IntroductionMeiosis in plants is the transition between the diploidsporophyte and haploid gametophyte generations which inlower plants exist as distinct free-living organisms. During thereproductive phase of development in flowering plants,specialized meiotic cells, the sporocytes, are formed in theanthers and ovules. In plants there is no separate germline asin animals, and the sporocytes are derived from the L2 layerof the meristem (Dawe and Freeling, 1990). The understandingof sporocyte specification and meiosis in plants has advancedsignificantly in recent years (reviewed by Yang andSundaresan, 2000; Bhatt et al., 2001). Genetic and molecularapproaches in Arabidopsisand maize have led to the isolationof mutants that are altered in sporocyte development or meiosis(Curtis and Doyle, 1991; McCormick, 1993; Chaudhury et al.,1994; Sheridan et al., 1996; Golubovskaya et al., 1997; Rosset al., 1997; Taylor et al., 1998; Sanders et al., 1999) as wellas to the identification of several genes that are required fordevelopment of the sporocyte or for meiosis. Many of the plantgenes that are responsible for basic components of the meioticmachinery that is common to all eukaryotes such aschromosome cohesion, synapsis, recombination andchromosome segregation show conservation with genes inyeast and other organisms (Klimyuk et al., 1997; Couteau et
al., 1999; Bai et al., 1999; Yang et al., 1999a; Grelon et al.,2001; Armstrong et al., 2002; Chen et al., 2002). Others appearto be unique to plants and do not have obvious homologues inyeast or animals (Byzova et al., 1999; Mercier et al., 2001;Azumi et al., 2002). The pathway leading to sporocytespecification is found only in plants and the two genes that havebeen identified in this pathway, SPOROCYTELESS/NOZZLEwhich encodes a nuclear protein related to MADS boxtranscription factors and is required for the initiation ofsporogenesis (Yang et al., 1999b; Schiefthaler et al., 1999) andEXCESS MICROSPOROCYTES1/EXTRA SPOROGENOUSCELLS, which encodes a putative LRR receptor kinaserequired for tapetum formation and control of male meiocytenumber (Canales et al., 2002; Zhao et al., 2002), do not havecorresponding homologues in yeast or animals.
The control of meiotic cell cycle progression in yeast isdependent upon checkpoints that monitor morphogenesis ofthe chromosomes during meiosis. Mutations that affectsynapsis and recombination lead to arrest of meioticprogression at the pachytene stage. The identification andanalysis of extragenic suppressors of pachytene arrest has ledto an understanding of the pachytene checkpoint (Roeder andBailis, 2000). The pachytene checkpoint has also been foundin animals (Edelmann et al., 1996), but remains to be identified
Progression through the meiotic cell cycle is an essentialpart of the developmental program of sporogenesis inplants. The duetmutant of Arabidopsiswas identified as amale sterile mutant that lacked pollen and underwent anaberrant male meiosis. Male meiocyte division resulted inthe formation of two cells instead of a normal tetrad. Inwild type, male meiosis extends across two successive budpositions in an inflorescence whereas in duet, meiotic stagescovered three to five bud positions indicating defectiveprogression. Normal microspores were absent in themutant and the products of the aberrant meiosis were uni-to tri-nucleate cells that later degenerated, resulting inanthers containing largely empty locules. Defects in malemeiotic chromosome organization were observed starting
from diplotene and extending to subsequent stages ofmeiosis. There was an accumulation of meiotic structuresat metaphase 1, suggesting an arrest in cell cycleprogression. Double mutant analysis revealed interactionwith dyad, a mutation causing chromosome cohesionduring female meiosis. Cloning and molecular analysis ofDUET indicated that it potentially encodes a PHD-fingerprotein and shows specific expression in male meiocytes.Taken together these data suggest that DUET is requiredfor male meiotic chromosome organization andprogression.
Key words: Chromatin, Male sterility, Checkpoint, Cohesion,Synapsis
Summary
The DUET gene is necessary for chromosome organization andprogression during male meiosis in Arabidopsis and encodes aPHD finger proteinThamalampudi Venkata Reddy 1,*, Jagreet Kaur 1,*, Bhavna Agashe 1,*, Venkatesan Sundaresan 2 andImran Siddiqi 1,†
1Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad – 500007, India2Department of Plant Biology and Agronomy, Life Sciences Addition 1002, University of California, Davis, CA95616, USA*These authors contributed equally to this work†Author for correspondence (e-mail: [email protected])
in plants (Couteau et al., 1999; Garcia et al., 2003). In additionto the pachytene checkpoint, which is specific to meiosis,chromosomal checkpoints that act during mitosis have alsobeen shown to function in meiosis (Lydall et al., 1996). Thisis consistent with the view that meiosis is a specialized cellcycle based on the mitotic cell cycle (reviewed by Lee andAmon, 2001).
There is limited information on the control of meioticprogression in plants. Arabidopsismutants that affect meioticprogression have been described (Siddiqi et al., 2000; Magnardet al., 2001) and several genes have also been characterized atthe molecular level. The ASK1gene is required for homologueseparation during male meiosis (Yang et al., 1999a). TheDYAD/SWI1 gene is required for meiotic chromosomeorganization and meiotic progression (Mercier et al., 2001;Agashe et al., 2002). The Swi1 protein has been shown to beexpressed in G1 and S phase of meiosis, and required for axialelement formation and initiation of recombination (Mercier etal., 2003). The SOLO DANCERSgene encodes a cyclin-likeprotein that is required for synapsis during meiosis (Azumi etal., 2002). Several of these genes appear to be associated withchanges in chromosome organization and dynamics, howeverthe mechanism by which these changes are related to theprogression of meiosis in plants remains unknown.
We describe the isolation and characterization of the duetmutant of Arabidopsisand molecular analysis of the DUETgene. We show that the duetmutant is defective in chromosomeorganization and progression during male meiosis. duetalsoshows a synergistic genetic interaction with the dyadmutant.The DUET gene encodes a plant homeo domain (PHD)-fingerprotein that is expressed in male meiocytes.
Materials and methodsPlant material and growth conditions Arabidopsisgrowth conditions were as described earlier (Siddiqi etal., 2000).
Light microscopyDevelopmental analysis of whole-mount anthers and ovules was doneafter clearing inflorescences in methyl benzoate as describedpreviously (Siddiqi et al., 2000). The anthers were dissected on a slideunder a stereo dissecting microscope, mounted with a coverslip andobserved on a Zeiss Axioplan imaging 2 microscope under DIC opticsusing ×40 and ×100 oil immersion objectives. Photographs werecaptured on a CCD camera (Axiocam, Carl Zeiss) using theAxiovision program (version 3.1). For a stage-wise comparison ofpollen development in the wild type and the duetmutant, the ovulesfrom the corresponding pistils were staged and used as a reference forpollen developmental stage. Scoring of ovule stages was based onexamination of all the ovules in a pistil. The pistil was mounted eitherintact or after separating the two carpels, and ovules were viewedthrough the carpel wall. At the early stages of ovule developmentcorresponding to pollen meiosis and gametogenesis, all ovules in apistil were at the same stage. Anthers were also bissected anddeveloping pollen was observed in optical sections taken through theanther wall. Pollen development in anthers was also found to besynchronous. The correspondence between ovule and pollen stagesacross different inflorescences was consistent. For plastic sections theinflorescences were fixed in 2% paraformaldehyde, 0.5%glutaraldehyde in 1× PBS overnight at 4°C. The inflorescences werethen washed with 1× PBS, treated with osmium tetroxide for 3 hours,followed by dehydration in a graded ethanol series (30%, 50%, 70%,
90%, 100% ×5) for 15 minutes each. Ethanol was replaced bypropylene oxide and the samples infiltrated with Araldite resinfollowed by embedding and curing for 3 days at 60°C. 2 µm sectionswere cut using a Reichert Ultracut E microtome. Sections were stainedwith 1% Toluidine Blue in 1% borax for 2-5 minutes and mounted inthe Araldite resin. Bright-field photographs of anther cross sectionswere taken using a Zeiss Axioplan imaging 2 microscope.Photographs were taken on Kodak Supra 100 ISO film using a bluefilter. All the photographs were edited using Adobe Photoshop 5.
cDNA isolation and expression analysisPoly(A)+ mRNA was isolated from young flower buds and leavesusing the PolyA tract mRNA isolation kit (Promega) according to themanufacturer’s protocol with an inclusion of RQ1 DNAase (Promega)treatment before mRNA precipitation. Pistils were dissected andstored in RNA Later (Ambion) and total RNA was isolated usingTrizol (Gibco BRL Life Sciences). The cDNA synthesis was carriedout with 150 ng of poly(A)+ RNA or 5 µg total RNA in the case ofdissected pistils, using the Superscript choice system for cDNAsynthesis (Gibco-BRL Life Sciences). Amplification of DUETcDNAwas carried out by using the gene-specific primers SETAF (5′-CGTCTCCATCGAAGCTAAAATC-3′) and SETR1 (5′-ATCTAC-AAAGTTTGATCCAAAAACTGAC-3′ ). The amplified cDNA wascloned into a pMOSBlue Vector (Amersham Pharmacia Biotech) andsequenced. DUET expression was examined by PCR using cDNAprepared from poly(A)+ mRNA as template and the primers SETF1(5′-CCAATCATCGAAACGTGTCGTAAGAG-3′) and SETR13 (5′-TCCGAGACTATTACAAAGCCGATCC-3′). GAPC expression wasdetected using the primers GAPC1 (5′-CTTGAAGGGTGGTGCC-AAGAAGG-3′) and GAPC2 (5′-CCTGTTGTCGCCAACGAAGT-CAG-3′). DYAD cDNA was amplified with the gene specific primers3RR1 (5′-CATGGAAGAGACCTTACCAGTTCACATCA-3′) andg2.2r7 (5′-AGCTAGTGATTATTGGAGAAACCTTGCG-3′).
In situ hybridization was carried out as described previously(Siddiqi et al., 2000). A 1.26 kb coding region of DUET cDNAlacking the PHD-finger domain was amplified using the primersSTMF1 (5′-GGTCATTTGGTATGTGTCAATGGTATGG-3′) andSTMR3 (5′-TCAATCTCAGTCACTACAAAATTTGACAAG-3′ ) andsubcloned into pGEM-T (Promega) for synthesis of RNA probes.
Molecular analysis of the duet mutant locusSouthern hybridization experiments using a Ds transposon-specificprobe derived from pWS31 (Sundaresan et al., 1995) was used toestablish copy number of the insertion. TAIL-PCR was used toamplify sequences flanking the transposon insertion (Parinov et al.,1999). The amplified product was sequenced. The site of insertion wasconfirmed by Southern analysis using as the probe a genomicfragment amplified using the primers Set3′(5′-TCTCGGAGCAA-GGTAATGGAG-3′) and R16 (5′-AAAGTTTGATCCAAAAACTG-ACTTTACAAA-3′ ) that were specific to At1g66170. To obtaingenomic sequences flanking the Ds element, Ds-specific primers fromboth the 5′and 3′ends, Ds5-2 (5′-CGTTCCGTTTTCGTTTTTTACC-3′) and Ds3-2 (5′-CCGGTATATCCCGTTTTCG-3′) were used incombination with gene-specific primers set5′(5′-GTAACTCAC-GTTCACGCGTTA-3′) and Set3′respectively.
Double mutant analysisduet(Ler) as the female parent was crossed to dyad(Col) as the maleparent. F1 and F2 were selected on MS plates containing kanamycinat 50 µg/ml. 96 F2 plants were transferred to soil; 49 of these weresterile. Plants homozygous for dyad were identified by examinationof ovules as described previously (Agashe et al., 2002). The presenceof the insertion and wild-type alleles at the duetlocus was examinedby PCR using a gene-specific primer R12 (5′-ATTCTCTGAACT-TGGAAACTCATACTTTGG-3′) in combination with Ds5-2 for theinsertion allele, and two gene-specific primers Dhf4 (5′-GTA-GTAGATGGCCTGTGAGGAGACTAAT-3′) and STR5 (5′-TCTGC-
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AAATTCTTCACAGCAATTCG-3′) on either side of the insertionsite for the wild-type allele. DNA was isolated from one or two rosetteleaves using the Nucleon Phytopure kit (Amersham). Pollen viabilitywas measured using fluorescein diacetate according to the method ofHeslop-Harrison and Heslop-Harrison (Heslop-Harrison and Heslop-Harrison, 1970). To determine the effect of duet/dueton femalefertility 10 plants that were homozygous for the duetallele and hadnormal ovules (dyad/+or +/+) were crossed as the female parent todyadas the male parent. All the siliques elongated and showed greaterthan 50% seed set after crossing, indicating no significant defect infemale fertility.
Fluorescence microscopy of meiotic chromosomesAnalysis of meiotic chromosome spreads of male meiocytes wascarried out according to the method of Ross et al. (Ross et al., 1996)with minor modifications (Agashe et al., 2002). Chromosomes wereobserved on a Zeiss Axioplan2 imaging microscope using a 365 nmexcitation, 420 nm long-pass emission filter and a 100× oil objective.The photographs were captured on an Axiocam CCD camera (CarlZeiss) using the Axiovision program (version 3.1) and were editedwith Adobe Photoshop 5.0.
ResultsThe duet mutant is male sterile because of a Dstransposon insertionThe duet mutant was identified as a sterile line, SET8286,carrying a Ds enhancer trap insertion (Parinov et al., 1999). Anexamination of flowers showed that the mutant anthers lackedpollen. To determine the reason for sterility, reciprocal crosseswere carried out to wild type. The results indicated that themutant was male sterile and that female fertility was normal(Table 1). The anther filament did not fully elongate and remainedbelow the level of the stigma (Fig. 1E), a feature that has alsobeen observed in other male sterile lines (Sanders et al., 1999).
Southern analysis indicated the presence of a single Dsinsertion in the duetmutant (data not shown). DNA flankingthe insertion site was isolated using TAIL PCR (Liu et al.,1995) and sequenced. The sequence indicated the insertion tobe within the putative gene At1g66170. Southern analysisusing a wild-type genomic clone as a probe confirmed the siteof insertion (data not shown). To test whether the phenotype
was caused by a transposon insertion, the mutant was crossedas the female parent to a line that was homozygous for aninsertion carrying the Ac transposase expressed under controlof the 35S promoter (Sundaresan et al., 1995). The F1 plantswere fertile and the segregation of the mutant phenotype in theF2 was consistent with it being a single gene recessive trait(Table 2). Out of a total of 51 sterile F2 plants, 17 were foundto contain a revertant sector bearing one or more elongatedsiliques (Fig. 1C). The reversion phenotype was confirmed fornine independent sectors in the F3 generation by the presenceof fertile plants among the progeny. Multiple plant progenyfrom seven of the nine sectors were tested by PCR for both thepresence and absence of the Ds at the original site of insertionin At1g66170. Three out of the seven sectors lacked Dsindicating that excision had occurred from both chromosomesand was associated with the reversion to fertility. GenomicDNA flanking the site of excision was amplified by PCR andsequenced for multiple plants from each of the three sectorslacking Ds (Fig. 1F). Two out of the three sectors containedone wild-type and one mutant allele carrying the same 7 bpfootprint at the excision site. The third sector contained one
Table 2. Segregation of the duetphenotypeWild type:mutant
*Mutant phenotype results from a single gene recessive mutation.†Two unlinked mutations are responsible for the mutant phenotype.
Table 1. The duet mutation causes male sterilityNumber of
Female parent Male parent seeds per silique
Wild type duet 0duet Wild type 25.8±2.8Wild type Wild type 27.2±1.8
Reciprocal crosses between duetand wild type, both in the Ler backgroundwere conducted to measure the seed yield. The results represent the mean andstandard deviation from a minimum of eight crosses.
Fig. 1.Wild-type (Ler), duetand revertant plants. (A) Wild-typeplant showing normal elongating siliques. (B) duetmutant plant withshort siliques. (C) Mutant plant with revertant sector showingelongating silique (arrow). (D) Wild-type flower with long antherfilaments and plentiful pollen. (E) duetflower with short filamentsand anthers lacking pollen. (F) Wild-type cDNA and derived aminoacid sequence of DUETnear the site of the Ds insertion. The duetmutant sequence shows a 8 bp duplication (bold underlined). Thetwo excision alleles have a 7 and 8 bp footprint respectively (bold).
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wild-type and two mutant alleles carrying the 7 bp and an 8 bpfootprint, respectively. We found one plant that washeteroallelic for the two mutant alleles and was sterile. This isconsistent with the development of the pollen that is carryingthe mutant excision allele being associated with a multipleevent that gave rise to the wild-type allele together with themutant allele in the sporophyte. These data show that the duetmutant phenotype is due to the Ds transposon insertion.
Microsporogenesis is defective in duetTo examine the basis of male sterility and lack of pollen in theduetmutant we carried out a stagewise analysis of anther andpollen development by examining cleared anthers as well asplastic sections. Early stages of pollen developmentcorresponding to anther stage 5 (Sanders et al., 1999) werenormal (Fig. 2). The endothecium and internal layers surroundingthe microsporocyte were indistinguishable from wild type as wasthe appearance of the microsporocyte prior to meiosis. Wecompared the time course of pollen development in wild type andduet by examination of cleared anthers and ovules fromsuccessive buds of inflorescences. Meiocyte division wasprolonged (Table 3) and extended across three to four successivebud positions in the mutant inflorescence whereas in wild type itcovered no more than two successive bud positions. The major
product of division of the meiocyte was a pair of cells instead ofa normal tetrad that is observed in wild type (Fig. 3). Tetrads wereseen only rarely (Fig. 2E). The pair of cells separated and formedenlarged cells that were uninucleate (Fig. 2H, Fig. 3I).Subsequently nuclear division took place and cells were seen tocontain one to three nuclei. Normal microspores were notobserved and the exine was not formed. The enlarged cells laterdegenerated, leaving an empty locule (Fig. 2I). Mutant meiocytesat the time of division had a prominent callose wall (Fig. 2C)
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Fig. 2.Anther and pollen development in wild type and duet. Plastic cross-sections of anthers. (A,D,G) Wild type; (B,C,E,F,H,I) duet.(A,B) Anthers with pollen mother cells at stage 5, all the layers of the anther are present in both genotypes. (C) duetmicrospore mother cell atmeiosis; pollen mother cells (PMCs) surrounded by a layer of callose. (D) Stage 7 anther showing tetrads held together by a layer of callose.(E) duetanther with dyads, triads, tetrads: products of a defective meiosis. (F) Products of aberrant meiosis separate out, enlarge and undergonuclear division (arrowheads). (G) Stage 12 anther containing mature pollen. (H) Enlarged microspore-like cells (arrowheads) with a singlenucleus and lacking exine. (I) Late stage anther showing empty locules. CL, callose layer; E, epidermis; En, endothecium; Ml, middle layer;PMC, pollen mother cell; T, tapetum; Tds, tetrads. Scale bars: 25 µm (A,B,C,F,G,I) and 12.5 µm (D,H,E).
Table 3. Pollen development in wild type and duetrelativeto ovule stages
Three inflorescences each for wild type and duetwere analyzed. Ovulestages are according to Schneitz et al., (Schneitz et al., 1995).
5979Arabidopsis meiotic chromosome organization
whereas the pair of cells formed by division of the meiocytelacked a thick callose wall and separated into single cells. Thisprobably corresponds to loss of the callose wall from tetrads atthe time of microspore release in wild type. The stage at whichthe pair of cells separated in the duetmutant corresponded toovule stage 2-4 which is later than the time of microspore releasein wild type (ovule stage 2-2; Table 3). Taken together these dataindicate that duetis defective in male meiotic progression.
DUET potentially encodes a PHD finger proteinBased on the predicted cDNA sequence of At1g66170, PCRprimers were designed to span the coding region. RT-PCR wascarried out using cDNA prepared from inflorescence mRNA.The sequence of the cDNA obtained (GenBank accession no.AY305007) indicated the presence of 3 exons potentiallyencoding a protein of 704 amino acids and having a mass of80.8 kDa (Fig. 4). The Ds insertion is in the third exon at aposition corresponding to aa 550 and hence likely to create anull allele. The putative protein was similar to that of the AGIannotation but contained an additional exon that was notpresent in the annotated sequence. A potential nuclearlocalization signal was found at amino acid residues 10-15.
A homology search using BLASTP 2.2.6 revealed thepresence of a PHD finger domain towards the C-terminal
portion of the predicted DUET protein, from aa 609-656. ThePHD domain is a modified zinc finger thought to be involvedin transcriptional regulation and chromatin organization(Aasland et al., 1995). The closest known protein to DUET wasthe MALE STERILITY 1 (MS1) protein (expectation value,E=4×10–83), which has been proposed to be a transcriptionalregulator of male gametogenesis and also contains a C-terminalPHD finger domain (Wilson et al., 2001). In addition, twopredicted genes from Arabidopsis(At1g33420, and At2g01810)and one from rice (GenBank ID AC090882) showed similarityto DUET. All three predicted proteins have a PHD domaintowards the C-terminal end. All five genes show similaritythroughout their length and hence constitute a gene family.Apart from the above genes, which showed strong similarity toDUET, weak similarity was detected to other Arabidopsisgenesincluding SWI1/DYAD(E=1×10–4). The SWI1/DYADgene hasbeen shown to be required for chromosome cohesion in meiosisand for female meiotic progression (Mercier et al., 2001;Agashe et al., 2002). The SWI1/DYADhomology residestowards the middle portion of the gene from aa 309 to 395.
DUET is expressed in male meiocytesExpression of the DUETgene was examined using RT-PCR.The presence of the transcript could be detected in the
Fig. 3.Stages of male meiosis and pollen development in wild type and duet. Optical sections of cleared anthers viewed under DIC optics.(A-D) Wild type; (E-K) duet. (A,E) Stage 5 anthers with pollen mother cells (arrowheads). (B) Stage 7 anther containing tetrads (arrowhead).(C) Microspores released from the tetrad generate an exine wall and become vacuolated (arrowhead). (D) Mature pollen. (F,G) Dyads(arrowhead in F) formed after a defective meiosis. (H,I) Cells of the dyad separate and enlarge (arrowhead in H). (J,K) The microspore-likecells released from the dyad enlarge and undergo nuclear division to form 2-3 nucleate cells (arrowhead in J), which later degenerate. Scalebars: 12.5 µm.
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inflorescence but not in leaves (Fig. 4C). The mutant did notshow expression under these conditions and hence is likely tobe a null allele. We have previously demonstrated the presenceof DYAD mRNA specifically in male and female meiocytes(Agashe et al., 2001). To test whether DUETexpression is malespecific, we compared the levels of DUETmessage with thatof DYADin dissected pistils using RT-PCR. We did not observeexpression of DUETwhereas DYADexpression could bedetected under the same conditions (Fig. 4D). To determine theDUET expression pattern in the inflorescence at the cellularlevel we carried out RNA in-situ hybridization using antisenseRNA complementary to DUET cDNA excluding the PHDdomain as a probe. Expression was first observed insporogenous cells at late anther stage 4 (Sanders et al., 1999)(Fig. 5A), reached a maximum in male meiocytes at antherstage 5, prior to meiosis (Fig. 5C). Lower expression was
observed at anther stage 6, during meiosis (Fig. 5D) andsubsequently declined. A weak signal could be seen in veryyoung pistils in the placenta, corresponding to the presumptivesite of ovule initiation (Fig. 5B,C). We did not see expressionin female meiocytes or in ovules (Fig. 5E,F). The lack offemale meiocyte expression as well as a phenotype in whatappears to be a null allele, would suggest that DUET does nothave a function in the female meiocyte. We also examinedGUS reporter gene expression in plants hemizygous andhomozygous for the insertion but did not observe expressionin anthers at stages 5 to 6.
Aberrant meiotic chromosome organization andmetaphase 1 arrest in the duet mutantMeiotic chromosome stages in wild type and duet wereanalyzed in spread chromosome preparations from anthers of
Development 130 (24) Research article
Fig. 4. (A) Schematic representation of the DUETgene. The exons are indicated with black boxes.Arrows indicate the primers used for cDNA isolationand expression analysis. Coordinates are with respectto BAC F15E12. (B) The predicted sequence of DUETprotein. The putative nuclear localization signal is inbold. The region showing homology to DYAD isunderlined and the PHD-finger domain is boxed. Theinverted triangle after amino acid 550 indicates the Dstransposon insertion site. (C) Analysis of DUETexpression by RT-PCR. Expression of the DUETgenewas examined in wild type (Wt) leaves (Lea),inflorescence (Inf) and duetmutant inflorescence byamplifying the cDNA synthesized from poly(A)+
mRNA. The shift in the size of the amplicon can beobserved when genomic DNA (gen) was used astemplate. The constitutive GAPC gene was used as thenormalization control. (D) Comparison of DUET andDYAD cDNA in pistils.
Fig. 5.Expression of DUETin male meiocytes: RNA insitu hybridization of DUETantisense RNA to sections offlower buds. (A) Expressionis first seen in sporogenouscells at anther stage 4.(B,C) Maximal expression isseen in microsporocytes atanther stage 5. (D) Antherstage 6, meiotic cells.(E,F) No expression is seenin the female meiocyte atstage 2-3 corresponding topre-meiosis. (E) Antisense;(F) sense control. Scale bars:50 µm.
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meiotic stage buds using the method of Ross et al. (Ross et al.,1996). Chromosome development in duet appeared normalthrough early stages of meiotic prophase up to pachytene.Abnormalities first became noticeable at diplotene when duetchromosomes started to appear somewhat diffuse in comparisonto wild type (Fig. 6D,N). During diplotene and diakinesis, duetchromosomes were observed to desynapse along much of theirlength including centromeres, to a greater extent than wasobserved for wild type (Fig. 6N-P). At diakinesis, duetchromosomes were irregular and less distinct than wild type
(Fig. 6P,Q). Metaphase 1 in duet varied from nearly normal(Fig. 6R) to a diffuse mass where individual chromosomescould not be clearly distinguished (Fig. 6T). In wild type, malemeiotic stages extended across no more than two successive budpositions in an inflorescence whereas in duet, meiotic stagescovered three to five bud positions. The total number of meioticstages in an inflorescence was also several times greater for duetthan for wild type. This could be approximately gauged fromthe fact that 364 meiotic stages were counted from sixinflorescences in the case of wild type and 602 from three
Fig. 6.Chromosome analysis in spreads of malemeiocytes in wild type and duet. (A-J) Wild type;(K-W) duet. (A,K) Chromosomes first become visibleas elongated strands during leptotene. (B,L) Synapsistakes place during zygotene. (C,M) Synapsis iscomplete at pachytene and chromosomes have a shorterand thicker appearance. (D) Diplotene stage, whenbivalents have undergone partial decondensation.(E) Late diakinesis showing five pairs of chromosomes
with chiasmata at their ends. (F) Metaphase I stage showing five condensed bivalents arranged on a metaphase plate. (G) Telophase I: fivechromosomes at each end are separated by an organelle band. (H) Metaphase II. (I) Anaphase II. (G-I) Arrows indicate the densely compactedorganelle band. (J) Telophase II where four groups of five chromosomes each have separated. (N) First apparent visible defect in duetatdiplotene. Chromosomes start to look diffuse and two bivalents have undergone partial desynapsis (arrowheads). (O) A more severe form ofdesynapsis can be observed in the majority of bivalents. (P,Q) Disorganized diakinesis in duet with diffuse chromosomes including thecentromeric region. (R,S,T) Metaphase I. (U) Anaphase I. (V) Defective anaphase I stage with fragmented chromosome and laggards.(W) Telophase I. The organelle band is absent. Scale bars: 12.5 µm.
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inflorescences for duet. In most of the buds there was an excessof metaphase 1 stages in the duetmutant (Fig. 7), whereas thesewere relatively few in wild type. The fraction of anaphase 1meiocytes was also greater for duet. A clear feature of duetmeiocytes was the absence of a band of organelles characteristicof wild type and prominently visible starting at telophase 1 inthe central portion of the meiocyte (Fig. 6G). Instead organelleswere dispersed throughout the cytoplasmic region of the malemeiocyte in the case of duet(Fig. 6W).
These data confirm and extend the analysis of male meiocytedivision in the duetmutant. The results indicate that duethasa defect in chromosome organization during male meiosis andis also defective in male meiotic progression. Many of themeiocytes arrest at metaphase 1.
The duet mutation interacts with dyadThe duetmutant phenotype is similar to what is seen for femalemeiosis in the dyadmutant (Siddiqi et al., 2000). In each casea pair of cells is formed after meiosis instead of a tetrad. Thepair of cells do not develop further into functional gametes. Forboth duet and dyad, the two-cell phenotype correlates withdefective progression through meiosis and the underlyingcause appears to be altered chromosome organization in bothcases. The DYAD/SWI1gene is required for female meioticprogression, and for chromosome cohesion in male and femalemeiosis (Mercier et al., 2001; Agashe et al., 2002). However,the dyad mutant allele is female specific and shows normalpollen development and male fertility.
To test if both mutants are affected in a related aspect ofchromosome organization during meiosis, we intercrossed duetand dyad as female and male parents respectively to test forgenetic interaction. The F1 were fully fertile and showednormal pollen and embryo sac development (data not shown).F2 plants were genotyped with respect to the alleles present atthe duet locus (duet/duet, duet/+, and +/+) and at the dyadlocus (dyad/dyad, and +/–), and various dose combinations ofduet and dyad were examined for evidence of geneticinteraction.
The duet dyaddouble mutant flowers lacked viable pollen.Stagewise analysis of pollen development in cleared anthers(Fig. 8) showed meiocytes followed by enlarged uninucleatecells that underwent further enlargement to produce binucleatecells. We did not observe clear cytological evidence of meioticdivisions. At a later stage anthers contained irregular enlargedcells containing multiple nuclei and surrounded by an irregularcell wall that resembled the exine. The exine-like structure wasnot observed in the duetsingle mutant. Buds from plants thatwere duet/+ dyad/dyadshowed reduced numbers of pollengrains compared to the corresponding single mutants. Anthersfrom freshly opened flowers were dissected and tested forpollen viability. The mean pollen viability for duet/+dyad/dyadwas 34±12% whereas for plants that were duet/++/– the pollen viability was 75±13%. The total number ofpollen grains was also lower for the interaction genotype. Arough indication of this could be obtained from the totalamount of pollen on the slide. For the duet/+ dyad/dyadgenotype the mean count of total pollen grains per flower was131±50 whereas for sibling plants that were duet/+ +/– themean pollen count was 326±171. Each individual genotypeduet/+ and dyad/dyadproduced numbers of viable pollen thatwere comparable to wild type (Siddiqi et al., 2000; data notshown). Analysis of pollen development in cleared anthersshowed that male meiocytes in duet/+ dyad/dyadplantsunderwent an aberrant division to produce mostly dyads andtriads, as well as some tetrads (Fig. 8). The majority of sporesproduced were defective and formed enlarged cells containing1-3 nuclei and that subsequently degenerated. The phenotypetherefore broadly resembled that of homozygous duet plantsthough it was less severe.
Examination of meiotic chromosome behavior revealeddifferences in duet/+ dyad/dyadplants from that observed inhomozygous duetplants. The early stages of meiotic prophasewere detected though aberrant chromosome morphology wasapparent at the pachytene stage (Fig. 9B). Diakinesis wasvariable with some being nearly normal and having all or amajority of the chromosomes retaining their bivalent structure
(Fig. 9C). In others, many of the chromosomesdissociated into univalents (Fig. 9D). A moreextreme phenotype observed at diakinesis wasthe loss of sister chromatid cohesion resulting indissociation of chromosomes into isolatedchromatids (Fig. 9E). The ensuing meiosis 1divisions spanned the range from a normalreductional division (these were relatively rareand not observed, but their existence could beinferred from the production of tetrads and viablepollen) to an equational mitotic-like division inwhich univalents separated into sister chromatids(Fig. 9G,H), to an unequal division arising fromrandom segregation of univalents and singlechromatids (Fig. 9I,J). We did not observe a highproportion of metaphase 1 arrest whichcorrelated with a reduction in the number of cellpairs that were seen compared to the duetsinglemutant. The homozygous double mutantcombination was also examined. Here toobivalents were observed to separate intounivalents at diakinesis (Fig. 9L) and in somemeiocytes chromosome cohesion was lost
Development 130 (24) Research article
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30
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L Z P DP DI M -I A-I T-I M-II A-II T-II TD
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Fig. 7. Quantitation of different stages of male meiosis in wild type and duet.(White bars) Wild type; (Black bars) duet; leptotene (L), zygotene (Z), pachytene(P), diplotene (DP), diakinesis (DI), metaphase I (M-I), anaphase I (A-I), telophaseI (T-I), metaphase II (M II), anaphase II (A-II), telophase (T-II) and tetrads (TD).Note the large increase in the proportion of metaphase I cells in duet.
5983Arabidopsis meiotic chromosome organization
resulting in isolated chromatids (Fig. 9M). At prometaphasebetween eight and ten thick and diffuse chromosomes couldgenerally be counted, most probably they were a mixture ofunivalents and bivalents (Fig. 9N). Male meiotic chromosomesin the dyadmutant showed no apparent differences from wildtype. In the case of duet/+also, the vast majority of meioticstructures observed were normal, in agreement with theessentially wild-type levels of viable pollen that were measuredin freshly opened flowers. However, in one inflorescence outof four examined we observed a single bud in which a minority(<10%) of meioses were aberrant. Chromosomes in this budshowed defects in diakinesis that were similar to thoseobserved in homozygous duet, and some meiocytes werearrested at metaphase 1 (data not shown). Separatedmicrospores were the major species present in this budindicating that the majority of meiocytes had completedmeiosis, and that the minority, showing defective meioticstructures, were defective in progression.
The increased severity of defects in pollen developmentresulting from defects in male meiosis that were observed induet/+ dyad/dyadplants, coupled with the loss of chromosomecohesion provide evidence of a synergistic interaction betweenthe duetand dyadmutant alleles.
To test for possible effects of dueton female meiosis weexamined cleared ovules as well as female meiosis in duet dyaddouble mutant plants (Fig. 9U-W). The ovule phenotype wasidentical to that of the dyadmutant. In both cases the majorityof ovules showed a single division meiosis and the presence oftwo enlarged cells in place of an embryo sac. Cytogeneticanalysis of female meiosis in duet dyadplants also indicatedthat chromosome behavior was the same as that describedearlier for dyad,which was shown to undergo an equationalmeiosis 1 division (Agashe et al., 2002). No additional effectswere observed from the presence of the duetmutant allele ina dyad background, on the integrity or appearance ofchromosomes. We also examined female fertility of duet/duet
Fig. 8. Interactions between duetand dyadduring male meiosis and pollen development. Optical sections of cleared anthers viewed under DICoptics. (A-D) duet dyaddouble mutant; (E-L) duet/+ dyad/dyad.(A,E) Normal-looking meiocytes (arrowheads). (B) Defective microsporeslacking exine (arrowhead). (C) Binucleate spores. (D) Late stage defective binucleate and multinucleate spores (arrow and arrowhead,respectively) surrounded by an uneven wall resembling the exine; not observed in the duetsingle mutant. (F) Dyad (arrowhead). (G) Undividedtriads (arrowhead). (H) Round spores containing 1-3 nuclei. (I) Normal-looking tetrads (arrowhead). (J) Normal-looking developing pollen.(K) Abnormal multinucleate structures undergoing shrinkage (arrowhead). (L) Shrunken pollen. Scale bar: 12.5 µm.
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dyad/+ plants and did not observe any reduction in fertility(data not shown). We therefore did not find any evidence tosupport a role for DUETin female meiosis.
DiscussionProgression through the meiotic cell cycle requiresorchestration of a set of events that includes the assembly ofchromosomes for reductional division, pairing of homologouschromosomes, recombination and segregation of homologues
to opposite poles of the meiosis 1 spindle (reviewed by Dawe,1998). Defects in meiotic chromosome organization can resultin delayed progression through the meiotic cell cycle. The roleof chromosomal checkpoints in monitoring and ordering thephases of the mitotic and meiotic cell cycle is well documentedin yeast and animals (Roeder and Bailis, 2000; Handel et al.,1999). In plants there is little information on the control ofmeiotic progression and the role of chromosomal checkpointsin meiosis (Garcia et al., 2003). Mutations in the yeast DMC1gene cause arrest as a result of activation of the pachytene
Development 130 (24) Research article
Fig. 9. Interactions between duetand dyad: meioticchromosome stages. (A-J) duet/+ dyad/dyad.(K-N,W) duet dyaddouble mutant. (O-Q) duet/+heterozygote. (R-T,V) dyadmutant. (A) Earlyzygotene. (B) Pachytene stage showing thickened butirregular chromosomes. (C) Diakinesis. Five bivalentsare visible. (D) Aberrant diakinesis in whichchromosomes have desynapsed to form ten univalents.(E) Extreme diakinesis in which both synapsis and
sister chromatid cohesion have been lost to yield single chromatids. (F) Early anaphase 1 undergoing mixed segregation in which bothunivalents and bivalents are involved. (G) Late anaphase 1 showing approximately equal separation of chromosomes. Eight to ten chromosomesare present at each pole indicating an equal division. (H) Telophase 1. Equal division. Ten chromosomes are present at each pole. (I) Telophase1. Unequal division. (J) Dyad formed after unequal division. (K) Zygotene. (L) Diakinesis involving 2 bivalents and 6 univalents. (M) Extremediakinesis containing mostly single chromatids. (N) Prometaphase 1 having eight to ten thick diffuse chromosomes. (O,R) Normal diakinesis.(P,S) Metaphase 1. (Q,T) Telophase 1. (U,V) Cleared ovules of dyad(U) and duet dyad(V). (W) Metaphase 1. Scale bars: 12.5 µm (A-T,W)and 25 µm (U,V).
5985Arabidopsis meiotic chromosome organization
checkpoint (Bishop et al., 1992; Lydall et al., 1996). However,a mutation in AtDMC1, the Arabidopsishomolog of DMC1does not cause arrest. Instead it leads to random chromosomesegregation in meiosis and the production of defective spores(Couteau et al., 1999). Hence the control of meioticprogression in plants may differ from that in yeast.
The properties of the DUETgene described above indicatethat it is required for male meiotic chromosome organizationand suggest that in the absence of DUET function, malemeiocytes undergo defective progression through the meioticcell cycle. In the duetmutant, male meiocytes went through asingle division to produce a pair of cells instead of a normaltetrad. The defective spores produced did not completegametogenesis and degenerated. The phenotype resembled thatobserved in the dyadmutant of Arabidopsisin which themajority of female meiocytes divide singly to give a dyadfollowed by an arrest in further development of the femalegametophyte (Siddiqi et al., 2000). Single division meiosis hasalso been described for the spo12, spo13and slk19mutants ofyeast (Klapholz and Esposito, 1980; Zeng and Saunders, 2000;Kamieniecki et al., 2000).
The DUET gene was cloned by transposon tagging with aDs element and found to encode a putative PHD finger domainprotein. The gene is closely related to the MS1gene, which hasbeen proposed to be a transcriptional regulator of malegametogenesis in Arabidopsis(Wilson et al., 2001), to twoother putative Arabidopsisgenes, At1g33420 and At2g01810,and to a putative rice gene (GenBank ID AC090882). The PHDfinger is a modified zinc finger and is found in a number ofproteins that play a role in chromatin organization andtranscriptional regulation and include members of theTrithorax and Polycomb groups (reviewed by Aasland et al.,1995). In plants the PHD finger has been found in atranscriptional regulator of genes involved in defense againstpathogens (Korfhage et al., 1994) and in genes that are requiredfor reproductive development and fertility: the PICKLEgeneis required to prevent re-expression of embryonic traits ingerminated seedlings and encodes a CHD3 domain protein thathas been proposed to act as a regulator to promote the transitionfrom embryonic to postembryonic development (Ogas et al.,1999); overexpression of the SHLgene has been show to leadto early flowering and defective reproductive developmentwhereas antisense inhibition caused dwarfism and delayedgrowth (Mussig et al., 2000). Hence, in both plants and animalsPHD finger genes play a role in developmental transitions.Recently the PHD finger domain has also been found inproteins that act as E3 ubiquitin ligases (Cosoy and Ganem,2003). This latter class of PHD finger proteins are localized tothe membrane or cytoplasm. The PHD finger in DUET is moresimilar to that found in proteins that act as chromatinremodeling factors or transcriptional regulators. The DUETgene also showed limited similarity to SWI1/DYADa gene thathas been demonstrated to be required for chromosomecohesion during meiosis in Arabidopsis(Mercier et al., 2001;Agashe et al., 2002).
Expression of the DUETgene in the inflorescence appearedto be specific to the male meiocyte. Earliest expression wasdetected in stage 4 anthers at a time that corresponds to thepresence of sporogenous cells. Maximal expression was seenat anther stage 5 prior to meiosis, after which the signaldeclined. Since the expression and phenotype for DUET and
the related MS1gene appears to be sex specific (Wilson et al.,2001; Ito and Shinozaki, 2002), it is possible that along withthe other two closely related putative genes At1g33420 andAt2g01810, they define a family of transcriptional regulatorsthat function during male meiosis and gametogenesis.
The appearance of chromosomes during early stages ofmeiotic prophase in the duetmutant was normal up topachytene. Differences from wild type first became noticeableat diplotene with chromosomes appearing more irregular anddiffuse in the mutant. This would suggest that either the timingof DUET action is at the onset of diplotene or else that DUETmay act earlier, but its absence may lead to visible changes inchromosome structure only at a later stage when thesynaptonemal complex is disassembled and most of the sisterchromatid cohesion is removed at diplotene. The differencebetween duetand wild type was accentuated at diakinesis andculminated in a high proportion of meiocytes showing arrest atmetaphase 1. The appearance of chromosomes at metaphase 1was variable and distinctly different from wild type. Themetaphase 1 phenotype ranged from nearly normal lookingstructures to ones in which the chromosomes appeared as anirregular mass towards the center of the cell in which individualchromosomes could not be clearly distinguished. A distinctcharacteristic of the mutant meioses was the absence of theorganelle band which is a prominent feature found at the centerof the cell of wild-type meiocytes at telophase 1. Instead, theorganelles in the mutant meiocytes appeared more evenlydistributed throughout the cytoplasm. The reason for this couldbe that the localization of mitochondria and plastids in thedividing meiocyte requires expression of specific genes duringmeiosis, and that the expression of these genes is adverselyaffected in the mutant. Alternatively the cause could be a moregeneral disruption of cytoplasmic and cytoskeletal organizationthat affects organelle transport and localization in the meiocyte.The finding of defects in chromosome organization in meiosiscaused by disruption of the DUET gene extends the role ofPHD finger proteins to include functions that are specific tomeiosis.
Double mutant combinations of duet with dyad revealedgenetic interaction manifesting in defects during male meiosis.The effect was most apparent in plants that were duet/+dyad/dyad. Whereas the individual dyad/dyad and duet/+plants showed no or very weak effects on pollen development,the combination resulted in strong defects in male meiosis.Microsporocytes showed defective division patterns and theproducts were dyads, triads and tetrads. Most of themicrospores produced were defective and degenerated.However a minority of spores did develop into viable pollen.At the cellular level, the defective divisions of the malemeiocyte were similar to those in duet/duet, although lesssevere. The duet dyaddouble mutant showed a progressiondefect that was more severe than in the duetsingle mutant asthe male meiocytes failed to divide. At the chromosomal levelthere were additional defects that were not observed in eithersingle mutant. In duet/+ dyad/dyadmeiocytes, chromosomeslost synapsis or cohesion prior to meiosis 1 and segregatedunequally in many cases. Loss of cohesion was not observedin either the dyador duetsingle mutants. dyadis a female-specific allele and shows normal male meiosis and pollendevelopment (Siddiqi et al., 2000). The stronger allele swi1-2is male sterile and shows loss of sister chromatid cohesion
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during prophase of male meiosis (Mercier et al., 2001). Hencethe loss in sister chromatid cohesion as a result of theinteraction may be interpreted to mean that hemizygous duet/+enhances the dyadmutant phenotype. The genetic interactionbetween duetand dyadcould be specific and the genes may actat the same level of chromosome architecture. The presence ofthe PHD finger in DUET implicates it as functioning in thecontrol of transcription at the level of chromatin organizationwhereas DYAD/SWI1 has been shown to function inchromosome organization and cohesion. If DUET does in factfunction as a transcriptional regulator, this would point to aclose connection between cohesion and the control oftranscription at the chromatin level during meiosis. Theformation of dyads and the fact that meiosis is more extendedin the duet mutant clearly suggests a defect in meioticprogression. Analysis of single division meiosis in the spo13mutant of yeast has shown that the basis for the progressiondefect is the activation of the spindle checkpoint. In the absenceof the spindle checkpoint, spo13 mutants undergo normalmeiotic progression and form four spores (Shonn et al., 2002;Lee et al., 2002). There is at present limited information on thecontrol of meiotic progression in plants. Immunolocalizationof a maize homologue of the yeast spindle checkpoint proteinMAD2 has shown that it is expressed in meiosis and localizedto the kinetochore where it functions through a tension-dependent mechanism (Yu et al., 1999). Hence the basicapparatus for the spindle checkpoint is conserved in plants. Arecent study has identified a mutant sog1 that suppressesgamma radiation-induced arrest and also affects pollendevelopment (Preuss and Britt, 2003). The further analysis ofmutants defective in meiotic progression should provideinformation on the existence of chromosomal checkpoints inplant meiosis.
This work was supported by the Council for Scientific andIndustrial Research, Government of India. T.V.R., B.A. and J.K. arerecipients of CSIR Research Fellowships. This work was also partlysupported by grants from NSTB Singapore to V.S. We thank Dr ShashiSingh for help with sectioning of plant material and Mehar Sultanafor synthesis of oligonucleotides. We also acknowledge the ABRC forDNA clones and seed material.
Note added in proofWhile the manuscript was under review, Yang et al. reportedthe cloning and expression analysis of the MMD gene, andanalysis of the mmdmutant. The MMDgene is identical toDUET (Yang, X. et al., 2003).
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