Rate of <i>clv3</i>-KO phenotypes among T2 progeny of individual pJF870, pJF871 and pJF872 T1 plants.

<p>KO: knock-out; WT: wildtype</p><p>Rate of <i>clv3</i>-KO phenotypes among T2 progeny of individual pJF870, pJF871 and pJF872 T1 plants.</p

(A) TALEN structure.

<p>The variant 34 amino acid (aa) repeats each binding a defined nucleotide as specified by their repeat-variable diresidues are depicted as cyan and magenta boxes. The invariant first and last repeats are marked in green. The part originating from the original TALEs is marked by an orange line. This part and the FokI domain are drawn to scale. For the first generation TALENs, one amino acid (alanine) has been inserted between aa1 and aa2 to create an NcoI site. The S-tag versions are 6 aa longer. (<b>B</b>) Binding sites of the TALENs used in this study. (<b>C</b>) Constructs for TALEN expression. pJF802 & pJF803 are built similarly to pJF752 & pJF753 but contain the <i>P16</i> promoter instead of the <i>CLV3</i> promoter and the <i>RBCS</i> terminator instead of the <i>CLV3</i> terminator. LB: left border. RB: right border. p: promoter. t: terminator. attB1/2: attachment sites from Gateway cloning. Basta^R: phosphinotricine acetyl transferase. Kan^R: neomycin phosphotransferase. NOS: nopaline synthase. NLS: nuclear localization signal.</p

(A) Origin of <i>clv3-10</i> to <i>clv3-14</i> alleles.

<p>Cyan square (WT): plant with no visible sign of <i>clv3</i> loss-of-function phenotype. Magenta square (KO): plant with full <i>clv3</i> loss-of-function phenotype. Dual-colored squares (pK): plants displaying partial loss-of-function phenotype. RIP: no surviving seedlings. (<b>B-E</b>) Inflorescence of Col-0 wildtype (<b>B</b>), <i>clv3-7</i> (<b>C</b>), a <i>clv3-10</i> T4 plant displaying the full loss-of-function phenotype (<b>D</b>) and a T4 descendant of the pJF752/753 “allele generator” T3 plant displaying a slow onset of the mutant phenotype (<b>E</b>). Green arrowheads: wild-type silique; red arrowheads <i>clv3</i> mutant silique.</p

Genome-wide identification of PIF3-binding sites and motifs.

<p>(A) Venn diagram depicting total numbers (parentheses) and reproducible presence (overlapping sectors) of statistically significant PIF3-binding peaks in ChIP-seq analysis of four biological replicates (Venn ovals) of dark-grown seedlings. (B) Relative binding-peak distribution across genomic regions. (C) MEME motif search identifies two dominant PIF3-binding motifs, defined as G-box (CACGTG) and PBE-box (CACATG) motifs. (D) Percentage of PIF3 binding sites containing designated motifs. Other E-box: Variants of E-box (CANNTG) motif other than G- or PBE-box. Unknown: Unknown and/or non-statistically-overrepresented motif. (E) Distribution of the G- and PBE-box motifs in the 1 kb regions surrounding the PIF3-binding peak-summits. (F) G- and PBE-box-motif coincidence with PIF3-binding peaks (% within 201, 101, and 51 bp centered at the peak-summits) is significantly higher than in other random genomic regions of the same size. Internal numbers indicate the relative fold motif-enrichment at PIF3 binding-sites. Error bars represent the standard deviation of 100 random simulations. (G) DPI-ELISA assays of <i>in vitro</i> binding of recombinant GST-PIF3 to the G- and PBE-box motifs. Binding activity (Relative Absorbance) for each DNA probe is expressed as a percentage of each reaction relative to GST-PIF3 binding to the <i>PIL1a</i> WT probe. Data represent the mean of independent duplicates +/− SEM. WT: wild-type competitor probes. mut: competitor probes mutated at the G-box and PBE-box motifs. GST: GST negative-control binding to the biotinylated WT probes.</p

A Quartet of PIF bHLH Factors Provides a Transcriptionally Centered Signaling Hub That Regulates Seedling Morphogenesis through Differential Expression-Patterning of Shared Target Genes in <em>Arabidopsis</em>

<div><p>Dark-grown seedlings exhibit skotomorphogenic development. Genetic and molecular evidence indicates that a quartet of <em>Arabidopsis</em> Phytochrome (phy)-Interacting bHLH Factors (PIF1, 3, 4, and 5) are critically necessary to maintaining this developmental state and that light activation of phy induces a switch to photomorphogenic development by inducing rapid degradation of the PIFs. Here, using integrated ChIP–seq and RNA–seq analyses, we have identified genes that are direct targets of PIF3 transcriptional regulation, exerted by sequence-specific binding to G-box (CACGTG) or PBE-box (CACATG) motifs in the target promoters genome-wide. In addition, expression analysis of selected genes in this set, in all triple <em>pif</em>-mutant combinations, provides evidence that the PIF quartet members collaborate to generate an expression pattern that is the product of a mosaic of differential transcriptional responsiveness of individual genes to the different PIFs and of differential regulatory activity of individual PIFs toward the different genes. Together with prior evidence that all four PIFs can bind to G-boxes, the data suggest that this collective activity may be exerted via shared occupancy of binding sites in target promoters.</p> </div

Expression patterns of PIF-encoding genes.

<p>(A–E) Representative images of histochemical staining of GUS activity in 2-d-old dark-grown transgenic seedlings. The <i>GUS</i> reporter gene is driven by <i>PIF1</i> (A), <i>PIF3</i> (B), <i>PIF4</i> (C), <i>PIF5</i> (D) and <i>CaMV 35S</i> (E) promoters, respectively. Data of biological triplicates were collected from two independent transgenic lines, and representative images are shown for each transgene. (F) Steady-state transcript levels of PIF-encoding genes defined by RNA-seq and RT-qPCR analyses in 2-d-old dark-grown WT seedlings. Data are presented as the mean of biological triplicates +/− SEM. (G) Relative expression of PIF-encoding genes in 2-d-old dark-grown <i>pifq</i> and <i>pif</i>-triple mutants. Expression was determined by RT-qPCR, normalized to an internal <i>PP2AA3</i> control, and presented relative to WT levels set at unity. Data are represented as the mean of biological triplicates +/− SEM.</p

Direct-target genes of PIF3-induced transcription (Class Z and YZ1.5 genes).

a<p>PIF3 loss-of-function (L.O.F.);</p>b<p>PIF3 gain-of function (G.O.F.);</p>c<p>PIF1/4/5-trio gain-of function (G.O.F.);</p>d<p>See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003244#pgen-1003244-g004" target="_blank">Figure 4</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003244#pgen.1003244.s003" target="_blank">Figure S3</a>;</p>e<p>See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003244#pgen.1003244-Leivar4" target="_blank">[17]</a>;</p>f<p>See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003244#pgen.1003244-Leivar3" target="_blank">[11]</a>;</p>g<p>Gene not represented on the Affymetrix ATH1 array.</p

Compiled ChIP–seq and RNA–seq data identify <i>PIL1</i> and <i>ATHB2</i> as direct targets of PIF3 transcriptional regulation.

<p>(A) Visualization of ChIP-seq and RNA-seq data in the genomic regions encompassing two representative genes, <i>PIL1</i> and <i>ATHB-2</i>. The ChIP and RNA tracks show the pile-up distribution of the combined raw reads from four biological replicates of ChIP-seq data and three replicates of RNA-seq data, respectively. P3M- and WT-ChIP: DNA immunoprecipitated from PIF3-Myc-expressing and from wild-type seedlings, respectively. WT-, <i>pif3-</i>, <i>pif145-</i> and <i>pifq</i>-RNA: RNA from genotypes used for expression analysis. Binding Site: 201 bp defined as the PIF3-binding site. Summit: predicted PIF3-binding center defined from the binding-peak maximum. G- and PBE-box: Vertical lines indicate motif positions. (B) ChIP-qPCR verification of specific PIF3 binding to the G-box-located promoter region of <i>PIL1</i>. The schematic diagram illustrates the genomic region around the <i>PIL1</i> locus. The short bars with numbers show 12 specific qPCR products. The black and white rectangular boxes represent CDS and UTR, respectively. Boxes labeled ‘G’ indicate the approximate locations of three G-box motifs in the <i>PIL1</i> promoter. The relative enrichment level is represented by the percentage of co-immunoprecipitated DNA to the input control in the P3M and WT samples. Data are represented as the mean of biological triplicates +/− SEM. 18S: <i>18S rRNA</i> as internal control.</p

PIFs direct differential light-signal channeling to the phy-regulated transcriptional network.

<p>Model depicting proposed quantitatively differential partitioning of transcriptional activation activity to shared direct-target genes, both by and between individual PIF-quartet members. Arrows represent the presence or absence and relative level (line thickness) of shared transcriptional activation of different direct-target genes by the individual PIFs in the dark. This representation is based on the data in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003244#pgen-1003244-g005" target="_blank">Figure 5A and 5B</a>. Light-activation of phy photoreceptors induces rapid proteolysis of the PIFs, reversing this transcriptional activity.</p

Differential regulation of PIF3 direct-target genes by individual PIF-quartet proteins.

<p>(A) Individual PIF-quartet members display diverse patterns of shared regulatory activity toward genes defined as direct targets of PIF3 transcriptional activation. Expression levels in the <i>pifq</i> and <i>pif</i>-triple mutants indicated, were determined by RT-qPCR, normalized to an internal <i>PP2AA3</i> control, and presented relative to WT levels set at unity. Data are represented as the mean of biological triplicates +/− SEM. (B) Matrix of relative contributions from individual PIF proteins toward the shared transcriptional activation of individual, potentially-shared direct-target genes. Percent contribution is calculated as the proportion of the total differential expression between <i>pifq</i> and WT, that is contributed by the differential expression between <i>pifq</i> and each <i>pif</i>-triple mutant. (C) <i>In planta PIL1</i> promoter activity requires both G-box motifs and PIF-quartet members. Left: Schematic of <i>pPIL1:LUC</i> constructs expressed transgenically in either WT or <i>pifq</i> plants, as indicated. Yellow and red stripes represent the locations of three native (<i>pPIL1</i>) and mutated (<i>mpPIL1</i>) G-box motifs, respectively, in variants of the <i>PIL1</i> promoter, as shown by the DNA sequences displayed below each construct. A contiguous 35S-promoter driven <i>RLUC</i> reporter was included as an internal control in each construct. Right: Mean expression of the <i>LUC</i> reporter gene is shown as LUC enzyme activity normalized to the RLUC control in the same transgenic plant. Data represent the means of 6 or 7 independent transgenic lines +/− SEM.</p