<i>NF-YB2-EDLL</i>, but not <i>NF-YB2</i>^<i>E65R</i><i>-EDLL</i>, rescues late flowering in an <i>FT</i>-dependent manner.

<p>Flowering time quantification for 15–20 randomly selected T1 plants of <i>p35S</i>:<i>NF-YB2</i>, <i>p35S</i>:<i>NF-YB2-EDLL</i>, and <i>p35S</i>:<i>NF-YB2</i>^<i>E65R</i><i>-EDLL</i> in A) Col-0 B) <i>co-2</i> C) <i>b2b3</i> D) short days E) <i>ft-10</i>. Asterisks represent significant differences derived from one-way ANOVA (P < 0.05) followed by Bonferroni’s multiple comparison tests (_*** P < 0.001; * P < 0.05).</p

<i>p35S</i>:<i>NF-YB2</i>^<i>E65R</i> cannot rescue the <i>nf-yb2 nf-yb3</i> late flowering phenotype.

<p>A) Flowering time quantification of 15–20 randomly selected T1 <i>p35S</i>:<i>NF-YB2</i> and <i>p35S</i>:<i>NF-YB2</i>^<i>E65R</i> plants in the Col-0 background. B) Flowering time quantification of 15–20 randomly selected T1 <i>p35S</i>:<i>NF-YB2</i> and <i>p35S</i>:<i>NF-YB2</i>^<i>E65R</i> plants in the <i>nf-yb2 nf-yb3</i> background. C) Flowering time quantification of two independent, stable T3 <i>p35S</i>:<i>NF-YB2</i> and <i>p35S</i>:<i>NF-YB2</i>^<i>E65R</i> plant lines in the <i>nf-yb2 nf-yb3</i> background (n≥12). D) Representative plants of <i>p35S</i>:<i>NF-YB2</i> and <i>p35S</i>:<i>NF-YB2</i>^<i>E65R</i> in the <i>nf-yb2 nf-yb3</i> background. E) Relative transcript abundance of <i>NF-YB2</i>, <i>FT</i> and <i>AP1</i> in stable T3 <i>p35S</i>:<i>NF-YB2</i> and <i>p35S</i>:<i>NF-YB2</i>^<i>E65R</i> plants in the <i>nf-yb2 nf-yb3</i> background. Asterisks in 3A, 3B and 3C represent significant differences derived from one-way ANOVA (P < 0.05) followed by Dunnett’s multiple comparison post hoc tests against <i>nf-yb2 nf-yb3</i>.</p

NF-YA2 is a positive regulator of photoperiod dependent flowering.

<p>A) Flowering time quantification of two independent plant lines each (plant lines 1 and 2) for <i>p35S</i>:<i>NF-YA2</i> (white bars), <i>p35S</i>:<i>NF-YA7</i> (light grey bars), <i>p35S</i>:<i>NF-YA8</i> (grey bars), and <i>p35S</i>:<i>NF-YA9</i> (dark grey bars) (n≥12/line). B) Flowering time quantification of two independent <i>pNF-YA2</i>:<i>NF-YA2</i> plant lines (n≥24). C) The expression pattern of <i>pNF-YA2</i>:<i>GUS</i> in leaves of 10 day old plants. D) Relative transcript abundance of <i>CO</i>, <i>FT</i>, and <i>AP1</i>. Asterisks in 1A and 1B represent significant differences derived from one-way ANOVA (P < 0.05) followed by Dunnett’s multiple comparison post hoc tests against Col-0. Asterisks in 1D represent significant differences derived from Student’s T-tests (P < 0.05). All experiments were repeated with identical results.</p

<i>pNF-YA2</i>:<i>NF-YA2-EDLL</i> can induce flowering in the absence of CO.

<p>Flowering time quantification for 15–20 randomly selected T1 plants in A) Col-0, B) <i>co-2</i>, and C) <i>ft-10</i>. Asterisks represent significant differences derived from one-way ANOVA (P < 0.05) followed by Bonferroni’s multiple comparison tests.</p

NF-YA2 and NF-YA6 bind the <i>FT</i> -5.3kb <i>CCAAT</i> box as a trimer with NF-YB2 and NF-YC3.

<p>NF-Y trimerization and <i>FT CCAAT</i> binding was assessed by EMSA analysis. An <i>FT CCAAT</i> probe was incubated with wild type (WT, lanes 2–8; 20) or E65R mutant (B2^E65R, lanes 15–18; 21) NF-YB2/NF-YC3 dimers (60 nM) in the presence of NF-YA2 (lanes 3–5; 16–18), or NF-YA6 (lanes 6–8) at increasing molar ratios (3, 4.5 or 6 fold), or CO (lanes 20, 21; 6 fold molar ratio). As controls, NF-YA2 (lane 9), NF-YA6 (lane 10), or CO (lane 22) were incubated alone with the probe, at the highest concentration of the dose curve (360 nM), in the absence of NF-YB2/NF-YC3. Lanes 1, 11, 14, 19: probe alone, without protein additions; lanes 12, 13: empty lanes. The NF-Y/DNA complex is indicated by a labelled arrowhead. fp: free probe.</p

Light perception is synergistically defective in <i>nf-yc triple hy5</i> mutants.

<p>Hypocotyl lengths are shown for five day old plants grown on B5 media in <b>A-B)</b> SD, <b>C)</b> cWL, and <b>D)</b> cD. Statistically significant differences were determined and described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006333#pgen.1006333.g002" target="_blank">Fig 2</a>. Scale bar in B) represents 2mm.</p

NF-YB2^E65R loses interaction with NF-YA subunits.

<p>A) Alignment of the core domain of human and Arabidopsis NF-YB subunits. * marks the position of the conserved glutamic acid required for interaction with NF-YA in humans [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006496#pgen.1006496.ref027" target="_blank">27</a>]. B) NF-YB2 and NF-YB2^E65R interact with NF-YC3, NF-YC4, and NF-YC9 in Y2H assays. C) NF-YB2, but not NF-YB2^E65R, interacts with NF-YA2 when NF-YC9 is expressed using a bridge vector in yeast three-hybrid assays. DBD: DNA binding domain, AD: activation domain, EV: empty vector control.</p

Multiple <i>cop1-4</i> mutant phenotypes are partially dependent on <i>NF-YC</i> genes.

<p>Partial suppression of <i>cop1</i> mutant phenotypes are quantified for <b>A)</b> dark-grown seedling hypocotyl elongation, <b>B)</b> rosette diameter, <b>C)</b> flowering time, and <b>D)</b> relative <i>FT</i> expression levels. Statistics and labeling as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006333#pgen.1006333.g002" target="_blank">Fig 2</a>, except <i>FT</i> expression statistics which were calculated using qBase software (Biogazelle).</p

NF-YC3, 4, and 9 are necessary for suppression of hypocotyl elongation in both cB and cR light.

<p>Hypocotyl lengths are shown for five day old plants grown on B5 media in <b>A)</b> cB (38μmol m^-2 s^-1), <b>B)</b> cFR (5μmol m^-2 s^-1), and <b>C)</b> cR (6μmol m^-2 s^-1). Statistically significant differences between groups (or lack thereof) are represented by lettering above bars (error bars represent 95% confidence intervals). Statistical differences were determined by standard ANOVA (p<0.01) when variances were not significantly different (cFR and cR) and Kruskal-Wallis ANOVA (non-parametric test, p<0.05) when variances were unequal (cB). Subsequent multiple comparisons were performed by either Tukey’s (cFR, cR) or Dunn’s (cB) procedures, respectively.</p

FRET-FLIM analysis shows a strong NF-YC9 by HY5 physical interaction.

<p>FRET experiments were conducted in tobacco leaves through transient 35S-driven overexpression of NF-YCs tagged with mCer3 and HY5 or NF-YB2 tagged with YFP. <b>A)</b> Nuclei expressing both mCer3 and YFP constructs were assayed for FRET through both FRAP and FLIM. <b>B)</b> A FRAP curve representative of a positive FRET result between two known interacting proteins, NF-YB2 and NF-YC9. Fluorescence intensity was calculated relative to the pre-photobleached intensity of each fluorescent protein. Yellow bars represent the timing of photobleaching events. <b>C)</b> FLIM was employed to detect FRET through lifetime measurements before and after acceptor photobleaching (FRAP) within the same nucleus. Each point is an independent combination of mCer3- and YFP-tagged proteins, and represents the shift in fluorescent lifetime elicited by acceptor photobleaching. Scale bar in A) represents 5μm. Error bars in B-C) represent 95% confidence intervals with an n ≥ 3.</p