The Arabidopsis Cdk1/Cdk2 homolog CDKA ;1 controls chromosome axis assembly during plant meiosis
2019; Springer Nature; Volume: 39; Issue: 3 Linguagem: Inglês
10.15252/embj.2019101625
ISSN1460-2075
AutoresChao Yang, Kostika Sofroni, Erik Wijnker, Yuki Hamamura, Lena Carstens, Hirofumi Harashima, Sara Christina Stolze, Daniel Vezon, Liudmila Chelysheva, Zsuzsanna Orbán-Németh, Gaëtan Pochon, Hirofumi Nakagami, Peter Schlögelhofer, Mathilde Grelon, Arp Schnittger,
Tópico(s)Plant Molecular Biology Research
ResumoArticle26 September 2019Open Access Source DataTransparent process The Arabidopsis Cdk1/Cdk2 homolog CDKA;1 controls chromosome axis assembly during plant meiosis Chao Yang Chao Yang Department of Developmental Biology, University of Hamburg, Hamburg, Germany Search for more papers by this author Kostika Sofroni Kostika Sofroni orcid.org/0000-0001-8648-4648 Department of Developmental Biology, University of Hamburg, Hamburg, Germany Search for more papers by this author Erik Wijnker Erik Wijnker Department of Developmental Biology, University of Hamburg, Hamburg, Germany Search for more papers by this author Yuki Hamamura Yuki Hamamura Department of Developmental Biology, University of Hamburg, Hamburg, Germany Search for more papers by this author Lena Carstens Lena Carstens Department of Developmental Biology, University of Hamburg, Hamburg, Germany Search for more papers by this author Hirofumi Harashima Hirofumi Harashima RIKEN Center for Sustainable Resource Science, Yokohama, Japan Search for more papers by this author Sara Christina Stolze Sara Christina Stolze Max-Planck-Institute for Plant Breeding Research, Cologne, Germany Search for more papers by this author Daniel Vezon Daniel Vezon Institut Jean-Pierre Bourgin, INRA, AgroParisTech, CNRS, Université Paris-Saclay, Versailles, France Search for more papers by this author Liudmila Chelysheva Liudmila Chelysheva orcid.org/0000-0002-7538-2184 Institut Jean-Pierre Bourgin, INRA, AgroParisTech, CNRS, Université Paris-Saclay, Versailles, France Search for more papers by this author Zsuzsanna Orban-Nemeth Zsuzsanna Orban-Nemeth Department of Chromosome Biology, Max F. Perutz Laboratories, Vienna Biocenter, University of Vienna, Vienna, Austria Search for more papers by this author Gaëtan Pochon Gaëtan Pochon Department of Developmental Biology, University of Hamburg, Hamburg, Germany Search for more papers by this author Hirofumi Nakagami Hirofumi Nakagami orcid.org/0000-0003-2569-7062 Max-Planck-Institute for Plant Breeding Research, Cologne, Germany Search for more papers by this author Peter Schlögelhofer Peter Schlögelhofer Department of Chromosome Biology, Max F. Perutz Laboratories, Vienna Biocenter, University of Vienna, Vienna, Austria Search for more papers by this author Mathilde Grelon Mathilde Grelon Institut Jean-Pierre Bourgin, INRA, AgroParisTech, CNRS, Université Paris-Saclay, Versailles, France Search for more papers by this author Arp Schnittger Corresponding Author Arp Schnittger [email protected] orcid.org/0000-0001-7067-0091 Department of Developmental Biology, University of Hamburg, Hamburg, Germany Search for more papers by this author Chao Yang Chao Yang Department of Developmental Biology, University of Hamburg, Hamburg, Germany Search for more papers by this author Kostika Sofroni Kostika Sofroni orcid.org/0000-0001-8648-4648 Department of Developmental Biology, University of Hamburg, Hamburg, Germany Search for more papers by this author Erik Wijnker Erik Wijnker Department of Developmental Biology, University of Hamburg, Hamburg, Germany Search for more papers by this author Yuki Hamamura Yuki Hamamura Department of Developmental Biology, University of Hamburg, Hamburg, Germany Search for more papers by this author Lena Carstens Lena Carstens Department of Developmental Biology, University of Hamburg, Hamburg, Germany Search for more papers by this author Hirofumi Harashima Hirofumi Harashima RIKEN Center for Sustainable Resource Science, Yokohama, Japan Search for more papers by this author Sara Christina Stolze Sara Christina Stolze Max-Planck-Institute for Plant Breeding Research, Cologne, Germany Search for more papers by this author Daniel Vezon Daniel Vezon Institut Jean-Pierre Bourgin, INRA, AgroParisTech, CNRS, Université Paris-Saclay, Versailles, France Search for more papers by this author Liudmila Chelysheva Liudmila Chelysheva orcid.org/0000-0002-7538-2184 Institut Jean-Pierre Bourgin, INRA, AgroParisTech, CNRS, Université Paris-Saclay, Versailles, France Search for more papers by this author Zsuzsanna Orban-Nemeth Zsuzsanna Orban-Nemeth Department of Chromosome Biology, Max F. Perutz Laboratories, Vienna Biocenter, University of Vienna, Vienna, Austria Search for more papers by this author Gaëtan Pochon Gaëtan Pochon Department of Developmental Biology, University of Hamburg, Hamburg, Germany Search for more papers by this author Hirofumi Nakagami Hirofumi Nakagami orcid.org/0000-0003-2569-7062 Max-Planck-Institute for Plant Breeding Research, Cologne, Germany Search for more papers by this author Peter Schlögelhofer Peter Schlögelhofer Department of Chromosome Biology, Max F. Perutz Laboratories, Vienna Biocenter, University of Vienna, Vienna, Austria Search for more papers by this author Mathilde Grelon Mathilde Grelon Institut Jean-Pierre Bourgin, INRA, AgroParisTech, CNRS, Université Paris-Saclay, Versailles, France Search for more papers by this author Arp Schnittger Corresponding Author Arp Schnittger [email protected] orcid.org/0000-0001-7067-0091 Department of Developmental Biology, University of Hamburg, Hamburg, Germany Search for more papers by this author Author Information Chao Yang1, Kostika Sofroni1, Erik Wijnker1,6, Yuki Hamamura1, Lena Carstens1,7, Hirofumi Harashima2,8, Sara Christina Stolze3, Daniel Vezon4, Liudmila Chelysheva4, Zsuzsanna Orban-Nemeth5,9, Gaëtan Pochon1, Hirofumi Nakagami3, Peter Schlögelhofer5, Mathilde Grelon4 and Arp Schnittger *,1 1Department of Developmental Biology, University of Hamburg, Hamburg, Germany 2RIKEN Center for Sustainable Resource Science, Yokohama, Japan 3Max-Planck-Institute for Plant Breeding Research, Cologne, Germany 4Institut Jean-Pierre Bourgin, INRA, AgroParisTech, CNRS, Université Paris-Saclay, Versailles, France 5Department of Chromosome Biology, Max F. Perutz Laboratories, Vienna Biocenter, University of Vienna, Vienna, Austria 6Present address: Laboratory of Genetics, Wageningen University & Research, Wageningen, The Netherlands 7Present address: Plant Developmental Biology & Plant Physiology, Kiel University, Kiel, Germany 8Present address: Solution Research Laboratory, AS ONE Corporation, Kawasakiku, Kawasaki, Japan 9Present address: Institute of Molecular Pathology, Vienna Biocenter, Vienna, Austria *Corresponding author. Tel: +49 40 428 16 502; Fax: +49 40 428 16 503; E-mail: [email protected] The EMBO Journal (2020)39:e101625https://doi.org/10.15252/embj.2019101625 [The copyright line of this article was changed on 16 December 2019 after original online publication.] PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Meiosis is key to sexual reproduction and genetic diversity. Here, we show that the Arabidopsis cyclin-dependent kinase Cdk1/Cdk2 homolog CDKA;1 is an important regulator of meiosis needed for several aspects of meiosis such as chromosome synapsis. We identify the chromosome axis protein ASYNAPTIC 1 (ASY1), the Arabidopsis homolog of Hop1 (homolog pairing 1), essential for synaptonemal complex formation, as a target of CDKA;1. The phosphorylation of ASY1 is required for its recruitment to the chromosome axis via ASYNAPTIC 3 (ASY3), the Arabidopsis reductional division 1 (Red1) homolog, counteracting the disassembly activity of the AAA+ ATPase PACHYTENE CHECKPOINT 2 (PCH2). Furthermore, we have identified the closure motif in ASY1, typical for HORMA domain proteins, and provide evidence that the phosphorylation of ASY1 regulates the putative self-polymerization of ASY1 along the chromosome axis. Hence, the phosphorylation of ASY1 by CDKA;1 appears to be a two-pronged mechanism to initiate chromosome axis formation in meiosis. Synopsis CDKA;1, the central cell cycle regulator in Arabidopsis and homolog of human Cdk1 and Cdk2, is essential for meiosis and especially needed for regulation of chromosome synapsis. In early meiosis, CDKA;1 promotes chromosome axis assembly via ASY1 phosphorylation. CDKA;1 and HORMA domain protein ASY1 co-localize on meiotic chromosomes, being released upon chromosome synapsis. ASY1 is phosphorylated by CDKA;1 in early meiosis. ASY1 phosphorylation antagonizes the releasing force of PCH2 ATPase, thereby enabling ASY1 localization on chromosomes in early meiosis. PCH2 is required for the efficient nuclear targeting of ASY1 in early meiosis. Introduction Cell division relies on a highly orchestrated order of events to allow the faithful distribution of chromosomes to daughter cells. Progression through the cell cycle is controlled by the activity of cyclin-dependent kinases (Cdks; Morgan, 1997; Malumbres et al, 2009; Harashima et al, 2013). Eukaryotes usually contain several different families of cyclins that are thought to provide substrate specificity to Cdk–cyclin complexes and guide their intracellular localization (Miller & Cross, 2001; Pagliuca et al, 2011). However, the absolute levels of kinase activity have been found to be of key importance for cell cycle control, and at least in fission yeast, a single Cdk–cyclin complex is sufficient to drive both mitosis and meiosis (Coudreuse & Nurse, 2010; Gutiérrez-Escribano & Nurse, 2015). In comparison with mitosis, much less is known about how Cdks control the progression of the two consecutive division events of meiosis. Meiosis II leads to the separation of sister chromatids that, at least formally, resembles a mitotic division and is thought to largely rely on similar control mechanisms as mitosis. In contrast, meiosis I holds many features that are not known from mitosis, foremost recombination between homologous chromosomes. Nonetheless, Cdk–cyclin complexes have been shown to control several aspects of meiosis I such as the formation of DNA double-strand breaks (DSBs) at the beginning of the meiotic recombination process by phosphorylating Mer2/Rec107 (meiotic recombination 2/recombination 107; Rockmill & Roeder, 1990; Henderson et al, 2006; Li et al, 2006). Furthermore, the repair of DSBs through meiotic recombination has been found to involve Cdks, namely to phosphorylate the nuclease Sae2/Com1 (sporulation in the absence of spo eleven 2/completion of meiotic recombination 1) and by that promotes its activity to generate 3′ overhangs at the DSB site (Huertas & Jackson, 2009; Anand et al, 2016; Cannavo et al, 2018). These DNA ends are further processed by the MRN/MRX complex comprising the subunits Mre11 (meiotic recombination 11), Rad50 (radiation 50), and Nbs1/Xrs2 (Nijmegen breakage syndrome 1/X-ray sensitive 2) (Mimitou and Symington, 2009; Manfrini et al, 2010). Subsequently, the single DNA strands are bound by the recombinases Rad51 (radiation 51) and Dmc1 (disrupted meiotic cDNA1) to promote strand invasion and formation of heteroduplex DNA (Shinohara et al, 1997; Kurzbauer et al, 2012; Da Ines et al, 2013). Depending on how the subsequently resulting double Holliday junctions are resolved, meiotic crossovers (COs) can be formed that lead to the reciprocal exchange of DNA segments between homologous chromosomes (Zickler & Kleckner, 2015; Lambing et al, 2017). Cdks were found to partially co-localize with Rad51 as well as other components acting downstream of Rad51 involved in CO formation (Baker et al, 1996; Zhu et al, 2010). This, together with the observation that inhibition of Cdk activity in early meiosis abolished the formation of Rad51 foci, led to the conclusion that the activity of Cdk is essential for DSB formation and/or processing (Henderson et al, 2006; Huertas et al, 2008; Zhu et al, 2010). In many species, the synaptonemal complex (SC) stabilizes the pairing of homologous chromosomes and plays an important role in promoting the interhomolog bias during recombination and in maturation of recombination intermediates into COs (Zickler & Kleckner, 1999; Mercier et al, 2015). The SC is formed by the two proteinaceous axes of homologous chromosomes that will become then the lateral elements of the SC after synapsis. A number of proteins have been identified that are required for the correct formation of the chromosome axis. These include Red1 in yeast and its orthologs such as ASY3 in Arabidopsis (Rockmill & Roeder, 1990; Smith & Roeder, 1997; Ferdous et al, 2012). Another key protein of the chromosome axis is the HORMA domain protein Hop1 in yeast and its ortholog ASY1 in Arabidopsis (Hollingsworth et al, 1990; Aravind & Koonin, 1998; Armstrong, 2002). The phosphorylation of Hop1 at an [S/T]Q cluster domain by Tel1 (Telomere maintenance 1) and Mec1 (mitosis entry checkpoint 1), the ATM (ataxia telangiectasia mutated) and ATR (ataxia telangiectasia and Rad3-related) orthologs, is essential for the interhomolog-biased recombination, but not for the chromosomal loading of Hop1 (Carballo et al, 2008). For the correct assembly of the SC, Hop1/ASY1 is recruited to the axis by interaction with Red1/ASY3 (Bailis & Roeder, 1998; de los Santos & Hollingsworth, 1999; Ferdous et al, 2012). Furthermore, it was recently proposed that Hop1 might build a homopolymer through its C-terminal closure motif and it was thought that this polymerization is likely crucial for its function and axis association since the point mutation K593A in the closure motif of Hop1 causes an 11-fold reduction in CO number and results in high spore lethality (Niu et al, 2005; West et al, 2018). In wild type, the chromosome axes (lateral elements) of homologs become connected in the SC via the central region formed by dimers of the Zip1/ZYP1 family of proteins along with other components (Zickler & Kleckner, 2015). SC assembly goes along with the coordinated release of Hop1/ASY1 from the chromosome axis, catalyzed by the triple AAA+ ATPase PCH2 (Wojtasz et al, 2009; Chen et al, 2014; Lambing et al, 2015). However, it is not clear how the dynamic localization Hop1/ASY1 on chromosomes is regulated. Cdk complexes have also been implicated in the assembly of the SC since mutations in their catalytic core, i.e., in Cdk2 in mice and in CDC28 (Cdk1 homolog) in budding yeast, resulted in defects in SC formation (Ortega et al, 2003; Zhu et al, 2010). However, although Zip1 has been shown to be phosphorylated by Cdk complexes in vitro, the molecular details of Cdk function for SC formation are still obscure since the SC is assembled normally in zip1 mutants in which the Cdk phosphorylation sites were exchanged with amino acids that cannot be phosphorylated (Zhu et al, 2010). The model plant Arabidopsis, similar to other multicellular eukaryotes, has several Cdks and cyclins with some of them having been assigned a function in meiosis (Wijnker & Schnittger, 2013). Six out of the 10 A- and one out of the nine B-type cyclins are expressed in meiosis including SOLO DANCERS (SDS), an atypical cyclin that has similarities to both A- and B-type cyclins (Azumi et al, 2002; Bulankova et al, 2013). However, of these eight cyclins potentially involved in meiosis, only the loss of either CYCA1;2, also known as TARDY ASYNCHRONOUS MEIOSIS (TAM), CYCB3;1, or SDS was found to result in meiotic defects (Magnard et al, 2001; Azumi et al, 2002; d'Erfurth et al, 2010; Bulankova et al, 2013; Prusicki et al, 2019). TAM is required for the repression of meiotic exit after the first meiotic division and the timely progression through meiosis II. SDS is necessary for crossover (CO) formation after DSBs have been induced, and the meiotic recombinase DMC1 does not localize to chromosomes in sds mutants (De Muyt et al, 2009). Mutants in CYCB3;1 have only a weak mutant phenotype and occasionally show premature and ectopic cell wall formation during meiosis I, a phenotype, however, that can be strongly enhanced in double mutants with sds demonstrating a redundant function of at least some of the meiotic cyclins in Arabidopsis (Bulankova et al, 2013). SDS and TAM build active kinase complexes with CDKA;1, the Arabidopsis Cdk1/Cdk2 homolog, that is the main cell cycle regulator in Arabidopsis (Cromer et al, 2012; Harashima & Schnittger, 2012; Nowack et al, 2012; Cifuentes et al, 2016). A function of CDKA;1 in meiosis is supported by the analysis of weak loss-of-function mutants, which are completely sterile (Dissmeyer et al, 2007, 2009). Next to CDKA;1, CDKG has been implicated in meiosis by controlling synapsis at ambient but not low temperatures (Zheng et al, 2014). However, CDKG, which is related to human Cdk10, is likely involved in transcriptional and posttranscriptional control of gene expression and presumably does not control structural components of chromosomes directly (Doonan & Kitsios, 2009; Tank & Thaker, 2011; Huang et al, 2013; Zabicki et al, 2013). Here, we demonstrate by detailed cytological and genetics studies that CDKA;1 is an important regulator of meiosis especially for chromosome synapsis and bivalent formation. We show that ASY1 is a phosphorylation target of CDKA;1 and that the phosphorylation of ASY1 is crucial for chromosomal axis formation in Arabidopsis by two, possibly interconnected mechanisms, involving the binding to ASY3 as well as to itself leading to ASY1 polymers assembling along the chromosome axis. Results Changes in subcellular distribution of CDKA;1 during meiosis For a detailed understanding of the role of CDKA;1 in meiosis, we first analyzed its localization pattern in male meiocytes. Previous studies using a functional fusion of CDKA;1 to mVenus have shown that CDKA;1 is present in both female meiosis and male meiosis (Nowack et al, 2007; Bulankova et al, 2010; Zhao et al, 2012). Since the previous reporter was subject to frequent silencing effects, a new CDKA;1 reporter was generated not relying on the cDNA, as in the previous construct. Instead, a 7 kb genomic fragment into which mVenus was introduced before the stop codon of CDKA;1 was used. The expression of this construct fully rescued the cdka;1 mutant phenotype and gave rise to stable CDKA;1:mVenus expression (Fig EV1A–C). Click here to expand this figure. Figure EV1. CDKA;1-mVenus fully complements the cdka;1 mutant phenotype The stems of a hypomorphic cdka;1 mutant CDKA;1T161D are completely sterile as indicated by short siliques in contrast to homozygous cdka;1 mutant expressing the CDKA;1:mVenus reporter construct that form long siliques and are full fertile. The siliques of hypomorphic CDKA;1T161D do not harbor viable seeds in contrast to homozygous cdka;1 mutant expressing CDKA;1:mVenus that develop healthy and plump seeds. Scale bars: 1 mm. Chromosome spread analysis of male meiocytes of a homozygous cdka;1 mutant expressing a functional CDKA;1:mVenus reporter reveals a wild type-like meiotic program. Scale bar: 20 μm. Chromosome spread analysis of the hypomorphic cdka;1 mutant CDKA;1T14D;Y15E. (a) zygotene-like stage; (b) pachytene-like stage; (c, d) diakinesis-like stages; and (e, f) end of meiosis I with two or three pools of chromosomes. Scale bar: 20 μm. Immunolocalization of ZYP1 (green) in wild-type (WT) and CDKA;1T161D mutants. Chromosomes are stained with DAPI (blue). Scale bars: 5 μm. Immunolocalization analysis of DMC1 (green) together with ASY1 (red) in late leptotene of male meiocytes of wild-type (WT) and CDKA;1T161D mutants. Scale bars: 5 μm. Chromosome spread analysis of rad51 and rad51 CDKA;1T161D mutants. (a, d) pachytene-like stage; (b, c, e, and f) anaphase I-like stage. Red arrowheads indicate the chromosomal fragments. Scale bars: 10 μm. Download figure Download PowerPoint By using this reporter, the subcellular localization pattern of CDKA;1 during male meiosis was revealed (Fig 1A and B, and Movie EV1). In early prophase, CDKA;1:mVenus is localized in both the nucleus (~60–70%) and the cytoplasm (~30–40%). As prophase progresses, CDKA;1 accumulates more strongly in the nucleus (~80%). Then, toward the end of prophase, CDKA;1 becomes more cytoplasmically localized (~50%). After nuclear envelope breakdown, CDKA;1 decorates the first meiotic spindle and later accumulates in the two forming nuclei. In metaphase II, CDKA;1 is uniformly present in the entire cell, then is enriched at the spindle, and subsequently accumulates in the nuclei of the four meiotic products, i.e., the microspores (Fig 1A). Figure 1. Changes in CDKA;1 distribution and meiotic defects in hypomorphic cdka;1 mutants in male meiocytes Confocal laser scanning micrographs showing the localization of a functional CDKA;1:mVenus fusion protein in the wild type (WT) and cartoons on top highlighting the changes in abundance of CDKA;1:mVenus in the nucleus and cytoplasm during the course of meiosis. The region colored in beige represents the cytoplasm, in green the nucleoplasm, and in white the nucleolus. Scale bar: 10 μm. Quantitative analysis of the signal distribution of the nuclear versus cytoplasmic fraction of CDKA;1:mVenus during prophase I of meiosis as revealed by live cell imaging (Movie EV1). Twenty cells at each time point were used for the analysis. Error bars represent mean ± SD, and two biological replicates were performed. Immunolocalization of CDKA;1 (green) and ASY1 (red) on spread chromosomes in leptotene and zygotene of wild-type plants expressing a functional PROCDKA;1:CDKA;1:Strep construct. The last lane shows a magnification of the region marked by the red rectangle. Arrowheads indicate synapsed regions of homologous chromosomes where CDKA;1 is no longer present. Scale bar: 5 μm. Chromosome spread analysis of the wild type and the hypomorphic cdka;1 mutant CDKA;1T161D. (a, h) zygotene or zygotene-like stages; (b, i) pachytene or pachytene-like stages; (c, j, k) diakinesis or diakinesis-like stages; (d) metaphase I; (e, i, m, n) end of meiosis I with two (e, m) or three (i) pools of chromosomes; (f) metaphase II; and (g) tetrad. Red arrowheads indicate the initiated formation of a phragmoplast. White arrowheads depict mitochondria. Scale bars: 10 μm. Download figure Download PowerPoint Due to the strong accumulation in the nucleoplasm, the presence of CDKA;1 at chromosomes, as reported for its mouse homolog Cdk2 or its yeast homolog Cdc28 (Ashley et al, 2001; Zhu et al, 2010), was difficult to judge. To address the chromosomal localization pattern of CDKA;1, we used plants that express a StrepIII-tag-CDKA;1 fusion construct known to completely rescue the cdka;1 mutant phenotype (Pusch et al, 2012), and followed the CDKA;1 localization in meiosis by immunolocalization using ASY1, a key component of the chromosome axis, for staging of meiosis. While Cdk2 and Cdc28 show a distinct punctuate staining in meiosis in mice and yeast (Ashley et al, 2001; Zhu et al, 2010), our experiments revealed that CDKA;1 co-localizes with ASY1 and forms a continuous signal along chromosomes at leptotene. At zygotene, when homologous chromosomes start to synapse, the fluorescent signals for both reporters, ASY1 and CDKA;1, concomitantly disappeared from the chromosome axes (Fig 1C). Since ASY1 is specifically removed from the synapsed chromosomes, we conclude from the similar patterns of CDKA;1 that CDKA;1 is excluded from the synapsed regions. These data suggest that CDKA;1 physically interacts with the chromosome axis during early meiotic prophase and might be important for chromosome pairing and synapsis. Meiosis is severely affected in hypomorphic cdka;1 mutants To assess the requirement of CDKA;1 for early stages of meiosis, we compared meiotic progression by chromosome spreads between wild-type plants and two previously described weak loss-of-function cdka;1 mutants (Figs 1D and EV1D). These alleles resulted from the complementation of a cdka;1 null mutant with CDKA;1 expression constructs, in which conserved amino acids have been replaced resulting in CDKA;1 variants with strongly reduced kinase activity: cdka;1 PROCDKA;1:CDKA;1T161D (in the following designated CDKA;1T161D) and cdka;1 PROCDKA;1:CDKA;1T14D;Y15E (in the following referred to as CDKA;1T14D;Y15E (Dissmeyer et al, 2007, 2009). Both mutants were found to exhibit similar meiotic phenotypes during male meiosis, because of which we focus on the description of one allele (CDKA;1T161D) in the following (Figs 1D and EV1D). In wild-type meiosis, chromosomes start to condense during early prophase, and initiate chromosome synapsis during zygotene, leading to full homolog synapsis at pachytene. Chromosome morphology becomes diffuse at diplotene followed by chromosome re-condensation toward diakinesis when bivalents become visible (Fig 1D a–c). In CDKA;1T161D, the first difference from the wild type becomes notable at zygotene-like stage manifested by the presence of clear thread-like chromosomes and the accumulation of mitochondria at the side of the meiocytes in which no homolog synapsis is observed (Fig 1D h) (58%; n = 120). The absence of synapsis was confirmed by the failure of ZYP1, a component of the central region of the synaptonemal complex, to localize to chromosomes of male meiocytes of CDKA;1T161D mutants as revealed by immunofluorescence analysis (Fig EV1E). Pachytene-like stages of CDKA;1T161D meiocytes show the characteristically even distribution of mitochondria as that in wild type through the cell, but have largely unpaired chromosomes (Fig 1D i). Like in the wild type, chromosomes in CDKA;1T161D then decondense at diplotene and recondense toward diakinesis with a major difference being the appearance of 10 univalents instead of five bivalents (Fig 1D d and k), which is the result of an achiasmatic meiosis (no bivalents found in nine out of nine meiocytes analyzed). These univalents are rod shaped and often show fuzzy borders that may indicate problems in chromosome condensation. The absence of synapsis and chiasmata can have several reasons, with one of the potentially earliest causes being the absence of SPO11-induced DSBs. However, the DSB repair recombinase DMC1 was localized correctly onto chromosomes with no significant reduction of foci, i.e., 138.5 ± 9.8 in CDKA;1T161D (n = 10) versus 169.9 ± 15.7 (n = 7) in WT (P = 0.09, two-tailed t-test). This suggested that DSBs are formed along the chromosome axis and that the achiasmatic meiosis in CDKA;1T161D results from defects in later steps of meiosis (Fig EV1F). The formation of DSBs was corroborated by the finding that a double mutant of CDKA;1T161D with rad51, which is required for DSB repair, showed chromosome fragmentation (44 out of 45 meiocytes analyzed) similar to the rad51 single mutant (39 out of 39 meiocytes; Fig EV1G). Therefore, we conclude that DSB processing, at least up to the loading of DMC1, is functional in CDKA;1T161D. With this, we conclude that the phenotype of the hypomorphic CDKA;1T161D mutants manifests after the meiotic DSB formation and initiation of repair but before synapsis. Meiotic progression in cdka;1 hypomorphic mutants is highly disturbed during meiotic stages after pachytene indicating additional roles of CDKA;1 in meiosis (Fig 1D j–n). At least a part of the cells give rise to interkinesis-like stages where two or more daughter nuclei are separated by a clear organelle band (Fig 1D l and m; 19%; n = 39). In such nuclei, up to 10 partially decondensed chromosomes are visible in two or more loosely organized groups, or as single chromosomes (Fig 1D l–n). A clear second meiotic division has not been observed in any cell (n = 206), and a phragmoplast occasionally becomes visible within the organelle band at interkinesis (in eight out of 39 cells), indicating that cytokinesis already begins at this stage (Fig 1D m). Taken together, these data suggest that CDKA;1 is an important regulator of meiosis especially for chromosome synapsis and bivalent formation. Phosphorylation of ASY1 by CDKA;1 promotes its recruitment to the chromosome axis Since in particular chromosome synapsis was affected in the weak loss-of-function cdka;1 mutants, we searched for possible phosphorylation targets of CDKA;1 involved in early chromosome engagement. Several meiotic regulators in yeast have been found to contain [S/T]P Cdk consensus phosphorylation sites (Zhu et al, 2010). Many of these regulators have homologs in Arabidopsis also harboring Cdk consensus sites. At the top of our list of putative CDKA;1 substrates was the Arabidopsis Hop1 homolog ASY1, especially also since asy1 mutants are known to be asynaptic, hence partially resembling the phenotype of the hypomorphic cdka;1 mutants (Armstrong, 2002). Moreover, a previous study identified the ASY1 ortholog of Brassica oleracea as a potential in vivo ATM/ATR and CDK phosphorylation target (Osman et al, 2017). In addition, Hop1 was found to be phosphorylated by Cdc28 in an in vitro screen for Cdk substrates in budding yeast (Ubersax et al, 2003), but the functional importance of the phosphorylation in both Brassica and yeast has remained unknown. The above-mentioned spatiotemporal co-localization of ASY1 with CDKA;1 on chromosomes revealed by immunolocalization is consistent with the idea that ASY1 could be a phosphorylation target of CDKA;1 (Fig 1C). To further test this, we generated two functional reporters for ASY1 (PROASY1:ASY1:GFP and PROASY1:ASY1:RFP), which both restored a wild type-like meiotic program when expressed in homozygous asy1 mutants (Appendix Fig S1A and C). As expected, and confirming our above-presented and previous immuno-detection studies (Ferdous et al, 2012; Lambing et al, 2015), ASY1 localizes to the chromosome axis at leptotene and is depleted during zygotene when the synaptonemal complex is formed as revealed by the concomitant analysis of ASY1:RFP together with a PROZYP1B:ZYP1B:GFP reporter (Figs 2A and EV2A). Figure 2. ASY1 is a phosphorylation target of CDKA;1 ASY1:GFP and ASY1T
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