Homolog interaction during meiotic prophase I in Arabidopsis requires the SOLO DANCERS gene encoding a novel cyclin-like protein
2002; Springer Nature; Volume: 21; Issue: 12 Linguagem: Inglês
10.1093/emboj/cdf285
ISSN1460-2075
Autores Tópico(s)Plant Genetic and Mutation Studies
ResumoArticle17 June 2002free access Homolog interaction during meiotic prophase I in Arabidopsis requires the SOLO DANCERS gene encoding a novel cyclin-like protein Yoshitaka Azumi Yoshitaka Azumi Department of Biology and the Life Sciences Consortium, The Pennsylvania State University, University Park, PA, 16802 USA Department of Biological Sciences, Kanagawa University, Hiratsuka, Kanagawa, 259-1293 Japan Search for more papers by this author Dehua Liu Dehua Liu Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 11724 USA Present address: SM Biotech, Inc., PO Box 1724, 380 Oakwood Road, Huntington Station, NY, 11746 USA Search for more papers by this author Dazhong Zhao Dazhong Zhao Department of Biology and the Life Sciences Consortium, The Pennsylvania State University, University Park, PA, 16802 USA Search for more papers by this author Wuxing Li Wuxing Li Department of Biology and the Life Sciences Consortium, The Pennsylvania State University, University Park, PA, 16802 USA Intercollegiate Graduate Program in Plant Physiology, The Pennsylvania State University, University Park, PA, 16802 USA Search for more papers by this author Guanfang Wang Guanfang Wang Department of Biology and the Life Sciences Consortium, The Pennsylvania State University, University Park, PA, 16802 USA Intercollegiate Graduate Program in Plant Physiology, The Pennsylvania State University, University Park, PA, 16802 USA Search for more papers by this author Yi Hu Yi Hu Department of Biology and the Life Sciences Consortium, The Pennsylvania State University, University Park, PA, 16802 USA Search for more papers by this author Hong Ma Corresponding Author Hong Ma Department of Biology and the Life Sciences Consortium, The Pennsylvania State University, University Park, PA, 16802 USA Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 11724 USA Intercollegiate Graduate Program in Plant Physiology, The Pennsylvania State University, University Park, PA, 16802 USA Search for more papers by this author Yoshitaka Azumi Yoshitaka Azumi Department of Biology and the Life Sciences Consortium, The Pennsylvania State University, University Park, PA, 16802 USA Department of Biological Sciences, Kanagawa University, Hiratsuka, Kanagawa, 259-1293 Japan Search for more papers by this author Dehua Liu Dehua Liu Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 11724 USA Present address: SM Biotech, Inc., PO Box 1724, 380 Oakwood Road, Huntington Station, NY, 11746 USA Search for more papers by this author Dazhong Zhao Dazhong Zhao Department of Biology and the Life Sciences Consortium, The Pennsylvania State University, University Park, PA, 16802 USA Search for more papers by this author Wuxing Li Wuxing Li Department of Biology and the Life Sciences Consortium, The Pennsylvania State University, University Park, PA, 16802 USA Intercollegiate Graduate Program in Plant Physiology, The Pennsylvania State University, University Park, PA, 16802 USA Search for more papers by this author Guanfang Wang Guanfang Wang Department of Biology and the Life Sciences Consortium, The Pennsylvania State University, University Park, PA, 16802 USA Intercollegiate Graduate Program in Plant Physiology, The Pennsylvania State University, University Park, PA, 16802 USA Search for more papers by this author Yi Hu Yi Hu Department of Biology and the Life Sciences Consortium, The Pennsylvania State University, University Park, PA, 16802 USA Search for more papers by this author Hong Ma Corresponding Author Hong Ma Department of Biology and the Life Sciences Consortium, The Pennsylvania State University, University Park, PA, 16802 USA Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 11724 USA Intercollegiate Graduate Program in Plant Physiology, The Pennsylvania State University, University Park, PA, 16802 USA Search for more papers by this author Author Information Yoshitaka Azumi1,2, Dehua Liu3,4, Dazhong Zhao1, Wuxing Li1,5, Guanfang Wang1,5, Yi Hu1 and Hong Ma 1,3,5 1Department of Biology and the Life Sciences Consortium, The Pennsylvania State University, University Park, PA, 16802 USA 2Department of Biological Sciences, Kanagawa University, Hiratsuka, Kanagawa, 259-1293 Japan 3Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 11724 USA 4Present address: SM Biotech, Inc., PO Box 1724, 380 Oakwood Road, Huntington Station, NY, 11746 USA 5Intercollegiate Graduate Program in Plant Physiology, The Pennsylvania State University, University Park, PA, 16802 USA *Corresponding author. E-mail: [email protected] The EMBO Journal (2002)21:3081-3095https://doi.org/10.1093/emboj/cdf285 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Interactions between homologs in meiotic prophase I, such as recombination and synapsis, are critical for proper homolog segregation and involve the coordination of several parallel events. However, few regulatory genes have been identified; in particular, it is not clear what roles the proteins similar to the mitotic cell cycle regulators might play during meiotic prophase I. We describe here the isolation and characterization of a new Arabidopsis mutant called solo dancers that exhibits a severe defect in homolog synapsis, recombination and bivalent formation in meiotic prophase I, subsequently resulting in seemingly random chromosome distribution and formation of abnormal meiotic products. We further demonstrate that the mutation affects a meiosis-specific gene encoding a novel protein of 578 amino acid residues with up to 31% amino acid sequence identity to known cyclins in the C-terminal portion. These results argue strongly that homolog interactions during meiotic prophase I require a novel meiosis-specific cyclin in Arabidopsis. Introduction Meiosis is required for eukaryotic sexual reproduction and provides an important mechanism for generating genetic diversity among individuals of a species. Cytological studies indicate that the meiotic prophase I is a long and complex stage that differs from the mitotic prophase (see reviews by Ashley and Plug, 1998; Dawe, 1998; Zickler and Kleckner, 1999). During prophase I, chromosomes condense and homologous chromosomes (homologs) pair. The prophase I continuum is divided into five substages based on chromosomal characteristics: first, in the leptotene stage, chromosomes begin to condense and can be seen as thin thread-like structures. Homolog pairing initiates in leptotene and continues extensively in the zygotene stage. In the pachytene stage, fully synapsed homologs are observed as thick thread-like structures under a light microscope. The synaptonemal complex (SC) forms at this stage and can be observed in detail using electron microscopy. During the diplotene stage, homologs become desynapsed along much of their length, but remain attached through chiasmata. Finally, during the diakinesis stage, chromosomes contract lengthwise to produce highly condensed bivalents. Therefore, an important outcome of prophase I is the formation of bivalents. Although the mechanisms may differ, it is universal that homologs are attached until the onset of anaphase I. Molecular genetic and biochemical studies in yeasts, Caenorhabditis elegans and Drosophila have provided many of the molecular insights into homolog interactions during prophase I (Roeder, 1997; Dernburg et al., 1998; Orr-Weaver, 1999; Zickler and Kleckner, 1999). Such interactions include pairing, synapsis and recombination, which are intimately associated with each other. Meiotic recombination is thought to begin in late leptotene or zygotene stages after homolog pairing has initiated, and continues in the pachytene stage while the homologs are synapsed. In budding yeast, double-stranded DNA breaks (DSBs) are generated by the Spo11p protein and are required for both recombination and SC formation. In C.elegans, DSBs also initiate homologous recombination, which is not required for SC formation (Dernburg et al., 1998). In budding yeast, the recA homologs Dmc1p and Rad51p are required for recombination and SC formation (Bishop et al., 1992; Rockmill et al., 1995; Shinohara et al., 1997). In addition, sister chromatids are closely associated due to sister chromatid cohesion during prophase I (Klein et al., 1999; Hirano, 2000). In budding yeast, sister chromatid cohesion depends on the cohesin complex, which contains at least four proteins: Smc1p, Smc3p, Scc1p and Scd3p (Michaelis et al., 1997; Nasmyth, 1999). Rec8p, a meiosis-specific Scc1p homolog, from both fission and budding yeasts, is essential for normal meiosis (Molnar et al., 1995; Orr-Weaver, 1999; StoopMyer and Amon, 1999; Watanabe and Nurse, 1999). During meiotic prophase I and metaphase I, sister chromatid cohesion along the chromosomal arms is required to maintain bivalents. The separation of sister chromatids along the arms at the metaphase I–anaphase I transition allows the homologs to separate. Sister chromatid cohesion at the centromere serves to maintain sister association until anaphase II, when the sisters separate. Therefore, both the recombinational crossover and sister chromatid cohesion contribute to the formation of chiasmata. Clearly, regulation of both recombination and sister chromatid cohesion is critical for proper segregation of homologs, although very little is known about such regulation at the molecular level, including the identity of genes that play regulatory roles. Numerous studies have been conducted to understand the molecular control of the mitotic cell cycle, particularly in animal and fungal systems (Futcher, 1991; Reed, 1991; Pines, 1993; Roberts et al., 1994). Among known mitotic cell cycle regulators, cyclins and cyclin-dependent protein kinases (CDKs) play central roles in controlling all major phases of the cell cycle, such as the G2/M and G1/S transitions (Murray, 1994; Andrews and Measday, 1998; Gitig and Koff, 2000). Cyclins were first identified as proteins showing a cyclical pattern of accumulation and destruction during early embryonic development in marine invertebrates (Evens et al., 1983; Swenson et al., 1986). In budding yeast, genetic analysis with various combinations of mutations in the B-type cyclins CLB1, CLB2, CBL3 and CLB4 indicates that CLB1 and CLB4 are required for meiosis II, but not for meiosis I (Grandin and Reed, 1993; Dahmann and Futcher, 1995). Furthermore, the budding yeast B-type cyclins CLB5 and CLB6 are required for pre-meiotic DNA replication, which seems indirectly to affect the initiation of recombination and SC formation (Dirick et al., 1998; Stuart and Wittenberg, 1998; Smith et al., 2001). Also, mouse cyclin A1 is required for the progression from pachytene to diplotene (Liu et al., 1998). However, whether there is a more direct requirement for cyclins in homolog synapsis and bivalent formation during prophase I is not known. Plants have been one of the most important sources of our knowledge about meiosis at the cytological level (Dawe, 1998; Zickler and Kleckner, 1999). Mutants with defects in homolog interactions have been described in several plants (Kaul and Murphy, 1985; Dawe, 1998). In particular, a wheat asynaptic mutant seemed to have normal leptotene but unpaired/unattached homologs throughout prophase I (La Cour and Wells, 1970). In addition, the maize desynaptic (dy) mutant was shown to form an SC, but could not fully maintain the SC at late pachytene; bivalents separate into univalents at a variable frequency during diplotene and diakinesis (Nelson and Clary, 1952; Maguire, 1978). However, the molecular nature of these mutant defects is as yet unknown. Recently, normal male meiosis in Arabidopsis has been described using fluorescence microscopy (Ross et al., 1996; Peirson et al., 1997), paving the way for molecular genetic analysis of Arabidopsis meiosis. For example, the Arabidopsis SYN1 gene (also called DIF1) encoding a protein similar to the yeast Rec8p cohesin subunit is essential for normal meiotic chromosome condensation and homolog interactions (Bai et al., 1999; Bhatt et al., 1999). In addition, disruption of an Arabidopsis SPO11 homolog (AtSPO11-1) causes defects in meiotic recombination, SC formation and bivalent formation (Grelon et al., 2001). Furthermore, recA homologs AtDMC1 and AtRAD51 are also required for meiosis in Arabidopsis (Klimyuk and Jones, 1997; Couteau et al., 1999), and the Rad51 protein has also been implicated in meiosis of maize and lily (Anderson et al., 1997; Franklin and Cande, 1998; Franklin et al., 1999). We have now isolated a meiotic mutant (solo dancers, or sds) that is severely defective in interactions between homologs in prophase I. Furthermore, we show that the SDS gene is expressed specifically in male and female meiocytes and encodes a protein that is similar to known cyclins. Our results demonstrate for the first time that a meiosis-specific novel cyclin-like protein is critical for normal homolog synapsis and bivalent formation, suggesting that a CDK may play a key role in controlling the normal chromosomal events during meiotic prophase I in Arabidopsis. Results Isolation and genetic characterization of a meiotic mutant In higher plants, male meiosis occurs inside the anther portion of the male reproductive organ, the stamen. To identify genes important for male meiosis, we screened for Ds insertional mutants with reduced fertility (see Materials and methods). Wild-type Arabidopsis plants (Figure 1A) produce large seedpods with dozens of seeds. We found that the progeny of one of the Ds lines segregated for normal and nearly sterile plants that had small seedpods (Figure 1B); furthermore, the normal and mutant plants were in a ratio of ∼3:1, suggesting that the parental plant was heterozygous for a recessive mutation. When the mutant plants also expressed the Ac transposase, they could produce fertile sectors due to the excision of the Ds element (Figure 1C). Whereas normal flowers produce many pollen grains (Figure 1D), the mutant flowers produced little or no normal pollen (Figure 1E). Nevertheless, the mutant was able to produce some seeds when pollinated with wild-type pollen, indicating that it was female fertile, although the seed set was much lower than normal. In addition, progeny of a cross from such a pollination were normal, confirming that the mutation was indeed recessive. We have named this new mutant solo dancers (sds) for its meiotic phenotype (see below). Figure 1.Wild-type and sds mutant plant, flower and pollen development. (A) A wild-type plant with normal seedpods (arrowheads). (B) An sds mutant plant, with small seedpods (arrows). (C) An sds mutant plant with small seedpods (arrows) and two large revertant sectors that have large seedpods (arrowheads). (D) A close-up view of the top portion of a wild-type flower, showing many pollen grains (p) on the stigma and along the side of the pistil. (E) Top of an sds flower, showing anthers and the stigma (s) that lack pollen. (F) A portion of a wild-type anther with functional pollen grains that stained red. (G) Wild-type microspores. (H) A normal tetrad with four spores (numbered). (I) A portion of an sds anther, showing many abnormal pollen grains stained blue. Several pollen grains are stained red and presumably are functional (arrows); some are larger than normal. (J) Microspores from an sds anther, showing a range of sizes. (K) Six spores from a meiosis in the sds mutant. Bar = 10 mm in (A–C), 250 μm in (D) and (E), 20 μm in (F), (G), (I) and (J), and 10 μm in (H) and (K). Download figure Download PowerPoint The sds mutant appeared to be normal in vegetative and flower development, producing normal floral organs (Figure 1E; other data not shown). Wild-type Arabidopsis anthers in an unopened floral bud contain normal pollen grains (Figure 1F). However, the sds mutant anthers had many abnormal pollen grains with variable sizes (Figure 1I). The defective pollen grains degenerated before the flower opened (data not shown). Analysis of immature anthers showed that the mutant microspores also had different sizes (Figure 1J), unlike normal microspores (Figure 1G). Further examination revealed that whereas a normal meiosis always produces four microspores of equal size in a tetrad (Figure 1H), the mutant male meiosis produced 2–8 microspores of variable sizes (Figure 1K shows a ‘hexad’ with six spores). From 565 sds meioses, we found six (1.1%) diads, 20 (3.5%) triads, 293 (51.9%) tetrads, 141 (25%) pentads, 80 (14.2%) hexads, 21 (3.7%) heptads and four (0.7%) octads. Therefore, the sds mutant is defective in male meiosis. The sds mutant is defective in homolog synapsis and bivalent formation in meiotic prophase I To determine meiotic defect(s) of the sds mutant, we compared wild-type and mutant male meiosis using a chromosome spread and 4′,6-diamino-2-phenylindole dihydrochloride (DAPI) staining procedure (Ross et al., 1996). During leptotene (Figure 2A) (Ross et al., 1996), chromosomes begin to condense and form visible thin lines. Chromosomes continue to condense and begin synapsis at zygotene (Figure 2B); at pachytene (Figure 2C), homologs have completed their synapsis, as indicated by the appearance of thick lines. In late diplotene (Figure 2D), homologs desynapse along much of the chromosome, leaving only limited association at the chiasmata; the chromosomes are also condensed further. At late diakinesis (Figure 2E), chromosomes are very highly condensed and the five bivalents can be easily recognized. Figure 2.Male meiosis I in wild-type and the sds mutant. Shown are images of DAPI-stained chromosomes. (A–E) Wild-type prophase I at leptotene, zygotene, pachytene, diplotene and diakinesis, respectively; note that (E) shows five brightly stained entities, representing five attached pairs of condensed homologs. (F–J) The sds mutant prophase I at similar stages; note that in (J), 10 staining bodies can be seen, indicating that the condensed homologs formed univalents. The arrows, as well as the two pairs of arrowheads, point to two strands of a partially separated chomosome, suggesting precocious separation of the arms of sister chromatids. (K–N) Wild-type early metaphase I, late metaphase I, anaphase I and telophase I, respectively. Chromosomes align at the equator (K), and are elongated presumably due to the forces of the spindle (L). Homologs separate (M) and form two clusters (N). (O–R) Meiosis I images from the sds mutant after prophase I. Chromosomes condense further (O), similarly to normal metaphase I chromosomes, but they did not all align at the equator. Chromosomes in (P) are elongated similarly to those in (L). Chromosomes are elongated further in (Q), suggesting that they might be pulled by the spindle as normal homologs are at anaphase I. Some chromosomes more distant from the center seem to have decondensed (R), resembling those in normal telophase I. Download figure Download PowerPoint In sds mutant cells, chromosomal patterns similar to the wild-type leptotene through pachytene stages were observed (Figures 2F–H). Although it is difficult to determine whether diplotene in the sds mutant (Figure 2I) was normal, it was obvious that by diakinesis the homologs were not attached in sds cells, as indicated by the observation of 10 univalents (Figure 2J). In addition, we noticed that the sds cells with chromosome images similar to that in Figure 2H were very infrequent, suggesting a defect in synapsis. To determine the distribution of prophase I cells, we examined hundreds of wild-type and sds meiotic cells. Because male meiosis in Arabidopsis is slightly asynchronous, a population of male meiotic cells from a single flower can cover a few adjacent meiotic stages or substages of the long prophase I. When data from samples containing only or largely prophase I cells were summarized (Figure 3), we found that among the wild-type prophase I cells (three samples, 717 cells), nearly half were pachytene cells, and relatively few were leptotene cells, with the other stages having moderate numbers of cells. In particular, pachytene was also the most frequent class in each individual sample (data not shown). In contrast, the sds mutant showed a dramatically different distribution for prophase I substages (six samples, 1241 cells); the number of cells at the leptotene and zygotene stages was higher than normal, whereas cells at the pachytene stage were very rare. In this case, pachytene-like cells were the least frequent in each sample. The sds distribution of prophase I stages further supports the idea that synapsis is defective in the mutant. Figure 3.Distribution of prophase I cells in wild type and the sds mutant. Chromosome spreads were examined as shown in Figure 2. The number of images at each stage is shown above the appropriate bar. The sds leptotene, zygotene, pachytene and diplotene stages were assigned on the basis on chromosome condensation and overall morphology, as shown in Figure 2. The sds zygotene and diplotene stages were not based on pairing or bivalents, respectively. The sds diakinesis images all had 10 univalents. Download figure Download PowerPoint Therefore, the sds mutant had a clear defect in homolog synapsis and bivalent formation in male meiosis, suggesting that normal homolog synapsis could not be achieved in the absence of SDS gene function, and chromosomes form univalents at the end of prophase I. On the other hand, chromosome condensation seems to be unaffected by the sds mutation, unlike the syn1 mutant (Bai et al., 1999; Bhatt et al., 1999). We likened the wild-type chromosome interactions and condensation to a highly choreographed duet dance in which homologs ‘dance’ in pairs. Therefore, the mutant had a clear phenotype of homologs behaving as 'solo dancers’. Next, we compared wild-type and sds meiosis from metaphase I to telophase I for possible additional mutant defects. In wild-type cells at metaphase I (Figure 2K and L), the five bivalents align at the equatorial plane. At anaphase I, homologs separate (Figure 2M), and move towards the opposite pole of the spindle (Figure 2N). In contrast, the univalents in the sds mutant did not align completely; some of them were quite far from the equator (Figure 2O and P). The abnormal distribution seemed to persist (Figure 2Q and R). It seemed that usually more than two clusters of chromosomes were formed. We examined chromosome distribution at late meiosis I. For wild-type cells at anaphase I, 62 out of 66 cells (93.9%) showed a 5:5 even distribution of chromosomes; the other four cells had <10 chromosomal DAPI spots, most probably due to a superimposition of some chromosomes. On the other hand, sds male meiotic cells exhibited many abnormal distribution patterns. Among 71 sds cells at anaphase I, 46 cells (64.8%) had two groups of chromosomes with the following distributions: 5:5 (nine cells), 6:4 (16 cells), 7:3 (14 cells), 8:2 (six cells) and 9:1 (one cell). The remaining cells had one (14 cells; 19.7%), two (eight cells; 11.3%) or three (three cells; 4.2%) chromosomes at the equator, with various distributions of other chromosomes (data not shown) on either side of the equator. Because the sds mutant also had reduced female fertility, we examined female meiosis in the wild-type and the sds mutant. As shown in Figure 4, wild-type prophase I at diakinesis displays five bivalents (Figure 4A), which become highly condensed at metaphase I (Figure 4B). The homologs separate at anaphase I, showing two groups with five chromosomes each (Figure 4C). In contrast, female meiosis in the sds mutant at diakinesis showed a variable number of univalents. Among 48 nuclei observed, 23 (47.9%) had only univalents (e.g. Figure 4D), similar to the sds male meiosis. Seven (14.6%) and eight (16.7%) nuclei had one (Figure 4G) or two bivalents (data not shown), respectively, with additional chromosomes forming univalents. Only a very small number of nuclei had four (2/48, or 4.2%) or five (2/48) bivalents (data not shown). These 8% of female meioses probably accounted for most of the female fertility when normal pollen was used. At metaphase I in the sds mutant, as expected, many female meioses had only univalents (Figure 4E) and others had some bivalents (Figure 4H). At anaphase I, one or more pairs of chromosomes were often observed, suggesting that they were homologs that had been associated in bivalents (Figure 4F and I). Other anaphase I images are consistent with an absence of bivalents (data not shown). These results indicate that in the sds mutant, female meiosis is defective in a way similar to male meiosis, but to a lesser extent. Figure 4.Female meiosis I in wild type and the sds mutant. Shown here are DAPI-stained images of chromosome spreads from the wild type (A–C) or the sds mutant (D–I). (A) A diakinesis image showing five bivalents. (B) At metaphase I, the five bivalents are aligned in parallel. (C) At anaphase I, homologs had segregated and formed two groups of five chromosome each. (D) At diakinesis, 10 univalents can be seen. (E) A metaphase I cell with 10 univalents. (F) An anaphase I cell with one pair of chromosomes elongated (arrowheads), suggesting that they were from a bivalent. (G) A diakinesis nucleus with one bivalent (arrows) and eight univalents (arrows point to two of them). (H) A metaphase I cell with two bivalents (arrows) and six univalents. (I) An anaphase I cell with three pairs of separating chromosomes that were probably from three bivalents. Three (arrowheads) of the four univalents were on one side of the equator, probably resulting in spores that have four or six chromosomes. Download figure Download PowerPoint During meiosis II in the wild-type male meiocytes (Figure 5), the two groups of chromosomes are separated by a band of organelles (arrows in Figure 5A–D). Partially condensed chromosomes were observed during prophase II (Figure 5A); at metaphase II, they congressed at the two equators (Figure 5B). Sister chromatids then separated at anaphase II (Figure 5C), moved to opposite poles at telophase II (Figure 5D) and decondensed to form four nuclei (Figure 5E). In the sds mutant, chromosome distribution was abnormal, most probably due to a defective meiosis I, but chromosome condensation and sister chromatid separation seemed to be quite normal. Chromosomes condensed at prophase II (Figure 5F and K), but alignment was abnormal (Figure 5G and L). Then sister chromatids separated at anaphase II (Figure 5H and M), and moved apart at telophase II (Figure 5I and N). Distributions of meiotic cells from metaphase I to the end of meiosis II were not dramatically different between the wild type and the sds mutant (data not shown). Figure 5.Male meiosis II in wild type and the sds mutant. (A–O) Images obtained from chromosome spreads. (A–D) Wild-type meiosis II at prophase, metaphase, anaphase and telophase, respectively. The arrows in these panels indicate the characteristic organellar band present during meiosis II. The DAPI staining patterns suggest the following events during normal meiosis II. First chromosomes condense (A) and move to the equatorial plain (B). Then sister chromatids separate (C) and move apart (D). The well-separated chromosomes form four nuclei (E). In the sds mutant, meiosis II seems to follow the normal course of events, although the distribution of initial groups of chromosomes is abnormal due to defects in meiosis I. Arrows in (F–H) and (K–N) indicate the organellar band. This band is also present in (I), but it is faint. Chromosomes condense (F and K) but they do not align into tight plates (G and L). Nevertheless, subsequent separation of sister chromatids seems normal (H, I, M and N). In some cases, even an isolated chromosome can separate into two sisters (arrowheads in H and M). The separated chromosomes then form clusters. In (I), there are two pairs of clusters with four chromosomes each, and a pair of clusters each with two chromosomes (arrowheads); in (N), one pair has four chromosomes in each cluster (arrowheads) and the other pair has six chromosomes in each cluster. These clusters then form nuclei (eight in J and six in O). Download figure Download PowerPoint Our results indicate that the sds mutant is defective in homolog synapsis and bivalent formation in prophase I. In addition, there was no obvious defect during later stages of meiosis I or during meiosis II other than those that are probable consequences of the failure to form bivalents. Furthermore, clear separation of sister chromatids at meiosis II indicates that meiotic DNA replication was not obviously affected in the mutant. Therefore, the sds mutant is specifically defective in homolog interaction during prophase I. The sds mutant is defective in meiotic recombination The phenotypes of the sds mutant were similar to those of the recombination mutants Atspo11 and Atdmc1, suggesting that sds might also be defective in recombination. To test such a defect in the sds mutant, we generated sds/sds plants that were heterozygous (L1L2/C1C2) for two molecular markers on each of chromosomes II and V (see Materials and methods). Although the sds mutant was nearly sterile, it was able to produce 50–100 seeds per plant (a normal plant can easily produce several thousand seeds). When the seeds from sds plants were planted, many of them developed to maturity and exhibited the same sds defects in fertility and meiosis (data not shown), indicating that the seeds from sds plants were not from cross-pollination. The progeny of sds/sds plants allowed the analysis of recombination frequency in the sds mutant. The sds/+ or +/+ siblings were also heterozyg
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