Overexpression of <i>PIF4</i> Increases the Expression of <i>YUC8</i> and Elevates Endogenous Free IAA Levels.

<p>(A) <i>YUC8</i> expression in wild type (Col-0) and <i>35S-PIF4</i> plants. Six-d-old Col-0 and <i>35S-PIF4</i> seedlings grown in normal growth conditions (22°C) were harvested at the same time for RNA extraction and qRT-PCR analyses. Transcript levels of <i>YUC8</i> were normalized to the <i>ACTIN7</i> expression and then were relative to those of Col-0 seedlings. Data shown are average and SD of triplicate reactions. Student's <i>t</i>-test between Col-0 and <i>35S-PIF4</i> seedlings was performed (**, P<0.01). Shown are representative data from one biological replicate; three biological replicates were conducted, yielding similar results. (B–C) <i>PIF4</i> and <i>YUC8</i> expression in transgenic plants containing a chemical-inducible construct <i>pMDC7:PIF4</i>. Eight-d-old <i>pMDC7:PIF4</i> seedlings were untreated or treated with 10 µM estradiol for 3 h before harvest for RNA extraction and qRT-PCR analyses. Transcript levels of target genes were normalized to the <i>ACTIN7</i> expression and then were relative to those of untreated seedlings (0 h). Data shown are average and SD of triplicate reactions. Student's <i>t</i>-test between estradiol-treated and untreated plants was performed (**, P<0.01). Shown are representative data from one biological replicate; three biological replicates were conducted, yielding similar results. (D) Overexpression of <i>PIF4</i> leads to increased free IAA levels. Eight-d-old seedlings of wild type and <i>35S-PIF4</i> seedlings grown in normal growth conditions (22°C) were harvested at the same time for free IAA measurement. Data shown are average±SD. Student's <i>t</i>-test between wild-type and <i>35S-PIF4</i> plants was performed (**, P<0.01). Shown are representative data from one biological replicate; this experiment was conducted for three biological replicates, yielding similar results.</p

Loss of PIF4 Function Disrupts the High Temperature–Induced Elevation of <i>YUC8</i> Transcripts and Free IAA Levels.

<p>(A–B) High temperature-induced expression patterns of <i>PIF4</i> and <i>YUC8</i> in wild type (Col-0) or the <i>pif4</i> mutant. Six-d-old Col-0 and <i>pif4</i> seedlings grown at 22°C in continuous light were transferred to 29°C in continuous light or were continually placed at 22°C for a 24 h time course, respectively. The 22°C-grown and 29°C-grown seedlings for each time point were harvested at the same time for RNA extraction and qRT-PCR analyses. Transcript levels of target genes were normalized to the <i>ACTIN7</i> expression and were relative to those of untreated Col-0 seedlings (0 h). Data shown are average and SD of triplicate reactions. Shown are representative data from one biological replicate; three biological replicates were conducted, yielding similar results. (C) High temperature-induced elevation of free IAA levels in hypocotyls of Col-0 and <i>pif4</i>. The hypocotyls of 6-d-old wild-type and <i>pif4</i> mutant seedlings grown at 22°C and 29°C in continuous light, respectively, were harvested for free IAA measurement. Data shown are average±SD. Student's <i>t</i>-test between 22°C and 29°C grown plants for each genotype was performed (**, P<0.01). Shown are representative data from one biological replicate; This experiment was conducted for three biological replicates, yielding similar results.</p

PIF4 Activates <i>YUC8</i> Expression, as Revealed by Transient Assays of <i>N. benthamiana</i> Leaves.

<p>(A) Transient expression assays showing that PIF4 activates the expression of <i>YUC8</i>. Representative images of <i>N. benthamiana</i> leaves 72 h after infiltration are shown. The right panel indicates the infiltrated constructs. (B) Quantitative analysis of luminescence intensity in (A). Five independent determinations were assessed. Error bars represent SD. Asterisks denote Student's <i>t</i>-test significance compared with control plants: ***, P<0.001. (C) qRT-PCR analysis of <i>PIF4</i> expression in the infiltrated leaf areas shown in (A). Total RNAs were extracted from leaves of <i>N. benthamiana</i> infiltrated with the constructs. Five independent determinations were assessed. Error bars represent SD.</p

The <i>shy2-2</i> Mutation Suppresses the Long-Hypocotyl Phenotype of <i>35S-PIF4</i> Seedlings.

<p>(A) Representative images showing that <i>shy2-2</i> suppresses the long-hypocotyl phenotype of <i>35S-PIF4</i>. Shown are 6-d-old seedlings of Col-0, L<i>er</i>, <i>shy2-2</i>, <i>35S-PIF4</i> and <i>35S-PIF4/shy2-2</i> grown at 22°C. (B) Hypocotyl length showing that <i>shy2-2</i> suppresses the long-hypocotyl phenotype of <i>35S-PIF4</i>. Hypocotyl length of six-d-old seedlings of the indicated genotypes grown at 22°C was measured. Data shown are average±SD. Student's <i>t</i>-test between mutant/transgenic and wild-type seedlings was performed (**, P<0.01). Shown are representative data from one biological replicate; three biological replicates were conducted, yielding similar results.</p

PIF4 Directly Binds to the Promoter Region of <i>YUC8</i>.

<p>(A) Illustration of the <i>YUC5</i>, <i>YUC8</i>, <i>YUC9</i> and <i>YUC10</i> promoter regions showing the presence of G-box DNA motifs. The arrows indicate positions of primers used for ChIP-PCR experiment. Shown are 2-kb upstream sequences of the <i>YUC</i> genes. The translational start site (ATG) is shown at position +1. (B) Gel photographs showing the amplified products from the ChIP assay. The ChIP assays were performed using 6-d-old seedlings expressing the PIF4-HA fusion protein untreated or treated with 29°C for 6 h. Antibody to the HA tag was used to immunoprecipitate PIF4-HA and associated DNA fragments. DNA was amplified by using primers specific to the region containing the G-box element or control regions in <i>ACT2</i> promoter as indicated. Shown are representative data from one biological replicate; this experiment was conducted for three biological replicates, yielding similar results. (C) EMSA assay showing that PIF4 binds the G-box motifs present in the <i>YUC8</i> promoter <i>in vitro</i>. The <i>YUC8</i> promoter fragments containing the G-box motifs were incubated with <i>in vitro</i> TNT-expressed PIF4 protein as indicated. Competition for PIF4 binding was performed with 10×, 20× and 50× cold <i>YUC8</i> probes containing G-box (G-wt, CACGTG) or mutated G-box (G-mut, CACGGG), respectively. FP, free probe. TnT indicates <i>in vitro</i>-expressed luciferase proteins used as a control.</p

The <i>yuc8</i> Mutation Reduces the Induction of Hypocotyl Elongation by High Temperature and <i>PIF4</i> Overexpression.

<p>(A) Hypocotyl length showing that the <i>yuc8</i> mutation reduces high temperature-induced hypocotyl elongation. Four-d-old seedlings grown at 22°C were transferred to 29°C in continuous light for additional 2 d before hypocotyl length measurement. Data shown are average±SD. Asterisks represent Student's <i>t</i>-test significance between 29°C and 22°C grown plants for each genotype or between pairs indicated with brackets (**, P<0.01). Shown are representative data from one biological replicate; three biological replicates were conducted, yielding similar results. (B) Hypocotyl length showing that the <i>yuc8</i> mutation partially suppresses the long-hypocotyl phenotype of <i>35S-PIF4</i> plants. The hypocotyl length of 6-d-old seedlings of the indicated genotypes grown at 22°C was measured. Data shown are average±SD. Asterisks represent Student's <i>t</i>-test significance between transgenic/mutant and wild-type plants or between pairs indicated with brackets (*, P<0.05; **, P<0.01). Shown are representative data from one biological replicate; three biological replicates were conducted, yielding similar results.</p

CHR729 Is a CHD3 Protein That Controls Seedling Development in Rice

<div><p>CHD3 is one of the chromatin-remodeling factors that contribute to controlling the expression of genes associated with plant development. Loss-of-function mutants display morphological and growth defects. However, the molecular mechanisms underlying CHD3 regulation of plant development remain unclear. In this study, a rice CHD3 protein, CHR729, was identified. The corresponding mutant line (<i>t483</i>) exhibited late seed germination, low germination rate, dwarfism, low tiller number, root growth inhibition, adaxial albino leaves, and short and narrow leaves. <i>CHR729</i> encoded a nuclear protein and was expressed in almost all organs. RNA-sequencing analysis showed that several plant hormone-related genes were up- or down-regulated in <i>t483</i> compared to wild type. In particular, expression of the gibberellin synthetase gibberellin 20 oxidase 4 gene was elevated in the mutant. Endogenous gibberellin assays demonstrated that the content of bioactive GA₃ was reduced in <i>t483</i> compared to wild type. Moreover, the seedling dwarfism, late seed germination, and short root length phenotypes of <i>t483</i> were partially rescued by treatment with exogenous GA₃. These results suggest that the rice CHD3 protein CHR729 plays an important role in many aspects of seedling development and controls this development via the gibberellin pathway.</p></div

Seed germination of WT and <i>t483</i>.

<p>A, Germination of WT and <i>t483</i> seeds after 3 days. B–E, Seedlings of WT and <i>t483</i> at 8 days post-germination. F, Seed germination rate. Values are means ±SD of three independent experiments. Significance of differences between WT and <i>t483</i> was determined by Student’s <i>t-</i>test (**<i>P</i><0.01). Scale bars: 2 cm (A–D); 1 cm (E).</p

Expression analysis of <i>CHR729</i> and subcellular localization of the encoding protein.

<p>A, qRT-PCR analysis of <i>CHR729</i> expression in WT roots (R), culms (C), leaves (L), leaf sheaths (Ls), and young 3 cm inflorescence (In). Dissected anthers (An), pistils (Pi), lemmas (Le) and paleas (Pa) at inflorescence stage 9 were also analyzed. Values are means ±SD of three replicates. B–D, <i>CaMV35S</i>:CHR729-GFP fusion protein localization in rice protoplast. B, Subcellular localization of CHR729-GFP fusion protein. C, Subcellular localization of MADS3-mCherry fusion protein (nuclear marker). D, Merged image of (B) and (C) in bright field. Scale bars: 10 μm (B–D).</p

Map-based cloning and confirmation of the <i>CHR729</i> gene.

<p>A, Map of the genomic region containing the <i>t483</i> mutant locus of interest. Numerals below the corresponding markers indicate the number of recombinants identified among F₂ plants with the mutant phenotype mutant. The mutated gene was located in a 27 kb region between markers IN27 and IN35. Three ORFs were predicted in the mapped region. Sequencing analysis revealed that an A to T substitution in the fifth exon of the ORF3 resulted in a stop codon in <i>t483</i>. B, Phenotypes of control and typical T₂ transgenic knockdown plants (RNAi-3) at the heading stage. C, Germination of control and RNAi-3 seeds at 3 day. D, Control and RNAi-3 seedlings 8 days post-germination. E, Two-week-old seedlings. F, Expression analysis of <i>CHR729</i> in leaves of control and RNAi-3 by qRT-PCR. G, Seed germination rates in control and RNAi-3 plants. H, Chlorophyll contents in control and RNAi-3 plants. Chlorophyll was extracted from above-ground parts of plants shown in (E). Values are means ±SD (n = 3, **<i>P</i><0.01). Scale bars: 25 cm (B); 2 cm (C, D); 5 cm (E).</p