- © 1999 American Society of Plant Physiologists
Abstract
In yeast, the DMC1 gene is required for interhomolog recombination, which is an essential step for bivalent formation and the correct partition of chromosomes during meiosis I. By using a reverse genetics approach, we were able to identify a T-DNA insertion in AtDMC1, the Arabidopsis homolog of DMC1. Homozygotes for the AtDMC1 insertion failed to express AtDMC1, and their residual fertility was 1.5% that of the wild type. Complete fertility was restored in mutant plants when a wild-type copy of the AtDMC1 gene was reintroduced. Cytogenetical analysis points to a correlation of the sterility phenotype with severely disturbed chromosome behavior during both male and female meiosis. In this study, our data demonstrate that AtDMC1 function is crucial for meiosis in Arabidopsis. However, meiosis can be completed in the Arabidopsis dmc1 mutant, which is not the case for mouse or some yeast mutants.
INTRODUCTION
Meiosis allows diploids to undergo sexual reproduction and leads to the genetic reassortment of characters. Meiosis differs from mitosis in that a single round of DNA replication precedes two sequential cell divisions so that an initially diploid cell generates four haploid cells. Homologous chromosomes segregate from one another during the first (reductional) division, and sister chromatids separate during the second (equational) division. Association of pairs of homologous chromosomes to form bivalents is a prerequisite to their accurate partitioning during the first division of meiosis. Bivalent formation relies on homologous recombination and synaptonemal complex polymerization, which physically bind the homologs via the establishment of crossovers and synapsis (Kleckner, 1996; Roeder, 1997; Zickler and Kleckner, 1998). The cytologically defined chiasmata structures result from this prophase I recombinational step (Carpenter, 1994).
In budding yeast, meiotic recombination is initiated by DNA double-strand breaks (DSBs) created by Spo11, a type II topoisomerase, at specific locations along the chromosomes. This early recombination step additionally requires that the Rad50, Mre11, Mre2, Rec102, Rec103, Rec104, and Rec114 functions be present. The DSBs are processed further by resection of their 5′ ends to yield long 3′ single-stranded tails, which can invade a homologous duplex (Kleckner, 1996; Smith and Nicolas, 1998). This recombination step depends on essential meiotic functions provided by Dmc1 and Rad51, two of the yeast homologs of the Escherichia coli protein RecA (Bishop et al., 1992; Shinohara et al., 1992). RecA promotes the invasion of duplex DNA by a single-stranded homologous DNA molecule. This activity is a key function in homologous recombination (Kowalczykowski and Eggleston, 1994). In vitro, the eukaryotic Rad51 and Dmc1 proteins also can catalyze strand exchange between homologous DNA molecules (Kowalczykowski and Eggleston, 1994; Li et al., 1997). This activity may explain the requirement for the yeast RecA homologs during meiosis: rad51 and some dmc1 mutants arrest before completion of the first division, at a stage when homologous recombination is crucial, and they accumulate hyperresected DSBs (Shinohara et al., 1997). Variation in the observed dmc1 phenotypes (meiotic arrest or not) may depend on secondary characters of the yeast strains that are in use in different laboratories (Rockmill et al., 1995). Although RAD51 also is involved in mitotic recombinational DNA repair, DMC1 is expressed and necessary only during meiotic prophase I in yeast (Bishop et al., 1992; Shinohara et al., 1992). Evidence indicates that Dmc1 is dedicated specifically to interhomolog recombination, which is an event that is specific to meiosis, whereas Rad51 is required for both interhomolog and intersister recombination (Schwacha and Kleckner, 1997; Shinohara et al., 1997).
Eukaryotic homologs of the RAD51 and DMC1 genes have been found in a number of organisms (Stassen et al., 1997). In fact, the yeast DMC1 (LIM15) gene was independently isolated when it was found to hybridize with a gene specifically induced at meiosis in Lilium longiflorum (Kobayashi et al., 1993). As occurs in yeast, the Rad51 and Dmc1 proteins of mouse and L. longiflorum colocalize as foci along the meiotic chromosomes during prophase I. During this stage, homologous recombination occurs (Bishop, 1994; Terasawa et al., 1995; Ikeya et al., 1996). More precisely, RecA homologs have been observed in early recombination nodules in L. longiflorum, rat, and mouse (Anderson et al., 1997; Moens et al., 1997; Plug et al., 1998). Finally, homozygous disruption of the DMC1 gene in mouse parallels some aspect of the yeast dmc1 mutations in that it also affects meiosis, although leading to apoptosis, which was not observed in yeast (Pittman et al., 1998; Yoshida et al., 1998). In Arabidopsis, an ortholog of the yeast DMC1 gene, AtDMC1, has been described (Sato et al., 1995; Klimyuk and Jones, 1997; Doutriaux et al., 1998). It is a single-copy gene that is expressed in young flower buds when cells are undergoing meiosis. To the difference of yeast and mouse, AtDMC1 also is found expressed in vegetative cells, at low level in leaves but to a high level in exponentially growing cells from a suspension culture (Klimyuk and Jones, 1997; Doutriaux et al., 1998). However, functional homology between the yeast, mouse, and Arabidopsis DMC1 genes remains to be proven. Besides, whereas plants, with the description of numerous meiotic mutants, have contributed significantly to the study of meiosis, identification of genes involved in this process has not been accomplished (Dawe, 1998).
In this study, we characterize an Arabidopsis dmc1 mutant. This mutant was recovered from a collection of T-DNA insertional lines by using a polymerase chain reaction (PCR)–based screening procedure. Homozygotes for the dmc1 insertional mutation showed reduced fertility in self-crosses and outcrosses. Sporogenesis was affected in both male and female organs, and abnormal meiosis was observed in pollen mother cells and in megaspore mother cells as well. Our results support the view that features essential to meiosis in yeast are conserved in plants. However, the dmc1 mutation in Arabidopsis does not lead to meiotic arrest, as has been observed in mouse and in some yeast mutants, a point that we discuss.
RESULTS
Isolation and Molecular Characterization of a dmc1 Insertional Mutation
The sequence of the Arabidopsis DMC1 gene was used to perform a PCR-based screen of a collection of 6000 T-DNA insertion lines (Feldmann, 1991; McKinney et al., 1995). Amplification was effective for one of six DNA pools (1000 lines per pool) in the reaction using the upstream primer a, which is located in the AtDMC1 gene coding sequence, and the downstream primer b, which is located at the left border of the T-DNA (Figure 1A). Subsequent fractionation of the positive pool allowed us to isolate a single line (3668), which carries an insertion in the AtDMC1 gene. Systematic characterization of plants derived from line 3668 by using PCR was performed with primers specific for either the disrupted (primers a and b) or the wild-type (primers c and d) AtDMC1 alleles, to define their genotypes early in growth (Figure 1B). Self-fertilized heterozygous dmc1+/- progeny typically produced 1:2:1 ratios of the wild-type/heterozygous dmc1+/-/homozygous dmc1-/- plants (χ2 = 1.121; P = 0.571). Although kanamycin-resistant plants were present among the progeny of line 3668, genetic analysis indicated that kanamycin resistance did not cosegregate with the mutant phenotype. Three independent T-DNA insertions, which could be effectively separated, were present in this line. Two of these insertions conferred kanamycin resistance and were not linked to any detectable phenotype; the third did not confer antibiotic resistance and proved to be the DMC1-disrupting insertion.
Molecular Characterization of the Arabidopsis dmc1 T-DNA Insertion.
(A) Schematic representation of the AtDMC1 genomic region. Exons are indicated as open boxes. Primers a, b, c, and d used to define plant genotypes are shown by small black boxes with arrows on top. The T-DNA insertion is located at the junction between intron 8 and exon 9. Dashed lines represent the 3′ region missing in the T-DNA; exon 9 lacks <20 bp in its 5′ border. The HpaI-SalI fragment (11 kb) was used for complementation.
(B) PCR determination of the genotypes of progeny deriving from a self-fertilized heterozygous plant. Amplification with primers a and b (935 bp) or primers c and d (1149 bp) specifies the mutant or wild-type allele, respectively, for each sample analyzed.
(C) RNA gel blot analysis of AtDMC1 and AtRAD51 expression in young flower buds. The arrow indicates the position of the AtDMC1 transcript. The blot was probed with EF-1α as a control for equal loading. +/+, +/-, and -/- indicate RNA extracted from wild-type, heterozygous, or dmc1 mutant plants, respectively.
Plants with a dmc1-/- genotype show reduced fertility. This phenotype exhibits a 3:1 segregation ratio (fertility/reduced fertility) in progeny of self-fertilized heterozygous dmc1+/- plants, indicating that the mutation is recessive and monogenic. Sequencing revealed the insertion to disrupt the AG consensus splice site between intron 8 and exon 9 (Figure 1A). Downstream, the right border of the T-DNA is missing, as was previously suggested by the kanamycin segregation data. A short deletion of exon 9 of AtDMC1 (<20 bp) accompanied the inserted T-DNA, as ascertained by PCR with different primers along exon 9 (data not shown). The insertion causes a disruption of the AtDmc1 protein in a well-conserved three–amino acid motif—QLP— that is found in the RecA domains of all Rad51 and Dmc1 homologs. RNA gel blot analysis (Figure 1C) shows that a full-length AtDMC1 transcript is absent from young flower buds of the mutant and that this transcript is less abundant in heterozygous than in wild-type plants. However, a shorter RNA that weakly hybridizes with the AtDMC1 probe may represent a transcript that initiated in the 5 ′ region of the gene, upstream of the insertion, and which probably terminates in the T-DNA. Both the short and the full-length transcripts are present in flower buds from heterozygous plants. AtRAD51 expression, also known to be induced in young flower buds, increased in both homozygous and heterozygous dmc1 plants when compared with the wild type (confirmed by reverse transcription–PCR; data not shown).
dmc1-/- Plants Show Reduced Fertility
Homozygous dmc1 plants develop normally but show strongly reduced fertility (Figure 2A) and produce only 3.15 ± 1.51 seeds per silique (versus 40 to 50 in a wild-type plant). Due to the low number of seeds that they contain, the siliques are short and curved, as is usually the case in Arabidopsis mutants that exhibit fertility deficiencies (Figures 2D and 2E). Twenty-nine percent of the seeds are dark, small, shrunken, and essentially nonviable, and another 24% are bigger but heterogeneous in size and shape. Of the apparently normal seeds (the remaining 47%), 75% germinate, often developing into arrested or abnormal plants, and only 40% grow into mature plants. Altogether, the residual fertility of the dmc1 mutant is 1.5% that of both wild-type and heterozygous plants, which are indistinguishable in this characteristic. Some examples of seeds germinating into aberrant seedlings are presented in Figures 3B to 3J; a wild-type seedling of the same age is shown in Figure 3A. Because of the abnormalities of the progeny, we always propagated the mutation through heterozygous plants. In the anthers of the mutant, only a few pollen grains of variable size are produced (Figures 2B, 2C, 2F, and 2G). However, some pollen grains that are capable of fertilization are produced because a few viable seeds eventually are released. The low fertility of dmc1 plants can be attributed to defects in female gametogenesis as well, because cross-fertilization of dmc1 plants with wild-type pollen does not improve seed set in the mutant flowers.
Complementation of the dmc1 Insertional Mutation
To definitely correlate the sterility phenotype with the DMC1 disruption, we conducted genetic complementation of heterozygous (dmc1+/-) plants. An 11-kb DNA fragment covering the entire Arabidopsis (ecotype Columbia) genomic DMC1 region (i.e., a HpaI-SalI fragment; see Figure 1A) was introduced into the T-DNA of an Agrobacterium binary vector. In addition to the coding region, this fragment includes the complete AtDMC1 promoter, as defined by Klimyuk and Jones (1997), which could be of functional significance for the precise timing of DMC1 expression. One heterozygous gentamycin-resistant transformant (T1) was recovered among the seeds of the infiltrated plants, and its T2 progeny was analyzed.
Among 80 gentamycin-resistant T2 plants, none was found to display the reduced fertility phenotype of the dmc1 mutant. When 55 of these gentamycin-resistant T2 plants were tested by using PCR, they all proved to carry a wild-type AtDMC1 allele; we were not able to differentiate the transgenic Columbia allele from the resident Wassilewskija allele. Although fertile, 41 of these plants also allowed PCR amplification with primers specific for the disrupted dmc1 allele. In the absence of complementation, such association of the wild-type phenotype with the disrupted allele would be observed only at a frequency below 10-7 ([2/3]41). After using PCR to characterize 10 independent T3 progeny, we ascertained that some of the T2 fertile plants that carried a mutant dmc1 allele were homozygous for the dmc1 insertion (that is, the mutant allele was present in all of the T3 plants derived from two out of four single T2 plants tested).
Meiosis Is Disturbed in the dmc1-/- Mutant Plants
To further characterize the Arabidopsis dmc1 mutant, we studied meiosis in male and female Arabidopsis plants because the dmc1 mutation clearly is associated with meiotic defects in yeast and mouse. In Arabidopsis, meiosis takes place in young flower buds 1 mm in size. Chromosomes of pollen mother cells in anthers or of megaspore mother cells in ovules were examined after 4′,6-diamidino-2-phenylindole or propidium iodide staining, respectively, and are shown in Figures 4 and 5. Due to their diffuse state during the early stages of meiosis, chromosome behavior was followed essentially from the point at which they become fully condensed, that is, metaphase I.
The dmc1 Mutant Phenotype.
(A) dmc1 plants at flowering time. Wild-type plant at left; dmc1 plant at right.
(B) to (E) Dissected flowers and siliques.(B) and (D) show wild type, and (C) and (E) show dmc1 plants. Pollen release and silique size are reduced in the dmc1 mutant anthers.
(F) and (G) Alexander staining of anthers. (F) shows wild type, and (G) shows dmc1 anthers with little viable pollen of irregular shape.
The Arabidopsis genome consists of five pairs of chromosomes (2n = 10), which become associated into five bivalents at metaphase I. Both male and female metaphase I or early anaphase I chromosomes can be seen in wild-type pollen mother cells and megaspore mother cells as five fully condensed cooriented bivalents (Figures 4A and 5A). After metaphase I, anaphase I proceeds, with the bivalents segregating into two groups of five chromosomes (two haploid genomes), which migrate to opposite poles. Metaphase II ensues, which shows two groups of five condensed univalents. This pattern was clearly observed in wild-type pollen mother cells and megaspore mother cells and is shown in Figures 4B and 5B.
In dmc1 pollen mother cells and megaspore mother cells at metaphase I, the chromosomes generally appear as 10 independent but fully condensed univalents scattered throughout the cytoplasm (Figures 4D and 5C to 5F). We did not detect proper metaphase II in dmc1 meiocytes. Instead, the univalents eventually partitioned randomly (Figures 4E to 4G and 5G to 5I). The maximum number of independent chromosomal structures that we could observe was always 10 (sometimes fewer when some chromosomes seemed to have associated into bivalents). Such variants never were observed in wild-type cells. This aberrant chromosomal behavior made it difficult to precisely define the stages of meiosis I in dmc1 meiocytes. Meiotic cells were interpreted as being in pseudometaphase I if the univalents seemed to have aggregated or to be evenly spread throughout the cell. They were classified as being in pseudometaphase II if the 10 univalents had segregated into subgroups. Sometimes one or more univalents seemed to lag in the middle, whereas others migrated to opposite poles of the cell, which can result in up to three discernible groups of univalents. These so-called laggards have been observed in other meiotic Arabidopsis mutants (e.g., syn1, Peirson et al., 1997; dsy1, Ross et al., 1997).
After metaphase II, in wild-type meiocytes, the univalents undergo sister chromatid separation and initiate a new poleward movement (anaphase II). At this time, chromosomes reorganize into four groups of five chromosomes and start to decondense during diakinesis, while four independent cells reconstitute. In pollen mother cells, the second division is perpendicular to the first, thus resulting in four meiotic products set at the four poles of the cell that will turn into a tetrad (see Figure 4C; Ross et al., 1996; Peirson et al., 1997). On the other hand, in megaspore mother cells, the second division may be parallel or perpendicular to the first, resulting in tetrads with a linear or a planar array of megaspores (Schneitz et al., 1995).
Progeny from dmc1 Plants.
Eight-day-old seedlings were grown on Murashige and Skoog medium.
(A) Wild-type seedling.
(B), (C), and (G) Variations in cotyledon number.
(D) Lack of cotyledon development.
(E), (F), (I), and (J) Variations in cotyledon shape. (E) and (J) show variation in symmetry of cotyledons.
(C) and (H) Lack or retardation of root development.
In dmc1 male and female meiocytes, meiosis II proceeds after the irregular meiosis I. In dmc1 pollen mother cells, during the early cytokinesis stage (at which organelles are distributed throughout the cytoplasm), it is clear that tetrad organization is characterized by the unequal partitioning of chromosomes to each pole and by the appearance of more than four groups of chromosomes in some instances (see Figure 4H). An early anaphase II configuration in a dmc1 megaspore mother cell, for which a stereopicture could be drawn (data not shown), is shown in Figure 5J (detail in Figure 5K). In this megaspore mother cell, the chromosomes seem to be organized into three groups: in one group (bottom right), three univalents already are positioned at one pole and show no sign of sister chromatid separation; in a second group (upper left), two chromosomes undergoing sister chromatid separation are set at the opposite pole; and finally, in the third group (center), five aligned chromosomes can be observed at the cell’s equator with their separating sister chromatids orientating to the previously defined opposite poles. At this point, it cannot be established whether such univalents will effectively disjoin or move undivided toward the same pole or whether they will fail to migrate. In this example, it is also interesting that two distinct planes of meiosis II division can be defined (Figure 5K, upper left and center), although one group of chromosomes (bottom right) is excluded from this operation.
Meiosis in 4′,6-Diamidino-2-Phenylindole–Stained Pollen Mother Cells.
(A) to (C) Wild-type pollen mother cells. (A) shows wild-type metaphase I with five aligned bivalents. (B) shows wild-type metaphase II with two groups of five condensed chromosomes. (C) shows a wild-type tetrad at the end of telophase II with four polar nuclei.
(D) to (H) dmc1 pollen mother cells. Precise staging of the pollen mother cells was difficult in the dmc1 mutant due to the absence of standard features, such as bivalents or fixed numbers of chromosome groupings. (D) shows dmc1 pseudometaphase I with 10 univalents scattered across the pollen mother cell. (E) to (G) show dmc1 pseudometaphase II with 10 univalents randomly separated into two groups. A laggard is visible in (F). (H) shows a dmc1 early tetrad at the issue of telophase II with irregular amounts of DNA in the nuclei and more than four nuclei in one pollen mother cell (arrow).
Bar in (H) = 10 μm for (A) to (H).
Meiosis in Propidium Iodide–Stained Megaspore Mother Cells.
(A) and (B) show wild-type megaspore mother cells; (C) to (J) show dmc1 megaspore mother cells. (K) is an enlargement of (J).
(A) Wild-type early anaphase I showing five bivalents.
(B) Wild-type metaphase II with two groups of five condensed chromosomes.
(C) to (F) dmc1 pseudometaphase I (no obvious bivalents; no obvious segregation).
(G) to (I) dmc1 pseudometaphase II with chromosomes randomly separated into two or three groups.
(J) Early anaphase II; chromosomes in two of the three subgroups (upper left and center) start separating into their sister chromatids according to two independent planes.
(K) Detail of (J), at ×3 magnification, showing the chromosomes separating into their sister chromatids. Arrows indicate movement of sister chromatids, migrating to the opposite poles of the cell in a defined plane.
Bars in (A) to (J) = 10 μm.
Gametophytic Development in dmc1-/- Plants
In Arabidopsis, the haploid products resulting from meiosis undergo a number of mitotic divisions and reorganizations to finally produce functional gametophytes. All four microspores in a tetrad (Figure 6A) ultimately differentiate into trinucleate mature pollen grains. On the other hand, only one of the four megaspores resulting from female meiosis will develop into a functional megaspore that generates the final eight-nuclear embryo sac (Figure 6E) after three successive nuclear divisons. The other three megaspores degenerate at an early stage. Layers of integuments derived from the parental sporophyte grow around the organizing embryo sac until a complete, mature ovule is formed. Integument growth progression currently is used as the criterion to determine the stage of megagametogenesis (Schneitz et al., 1995).
In dmc1 anthers, tetrads contain variable numbers of heterogeneously sized microspores (Figure 6B) instead of the usual four (Figure 6A). Although young anthers from dmc1 plants contain as many microspores as do wild-type anthers (Figures 6C and 6D), pollen release is largely reduced in dmc1 plants (see Figures 2B and 2C). In the majority of ovules from dmc1 plants, gametogenesis does not result in a normal eight-nuclear embryo sac (Figure 6E). Most of the megaspore mother cells of the mutant arrest just after meiosis, with only degenerated cells being visible (Figure 6F); in a few cells, partial differentiation of the embryo sac takes place (in these megaspore mother cells, two nuclei are visible; data not shown). However, there are some ovules in which a functional embryo sac is present because fertility is not totally abolished in dmc1 mutant plants. During the early stages of megagametogenesis, integument growth around the female gametophyte is normal in mutant plants, allowing for the precise staging of the ovules (Figures 6E and 6F).
Fertilization Events in dmc1-/- Plants
Callose deposition is associated with pollen tubes as they make their way through the pistil toward the ovules before fertilization (Hulskamp et al., 1995). Callose in plant cells can be visualized by staining with aniline blue, which results in a bright fluorescent product, as shown in Figure 7. Slightly before fertilization (Figures 7A and 7B), a strong aniline blue fluorescence signal is observable in the dmc1 ovules, whereas no such signal can be detected in wild-type ovules of the same age (as ascertained by their identical size). Natural pollen release and germination also were examined. Figure 7D shows that by contrast to the wild type (Figure 7C) very few dmc1 pollen grains germinate on dmc1 stigmas. When wild-type pollen is deposited manually on dmc1 stigmas, the pollen readily germinates (data not shown). This confirms that the floral reproductive organs, which are derived from the parental sporophyte, are not functionally impaired by the dmc1 mutation. Although no fluorescence was detected in wild-type ovules (Figures 7E and 7G), bright fluorescent spots often were visible at fertilization in dmc1 ovules, which appeared to be of varied sizes (Figures 7F and 7H). Some rare fertilization events are visible in this last example (wild-type pollen × dmc1 pistil; Figure 7H). But, interestingly, other pollen tubes also can be observed to grow in an erratic manner, seemingly incapable of penetrating the ovules (see Figure 7H, bottom arrow).
Female and Male Gametogenesis in the Ovules and Anthers of Wild-Type and dmc1 Plants.
(A) and (B) Microspore tetrads. In the wild type, shown in (A), four microspores are grouped in tetrads. In the dmc1 mutant, shown in (B), tetrads are composed of two to six microspores (arrow) of irregular sizes. Bars in (A) and (B) = 10 μm.
(C) and (D) At a later stage, the released microspores are regular in the wild type ([C]) but heterogeneous in size and shape in the dmc1 ([D]) anthers. Bars in (C) and (D) = 28 μm.
(E) and (F) Wild-type and dmc1 ovules containing embryo sacs. Shape and integument development are indicative of identical developmental stages. In the wild type, shown in (E), a functional embryo sac with the egg cell (ec) and the central cell (cc; 2 nuclei) are visible. In the dmc1 ovules at the same stage, shown in (F), only the degenerated cells (dc) are visible. Bars in (E) and (F) = 50 μm.
DISCUSSION
The Arabidopsis dmc1 mutant is a novel meiotic mutant that was isolated by using reverse genetics. That the same meiotic defect affects both male and female sporogenesis in dmc1 plants supports the hypothesis that DMC1 possesses a central and fundamental function in plant meiosis, as was previously shown for the yeast (Bishop et al., 1992) and mouse (Pittman et al., 1998; Yoshida et al., 1998) homologs. Other mutations that might disturb general meiotic function have been described in Arabidopsis that are both male and female partial sterile, but female meiosis was not cytologically examined (Peirson et al., 1997; Ross et al., 1997). Furthermore, none of the genes responsible for these sterility phenotypes has been molecularly characterized.
Meiosis is profoundly disturbed in Arabidopsis dmc1 meiocytes. Ten univalents were regularly found in mutant pollen mother cells and megaspore mother cells undergoing meiosis instead of the expected five metaphase I bivalents. All ensuing aberrant behavior of Arabidopsis dmc1 meiotic chromosomes is likely to result from this initial failure to constitute bivalents. In yeast dmc1 mutants, interhomolog recombination is markedly reduced, and synaptonemal complex establishment is strongly delayed (Bishop et al., 1992; Rockmill et al., 1995). Similarly, in mouse dmc1 mutants, mature synaptonemal complexes cannot be detected, and meiosis arrest occurs at zygotene (Pittman et al., 1998; Yoshida et al., 1998). Bivalent stabilization is essential to correct segregation and depends on the presence of chiasmata, which are structures resulting from crossover recombinational events between pairs of homologous chromosomes (Carpenter, 1994). In prophase I of meiosis, Dmc1 specifically participates in interhomolog recombination, which results in bivalent stabilization and chiasma formation. Our cytogenetical observations indicate that the Dmc1 protein is necessary for bivalent formation and/or stabilization at meiosis.
The characteristics of the Arabidopsis dmc1 mutant— reduced fertility and disturbed meiosis—argue that the Dmc1 function is conserved in plants. Nevertheless, the meiotic disorders caused by the dmc1 mutation do not lead to the same severe phenotype in Arabidopsis as in mouse or in some yeast strains. In particular, meiosis is completed in the Arabidopsis mutants, whereas dmc1 mutations can trigger meiotic arrest during early prophase in yeast and meiotic arrest and apoptotic cell death in mouse (Bishop et al., 1992; Pittman et al., 1998; Yoshida et al., 1998). Meiosis arrest is also not observed in the dmc1 mutants of Rockmill et al. (1995), but, although delayed, chromosome synapsis occurs in these strains. The absence of bivalents excludes the idea that efficient synapsis takes place in dmc1 plants. Our observations may highlight some aspects of meiosis that are unique to Arabidopsis or plants in general. Meiosis completion is not specific to the Arabidopsis dmc1 mutant; other aberrant meioses have been described for plant mutants, and they all seem to go through meiosis without arresting (Neuffer et al., 1997; Peirson et al., 1997; Ross et al., 1997).
In yeast, the DSBs that initiate meiotic recombination are repaired by a Dmc1-dependent homologous recombination process, and this conditions further progression of the cells through meiosis. Dmc1 mutants that do not form DSBs do not undergo meiotic arrest (Bishop et al., 1992). The persistence of unrepaired DSBs in dmc1 mutants probably causes meiotic arrest by triggering specific checkpoint functions (mediated by the MEC1, RAD17, or RAD24 genes; Lydall et al., 1996). Similar checkpoint genes may not exist or be as stringent in Arabidopsis. When meiosis is allowed to proceed in a yeast checkpoint dmc1 double mutant, DNA is found fragmented in the meiotic products, which may be a consequence of unrepaired DSBs (Lydall et al., 1996). Whereas two mutants have been described in Arabidopsis that exhibit early meiotic chromosome fragmentation (syn1, Peirson et al., 1997; mcd1, Ross et al., 1997), we did not observe fragmented DNA. Consequently, we suggest that a consistent signal for meiotic arrest, that is, a DNA lesion, is not present in the Arabidopsis dmc1 mutant.
Two hypotheses might explain how meiosis might be completed without chromosome breakage in the Arabidopsis dmc1 mutant: either no meiotic DSBs are formed in the dmc1 mutant or DSBs are created but efficiently repaired. Meiotic DSBs are specifically created in yeast by Spo11, a type II topoisomerase (Bergerat et al., 1997; Keeney et al., 1997). A requirement for Spo11-dependent DSB formation to initiate meiotic recombination extends to multicellular organisms because, as in yeast, radiation-induced DNA breaks can partially replace Spo11, thereby restoring proper chromosome segregation at meiosis in Caenorhabditis elegans (Thorne and Byers, 1993; Dernburg et al., 1998). A potential SPO11 homolog is present in the rice genome (GenBank accession number AQ159537).
It is possible that DSBs are not formed in Arabidopsis dmc1 plants. We think it unlikely that Dmc1 directly promotes the formation of DSBs because this has not been observed for Dmc1 or any RecA homologs in other species. As for the second hypothesis, if meiotic DSBs are created in the Arabidopsis dmc1 mutant, they are not left unrepaired. Two pathways are known to be involved in DNA DSB repair: one relies on homologous recombination, and the other acts via DNA end joining (Moore and Haber, 1996; Kanaar et al., 1998). DSB repair via homologous recombination with the sister chromatid could be mediated by Arabidopsis Rad51 and associated proteins, in which case the broken chromosome would be healed but no chiasmata would be established. Indeed, a way to rescue meiotically arrested yeast dmc1 mutants is to return them to vegetative growth. The idea is that at meiosis, DSB repair is forced to the homolog only (via Dmc1 or some Dmc1 cofactor), whereas at mitosis, it is allowed to proceed from the sister chromatid (via Rad51 and/or other DSB repair proteins; Schwacha and Kleckner, 1997; Shinohara et al., 1997; Zenvirth et al., 1997). Repair from the sister chromatid is similarly invoked to explain meiotic progression of some yeast dmc1 mutants (Rockmill et al., 1995).
It is known that in higher eukaryotes, more than in yeast, DNA end joining significantly participates in DSB repair (Liang et al., 1998; Salomon and Puchta, 1998; Takata et al., 1998). Any of these pathways could provide a means for repairing chromosomes at meiosis in Arabidopsis. Consistent with this idea, AtRAD51 is transcriptionally activated both after γ ray–induced DNA damage and in dmc1 flowers (Doutriaux et al., 1998).
Aniline Blue Staining of Callose in the Ovules and Pollen Tubes.
(A) and (B) Just before pollen release, in the wild-type ovules and anthers shown in (A), no fluorescence is visible; in the dmc1 mutant, shown in (B), numerous callose clusters are visible in the ovules and anthers.
(C) and (D) When pollen is released, the stigma is covered with germinating pollen grains in the self-fertilized wild-type plants shown in (C). In self-fertilized dmc1 flowers, shown in (D), only a few pollen reach the stigma and germinate.
(E) and (F) Mature wild-type and dmc1 ovules are shown at a higher magnification. In wild-type ovules, a fertilizing pollen tube is visible (arrow in [E]); in most dmc1 ovules, irregular callose clusters (arrows in [F]) are present.
(G) and (H) Artificial pollination with wild-type pollen. In (G), wild-type pollen is deposited on the wild-type pistil. In (H), wild-type pollen is deposited on dmc1 pistils. Aniline blue staining reveals the pollen tubes as they grow toward the ovules. In (G), most wild-type ovules are directly reached by a pollen tube (arrows) emerging perpendicular to the ovary axis. In (H), wild-type pollen tube growth is erratic in the dmc1 pistil. The only ovules to be fertilized are devoid of fluorescence and are well developed (top arrows show fertilization of regular-looking ovules; the bottom arrow shows a pollen tube turning around an unfit ovule without fertilizing it).
Bars in (A) to (D), (G), and (H) = 100 μm; bars in (E) and (F) = 34 μm.
The meiotic products issued from an aberrant meiosis cannot contain a normal complement of chromosomes. This likely explains why microspores produced by the dmc1 anthers are heterogeneous in size, which is a common characteristic of meiotic mutants and is considered to reflect variable chromosomal DNA content. Haplosufficiency is considered critical for the development of a functional gametophyte from the microspores and megaspores (Chasan and Walbot, 1993). Whereas young dmc1 anthers contain as many microspores as do wild-type anthers, only a few pollen grains survive to maturity in mutant anthers.
We also observed a strong clustering of callose in most of the mutant ovules at the time of fertilization. Callose deposition has been proposed to be associated with cell death in plants (Mittler and Lam, 1995; Ray et al., 1997). If cell death or arrest does in fact occur in dmc1 plants, it occurs after the completion of meiosis. Therefore, fertilization would involve only gametophytes with favorable ploidies, as exemplified by the patterns of fertilization that we observe in mutant pistils. It has been proposed that the embryo sac guides pollen tube movements by chemotaxis (Ray et al., 1997). In dmc1 plants, the only ovules that are penetrated by a pollen tube must be those that contain a functional embryo sac.
It is intriguing that a relatively high level of viable progeny is produced by the Arabidopsis dmc1 mutant. How can chromosomes that did not form bivalents partition correctly? The residual fertility in the dmc1 mutant (1.5%) closely correlates with the random segregation of five (n = 5) chromosomes (P n = 5 = [1/2]5 = 3%). This excludes that massive lethality or arrest would result from a failure of bivalent formation per se. Therefore, its low chromosome number offers a pathway for Arabidopsis to partly escape the consequences of a dmc1 mutation. The altered progeny issued from dmc1 mutant plants would thus represent various surviving aneuploids. Yeast, on the other hand, with 16 chromosome pairs, is much less likely to end up with viable spores after their random segregation.
METHODS
Strains
Arabidopsis ecotype Wassilewskaja T3 (F2) seeds of the T-DNA–transformed line 3668 from Feldmann’s collection (Feldmann, 1991) were provided by the Arabidopsis Biological Resource Center (Columbus, OH). Plants were grown in a greenhouse under standard conditions, although they were closely protected and allowed to self-fertilize. Flower buds for meiotic tissue examination were picked early in the morning on sunny days.
Nucleic Acid Procedures
For plant genotype determination, DNA for polymerase chain reactions (PCRs) was ethanol precipitated from the supernatant of leaf fragments homogenized in a buffer consisting of 200 mM Tris-HCl, pH 7.5, 250 mM NaCl, 25 mM EDTA, and 0.5% SDS. PCR conditions were 94°C for 2 min followed by 30 cycles (94°C for 30 sec, 60°C for 30 sec, 72°C for 1 min) in the presence of Tfl DNA polymerase (Promega) and according to the manufacturer’s protocol. Primer sequences are as follows: primer a, 5′-CCTGCAATGGTCTCATGATGCATAC-3′; primer b, 5′-GATGCAATCGATATCAGCCAATTTTAGAC-3′ (LB102, as in McKinney et al., 1995); primer c, 5 ′-AGGTACTCTGTCTCTCAATG-3′; and primer d, 5′-ACTAATCCTTCGCGTCAGCAATGC-3′.
For RNA gel blot analysis, RNA was extracted in Trizol (Gibco BRL), according to the manufacturer’s protocol. Twenty-five micrograms of total RNA was loaded on a denaturing gel. Electrophoresis, transfer to Hybond N+ membranes (Amersham), and 32P radiolabeling of probes were done according to standard techniques (Sambrook et al., 1989). Hybridization was with the following probes: the AtDMC1 coding sequence, the AtRAD51 coding sequence, and the bean translation elongation factor EF-1α cDNA (Doutriaux et al., 1998). The complementing construct was derived from a 14-kb AtDMC1 genomic sequence (including 7.5 kb of 5′ sequences upstream of the ATG codon), isolated from a previously described genomic library (Doutriaux et al., 1998). An 11-kb HpaI-SalI fragment containing the complete promoter and coding sequence of AtDMC1 was subcloned into a SmaI-SalI restriction-digested pPZP121 binary vector (Hajdukiewicz et al., 1994). The final construct was electroporated into Agrobacterium LBA4404 strain for plant transformation (Bevan, 1984).
Plant Transformation
Plant transformation was performed as described previously (Bechtold et al., 1993). Only plants predetermined as heterozygous for the AtDMC1 insertional mutation were transformed. Seeds from the Agrobacterium-treated plants were selected on Murashige and Skoog medium containing 100 mg L-1 gentamycin (Carrer et al., 1991). Gentamycin-resistant plantlets (T1) were transfered to the greenhouse and then characterized by using PCR. Seeds from one T1 transformed plant that was PCR-determined as heterozygous were harvested and grown to establish the fertility or sterility phenotype of its T2 progeny.
Cytological Analysis
Pollen viability was assayed by Alexander staining (Alexander, 1969). Microspores were cleared (Herr, 1971) and then observed by using differential interference contrast microscopy. Ovule preparation for differential interference contrast microscopy was as follows: pistils were fixed in FPA50 (formaldehyde/propionic acid/50% ethanol, 5:5:90 [v/v]) for 3 hr at 20°C and then cleared in LAP (85% lactic acid/phenol, 2:1 [v/v]) for 30 min at 20°C before mounting in a drop of LAP. Aniline blue staining of callose in the pistils was done according to Ruffio-Chable (1992) before observation by fluorescence microscopy (BP 340-380 excitation filter and LP 430 barrier filter; Leitz, Wetzlar, Germany).
Chromosomes in pollen mother cells were observed by fluorescence microscopy (Axiophot, Zeiss, Germany) after 4′,6-diamidino-2-phenylindole staining (Maluszynska and Heslop-Harrison, 1991). Chromosomes in megaspore mother cells were examined after propidium iodide treatment, as described by Clark et al. (1993), except for the following modifications. Pistils were treated for 30 min at 37°C with 20 μg mL-1 RNase A (Boehringer Mannheim) in 2.5 M NaCl, 50 mM Tris-HCl, and 5 mM EDTA, pH 8.0, and then stained in propidium iodide (Sigma) 5 mg mL-1 in 0.1 M arginine (Research Organics Inc., Cleveland, OH), pH 12.4, at 4°C overnight. Pistils then were washed twice in 0.1 M arginine, pH 8.0, and dissected to free the ovules. The slides were prepared by adding a drop of citifluor glycerol/PBS (Oxford Institute, Orsay, France). Observations were performed on a Leica TCS-NT confocal laser scanning microscope (Leica Microsystems, Heidelberg, Germany) with an argon/krypton laser (Omnichrome, Chino, CA), BP 568 excitation filter, and LP 590 barrier filter. Slow-scan (220 lines per sec) images were generated using a 40×/1.00 to 0.50 PL Fluotar objective combined with an electronic zoom (Leica Microsystems). Chromosomes in megaspore mother cells were examined with a maximum projection of optical sections.
Graphics
Slides were scanned with a Polaroid 35 LE Sprint Scanner. Pictures were processed using Adobe Photoshop 4.0 and Power Point 4.0. Printouts were generated on an Epson color printer.
Acknowledgments
We are grateful to Drs. Raymond Devoret, Kathleen Smith, Alain Tissier, and Denise Zickler for critical reading of the manuscript; members of Dr. Catherine Bergounioux’s laboratory for helpful discussions; Dr. Bernard Hugueny for his contribution; Thi Hai Phan for excellent technical assistance; Dr. Kenneth Feldmann for providing access to the T-DNA mutant collection; and Dr. Pal Maliga for kindly providing the pPZP binary vectors. We also thank Jean-Paul Bares and Gilles Santé for excellent plant maintenance and Roland Boyer for photographic work. This work was supported by Centre National de la Recherche Scientifique, Rhône-Poulenc, Biogemma and Tepral (M.-P.D. and F.C.), Institut National de la Recherche Agronomique (C.H., O.G., and D.V.) and Natural Sciences and Engineering Research Council of Canada (F.B.). F.C. is the recipient of a Ministère de l’Education Nationale, de la Recherche et de la Technologie fellowship.
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