Diurnal expression profiles of <i>CO</i>, <i>FT</i>, <i>miP1a</i> and <i>miP1b</i>.

<p>Quantitative RT-PCR analysis of <i>CO</i> and <i>FT</i> (<b><i>A-F</i></b>), <i>miP1a</i> (<b><i>G</i>,<i>H</i></b>) and <i>miP1b</i> (<b>I,J</b>). Plants were grown in 16-hour long days (<b><i>A</i>, <i>C</i>, <i>E</i>, <i>G</i>, <i>H</i></b>) or 8-hour short day conditions (<b><i>B</i>, <i>D</i>, <i>F</i>, <i>H</i>, <i>J</i></b>). Samples were harvested every 3 h over a time period of 24 h. Expression levels are relative to <i>GAPDH</i> and the error bars represent the standard deviation of four technical replicates. (<b><i>A</i>,<i>B</i></b>) <i>CO</i> and <i>FT</i> expression in Col-0 wild type plants. (<b><i>C</i>,<i>D</i></b>) <i>CO</i> and <i>FT</i> expression in transgenic <i>35S</i>::<i>FLAG-miP1a</i> plants. (<b><i>E</i>,<i>F</i></b>) <i>CO</i> and <i>FT</i> expression in transgenic <i>35S</i>::<i>FLAG-miP1b</i> plants. (<b><i>G</i>,<i>H</i></b>) Expression profile of <i>miP1a</i> in LD and SD. (<b><i>I</i>,<i>J</i></b>) Expression profile of <i>miP1b</i> in LD and SD.</p

Model depicting the role of microProteins in flowering time regulation.

<p>The circadian clock is entrained by day/night cycles. In response to long days, <i>CO</i> is activated by GI/FKF1. Increasing levels of CO cause induction of <i>FT</i>, which triggers the transition from vegetative to reproductive growth. MiP1a/b act by controlling CO activity. If miP1a/b levels are ectopically high, CO activity is low and flowering is delayed.</p

The microProteins miP1a/b act by engaging CO in a TOPLESS/TOPLESS-like co-repressor complex.

<p>(<b><i>A</i></b>) Representative image series of co-localization studies of GFP-CO and RFP-TPL co transformed with either miP1a (n = 15), the B-Box-dead version miP1a* (n = 16) or miP1aΔPFVFL (n = 9) that is lacking the TPL-interaction motif. (<b><i>B</i></b>) Yeast-three-hybrid demonstrating the formation of a CO-TPL-miP1a trimeric complex. Growth of serial dilutions on non-selective SD-medium lacking leucine, tryptophan and uracil (-L/-W/-U) show normal yeast growth. Only positive interactions were able to grow on restrictive growth medium supplemented with 10mM 3-Aminotriazole (3-AT) and lacking histidine. (<b><i>C</i></b>) <i>In vitro</i> pull-down experiments. Recombinant MBP-CO, GST-miP1a, GST-miP1aΔPFVFL, GST-ZPR3 and HIS-TPL proteins were produced in <i>E</i>. <i>coli</i>. After cell lysis, cell extracts of MBP-CO and HIS-TPL were mixed with GST-miP1a, GST-miP1aΔPFVFL or GST-ZPR3 and incubated with magnetic anti-GST coupled magnetic beads (Promega). GST-miP1a, GST-miP1aΔPFVFL and GST-ZPR3 complexes were precipitated and washed using a magnetic stand, eluted by boiling in SDS-loading buffer and separated by SDS-PAGE. HIS-TPL and MBP-CO Proteins were detected by immunoblotting. (<b><i>D</i></b>) <i>In vitro</i> pull-down experiment of the trimeric TPL-miP1a-CO complex. MBP-CO and HIS-TPL were mixed with either miP1a or miP1a* proteins. After immunoprecipitation of MBP-CO with an amylose resin proteins were detected by immunoblotting.</p

Gene-ontology analysis of KAN1 targets.

<p><b>A</b>) KAN1 binds to the <i>ASYMMETRIC </i><i>LEAVES2</i> (AS2) promoter. Three distinct binding regions were identified but only the second peak contains the VGAATAW motif. The guanine depicted in red is mutated to adenine in the <i>as2-5d</i> mutant. <b>B</b>) and <b>C</b>) Enrichment of GO terms identified in the set of genes located downstream of the KAN1-binding site. Over-representation of genes involved in multicellular organismal development and in the response to stimuli targeted by KAN1.</p

Identification of KAN1 target genes.

<p><b>A</b>) Constructing an inducible KAN1 expression system. <b>B</b>) Sequence logos for the <i>cis</i>-element, forward and reverse orientation, enriched in the ChIP-Seq dataset <b>C</b>) Distribution of KAN1 binding sites across the five <i>Arabidopsis</i> chromosomes. <b>D</b>) Location of peaks identified by ChIP-Seq. About 25% of all peaks are located in the first 1000bp upstream of the transcriptional start site.</p

Genome-wide comparison of genes bound and regulated by KAN1.

<p><b>A</b>) Venn-diagram showing numbers of genes bound by KAN1 and regulated by KAN1. The overlap contains 211 genes that are both bound and also regulated by KAN1. <b>B</b>) Gene ontology analysis of 211 potential direct KAN1 targets reveals a strong enrichment for genes involved in shoot patterning and the auxin response. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077341#pone-0077341-t001" target="_blank">Tables 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077341#pone-0077341-t002" target="_blank">2</a> contain these genes including the binding site information.</p