Arabidopsis MSI1 is a component of the MEA/FIE Polycomb group complex and required for seed development

Abstract

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.

Fig. 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.

Fig. 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).

Fig. 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.

Fig. 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.

Fig. 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).

Fig. 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).

Fig. 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%).