Altered auxin accumulation and transport in gain- and loss-of-function <i>idd</i> mutants.

<p>(A) Auxin accumulation assayed with the <i>DR5∶GUS</i> reporter in WT, <i>idd14-1D</i>, and <i>idd</i> triple mutant leaves. The leaves at 3, 5, and 15 days after initiation (from top to bottom panels) were subjected to GUS staining. The scale bars represent 50 µm in the top and middle panels and 5 mm in the bottom panel. (B) Expression of the <i>DR5∶GUS</i> reporter in inflorescence stems of WT, <i>idd14-1D</i>, and <i>idd</i> triple mutant plants. The scale bars represent 2 mm in the top panel and 100 µm in the bottom panel. (C) GFP fluorescence signals in the primary roots of WT, <i>idd14-1D</i>, and <i>idd</i> triple mutant carrying a <i>DR5∶GFP</i> reporter. The scale bar represents 50 µm. (D) Quantification of the GUS activity in the organs of the WT, <i>idd14-1D</i>, and <i>idd</i> triple mutant carrying a <i>DR5∶GUS</i> reporter. Data are from three biological replicates and shown as mean values ± one SD (Student's <i>t</i>-test, *P<0.05 and **P<0.01). (E) Endogenous free IAA levels in WT, <i>idd14-1D</i>, and <i>idd</i> triple mutant plants. Aerial organs of 15-day-old plants were used for measurement of free IAA levels. Data are from four biological replicates and are shown as mean values ± one SD (Student's <i>t</i>-test, *P<0.05 and **P<0.01). (F) Polar auxin transport capability of WT, <i>idd14-1D</i>, and <i>idd</i> triple mutant stems. The inflorescence stem segments of 5-week-old plants were used to determine the basipetal IAA transport efficiency and the background acropetal movement. Data are from four biological replicates and shown as mean values ± one SD (Student's <i>t</i>-test, *P<0.05 and **P<0.01).</p

Pleiotropic organ phenotypes in loss-of-function <i>idd</i> mutants.

<p>(A) Schematic illustration of the <i>idd14-1</i>, <i>idd15-5</i>, and the <i>IDD16-RNAi</i> construct. A 248-bp specific <i>IDD16</i> cDNA fragment was used for construction of <i>p35S::IDD16-RNAi</i>. (B) Semi-quantitative RT-PCR analysis of <i>IDD</i> genes in WT and <i>idd</i> mutants. The transcripts of <i>IDD14</i>, <i>IDD15</i>, and <i>IDD16</i> in the <i>idd</i> triple mutant are shown as representatives. (C) 36-day-old plants of <i>idd</i> single, double, and triple mutants. Arrows indicate the infertile siliques. The arrowheads show the increased angles between the inflorescence stems and branches. The scale bar represents 2 cm. (D) Morphology of sixth leaves in 25-day-old <i>idd</i> mutants. The longitudinal curvature (LC) index and leaf index were determined from at least 10 leaves in each genotype. The data are shown as mean values ± one SD (Student's <i>t</i>-test, *P<0.05 and ***P<0.001). (E) Enlarged floral organs and infertile siliques in the <i>idd</i> triple mutant. The arrow indicates the stigma lacking pollen. The arrowheads show the unfertilized ovules.</p

Phenotype of <i>cuf1-D</i> plants.

<p>(A) Morphology of 4-week-old wild-type (WT), <i>cuf1-D/+</i>, and <i>cuf1-D</i> plants. The scale bar represents 1 cm. (B) Blade areas of WT, <i>cuf1-D/+</i>, and <i>cuf1-D</i> leaves. (C) Transverse curvature (TC) index of WT, <i>cuf1-D/+</i>, and <i>cuf1-D</i> leaves. (D) Leaf index (ratio of length to width) in WT, <i>cuf1-D/+</i>, and <i>cuf1-D</i> plants. At least 10 sixth leaves from each genotype were used for determination of the leaf area, TC index, and leaf index as described in the methods section, respectively. Data are shown as mean values ± one SD, and the letters (a to c) indicate the significance (P<0.05) among the genotypes according to one-way ANOVA test (SPSS 13.0, Chicago, IL, USA).</p

Differential and overlapping expression of <i>IDD14</i>, <i>IDD15</i>, and <i>IDD16</i>.

<p>(A) Expression of <i>IDD14</i>, <i>IDD15</i>, and <i>IDD16</i> in seedlings assayed by GUS staining. The 12-day-old transgenic seedlings carrying <i>pIDD14::GUS</i>, <i>pIDD15::GUS</i>, or <i>pIDD16::GUS</i> constructs were subjected to GUS staining assays. Insets show the primary roots. The scale bar represents 1 mm. (B) GUS staining of the florescence stems of <i>pIDD14::GUS</i>, <i>pIDD15::GUS</i>, and <i>pIDD16::GUS</i> transgenic plants. Insets show the transverse sections of inflorescence stems. The scale bar represents 2 mm. (C) GUS staining of the floral organs of <i>pIDD14::GUS</i>, <i>pIDD15::GUS</i>, and <i>pIDD16::GUS</i> transgenic plants. The scale bar represents 1 mm. (D) Expression of <i>IDD14</i>, <i>IDD15</i>, and <i>IDD16</i> in inflorescence stems assayed by RNA <i>in situ</i> hybridization. A section hybridized with an <i>IDD16</i> sense probe is shown as a control. The scale bar represents 50 µm.</p

Molecular characterization of <i>CUF1</i>/<i>IDD14</i>.

<p>(A) Scheme of the genomic region flanking T-DNA insertion in <i>cuf1-D</i>. Genes are shown as thick arrows and intergenic regions are shown as lines. The orientation of T-DNA left border (LB), right border (RB), and the four CaMV 35S enhancers (4×35S) are indicated. (B) Linkage analysis of the T-DNA and <i>cuf1-D</i> phenotype. The 954-bp genomic fragments in WT and <i>cuf1-D/+</i> plants were amplified with primers in the genomic region flanking the T-DNA insertion site, and the 894-bp fragments in <i>cuf1-D/+</i> and <i>cuf1-D</i> were amplified with an LB primer and a downstream genomic primer. (C) Expression analysis of the genes flanking the T-DNA in WT and <i>cuf1-D</i> plants. <i>ACTIN2</i> (<i>ACT2</i>) was used as an internal control. Note that transcripts of At1g68130 (<i>IDD14</i>) are highly elevated in <i>cuf1-D</i>. (D) Morphology of 25-days-old WT, <i>cuf1-D</i> carrying <i>p35S::anti-IDD14</i>, and <i>p35S::IDD14</i> plants. The scale bar represents 2 cm. (E) Alignment of the ID domains in IDD14, IDD15, and IDD16. ZF1-ZF4 represents the four C2H2-type zinc finger motifs. The arrowheads indicate the conserved cysteine and histidine residues. (F) Morphology of 4-week-old transgenic plants overexpressing <i>IDD15</i> or <i>IDD16</i>. The scale bar represents 1 cm.</p

Genetic interaction of auxin biosynthesis and IDD-mediated organ morphogenesis and gravitropism.

<p>(A) Aerial organ morphology of 25-day-old <i>idd14-1D</i>, <i>yuc2 yuc6</i>, and <i>idd14-1D yuc2 yuc6</i> plants. The scale bar represents 2 cm. (B) Gravitropic responses of WT, <i>idd14-1D</i>, <i>yuc2 yuc6</i>, and <i>idd14-1D yuc2 yuc6</i> inflorescence stems. (C) 45-day-old plants of the <i>idd</i> triple mutant, <i>35S</i>-<i>YUC2</i>, and <i>idd</i> triple mutant carrying a <i>p35S::YUC2</i> construct. The scale bar represents 2 cm. (D) The silique orientation of WT, <i>idd</i> triple mutant, <i>35S</i>-<i>YUC2</i>, and <i>idd</i> triple mutant carrying a <i>p35S::YUC2</i> construct (one-way ANOVA test, P<0.05).</p

Silique orientation and gravitropic response in gain- and loss-of-function <i>idd</i> mutants.

<p>(A) Silique orientation in the <i>idd</i> single, double, and triple mutants. The angles between siliques and inflorescence stems were measured in 45-day-old WT and <i>idd</i> mutant plants. The data are shown as mean values ± one SD (Student's <i>t</i>-test, **P<0.01 and ***P<0.001). (B) Gravitropic responses of inflorescence stems in <i>idd14-1D</i> and loss-of-function <i>idd</i> mutants. The degree of curvature was determined from at least 18 inflorescence stems in each genotype after gravistimulation at the indicated times. The data are shown as mean values ± one SE.</p