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First published online 12 September 2007
doi: 10.1242/dev.009027

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Polycomb group proteins function in the female gametophyte to determine seed development in plants

¹ Institute of Plant Sciences and Zürich-Base Plant Science Center, Swiss Federal Institute of Technology, ETH Centre, CH-8092 Zürich, Switzerland.
² Institute of Biology II, University of Freiburg, Schänzlestr. 1, 79104 Freiburg, Germany.

View larger version (43K): [in this window] [in a new window]   Fig. 1. Homozygous msi1 mutant Arabidopsis seeds show an early developmental arrest. (A) Cleared seeds derived from the same silique that arrested at different developmental stages. Homozygous (left) and heterozygous (middle) msi1 seeds and wild-type seeds (right). (B) Self-pollinated msi1/MSI1 plants form small aborting seeds (arrows, middle). No small aborting seeds are formed after pollination of msi1/MSI1 plants with wild-type pollen (right). A wild-type (wt) silique is shown as a control in the left panel. (C) Quantification of seed abortion observed after crosses of wtxmsi1/MSI1 (n=212), msi/MSI1 xmsi1/MSI1 (n=583) and msi1/MSI1 xwt (n=487). Scale bars: 50 µm in A; 200 µm in B.

View larger version (36K): [in this window] [in a new window]   Fig. 2. The paternal MSI1 allele is expressed in the embryo and endosperm. (A) Schematic of the PCR assay used to amplify specifically either the MSI1 or MSI1* allele. Primer combination s1-as1 amplifies only the MSI1 allele (as shown beneath, left), whereas primer combination s2-as1 amplifies only the MSI1* allele (beneath, right). (B) Time-course analysis of maternal and paternal MSI1 expression. Reciprocal crosses of wild-type (MSI1) and MSI1* Arabidopsis plants were performed, and expression of maternal and paternal alleles was analyzed by RT-PCR in siliques derived from these crosses. The upper panel shows results from primer combination s1-as1, the lower panel from primer combination s2-as1. (C) Seeds derived from a cross of MSI and MSI1* plants were dissected 6 days after pollination (DAP). Embryos and endosperm plus seed coat fractions were analyzed for expression of maternal (MSI1) and paternal MSI1* alleles by RT-PCR. ACTIN provided a positive control.

View larger version (69K): [in this window] [in a new window]   Fig. 3. Markers for embryo pattern formation are similarly expressed in wild-type and msi1 mutant embryos. Expression of DR5::GFP, WOX8::YFP, Q0990, SCR::YFP, M0223 and M0221 in msi1 mutant and wild-type Arabidopsis embryos. Corresponding bright-field images of embryos are shown in the right panel of each pair. Scale bars: 50 µm.

View larger version (29K): [in this window] [in a new window]   Fig. 4. Early paternal MSI1 expression does not rescue the msi1 mutant phenotype. (A) Early paternal expression of the PHE1::MSI1 transgene was tested by RT-PCR in Arabidopsis seeds derived from msi1 mutants pollinated with PHE1::MSI1 pollen. The primers specifically detect only the transgene-derived MSI1 transcript. Paternal MSI1 expression in seeds derived from MSI1* plants pollinated with wild-type pollen is shown as a control. `Standard' refers to a dilution series of the PHE1::MSI1 plasmid, with 1-4 containing 0.48, 2.4, 12 and 60 ng DNA, respectively. (B) msi1 mutants pollinated with PHE1::MSI1 pollen have 50% aborted seeds. (C) Cleared seeds derived from the same silique of an msi1 mutant pollinated with PHE1::MSI1 pollen. Wild-type (wt) seed (left), msi1 mutant seed (right). (D) Quantification of seed abortion observed after crosses of msi/MSI1 xmsi1/MSI1 (n=583) and msi1/MSI1 xPHE1::MSI1; msi1/MSI1 #1 (n=278), msi1/MSI1 xPHE1::MSI1; msi1/MSI1 #2 (n=498), msi1/MSI1 xPHE1::MSI1; msi1/MSI1 #3 (n=166). Scale bars: 200 µm in B; 50 µm in C.

View larger version (42K): [in this window] [in a new window]   Fig. 5. Expression of MSI1 before and shortly after fertilization can rescue the female gametophytic msi1 mutant phenotype. (A) DD46::GUS expression can be detected in the female gametophyte (left). Residual expression of DD46::GUS in seeds at 2 DAP (right). (B) Expression of DD46 on microarrays. DD46 is expressed before fertilization (stage I) and is reduced to baseline (dashed line) levels after fertilization (stage II) and during seed development (stage III) [data from Hennig et al. (Hennig et al., 2005)]. (C) msi1; DD46::MSI1 plants pollinated with wild-type pollen do not form aborting seeds. (D) Quantification of seed abortion observed after crosses of wild type (wt)xwt (n=269), msi/MSI1 xwt (n=487), msi1/MSI1; DD46::MSI1 #1 xwt (n=369), msi1/MSI1; DD46::MSI1 #2 xwt (n=475). (E) Cleared seeds of msi1/MSI1; DD46::MSI1 pollinated with wild-type pollen. Seeds of this cross (right) are indistinguishable from wild-type seeds (left). (F) msi1/MSI1; DD46::MSI1 plants do not form endosperm without fertilization (right). Autonomous endosperm development in msi1 mutants at a similar time point (left). Scale bars: 50 µm in A,E,F; 200 µm in C.

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