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. 2013 Jan;25(1):167-86.
doi: 10.1105/tpc.112.103051. Epub 2013 Jan 4.

Repression of essential chloroplast genes reveals new signaling pathways and regulatory feedback loops in chlamydomonas

Silvia Ramundo  1 , Michèle Rahire, Olivier Schaad, Jean-David Rochaix
Affiliations

Repression of essential chloroplast genes reveals new signaling pathways and regulatory feedback loops in chlamydomonas

Silvia Ramundo et al. Plant Cell. 2013 Jan.

Abstract

Although reverse genetics has been used to elucidate the function of numerous chloroplast proteins, the characterization of essential plastid genes and their role in chloroplast biogenesis and cell survival has not yet been achieved. Therefore, we developed a robust repressible chloroplast gene expression system in the unicellular alga Chlamydomonas reinhardtii based mainly on a vitamin-repressible riboswitch, and we used this system to study the role of two essential chloroplast genes: ribosomal protein S12 (rps12), encoding a plastid ribosomal protein, and rpoA, encoding the α-subunit of chloroplast bacterial-like RNA polymerase. Repression of either of these two genes leads to the arrest of cell growth, and it induces a response that involves changes in expression of nuclear genes implicated in chloroplast biogenesis, protein turnover, and stress. This response also leads to the overaccumulation of several plastid transcripts and reveals the existence of multiple negative regulatory feedback loops in the chloroplast gene circuitry.

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Figures

Figure 1.
Figure 1.
Reversible Vitamin-Controlled Repression of Chloroplast Gene Expression. (A) Scheme of the vitamin-controlled repressible chloroplast gene expression system. The Nac2 gene, which is specifically required for the accumulation of the chloroplast psbD mRNA, is fused to the Thi4 5′UTR containing the TPP-responsive riboswitch, and the MetE upstream region is used as promoter. Addition of thiamine causes alternative splicing in the riboswitch region, which results in translation termination due to the inclusion of a stop codon (red flag) (Croft et al., 2007). The yellow box below the second exon indicates the genomic location of the TPP riboswitch. Change of the box color represents in a schematic way the conformational change of the riboswitch upon binding of TPP (green, no TPP binding; red, TPP binding). Because Nac2 acts specifically on the psbD 5′UTR, it is possible to render the expression of any chloroplast gene dependent on Nac2 by fusing its coding sequence to the psbD 5′UTR. To allow for phototrophic growth in the presence of the vitamins, the psbD 5′UTR of the psbD gene was replaced by the psaA 5′UTR, thus making psbD expression independent of Nac2. (B) Growth patterns of the wild-type (WT), nac2-26, Rep112, and A31 strains. Cells were spotted on TAP and HSM (minimal medium) plates in the absence or presence of vitamins B12 (20 μg/L) and thiamine (20 μM) under an illumination of 60 μmol m−2 s−1. (C) Decrease of PSII activity of Rep112 after addition of vitamins. Fv/Fm was measured for the indicated strains at different times after addition of vitamins (20 μM thiamine and 20 μg/L B12). The experiment was repeated three times with similar results. (D) Growth curves of the indicated strains after addition of vitamins. To maintain cells in exponential growth during the entire time course, they were diluted to 0.5 × 106 cells/mL when they reached a concentration between 2 and 4 × 106 cells/mL. The experiment was repeated three times with similar results. div, divisions. (E) Recovery of photosynthetic activity (Fv/Fm) upon transfer of Rep112 from vitamin-containing medium to vitamin-free medium. (F) Growth of Rep112 under the same conditions as in (E). To maintain cells in exponential growth during the time course, they were diluted to 0.5 × 106 cells/mL when they reached a concentration between 2 and 4 × 106 cells/mL. The experiment was repeated three times with similar results.
Figure 2.
Figure 2.
Vitamin-Mediated Repression and Induction of psbD Expression. (A) RNA gel blot analysis of psbD in the indicated strains in the absence and presence of vitamins (vit). The psaB mRNA was used as control. A dilution series of wild-type (WT) extracts was used for estimating the amount of RNA. (B) Immunoblot analysis of the chloroplast proteins D2, PRK, PsaD, and ClpP1 in the indicated strains. (C) Immunoblot analysis of Nac2, ThiC, and Hsp90 in Rep112. Protein samples were examined at the indicated times after addition of vitamins. (D) RNA gel blot analysis of psbD RNA in Rep112. The atpB mRNA was used as a loading control. RNA samples were examined at the indicated times (hours) after addition of vitamins. (E) Immunoblot analysis of D2, D1, PRK, and ClpP1 in Rep112. Protein samples were examined at the indicated times (hours) after addition of vitamins. (F) Time course of psbD mRNA recovery after removal of vitamins. RNA gel blot analysis was performed at different times after removal of vitamins with the indicated probes. (G) Time course of D2 recovery after removal of vitamins. Immunoblot analysis was performed at different times with antibodies against D2 and the indicated proteins. (H) Accumulation of D2 in the absence (−) or presence of thiamine (T) and vitamin B12 (B12) in the nac2 and Rep112 strains.
Figure 3.
Figure 3.
Growth Patterns upon Repression of Chloroplast Transcription in DPF1 and Translation in RR5. (A) Growth patterns of DPF1, RR5, A31, and Rep112. Cells were spotted on TAP and HSM plates in the absence or presence of vitamins B12 (20 μg/L) and thiamine (20 μM) under an illumination of 60 μmol m−2 s−1. (B) Growth curves and Fv/Fm measurements in the presence of vitamins. Cell concentration and Fv/Fm of A31, RR5, and DPF1 were measured at different times after addition of vitamins. To maintain cells in exponential growth during the time course, they were diluted to 0.5 × 106 cells/mL when they reached a concentration between 2 and 4 × 106 cells/mL and growth was continued. The experiment was repeated three times with similar results. (C) Light- and dark-grown cell cultures of A31, DPF1, and RR5 in the presence (+) or absence (−) of vitamins. (D) Light microscopy of cells from A31, DPF1, and RR5 in the presence (+) or absence (−) of vitamins. Cells were stained with Trypan blue after 6 d in the light (60 μmol m−2 s−1) or 12 d in the dark.
Figure 4.
Figure 4.
Polysome Profiles and Accumulation of Chloroplast Proteins in RR5 upon Repression of rps12 with Vitamins. (A) Polysome isolation. Polysomes were isolated from untreated RR5 cells or RR5 cells treated with the vitamins for 48 h and subjected to Suc density gradient centrifugation. Gradients containing EDTA to disrupt the polysomes are indicated. RNA was isolated from each fraction and hybridized to the chloroplast psaB, psbD, and tufA probes. ORM encoding a cytosolic protein was used as control. (B) Immunoblot analysis of Rps12, ribosomal proteins, and TufA in RR5 and A31. Proteins were examined at different time points after addition of the vitamins using antibodies against the indicated proteins. (C) Immunoblot analysis of ClpP1 and proteins involved in photosynthesis. Conditions were as in (B). (D) Immunoblot analysis of Vipp1, Alb3.2, Hsp90C, DnaJ, and Hsp70B. Conditions were as in (B).
Figure 5.
Figure 5.
RNA Analysis of RR5 and DPF1. (A) RNA samples were examined at the indicated times after addition of vitamins. The size difference of rps12 mRNA in RR5 and A31 is due to the fusion of the psbD 5′UTR to the rps12 coding sequence in RR5. The hybridization probes for detecting other chloroplast RNAs are indicated. Cytoplasmic 25S rRNA was not affected by the vitamin treatment and used as a loading control. (B) RNA samples from A31, RR5, and clpP-AUU were examined after 120 h of vitamin treatment and hybridized with clpP1 and rpoA probes. Equal loading was checked by rRNA staining with ethidium bromide. (C) and (D) RNA gel blot analysis of rpoA mRNA in DPF1. RNA samples were examined at different times after addition of the vitamins using the indicated hybridization probes. (E) and (F) Chloroplast transcriptional activity is reduced in the DPF1 strain grown with vitamins. Cells were grown for 48 (E) and 144 h (F) in the absence or presence of the vitamins, permeabilized through one freezing-defreezing cycle, and pulse labeled with 32P-UTP for 15 min. RNA was isolated and hybridized with a filter containing the indicated gene probes.
Figure 6.
Figure 6.
Hierarchical Cluster Analysis of the Relative RNA Levels of RR5 and DPF1 upon Arrest of Chloroplast Translation and Transcription. RNA was isolated from A31, RR5, and DPF1 at 0, 12, 48, and 146 h after addition of vitamins. RNA levels were quantified with a NanoString nCounter. The genes were first grouped by class and then within each class by Euclidian distance using the statistical function in Matlab 2012b (MathWorks). Genes are listed on the right with a different color for each class. The scale is in log2 relative to A31 at each time point.
Figure 7.
Figure 7.
Immunoblot Analysis of RpoA and Other Chloroplast and Cytoplasmic Proteins in DPF1. Proteins were examined at different times after addition of the vitamins using antibodies against FLAG and the indicated proteins.
Figure 8.
Figure 8.
Expression of Nuclear LHC Genes in A31, DPF1, and RR5. The levels of the indicated mRNAs were determined by qRT-PCR from cells grown for 6 d in the light or 12 d in the dark in the presence of vitamins.
Figure 9.
Figure 9.
Negative Regulatory Feedback Loops in the Chloroplast Gene Circuitry. Left, chloroplast compartment; right, nucleo-cytosol. A selected set of chloroplast mRNAs examined in this work is shown. They comprise mRNAs of ClpP1, chloroplast ribosomal proteins (r-prot), subunits of chloroplast RNA polymerase (rpo), elongation factor TufA (tufA), and subunits of the light-independent protochlorophyllide reductase (chl). Nucleus-encoded mRNA genes coding for chloroplast and cytoplasmic proteins are shown in red and blue, respectively, on the right. Negative regulatory feedback loops are revealed through repression of transcription (green lines) or translation (red lines). Factors involved are still unknown (X, Y, and Z) except for the ClpP1 protein, which represses accumulation of its own mRNA directly or indirectly (green circular line). The feedback loops act mostly at the level of RNA accumulation in contrast with CES (for Control of Epistasy of Synthesis), an assembly-dependent feedback process in which unassembled CES subunits inhibit directly or indirectly their own translation (green circular lines) (reviewed in Choquet and Wollman, 2002).
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