BAF60 controls histone modifications and RNA Pol II occupancy at the <i>IPT3</i> and <i>IPT7</i> loci.

<p>(A) Schematic representation of the regions of the <i>IPT3</i> and <i>IPT7</i> loci analysed. Black boxes correspond to exons, the arrow indicates the site of translation initiation, numbers indicate the positions of primer pairs used. (B) Quantification data of the chromatin immunoprecipitation results. Nuclei were extracted from 14-day-old roots and immunoprecipited with antibodies specific for H3K27me3, or H3K4me3 or RNA polymerase II. Average relative quantities ± sd are shown for each sample. Data [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138276#pone.0138276.ref012" target="_blank">12</a>]significant difference from the wild type (WT) (P < 0.05, Student’s t test).</p

BAF60 control cell cycle progression.

<p>(A) Mitotic index in root tips of 7-day-old wild-type and <i>BAF60</i> RNAi plantlets grown vertically on MS medium. Values are average +/- standard deviation of biological triplicates. Asterisks indicate significant difference from the wild type (WT) (P < 0.01, Student’s t test). (B) Histochemical staining of the GUS activity in wild-type and <i>BAF60_2</i> RNAi roots harbouring the CYCB1;1::DB-GUS construct. The number of stained cells reflects the number of cells in G2/M. Plantlets were grown vertically for 7 days on MS medium. Bars = 100 μm. (C) Proportion of EdU positive cells in the root tips of seven-day-old wild-type and <i>BAF60</i> RNAi lines grown vertically on MS medium. Values are average +/- standard deviation of biological triplicates. Asterisks indicate significant difference from the wild type (WT) (P < 0.01, Student’s t test). (D) Cell cycle progression was examined by flow cytometry. Histograms show the percentage of cells in G1, S and G2/M phases in wild-type and <i>BAF60</i> RNAi root-derived protoplasts. Plantlets were grown vertically for 7 days on MS medium before protoplast preparation. Data presented are representative of three biological replicates. Asterisks indicate significant difference from the wild type (WT) (P < 0.05, Student’s t test).</p

BAF60 regulates cytokinin production.

<p>(A) Zeatin content in the roots of 14-day-old wild-type and <i>BAF60</i> RNAi plantlets grown vertically on MS medium. Values are average +/- standard deviation of four biological replicates. Asterisks indicate significant difference from the wild type (WT) (P < 0.01, Student’s t test). (B) qRT-PCR data show the relative expression of the indicated genes in wild-type and <i>BAF60</i> RNAi lines. Total RNA samples were collected from 14-day-old roots. mRNA abundance was quantified by qRT-PCR and expressed relative to the abundance of <i>UBQ10</i> transcripts. Values are average +/- standard deviation of triplicates. Data presented are representative of three biological replicates. Asterisks indicate significant difference from the wild type (WT) (P < 0.01, Student’s t test).</p

BAF60 binds <i>IPT3</i> and <i>IPT7</i> loci to regulate gene loops formation.

<p>(A) Top: Schematic representation of the <i>IPT3</i> and <i>IPT7</i> loci, the position of each primer pair used for ChIP-qPCR is indicated. The arrow indicates the position of the transcription start site. Exons are represented as black boxes and introns as black lines. Bottom: Quantification data of chromatin immunoprecipitation results. Nuclei were extracted after cross-linking from 14-day-old roots expressing the BAF60-CFP transgene. Chromatin-protein complexes were isolated and immuno-precipitated with antibodies specific for GFP or IgG. Average relative quantities ± sd are shown for each sample. Data presented are representative of three biological replicates. Asterisks indicate significant difference from the wild type (WT) (P < 0.05, Student’s t test). (B) Quantitative 3C of the <i>IPT3</i> and <i>IPT7</i> loci using region I as the anchor region in 14-day-old WT and <i>BAF60</i> RNAi roots. Relative interaction frequencies were calculated as described in Materials and Methods. Data are the average of three biological replicates (each performed on three technical replicates). In the graph, NheI-HF and BglII restriction sites are indicated with vertical dotted lines for <i>IPT3</i> and <i>IPT7</i> respectively. A schematic representation of these loci is shown above with the position of primers used for the 3C analysis represented by grey arrowheads. Grey Asterisks indicate significant difference between the crosslinked and the not crosslinked (P < 0.05, Student’s t test). Black Asterisks indicate significant difference from the wild type (WT) (P < 0.05, Student’s t test).</p

BAF60 regulates root development.

<p>(A) Fourteen-day-old wild type (WT) and <i>BAF60</i> RNAi lines grown vertically under LD conditions. Bar = 10 mm. (B) Time course analysis of root length (left) and lateral root density (right) in wild type (black) and <i>BAF60</i> RNAi plantlets (red) grown vertically on MS medium. Values are average +/- standard deviation (n = 100). Asterisks indicate significant difference from the wild type (WT) (P < 0.01, Student’s t test). (C) Quantification of root meristem length of fourteen-day-old wild type and <i>BAF60</i> RNAi lines grown vertically on half MS medium. Values are average +/- standard deviation (n > 15). (D) Confocal images of mPS-PI–stained root tips of fourteen-day-old wild type and <i>BAF60</i> RNAi plantlets growth vertically on MS medium. Bars = 50 μm.</p

Model for the role of BAF60 in root development.

<p>In the wild-type root tip, BAF60 binds <i>IPT3</i>, <i>IPT7</i> and <i>KRP7</i> and inhibits their expression during root development. In contrast, in <i>BAF60</i> RNAi lines, these genes are overexpressed due to formation of gene loops and augmentation of H3K4me3 level. This blocks the cell cycle in G1 phase and inhibits root development.</p

MIK2 is required for resistance to the fungal root pathogen <i>Fusarium oxysporum</i> in a THE1-independent manner.

<p>(A,B) Percentage of chlorotic leaves per plant (A), and percentage of decayed plants (B) after infection of the roots with <i>F</i>. <i>oxysporum</i> isolate Fo5176. (A) The percentage of chlorotic leaves per plant was counted 10 days after inoculation with <i>F</i>. <i>oxysporum</i> spores. (B) The number of decayed plants was counted 3 weeks after inoculation with <i>F</i>. <i>oxysporum</i> spores. (A,B) The bars represent the average of four independent experiments, each consisting of n = 20–40 plants per genotype. Error bars represent the standard error of n = 4 experiments. Different letters indicate statistically significant differences between genotypes (ANOVA and Holm-Sidak test (<i>p</i> < 0.05)). No disease symptoms were observed on mock-inoculated plants for any of the genotypes (n = 10). (C) Representative pictures of the different genotypes in (A) and (B) after <i>F</i>. <i>oxysporum</i> infection.</p

MIK2 controls root angle in a THE1- and cellulose synthase complex-dependent manner.

<p>(A-D) Nine-day-old Arabidopsis seedlings grown in an upright position (under a 10° angle relative to the direction of gravity) on MS agar medium with 1% sucrose. Pictures were taken from the front of the plate. (A-C) The growth medium contained DMSO (mock) (A), 2 nM ISX (B), or 25 μM DCB (C). (A) The white arrow indicates skewing of <i>mik2-1</i> roots relative to the vertical growth axis. (A-D) Root angle was quantified; a positive value indicates skewing to the left, while a negative value indicates skewing to the right. Error bars represent standard error of n = 15 biological replicas. Different letters indicate statistically significant differences between genotypes (ANOVA and Holm-Sidak test (<i>p</i> < 0.05)). The experiments were repeated at least three times with similar results.</p

MIK2 is required for salt stress tolerance in a THE1-dependent manner.

<p>(A) Ten-day-old Arabidopsis seedlings were grown in an upright position on ½ MS agar medium without sucrose, supplemented with or without 75 mM NaCl or 150 mM sorbitol. Depicted is the change in the angle of the root after NaCl or sorbitol treatment compared to mock treatment; the negative value indicates a change to the right. Error bars represent standard error of n = 20 biological replicas. The experiment was repeated three times with similar results. (B) Dry weight of NaCl-treated plants as percentage of the dry weight of untreated plants. (Absolute dry weight is depicted in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006832#pgen.1006832.s010" target="_blank">S10 Fig</a>). One week after germination, plants were transferred to pots with soil watered from below with or without 75 mM of NaCl in rainwater. After 4 weeks of treatment the rosettes were cut, and dry weight was determined. The experiment was repeated three times with similar results, data were pooled and the average is depicted. Error bars represent the standard error of n = 60 plants. (A,B) Different letters indicate statistically significant differences between genotypes (Kruskal-Wallis ANOVA on ranks followed by Dunn’s multiple comparison procedures (<i>p</i> <0.05)).</p

The LRR-RK MIK2 and CrRLK1L THE1 are major regulators of responses triggered by cellulose biosynthesis inhibition.

<p>(A) Immune marker gene expression in 13-day-old Arabidopsis seedlings determined by qRT-PCR. Seedlings were mock treated, or treated with 0.6 μM ISX, 6 μM DCB, or 0.4 μM TXT for 9 h. Expression of the immune marker genes <i>FRK1</i>, <i>At1g51890</i>, and <i>CYP81F2</i> was normalized relative to <i>U-box</i> expression values. Depicted is the fold change in expression relative to mock treatment. Error bars represent standard error of three technical replicas. (B-E) JA (B) and SA production (C) and lignin-deposition (D,E) in 6-day-old Arabidopsis seedlings, mock treated or treated with 0.6 μM ISX for 7 h (B,C) and 12 h (D,E). Error bars represent standard error of n = 4 (B,C) or n = 20 (E) biological replicas. (B) The upper and lower panel display the same data, yet in the lower panel, the y-axis has been adjusted to visualize the JA levels in mock-treated samples. (D) The size bar represents 100 μm. (A-E) Asterisks indicate a statistically significant difference relative to Col-0, as determined by a two-tailed Student’s <i>T</i>-test (<i>p</i> < 0.05). Experiments were repeated at least three times with similar results.</p