<i>STK</i> has a pivotal role in the control of PA production.

<p>Schematic representation of the pathways for PA production. The genes involved in these pathways are shown in boxes. <i>BAN</i>, that was found to be up-regulated in RNA-Seq, has been analysed by <i>in situ</i> hybridization, qRT-PCR and ChIP assay. The ChIP assay demonstrated that STK directly regulates <i>BAN</i>, <i>ABS</i> and <i>EGL3</i> (solid line). The ChIP assay revealed that STK function negatively correlates with the level of the H3K9ac mark on the <i>BAN</i> promoter (solid line with nucleosome).</p

Solvent soluble PAs analysed by LC-MS.

<p>Soluble PAs were detected in wild type (black bars) and the <i>stk</i> mutant (grey bars) at the immature (6 DAP; <i>A</i>) and mature (<i>B</i>) stages of seed development. Error bars represent SD of three independent measurements. Asterisks indicate statistically significant differences as determined by Student's <i>t</i> test (* <i>P</i><0.05, ** <i>P</i><0.01).</p

<i>stk</i> mutant seeds present defects in seed coat PA accumulation.

<p>(<i>A</i>) Sections of wild-type seeds stained with the toluidine blue O revealed the presence of phenolic compounds in the endothelium (ii1). (<i>B</i>) Scheme of Arabidopsis seed coat anatomy. (<i>C</i>) In the <i>stk</i> mutant phenolic compounds are accumulated in the endothelium (ii1) and also in the second layer of the inner integument (ii2, asterisk). (<i>D</i>) Whole-mount vanillin staining confirmed the presence of PAs in the wild-type and (<i>E</i>) in the <i>stk</i> mutant endothelium. In the <i>stk</i> mutant PAs are also accumulated outside the endothelium in the second layer of the inner integument (asterisk). mi, micropyle; en, endothelium. Scale bars = 30 µm (<i>A–E</i>).</p

Confocal laser-scanning images of <i>pSTK::STK-GFP</i> expression patterns during ovule and seed development.

<p>(<i>A</i>) Early ovule development: STK-GFP nuclear protein is expressed in the placenta and in the ovule primordia. (<i>B</i>) When integuments arise STK-GFP signal is localized in the nucellus and in the funiculus. (<i>C</i>, <i>D</i>) Mature ovule development: GFP can be detected throughout the integuments, funiculus and the adjacent placental region. (<i>E</i>) After fertilization the STK-GFP signal is present in the outer integuments and funiculus of developing seeds. (<i>F</i>) Magnification of figure E with an overlay projection images of specific PI staining to determine the presence of the GFP signal in the integuments. Protein can be detected in the two layers of the outer integument and also in the more external layer of the inner integument. op, ovule primordia; pl, placenta; nu, nucellus; i, integuments; fu, funiculus; mi, micropyle; ii2, internal layer of inner integument. Scale bars = 50 µm (<i>A</i> and <i>B</i>), 40 µm (<i>C</i>, <i>D</i> and <i>E</i>) and 20 µm (<i>F</i>).</p

STK directly regulates <i>BAN</i> expression through the modification of chromatin state.

<p>(<i>A</i>) <i>In situ</i> hybridization experiments illustrating the expression of the <i>BAN</i> transcript: in wild-type seeds at 1 DAP, <i>BAN</i> is expressed only in the endothelium layer. (<i>B</i>) <i>BAN</i> expression in the <i>stk</i> mutant background at 1 DAP and (<i>C</i>) at 4 DAP. In the <i>stk</i> mutant background, <i>BAN</i> expression is affected: the <i>BAN</i> transcript was detected not only in the endothelium layer but also in the ii2 layer. (<i>D</i>) Schematic representation of CArG box positions indicating the regions analysed by the ChIP experiment (black bars). Black boxes: exons; white boxes: promoter, introns, 3′ and 5′ UTRs. Asterisks indicate CArG boxes. (<i>E</i>) ChIP enrichment tests by qRT-PCR show that STK binds to the selected region of <i>BAN</i>. Fold enrichment was calculated over the negative controls. Error bars represent the propagated error value using three replicates. (<i>F</i>) ChIP enrichment tests by qRT-PCR show that STK negatively correlates with the H3K9ac acetylation mark at the <i>BAN</i> translational start site. qRT-PCR quantification of <i>BAN</i> sequences in precipitated chromatin was used to infer the acetylated histone H3 and total histone H3 representation at the STK-binding site. Levels of histone modification were normalized to total histone H3. Ct values were used to calculate the IP/IN signal. ChIP enrichments are presented as the percentage (%) of bound/input signal. ChIP enrichments for H3K9ac were normalized to histone H3 density. We tested the efficiency of IP by quantifying the presence of the H3K9ac mark in IAA8 <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004856#pgen.1004856-Zhou1" target="_blank">[49]</a> which was shown to be strongly and equally expressed in both samples and yielded equal enrichment ratios. mi, micropyle; ii1, endothelium; ii2, internal layer of inner integument. Scale bars = 40 µm (<i>A–C</i>).</p

<i>BAN</i> regulators are direct targets of STK.

<p>(<i>A</i>) qRT-PCR performed on cDNA obtained from siliques from 3 to 4 DAP and from unpollinated flowers for <i>ABS</i>. Relative mRNA levels indicate that the expression of all the genes is up-regulated in the absence of the STK protein at 3–4 DAP; these differences are statistically significant as determined by Statistical Student's <i>t</i> test (<i>P</i><0.01). The expression level of <i>ABS</i> is not affected in unpollinated flowers in the absence of the STK protein. Error bars represent the propagated error value using three replicates. (<i>B</i>) Schematic representation of CArG box positions. Schematic diagrams of EGL3, ABS and TT8 loci indicating the regions analysed by the ChIP experiment (black bars). Black boxes: exons; white boxes: promoters, introns, 3′ and 5′ UTRs. Asterisks indicate CArG boxes. (<i>C</i>) ChIP enrichment tests by qRT-PCR show that STK binds to the selected regions of <i>ABS</i> and <i>EGL3</i>. Fold enrichment was calculated over the negative controls. Error bars represent the propagated error value using three replicates.</p

Histogram of functional gene ontology analysis of differentially expressed genes.

<p>Slim Plant term enrichment - up and down-regulated genes. Genes with higher expression in <i>stk</i> in the Biological Process (<i>A</i>), Molecular Function (<i>B</i>) and Cellular Component (<i>C</i>) categories. Genes with lower expression in <i>stk</i> Molecular Function category (<i>D</i>).</p

The cytokinin signaling repressors <i>AHP6</i> and <i>ARR16</i> likely block cytokinin responses in lateral tissues.

<p><b>(A-D)</b> Expression of the transcriptional reporter <i>AHP6</i>::<i>GFP</i> in transverse sections of stage 7, 8, 9, and 12 gynoecia. <b>(E, F)</b> Expression of the cytokinin response reporter <i>TCS</i>::<i>GFP</i> in transverse sections of stage 9 and 12 gynoecia in an <i>ahp6-1</i> mutant background. Arrowheads indicate the absence of GFP signal in the epidermis of the valves. <b>(G, H)</b> Phenotypes of wild-type (G) and <i>ahp6-1</i> (H) gynoecia one week after receiving BAP treatment for two weeks. <b>(I-L)</b> Expression of the transcriptional reporter <i>ARR16</i>::<i>GUS</i> (type-A <i>ARR</i>) in transverse sections of stage 7, 8, 9, and 12 gynoecia. Scale bars: 10 μm (A-C, E), 20 μm (D, F), 1 mm (G, H), 100 μm (I-L).</p

The auxin transporter <i>PIN3</i> is coordinately activated by cytokinin and SPT.

<p><b>(A-C)</b> PIN3 expression in stage 9 <i>PIN3</i>::<i>PIN3-GFP</i> gynoecia that either received mock (<b>A,</b> transverse section) or BAP treatment for 48 hours (<b>B</b>, transverse section and <b>C</b>, longitudinal view). The inset in <b>(C)</b> shows a magnified view of the proliferating tissue. Arrows indicate the possible auxin flow. <b>(D-F)</b> PIN3 expression in transverse sections of stage 9 <i>PIN3</i>::<i>PIN3-GFP</i> gynoecia in <i>spt-2</i> <b>(D)</b>, <i>35S</i>::<i>SPT</i> <b>(E)</b>, and in <i>spt-2</i> treated for 48 hours with BAP <b>(F)</b>. <b>(G-J)</b> Transverse sections of stage 12 gynoecia of wild-type <b>(G, H)</b> and <i>pin3-4</i> <b>(I, J)</b>. Gynoecia phenotypes after three to four weeks of mock <b>(G, I)</b> or BAP treatment for five days <b>(H, J)</b>. Insets show a scanning electron microscopy image of the gynoecium. <b>(K)</b> Luciferase reporter assay in <i>N</i>. <i>benthamiana</i> leaves co-transformed with <i>35S</i>::<i>ARR1</i> and <i>pPIN3</i>::<i>LUC</i>. Ratio of LUC/REN activity. <b>(L)</b> ChIP experiments against the <i>PIN3</i> promoter regions (indicated by “a” and “b” in the scheme above) using an inducible <i>35S</i>::<i>ARR1ΔDDK</i>:<i>GR</i> line treated with dexamethasone or mock. <i>ACT2/7</i> served as a negative control. <b>(M)</b> Luciferase reporter assay in <i>N</i>. <i>benthamiana</i> leaves co-transformed with <i>35S</i>::<i>SPT</i> and <i>pPIN3</i>::<i>LUC</i>. Ratio of LUC/REN activity. <b>(N)</b> ChIP experiments against the <i>PIN3</i> promoter regions (indicated by “a” and “b” in the scheme above) using a <i>35S</i>::<i>SPT-HA</i> line and wild-type. <i>ACT2/7</i> served as a negative control. Error bars represent the SD for the LUC assays based on three biological replicates. ChIP results of one representative experiment is shown and the error bars represent the SD of the technical replicates. *<i>P</i> < 0.05 (LUC: Student-t test; qPCR: ANOVA). Scale bars: 10 μm (A-F), 100 μm (G-J, G-J insets). Ovule primordium (op).</p

Phenotypes of the type-B <i>arr</i> mutants and of the <i>spt</i> mutant.

<p><b>(A)</b> Mature gynoecium size of wild-type, <i>arr1</i>, <i>arr10</i>, <i>arr12</i>, <i>arr1 arr10</i>, <i>arr10 arr12</i>, <i>arr1 arr12</i>, and <i>arr1 arr10 arr12</i>. <b>(B)</b> Mature fruit size of wild-type, <i>arr1</i>, <i>arr10</i>, <i>arr12</i>, <i>arr1 arr10</i>, <i>arr10 arr12</i>, <i>arr1 arr12</i>, and <i>arr1 arr10 arr12</i>. <b>(C-F)</b> Phenotypes of the type-B <i>arr1 arr10 arr12</i> triple mutant compared to wild-type (WT): fruit length <b>(C)</b>, ovule number <b>(D)</b>, replum width <b>(E)</b>, and replum cell number <b>(F)</b>. <b>(G-I)</b> Transverse sections of stage 12 gynoecia of wild-type <b>(G)</b>, <i>arr1 arr10 arr12</i> (with transmitting tract and septum fusion defects) <b>(H)</b>, and <i>spt-2</i> <b>(I)</b>. Scale bars: 1 mm (A), 5 mm (B), 50 μm (G-I). Error bars represent SD. *<i>P</i> < 0.05 (Student-t test). Sample numbers: (C, D) WT, n = 14 and <i>arr1 arr10 arr12</i>, n = 19; (E, F) WT, n = 20 and <i>arr1 arr10 arr12</i>, n = 19.</p