Article Figures & Data
Figures
- Figure 1.
Root Phototropism in Arabidopsis Wild-Type Seedlings and in rpt Mutants.
Angles of root-growing direction against gravity (–χ°) were measured after 48 hr of exposure to blue light at the indicated fluence rates. Experiments were repeated independently three times, and the angles of root-growing direction of eight to 16 seedlings were measured each time. The average of degrees was calculated. Data and error bars represent the mean ±sd from the average values of the three repeated experiments. Wild type is Ler.
- Figure 2.
Second-Positive Hypocotyl Phototropism in Etiolated Arabidopsis Wild-Type Seedlings and in Etiolated rpt Single and Double Mutants.
The curvature of hypocotyls (χ°) was measured after 12 hr of exposure to blue light at the indicated fluence rates. Experiments were repeated independently three times, and the curvatures of hypocotyls of eight to 15 seedlings were measured each time. The average of curvature degrees was calculated. Data and error bars represent the mean ±sd from the average values of the three repeated experiments. Wild type is Ler.
- Figure 3.
In Vitro Blue Light–Induced Phosphorylation of a 120-kD Protein in Microsomal Membranes.
Microsomal membranes (20 μg) prepared from 5-day-old etiolated wild-type Ler seedlings (WT) and rpt2-1 seedlings (rpt2) were irradiated by blue light, incubated with γ-32P-ATP, electrophoresed on an 8% SDS–polyacrylamide gel, and autoradiographed. D, mock-irradiated control; L, blue light–irradiated microsomal membrane fraction.
- Figure 4.
Map Position and Genomic Structure of the RPT2 Gene.
(A) Map position of the RPT2 gene. Numerals under vertical arrows indicate the number of recombinant chromosomes found in 1116 chromosomes of F2 progeny exhibiting the rpt2-1 mutant genotype. Horizontal arrows indicate genes predicted from sequencing and annotation by the TIGR group. Regions covered by restriction DNA fragments isolated from bacterial artificial chromosome (BAC) clone T6B20, which were cloned in the pBI101 binary vector for use in the complementation assay, are shown with thick lines. cM, centimorgan.
(B) Genome structure of the RPT2 gene. Black and white rectangles indicate the protein coding regions and noncoding regions, respectively. The cause of the rpt2-1 mutation was a base substitution from G to A at the splice acceptor site of the first intron. Nucleotides in the intron are shown in italic.
- Figure 5.
Complementation of the rpt2 Mutation in Transgenic Plants.
(A) Root phototropic response. Three-day-old seedlings were photographed after continuous irradiation of the right side. rpt2PmaCI shows the phototropic response of the rpt2 plant transformed with a pBI-PmaCI construct. Wild type is Ler.
(B) Hypocotyl phototropic response. The degree of curvature was measured as described in Figure 2. rpt2Bst1107I and rpt2PmaCI show the phototropic response of the rpt2 plant transformed with a pBI-Bst1107I and a pBI-PmaCI construct, respectively. Error bars represent the mean ±sd from the average values of the three repeated experiments. Wild type is Ler.
- Figure 6.
Nucleotide Sequence of the RPT2 cDNA and Amino Acid Sequences of the Conserved Regions Found in Members of a Gene Family That Includes RPT2 and NPH3.
(A) cDNA sequence and deduced amino acid sequence of RPT2 (GenBank accession number AF181683). An asterisk indicates the stop codon. Underlined and double-underlined regions show conserved regions I and II, respectively, as shown in (B) and (C). The last amino acid residue before the site of the frameshift mutation in the rpt2-1 mutant (position 24) and the putative new stop codon are shown in white-in-black boxes. The putative phosphorylation sites recognized by protein kinase A ([RK]-[RK]-x-[ST], where x can be any amino acid) and protein kinase C ([ST]-x-[RK]), whose kinase domains are similar to that of the NPH1 protein, are boxed.
(B) Alignment of amino acid sequences of RPT2, NPH3, and the other predicted genes of Arabidopsis at regions I and II. Amino acid residues identical to those of RPT2 are shown in white-in-black boxes. Numbers shown in RPT2 and NPH3 are amino acid residue numbers. Other putative genes are designated by the BAC or P1 clone from which the genes were predicted. Numbers are nucleotide numbers registered in GenBank. Percentages of amino acid identity between deduced amino acid sequence of each gene and that of RPT2 are shown in parentheses.
(C) Structure of RPT2 and NPH3 proteins. Boxes I and II indicate conserved regions I and II, respectively, as shown in (B).
- Figure 7.
RNA Gel Blot Analysis of RPT2 Gene Expression.
Etiolated seedlings were irradiated with various types of light before preparation of total RNA. RNA gel blot hybridization was performed with the 32P-radiolabeled RPT2 cDNA and 18S rDNA as probes against 5 μg of total RNA.
(A) Independent expression of the RPT2 gene with respect to light quality. Lanes 1 and 2, mock-irradiated control (kept in the dark) for 4 and 24 hr, respectively; lanes 3 and 4, irradiated by white light for 4 and 24 hr, respectively; lanes 5 and 6, irradiated by blue light for 4 and 24 hr, respectively; lanes 7 and 8, irradiated by green light for 4 and 24 hr, respectively; and lanes 9 and 10, irradiated by red light for 4 and 24 hr, respectively. The graph shows the relative abundance of RPT2 mRNA under various light conditions. The value was calculated relative to that of the sample mock irradiated for 4 hr (lane 1) after normalization versus the 18S rRNA signal.
(B) Dependence of expression of the RPT2 gene on fluence rate. Lanes 1 to 6, irradiated by blue light for 4 hr at the fluence rate of 0, 0.01, 0.1, 1, 10, and 100 μmol m–2 sec–1, respectively. The graph shows the relative abundance of RPT2 mRNA under various fluence rates. The value was calculated against mock irradiation (lane 1) after normalization by the 18S rRNA.
- Figure 8.
Models of the Signaling Pathways of the Phototropic Response in Hypocotyls of Arabidopsis Seedlings.
The pathway marked by an asterisk indicates the inhibitory effect of NPH1 to the RPT2-independent pathway by adaptation or unknown mechanisms at high fluence rates. See the text for more details.
