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doi: 10.1242/10.1242/dev.00470

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Arabidopsis MSI1 is required for epigenetic maintenance of reproductive development

Lars Hennig1,*, Patti Taranto2,*, Marcel Walser1, Nicole Schönrock1 and Wilhelm Gruissem1,2,{dagger}

1 Institute of Plant Sciences, Swiss Federal Institute of Technology, ETH Centre, CH-8092 Zürich, Switzerland
2 Department of Plant and Microbial Biology, University of California, Berkeley, CA 94720, USA



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  		Fig. 1. Phylogenetic relationship of MSI1-like proteins. Arabidopsis thaliana AtMSI1-5, maize ZmCAF-C, maize ZmCAF-C2, Saccharomyces cerevisiae MSI1, S. cerevisiae HAT2p, human RbAp46 and RbAp48, Drosophila melanogaster p55 and Caenorhabditis elegans LIN-53 were used to generate a multiple alignment and the unrooted phylogenetic tree. Numbers represent bootstrap values of 100 trials.

 


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  		Fig. 2. Expression of AtMSI1. (A) Seedlings were grown for 7 days on plates containing MS medium, otherwise plants were grown on soil under a long-day light regime. RNA was extracted from different plant organs and RNA blots containing 10 µg of total RNA were probed with an AtMSI1 probe (upper panel). The ethidium bromide-stained agarose gel is shown as a loading control (lower panel). (B) HA-tagged AtMSI1-5 were produced in vitro. Immunoblots containing similar fractions of the total reaction mixture were tested with either affinity-purified a-MSI1 or with a-HA antisera. Extract not supplemented with an AtMSI cDNA served as control (last lane). (C) Protein extracts were prepared from different plant organs. Leaves were harvested just after emergence from the shoot apical meristem and before they were completely expanded (young leaves) or after many other vegetative leaves had developed (mature leaves). Seedlings were grown on plates containing 50% MS medium. 10 µg protein was loaded in each lane. Blots were probed with affinity-purified, a-MSI1-specific antisera. (D) Protein was extracted from leaves of wild-type control plants (Col) and siblings of a segregating progeny of an AtMSI1 overexpression (OE) line (1OEa3). Ten µg protein per sample was subjected to immunoblotting with affinity-purified, a-MSI1-specific antiserum (upper panel). Ponceau red-staining of the blot is shown as a loading control (lower panel). (E) RNA was isolated from leaves of wild-type and AtMSI1-CS plants before bolting. After treatment with DNaseI, RNA was subjected to reverse transcription in the presence or absence of reverse transcriptase using oligo(dT) primers. PCR with different cDNA-specific primers was performed on aliquots of the produced cDNA.

 


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  		Fig. 3. Developmental alterations in AtMSI1-CS plants. (A) Rosettes of 3-week-old wild-type and AtMSI1-CS plants. (B) Rosette leaves of wild-type and AtMSI1-CS plants at time of bolting (scale bar: 10 mm). (C) Phenotype of the primary inflorescence shoot shortly after bolting in wild-type (Col) and AtMSI1-CS plants. (D) Close-up view of an arrested primary inflorescence shoot of an AtMSI1-CS plant. (E) Appearance of mature, flowering wild-type and AtMSI1-CS plants.

 


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  		Fig. 4. Flower morphology of AtMSI1-CS plants. (A) A flower of a wild-type plant (Col) and a flower formed early on a secondary inflorescence shoot of an AtMSI1-CS plant (right). (B) A flower that developed later on the same AtMSI1-CS secondary inflorescence shoot. (C) Latest flowers that developed on this AtMSI1-CS secondary inflorescence shoot.

 


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  		Fig. 5. Irregular ovule development in AtMSI1-CS plants. Floral buds of stage 11-12 were either embedded, sectioned and stained with Toluidine Blue or cleared and observed with differential interference contrast (DIC) optics. (A) Section of a floral bud from a AtMSI1-CS plant. (B,C) Longitudinal and transverse sections of mature ovules of a wild-type plant. (D-G) Sections of ovules from AtMSI1-CS plants. (H) DIC image of a wild-type ovule. (I) DIC images of ovules from an AtMSI1-CS plant. (K,L) DIC images of ovules from stage 16 flower from AtMSI1-CS plants. Scale bars: 50 µm (C-G,I,K,L) and 100 µm (B,H). es, embryo sac; f, funiculus; i.i, inner integument; ms, megaspore; n, nucellus; o.i, outer integument.

 


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  		Fig. 6. Ectopic expression of floral homeotic genes in leaves of AtMSI1-CS plants. RNA was isolated from (A) floral buds and open flowers of wild-type plants and leaves of wild-type and AtMSI1-CS, and (B) flowers of wild-type and leaves of fas1, fas2 and fas1 fas2 plants before bolting. After treatment with DNAseI, RNA was subjected to reverse transcription in the presence (+) or absence (-) of reverse transcriptase using oligo(dT) primers. PCR with different cDNA-specific primers was performed on aliquots of the produced cDNA (10 ng total RNA from flowers and 50 ng total RNA from leaves).

 


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  		Fig. 7. Spatial expression patterns of AGAMOUS in flowers of AtMSI1-CS plants. AtMSI1-CS plants were crossed with an AG::GUS reporter line. Flowers of segregating wild-type plants carrying the reporter (A) and of AtMSI1-CS AG::GUS plants (B) were stained for GUS activity. Shown are (from left to right) whole flowers, stamens (x2), petals (x2) and sepals (x2). Scale bars: 100 µm.

 


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  		Fig. 8. Reduced amounts of heterochromatin in AtMSI1-CS plants. (A) Phenotypes of representative DAPI-stained petal interphase nuclei. Chromocentres are smaller and staining is weaker in AtMSI1-CS than in wild-type nuclei. (B) Quantification of observations shown in A. Number of chromocentres (left), relative nuclear size (middle) and the ratio of euchromatin: heterochromatin (right) are plotted for wild type and AtMSI1-CS. Shown are the mean±s.e.m. of 40 nuclei each.

 

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