Effect of <i>OsWOX4</i> knockdown on the expression of cell cycle-related genes.

<p>(A) Expression levels of <i>CDKB2;1</i>, <i>CYCA3;1</i>, <i>CYCB1;3</i>, <i>CYCD3;1</i>, <i>OsE2F2</i>, <i>OsDP1</i> and <i>OsRBR2</i> relative to mock-treated samples in microarray analysis. (B) to (E) Expression pattern of <i>HISTONE H4</i> ([B] and [C]) and <i>CDKB2</i> ([D] and [E]) genes. Transgenic plants (5 dag) carrying <i>pACT1-GVG>OsWOX4</i>:<i>RNAi</i> were treated as indicated in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007365#pgen.1007365.g004" target="_blank">Fig 4A</a>. M, SAM. Bars = 50 μm.</p

Effects of pulse downregulation of <i>OsWOX4</i> on vascular development in leaves.

<p>(A) to (D) Transverse sections of the central large vascular bundle (LVB) of a P4 leaf primordium in mock-treated (A) and DEX-treated (B and C) plants, and a P3 leaf primordium in a 5-dag plant before DEX treatment (D). The tissues were stained with toluidine blue. Bars = 10 μm. P, phloem; X, xylem. (E) Number of differentiated xylem cells in the central LVB of P4. (F) Area corresponding to xylem cells in the central LVB of P4. (G) Number of normal and incomplete LVBs in P4. Transgenic plants carrying <i>pACT1-GVG>OsWOX4</i>:<i>RNAi</i> were treated as indicated in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007365#pgen.1007365.g002" target="_blank">Fig 2A</a>. In (E) to (G), data are the mean ± SE (<i>n</i> = 12 [mock]; <i>n</i> = 13 [DEX]). Student’s t-test, *P < 0.01. ns, not significant.</p

Boxplots of (left panel) and log (right panel) in each functional category

The empty box indicates the interquartile (25–75%) range. Bars across the boxes represent the median value. Whiskers below and above the box indicate the range of values within 1.5 times the value of the upper or lower edge of the box. Circles represent outliers. The statistical significance of differences was tested by Dunnett's multiple comparison tests. Asterisks indicate significant differences with average values of all Cu-responsive genes (* <p><b>Copyright information:</b></p><p>Taken from "Gene expression and sensitivity in response to copper stress in rice leaves"</p><p></p><p>Journal of Experimental Botany 2008;59(12):3465-3474.</p><p>Published online 1 Aug 2008</p><p>PMCID:PMC2529235.</p><p></p

Effect of <i>OsWOX4</i> knockdown on the morphology of leaf primordia.

<p>(A) to (C) Transverse section of the shoot apex in a wild-type plant (A), and mock-treated (B) and DEX-treated (C) transgenic plants carrying <i>pACT1-GVG>OsWOX4</i>:<i>RNAi</i>. Tissues were stained with toluidine blue. M, SAM. (D) to (F) Central region of a P4 leaf primordium. Cells in the central file are indicated by false coloring and asterisks. (G) Number of cells in the central file of a P4 leaf primordium. Data are the mean ± SE (<i>n</i> = 12 [mock]; <i>n</i> = 13 [DEX]). Student’s t-test, *P < 10⁻⁴. (H) and (I) In situ localization of <i>DL</i> transcript. The SAM is indicated by the dotted line. Transgenic plants carrying <i>pACT1-GVG>OsWOX4</i>:<i>RNAi</i> were treated as indicated in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007365#pgen.1007365.g002" target="_blank">Fig 2A</a> (A to G) and in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007365#pgen.1007365.g004" target="_blank">Fig 4A</a> (H and I). Bars = 50 μm in (A) to (C), (H) and (I); 10 μm in (D) to (F).</p

Spatial expression pattern of <i>OsWOX4</i> detected by in situ hybridization and the effect of continuous downregulation of <i>OsWOX4</i> on leaf growth.

<p>(A) to (E) Spatial expression pattern of <i>OsWOX4</i> in the wild-type shoot apex. Cross-sections (A to D) and a longitudinal section (E). Cross-section A and C lies above and below the section containing the meristem (B), respectively. Arrowheads indicate the region where differentiation of large vascular bundles should initiate, and arrows indicate developing large vascular bundles. M, SAM; LV, large vascular bundle; SV, small vascular bundle. (F) and (G) Phenotype of the mock- (F) and DEX- (G) treated seedlings. Transgenic plants carrying <i>pACT1-GVG>OsWOX4</i>:<i>RNAi</i> were treated with DEX for 5 days from germination. Bars = 20 μm in (A) to (C); 50 μm in (D) and (E); 1 cm in (F) and (G).</p

Transcriptome analysis to examine the effect of <i>OsWOX4</i> knockdown.

<p>(A) Number of upregulated and downregulated genes (fold change ≥ 2.0, P < 0.01). (B) Selected terms in gene ontology enrichment analysis of genes that were up- or downregulated genes after 12 h of DEX treatment. Transgenic plants carrying <i>pACT1-GVG>OsWOX4</i>:<i>RNAi</i> were treated with DEX for 3 or 12 h, and then microarray analysis was performed as described in Materials and methods.</p

Effects of longer <i>OsWOX4</i> knockdown on cell activity.

<p>(A) Schematic representation showing the protocol for longer <i>OsWOX4</i> knockdown by DEX treatment. (B) and (C) Phenotype of seedlings after DEX or mock treatment. (D) Length of the 3^rd and 4^th leaves (total length of leaf blade and leaf sheath) of DEX- and mock-treated plants. Data are the mean ± SE (<i>n</i> = 6). Student’s t-test, *P < 10⁻⁴, **P < 10⁻⁵. (E) and (F) Shoot apex containing the SAM and leaf primordia (basal region). The SAM (M) and leaf primordia are outlined. (G) and (H) Shoot apex containing the leaf primordia above the SAM (apical region). (I), (K) and (M) Magnified views of the colored boxed regions shown in (G). (J), (L), and (N) Magnified views of the colored boxed regions shown in (H). The vascular bundle is outlined in (I) and (J). Transgenic plants carrying <i>pACT1-GVG>OsWOX4</i>:<i>RNAi</i> were treated as indicated in (A). Tissues were embedded in resin (Technovit 7100). Thin sections (0.7 μm) were generated and stained with toluidine blue. Bars = 1 cm in (B) and (C); 50 μm in (E) to (H); 5 μm in (I) to (N).</p

Reduced VLCFA synthesis increases the expression of <i>IPT3</i>.

<p>Expression patterns of <i>ProIPT3:GUS</i>. Aerial parts of 5-d-old seedlings grown in the absence or presence of cafenstrole (A), and transverse sections of shoot apices (B) and cotyledons (C). <i>pas2-1</i> grown in the absence of cafenstrole is shown for comparison. <i>ad</i>, adaxial side of cotyledons. Bars, 1 mm (A) and 100 µm (B, C).</p

<i>PAS2</i> expression in the epidermis is essential for plant development.

<p>(A) Quantification of <i>PAS2</i> expression levels in 5-d-old seedlings. The mRNA levels were normalized to <i>TUBULIN4</i>. The expression level in wild-type expressing <i>ProATML1:PAS2RNAi</i> is indicated as a relative value, with that in wild-type set to 1. Data are presented as mean ± SD (<i>n</i> = 3). (B) 5-d-old seedlings of wild-type and wild-type expressing <i>ProATML1:PAS2RNAi</i> with a severe phenotype. Transverse sections of shoot apices of 7-d-old seedlings are shown in (C). (D) 5-d-old seedlings of <i>pas2-1</i> and <i>pas2-1</i> expressing <i>ProATML1:PAS2–GUS</i>. (E) Transverse section of the shoot apex of a 5-d-old <i>pas2-1</i> seedling expressing <i>ProATML1:PAS2–GUS</i>. Bars, 1 mm (B, D), 200 µm (C), and 100 µm (E).</p

Overproliferation phenotypes of <i>pas2-1</i> mutants.

<p>(A) True leaf of a 2-wk-old <i>pas2-1</i> mutant seedling. (B) 5-d-old wild-type and <i>pas2-1</i> seedlings. (C) Cross sections of 5-d-old hypocotyls. (D) Transverse sections of shoot apices of 7-d-old seedlings. Bars, 1 mm (A), 500 µm (B), and 100 µm (C, D).</p