Article Figures & Data
Figures
- Figure 1.
Immunoblot Analysis of Photosynthetic Membrane Proteins from hcf152-1, hcf152-2, and the Wild Type.
Chloroplast proteins (20 μg or the indicated dilution of the wild-type sample) of 3-week-old plants were used for the analysis. Plants were grown under low light (5 to 10 μmol·m−2·s−1) in a growth chamber. Samples were fractionated on SDS-polyacrylamide gels, transferred to nitrocellulose, and immunodetected with the indicated antisera.
- Figure 2.
Transcript Pattern of the psbB-psbT-psbH-petB-petD Operon in the Mutants hcf152-1 and hcf152-2.
Eight micrograms of total leaf RNA from 3-week-old mutant and wild-type (WT) seedlings was analyzed by RNA gel blot hybridization. The nylon filters were hybridized with the gene-specific probes indicated with arrows and letters (A to H). Introns of the petB and petD genes are designated IB and ID, respectively. The sizes (in nucleotides) and identities of the transcripts are indicated.
- Figure 3.
RNase Protection Assay for Processed and Spliced RNAs of the psbB-psbT-psbH-petB-petD Operon in hcf152-1, hcf152-2, and the Wild Type.
RNase protection assay for spliced and processed petB, petD (A), and psbH (B) RNAs. Total leaf RNA isolated from the wild type (WT) or the mutants (hcf152-1 and hcf152-2) was annealed with uniformly labeled probes spanning the petB, petD, and psbH splice junction and processing sites. The transcribed probes included a small amount of vector sequence on both sides to be separated from the protected fragment of the unspliced RNA. A parallel control reaction contained no leaf RNA but an equivalent amount of yeast tRNA (tRNA). The RNA was digested with RNase T1, and the protected fragments were analyzed on sequencing gels and by autoradiography. The locations of the probes are indicated in the schemes (thick lines). The sizes of the protected fragments (thin lines and numbers in boldface to the left of the lanes) were determined by coelectrophoresis with a DNA sequence ladder. For subsequent identification of protected bands, a 100-bp DNA sequence ladder was used that was radioactively labeled (M) (sizes of the marker fragments are indicated with lightface numbers). Protected probe fragments correspond to unspliced (u), spliced (s), spliced intron (si), unspliced processed (u,p), or processed (p) RNA and are indicated at right. A protected band of the petD probe of ∼220 nucleotides of unknown origin is indicated with an asterisk. S1 protection experiments revealed a single protected fragment of 430 nucleotides for the unspliced petD RNAs, indicating that the triplet detected here reflects heterogeneous cutting by RNase T1.
- Figure 4.
Map of the Genomic Region Around the T-DNA Insertion in hcf152-1 and Analysis of Transcript Levels of the Corresponding Genomic Region.
In the scheme at top, the T-DNA insertion is located in a genomic segment of BAC F11F8. Genes adjacent to the insertion point are shown and designated according to the MIPS database code. Black bars represent exon sequences, and white bars represent intron sequences. Arrows indicate gene orientation. The probes used in RNA gel blot hybridization experiments are depicted below the scheme.
(A) to (C) Transcript levels of the At3g09650 (A), At3g09660 (B), and At3g09670 (C) genes in wild-type (WT) and hcf152-1/2 detected with double-stranded probes. Five micrograms of poly(A) RNA was loaded in each lane.
(D) Wild-type and mutant RNA hybridized with an antisense probe of the At3g09650 gene. The transcript of HCF152 is indicated by the arrow. Two micrograms of poly(A) RNA was loaded in each lane.
- Figure 5.
Characterization of Complemented hcf152 Transformants.
(A) Chlorophyll fluorescence induction kinetics of the wild type, hcf152-1 mutants, and hcf152-1 transformants complemented with the open reading frame of the At3g09650 gene. A representative curve for each plant is shown (for details, see Methods). Fm, maximal fluorescence; Fo, initial fluorescence.
(B) Transcript pattern of the psbB-psbT-psbH-petB-petD operon in the T1 generation of complemented hcf152-1. The RNA gel blot was hybridized with the probe against the petD gene. Arrows indicate significant differences between the mutants and the transformants.
- Figure 6.
Predicted Amino Acid Sequence of HCF152 and Alignments of Its PPR Motifs.
(A) Protein sequence of HCF152 with the putative plastid transit sequence underlined. The dotted line indicates the sequence translated from the upstream ATG start codon. The amino acid that is altered in the EMS allele HCF152-2 (Pro → Leu) is boxed.
(B) Alignment of the 12 putative PPR motifs. Residues identical to the consensus motif (shown at bottom) are shaded in black, and similar residues are shaded in gray.
(C) Comparison of the PPR motif arrangement in HCF152 with that of CRP1 of maize. The PPR motifs are indicated as open boxes. aa, amino acids.
- Figure 7.
Detection of HCF152 in Arabidopsis Chloroplasts.
(A) Detection of HCF152 in the wild type (WT) and hcf152-1 mutants. The protein was detected by immunoblot analysis using a specific HCF152 antibody (top gel). Total leaf proteins (50 μg) were analyzed. Pounceau S staining of the large subunit of ribulose bisphosphate carboxylase at 55 kD is presented in the bottom gel.
(B) Immunolocalization of HCF152. Chloroplasts were purified from Arabidopsis leaves and were fractionated further into the stroma and membrane fractions. Proteins (100 μg) of these fractions and total soluble leaf proteins were examined by immunoblot analysis using the specific antibodies to HCF152, the cytoplasm- and nucleus-located NAP-1, the stroma-located large subunit of ribulose bisphosphate carboxylase (RuBPcase), and the thylakoid protein D1.
(C) Quantification of HCF152 in the stroma fraction. A total of 100 μg of chloroplast stromal proteins as well as the indicated amounts of glutathione S-transferase fused to the N terminus of the HCF152 protein (GST-HCF152) was examined by immunoblot analysis. The native HCF152 and the GST-fused HCF152 are indicated by arrows. The HCF152 antibodies do not cross-react with the GST part of the fusion protein used for quantification.
(D) Fractionation of HCF152 by size-exclusion chromatography. Chloroplast-soluble proteins were fractionated on a Superdex 200 size-exclusion column. The proteins of each fraction were examined for the presence of HCF152 by immunoblot analysis with specific antibodies. The elution profiles of several molecular mass markers are indicated at top. Extensive treatment with ribonucleases did not change the elution profile.
- Figure 8.
RNA Binding Characteristics of HCF152.
(A) Scheme of the Arabidopsis psbB-petB region. The RNA probes for the binding assays are indicated by arrows and letters (BDb to BDf). The length of each arrow indicates the length of the probe, and a scale bar for 400 nucleotides is indicated. Stars indicate the high-affinity binding sites for HCF152.
(B) RNA binding of HCF152-F to several RNAs derived from the psbB-petD operon was analyzed with the UV cross-linking assay.
(C) Competition UV cross-linking assay. The indicated RNAs competed with the BDd RNA binding to the recombinant HCF152-F. The experiments were performed with radiolabeled BDd RNA and 25-, 50-, or 100-fold molar excess of the nonradioactive RNAs BDb to BDf. The IC50 values calculated from the graphs shown in (D) of at least three independent experiments are shown.
(D) Intensities of the bands in (C) were quantified and plotted. The intensity without a competitor RNA was defined as 100%. Closed circle, BDb; closed triangle, BDc; closed square, BDd; open circle, BDe; open square, BDf.
