Effects of suppressor mutants upon flowering time.

<p>Flowering time was recorded as the number of rosette leaves at bolting, thus late flowering plants have more rosette leaves. Plants were grown in long days unless otherwise stated. Error bars show standard error of mean calculated from at least 10 plants. (<b>A</b>) The <i>clf-50 fpa-10</i> mutant shows a strong vernalization reponse. (<b>B</b>) The <i>clf-50 ft-12</i> mutant does not respond to vernalization treatment. (<b>C</b>) The <i>clf-50 sep3-7</i> mutant flowers at similar time to wild type, thus <i>SEP3</i>⁺ activity is needed for early flowering in the <i>clf</i> background. (<b>D</b>) The <i>flc-3</i> mutation enhances the early flowering of <i>clf-28</i> mutants, revealing that <i>FLC</i> activity delays flowering in the <i>clf</i> background. Plants grown in short days, where the effects of <i>clf</i> on flowering time are most obvious (<b>E</b>) <i>clf-28 ft-10</i> mutants flower later than <i>ft-10</i> mutants due to <i>FLC</i>⁺ activity.</p

Effect of the <i>clf</i> mutation on histone methylation.

<p>ChIP analysis using 12 day old seedlings. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030715#s3" target="_blank">Results</a> show H3K27me3 levels at different genes, other than the rightmost bars which show H3K4me3 levels at the <i>AG</i> gene. The SEP3-2 primers amplify a region of the <i>SEP3</i> promoter previously implicated in regulation of <i>SEP3</i> by Pc-G proteins <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030715#pone.0030715-Liu4" target="_blank">[63]</a>, SEP3-M amplify part of the large first intron of <i>SEP3</i>. Error bars are standard error of mean of three technical replicates. The experiment was repeated on independent samples with very similar results as shown in supplementary data <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030715#pone.0030715.s003" target="_blank">Figure S3</a>.</p

Gene expression in suppressor mutants.

<p>(<b>A</b>) Western blot analysis of FPA protein levels. Three independent <i>clf-50 fpa-10</i> samples were processed. Note that no protein is detected in the null <i>fpa-7</i> control, whereas in extracts from a <i>35S::FPA-YPA</i> transgenic line a larger product corresponding to the <i>FPA-YFP</i> fusion protein is detected, confirming the specificity of the antibody for FPA. No FPA protein is detectable in <i>fpa-10</i> extracts, indicating that <i>fpa-10</i> is likely a null allele. (<b>B</b>) Real time PCR analysis of <i>FLC</i> expression. (<b>C</b>) Real time PCR analysis of <i>AG</i> expression, showing high <i>AG</i> expression in suppressor mutants. (<b>D</b>) Western blot analysis of AG protein expression. The AG antibody detects two proteins of about 29 kDa that are specific for AG, the smaller band possibly representing a truncated product or spurious translation initiation event (Riechmann et al., 1999). AG protein is strongly detected in wild type flowers but not in leaves. Weak expression is found in <i>clf-50</i> and <i>clf-50 fpa-10</i> leaves. (<b>E</b>) Real time PCR analysis of <i>SEP3</i> expression. (<b>F</b>) Histochemical staining of GUS reporter gene activity. <i>SEP3::GUS</i> is not expressed in wild type leaves but shows weak expression in vasculature of <i>clf-81</i> leaves (enlarged in inset). (<b>G</b>) Real time PCR analysis of <i>SEP2</i> expression. (<b>H</b>) Real time PCR analysis of <i>FT</i> expression (<b>I</b>) Real time PCR analysis of <i>SEP3</i> expression. Error bars in real time PCR experiments represent standard error of mean of three independent samples (biological replicates). Expression was normalised relative to the <i>EiF4A</i> gene, and is expressed relative to expression in wild type. In <b>B</b>, <b>C</b>, <b>E</b>, <b>G</b>, <b>H</b> whole seedlings less roots of 20 day old short day grown seedlings were used. In <b>I</b> rosette leaves of long day plants at 21 days were used.</p

ALP1 interacts with PRC2.

<p>(A) Western blot of seedling protein extracts analysed using anti-CLF antibodies. The left and right panels show blots with short (right panel) and longer (left panel) chemiluminescent detection times as the two extracts from <i>35S</i>::<i>GFP-CLF</i> transgenic plants show much higher expression of GFP-CLF than native CLF. The positions of the size markers in the ladder lane have been marked on the image. Both CLF (≈125kD) and GFP-CLF (≈155 kD) migrate as larger proteins than their predicted sizes (102 and 129 kD, respectively). When the CLF protein was expressed in <i>E</i>. <i>coli</i> it also migrated larger than predicted, possibly because of the high lysine and arginine content in the N-terminal portion. (B-C) Co-immunoprecipitation experiments in which protein extracts were immunoprecipitated using anti-GFP antibodies, immunoblotted and analysed using anti-CLF (B) or anti-MSI1 (C) antibodies. (D-E) Immunoprecipitation of chromatin prepared from 12-day old Ws, <i>clf-5</i>0, <i>alp1-4</i> and <i>clf-50 alp1-4</i> seedlings using anti-H3K27me3 (D) or anti-H3K36me3 (E) antibodies. Precipitated DNA was quantified using real time PCR and is displayed as percentage of input. PCR fragments were located in promoter (pro), transcriptional start site (TSS), exon (ex), intron (in) and at end of interrogated genes as indicated. Error bars indicate the mean and standard error of three separate experiments, each with three technical replicates. The differences between <i>alp1</i> and wild-type or between <i>alp1 clf</i> and <i>clf</i> were not statistically significant (Tukey multiple comparison of means test) in any of the regions examined.</p

Phylogenetic analysis of ALP1 sequences from land plants and green algae.

<p>Molecular phylogenetic analysis by maximum likelihood (ML) method implemented in MEGA6 [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005660#pgen.1005660.ref074" target="_blank">74</a>]. The bootstrap consensus tree inferred from 200 replicates is taken to represent the evolutionary history of the taxa analyzed. Branches corresponding to partitions reproduced in less than 50% bootstrap replicates are collapsed. The composition of the DDE catalytic triad is indicated on the tips of the branches. The tree is unrooted. The species indicated are <i>Arabidopsis thaliana</i>, <i>Glycine max</i> (soybean), <i>Vitis vinifera</i> (grape), <i>Prunus persica</i> (peach), <i>Theobroma cacao</i> (cacao), <i>Ricinus communis</i> (castor bean), <i>Populus trichocarpa</i> (poplar), <i>Solanum lycopersicon</i> (tomato), <i>Oryza sativa</i> (rice), <i>Phoenix dactylifera</i> (date palm), <i>Amborella trichopoda</i>, <i>Cycas micholitzii</i>, <i>Picea sitchensis</i>, <i>Ginkgo biloba</i>, <i>Cyathea spinulosa</i>, <i>Psilotum nudum</i>, <i>Marchantia paleacea</i>, <i>Diphyscium foliosum</i>, <i>Nothoceros vincentianus</i>, <i>Chara braunii</i> and <i>Zea mays</i> (maize). PIF/Harbinger transposase branches are coloured in black, those of green algae in red, bryophytes in blue, pteridophytes in orange, gymnosperms in magenta, the angiosperm ALP1 clade in green, the angiosperm At3g55350 clade in light blue. Genbank accession numbers are prefixed GI, others are accession numbers for sequence retrieved from the 1000 plant genomes website (<a href="http://www.onekp.com" target="_blank">www.onekp.com</a>) with the exception of the <i>Chara braunii</i> sequence which is given the contig number in the transcriptome assembly. The analysis involved 34 amino acid sequences. All positions with less than 95% site coverage were eliminated. That is, fewer than 5% alignment gaps, missing data, and ambiguous residues were allowed at any position. There were a total of 323 positions in the final dataset.</p

<i>ALP1</i> is widely expressed and its protein product is nuclear-localised.

<p>(A) Complementation assay in <i>clf-50 alp1-4</i> background. The <i>pALP1</i>::<i>ALP1-GFP</i> transgene fully complements <i>alp1-4</i> and restores the clf phenotype, whereas <i>pALP1</i>::<i>ALP1-GUS</i> gives weaker complementation so that plants retain a partially suppressed clf phenotype. (B-G) Histochemical staining showing <i>pALP1</i>::<i>ALP1-GUS</i> activity in rosettes (B), leaves (C), roots (D) and inflorescences (E). (F–G) <i>pALP1</i>::<i>ALP1-GFP</i> is nuclear localised in roots (G), whereas a control <i>35S</i>:<i>GFP</i> construct shows more diffuse localisation in cytoplasm and nucleus (F). Scale bars are 1cm in A, 1mm in B-E and 20μm in F,G.</p

<i>ALP1</i> is required to activate PcG target gene expression.

<p>(A) T1 plants transformed with <i>35S</i>::<i>AG</i> transgene. The <i>alp1-1</i> mutation did not suppress the characteristic phenotype of small, early flowering plants with narrow, curled leaves. (B) Real time RT-PCR analysis of <i>SEP3</i>, <i>FT</i>, <i>FLC</i> and <i>AG</i> expression in seedlings of 12 day old short day grown plants. Relative expression was first normalised relative to the <i>EiF4A</i> reference gene and then calculated relative to the wild type value. Error bars indicate the standard error of the mean of three biological replicates. All four genes are upregulated in <i>clf-50</i> but show reduced expression in <i>clf-50 alp1-4</i> double mutants. One way ANOVA tests indicate that the differences are significant (p<0.05) between <i>clf-50</i> and <i>clf-50 alp1-4</i> for <i>SEP3</i>, <i>FT</i> and <i>FLC</i> but not <i>AG</i>. (C) Venn diagram comparing the number of genes mis-regulated relative to wild type (Ws) in 12 day old seedlings. Misregulated genes showed Log₂(FoldChange)>2 and False Discovery Rate <0.05. (D) Bar charts comparing the number of genes downregulated (blue) and up-regulated (red) relative to wild-type. Numbers above the bars indicate the proportion of up-regulated genes. (E) Pie chart showing that the bulk of genes mis-expressed in <i>clf-50</i> relative to wild-type are no longer mis-expressed (restored) in <i>clf-50 alp1-4</i> relative to wild-type. (F-G) Inflorescences (F) and flowers (G) illustrating the enhancement of the weak <i>lfy-5</i> phenotype by <i>alp1-3</i>. In <i>lfy-5</i> flowers, fewer petals and stamens are produced than in wild-type whereas <i>lfy-5 alp1-3</i> flowers from similar position on the inflorescence had much more severe phenotype with petals and stamens usually lacking and replaced with sepals and carpels, respectively (G). (H) Real time RT-PCR analysis of <i>AP3</i> and <i>PI</i> expression in inflorescences shows reduced expression of both genes in <i>lfy-5</i> compared to wild type and a more severe reduction in <i>lfy-5 alp1-3</i> consistent with the enhanced phenotype. Expression is normalised relative to the reference gene <i>EIF4A</i>. Error bars indicate standard error of mean of three biological replicates. Scale bars are 5mm in A and F, 500μm in G.</p

ALP1 co-purifies with Pc-G proteins.

<p>The table summarises the results from three independent replicate experiments (IP1-IP2-IP3) and lists the number of uniquely identified peptides from each protein. The total number of peptides identified in each experiment is also shown (all peptides). In IP1 some of the <i>35S</i>::<i>GFP</i> lysate was lost during filtration, in IP3 there was considerable loss of all samples except <i>35S</i>::<i>GFP-CLF</i> during the stage tip purification of in gel tryptic digests, hence the lower total number of peptides. The full list of proteins identified is presented as an excel sheet in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005660#pgen.1005660.s010" target="_blank">S3 Table</a>.</p