Full length NF-YB2 is required for the ABF3 interaction.

<p>Y2H assays were performed between AD:ABF3 and DBD fused to: A) Full length NF-YB2 (AA 1–190), B2HFM (AA 26–121), B2N (AA 1–25), B2C (AA 122–190), B2N+B2HFM (AA 1–122), and B2HFM+B2C (AA 122–190); B) Chimeric constructs - full length NF-YB10 (AA1–228), NF-YB2/NF-YB10, NF-YB10/NF-YB2, NF-YB10N (AA1–27) and NF-YB10C (AA 123–228).</p

NF-YC3, NF-YC4 and NF-YC9 protein-protein interaction network.

<p>Both demonstrated and GeneMANIA predicted protein-protein interaction data for NF-YC3, NF-YC4, and NF-YC9 were visualized using Cytoscape <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059481#pone.0059481-Cline1" target="_blank">[46]</a>. Predicted physical interactions are depicted as dashed lines, while demonstrated interactions (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059481#pone.0059481-Wenkel1" target="_blank">[6]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059481#pone.0059481-Kumimoto2" target="_blank">[16]</a> and this work) are depicted as solid lines. Input nodes NF-YC3, NF-YC4 and NF-YC9 are shown as squares. Circle nodes are those predicted data from GeneMANIA <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059481#pone.0059481-WardeFarley1" target="_blank">[44]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059481#pone.0059481-Mostafavi1" target="_blank">[45]</a>. Octagonal nodes represent demonstrated physical interactions (e.g., Y2H, some shown in this report - see below). Related protein nodes are color coded as follows: red-bZIP; blue – CCT; green - H2A; orange/tan - NF-YB; yellow – NF-YA; gray – unclassified interacting proteins. A Microsoft Excel file is available to recreate/manipulate this data (File S1). Common names were used where available - File S1 contains all AGI numbers and references for sources of data.</p

Misregulated genes in the <i>nf-yc triple</i> mutant have ABRE-like promoter elements.

<p>A) ABRE-like motif discovered through MEME analysis. B) Positional distribution of MEME motif within the promoter set. TSS - transcriptional start site. To help assess the relationships between Arabidopsis NF-Y proteins discussed here and below, note that phylogenetic trees were previously published <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059481#pone.0059481-Siefers1" target="_blank">[9]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059481#pone.0059481-Gusmaroli1" target="_blank">[76]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059481#pone.0059481-Gusmaroli2" target="_blank">[77]</a>.</p

ABA related genes are misregulated in <i>NF-Y</i> mutant lines.

<p>Gene expression in 24 hr post light incubation seeds analyzed by quantitative RT-PCR for A) <i>ABI3</i>, B) <i>ABI5</i>, C) <i>AIA</i>, D) <i>AIL</i>, E) <i>RAB18</i>, and F) <i>RD29B</i>. For each gene, the expression level in Col-0 was defined as 1. Data represent means and standard deviation of three replicates.</p

<i>NF-YC</i> mutants show opposing phenotypes in response to ABA.

<p>A–C) Germination of <i>nf-yc triple</i> mutants on B5 media, B5 media with 1 µM ABA, and in response to variable ABA dosage at 84 hours, respectively. D–F) As in A–C for all <i>nf-yc</i> double mutant combinations and G–I) single mutants. J) Percent germination for all <i>nf-yc</i> mutant combinations on 1 µM ABA at 84 hours. K) Percentage of plants with open green cotyledons at 10 days for all combinations of <i>nf-yc</i> mutants. L) Picture of 10 day old seedlings grown on 1 µM ABA; 1 = Col-0, 2 = <i>nf-yc3</i>, 3 = <i>nf-yc4</i>, 4 = <i>nf-yc9</i>, 5 = <i>nf-yc3/c4,</i> 6 = <i>nf-yc3/c9,</i> 7 = <i>nf-yc4/c9,</i> 8 = <i>nf-yc triple</i>. Asterisks for J–K represent Fisher’s Exact Test p-values; *p<0.01, **p<0.001, ***p<0.0001. Germination data is a compilation of two experiments (n = 6 replicates per genotype) using independent sets of matched seeds. Each replicate contained at least 30 seeds.</p

<i>NF-YB</i> overexpression results in late germination.

<p>A–B) <i>nf-yb2</i>, <i>nf-yb3</i> and <i>nf-yb2/b3</i> double mutants on B5 and B5+1 µM ABA media. C–D) <i>p35S::NF-YB2</i> and <i>p35S::NF-YB3</i> on B5 and B5+1 uM ABA media. Germination data is compilation of two experiments (total of n = 6 replicates per genotype) using independent sets of matched seeds. Each replicate contained at least 30 seeds. A Fisher’s Exact Test was performed for both mutants (no difference) and overexpression lines at 84 hrs post-incubation. Both overexpression lines were significantly different (p<0.01) from parental Col-0. Separate, independent <i>NF-YB</i> overexpression lines had similar results.</p

<i>NF-YC</i> are strongly expressed in embryos and the endosperm 24 hours post incubation in light.

<p>A,G) Col-0, B,H) NF-YB2, C,I) NF-YB3, D,J) NF-YC3 E,K) NF-YC4 F,L) NF-YC9. Scale bar in (A) equals 200 µm.</p

NF-YB and NF-YC subunits interact with bZIP transcription factors.

<p>Directed Y2H interactions between NF-YB or NF-YC subunits fused to DNA binding domains (DBD) and select bZIP proteins fused to activation domains (AD). Two independent colonies are shown for the activation of a β-galactosidase reporter gene (similar activation seen for two other reporters). A) NF-YC3, NF-YC4 and NF-YC9 interactions with ABF1-4 and HY5. B) NF-YB1, NF-YB2 and NF-YB3 interactions with ABF1-4 and HY5. MC = manufacturer’s controls (+ = strong positive, +/− = intermediate positive, − = negative), EV = empty vector.</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

<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