Abstract

Article15 September 2003free access Arabidopsis MSI1 is a component of the MEA/FIE Polycomb group complex and required for seed development Claudia Köhler Claudia Köhler Institute of Plant Biology and Zurich–Basel Plant Science Centre, University of Zurich, Zollikerstrasse 107, CH-8050 Zurich, Switzerland Search for more papers by this author Lars Hennig Lars Hennig Institute of Plant Sciences and Zurich–Basel Plant Science Centre, Swiss Federal Institute of Technology, ETH Center, CH-8092 Zurich, Switzerland Search for more papers by this author Romaric Bouveret Romaric Bouveret Institute of Plant Sciences and Zurich–Basel Plant Science Centre, Swiss Federal Institute of Technology, ETH Center, CH-8092 Zurich, Switzerland Search for more papers by this author Jacqueline Gheyselinck Jacqueline Gheyselinck Institute of Plant Biology and Zurich–Basel Plant Science Centre, University of Zurich, Zollikerstrasse 107, CH-8050 Zurich, Switzerland Search for more papers by this author Ueli Grossniklaus Ueli Grossniklaus Institute of Plant Biology and Zurich–Basel Plant Science Centre, University of Zurich, Zollikerstrasse 107, CH-8050 Zurich, Switzerland Search for more papers by this author Wilhelm Gruissem Corresponding Author Wilhelm Gruissem Institute of Plant Sciences and Zurich–Basel Plant Science Centre, Swiss Federal Institute of Technology, ETH Center, CH-8092 Zurich, Switzerland Search for more papers by this author Claudia Köhler Claudia Köhler Institute of Plant Biology and Zurich–Basel Plant Science Centre, University of Zurich, Zollikerstrasse 107, CH-8050 Zurich, Switzerland Search for more papers by this author Lars Hennig Lars Hennig Institute of Plant Sciences and Zurich–Basel Plant Science Centre, Swiss Federal Institute of Technology, ETH Center, CH-8092 Zurich, Switzerland Search for more papers by this author Romaric Bouveret Romaric Bouveret Institute of Plant Sciences and Zurich–Basel Plant Science Centre, Swiss Federal Institute of Technology, ETH Center, CH-8092 Zurich, Switzerland Search for more papers by this author Jacqueline Gheyselinck Jacqueline Gheyselinck Institute of Plant Biology and Zurich–Basel Plant Science Centre, University of Zurich, Zollikerstrasse 107, CH-8050 Zurich, Switzerland Search for more papers by this author Ueli Grossniklaus Ueli Grossniklaus Institute of Plant Biology and Zurich–Basel Plant Science Centre, University of Zurich, Zollikerstrasse 107, CH-8050 Zurich, Switzerland Search for more papers by this author Wilhelm Gruissem Corresponding Author Wilhelm Gruissem Institute of Plant Sciences and Zurich–Basel Plant Science Centre, Swiss Federal Institute of Technology, ETH Center, CH-8092 Zurich, Switzerland Search for more papers by this author Author Information Claudia Köhler1, Lars Hennig2, Romaric Bouveret2, Jacqueline Gheyselinck1, Ueli Grossniklaus1 and Wilhelm Gruissem 2 1Institute of Plant Biology and Zurich–Basel Plant Science Centre, University of Zurich, Zollikerstrasse 107, CH-8050 Zurich, Switzerland 2Institute of Plant Sciences and Zurich–Basel Plant Science Centre, Swiss Federal Institute of Technology, ETH Center, CH-8092 Zurich, Switzerland *Corresponding author. E-mail: [email protected] The EMBO Journal (2003)22:4804-4814https://doi.org/10.1093/emboj/cdg444 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Seed development in angiosperms initiates after double fertilization, leading to the formation of a diploid embryo and a triploid endosperm. The active repression of precocious initiation of certain aspects of seed development in the absence of fertilization requires the Polycomb group proteins MEDEA (MEA), FERTILIZATION-INDEPENDENT ENDOSPERM (FIE) and FERTILIZATION-INDEPENDENT SEED2. Here we show that the Arabidopsis WD-40 domain protein MSI1 is present together with MEA and FIE in a 600 kDa complex and interacts directly with FIE. Mutant plants heterozygous for msi1 show a seed abortion ratio of 50% with seeds aborting when the mutant allele is maternally inherited, irrespective of a paternal wild-type or mutant MSI1 allele. Further more, msi1 mutant gametophytes initiate endosperm development in the absence of fertilization at a high penetrance. After pollination, only the egg cell becomes fertilized, the central cell starts dividing prior to fertilization, resulting in the formation of seeds containing embryos surrounded by diploid endosperm. Our results establish that MSI1 has an essential function in the correct initiation and progression of seed development. Introduction In contrast with animals, plant gametes do not differentiate directly after meiosis but are produced by multicellular gametophytes that develop from meiotically derived spores. In flowering plants the female gametophyte is formed through a well-defined programme of nuclear divisions, nuclear migration and cellularization that gives rise to a multicellular structure consisting of two synergids, three antipodals, a central cell and an egg cell (for review see Grossniklaus and Schneitz, 1998). Seed development is initiated by double fertilization in which two sperm cells fuse with the egg and the central cell of the female gametophyte, respectively. The fertilized egg cell develops into the embryo, while the fertilized central cell forms the nourishing endosperm. In addition, maternal tissue of the ovule, in which the female gametophyte is embedded, contributes to the developing seed by forming the seed coat (testa). Interestingly, in most angiosperms, including Arabidopsis, embryo and testa are diploid but the endosperm is triploid with two maternal copies and one paternal copy of the genome. Double fertilization and formation of the endosperm are key events in angiosperm evolution; however, the origin and evolutionary advantages of these two processes are not yet fully understood (for review see Chaudhury et al., 1998; Berger, 1999, 2003; Grossniklaus et al., 2001; Baroux et al, 2002). After fertilization, development of testa, endosperm and embryo are highly coordinated. Genetic studies have shown that seed and fruit development are actively repressed in the absence of fertilization and that early phases of embryo and endosperm development are largely under maternal control (Vielle-Calzada et al., 2000; Vivian-Smith et al., 2001; Walbot and Evans, 2003). Several genes that encode regulators of early seed development are also required to repress fertilization-independent seed development. With reference to the Greek priestess Medea, who killed her own children in revenge for the unfaithfulness of her husband Jason, one of these genes was named MEDEA (MEA) because a mutant allele causes seed abortion only when inherited maternally (Grossniklaus et al., 1998). FERTILIZATION-INDEPENDENT ENDOSPERM (FIE) and FERTILI ZATION-INDEPENDENT SEED2 (FIS2) are two other genes that are required to repress seed development in the absence of fertilization (Luo et al., 1999; Ohad et al., 1999), a phenotype also shared by mea (Grossniklaus and Vielle-Calzada, 1998; Kiyosue et al., 1999). All three fis-class mutants show a gametophytic maternal effect: the mutant phenotype can only be observed when the mutation is inherited through the female gametophyte. Early seed development of fis mutant seeds is indistinguishable from that of wild-type seeds. However, from the globular stage onwards fis embryo development is delayed and eventually arrests with oversized heart-shaped embryos and an abnormally proliferated endosperm (Grossniklaus et al., 2001). MEA encodes a protein similar to the Drosophila Polycomb group (PcG) protein Enhancer of Zeste [E(Z)] (Grossniklaus et al., 1998). In Drosophila, E(Z) interacts with ESC, a protein sharing similarity with FIE (Jones et al., 1998; Ohad et al., 1999; Ng et al., 2000; Tie et al., 2001). MEA and FIE, as well as the mammalian homologues ENX1 and EED, interact, indicating a strong evolutionary conservation of PcG proteins and their interactions (Sewalt et al., 1998; Luo et al, 2000; Spillane et al., 2000; Yadegari et al., 2000). The E(Z)–ESC complex has a molecular mass of about 600 kDa and contains additional subunits, including p55 and the FIS2 homologue Su(Z)12 (Luo et al., 1999; Tie et al., 2001; Müller et al., 2002). Because many SET domain proteins like E(Z) have histone methyltransferase activity, it has been proposed that PcG complexes establish repressive chromatin environments at certain target loci through histone deacetylation and histone methylation (for review see Francis and Kingston, 2001). It is now increasingly appreciated that maintenance of intact chromatin states is critical for normal development and the maintenance of developmental decisions (for review see Muller and Leutz, 2001; Köhler and Grossniklaus, 2002; Reyes et al., 2002; Reyes and Grossniklaus, 2003). WD-40 proteins similar to yeast MSI1 have key functions in the maintenance and modulation of chromatin. These proteins exist in all eukaryotes and participate in various complexes involved in chromatin dynamics, including the above mentioned PcG complexes, as well as chromatin assembly factor CAF-1, nucleosome remodelling factor NURF, histone acetyl transferase and histone deacetylase complexes, and they interact with the retinoblastoma tumor suppressor protein (Qian et al., 1993; Parthun et al., 1996; Taunton et al., 1996; Verreault et al., 1996). Members of this family include p55 in Drosophila, the retinoblastoma-associated proteins RbAp46 and RbAp48 in vertebrates and CAC3p in Saccharomyces cerevisiae. Despite their ubiquitous presence, very little is known about the in vivo function of MSI1-like proteins. In yeast, CAC3p is required for efficient gene silencing at telomeres and the mating type loci, and cac3 mutants show enhanced sensitivity to ultraviolet radiation (Kaufman et al., 1997). In Caenorhabditis elegans, dominant negative alleles of the MSI1-like LIN-53 gene cause defects in vulva differentiation (Lu and Horvitz, 1998), but no other functions of MSI1-like proteins in multicellular eukaryotes have been reported. Arabidopsis has five MSI1-like genes (MSI1–5) (Ach et al., 1997; Kenzior and Folk, 1998; Hennig et al, 2003), and reducing Arabidopsis MSI1 levels by co-suppression causes ectopic expression of floral homeotic genes and strongly interferes with cellular differentiation (Hennig et al., 2003). Because the severity of phenotypic alterations increased during development, we suggested that MSI1 is required for the inheritance of epigenetic states during mitosis. Here we describe an Arabidopsis MSI1 insertion allele that causes seed abortion early in development when maternally inherited. Heterozygous msi1 mutants also show a high penetrance of fertilization-independent seed development. We demonstrate that Arabidopsis MSI1 physically interacts with FIE and that MSI1, FIE and MEA are part of a 600 kDa protein complex in vivo that is required for seed development. Results Identification of an msi1 T-DNA insertion mutant To analyse the function of Arabidopsis MSI1 we searched several collections of T-DNA insertion mutants for a disruption of the MSI1 gene and identified one candidate in the SAIL collection (Sessions et al., 2002). Genomic DNA blots revealed a complex insertion of two T-DNAs at the same locus. PCR analysis and sequencing confirmed the insertion into the second exon of the MSI1 gene (data not shown). Heterozygous plants developed normally from the seedling stage to maturity. The T-DNA insertion allele in Arabidopsis MSI1 was termed msi1 and was used in all subsequent experiments. Loss of MSI1 causes a maternal effect seed abortion phenotype We were unable to obtain homozygous msi1 plants, suggesting that seed development is defective in MSI1/msi1 mutants. In contrast with wild-type plants that contained only normally developing seeds, MSI1/msi1 siliques had aborted seeds that appeared brown and shrunken (Figure 1). Quantification of seed abortion revealed that 50% of the seeds from heterozygous msi1 mutants aborted, in contrast with less than 1% abortion observed in the wild type (Table I). Arabidopsis MSI1 was previously reported to participate in CAF-1 (Kaya et al., 2001), but we found no striking overlapping phenotypes between the Arabidopsis CAF-1 mutants fasciata1 (fas1) and fasciata2 (fas2) and MSI1 co-suppression plants (Hennig et al., 2003). Therefore we assayed seed abortion in fas1 and fas2, but, in contrast with MSI1/msi1 plants, seed development was not impaired in the CAF-1 mutants (Table I). Figure 1.Siliques of MSI1/msi1 plants contain 50% normal and 50% aborted seeds. Opened siliques of (A) wild-type (WT), selfed, (B) MSI1/msi1, selfed, (C) WT × MSI1/msi1 and (D) MSI1/msi1 × WT. Scale bars, 500 μm. Download figure Download PowerPoint Table 1. Seed abortion in msi1 and fasciata mutants Normal Aborted Expected p value WT 549 1 (0.2%) NA NA MSI1/msi1 ×MSI1/msi1 268 261 (49.3%) 50 0.76 fas1 162 2 (1.2%) NA NA fas2 176 4 (2.2%) NA NA msi1 compl-1 184 66 (26.4%) 25 0.61 MSI1/msi1 ×MSI1/MSI1 93 99 (48.4%) 50 0.66 MSI1/MSI1 × MSI1/msi1 262 0 (0.0%) 0 NA NA, not applicable. A single-locus recessive embryo-lethal mutant is expected to give rise to 25% aborted seeds. However, seed abortion in MSI1/msi1 occurred at a ratio of 50%. Because 50% seed abortion is a strong indication for a defect that is under female gametophytic control, we performed reciprocal crosses between MSI1/msi1 and wild-type plants. A paternally derived mutant allele (msi1 pollen) did not impair seed development, while the msi1 phenotype could not be complemented by fertilization of msi1 mutant gametophytes with wild-type pollen (Figure 1 and Table I). These observations suggest that the fate of seeds is determined only by the maternally derived MSI1 allele. Seeds derived from ovules with an msi1 gametophyte (hereafter referred to as msi1 seeds) abort regardless of the paternal genotype. This hypothesis predicts that the msi1 mutant allele can be transmitted only paternally. We tested for the presence of the msi1 allele in the progeny of selfed msi1 heterozygous plants and of reciprocal crosses between MSI1/msi1 and wild-type plants. The results shown in Table II confirm this hypothesis. Table 2. Transmission of the msi1 allele Phosphinotrocin sensitive Phosphinotrocin resistant Expected p value MSI1/msi1 × MSI1/msi1 113 125 (52.5%) 50 0.44 75 0.05 MSI1/msi1 × MSI1/MSI1 51 0 (0.0%) 0 NA msi1 compl-1 × MSI1/MSI1 48 26 (36.8%) 33 0.696 NA, not applicable. Genetic complementation of msi1 T-DNA insertion mutagenesis can also result in mutations that are not tagged by the inserted T-DNA (Azpiroz-Leehan and Feldman, 1997). Therefore we tested whether the observed msi1 phenotype is indeed caused by the insertion into the MSI1 gene. Plants heterozygous for msi1 were transformed with the MSI1 cDNA under control of the endogenous MSI1 promoter consisting of a 2 kbp DNA fragment upstream of the translation start codon. Two randomly chosen transgenic lines showed less seed abortion than the parental MSI1/msi1 mutant. We selected a transgenic line with a single insert of the transgene (msi1 compl-1, data not shown) for further analysis. As msi1 is a gametophytic maternal effect mutant, the expected ratio of aborted seeds in an MSI1/msi1 plant is 50%. An unlinked single copy MSI1 transgene will segregate randomly in the gametes such that half of the msi1 seeds are expected to contain the transgene rescuing the msi1 phenotype. This will reduce the ratio of aborted seeds to 25% (see below). As expected, the fraction of aborting seeds dropped from 49.3% in MSI1/msi1 to 26.4% in MSI1/msi1 compl-1 (Table I). Because seed abortion can be complemented by an intact MSI1 cDNA, we conclude that this phenotype of MSI1/msi1 plants is indeed caused by loss of MSI1 function. Aborting seeds in msi1 arrest early during embryo development We analyzed the morphology of mutant and wild-type seeds in cleared whole-mount and semi-thin sectioned specimens to characterize the defects of msi1 mutant seeds in more detail (Figure 2). Mutant msi1 seeds were developmentally delayed compared with their wild-type siblings. The developmental arrest occurred at different developmental stages and was highly variable among different siliques. Thus, when about half of the seeds of MSI1/msi1 plants contained transition or heart-stage embryos (Figure 2A and D), about 36% (n = 361) of the seeds contained preglobular and globular stage embryos (Figures 2B and E). The remaining seeds aborted shortly after fertilization without the formation of any embryo. In contrast with the well-organized structure of preglobular embryos in wild-type seeds (Figure 2C and F), delayed preglobular seeds in MSI1/msi1 siliques often contained abnormal embryos with irregular orientation of cell division planes. In fact, there was no recognizable separation between the embryo proper and the suspensor (Figure 2B and E). Very few msi1 embryos continued development until the heart stage. However, msi1 heart-stage embryos showed overproliferation and the seeds contained an enlarged chalazal endosperm (Figure 2H and L). When developing wild-type seeds had reached the late torpedo stage (Figure 2G and K), the majority of the mutant siblings were already aborted and only very few contained overproliferated heart-stage embryos. Consequently, seed development is initiated in msi1 female gametophytes but suffers from early defects in embryo and endosperm development, eventually leading to seed abortion. Figure 2.Mutant msi1 seeds contain abnormal embryos arrested at different developmental stages: (A), (D) Wild-type seeds with transition stage embryos; (B), (E) mutant seeds from the same silique as in (A) and (D); (C), (F) preglobular wild-type embryos for comparison; (G), (K) wild-type seeds with torpedo stage embryos; (H), (L) mutant seeds from same silique as (G) and (K) containing enlarged mutant embryo at the heart stage and chalazal endosperm that is overproliferated; (I), (M) wild-type seeds with late heart-stage embryos for comparison. Abbreviations: CZE, chalazal endosperm; E, endosperm; EM, embryo; EP, embryo proper; S, suspensor. Scale bars, 100 μm in (A), (B), (C), (G), (H), (K), (I) and (M); 50 μm in (D), (E), (F) and (L). Download figure Download PowerPoint MSI1 is strongly expressed in the female gametophyte and the embryo MSI1 is strongly expressed in floral buds and flowers (Hennig et al., 2003), but seed abortion in the absence of an intact maternal MSI1 allele suggested expression of MSI1 in fruits also. RT-PCR analysis demonstrated that MSI1 is strongly expressed in siliques during wild-type seed development from 0 to 4 DAP (Figure 3A). In order to characterize the tissue-specific MSI1 expression, we performed in situ hybridization experiments (Figure 3B). We obtained a strong signal with the antisense RNA probe in both the female gametophyte and the sporophytic tissue of the ovules (e.g. in integuments). After fertilization, the strongest expression was detected in developing embryos. We also observed specific hybridization signals in pollen sacs and pollen (data not shown). Together, these results support the view that MSI1 has a pivotal role in gametophyte and embryo development. Figure 3.MSI1 is expressed in the female gametophyte and during different stages of seed development. (A) RNA was isolated from wild-type flowers before fertilization (0 DAP), siliques containing embryos at preglobular stage (1–2 DAP) and siliques containing embryos at late globular stage (3–4 DAP). After treatment with DNase I, RNA was subjected to reverse transcription in the presence (+) or absence (−) of reverse transcriptase using oligodT primers. PCR with cDNA-specific primers was performed on aliquots of the produced cDNA, which equalled 50 ng total RNA. (B) Localization of MSI1 expression in gametophytes and developing seeds of wild-type Arabidopsis plants. Sections were hybridized with a sense (left) or antisense (right) MSI1 probe. Top, expression in female gametophytes; middle, expression in globular embryos; bottom, expression in heart-stage embryos. Abbreviations: EM, embryo; MG, megagametophyte; IN, integuments. Scale bars: top, 30 μm; middle and bottom, 50 μm. Download figure Download PowerPoint MSI1, MEA and FIE participate in a high molecular weight complex in vivo Similar to msi1, the maternal effect mutants mea, fie and fis2 also cause 50% seed abortion. Since MEA and FIE interact in vitro and in vivo (Luo et al., 2000; Spillane et al., 2000; Yadegari et al., 2000), we tested whether MSI1 is also a subunit of the MEA–FIE complex. In Drosophila, E(Z) and ESC are subunits of a large complex with a molecular mass of about 500–600 kDa (Ng et al., 2000; Tie et al., 2001). In C.elegans, however, homologues of E(Z) and ESC are found in a complex with a molecular weight of only 255 kDa (Xu et al., 2001). The molecular weight and subunit composition of the Arabidopsis MEA–FIE complex is currently unknown. Therefore we investigated the MEA–FIE complex using size exclusion chromatography (SEC) of protein extracts from Arabidopsis flowers and young siliques and tested the fractions on protein blots. The antisera used in these experiments had previously been shown to recognize the corresponding antigen and do not cross-react with other proteins of the same size (Hennig et al., 2003; Köhler et al., 2003). Figure 4A shows that MSI1, MEA and FIE co-elute at a molecular weight of about 600 kDa, suggesting that they are part of a complex similar to the PcG complex in Drosophila. Interestingly, a large amount of FIE, but not MEA and MSI1, could also be detected in a monomeric form. To confirm that MSI1 is also a subunit of the MEA–FIE complex, we performed immunoprecipitation experiments with Arabidopsis nuclear extracts. Both anti-MEA and anti-FIE, but not the preimmune sera, coprecipitated MSI1 (Figure 4B). Figure 4.MEA, FIE and MSI1 are part of a large protein complex. (A) Gel filtration analysis of nuclear extracts of plant inflorescences. Nuclear extracts were loaded onto a 14 ml Bio-SEC-250 column. Fractions were separated by SDS–PAGE and tested on protein blots. Fraction numbers are indicated at the top and arrows indicate elution positions of molecular mass standards. (B) MSI1, MEA and FIE reside in one protein complex in vivo. Co-immunoprecipitations were performed on nuclear extracts prepared from plant inflorescences. Immunoprecipitated proteins were analyzed on protein blots with the antibodies indicated at top. Input contains 3% of the protein used for the co-immunoprecipitation assay. (C) MSI1 and FIE interact physically in vitro. Bacterial extracts containing GST-MSI, MEA or FIE were mixed, incubated together and bound to glutathione beads. GST alone was used as a negative control. After extensive washing, proteins were analyzed by SDS–PAGE and Coomassie staining (left panel) or protein blotting (right panel). Input contains 4% of the protein used for the pulldown assay. Download figure Download PowerPoint To characterize the protein–protein interactions between MSI1, MEA and FIE in more detail, an MSI1–GST fusion protein was expressed in Escherichia coli and incubated with MEA or FIE proteins before binding to gluthathione beads (Figure 4C). The binding assay revealed that MSI1 can efficiently bind to FIE, but not to MEA, when it is presented as a single binding partner. These results suggest that MSI1 and FIE interact directly in vivo, but that the interaction of MEA and MSI1 requires either post-translational modifications of at least one partner or additional proteins like FIE to mediate the interaction. Loss of MSI1 leads to fertilization-independent seed development Both mea and fie belong to the fis class of mutants, and the current model suggests that the MEA–FIE complex prevents seed development in the absence of a fertilization signal (for review see Grossniklaus et al., 2001). If MSI1 is an integral part of the MEA–FIE complex, we would also expect autonomous endosperm or seed development in MSI1/msi1 mutants. Floral buds of wild-type and MSI1/msi1 mutant plants were emasculated and compared with manually pollinated gynoecia. Pollinated gynoecia of wild-type and MSI1/msi1 mutant plants were indistinguishable after 6 days and reached a length of about 13 mm. In contrast, after 6 days without pollination, gynoecia of wild-type plants were only 2–3 mm long, whereas in MSI1/msi1 mutant plants unpollinated gynoecia formed siliques of 10 mm (Figure 5). Figure 5.MSI1/msi1 plants undergo fertilization-independent silique elongation. (A) Representative siliques of wild-type and MSI1/msi1 plants at 6 DAP or non-pollinated (np). Scale bar, 5 mm. (B) Average silique length of wild-type or MSI1/msi1 plants (mean ± SE, n = 10) at 6 DAP or 6 days after emasculation (MSI1/msi1). Download figure Download PowerPoint Since silique elongation in the absence of fertilization suggests autonomous endosperm and seed development we investigated the developing seeds in MSI1/msi1 plants in more detail. Figure 6 shows that unfertilized wild-type ovules did not develop (Figure 6B), whereas many unfertilized msi1 ovules initiated seed development (Figure 6C and D). Optical and semi-thin sections through such fertilization-independent seeds revealed the presence of a multinuclear endosperm but no embryo. Sometimes embryo-like structures could be observed at the micropylar end of the embryo sac (Figure 6D). However, in contrast with embryonic cells that contain small vacuoles, these cells were highly vacuolated, suggesting that they originated from the endosperm. Only 50% of the ovules carry a mutant msi1 allele and thus can be expected to initiate fertilization-independent seed development. Quantification revealed that 41.2% of the ovules displayed the FIS phenotype, demonstrating a penetrance of more than 80%. This is significantly higher than the reported penetrance of 15–20% in the mea mutants (Grossniklaus and Vielle-Calzada, 1998; Kiyosue et al., 1999). Figure 6.Fertilization-independent seed development in MSI1/msi1 plants. (A) Wild-type seed at 6 DAP containing a dermatogen-stage embryo. (B) Unfertilized wild-type ovule at 6 DAP. (C), (D) Unfertilized msi1 ovules that started seed development and contain multinuclear endosperm. Abbreviations: E, endosperm; EM, embryo; ELS, embryo-like structure; MG, megagametophyte. Scale bars: 100 μm in (A), (C) and (D); 50 μm in (B). Download figure Download PowerPoint Double fertilization is impaired in msi1 The high penetrance of the FIS phenotype in MSI1/msi1 plants suggests that endosperm development fails to arrest and starts without fertilization even after pollination. We tested this hypothesis by performing ploidy analysis using flow cytometry of nuclei from developing seeds (Figure 7). This assay was previously used to demonstrate that endoreduplication produces 6C and 12C nuclei in the endosperm and 4C, 8C and 16C nuclei in the embryos of several species (Matzk et al., 2000). We observed up to 40% 3C or 6C nuclei derived from the endosperm in wild-type seeds at 6 DAP. As expected, seeds developing in unpollinated MSI1/msi1 siliques did not yield any 3C or 6C signal, demonstrating that these seeds contain only nuclei derived from the diploid central cell. Surprisingly, 3C or 6C signals were also largely absent in msi1 mutant seeds derived from pollinated gynoecia. The small percentage of 3C and 6C nuclei detected in some preparations (compare Figure 7A, bottom, and B) suggests that double fertilization can occur in a small fraction of msi1 seeds. Because we observed embryo formation in msi1 mutant seeds only after pollination, fertilization of the egg cell appears to be required for development of the embryo but not the endosperm. Figure 7.Mutant msi1 seeds contain diploid endosperm. (A) Ploidy analysis of nuclei from wild-type seeds at 6 DAP (WT), msi1 seeds developing without fertilization (msi1, np) and msi1 seeds at 6 DAP (msi1). (B) Quantified results from ploidy analysis (n ≥ 4, SD < 5%). Download figure Download PowerPoint Discussion MSI1-like proteins are components of various chromatin-modifying protein complexes, but their biological function is not well understood at present. In yeast, MSI1 is required for gene silencing at telomeres and mating type loci (Kaufman et al., 1997; Enomoto and Berman, 1998). In C.elegans, LIN53 is repressing genes required for the development of vulval cell fates (Lu and Horvitz, 1998). Recently, we have shown that Arabidopsis MSI1 is required for the maintenance of differentiation processes in vegetative and reproductive development (Hennig et al., 2003). In many organisms, including yeast, vertebrates and many plants, several MSI1-like proteins exist that are functionally distinct (Verreault et al., 1996; Ach et al., 1997; Kaufman et al., 1997; Hennig et al., 2003). However, insects like Drosophila and Anopheles contain only a single MSI1-like gene. Sequence similarities suggest that MSI1-like genes in different clades diverged independently during evolution. However, the advantages or costs of MSI1 diversification are not well understood. The Arabidopsis genome encodes five MSI1-like proteins (MSI1–5). MSI2 and MSI3, as well as MSI4 and MSI5, are pairs of closely related genes, while MSI1 is more distantly related and has no close homologue (Hennig et al., 2003). We isolated an insertion mutant in