Coordination between bZIP28 and HSFA2 in the regulation of heat response signals in Arabidopsis

<p>Heat stress can have detrimental effects on yield production worldwide. Although bZIP28 and HSFA2 were identified as putative heat sensors in plants, coordination between them has not been uncovered. In this study, the deficiency in bZIP28 did not affect heat tolerance in plants. However, the plants lacking bZIP28 showed enhanced activation of APXs-, MBF1c-and HSPs-dependent pathways as well as higher level of <i>HsfA2</i> transcripts and H₂O₂ accumulation, suggesting that these pathways might compensate for the deficiency in bZIP28 during heat stress. In addition, requirement of HSFA2 for the activation of APXs-dependent pathway during heat stress was supported by the analyses of plants lacking HSFA2. Our study demonstrated the flexible mode of heat response pathways involving bZIP28, HSFA2 and ROS-dependent signals.</p

Correction: A Maternal System Initiating the Zygotic Developmental Program through Combinatorial Repression in the Ascidian Embryo

<p>Correction: A Maternal System Initiating the Zygotic Developmental Program through Combinatorial Repression in the Ascidian Embryo</p

Tcf7-binding sites are critical for genes expressed specifically in the posterior vegetal hemisphere domain (PVD).

<p>(A–C) Analysis of a regulatory region of <i>Tbx6</i>.<i>b</i>. (A) Illustrations on the left depict the constructs. The numbers indicate the relative nucleotide positions from the transcription start site of <i>Tbx6</i>.<i>b</i>. Mutated Tcf7-binding sites are indicated by X. Graphs show the percentage of blastomeres expressing the reporter in the anterior vegetal blastomeres, in the posterior vegetal blastomeres, and in the animal blastomeres. Note that not all cells or embryos could express the reporter because of mosaic incorporation of the electroporated plasmid. (B, C) Images showing <i>Gfp</i> expression in embryos electroporated with the fourth and last constructs shown in (A). Scale bar, 100 μm. (D–F) Mapping of the Tcf7 and Zic-r.a ChIP data onto genomic regions consisting of the exons and upstream regions of (D) <i>Tbx6</i>.<i>b</i>, (E) <i>Wnttun5</i>, and (F) <i>Admp</i>. The ChIP-chip data are shown in bars, and the ChIP-seq data are shown as magenta lines. Each graph shows the fold enrichment (y-axis) for the chromosomal regions (x-axis). Green and yellow boxes indicate the regions essential for specific expression, which were revealed by the reporter gene assays shown in (A), and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006045#pgen.1006045.s006" target="_blank">S5 Fig</a>. Regions indicated by green boxes overlap peak regions identified by the peak caller programs for ChIP-seq and ChIP-chip, while the peak caller programs did not identify peaks within regions indicated by yellow boxes. (G) Gel-shift analysis showing that the proximal Tcf7-binding site did not bind GST protein but bound the Tcf7-GST fusion protein. The shifted band disappeared by incubation with a specific competitor, but not a competitor with a mutant Tcf7-binding site.</p

Gata-binding sites are critical for animal hemisphere-specific gene expression.

<p>(A) Analysis of a regulatory region of <i>Efna</i>.<i>d</i>. Illustrations on the left depict the constructs. The numbers indicate the relative nucleotide positions from the transcription start site of <i>Efna</i>.<i>d</i>. Mutant Tcf7-binding sites are indicated by X. Graphs show the percentage of blastomeres expressing the reporter in the vegetal blastomeres and in the animal blastomeres. Note that not all cells could express the reporter because of mosaic incorporation of the electroporated plasmid, and not all embryos could express the reporter either. (B, C) Images showing <i>Gfp</i> expression in embryos electroporated with the second and sixth constructs shown in (A). (D) Mapping of the Gata.a and Tcf7 ChIP data onto genomic regions consisting of the exons and upstream region of <i>Efna</i>.<i>d</i>. The ChIP-chip data are shown in bars. The ChIP-seq data are shown as a magenta line. Each graph shows the fold enrichment (y-axis) for the chromosomal regions (x-axis). Green and yellow boxes indicate the region essential for specific expression, which was revealed by the reporter gene assays. The green box indicates that a peak was identified by the peak caller programs for ChIP-seq and ChIP-chip in the region, while the yellow box indicates that no peaks were identified in the region. (E–I) Expression of <i>Gfp</i> in embryos injected with constructs containing the essential 419 bp region of <i>Efna</i>.<i>d</i>. Coinjection of the β-catenin MO evoked ectopic expression of the reporter with intact Gata-binding sites in the vegetal hemisphere, while it did not evoke expression of the reporter with mutant Gata-binding sites. (E) Graphs show the percentage of blastomeres expressing the reporter in the vegetal blastomeres and in the animal blastomeres. (F–I) Images showing <i>Gfp</i> expression in embryos injected with (F, G) the reporter construct with intact Gata sites or (H, I) mutant Gata sites. The embryos shown in (G, I) were co-injected with the <i>β-catenin</i> MO. Animal views (ani) and vegetal views (veg) are shown in each panel. White and magenta arrowheads indicate loss of expression and ectopic expression of the reporter, respectively. Scale bar, 100 μm.</p

Interactions between Tcf7, Gata.a, and β-catenin.

<p>(A–D) Using the upstream sequence of <i>Dlx</i>.<i>b</i>, (A) 3xmyc-tagged Tcf7, (B) 3xmyc-tagged Gfp, (C) 3xmyc-tagged Gata.a, and (D) 3xmyc-tagged Tcf7 and 3xflag-tagged Gata.a were misexpressed in epidermal cells. Note that Gfp protein expressed using the upstream sequence of <i>Dlx</i>.<i>b</i> is present in both the nucleus and cytoplasm (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006045#pgen.1006045.s011" target="_blank">S10 Fig</a>). Lysates of embryos were used for immunoprecipitation assays with an anti-myc antibody. Western blotting was performed with an (A–C) anti-β-catenin antibody and (D) anti-flag antibody. (E) Immunoprecipitation assay to examine interactions among recombinant β-catenin, Tcf7, and Gata.a proteins produced in <i>E</i>. <i>coli</i>.</p

Gata.a binding activity is suppressed in a ternary complex with Tcf7 and β-catenin.

<p>(A) Gata.a protein produced <i>in vitro</i> specifically recognized the Gata site critical for <i>Efna</i>.<i>d</i> expression. As a negative control, we used a rabbit reticulocyte lysate that did not contain template plasmids (NT control). (B) Co-incubation of Gata.a with either β-catenin or Tcf7 did not affect the binding activity of Gata.a for the proximal Gata site (Gata site 1) in the upstream sequence of <i>Efna</i>.<i>d</i>, whereas co-incubation of Gata.a with β-catenin and Tcf7 reduced the binding activity of Gata.a. (C) Co-incubation of Gata.a with β-catenin and Tcf7 reduced the binding activity of Gata.a for Gata sites in the upstream sequences of <i>Efna</i>.<i>d</i> and <i>Gdf1/3-r</i>. (D) Expression of <i>Efna</i>.<i>d</i> was suppressed in embryos injected with β<i>-catenin</i> mRNA. (E) ChIP followed by quantitative PCR revealed that BIO treatment reduced Gata-a binding to the <i>Efna</i>.<i>d</i> upstream region. Error bars indicate standard errors of three independent experiments.</p

Distribution of Gata.a, Tcf7, and Zic-r.a at the 16-cell stage.

<p>Immunostaining of (A, A’) Gata.a, (B, B’) Tcf7, and (C, C’) Zic-r.a with specific antibodies. (A–C) Animal views and (A’–C’) vegetal views are shown. Cell nuclei were stained with these antibodies (arrows). Areas with maternal <i>Zic-r</i>.<i>a</i> mRNA expression [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006045#pgen.1006045.ref014" target="_blank">14</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006045#pgen.1006045.ref015" target="_blank">15</a>] were stained with the anti-Zic-r.a antibody (arrowheads). Photographs are Z-projected image stacks overlaid in pseudocolor. Signals detected in cell nuclei are depicted by black dots in (A”–C”). Scale bar, 100 μm.</p

Regulatory system for the initial zygotic gene expression.

<p>Interactions among Gata.a, β-catenin, Tcf7, and Zic-r.a establish three distinct expression domains. See text for details.</p

Tcf7-binding sites are critical for expression in the vegetal hemisphere domain (VD).

<p>(A) Analysis of a regulatory region of <i>Fgf9/16/20</i>. Illustrations on the left depict the constructs. Green boxes indicate the <i>Gfp</i> gene and SV40 polyadenylation signal. The numbers indicate the relative nucleotide positions from the transcription start site of <i>Fgf9/16/20</i>. Mutant Tcf7-binding sites are indicated by X. The graphs show the percentage of blastomeres expressing the reporter in the anterior vegetal blastomeres, posterior vegetal blastomeres, and animal blastomeres. Note that not all cells or embryos could express the reporter because of mosaic incorporation of the electroporated plasmid. (B, C) Images showing <i>Gfp</i> expression, which was revealed by in situ hybridization, in embryos electroporated with the fourth and eighth constructs shown in (A). Scale bar, 100 μm. (D) Mapping of the Tcf7 ChIP data onto genomic regions consisting of the exons and upstream region of <i>Fgf9/16/20</i>. ChIP-chip data are shown in bars. ChIP-seq data are shown as a magenta line. Each graph shows the fold enrichment (y-axis) for the chromosomal region over <i>Fgf9/16/20</i> (x-axis). A green box indicates the region essential for specific expression, which was revealed by reporter gene assays. This region overlapped peaks identified by the peak caller programs for ChIP-seq and ChIP-chip. (E) Gel-shift analysis showing that Tcf7-binding site b did not bind the GST protein but bound the Tcf7-GST fusion protein. The shifted band was greatly reduced by incubation with a specific competitor, but not a competitor with a mutant Tcf7-binding site b.</p

A repressive regulatory element required for specific expression of <i>Tbx6</i>.<i>b</i>.

<p>(A, B) Co-immunoprecipitation assays showing the interaction between Tcf7 and Zic-r.a. (A) 3xmyc-tagged Tcf7 and 3xflag-tagged Zic-r.a were misexpressed in epidermal cells using the <i>Dlx</i> upstream sequence, and a lysate of misexpressed embryos was used for the immunoprecipitation assay. (B) Recombinant 3xmyc-tagged Tcf7 and 3xflag-tagged Zic-r.a were produced in <i>E</i>.<i>coli</i>, and applied to the co-immunoprecipitation assay. (C) Replacing the Tcf7 sites critical for expression of <i>Fgf9/16/20</i> in the anterior and posterior vegetal cells with the proximal Tcf7 site critical for expression of <i>Tbx6</i>.<i>b</i> in the posterior vegetal cells did not affect specificity of the reporter gene expression. (D) A series of mutant constructs to identify the region required for specific expression in posterior vegetal cells is shown on the left. Four repeats of the critical upstream sequence of <i>Tbx6</i>.<i>b</i> with or without mutations were ligated to the <i>Brachyury</i> basal promoter. Graphs show the percentage of blastomeres expressing the reporter among the anterior vegetal blastomeres and among the posterior vegetal blastomeres. A graph showing the percentage in the animal hemisphere is not shown, because no embryos expressed the reporter in the animal hemisphere. Note that not all cells or embryos could express the reporter because of mosaic incorporation of the electroporated plasmid. (E, F) Images showing the reporter gene expression in embryos electroporated with the (E) second and (F) fifth constructs shown in (D). (G) Illustrations on the left depict the constructs. A potential repressive element in the upstream sequence of <i>Tbx6</i>.<i>b</i> was inserted into the upstream sequences of <i>Fgf9/16/20</i> and <i>Foxd</i>.<i>b</i>. Graphs on the right show the percentage of blastomeres expressing the reporter gene in the anterior vegetal blastomeres and in the posterior vegetal blastomeres. A graph showing the percentage in the animal hemisphere is not shown, because no embryos expressed the reporter in the animal hemisphere. (H–L) The repressive element of <i>Tbx6</i>.<i>b</i> directed specific expression in the posterior vegetal cells in a manner dependent on Zic-r.a activity. (H) Constructs depicted in the illustrations on the left were injected with or without an MO against <i>Zic-r</i>.<i>a</i>. Graphs on the right show the percentage of blastomeres expressing the reporter gene in the anterior vegetal blastomeres and in the posterior vegetal blastomeres. (I–L) Photographs of embryos injected with the <i>Fgf9/16/20</i> reporter construct with the repressive element of <i>Tbx6</i>.<i>b</i> (I, J) and the intact <i>Fgf9/16/20</i> reporter construct (K, L). The embryos shown in (J, L) were co-injected with the <i>Zic-r</i>.<i>a</i> MO.</p