RESEARCH ARTICLE

A wide reprogramming of histone H3 modifications during malemeiosis I in rice is dependent on the Argonaute protein MEL1Hua Liu1 and Ken-Ichi Nonomura1,2,*

ABSTRACTThe roles of epigenetic mechanisms, including small-RNA-mediatedsilencing, in plant meiosis largely remain unclear, despite theirimportance in plant reproduction. This study unveiled that ricechromosomes are reprogrammed during the premeiosis-to-meiosistransition in pollen mother cells (PMCs). This large-scale meioticchromosome reprogramming (LMR) continued throughout meiosis I,during which time H3K9 dimethylation (H3K9me2) was increased,and H3K9 acetylation and H3S10 phosphorylation were broadlydecreased, with an accompanying immunostaining pattern shift ofRNA polymerase II. LMR was dependent on the rice Argonauteprotein, MEIOSIS ARRESTED AT LEPTOTENE1 (MEL1), which isspecifically expressed in germ cells prior to meiosis, because LMRwas severely diminished in mel1 mutant anthers. Pivotal meioticevents, such as pre-synaptic centromere association, DNA double-strand break initiation and synapsis of homologous chromosomes,were also disrupted in this mutant. Interestingly, and as opposed tothe LMR loss in most chromosomal regions, aberrant meiotic proteinloading and hypermethylation of H3K9 emerged on the nucleolarorganizing region in the mel1 PMCs. These results suggest thatMEL1 plays important roles in epigenetic LMR to promote faithfulhomologous recombination and synapsis during rice meiosis.

KEY WORDS: Argonaute, Meiosis, Silencing, Histone modification,Plant, Rice

INTRODUCTIONArgonaute (AGO) family proteins are highly conserved ineukaryotes. AGOs, in association with non-coding small RNAs,modulate both transcriptional and post-transcriptional genesilencing during various developmental events (reviewed byVaucheret, 2008). AGO proteins are classified into twosubfamilies, AGO and PIWI. AGO subfamily proteins areexpressed ubiquitously, and associate with microRNAs (miRNAs)and small interfering RNAs (siRNAs). The PIWI subfamily proteinsbind a distinct class of small RNAs, termed PIWI-interacting smallRNA (piRNA; Kim et al., 2009). The piRNA-induced silencingcomplex plays important roles in germline development in manyanimals (Aravin et al., 2006; Girard et al., 2006; Lau et al., 2006).In contrast, plant genomes lack PIWI-clade AGOs (Kapoor et al.,

2008). However, several plant AGO proteins specifically acting ingerm cell development have been reported. MEIOSIS ARRESTED

AT LEPTOTENE1 (MEL1) is a rice AGO protein, which functionsin germ-cell development and meiosis (Nonomura et al., 2007).MEL1-clade AGOs include Arabidopsis thaliana AGO5 (Kapooret al., 2008). Of ten Arabidopsis AGOs, AGO2 and AGO9 areresponsible for chiasma frequency and dissolution of meioticchromosome interlock, respectively, but AGO5 is not required formeiotic events (Oliver et al., 2014). Thus, the meiotic roles ofMEL1-clade AGOs are still largely ambiguous. MEL1 is expressedin premeiotic germ cells in a gender-neutral fashion; the mutantphenotypes confirm that the wild-type MEL1 function in faithfulprogression of premeiotic mitosis and meiotic homolog synapsis(Nonomura et al., 2007; Komiya et al., 2014). Recently, deepsequencing of MEL1-associated small RNAs has revealed thatMEL1 prefers a unique class of 21-nucleotide (nt) siRNAs, whichhave a conserved cytosine residue at the 5′-terminus and areexpressed abundantly in reproductive organs of rice (Komiya et al.,2014). The precursors of the MEL1-associating siRNAs(masiRNAs) are transcribed specifically in the reproductive phasefrom >1000 intergenic loci (Komiya et al., 2014), mostlycorresponding to the 21-nt phased siRNA (phasiRNA) precursor(PHAS) loci, reported previously (Johnson et al., 2009). Both 21-ntmasiRNAs and phasiRNAs supposedly target few transposons, butlack obvious target RNAs (Song et al., 2012; Komiya et al., 2014;Zhai et al., 2015). The miR2118, which targets the 22-nt conservedmotifs in most of the 21-nt PHAS loci, is broadly conserved inangiosperms and gymnosperms (Zhai et al., 2011). In contrast tograss species, the dicot plants retain the PHAS loci as overlappingwith protein-coding regions; the PPR family genes in Arabidopsis(Chen et al., 2007; Howell et al., 2007) and NB-LRR defense familygenes in the legume Medicago (Zhai et al., 2011). Thus, thebiological functions of the MEL1–masiRNA complex have largelyremained elusive. Furthermore, the existence of thousands or moreof 21-nt masiRNA and phasiRNA species has made it difficult tonarrow down their target RNAs to date.

Dynamic chromatin reprogramming is indispensable for faithfulprogression of meiosis in most eukaryotes, including plants. Forexample, meiotic double-strand break (DSB) positions are highlycorrelated with specific chromatin marks, such as trimethylation ofhistone H3 lysine 9 (H3K9) in yeast (Pan et al., 2011) and mammals(Brick et al., 2012), and recruitment of histone H2AZ, an H2Avariant, in Arabidopsis (Choi et al., 2013). In Arabidopsismegaspores, a highly dynamic chromatin reprogramming,coinciding with meiotic DNA replication, is required for thetransition of cell fate from somatic to germline (She et al., 2013).Transcriptional inactivation, represented by rapid H3K9deacetylation and removal of active RNA polymerase II (RNAPII)from meiotic chromosomes, takes place at early meiotic stages inmouse meiocytes (Page et al., 2012). During these stages, meioticDSB initiation and repair, and homologous chromosome synapsisprogress. However, the biological roles of the dynamic chromatinremodeling during plant meiosis have been ambiguous.Received 17 December 2015; Accepted 5 August 2016

1Experimental Farm, National Institute of Genetics, Yata 1111, Mishima, Shizuoka411-8540, Japan. 2Department of Life Science, Graduate University for AdvancedStudies/SOKENDAI, Yata 1111, Mishima, Shizuoka 411-8540, Japan.

*Author for correspondence (knonomur@nig.ac.jp)

K.-I.N., 0000-0002-2057-543X

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In the present study, we performed immunocytological analysesof Oryza sativa pollen mother cells (PMCs) in the mel1mutant, andinvestigated the possibility of MEL1 being involved in the globalcontrol of histone modifications in meiotic chromosomes, prior tohomologous recombination and pairing. The nucleolar organizingregion (NOR), encoding hundreds of tandem-repeated 45Sribosomal RNA (rRNA) genes (Oono and Sugiura, 1980), wasaffected by the mel1 mutation differently from most chromosomalregions. This study provides new insights in the relationshipbetween the AGO-mediated epigenetic control and the pivotalmeiotic events in plants, such as pre-synaptic centromereassociation, meiotic DSB initiation and homologous chromosomesynapsis.

RESULTSMeiotic DSB formation and homologous chromosomesynapsis are disrupted in mel1 mutantsGenerally, commitment to meiosis, including meiosis-specificDNA replication, occurs during premeiotic interphase. Meioticprophase I is divided into five substages; leptotene, zygotene,pachytene, diplotene and diakinesis. In flowering plants,programmed DSBs during meiosis are initiated and repaired, andhomologous pairs are synapsed from zygotene to pachytene(Hamant et al., 2006). To determine the meiotic stages precisely,two synaptonemal complex components were used: PAIR2 as anaxial component (Nonomura et al., 2006) and ZEP1 as a centraltransverse filament (Wang et al., 2010) (Fig. S1A, see Materials andMethods for the details).In the mel1 mutants of Oryza sativa, the development of germ

cells was arrested at the meiotic leptotene stage, despite the displayof meiotic cell features, specified by their enlarged volume andmeiosis-specific gene expression (Nonomura et al., 2007; Komiyaet al., 2014). To get further insights into the mel1 phenotype,meiotic DSB formation was examined in PMCs by immunostainingof phosphorylated histone H2AX (γH2AX). γH2AXwas transientlypresent in the DSB-containing chromatin stretches (Kuo and Yang,2008). In leptotene and zygotene, the signal was widespread alongthe wild-type rice chromosomes (Fig. 1A,B), but almost

disappeared during the stages from zygotene to pachytene(Fig. 1C). In contrast, it was hardly detectable in mel1 mutantPMCs (Fig. 1D,E), indicating that meiotic DSB initiation waslargely impaired.

Centromere (CEN) association is another pivotal meiotic event.CEN association often occurs among the nonhomologousCENs, prior to homolog synapsis, and is required for properhomolog recognition and pairing in eukaryotes, including manyplant species (Aragón-Alcaide et al., 1997; Martinez-Perezet al., 2001; Prieto et al., 2004; Zhang et al., 2013). As meiosisproceeds, CEN association is replaced by homologous CENpairing by pachytene (Tsubouchi and Roeder, 2005; Bisiget al., 2012; Obeso et al., 2014). It was observed that the numberof CEN foci was reduced in both the wild-type andmel1 PMCs withthe progression from pre-leptotene to meiotic leptotene (Fig. 2A).This reduction took place at leptotene, prior to the homologsynapsis, and was defined as presynaptic centromere association.The CEN number seemed unchanged in the subsequentpachytene (Fig. 2A), although, from cytological observations, wejudged that CEN associations were switching to the homologous

Fig. 1. Disruption of meiotic DSB initiation in mel1 mutant PMCs. PMCnuclei from wild-type (A–C) andmel1mutant (D,E) strains were visualized withanti-γH2AX (red) and anti-CenH3 antibodies (green). Chromosomes werecounterstained with DAPI (blue). (A,D) Leptotene, (B) zygotene,(C) pachytene, and (E) pachytene-like stages. Scale bars: 5 µm.

Fig. 2. Failed exit from centromere association in themel1mutant. (A) Thenumber of centromere (CenH3) foci in wild-type (WT) andmel1mutant strains.PAIR2was used as amarker for meiotic-stage progression. Pre, pre-leptotene;Lep, meiotic leptotene; Pac, meiotic pachytene. Results are average±s.d.(B) Box plots of areas of the CenH3 foci in wild-type (WT) and mel1 mutantPMCs. The area of the largest focus was measured in each cell, and plotted.The box represents the 25–75th percentiles, and the median is indicated. Thewhiskers show the 0–100th percentiles. ***P<0.001, **P<0.01 (two-samplet-test). The bottom images are examples of an ordinary-sized CenH3 focus(green) in WT (left) and an extraordinarily enlarged focus found in the mel1mutants (right). Chromosomes were counterstained with DAPI (blue).

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CEN pairing in the wild type. In contrast, in the mel1 mutant,CEN association was unresolved and the CEN number furtherdecreased from leptotene to pachytene (Fig. 2A). UnresolvedCEN association frequently resulted in the appearance ofextremely enlarged CEN foci (Fig. 2B). These results imply thatMEL1 has some functions in establishing the meiosis-specificCEN structure that is essential for facilitating the dissociationof CEN associations.Meiotic chromosome synapsis, represented by ZEP1 loading

between the homologous pairs, was fully disrupted in the mel1mutant (Fig. S1B), as reported previously (Komiya et al., 2014).Taken together, we conclude that MEL1 is required for pivotalmeiotic events, such as centromere association, DSB initiation andhomolog synapsis.

Large-scale alteration of H3K9 modifications duringwild-type meiosis ILoss of pivotal meiotic events indicated the possibility that thestructure and modification of meiotic chromosomes was globallyaltered in themel1mutant. To confirm this, we focused on the H3K9dimethylation (H3K9me2) and acetylation (H3K9ac). H3K9me2 isassociated with gene silencing and heterochromatin structure(Soppe et al., 2002). H3K9ac is a histone mark often associatedwith active transcription and is antagonistic to H3K9 methylation(Rando, 2007).

During pre-leptotene, H3K9me2 was maintained at a low level inthe wild-type PMCs (Fig. 3A). However, it was remarkablyintensified in the late leptotene (Fig. 3B). The intense signal wasalso observed in pachytene and metaphase I (Fig. 3C,D) andeventually declined during anaphase and telophase I (Fig. 3E). Themedian of overall H3K9me2 intensity was significantly increasedby 5.5-fold during the premeiosis-to-meiosis transition (Fig. 3F).These results were obtained with a monoclonal anti-H3K9me2antibody product, mAbcam1220 (Abcam). A similar H3K9me2dynamism in wild-type PMCs was reproduced with anotherantibody product, 05-1249 (Upstate) (Fig. S2A–E), although theoverall intensity was weaker than with the mAbcam1220 antibody.

The H3K9ac staining pattern was in contrast to that of theH3K9me2. The H3K9ac intensity was maintained at a moderatelevel at pre-leptotene (Fig. 3G), but was extremely reduced in lateleptotene and pachytene (Fig. 3H,I). Although the signal sometimesrecovered (data not shown), it weakened again by metaphase I(Fig. 3J) and eventually recovered at telophase I (Fig. 3K). Thetransient signal recovery at diakinesis was seemingly because of therapid chromosome compaction. The median of H3K9ac intensitywas significantly reduced by 2.7-fold during the premeiosis-to-meiosis transition (Fig. 3L).

These results suggest that H3K9s were reprogrammed broadly,and dimethylated and deacetylated during the meiotic prophase I inthe wild-type PMCs.

Fig. 3. Large-scale meiotic reprogramming of H3K9 in wild-type PMCs. (A–E,G–K) In A–E, the nuclei were stained with anti-PAIR2 (green) and anti-H3K9me2 (mAbcam1220) antibodies (red), and in G–K, with anti-ZEP1 (red) and anti-H3K9ac antibodies (green). Chromosomes were counterstained with DAPI(blue). (A,G) Pre-leptotene (premeiosis), (B,H) meiotic late leptotene, (C,I) pachytene, (D,J) metaphase I, and (E,K) telophase I. Scale bars: 5 µm. (F,L) Box plotsof relative intensity values of H3K9me2 (F) and H3K9ac (L), normalized to DAPI intensity. The box represents the 25–75th percentiles, and the median isindicated. The whiskers show the 0–100th percentiles. Pre, pre-leptotene; Le-Pa, leptotene-pachytene. ***P<0.001 (two-sample t-test).

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Meiotic alteration of H3K9 modifications depends on MEL1functionWe next examined the large-scale meiotic alteration of H3K9modifications in the mel1 mutant PMCs. As assessed by use of theantibody Abcam1220, the mel1 PMCs exhibited lower H3K9me2levels at pre-leptotene (Fig. 4A), as did wild-type cells (Fig. 3A). Inleptotene-like PMCs arrested by the mel1 mutation, unlike in thewild type, the reduced H3K9me2 levels continued even aftermeiotic entry, which was indicated by PAIR2 loading onto thechromosomes (Fig. 4B). H3K9me2 signals disappeared from mostof the chromatin regions in the later stages (Fig. 4C). The intensitywas significantly lower than that of the wild type in both pre-leptotene and early meiotic stages (Fig. 3F). The same tendency wasalso observed upon use of the 05-1249 antibody (Fig. S2). Incontrast, unexpected hyperacetylation of H3K9 was frequentlyobserved at pre-leptotene in themel1 PMCs (Figs 3L and 4D), morethan in wild-type PMCs. The hyperacetylation continued in cells atleptotene-like or later stages (Fig. 4E). The median of the H3K9acintensity was not reduced during premeiosis-to-meiosis transition,but rather increased in the mel1 mutant (Fig. 3L). The aboveobservations indicate that the reprogramming events are dependenton MEL1 function.

MEL1-dependent reduction of H3S10 phosphorylation duringmale meiosis ICrosstalk between phosphorylation and other modifications at thehistone H3 N-terminal tail has been reported. In mammals andyeast, H3K9ac is promoted by the acetyltransferase recruited ontoH3 phosphorylated at S10 (H3S10ph), which is adjacent to the K9acetylation site, resulting in transcriptional activation and K9hypomethylation (Cheung et al., 2000; Lo et al., 2001). In wild-type rice PMCs, H3S10ph was maintained at moderate levelsduring pre-leptotene to pachytene and was reduced duringzygotene to metaphase I (Fig. 5A–E). In contrast, extremelyintense H3S10ph signals were observed in the pre-leptotene mel1PMCs (Fig. 5F), with a median significantly higher (2.3-fold) thanin the wild-type pre-leptotene PMCs (Fig. 5I). Different from inthe wild type, the H3S10ph levels in pre-leptotene PMCs did notdecline, but rather increased at leptotene and pachytene in the

mel1 mutant (Fig. 5G–I). This pattern was largely consistent withthe H3K9ac pattern (Fig. 3G–L), suggesting that MEL1 functionsto repress H3S10ph, as well as H3K9ac, in the context of thelarge-scale meiotic chromosome reprogramming observed abovein PMCs.

RNApolymerase II is inactivated duringmeiotic prophase I ina manner that is dependent on MEL1Generally, H3K9me2 is known as a silencing mark in plants (Soppeet al., 2002). Thus, H3K9me2 accumulation onmeiotic chromosomessuggests that transcription is inactivated widely in wild-type ricePMCs. However, a previous analysis using premeiotic flowerssuggests that mRNA transcription patterns are almost comparablebetween the wild type and mel1mutant (Komiya et al., 2014). In themeiotic anthers used in this study, no difference was detected inamounts of 18S and 25S ribosomal RNAs by quantitative RT-PCR(Fig. S3A, see legend for further details regarding quantitativeRT-PCR). Given that flowers and anthers contain numerous somaticcells, transcriptional differences due to the mutation in PMCs, if any,could be masked by the larger amounts of unaffected transcripts fromsomatic cells. The results suggest that somatic anther cells areunaffected by the mel1 mutation, as expected.

Thus, the transcription status in meiotic PMCs was estimated byanalyzing the immunostaining pattern of phosphorylated Rpb1, thelargest subunit of RNAPII. Phosphorylation of Rpb1 at differentresidues in its C-terminal domain (CTD) correlates with the RNAPIItranscriptional activity; phosphorylation at S5 (S5ph) is associatedwith initiation and elongation of RNAPII-dependent transcripts, andthat at S2 (S2ph) with the termination of transcription (Komarnitskyet al., 2000). In cultured mammalian cells, numerous minuteRNAPII foci are linked with active transcription, and enlargedspeckles are associated with the inactive state (Bregman et al., 1995;Zeng et al., 1997).

In the wild-type rice PMCs, numerous minute signals of bothS5ph and S2ph were scattered in the pre-leptotene nuclei(Fig. 6A; Fig. S3B). However, from leptotene to zygotene, thenumber of scattered foci was reduced, and many large speckle-like structures emerged in all ten PMCs observed (Fig. 6B-D;Fig. S3B). These signals disappeared by metaphase I (Fig. 6E;Fig. S3B). Few S5ph signals overlapped with S2ph signalsduring the meiosis I stages (Fig. S3B), suggesting that the twotypes of CTD phosphorylation are also associated with differenttranscriptional steps in rice cells. Taken together, the appearanceof enlarged speckled-foci of S5ph and S2ph in the wild-typePMCs (Fig. 6C,J) suggests that large-scale meiotic alteration ofhistone modifications is accompanied by the inactivation oftranscription in rice.

At pre-leptotene, the staining pattern of both S5ph and S2ph inthemel1mutant PMCs was largely comparable with that of the wildtype (Fig. 6F; Fig. S3B), except in the ribosomal DNA (rDNA)repeat regions (see below). However, unlike in the wild type,numerous minute foci of phosphorylated RNAPII continued toappear during meiosis I in all ten PMCs observed (Fig. 6G–I,K),although the signal eventually disappeared, as in the wild type (datanot shown). The frequency of the appearance of enlarged S5phspeckles was slightly decreased in mutant PMCs (27.1% of all focicounted) compared to that in wild-type PMCs (13.2%) (Fig. 6L). Inaddition, the size and intensity of foci were significantly reduced inthe mel1 mutant (Fig. 6M). These results indicate that theinactivation of transcription is accompanied by large-scale histonemodifications during wild-type meiosis I, and that it is alsodependent on the MEL1 function.

Fig. 4. Disrupted meiotic H3K9 reprogramming in the mel1 mutant.(A–C) PMC nuclei were stained with anti-PAIR2 (green) and anti-H3K9me2antibodies (mAbcam1220) (red). Chromosomes were counterstained withDAPI (blue). (A) Pre-leptotene, (B) zygotene-like stage, and (C) pachytene-likestages. (D,E) PMC nuclei were stained with anti-ZEP1 (red) and anti-H3K9acantibodies (green). Chromosomes were counterstained with DAPI (blue).(D) Leptotene, and (E) zygotene-like stages. In each panel, the nucleolus isidentifiable by an immunofluorescence-negative ‘black hole’. Arrows indicateaberrant accumulation of PAIR2 onNORs, often observed as adhering to outernucleolar periphery. Scale bars: 5 µm.

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Chromatin structureof rDNA regionsareaffectedby themel1mutationNonomura et al. (2007) have demonstrated that there is an aberrantaggregation of a meiotic protein PAIR2 on the NOR, composed ofthe tandem-repeated ribosomal 45S RNA genes (rDNA). Thepresent study reconfirmed the previous result (Fig. 4B,C). Inaddition to PAIR2 accumulation, excess amounts of RNAPII S5phand S2ph were enriched on NORs in the mel1 mutant PMCs(Fig. 6H,I). The aberrant PAIR2- and active RNAPII-enrichedNORs were often observed as adhering to the external periphery ofthe nucleolar region, which was detectable as a ‘black hole’without4′,6-diamidino-2-phenylindole (DAPI) staining (Fig. 4B,C;

Fig. S3C). When using the 05-1249 antibody, an intenseH3K9me2 signal was observed as overlapping PAIR2 andRNAPII signals on NORs (Figs S2F–H and S3C). Similar NORsignals were also observed upon use of the Abcam1220 antibody,but the intensity was much fainter than with 05-1249 (Fig. S3C),although the reason was unclear. It was clear that compared to wild-type PMCs, more H3K9s were dimethylated in rDNA regions inmel1 mutant PMCs, but further analyses will be necessary.

Interestingly, a hypermethylation at H3K9 was also detected atCEN regions in mel1 mutant PMCs (Fig. S2H). The reason isambiguous, but the result might relate to the mutant phenotype ofcentromere dissociation in mel1 PMCs (Fig. 2).

Fig. 5. MEL1-dependent meiotic reprogramming of H3S10ph. PMC nuclei of wild-type (A–E) and mel1 mutant (F–H) strains were stained with anti-PAIR2(green) and anti-H3S10ph antibodies (red). Chromosomes were counterstained with DAPI (blue). (A,F) Leptotene, (B,G) zygotene, (C) early pachytene, (D) latepachytene, (E) metaphase I, and (H) pachytene-like stages. Scale bars: 5 µm. (I) Box plots of H3S10ph signal intensity normalized to DAPI intensity. The boxrepresents the 25–75th percentiles, and the median is indicated. The whiskers show the 0–100th percentiles. Pre, pre-leptotene; Le-Pa, leptotene-pachytene.***P<0.001 (two-sample t-test).

Fig. 6. Staining pattern of active RNA polymerase II during meiotic silencing. (A–I) Nuclei of wild-type (A–E) and mel1 mutant male meiocytes (F–I) werestained with anti-RNAPII S5ph antibody (red). Chromosomes were counterstained with DAPI (blue). Cellular stages were determined by subnuclearlocalization of PAIR2 (data not shown). (A,F) Pre-leptotene, (B,G) leptotene, (C) zygotene, (D) pachytene, (E) metaphase I, (H) zygotene-like, and (I) pachytene-like stages. (J,K) Magnified view of the indicated areas in C and H, respectively. Arrows indicate silenced NORs. Scale bars: 5 µm. (L) A bar graph showing theaverage±s.d. percentage of enlarged RNAPII S5ph speckles in PMCs at the zygotene or zygotene-like stage. The foci numbers were counted in ten PMCseach for thewild type and themel1mutant. Here, foci larger than the 75th percentile (>2644 integral density) of all foci counted were defined as enlarged speckles.(M) Box plots of the size of all RNAPII S5ph foci counted in L. The box represents the 25–75th percentiles, and the median is indicated. The whiskers show the0–100th percentiles. ***P<0.001 (two-sample t-test).

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DISCUSSIONLarge-scale meiotic remodeling occurring in rice meiosis IThe present study demonstrates that large-scale andmeiosis-specificchromatin remodeling occurs during rice meiosis I. In the wild-typePMCs, most of the meiotic chromosomal regions except for therDNA repeat regions were reprogrammed as indicated by increasedH3K9me2, and decreased H3K9ac and H3S10ph (Figs 3 and 5).This is consistent with the results observed in the Arabidopsisfemale megaspore mother cell (MMC), in which cell fate transitionwas accompanied by large-scale chromatin reprogramming,including increased H3K9me2 and a reduction in active RNAPII(She et al., 2013). Hereafter, we designate the MEL1-dependentmeiotic events demonstrated in this study as large-scale meioticchromosome reprogramming (LMR). LMR would also be expectedto occur in riceMMCs, becauseMEL1 functions in a gender-neutralfashion (Nonomura et al., 2007).The rice LMR was concomitant with the alteration of the active

RNAPII distribution pattern during the premeiosis-to-meiosistransition (Fig. 6), likely representing global transcriptionalinactivation of meiotic chromosomes in PMCs. This finding isreminiscent of meiotic silencing of unpaired chromosomes (MSUC)in mice (Barchi et al., 2005; Bellani et al., 2005; Turner et al., 2005),which is accompanied by reduced H3K9ac and inactive RNAPII(Page et al., 2012). In male mice, unpaired regions of X and Ychromosomes are compartmentalized in the sex-body and aresilenced (Turner, 2007). In addition, unpaired autosomal regionsare accompanied by pseudo-sex body formation and aretranscriptionally silenced by MSUC, leading to meiocytesundergoing apoptosis due to a ‘pachytene checkpoint’, which iswidely present in the non-plant species (Roeder and Bailis, 2000).Pseudo-sex bodies are formed at pachytene, when meiotictranscription is resumed at the paired regions. Thus, MSUC mightbe a surveillance mechanism to remove meiocytes that couldaccidentally cause unpairing of the autosomes. In other words,global transcriptional reactivation at mid-pachytene exposessilenced MSUC regions to the checkpoint machineries.However, the timing of relieving rice LMR (anaphase or

telophase I; Figs 3 and 5) was different from that of mice MSUC(mid-pachytene) (Page et al., 2012). This distinction might be due tothe difference in surveillance systems between plant and non-plantspecies. It is widely accepted that the pachytene checkpoints do notoccur in plants (Caryl et al., 2003). The absence of the pachytenecheckpoint might facilitate the survival of unreduced gametes andthe long-term persistence of the polyploidy (Li et al., 2009), whichis generally thought to be advantageous for inheritance of geneticdiversity and heterosis in plant species (Comai, 2005). Rice LMRwas observed almost throughout the meiosis-I stages (Figs 3 and 5),and, thus, plant meiocytes cannot be discriminated by thetranscriptional state of the unpaired regions. Therefore, plantsmight have evolved a transcription-independent system to monitorthe homologous chromosome synapsis for survival.Another possibility is that LMR determines the proper positions

of meiotic DSBs rather than controlling the transcriptional state. InArabidopsis, meiotic crossover hotspots overlap with histone-H2AZ-enriched nucleosomes at the promoter regions of genes(Choi et al., 2013). H2AZ activates transcription by protecting thepromoters from DNA methylation (Zilberman et al., 2008). Itsdepletion caused by the loss of ARP6, a nucleosome-remodelingcomplex, reduces the foci of meiotic proteins, such as RAD51,DMC1 and MLH1 during meiosis I (Choi et al., 2013). Thesestudies suggest that H2AZ-enriched chromatins are required forboth active transcription and meiotic DSB formation. H3K9ac is

also found at the 5′-end of coding regions, in addition to H2AZ andH3K4me3, and their levels correlate with the transcription rate(Rando, 2007; Choi et al., 2013). Thus, it is possible that the MEL1-dependent LMR restricts transcriptionally active genes to determinethe sites for meiotic DSB initiation. In this case, the increasedH3K9ac level in the mel1 mutant PMCs might cause too manyregions that can be preferred for the meiotic DSBs, therebydisturbing proper DSB formation (Fig. 1).

A possible MEL1 role in rRNA gene repeatsDifferent from most of the other chromosomal regions, theH3K9me2 level was exceptionally increased at the NOR in themel1 mutant, in addition to the aberrant loading of PAIR2 andphosphorylated RNAPII (Figs 4 and 6; Figs S2 and S3B,C). Theaberrant PAIR2 and RNAPII accumulations were never observed insomatic cells in mutant anthers (Fig. S4), indicating that MEL1-dependent reprograming events happened only in meiotic PMCs. InArabidopsis, silenced and heterochromatic rDNA sequences areepigenetically allocated at the nucleoplasm, towards the outside ofthe nucleolar territory (Lawrence and Pikaard, 2004; Earley et al.,2006, 2010), as observed in this study (Fig. S3C). Thus, PAIR2-,RNAPII- and H3K9me2-enriched NORs in the mel1 mutant PMCsare supposed to be highly heterochromatinized and silenced. Thissituation seemed to be the opposite of that in the other chromosomalregions, in which the H3K9me2 level was extremely reduced in themutant (Figs 3A–F and 4).

One possibility to explain this discrepancy is that the hyper-methylation at H3K9 in NORs in the mel1mutant is a by-product tocompensate for reduced H3K9me2 on most chromosomal regions.Another possibility is that the rDNA repeat region is prevented fromundergoing LMR during wild-type meiosis I in a manner that isdependent on MEL1 function. In this context, concomitantaggregation of PAIR2 on the NOR is suggestive. Rice PAIR2 is aHORMA domain protein (Nonomura et al., 2004, 2006), afunctional homolog of yeast HOP1 and Arabidopsis ASY1(Hollingsworth et al., 1990; Sanchez-Moran et al., 2007), both ofwhich are essential for establishment of homologous chromosomesynapsis and chiasma formation. In yeast, HOP1 is prevented frominvading nucleolar territory and NOR by the meiosis-specificAAA+ family ATPase PCH2 to suppress non-allelic homologousrecombination (NAHR) between rDNA repeats (San-Segundo andRoeder, 1999; Vader et al., 2011). NAHR in repetitive regionsresults in structural or copy-number changes, such as deletion,duplication and inversion, leading to genome destabilization (Sasakiet al., 2010). Taken together with H3K9me2 accumulation resultingin heterochromatinization of NORs (Earley et al., 2006), keepingNORs away from the LMR or heterochromatinization duringmeiosis I might be required for excluding meiotic machineriesfrom the NOR, probably resulting in NAHR inhibition andmaintenance of genome stability, as well as for protecting rRNAgenes from silencing to secure meiotic cell homeostasis.

A recent study deciphered that Dcr1, the sole Dicer protein infission yeast, promotes termination of RNAPII-dependenttranscription to avoid collision with DNA replication (Castelet al., 2014). RNA-interference-mediated knockdown of Dcr1caused an alteration in RNAPII loading in broad genomic regions,including those for rDNA. The rDNA repetitive regions are knownas ‘difficult-to-replicate’ regions, because passage of transcriptioncomplexes frequently stalls DNA replication (Alzu et al., 2012;Sabouri et al., 2012). Interestingly, in the dcr1mutant, H3K9me2 isslightly enriched in rDNA, which is different from other genomicregions, and the rDNA copy number is extremely reduced through

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meiotic NAHR (Castel et al., 2014). In addition, RNAPII S5ph andS2ph are aberrantly accumulated on rDNA in the dcr1 mutants(Castel et al., 2014). Thus, the phenotypes of the yeast dcr1 mutantresemble those of the rice mel1 mutant, as demonstrated in thisstudy. In fission yeast, the RNA silencing complex, includingAGO1, induces H3K9methylation in an siRNA-dependent manner,to create binding sites for SWI6, the heterochromatin protein 1(HP1) homolog (Hutvagner and Simard, 2008). These findingsprompted us to hypothesize that MEL1 is involved in themaintenance of genome stability and rDNA copy number throughtranscriptional control during rice meiosis. Although the effect ofMEL1 in rRNA transcription could not directly be verified in thisstudy (Fig. S3A), how MEL1 balances the transcriptional activitybetween NORs and non-NORs will be an important question.

How does the MEL1 AGO control the H3 N-terminal tailmodification?Subcellular localization is important for understanding themolecular action of AGOs. Cytoplasmic AGOs are generallythought to function in post-transcriptional gene silencing throughmRNA degradation and translational inhibition (Baumberger andBaulcombe, 2005; Leung et al., 2006; Lanet et al., 2009; Li et al.,2013). The cytoplasmic MEL1 AGO is also supposed to havesimilar functions (Komiya et al., 2014). If it is so, the meiosis-specific control of chromatin modifications, as demonstrated in thisstudy, should be mediated by unidentified genes and proteins,downstream of the MEL1 pathway.However, it is also possible that MEL1 is transported into the

meiotic nuclei and directly engages with the dynamic control ofmeiotic chromatin modifications. In fact, nuclear localization ofMEL1 has been reported in leptotene meiocytes, althoughinfrequently (Komiya et al., 2014). Arabidopsis AGO4 associateswith heterochromatic-siRNAs (hc-siRNAs) at the cytoplasm andmediates RNA-directed DNA methylation (RdDM), a nuclearprocess (Law and Jacobsen, 2010; Li et al., 2006; Pontes et al.,2006). The cytoplasmic Arabidopsis AGO4 assembly with hc-siRNAs is thought to induce conformational change in the AGO4protein around the nuclear localization signal and promotes thenuclear import of the AGO4–hc-siRNA complex (Ye et al., 2012).The relationship of LMR to nuclear MEL1 function warrants furtherexamination.In conclusion, the present study highlights the importance of

meiosis-specific chromatin remodeling, LMR, for pivotal meioticevents during rice meiosis I. LMR accompanies the dynamicalteration of chromatin modifications, such as H3K9me2, H3K9acand H3S10ph, and transcription inactivation represented by theRNAPII-loading pattern in the meiotic nuclei. Cytologicalcharacterization of mel1 phenotypes suggests that LMR is ahighly controlled epigenetic process and that the MEL1 protein isa key regulator of LMR. Furthermore, the impact of rRNAtranscriptional control on meiosis is strongly suggested in rice.This finding implies the existence of unknown epigeneticmechanisms to protect repetitive rDNA regions from thedeleterious NAHR during plant meiosis. This system might beshared with other genomic repeats, because meiotic behavior ofcentromeres was somewhat affected by the mel1 mutation (Fig. 2;Fig. S2H). MEL1 localizes in the cytoplasm in premeiosis, but iscompetent to transfer into the nucleus at the onset of meiosis(Komiya et al., 2014). Generally, cytoplasmic AGOs are engagedin translational control, whereas nuclear AGOs are involvedin transcriptional silencing. Whether cytoplasmic or nuclearlocalization of MEL1 or both is more essential for LMR is an

important question. Reproductive phasiRNAs might affect MEL1subcellular localization, because the 21-nt and 24-nt phasiRNAs areexpressed preferentially during premeiosis and meiosis,respectively (Zhai et al., 2015), and MEL1 is able to bind bothclasses (Komiya et al., 2014). Further studies on MEL1 will shedmore light on the unidentified epigenetic mechanisms to assurefaithful meiosis progression in plants.

MATERIALS AND METHODSPlant materials and growth conditionsThe rice (Oryza sativa L. subspecies japonica) cultivar Nipponbare wasused as wild-type plants. Themel1 homozygous mutants were selected fromselfed progenies of Tos17-inserted heterozygous plants (Nonomura et al.,2007), which were backcrossed five times with cultivar Nipponbare.Genotyping in the MEL1 locus was performed as described by Nonomuraet al. (2007). All plants were grown in a field in the city of Mishima,Shizuoka, Japan.

Sampling of flowers and anthersRice flowers at premeiosis andmeiosis were fixed and stored as described byNonomura et al. (2006). The flower length and/or anther length werereferred to for estimating meiotic stages of flowers and anthers, according toItoh et al. (2005).

Meiosis markersRice proteins PAIR2 and ZEP1 were used as intracellular meiotic markers.PAIR2 begins to accumulate in the nucleoplasm just following meioticDNA replication, and subsequently, at meiotic leptotene, associates with theaxial element of the synaptonemal complex (Nonomura et al., 2006)(Fig. S1A). During zygotene and pachytene, axial PAIR2 proteins aregradually removed from chromosomes and replaced by ZEP1 loadingbetween homolog pairs (Wang et al., 2010) (Fig. S1A). In this study,‘premeiosis’ or ‘pre-leptotene’ is defined as the cellular stage at whichPMCs retain decondensed chromatins and accumulate PAIR2 proteinsthrough the nucleoplasm, but not on chromatin.

Indirect immunofluorescence and antibodiesSubcellular localization of MEL1, PAIR2, ZEP1 and CenH3 was observedby indirect immunofluorescent staining of PMCs, as described previously(Nonomura et al., 2006, 2007; Komiya et al., 2014). The antibody againstrice γH2AXwas produced according to the method described by Miao et al.(2013) and used at 1:150. To examine histone H3 modification, mousemonoclonal anti-H3S10ph antibody (1:150, cat. no. 05-598, Upstate) andrabbit polyclonal anti-H3K9ac antibody (1:150, cat. no. 06-942, Upstate)were used. For H3K9me2 detection, two different products of the mousemonoclonal anti-H3K9me2 antibody were used; cat. no. 05-1249 (Upstate)and mAbcam1220 (Abcam) at 1:75 and 1:150, respectively. Anti-PIIS2phantibody (1:150, cat. no. ab5095, Abcam) and anti-PIIS5ph antibody(1:150, cat. no. ab5408, Abcam) were used to stain the differentphosphorylated forms of RNA polymerase II. Antibodies for PAIR2 andZEP1 were used as meiotic stage markers (Nonomura et al., 2006; Wanget al., 2010). All immunofluorescent images were obtained by confocal laserscanning microscopy with a FV300 microscope (Olympus). To make thesubcellular localization of immunofluorescent foci clearer, a photo of asingle optical section was taken for each nuclear image, except for countingCenH3 foci (see below), and processed by the software Photoshop (Adobe).

Cytological validation of centromere associationFor validation of the centromere association, stereo 3D images of a PMCwere viewed through an Olympus FV1000-D microscope. PMCs werestained with antibodies against CenH3 and PAIR2. CenH3, a histone H3variant specifically involved in centromeric chromatins, was used tomonitorthe centromere. PAIR2 was used as a marker for meiotic-stage progression.In Fig. 2A, the number of CenH3 foci was counted on a merged image ofmultiple optical sections from the same meiocyte. The largest CenH3 focuswas selected in each meiocyte, and the pixel value of each focus was

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calculated by ImageJ (Schneider et al., 2012) and plotted in the graph shownin Fig. 2B.

Quantification of immunofluorescent signal intensityThe signal intensity of H3K9me2, H3K9ac and H3S10ph was quantifiedaccording to a previous method (She et al., 2013) with slight modifications.Briefly, PMCs were immunostained with antibodies for H3K9me2, H3K9acor H3S10ph, and counterstained with 4′,6-diamidino-2-phenylindole(DAPI). Antibody against PAIR2 or ZEP1 was used as a marker formeiotic-stage progression. A nuclear midplane image was taken in eachH3K9- and DAPI-stained PMC observed, and their pixel values werecalculated by ImageJ. The intensity of H3K9me2, H3K9ac or H3S10ph wasdivided by that of DAPI for normalization, and plotted (Figs 3F,L and 5I). Inthe figures, each plot was obtained by combining results from two or threebiological replicates together, because in all wild-type samples, thereplicates showed a similar variance of the pixel values to each other inaccordance with one-way ANOVA (data not shown).

To investigate RNAPII activity during meiosis I, PMCs wereimmunostained with anti-PIIS5ph or PIIS2ph antibodies and counterstainedwithDAPI. A nuclear midplane imagewas taken for each PMCobserved. Theimages of RNAPII S5ph foci were binarized in ten PMCs each for the wildtype and themel1mutant, to determine the areas to be analyzed, and the pixeldensity of each focus area was measured by ImageJ.

AcknowledgementsWe thank Mitsugu Eiguchi (NIG) for great help in growing plant materials. We alsothank Dr Robert A. Martienssen (Cold Spring Harbor, USA) for useful comments anddiscussions. We are grateful to Drs Takahiro Kusakabe (Kyushu U., Japan) andKatsutoshi Tsuda (NIG, Japan) for useful comments and reading the manuscript.The mel1 mutant seeds were kindly provided by National Bioresource Project(NBRP) Rice, Japan Agency for Medical Research and Development (AMED).

Competing interestsThe authors declare no competing or financial interests.

Author contributionsK.-I.N. designed the research. H.L. performed all experiments. H.L. and K.-I.N.analyzed data and wrote the article.

FundingThis study is supported by KAKENHI grants from the Japan Society for thePromotion of Science (JSPS) [grant numbers 25252004, 15K14630]; and by apostdoctoral fellowship from the National Institute of Genetics, Japan.

Supplementary informationSupplementary information available online athttp://jcs.biologists.org/lookup/doi/10.1242/jcs.184937.supplemental

ReferencesAlzu, A., Bermejo, R., Begnis, M., Lucca, C., Piccini, D., Carotenuto, W.,Saponaro, M., Brambati, A., Cocito, A., Foiani, M. et al. (2012). Senataxinassociates with replication forks to protect fork integrity across RNA-polymerase-II-transcribed genes. Cell 151, 835-846.

Aragon-Alcaide, L., Reader, S., Beven, A., Shaw, P., Miller, T. and Moore, G.(1997). Association of homologous chromosomes during floral development.Curr. Biol. 7, 905-908.

Aravin, A., Gaidatzis, D., Pfeffer, S., Lagos-Quintana, M., Landgraf, P., Iovino,N., Morris, P., Brownstein, M. J., Kuramochi-Miyagawa, S., Nakano, T. et al.(2006). A novel class of small RNAs bind to MILI protein in mouse testes. Nature442, 203-207.

Barchi, M., Mahadevaiah, S., Di Giacomo, M., Baudat, F., de Rooij, D. G.,Burgoyne, P. S., Jasin, M. and Keeney, S. (2005). Surveillance of differentrecombination defects in mouse spermatocytes yields distinct responses despiteelimination at an identical developmental stage. Mol. Cell. Biol. 25, 7203-7215.

Baumberger, N. and Baulcombe, D. C. (2005). Arabidopsis ARGONAUTE1 is anRNA Slicer that selectively recruits microRNAs and short interfering RNAs. Proc.Natl. Acad. Sci. USA 102, 11928-11933.

Bellani, M. A., Romanienko, P. J., Cairatti, D. A. and Camerini-Otero, R. D.(2005). SPO11 is required for sex-body formation, and Spo11 heterozygosityrescues the prophase arrest ofAtm−/− spermatocytes. J. Cell Sci. 118, 3233-3245.

Bisig, C. G., Guiraldelli, M. F., Kouznetsova, A., Scherthan, H., Hoog, C.,Dawson, D. S. and Pezza, R. J. (2012). Synaptonemal complex components

persist at centromeres and are required for homologous centromere pairing inmouse spermatocytes. PLoS Genet. 8, e1002701.

Bregman, D. B., Du, L., van der Zee, S. and Warren, S. L. (1995). Transcription-dependent redistribution of the large subunit of RNA polymerase II to discretenuclear domains. J. Cell Biol. 129, 287-298.

Brick, K., Smagulova, F., Khil, P., Camerini-Otero, R. D. and Petukhova, G. V.(2012). Genetic recombination is directed away from functional genomic elementsin mice. Nature 485, 642-645.

Caryl, A. P., Jones, G. H. and Franklin, F. C. H. (2003). Dissecting plant meiosisusing Arabidopsis thaliana mutants. J. Exp. Bot. 54, 25-38.

Castel, S. E., Ren, J., Bhattacharjee, S., Chang, A.-Y., Sanchez, M., Valbuena,A., Antequera, F. and Martienssen, R. A. (2014). Dicer promotes transcriptiontermination at sites of replication stress to maintain genome stability. Cell 159,572-583.

Chen, H.-M., Li, Y.-H. and Wu, S.-H. (2007). Bioinformatic prediction andexperimental validation of a microRNA-directed tandem trans-acting siRNAcascade in Arabidopsis. Proc. Natl. Acad. Sci. USA 104, 3318-3323.

Cheung, P., Tanner, K. G., Cheung, W. L., Sassone-Corsi, P., Denu, J. M. andAllis, C. D. (2000). Synergistic coupling of histone H3 phosphorylation andacetylation in response to epidermal growth factor stimulation. Mol. Cell 5,905-915.

Choi, K., Zhao, X., Kelly, K. A., Venn, O., Higgins, J. D., Yelina, N. E., Hardcastle,T. J., Ziolkowski, P. A., Copenhaver, G. P., Franklin, F. C. H. et al. (2013).Arabidopsismeiotic crossover hot spots overlap with H2A.Z nucleosomes at genepromoters. Nat. Genet. 45, 1327-1336.

Comai, L. (2005). The advantages and disadvantages of being polyploid. Nat. Rev.Genet. 6, 836-846.

Earley, K., Lawrence, R. J., Pontes, O., Reuther, R., Enciso, A. J., Silva, M.,Neves, N., Gross, M., Viegas, W. and Pikaard, C. S. (2006). Erasure of histoneacetylation byArabidopsis HDA6mediates large-scale gene silencing in nucleolardominance. Genes Dev. 20, 1283-1293.

Earley, K. W., Pontvianne, F., Wierzbicki, A. T., Blevins, T., Tucker, S., Costa-Nunes, P., Pontes, O. and Pikaard, C. S. (2010). Mechanisms of HDA6-mediated rRNA gene silencing: suppression of intergenic Pol II transcription anddifferential effects on maintenance versus siRNA-directed cytosine methylation.Genes Dev. 24, 1119-1132.

Girard, A., Sachidanandam, R., Hannon, G. J. and Carmell, M. A. (2006). Agermline-specific class of small RNAs binds mammalian Piwi proteins. Nature442, 199-202.

Hamant, O., Ma, H. and Cande, W. Z. (2006). Genetics of meiotic prophase I inplants. Annu. Rev. Plant Biol. 57, 267-302.

Hollingsworth, N. M., Goetsch, L. and Byers, B. (1990). TheHOP1 gene encodesa meiosis-specific component of yeast chromosomes. Cell 61, 73-84.

Howell, M. D., Fahlgren, N., Chapman, E. J., Cumbie, J. S., Sullivan, C. M.,Givan, S. A., Kasschau, K. D. and Carrington, J. C. (2007). Genome-wideanalysis of the RNA-DEPENDENTRNAPOLYMERASE6/DICER-LIKE4 pathwayin Arabidopsis reveals dependency on miRNA- and tasiRNA-directed targeting.Plant Cell 19, 926-942.

Hutvagner, G. and Simard, M. J. (2008). Argonaute proteins: key players in RNAsilencing. Nat. Rev. Mol. Cell Biol. 9, 22-32.

Itoh, J.-I., Nonomura, K.-I., Ikeda, K., Yamaki, S., Inukai, Y., Yamagishi, H.,Kitano, H. and Nagato, Y. (2005). Rice plant development: from zygote tospikelet. Plant Cell Physiol. 46, 23-47.

Johnson, C., Kasprzewska, A., Tennessen, K., Fernandes, J., Nan, G.-L.,Walbot, V., Sundaresan, V., Vance, V. and Bowman, L. H. (2009). Clusters andsuperclusters of phased small RNAs in the developing inflorescence of rice.Genome Res. 19, 1429-1440.

Kapoor, M., Arora, R., Lama, T., Nijhawan, A., Khurana, J. P., Tyagi, A. K. andKapoor, S. (2008). Genome-wide identification, organization and phylogeneticanalysis of Dicer-like, Argonaute and RNA-dependent RNA Polymerase genefamilies and their expression analysis during reproductive development and stressin rice. BMC Genomics 9, 451.

Kim, V. N., Han, J. and Siomi, M. C. (2009). Biogenesis of small RNAs in animals.Nat. Rev. Mol. Cell Biol. 10, 126-139.

Komarnitsky, P., Cho, E.-J. and Buratowski, S. (2000). Different phosphorylatedforms of RNA polymerase II and associated mRNA processing factors duringtranscription. Genes Dev. 14, 2452-2460.

Komiya, R., Ohyanagi, H., Niihama, M., Watanabe, T., Nakano, M., Kurata, N.and Nonomura, K.-I. (2014). Rice germline-specific Argonaute MEL1 proteinbinds to phasiRNAs generated from more than 700 lincRNAs. Plant J. 78,385-397.

Kuo, L. J. and Yang, L. X. (2008). Gamma-H2AX - a novel biomarker for DNAdouble-strand breaks. In Vivo 22, 305-309.

Lanet, E., Delannoy, E., Sormani, R., Floris, M., Brodersen, P., Crete, P.,Voinnet, O. and Robaglia, C. (2009). Biochemical evidence for translationalrepression by Arabidopsis microRNAs. Plant Cell 21, 1762-1768.

Lau, N. C., Seto, A. G., Kim, J., Kuramochi-Miyagawa, S., Nakano, T., Bartel,D. P. and Kingston, R. E. (2006). Characterization of the piRNA complex from rattestes. Science 313, 363-367.

3560

RESEARCH ARTICLE Journal of Cell Science (2016) 129, 3553-3561 doi:10.1242/jcs.184937

Journal

ofCe

llScience

Law, J. A. and Jacobsen, S. E. (2010). Establishing, maintaining and modifyingDNA methylation patterns in plants and animals. Nat. Rev. Genet. 11, 204-220.

Lawrence, R. J. and Pikaard, C. S. (2004). Chromatin turn ons and turn offs ofribosomal RNA genes. Cell Cycle 3, 878-881.

Leung, A. K. L., Calabrese, J. M. and Sharp, P. A. (2006). Quantitative analysis ofArgonaute protein reveals microRNA-dependent localization to stress granules.Proc. Natl. Acad. Sci. USA 103, 18125-18130.

Li, C. F., Pontes, O., El-Shami, M., Henderson, I. R., Bernatavichute, Y. V., Chan,S. W.-L., Lagrange, T., Pikaard, C. S. and Jacobsen, S. E. (2006). AnARGONAUTE4-containing nuclear processing center colocalized with Cajalbodies in Arabidopsis thaliana. Cell 126, 93-106.

Li, X. C., Barringer, B. C. and Barbash, D. A. (2009). The pachytene checkpointand its relationship to evolutionary patterns of polyploidization and hybrid sterility.Heredity 102, 24-30.

Li, S., Liu, L., Zhuang, X., Yu, Y., Liu, X., Cui, X., Ji, L., Pan, Z., Cao, X., Mo, B.et al. (2013). MicroRNAs inhibit the translation of target mRNAs on theendoplasmic reticulum in Arabidopsis. Cell 153, 562-574.

Lo, W.-S., Duggan, L., Emre, N. C., Belotserkovskya, R., Lane, W. S.,Shiekhattar, R. and Berger, S. L. (2001). Snf1–a histone kinase that works inconcert with the histone acetyltransferase Gcn5 to regulate transcription. Science293, 1142-1146.

Martinez-Perez, E., Shaw, P. and Moore, G. (2001). The Ph1 locus is needed toensure specific somatic and meiotic centromere association. Nature 411,204-207.

Miao, C., Tang, D., Zhang, H., Wang, M., Li, Y., Tang, S., Yu, H., Gu, M. andCheng, Z. (2013). CENTRAL REGION COMPONENT1, a novel synaptonemalcomplex component, is essential for meiotic recombination initiation in rice. PlantCell 25, 2998-3009.

Nonomura, K.-I., Nakano, M., Murata, K., Miyoshi, K., Eiguchi, M., Miyao, A.,Hirochika, H. and Kurata, N. (2004). An insertional mutation in the rice PAIR2gene, the ortholog of Arabidopsis ASY1, results in a defect in homologouschromosome pairing during meiosis. Mol. Genet. Genomics 271, 121-129.

Nonomura, K.-I., Nakano, M., Eiguchi, M., Suzuki, T. and Kurata, N. (2006).PAIR2 is essential for homologous chromosome synapsis in rice meiosis I. J. CellSci. 119, 217-225.

Nonomura, K.-I., Morohoshi, A., Nakano, M., Eiguchi, M., Miyao, A., Hirochika,H. and Kurata, N. (2007). A germ cell specific gene of the ARGONAUTE family isessential for the progression of premeiotic mitosis and meiosis duringsporogenesis in rice. Plant Cell 19, 2583-2594.

Obeso, D., Pezza, R. J. and Dawson, D. (2014). Couples, pairs, and clusters:mechanisms and implications of centromere associations in meiosis.Chromosoma 123, 43-55.

Oliver, C., Santos, J. L. and Pradillo, M. (2014). On the role of someARGONAUTEproteins in meiosis and DNA repair in Arabidopsis thaliana. Front. Plant Sci. 5,177.

Oono, K. and Sugiura, M. (1980). Heterogeneity of the ribosomal RNA geneclusters in rice. Chromosoma 76, 85-89.

Page, J., de la Fuente, R., Manterola, M., Parra, M. T., Viera, A., Berrıos, S.,Fernandez-Donoso, R. and Rufas, J. S. (2012). Inactivation or non-reactivation:what accounts better for the silence of sex chromosomes during mammalian malemeiosis? Chromosoma 121, 307-326.

Pan, J., Sasaki, M., Kniewel, R., Murakami, H., Blitzblau, H. G., Tischfield, S. E.,Zhu, X., Neale, M. J., Jasin, M., Socci, N. D. et al. (2011). A hierarchicalcombination of factors shapes the genome-wide topography of yeast meioticrecombination initiation. Cell 144, 719-731.

Pontes, O., Li, C. F., Nunes, P. C., Haag, J., Ream, T., Vitins, A., Jacobsen, S. E.and Pikaard, C. S. (2006). The Arabidopsis chromatin-modifying nuclear siRNApathway involves a nucleolar RNA processing center. Cell 126, 79-92.

Prieto, P., Santos, A. P., Moore, G. and Shaw, P. (2004). Chromosomes associatepremeiotically and in xylem vessel cells via their telomeres and centromeres indiploid rice (Oryza sativa). Chromosoma 112, 300-307.

Rando, O. J. (2007). Global patterns of histone modifications. Curr. Opin. Genet.Dev. 17, 94-99.

Roeder, G. S. and Bailis, J. M. (2000). The pachytene checkpoint. Trends Genet.16, 395-403.

Sabouri, N., McDonald, K. R., Webb, C. J., Cristea, I. M. and Zakian, V. A. (2012).DNA replication through hard-to-replicate sites, including both highly transcribedRNAPol II and Pol III genes, requires theS. pombe Pfh1 helicase.Genes Dev. 26,581-593.

Sanchez-Moran, E., Santos, J.-L., Jones, G. H. and Franklin, F. C. H. (2007).ASY1 mediates AtDMC1-dependent interhomolog recombination during meiosisin Arabidopsis. Genes Dev. 21, 2220-2233.

San-Segundo, P. A. and Roeder, G. S. (1999). Pch2 links chromatin silencing tomeiotic checkpoint control. Cell 97, 313-324.

Sasaki, M., Lange, J. and Keeney, S. (2010). Genome destabilization byhomologous recombination in the germ line. Nat. Rev. Mol. Cell Biol. 11, 182-195.

Schneider, C. A., Rasband,W. S. and Eliceiri, K. W. (2012). NIH Image to ImageJ:25 years of image analysis. Nat. Methods 9, 671-675.

She, W., Grimanelli, D., Rutowicz, K., Whitehead, M. W. J., Puzio, M., Kotlinski,M., Jerzmanowski, A. and Baroux, C. (2013). Chromatin reprogramming duringthe somatic-to-reproductive cell fate transition in plants. Development 140,4008-4019.

Song, X., Li, P., Zhai, J., Zhou, M., Ma, L., Liu, B., Jeong, D.-H., Nakano, M., Cao,S., Liu, C. et al. (2012). Roles of DCL4 and DCL3b in rice phased small RNAbiogenesis. Plant J. 69, 462-474.

Soppe, W. J., Jasencakova, Z., Houben, A., Kakutani, T., Meister, A., Huang,M. S., Jacobsen, S. E., Schubert, I. and Fransz, P. F. (2002). DNA methylationcontrols histone H3 lysine 9 methylation and heterochromatin assembly inArabidopsis. EMBO J. 21, 6549-6559.

Tsubouchi, T. and Roeder, G. S. (2005). A synaptonemal complex proteinpromotes homology-independent centromere coupling. Science 308, 870-873.

Turner, J. M. A. (2007). Meiotic sex chromosome inactivation. Development 134,1823-1831.

Turner, J. M., Mahadevaiah, S. K., Fernandez-Capetillo, O., Nussenzweig, A.,Xu, X., Deng, C. X. and Burgoyne, P. S. (2005). Silencing of unsynapsed meioticchromosomes in the mouse. Nat. Genet. 37, 41-47.

Vader, G., Blitzblau, H. G., Tame, M. A., Falk, J. E., Curtin, L. and Hochwagen, A.(2011). Protection of repetitive DNA borders from self-induced meiotic instability.Nature 477, 115-119.

Vaucheret, H. (2008). Plant ARGONAUTES. Trends Plant Sci. 13, 350-358.Wang,M.,Wang, K., Tang,D.,Wei, C., Li, M., Shen, Y., Chi, Z., Gu,M. andCheng,

Z. (2010). The central element protein ZEP1 of the synaptonemal complexregulates the number of crossovers during meiosis in rice. Plant Cell 22, 417-430.

Ye, R., Wang, W., Iki, T., Liu, C., Wu, Y., Ishikawa, M., Zhou, X. and Qi, Y. (2012).Cytoplasmic assembly and selective nuclear import of Arabidopsis Argonaute4/siRNA complexes. Mol. Cell 46, 859-870.

Zeng, C., Kim, E., Warren, S. L. and Berget, S. M. (1997). Dynamic relocation oftranscription and splicing factors dependent upon transcriptional activity.EMBO J.16, 1401-1412.

Zhai, J., Jeong, D.-H., De Paoli, E., Park, S., Rosen, B. D., Li, Y., Gonzalez, A. J.,Yan, Z., Kitto, S. L., Grusak, M. A. et al. (2011). MicroRNAs as master regulatorsof the plant NB-LRR defense gene family via the production of phased, trans-acting siRNAs. Genes Dev. 25, 2540-2553.

Zhai, J., Zhang, H., Arikit, S., Huang, K., Nan, G.-L., Walbot, V. andMeyers, B. C.(2015). Spatiotemporally dynamic, cell-type–dependent premeiotic and meioticphasiRNAs in maize anthers. Proc. Natl. Acad. Sci. USA 112, 3146-3151.

Zhang, J., Pawlowski, W. P. and Han, F. (2013). Centromere pairing in earlymeiotic prophase requires active centromeres and precedes installation of thesynaptonemal complex in maize. Plant Cell 25, 3900-3909.

Zilberman, D., Coleman-Derr, D., Ballinger, T. and Henikoff, S. (2008). HistoneH2A.Z and DNA methylation are mutually antagonistic chromatin marks. Nature456, 125-129.

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RESEARCH ARTICLE Journal of Cell Science (2016) 129, 3553-3561 doi:10.1242/jcs.184937

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