Article Figures & Data
Figures
- Figure 1.
Type-A ARR Phylogeny and Positions of T-DNA Insertions.
(A) An unrooted phylogenetic tree was made using receiver domain sequences of type-A and type-B response regulators from Arabidopsis (ARR), maize (ZmRR), and rice (Os with accession numbers). Full-length protein sequences of the response regulators were obtained from Entrez Protein Database (National Center for Biotechnology Information [NCBI]), and their receiver domain sequences were identified by searching Conserved Domain Database (version 1.62; NCBI). Receiver domain sequences were aligned using the CLUSTAL W program (version 1.81; University of Nijmegen, http://www.cmbi.kun.nl/bioinf/tools/clustalw.shtml), and the phylogenetic tree was constructed with 1000 bootstrapping replicates. The unrooted tree is presented in TreeView (version 1.6.6, 2001; Page, 1996). The bootstrap values are indicated on the tree. Scale bar represents 0.1 amino acid substitution per site.
(B) Positions of T-DNA insertions in the type-A arr mutants. The insertional mutants were identified by PCR screening, and the site of insertion determined by DNA sequencing of the border fragment. Boxes represent exons, lines represent introns, and inverted triangles indicate T-DNA insertions. Receiver domains are shaded. The DDK residues that are conserved in two-component receiver domains are indicated.
(C) Expression of type-A ARRs in insertional mutants. RNA from 3-d-old seedlings was either blotted to nylon for RNA gel blot analysis (left) or transcribed in vitro to cDNA for use in an RT-PCR reaction (right) as described in Methods. For the RNA gel blot, different cDNA clones were used as hybridization probes, as indicated above the figure, and the ethidium bromide–stained agarose gel is shown below. For RT-PCR, primers were designed to amplify the first three exons of ARR3 or the entire β-tubulin gene as a control. Col, wild-type Columbia.
- Figure 2.
arr Mutant Phenotypes.
(A) and (B) arr adult plants are affected in short-day conditions. Plants of the genotypes noted were grown in short-day conditions (8-h-light/16-h-dark) for 9 weeks. At least eight plants per genotype were examined, and photographs of representative plants for each line are shown. The experiment was conducted three times with similar results. The red scale bar in each photograph corresponds to 3 cm. Plants in (A) and (B) are from two separate experiments.
(C) arr seedlings are more sensitive to cytokinin. Seedlings were grown vertically on plates supplemented with the specified concentrations of BA or a DMSO vehicle control under constant light conditions at 23°C. Seedlings were photographed at 10 d.
(D) arr mutants form elaborate shoot structures on low cytokinin concentrations and fewer roots on high auxin concentrations in shoot initiation assay. Hypocotyls were excised from seedlings grown for 3 d in the dark, followed by 3 d in dim light, and transferred to media containing various concentrations of auxin (NAA) and cytokinin (kinetin) for 4 weeks under constant light. Five hypocotyls of each genotype were examined at each concentration. One hypocotyl representative of the response at each concentration was selected and arranged to create a composite photograph for each genotype.
- Figure 3.
arr Seedlings Are More Sensitive to Cytokinin Inhibition of Root Elongation.
(A) to (E) Seedlings were grown vertically on plates supplemented with the specified concentrations of BA or a DMSO vehicle control under constant light conditions at 23°C. Root elongation between days 4 and 9 was measured as described in Methods. Results shown were pooled from an experimental set of three independent samples of 10 to 15 individual seedlings. Error bars represent se (n > 30). Each experiment was repeated at least twice with consistent results.
(F) Complementation of arr3,4,5,6 phenotype with ARR5. A construct containing a wild-type ARR5 cDNA driven by the ARR5 promoter was transformed into arr3,4,5,6. Wild-type seedlings, various arr mutant seedlings, and 10 transformed lines were grown as in (A) to (E) in the presence of 5 nM BA (black bars), 10 nM BA (shaded bars), or a DMSO vehicle control (open bars). Ten independent T1 lines are denoted 1 to 10. Error bars represent se (n = 15).
- Figure 4.
arr Seedlings Are More Sensitive to Cytokinin Inhibition of Lateral Root Formation.
(A) to (E) Seedlings were grown vertically on plates supplemented with the specified concentrations of BA or a DMSO vehicle control under constant light conditions at 23°C. The total number of lateral roots was quantified at 9 d. Results shown were collected from the same experimental sets as in Figure 2. Error bars represent SE (n > 30).
(F) Complementation of arr3,4,5,6 phenotype with ARR5. A construct containing a wild-type ARR5 cDNA driven by the ARR5 promoter was transformed into arr3,4,5,6. Wild-type seedlings, various arr mutant seedlings, and seven transformed lines were grown as in (A) to (E) in the presence of 5 nM BA (black bars), 10 nM BA (shaded bars), or a DMSO vehicle control (open bars). Ten independent T1 lines are denoted 1 to 10. Error bars represent SE (n = 15).
- Figure 5.
arr3,4,5,6 Shows Delayed Leaf Senescence.
Fully expanded leaves were excised from 3.5-week-old plants and floated on water supplemented with various concentrations of cytokinin for 10 d in the dark. Chlorophyll content was determined spectrophotometrically as described in Methods. Three independent plates with six leaves per plate were examined at each concentration. Two chlorophyll measurements were taken per plate. Results shown are pooled from three independent experiments ±se (n = 18).
- Figure 6.
Expression Analysis of ARR Gene Promoters.
ARR promoter–driven GUS constructs were generated and introduced into wild-type Columbia background. Transgenic seedlings were grown on MS media (−BA) or media supplemented with 10 nM BA (+BA) for 9 d and assayed for GUS activity. Ten transformed lines were examined, and one representative line for each construct was photographed. With the exception of ARR8:GUS, close-up images show the relative GUS activity at the primary root tip. For ARR8:GUS, the close-up images show lateral root junctions on the primary root. Scale bars = 1 mm for aerial tissues, 250 μm for roots.
- Figure 7.
arr Mutants Are Affected in the Cytokinin Primary Response Pathway.
RNA was extracted from 10-d-old light-grown seedlings treated with 10 nM BA in liquid MS with 1% sucrose for the indicated time. The RNA was analyzed by RNA gel blotting. The blots were probed with either an ARR7, SST1, or β-tubulin radiolabeled probe. The signal obtained for each was quantified using a PhosphorImager, and the ARR7 and SST1 signals were normalized to the β-tubulin signal. The experiment was conducted twice with similar results.
- Figure 8.
arr Seedlings Exhibit Altered Hypocotyl Growth Response to Red Light.
Mutant and wild-type seeds were stratified and pretreated with fluorescent light before incubation under various red light intensities for 3 d ([A], [B], and [D]) or directly irradiated with red light after stratification (C). Mean hypocotyl lengths at various light intensities are normalized to the mean value of the etiolated seedlings of the respective genotypes. Mean etiolated hypocotyl heights (mm) are 9.7, 8.5, 8.7, 9.3, and 10 for arr3, arr4, arr5, arr6, and the wild type in (A); 8.6, 8.8, 8.2, 9.7, and 9.6 for arr3,4, arr4,6, arr4,5, arr5,6, and the wild type in (B); 8.6, 7.8, 6.4, 7.0, and 6.7 for arr3, arr4, arr5, arr6, and the wild type in (C); and 9.4 and 9.2 for arr3,4,5,6,8,9 and the wild type in (D), respectively. Bars represent SE (n > 13). The experiment was conducted twice with consistent results.
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