Fig 1.
Chilling sensitivity of the RNP mutants.
(A) Shown are wild type Col-0, orrm1, and aml1 mutants grown for 2 months at 4°C on the plate under a 16 h light/day photoperiod. Representative chilling sensitive phenotypes are shown. (B) Shown are wild type Col-0, rbd1-1, orrm1, aml1, cp29a, and cp31a mutants grown at 22°C and 4°C. Top row: Plants grown for 3 weeks at 22°C under a 12 hour (h) light photoperiod. Bottom row: Plants grown for 3 weeks at 22°C, then shifted to 4°C for additional 4 weeks under a 12 h light photoperiod. (C) Shown are fresh weights of the 5 RNP mutants grown at 22°C and 4°C for 3 weeks and 10 weeks respectively under a 12 h light photoperiod.
Table 1.
Chilling sensitive mutants potentially defective in chloroplast-localized proteins.
Fig 2.
Characterization of the rbd1 mutants.
(A) Structure diagram of the RBD1 protein. It contains one RNA recognition motif/ RNA-binding domain between 188–267 amino acid (aa) predicted at SMART (http://smart.embl-heidelberg.de/). (B) Diagram of T-DNA insertion mutants of the RBD1 gene. Arrows indicate the translation start (ATG) and stop (TAA) codons. Triangles indicate the site of T-DNA insertion in the SALK_041100 (rbd1-1) and SALK_012657 (rbd1-2) mutants. ‘a’ and ‘b’ mark the regions for RT-PCR analysis of RBD1 gene expression. (C) Semi-quantitative RT-PCR analysis of RBD1 expression in rbd1 mutants. The Actin gene was used as a normalization control. Bands of each row are cut from the same gel. (D) Phenotype of rbd1 mutants, rbd1 RNAi and complementation lines. Top row: plants grown for 3 weeks at 22°C with 12h light /12h dark. Bottom row: plants grown for 3 weeks at 22°C, then shifted to 4°C for additional 4 weeks with 12h light /12h dark. (E) The rbd1-1 plants at 2 days of 22°C recovery after 3 weeks of 4°C treatment. (F) The accumulation of pigments in rbd1 mutants and complementation lines. Shown are contents of chlorophyll a, chlorophyll b and carotenoid from the fifth and sixth leaves of 2 weeks old plants grown at 22°C with constant light. Means ± SD, n = 3, * represents significant differences between means at P < 0.05 according to Student’s t test.
Fig 3.
Expression of CBF and COR genes in rbd1-1 mutant.
(A-B) Expression level of CBF1, CBF2 and CBF3 (A) and COR15A, KIN1 and COR47 (B) after 4°C treatment at different time points. RNAs were isolated from five newly emerging leaves from 2 weeks old plants grown at normal condition followed by 4°C treatment. The Actin gene was used as a normalization control and the experiments were repeated three times with similar results (A). Equal loading was controlled by cytosolic 25S rRNA (25S) stained with ethidium bromide (B). (C) The expression patterns of RBD1 under 4°C. Total RNA was extracted from two weeks old plants chilling-treated for 3, 7, 14 and 21 days. The Actin gene was used as a normalization control. The experiments were repeated three times with similar results.
Fig 4.
Gene expression and protein localization of RBD1.
(A) Detection of RBD1 transcripts in various tissues. Total RNA was extracted from the indicated tissues from plants grown at 22°C with constant light. The Actin gene was used as a normalization control. (B) GUS staining of pRBD1::GUS transgenic plants. (C) Shown are confocal microscope images of protoplasts isolated from wild-type Arabidopsis leaves either untransformed or transformed with GFP or RBD1:GFP. A representative image of a single protoplast is shown in each panel. Left panels show the GFP signals, middle panels show the chlorophyll signals, and the right panels show the merged signals.
Fig 5.
Chloroplast translation is inhibited in the rbd1 mutants.
(A) Total RNAs were fractionated on 1.2% formaldehyde gels and stained with ethidium bromide. (B) RNA blotting analysis of 23S rRNA processing. Total RNAs extracted from leaves were fractionated on 1.2% formaldehyde gels and hybridized to a probe (indicated by a black bar) detecting the 3’-end of the 23S rRNA. Equal loading was controlled by the cytosolic 25S rRNA (25S) stained with ethidium bromide. Processing sites in the 23S rRNA precursor were indicated by scissors. Marked numbers indicate the relative amount in the mutant compared to that in Col-0 quantified by Image J. (C) Coomassie Blue staining of total proteins from the wild type and the rbd1 mutants after SDS-PAGE separation. Accumulation of Rubisco Large Subunit (RbcL) is indicated by an arrow. (D) Shown are Col-0, rbd1 mutants and complementation lines grown on solid media in the presence or absence of spectinomycin at 3 mg/L for 7 days. For A, B, and C, plants were grown for 3 weeks at normal condition followed by 4°C for 4 weeks, and tissues were collected from five newly emerged leaves.
Fig 6.
RBD1 binds to the 23S rRNA in a temperature dependent manner.
(A) Shown are relative amounts of 23S rRNA transcripts co-immunoprecipitated (co-IPed) from RBD1 versus sORRM1 analyzed by qRT-PCR. Primer pairs for 5’-terminal (5’), middle part (M), and 3’-terminal (3’) of 23S rRNA were respectively used to amplify RNAs from immuno-precipitates after reverse transcription. DNase I was used to treat the precipitated samples before reverse transcription (RT), and samples not subject to RT were used as control in PCR to ensure there is no genomic DNA contamination. (B) Shown are relative amounts of transcripts of 16S rRNA, psbF and rbcL co-IPed from RBD1 versus sORRM1 analyzed by qRT-PCR. The GFP fusions of RBD1 (RBD1:GFP) and the signal peptide of ORRM1 (sORRM1:GFP) were expressed in protoplasts of wild-type Arabidopsis leaves at 22°C and 4°C, and anti-GFP antibodies were used to precipitate the GFP fusions. ‘-’ and ‘+’ mean no antibodies and added antibodies respectively.
Fig 7.
Analyses of chloroplast translation in the 5 RNP mutants.
(A) Total RNAs extracted from the 5 RNP mutants were fractionated on 1.2% formaldehyde gels and stained with ethidium bromide. (B) RNA blotting analysis of 23S rRNA processing in the 5 RNP mutants. Total RNAs extracted from leaves were fractionated on 1.2% formaldehyde gels and hybridized to a probe described in Fig 5B. Equal loading was controlled by the cytosolic 25S rRNA (25S) stained with ethidium bromide. (C) Ponceau S Staining of total proteins from Col-0, rbd1-1, aml1, orrm1, cp29a and cp31a after SDS-PAGE separation. Accumulation of RbcL is indicated by an arrow. (D) TOC75 protein was detected by immune-blotting using specific monoclonal antibodies, loading control is shown in Fig 7C. (E) Shown are Col-0, rbd1-1, aml1, orrm1, cp29a, and cp31a mutants grown on solid media in the presence or absence of spectinomycin (spec) at 3 mg/L for 10 days. Plants were grown for 3 weeks at normal condition followed by 4°C for 4 weeks (A and B) or 2 weeks (C and D), and tissues were collected from five newly emerged leaves. Marked numbers indicate the relative amount in the mutant compared to that in Col-0 quantified by Image J.