regulatory network with TDM and CYCA1;2/TAM. PLoS Genetics 8: e1002865. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Cromer L, Jolivet S, Horlow C, Chelysheva L, Heyman J, De Jaeger G, Koncz C, De Veylder L, Mercier R. 2013. Centromeric cohesion is protected twice at meiosis, by SHUGOSHINs at anaphase 1 and by PATRONUS at interkinesis. Current Biology 23: 2090–2099. [DOI] [PubMed] [Google Scholar]
  14. Cullen CF, May KM, Hagan IM, Glover DM, Ohkura H. 2000. A new genetic method for isolating functionally interacting genes: high plo1+‐dependent mutants and their suppressors define genes in mitotic and septation pathways in fission yeast. Genetics 155: 1521–1534. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Di Fiore B, Wurzenberger C, Davey NE, Pines J. 2016. The mitotic checkpoint complex requires an evolutionary conserved cassette to bind and inhibit active APC/C. Molecular Cell 64: 1144–1153. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Duro E, Marston AL. 2015. From equator to pole: splitting chromosomes in mitosis and meiosis. Genes & Development 29: 109–122. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Eloy NB, Lima MD, Van Damme D, Vanhaeren H, Gonzalez N, De Milde L, Hemerly AS, Beemster GTS, Inze D, Ferreira PCG. 2011. The APC/C subunit 10 plays an essential role in cell proliferation during leaf development. The Plant Journal 68: 351–363. [DOI] [PubMed] [Google Scholar]
  18. d'Erfurth I, Cromer L, Jolivet S, Girard C, Horlow C, Sun YJ, To JPC, Berchowitz LE, Copenhaver GP, Mercier R. 2010. The CYCLIN‐A CYCA1;2/TAM is required for the meiosis I to meiosis II transition and cooperates with OSD1 for the prophase to first meiotic division transition. PLoS Genetics 6: e1000989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Fülöp K, Tarayre S, Kelemen Z, Horvath G, Kevei Z, Nikovics K, Bako L, Brown S, Kondorosi A, Kondorosi E. 2005. Arabidopsis anaphase‐promoting complexes: multiple activators and wide range of substrates might keep APC perpetually busy. Cell Cycle 4: 1084–1092. [PubMed] [Google Scholar]
  20. Gao J, Yang S, Cheng W, Fu Y, Leng J, Yuan X, Jiang N, Ma J, Feng X. 2017. GmILPA1, encoding an APC8‐like protein, controls leaf petiole angle in soybean. Plant Physiology 174: 1167–1176. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Garbe D, Doto JB, Sundaram MV. 2004. Caenorhabditis elegans lin‐35/Rb, efl‐1/E2F and other synthetic multivulva genes negatively regulate the anaphase‐promoting complex gene mat‐3/APC8. Genetics 167: 663–672. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Guo L, Jiang L, Zhang Y, Lu XL, Xie Q, Weijers D, Liu CM. 2016. The anaphase‐promoting complex initiates zygote division in Arabidopsis through degradation of cyclin B1. The Plant Journal 86: 161–174. [DOI] [PubMed] [Google Scholar]
  23. Hellmuth S, Pohlmann C, Brown A, Bottger F, Sprinzl M, Stemmann O. 2015. Positive and negative regulation of vertebrate separase by Cdk1‐cyclin B1 may explain why securin is dispensable. Journal of Biological Chemistry 290: 8002–8010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Heyman J, De Veylder L. 2012. The anaphase‐promoting complex/cyclosome in control of plant development. Molecular Plant 5: 1182–1194. [DOI] [PubMed] [Google Scholar]
  25. Holt JE, Pye V, Boon E, Stewart JL, Garcia‐Higuera I, Moreno S, Rodriguez R, Jones KT, McLaughlin EA. 2014. The APC/C activator FZR1 is essential for meiotic prophase I in mice. Development 141: 1354–1365. [DOI] [PubMed] [Google Scholar]
  26. Homer H. 2013. The APC/C in female mammalian meiosis I. Reproduction 146: R61–R71. [DOI] [PubMed] [Google Scholar]
  27. Ji W, Luo Y, Ahmad E, Liu S. 2018. Direct interactions of mitotic arrest deficient 1 (MAD1) domains with each other and MAD2 conformers are required for mitotic checkpoint signaling. Journal of Biological Chemistry 293: 484–496. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Jin F, Hamada M, Malureanu L, Jeganathan KB, Zhou W, Morbeck DE, van Deursen JM. 2010. Cdc20 is critical for meiosis I and fertility of female mice. PLoS Genetics 6: e1001147. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Kashevsky H, Wallace JA, Reed BH, Lai C, Hayashi‐Hagihara A, Orr‐Weaver TL. 2002. The anaphase promoting complex/cyclosome is required during development for modified cell cycles. Proceedings of the National Academy of Sciences, USA 99: 11217–11222. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Kelley LA, Mezulis S, Yates CM, Wass MN, Sternberg MJ. 2015. The Phyre2 web portal for protein modeling, prediction and analysis. Nature Protocols 10: 845–858. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Kevei Z, Baloban M, Da Ines O, Tiricz H, Kroll A, Regulski K, Mergaert P, Kondorosi E. 2011. Conserved CDC20 cell cycle functions are carried out by two of the five isoforms in Arabidopsis thaliana . PLoS ONE 6: e20618. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Kimata Y, Kitamura K, Fenner N, Yamano H. 2011. Mes1 controls the meiosis I to meiosis II transition by distinctly regulating the anaphase‐promoting complex/cyclosome coactivators Fzr1/Mfr1 and Slp1 in fission yeast. Molecular Biology of the Cell 22: 1486–1494. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Klein F, Mahr P, Galova M, Buonomo SBC, Michaelis C, Nairz K, Nasmyth K. 1999. A central role for cohesins in sister chromatid cohesion, formation of axial elements, and recombination during yeast meiosis. Cell 98: 91–103. [DOI] [PubMed] [Google Scholar]
  34. Komaki S, Schnittger A. 2017. The spindle assembly checkpoint in Arabidopsis is rapidly shut off during severe stress. Developmental Cell 43: 172–185. [DOI] [PubMed] [Google Scholar]
  35. Kwee HS, Sundaresan V. 2003. The NOMEGA gene required for female gametophyte development encodes the putative APC6/CDC16 component of the Anaphase Promoting Complex in Arabidopsis. The Plant Journal 36: 853–866. [DOI] [PubMed] [Google Scholar]
  36. Lam WS, Yang XH, Makaroff CA. 2005. Characterization of Arabidopsis thaliana SMC1 and SMC3: evidence that AtSMC3 may function beyond chromosome cohesion. Journal of Cell Science 118: 3037–3048. [DOI] [PubMed] [Google Scholar]
  37. McGuinness BE, Anger M, Kouznetsova A, Gil‐Bernabe AM, Helmhart W, Kudo NR, Wuensche A, Taylor S, Hoog C, Novak B et al 2009. Regulation of APC/C activity in oocytes by a Bub1‐dependent spindle assembly checkpoint. Current Biology 19: 369–380. [DOI] [PubMed] [Google Scholar]
  38. Nagasaka K, Gallego‐Paez LM, Hirota T. 2011. Mitosis and meiosis: molecular control of chromosome separation. In: eLS. Chichester, UK: John Wiley & Sons, Ltd. [Google Scholar]
  39. Niu BX, Wang LD, Zhang LS, Ren D, Ren R, Copenhaver GP, Ma H, Wang YX. 2015. Arabidopsis cell division cycle 20.1 is required for normal meiotic spindle assembly and chromosome segregation. Plant Cell 27: 3367–3382. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Okaz E, Arguello‐Miranda O, Bogdanova A, Vinod PK, Lipp JJ, Markova Z, Zagoriy I, Novak B, Zachariae W. 2012. Meiotic prophase requires proteolysis of M phase regulators mediated by the meiosis‐specific APC/CAma1. Cell 151: 603–618. [DOI] [PubMed] [Google Scholar]
  41. Pal M, Nagy O, Menesi D, Udvardy A, Deak P. 2007. Structurally related TPR subunits contribute differently to the function of the anaphase‐promoting complex in Drosophila melanogaster . Journal of Cell Science 120: 3238–3248. [DOI] [PubMed] [Google Scholar]
  42. Perez‐Perez JM, Serralbo O, Vanstraelen M, Gonzalez C, Criqui MC, Genschik P, Kondorosi E, Scheres B. 2008. Specialization of CDC27 function in the Arabidopsis thaliana anaphase‐promoting complex (APC/C). The Plant Journal 53: 78–89. [DOI] [PubMed] [Google Scholar]
  43. Pesin JA, Orr‐Weaver TL. 2008. Regulation of APC/C activators in mitosis and meiosis. Annual Review of Cell and Developmental Biology 24: 475–499. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Peter M, Castro A, Lorca T, Le Peuch C, Magnaghi‐Jaulin L, Doree M, Labbe JC. 2001. The APC is dispensable for first meiotic anaphase in Xenopus oocytes. Nature Cell Biology 3: 83–87. [DOI] [PubMed] [Google Scholar]
  45. Peters JM. 2002. The anaphase‐promoting complex: proteolysis in mitosis and beyond. Molecular Cell 9: 931–943. [DOI] [PubMed] [Google Scholar]
  46. Peters JM. 2006. The anaphase promoting complex/cyclosome: a machine designed to destroy. Nature Reviews Molecular Cell Biology 7: 644–656. [DOI] [PubMed] [Google Scholar]
  47. Peterson R, Slovin JP, Chen C. 2010. A simplified method for differential staining of aborted and nonaborted pollen grains. International Journal of Plant Biology 1: e13. [Google Scholar]
  48. Pines J. 2011. Cubism and the cell cycle: the many faces of the APC/C. Nature Reviews Molecular Cell Biology 12: 427–438. [DOI] [PubMed] [Google Scholar]
  49. Qiao R, Weissmann F, Yamaguchi M, Brown NG, VanderLinden R, Imre R, Jarvis MA, Brunner MR, Davidson IF, Litos G et al 2016. Mechanism of APC/CCDC20 activation by mitotic phosphorylation. Proceedings of the National Academy of Sciences, USA 113: E2570–E2578. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Rankin S, Dawson DS. 2016. Recent advances in cohesin biology. F1000Research 5. doi: 10.12688/f1000research.8881.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Sarangapani KK, Duro E, Deng Y, Alves FD, Ye QZ, Opoku KN, Ceto S, Rappsilber J, Corbett KD, Biggins S et al 2014. Sister kinetochores are mechanically fused during meiosis I in yeast. Science 346: 248–251. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Schmittgen TD, Livak KJ. 2008. Analyzing real‐time PCR data by the comparative C(T) method. Nature Protocols 3: 1101–1108. [DOI] [PubMed] [Google Scholar]
  53. Schreiber A, Stengel F, Zhang Z, Enchev RI, Kong EH, Morris EP, Robinson CV, da Fonseca PC, Barford D. 2011. Structural basis for the subunit assembly of the anaphase‐promoting complex. Nature 470: 227–232. [DOI] [PubMed] [Google Scholar]
  54. Severson AF, von Dassow G, Bowerman B. 2016. Oocyte meiotic spindle assembly and function. Current Topics in Developmental Biology 116: 65–98. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Singh DK, Spillane C, Siddiqi I. 2015. PATRONUS1 is expressed in meiotic prophase I to regulate centromeric cohesion in Arabidopsis and shows synthetic lethality with OSD1. BMC Plant Biology 15: 201. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Sivakumar S, Gorbsky GJ. 2015. Spatiotemporal regulation of the anaphase‐promoting complex in mitosis. Nature Reviews Molecular Cell Biology 16: 82–94. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Wang Y, Cheng Z, Huang J, Shi Q, Hong Y, Copenhaver GP, Gong ZZ, Ma H. 2012a. The DNA replication factor RFC1 is required for interference‐sensitive meiotic crossovers in Arabidopsis thaliana . PLoS Genetics 8: e1003039. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Wang Y, Cheng Z, Lu P, Timofejeva L, Ma H. 2014. Molecular cell biology of male meiotic chromosomes and isolation of male meiocytes in Arabidopsis thaliana . Methods in Molecular Biology 1110: 217–230. [DOI] [PubMed] [Google Scholar]
  59. Wang Y, Hou YN, Gu HY, Kang DM, Chen ZL, Liu JJ, Qu LJ. 2012b. The Arabidopsis APC4 subunit of the anaphase‐promoting complex/cyclosome (APC/C) is critical for both female gametogenesis and embryogenesis. The Plant Journal 69: 227–240. [DOI] [PubMed] [Google Scholar]
  60. Wang YB, Hou YN, Gu HY, Kang DM, Chen ZL, Liu JJ, Qu LJ. 2013. The Arabidopsis anaphase‐promoting complex/cyclosome subunit 1 is critical for both female gametogenesis and embryogenesis. Journal of Integrative Plant Biology 55: 64–74. [DOI] [PubMed] [Google Scholar]
  61. Wang J, Niu B, Huang J, Wang H, Yang X, Dong A, Makaroff C, Ma H, Wang Y. 2016. The PHD finger protein MMD1/DUET ensures the progression of male meiotic chromosome condensation and directly regulates the expression of the condensin gene CAP‐D3. Plant Cell 28: 1894–1909. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Watanabe Y. 2004. Modifying sister chromatid cohesion for meiosis. Journal of Cell Science 117: 4017–4023. [DOI] [PubMed] [Google Scholar]
  63. Waterhouse A, Bertoni M, Bienert S, Studer G, Tauriello G, Gumienny R, Heer FT, de Beer TAP, Rempfer C, Bordoli L et al 2018. SWISS‐MODEL: homology modelling of protein structures and complexes. Nucleic Acids Research 46: W296–W303. [DOI] [PMC free article] [PubMed] [Google Scholar]
  64. Yamashita YM, Nakaseko Y, Kumada K, Nakagawa T, Yanagida M. 1999. Fission yeast APC/cyclosome subunits, Cut20/Apc4 and Cut23/Apc8, in regulating metaphase‐anaphase progression and cellular stress responses. Genes to Cells 4: 445–463. [DOI] [PubMed] [Google Scholar]
  65. Yu HT, Peters JM, King RW, Page AM, Hieter P, Kirschner MW. 1998. Identification of a cullin homology region in a subunit of the anaphase‐promoting complex. Science 279: 1219–1222. [DOI] [PubMed] [Google Scholar]
  66. Zamariola L, De Storme N, Vannerum K, Vandepoele K, Armstrong SJ, Franklin FCH, Geelen D. 2014a. SHUGOSHINs and PATRONUS protect meiotic centromere cohesion in Arabidopsis thaliana . The Plant Journal 77: 782–794. [DOI] [PubMed] [Google Scholar]
  67. Zamariola L, Tiang CL, De Storme N, Pawlowski W, Geelen D. 2014b. Chromosome segregation in plant meiosis. Frontiers in Plant Science 5. doi: 10.3389/fpls.2014.00279. [DOI] [PMC free article] [PubMed] [Google Scholar]
  68. Zhang JF, Wan LX, Dai XP, Sun Y, Wei WY. 2014. Functional characterization of Anaphase Promoting Complex/Cyclosome (APC/C) E3 ubiquitin ligases in tumorigenesis. Biochimica Et Biophysica Acta–Reviews on Cancer 1845: 277–293. [DOI] [PMC free article] [PubMed] [Google Scholar]
  69. Zheng BL, Chen XM, McCormick S. 2011. The anaphase‐promoting complex is a dual integrator that regulates both MicroRNA‐mediated transcriptional regulation of Cyclin B1 and degradation of Cyclin B1 during Arabidopsis male gametophyte development. Plant Cell 23: 1033–1046. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Please note: Wiley Blackwell are not responsible for the content or functionality of any Supporting Information supplied by the authors. Any queries (other than missing material) should be directed to the New Phytologist Central Office.

Fig. S1 Silique images and seed numbers in wild‐type, atapc mutants and the APC8‐complementated plants.

Fig. S2 Relative transcript levels of AtAPC8, AtOSD1, AtTDM1 and AtPANS1 in wild‐type (Col‐0), atapc8‐1 and APC8‐complementated plant.

Fig. S3 FISH analysis of meiotic chromosomes in APC8‐complementated plants using a centromere probe.

Fig. S4 Mitotic chromosome behaviours in wild‐type, atapc8‐1 and APC8‐complementated plants.

Fig. S5 Immunolocalisation of AtSMC3 in wild‐type and atapc8‐1.

Fig. S6 Immunolocalisation of microtubules during mitosis in wild‐type and atapc8‐1 mutant.

Fig. S7 Examination of protein−protein interaction by yeast two‐hybrid assay and modelling the APC8 protein structure.

Table S1 Primers used in this study.

Click here for additional data file. (1.4MB, pdf)

Articles from The New Phytologist are provided here courtesy of Wiley

RESOURCES