Reduced meiotic crossovers and delayed prophase I progression in AtMLH3-deficient Arabidopsis
2006; Springer Nature; Volume: 25; Issue: 6 Linguagem: Inglês
10.1038/sj.emboj.7600992
ISSN1460-2075
AutoresNeil Jackson, Eugenio Sánchez‐Morán, Ewen F. Buckling, Susan J. Armstrong, Gareth H. Jones, F. Chris H. Franklin,
Tópico(s)RNA Research and Splicing
ResumoArticle9 February 2006free access Reduced meiotic crossovers and delayed prophase I progression in AtMLH3-deficient Arabidopsis Neil Jackson Neil Jackson Search for more papers by this author Eugenio Sanchez-Moran Eugenio Sanchez-Moran Search for more papers by this author Ewen Buckling Ewen Buckling Search for more papers by this author Susan J Armstrong Susan J Armstrong Search for more papers by this author Gareth H Jones Gareth H Jones Search for more papers by this author Frederick Christopher Hugh Franklin Corresponding Author Frederick Christopher Hugh Franklin The School of Biosciences, University of Birmingham, Edgbaston, Birmingham, UK Search for more papers by this author Neil Jackson Neil Jackson Search for more papers by this author Eugenio Sanchez-Moran Eugenio Sanchez-Moran Search for more papers by this author Ewen Buckling Ewen Buckling Search for more papers by this author Susan J Armstrong Susan J Armstrong Search for more papers by this author Gareth H Jones Gareth H Jones Search for more papers by this author Frederick Christopher Hugh Franklin Corresponding Author Frederick Christopher Hugh Franklin The School of Biosciences, University of Birmingham, Edgbaston, Birmingham, UK Search for more papers by this author Author Information Neil Jackson‡, Eugenio Sanchez-Moran‡, Ewen Buckling, Susan J Armstrong, Gareth H Jones and Frederick Christopher Hugh Franklin 1 1The School of Biosciences, University of Birmingham, Edgbaston, Birmingham, UK ‡These authors contributed equally to this work *Corresponding author. The School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK. Tel.:+44 121 414 5910; Fax: +44 121 414 5925; E-mail: [email protected] The EMBO Journal (2006)25:1315-1323https://doi.org/10.1038/sj.emboj.7600992 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Characterization of AtMLH3, the Arabidopsis homologue of the prokaryotic MutL mismatch repair gene, reveals that it is expressed in reproductive tissue where it is required for normal levels of meiotic crossovers (COs). Immunocytological studies in an Atmlh3 mutant indicate that chromosome pairing and synapsis proceed with normal distribution of the early recombination pathway proteins. Localization of the MutS homologue AtMSH4 occurs, suggesting that double Holliday junctions (dHjs) are formed, but the MutL homologue AtMLH1, which forms a heterocomplex with AtMLH3, fails to localize normally. Loss of AtMLH3 results in an ∼60% reduction in COs and is accompanied by a substantial delay of ∼25 h in prophase I progression. Analysis of the chiasma distribution in Atmlh3 suggests that dHj resolution can occur, but in contrast to wild type where most or all dHjs are directed to form COs the outcome is biased in favour of a non-CO outcome by a ratio of around 2 to 1. The data are compatible with a model whereby the MutL complex imposes a dHj conformation that ensures CO formation. Introduction The eukaryotic homologues of the Escherichia coli MutS and MutL mismatch repair (MMR) proteins play important roles in maintaining genome stability during both mitosis and meiosis (Kolodner and Marsischky, 1999; Hoffmann and Borts, 2004; Svetlanov and Cohen, 2004). Studies in yeast have identified four MutL homologues that form functionally distinct heterodimers. Two of these, Mlh1/Pms1 and Mlh1/Mlh2, are proposed to have roles in the correction of different classes of DNA mismatch, whereas the Mlh1/Mlh3 heterodimer appears to play an important role in promoting meiotic crossovers (COs) (Wang et al, 1999). Studies in Mlh1-deficient yeast have revealed that they exhibit reduced spore viability and a reduction in COs such that map distance in an interval on chromosome III is reduced by 21–33% (Wang et al, 1999). Mouse knockouts in MLH1 disrupt meiotic recombination in both male and female animals, resulting in the formation of unpaired univalent chromosomes at the first meiotic division (Baker et al, 1996; Woods et al, 1999). The early stages of prophase I appear normal in the knockout mice, but as the synaptonemal complex disassembles at the onset of diplotene the absence of chiasmata is revealed. An mlh3 mouse knockout has a similar, although not identical effect on meiotic progression to that of the mlh1 knockout (Lipkin et al, 2002). Chromosome synapsis is normal, but very few bivalents persist beyond pachytene. However, in contrast to the mlh1 knockout where spermatocyte apoptosis is induced swiftly at diplotene, a substantial proportion of mlh3 knockout spermatocytes progress through to metaphase I/anaphase I, where following chromosome missegregation apoptosis occurs. Female mice are infertile, failing to complete meiosis I after fertilization. In accord with these findings, immunolocalization studies using light and electron microscopy have revealed that MLH1 and MLH3 proteins colocalize as foci on mouse chromosomes during pachytene and that their distribution is consistent with each being a component of the late recombination nodules (RNs) (Moens et al, 2002; Marcon and Moens, 2003). Moreover a recent study, in which the phosphatase inhibitor okadaic acid was used to induce the precocious onset of diplotene in mouse spermatocytes, succeeded in demonstrating that the MLH1/MLH3-containing RNs are localized to the chiasmata (Marcon and Moens, 2003). Studies of MMR genes in Arabidopsis thaliana have identified several homologues of the E. coli MutS gene, namely AtMSH2, AtMSH3, AtMSH4, AtMSH5, AtMSH6-1, AtMSH6-2 and AtMSH7 (Ade et al, 1999; Culligan and Hays, 2000). Of the Arabidopsis MutL homologues, AtMLH1 has been studied to a limited extent (Jean et al, 1999). The predicted protein shares 37% identity, 55% similarity over its entire length to MLH1 from mouse and 31% identity, 48% similarity to the yeast protein. However, a corresponding mutant was not isolated, hence a functional analysis of this gene was not conducted. In addition to AtMLH1, the study revealed two other MutL homologues in the Arabidopsis genome. Phylogenetic analysis indicated that one of these is likely to be the Arabidopsis homologue of PMS1. Although the other, originally designated AtMLHx, was also considered to be a member of the PMS1 family, it appeared somewhat distinct, and was placed in an intermediate position between PMS1 and MLH1 (Jean et al, 1999). Recently, AtMLHx has been redesignated as AtMLH3 (Figure 1A) (Alou et al, 2004). The AtMLH3 protein contains a predicted MutL domain in the NH2-terminal region of the protein, which exhibits 32% amino-acid identity over a region of 228 aa to that present in the Arabidopsis AtMLH1 protein. However, AtMLH3 (1151 aa) is considerably larger than MLH1 (737 aa) and contains an additional MutL domain. This does not exhibit any significant homology to the other MutL domain in the protein or with that in AtMLH1. However, it shares significant (37%) similarity with the MutL domain found in the mouse MLH3 protein. Phylogenetic analysis clearly places AtMLH3 within the MLH3 group (Alou et al, 2004). Despite discrepancies in their sizes, the mouse and yeast MLH3 proteins (192 and 715 aa, respectively) are thought to perform a similar role in meiotic recombination. Hence, this suggests that although AtMLH3 is larger than either, it might also function in meiosis. The work described below has revealed that AtMLH3 is specifically expressed in reproductive tissues of Arabidopsis and that the protein localizes to foci associated with the chromosome axes during prophase I of meiosis. Analysis of two independent T-DNA insertion mutants of the gene confirms a role in the formation of meiotic COs and provides new insight into the role played by the MutL homologues in CO/non-CO resolution of double Holliday junctions (dHjs). Figure 1.(A) Map of the 6.3 kb At4g35520 locus showing the exon/intron organization of AtMLH3. The exons are shown as numbered black boxes. The triangles indicate the T-DNA insertion sites in Atmlh3-1 and Atmlh3-2. (B) RT–PCR expression analysis revealing that in contrast to AtMLH1, expression of AtMLH3 is restricted to reproductive tissue. Download figure Download PowerPoint Results AtMLH3 is expressed in reproductive tissue Studies in yeast and mouse indicate that MLH3 and MLH1 function as a heterocomplex during meiosis, but unlike MLH1, the MLH3 protein is thought to have only a limited role in DNA MMR in mitotic cells (Flores-Rozas and Kolodner, 1998; Kolas and Cohen, 2004). This suggested that expression of an Arabidopsis homologue of MLH3 may be restricted to or more abundant in reproductive tissue. To explore this possibility, we carried out RT–PCR using AtMLH3- and AtMLH1-specific primers with mRNA from a range of plant tissues (Figure 1B). This clearly revealed that AtMLH3 was expressed in bud tissue but was not detectable in vegetative tissues, whereas expression of AtMLH1 was detected in all the tissues tested. This finding is consistent with a role for AtMLH3 during meiosis in Arabidopsis, but cannot be absolutely definitive, as buds are comprised of a mixture of reproductive and vegetative cells. AtMLH3 localizes to meiotic chromosome axes in mid–late prophase I An antibody was raised to a 347 aa region comprising residues 804–1151 of the AtMLH3 protein. FITC-labelled anti-AtMLH3 Ab was used to immunolocalize AtMLH3 on meiotic chromosome spreads prepared from Arabidopsis pollen mother cells (PMCs) at different stages of meiosis (Figure 2A–C). In order to accurately establish when AtMLH3 is initially detectable, dual immunolocalization was performed with antibodies that recognize the meiotic proteins ASY1, AtMSH4 and AtMLH1, which may be used to monitor prophase I progression from early leptotene through to pachytene (Armstrong et al, 2002; Higgins et al, 2004, 2005). ASY1 is associated with the chromosome axes and its first appearance identifies the onset of leptotene (Armstrong et al, 2002). At this stage, AtMLH3 was not detectable. As the meiocytes progressed to zygotene, AtMLH3 foci gradually appeared. At pachytene, the mean number of foci per nucleus was 9.4 (n=42), which is in close agreement with the mean chiasmata frequency of 9.86 previously reported for Arabidopsis (Higgins et al, 2004). The AtMLH3 foci continued to be detectable throughout pachytene, where they were found to colocalize with AtMLH1 (Figure 2D–F). The foci remained present as the chromosomes began to desynapse during diplotene, but by metaphase I they could no longer be detected. Thus, the timing and frequency of the AtMLH3 foci are consistent with a role for the protein in the later stages of meiotic recombination. Figure 2.(A–C) Dual immunolocalization of AtMLH3 (red) and ASY1 (green). (A) At leptotene, ASY1 is localized to the developing chromosome axes. (B) AtMLH3 foci first become detectable during zygotene. (C) At pachytene 9–10, AtMLH3 foci are found in association with the chromosome axes. (D–F) Colocalization of the MutL homologues at pachytene. (D) AtMLH3 (red), (E) AtMLH1 (green) and (F) merged image. (G–I) Limited colocalization of AtMLH3 and AtMSH4. At early zygotene, AtMSH4 foci (red) are abundant with few AtMLH3 foci (green) detectable (G). At mid-zygotene (H) through to late zygotene/early pachytene (I), there is an increase in AtMLH3 foci and a reduction in AtMSH4 foci. Colocalization between the foci is consistently observed, but is limited to only few foci (1–2) per nucleus. Bar=10 μm. Download figure Download PowerPoint Dual-immunolocalization studies were conducted to establish the relationship of AtMLH3 and AtMSH4 during zygotene (Figure 2G–I). At early zygotene, numerous AtMSH4 foci are detected with few if any AtMLH3 foci present. During mid-zygotene through to late zygotene/early pachytene, there was a continual decrease in the number of AtMSH4 foci, whereas the number of AtMLH3 foci increased to ∼10 per nucleus. Although most AtMLH3/AtMSH4 foci did not appear to colocalize, we consistently observed colocalization of 1–2 foci in each nucleus (N=20). AtMLH3-deficient Arabidopsis exhibit reduced fertility and meiotic defects To determine if AtMLH3 was required for meiosis, we identified two Atmlh3 mutant lines among the Salk Institute T-DNA insertion collection (Figure 1A). The position of the T-DNA within AtMLH3 was determined in each case using PCR and nucleotide sequencing. The first line, Atmlh3-1, was found to carry a T-DNA insert 114 bp into exon 9 of the gene. Cytological analysis using fluorescence in situ hybridization (FISH) with a T-DNA probe indicated that in addition to the insertion on chromosome 4 in AtMLH3 the line possessed a second insertion on chromosome 5. Crosses were therefore made to obtain a single insertion line, which was confirmed by FISH (Supplementary Figure 1). As it is predicted that an insertion in exon 9 would result in a truncation of the AtMLH3 protein to an NH2-terminal peptide of just 262 aa of the total 1151 aa, it seems likely that Atmlh3-1 is a null mutant. This is supported by the absence of the corresponding RNA transcript and protein, as determined by RT–PCR (data not shown) and immunolocalization respectively (Supplementary Figure 2A). Analysis of the second line (Atmlh3-2) revealed that the T-DNA was inserted within the coding region, 279 bp from the 3′ end of the gene. Although this mutation could have resulted in the production of a truncated AtMLH3 protein, immunolocalization using anti-MLH3 Ab failed to detect any evidence of residual protein during prophase I (Supplementary Figure 2B). Vegetative growth of both T-DNA insertion mutants was indistinguishable from wild-type plants, with no apparent somatic abnormalities. However, differences became apparent when the plants began to set seed. The wild-type plants developed normal length siliques of a more or less consistent length containing an average of 52.8 seeds (n=32 siliques), whereas Atmlh3-1 and Atmlh3-2 siliques contained means of 26 (n=50 siliques) and 22.8 (n=32) seeds, respectively. A reduced fertility phenotype is typical of that found for other meiotic mutants in Arabidopsis (Caryl et al, 2003), although in those characterized to date the reduction in seed set is more dramatic than that observed for the Atmlh3 mutant lines. Nevertheless, this observation further suggested a role for AtMLH3 in meiosis. Cytological analysis of Atmlh3-deficient lines reveals defects in recombination The reduced fertility phenotype of the mutant lines is consistent with a role for AtMLH3 in meiosis. To investigate this further, meiotic chromosome spreads were prepared using PMCs isolated from the mutants Atmlh3-1 and Atmlh3-2 and a wild-type control. These were then examined using fluorescence microscopy. Figure 3 shows a cytological profile of Atmlh3-1. Early prophase I from leptotene through to pachytene was indistinguishable from wild type with apparently normal chromosome pairing, leading to full synapsis at pachytene (Figure 3A). However, as the chromosomes desynapsed towards the end of prophase I and began to condense during late diplotene/diakinesis, it became clear that a proportion (see later for details) of the homologous chromosome pairs lack chiasmata and are present as univalents at metaphase I (Figure 3B and C). This resulted in missegregation at the first meiotic division, leading to the formation of dyads containing aberrant chromosome numbers. This in turn resulted in aneuploid tetrads following the second meiotic division (Figure 3D). Analysis of mlh3-2 revealed an identical effect on meiosis with a reduction in the average number of meiotic COs leading to aneuploidy at the second division (Supplementary Figure 3). These observations clearly demonstrate that AtMLH3 is required for normal progression through meiosis and thus plays a crucial role in meiosis. Figure 3.(A–D) Cytological analysis of Atmlh3-1. Prophase I appears normal with fully synapsed homologous chromosomes at pachytene (A). However, as the chromosomes desynapse at the end of prophase I and begin to condense during diplotene/diakinesis (B) before aligning at metaphase I (C), the presence of univalents becomes clear. This leads to subsequent missegregation and the formation of unbalanced tetrads (D). (E–H) Complementation of Atmlh3-1 with a genomic fragment encoding AtMLH3 results in the restoration of normal meiosis. Fully synapsed chromosomes are observed at pachytene (E) with five bivalents at diplotene/diakinesis and metaphase I (F, G) leading to the formation of balanced tetrads containing five chromosomes (H). (I–L) Crossing Atmlh3-1 and Atmlh3-2 fails to restore normal meiosis, indicating that the mutants are allelic. Cytologically, the line is indistinguishable from the parental single knockout lines, apparently normal at pachytene (I) but with univalents at diplotene/diakinesis and metaphase I (J, K) leading to missegregation at the dyad stage. Bar=10 μm. Download figure Download PowerPoint Complementation of Atmlh3-1 and allelism with Atmlh3-2 To confirm that mutation of the AtMLH3 gene was responsible for the reduced fertility phenotype, an 11.42 kb genomic XbaI fragment encoding the full-length gene was cloned from BAC F8D20 into pCAMBIA 1302. The construct was then transformed into Atmlh3-1. Transformed lines were selected on hygromycin medium and transferred to soil. Analysis of 50 independent transformants revealed that fertility was fully restored (50–55 seeds/silique) (Supplementary Figure 4). Cytological analysis of two lines revealed that meiosis was entirely normal (Figure 3E–H). In addition to the complementation test, an allelism test was carried out by reciprocally crossing heterozygous Atmlh3-1 and Atmlh3-2 lines. All the progeny genotyped as Atmlh3-1/Atmlh3-2 (N=12) exhibited a reduced fertility phenotype and univalent chromosomes were present at metaphase I (Figure 3I–L and Supplementary Figure 4) Immunolocalization of meiotic proteins in Atmlh3 mutants To further characterize the effect of the Atmlh3 mutation on meiosis, fluorescence immunolocalization studies were carried out on spread preparations of PMCs at prophase I from the Atmlh3 mutants. The localization of ASY1 and the synaptonemal complex transverse filament protein ZYP1 (Higgins et al, 2005) in Atmlh3-1 was indistinguishable from that in wild-type PMCs, leading us to believe that axis formation, chromosome pairing and synapsis are unaffected in the mutant (Figure 4A and B; the results for Atmlh3-2 were identical and are presented in Supplementary Figure 2C and D). This finding suggested that early steps in the recombination pathway proceed as normal in Atmlh3-1. This was supported by the observation that distribution of AtRAD51, a component of early RNs, was unaltered in the mutant line. Immunolocalization using anti-AtRAD51 Ab revealed that the protein is first detectable as numerous punctate foci associated with the developing chromosome axes during early leptotene (Figure 4C). These remain throughout zygotene/early pachytene until mid-pachytene when they rapidly decline in number. The disappearance of the AtRAD51 foci is associated with the removal of the early RNs and the emergence of the late RNs that correspond to sites of the chiasmata (Moens et al, 2002). Although not proven, it is postulated that the late RNs represent a subset of early RNs that have been 'marked' as the future sites of COs/chiasmata. Figure 4.Immunolocalization of meiotic proteins in spread preparations of PMCs in an Atmlh3-1 mutant. Localization of the axis-associated protein ASY1 (green) at zygotene (A) and the SC protein ZYP1 (green) at pachytene (B) appears normal, indicating that chromosome pairing and synapsis occur in the absence of AtMLH3. Dual localization of ASY1 (green) with the recombination protein RAD51 (red) (C) and ZYP1 (green) with AtMSH4 (red) (D) suggests that recombination progresses to the dHj stage. However, normal localization of AtMLH1 fails to occur. Dual localization with ASY1 (green) at leptotene reveals aberrant nucleolar localization of AtMLH1 (red) (E). At pachytene, ZYP1 (green) is present but there is a complete absence of AtMLH1 foci (F). Bar=10 μm. Download figure Download PowerPoint The MutS homologue, AtMSH4, is proposed to play a crucial role in this transitional period (Moens et al, 2002). We therefore used an anti-AtMSH4 antibody to investigate localization of the protein in Atmlh3-1. This was found to be indistinguishable from that previously reported in wild-type Arabidopsis (Higgins et al, 2004). Numerous MSH4 foci were first detectable at late leptotene. As prophase I progressed, the number of foci gradually reduced in number, with a few ( 0.1) or 12 (χ2(7)=11.15; P>0.1) (Figure 5D). As up to 12 chiasmata per cell are observed in wild-type Arabidopsis, these values of k are quite reasonable. Of course, this is likely a gross simplification because, for example, k (the number of RIs) may differ from cell to cell. Additionally, it seems highly probable that Arabidopsis has a second recombination pathway that accounts for an average of 1.5 chiasmata per cell (Higgins et al, 2004) that would be unaffected by the Atmlh3 mutation. However, this subset of COs do not exhibit interference and thus may occur too close to other COs to be distinguished separately as chiasmata. An alternative approach that avoids some of these difficulties is to compare the chiasma distribution to the Poisson distribution that is related to the binomial distribution. The practical advantage of this is that there is no necessity to find values for p and k, as the Poisson distribution is specified by the mean (μ) that is derived directly from the data. When this comparison was carried out, the Atmlh3 cell chiasma frequency distribution did not differ significantly from a Poisson distribution (χ2(6)=5.84; P>0.3) (Figure 5E). In this context, the possible presence of a subset of interference free (random) chiasmata originating from a second recombination pathway, which also follow a Poisson distribution (Higgins et al, 2004), is not a problem because when two Poisson distributions are combined the resulting distribution is itself Poissonian (http://mathworld.wolfram.com/PoissonDistribution.html; Eric W Weisstein 'Poisson distribution'). Discussion MLH1 and MLH3 are eukaryotic homologues of the bacterial MMR gene MutL. A previous investigation in Arabidopsis resulted in the identification of three MutL homologues, AtMLH1, AtPMS1 and AtMLHx (Jean et al, 1999). Subsequently, the same authors redesignated AtMLHx as AtMLH3 on the basis of sequence homology to the yeast and mammalian genes (Alou et al, 2004). The investigation reported here provides strong functional evidence that the AtMLH3 protein is required for normal meiotic recombination in Arabidopsis. Immunolocalization studies indicate that in an Atmlh3 mutant, recombination progresses to the later stages. Based on this, we propose that resolution of dHjs can occur in the absence of the MLH3 protein, but that resolution is biased in favour of a non-CO outcome. AtMLH3 localizes to homologous chromosomes during meiotic prophase I Studies in mouse have revealed that MLH3 protein localizes as foci to the chromosome axes at early to mid-pachytene, persisting until early diplotene (Lipkin et al, 2002). At mid- to late pachytene, one to two foci were observed per chromosome, colocalizing with MLH1. It has been proposed that these foci mark the positions of meiotic COs that will subsequently appear as chiasmata in diplotene, diakinesis and metaphase I. This has recently been confirmed in mouse where both MLH1 and MLH3 have been shown to localize to the sites of chiasmata precociously induced using okadaic acid treatment of spermatocytes (Marcon and Moens, 2003). Our immunolocalization studies have revealed that AtMLH3 localizes as discrete foci during prophase I of meiosis. The protein is first detected at mid-zygotene. By pachytene, the number of foci detected is ∼10 and corresponds closely to the number of COs. In the absence of AtMLH3, the early recombination pathway remains, as far as can be judged, unaltered. However, in agreement with the mouse, the loading of AtMLH1 is dependent on AtMLH3, as AtMLH1 foci are not observed in the Atmlh3 mutants. Instead, the protein remains associated with the nucleolus. The basis of the nucleolar accumulation is unknown, but it has previously been noted in the case of the meiotic protein SWI1 and it has been suggested that nucleolus may be a reservoir that somehow regulates protein availability (Visintin and Amon, 2000; Mercier et al, 2003). Additionally, AtMLH3 foci are not detected in an Atmsh4 background (Higgins et al, 2004), whereas the number and distribution of AtMSH4 foci in Atmlh3 is indistinguishable from that observed in wild-type meiocytes. Thu
Referência(s)