Nucleus-independent control of the Rubisco operon by the plastid-encoded transcription factor Ycf30 in the red alga Cyanidioschyzon merolae

Chloroplasts originated from a cyanobacterium, which was engulfed by a primitive eukaryotic host cell. During evolution, chloroplasts have largely lost their autonomy due to loss of many 3 genes from their own genomes. Consequently, expression of genes encoded in the chloroplast 4 genome is mainly controlled by the factors transferred from the cytosol to chloroplasts. However, chloroplast genomes of glaucophytes and red algae have retained some 6 transcription factors (hypothetical chloroplast open reading frame 27-30, Ycf27-30) that are 7 absent from green algae and land plants. Here we show that the red algal chloroplast 8 up-regulates transcription of the Rubisco operon ( rbcLS-cbbX ) via Ycf30 independently of 9 nuclear control. Light-induced transcriptional activation of the Rubisco operon was observed 10 in chloroplasts isolated from the red alga Cyanidioschyzon merolae. The activation was 11 suppressed by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). These results suggest that chloroplast autonomously regulates transcription of the Rubisco operon in response to activation of photosynthesis driven by the light. Transcriptional activation of the Rubisco operon was specifically repressed by addition of ani-Ycf30 antibodies. Furthermore, NADPH, transcripts activation of operon dark-light shift. antibodies chloroplasts in rbcL transcript levels by 46 ± 15%, additions of anti-RbcL or RbcS affect rbcL transcript levels. rbcL transcripts, the level of clpC transcripts after the dark-light shift in permeabilized chloroplasts, as addition of antibodies not affect light-induced transcription of clpC . These results suggest that Ycf30 is specifically required for activation of Rubisco transcription. µl reaction mixture consisting of transcription buffer (5 mM Hepes-KOH, pH 7.6, 5 mM KCl, 10 U RNase inhibitor, 25 µg/ml BSA, 0.5 mM each of ATP, GTP, and CTP, 2 and 50 µM UTP containing 10 µCi [ α - 32 P]UTP (800 Ci/mmol, MP Biomedicals, Inc., 3 U.S.A.) and permeabilized chloroplasts (3.5 mg/ml of total proteins). To examine 4 light-induced transcription, the reaction mixture without transcription substrates (0.5 5 mM each of ATP, GTP, and CTP, and 50 µM UTP containing 10 µCi [ α - 32 P]UTP) was 6 pre-incubated at 42°C for 2 min in the dark. After addition of transcription substrates, 7 the reaction mixture was incubated at 42°C for 10 min in the light (40 µE/m 2. s). 8 Inhibitors and antibodies (5 µg/ml) were added just before the dark-light shift. To 9 examine the effects of metabolites, transcription substrates were added after 2 min of 10 dark pre-incubation and the reaction mixture was incubated for a further 10 min in the 11 dark. Metabolites (0.5 mM) and antibodies (5 µg/ ml) were added at the onset of 12 pre-incubation. After the reaction, RNA was extracted and hybridized at 48°C for 48 h to single-strand DNA fragments (0.4 µg) dot-blotted onto a nylon membrane. For hybridizations, one-fifth of the total RNA was used for rbcL , and the rest was used for clpC . The DNA blots were washed five times with 0.1 × SSC and 0.1% SDS at 65°C for 15 min. Signals were measured by liquid scintillation (Beckman Coulter LS6500, U.S.A.).

1 cyanobacterium and a mitochondriate eukaryote, called the primary endosymbiosis, which 2 introduced photosynthesis into eukaryotes (Rodríguez-Ezpelta et al., 2005;Deusch et al., 3 2008). Over time, many genes of the endosymbiont have been either lost or relocated to the 4 nucleus. Consequently, chloroplasts almost lost their autonomy to proliferate and respond to 5 environmental changes. Now, chloroplast biogenesis and homeostasis largely rely on cell 6 signaling pathways of the host cell, which are composed of nuclear-encoded factors (host cell 7 signaling pathways).

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In autonomous bacteria including cyanobacteria, regulation of transcription is a major 9 strategy to acclimate to environmental changes. However, chloroplasts have almost lost 10 autonomous transcriptional regulation due to loss of genes for regulatory factors, including 11 transcription factors and sensory histidine kinases, from their own genomes. As a result, 12 expression of chloroplast genes in green algae and land plants is governed by nuclear factors at 13 multiple steps after transcription (e.g. post-transcription, translation, and protein import steps) 14 (Bock R, 2007). As an exception, it is known that the redox state of plastoquinone pool 15 controls the rate of transcription of chloroplast genes encoding reaction-center apoproteins of 16 photosystems (Pfannschmidt et al., 1999). In contrast, it appears that genes in red algal 17 chloroplasts are still controlled mainly at the transcriptional level (Apt and Grossman, 1993; 18 Minoda et al., 2005). Genes for transcription factors Ycf27-30 are retained in currently known 7 plants (Viridiplantae) (Reith, 1995;Martin et al., 1998;Sánchez et al., 2005). Thus, the 1 transcription systems in red algae and glaucophyte chloroplasts still retain relics of bacterial 2 transcriptional regulation.

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In α-proteobacteria, CbbR regulates the expression of genes encoding CBB cycle 16 enzymes including Rubisco (Tabita, 1999). On the other hand, cyanobacterial genomes 17 encode several CbbR proteins that regulate distinct target genes (e.g. nitrate assimilation, 18 adaptation to osmotic stress, and uptake of inorganic carbon) (Maeda et al., 1998;Figge et al., 8 conserved in all cyanobacterial genomes and is most closely related to the 1 chloroplast-encoded Ycf30 (Maier et al., 2000). Therefore, RbcR is a strong candidate as the 2 regulator for Rubisco transcription. However, the function of RbcR or Ycf30 remains 3 unknown, because the gene disruptants are lethal. LTTR has a conserved structure with an 4 N-terminal helix-turn-helix motif for DNA binding and a C-terminal co-inducer-binding 5 domain. Co-inducers (often a metabolite of the LTTR-regulated pathway) are important for 6 activation of transcription, as they make proper interaction between LTTR and RNA 7 polymerase (Schell, 1993). In addition, studies using α-proteobacteria demonstrated that 8 metabolites of the CBB cycle are co-inducers of CbbR (Tabita, 1999). Given that Ycf30 (a 9 CbbR ortholog) is still encoded in chloroplast genomes of glaucophytes and the red lineage of 10 algae, Ycf30 might be a remnant of the autonomous transcriptional regulation in chloroplasts 11 of these organisms. Therefore, elucidating the relationship between Ycf30 and Rubisco 12 expression in response to environmental changes will give insight into chloroplast evolution.

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To examine the role of Ycf30 and effects of environmental changes on 14 chloroplast-encoded Rubisco transcription, we chose C. merolae (Matsuzaki et al, 2004). As a 15 study organism, C. merolae has several advantages, such as availability of chloroplast DNA 16 microarray analyses and established techniques for cultivation and genetic manipulation 17 (Minoda et al., 2004;Minoda et al., 2005;Imamura et al., 2010)

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Transcription activation of Rubisco operon in isolated chloroplsts.
2 rbcL and rbcS genes encode the large and small subunits of Rubisco, 3 respectively. In green algae and land plants, rbcL is encoded in the chloroplast genome 4 and rbcS in the nuclear genome. In contrast, red algal rbcLS genes form the Rubisco 5 operon, rbcLS-cbbX, together with cbbX, an AAA + class protein in the chaperon-like 6 ATPase family in the chloroplast genome (Bowien and Kusian, 2002;Fujita et al., 7 2008a; Figure 1A). 8 When C. merolae cells were incubated for 16 h in the dark and transferred into 9 light conditions (dark-light shift), the increase of three Rubisco transcripts was observed 10 (Minoda et al., 2005). The RNA gel blot analyses by using rbcL probe also gave 11 information on the structure of the Rubisco operon: transcripts initiated from the rbcL 12 upstream promoter partially terminated in rbcS and cbbX genes, but some continued to 13 the 3'-end of cbbX ((Minoda et al., 2005; mRNA a-c in Figure 1A). Using primer 14 extension mapping, a unique transcription initiation site was identified under dark and 15 light conditions (Fujita et al., 2008b). Thus, the transcription from the rbcL promoter 16 was increased in response to the light.

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To further investigate the effect of light on Rubisco transcript levels, isolated chloroplasts were subjected to a dark-light shift. Intact chloroplasts (>90%) were 1 isolated from dark-grown C. merolae cells ( Figure S1) and then transferred into light 2 conditions (40 µE/m 2. s) at 42°C (optimal growth temperature for C. merolae cells). In 3 these conditions, the dark-light shift resulted in increased levels of rbcL transcripts 4 ( Figure 1B). This increase was blocked by addition of 250 µg/ml rifampicin, an 5 inhibitor of bacterial-type RNA polymerases, suggesting that the increase in rbcL 6 transcript levels is mainly caused by up-regulation of transcription rather than 7 down-regulation of degradation. In addition, rifampicin inhibits the initiation of 8 transcription, but not transcriptional elongation (McClure and Cech, 1978). Therefore, 9 the increase in the levels of rbcL transcripts is mainly due to activation of Rubisco   Figure 3A) for the LTTR recognition sequence, 16 T-N 11 -A (Schell, 1993). All three oligonucleotides competed with the 90-bp probe in the 17 binding to Ycf30 with only slight differences in binding affinity ( Figure S5). This result indicates that the consensus among the three sequences (ATN 7 ANANAN) was sufficient 1 for recognition by Ycf30 ( Figure 3D). In addition, this consensus sequence exists 2 upstream of the rbcL gene in the chloroplasts of red algae, cryptophytes, haptophytes, 3 diatoms, glaucophytes and cyanobacteria ( Figure 3E, F). Taken together, the above 4 results suggest that Ycf30 binds to the rbcL-upstream sequence, which is well conserved 5 in cyanobacteria and chloroplasts.

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Role of Ycf30 in the autonomous signaling pathway of the C. merolae chloroplast.

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Given that the Ycf30 protein levels remained constant during transcriptional 8 activation of the Rubisco operon ( Figure S2), it is likely that factors other than Ycf30 9 trigger the activation of Rubisco transcription via Ycf30. In α-proteobacteria and 10 cyanobacteria, binding affinities of the CbbRs to the target DNA fragments are 11 enhanced by NADP, NADPH, ADP, ATP and metabolites derived from the CBB cycle 12 (Nishimara T et al., 2008;van Keulen et al., 1998;Dubbs et al., 2004). Therefore, these 13 metabolites are candidates for co-inducers of Ycf30 to enable autonomous the Rubisco operon by NADPH, 3PGA, or RuBP was significantly inhibited by addition 1 of anti-Ycf30 antibodies, whereas it was not affected by addition of anti-rbcL antibodies 2 ( Figure 4B). These results suggest that NADPH, 3PGA, and RuBP can trigger the 3 activation of Rubisco transcription via Ycf30.

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To further examine whether NADPH, 3PGA, and RuBP affect the binding of 5 Ycf30 to the rbcL promoter region, these metabolites were added into EMSA using the 6 recombinant Ycf30 and 90-bp DNA probes (the same probe as in Figure 3A). We could

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Of the three molecules, RuBP is a substrate for Rubisco. The amount of RuBP almost same when photosynthesis was partially suppressed after a shift to 1 photomixotrophic conditions (Takahashi et al., 2008). On the other hand, the level of 2 RuBP increased in response to light in both whole leaves and isolated chloroplasts of 3 land plants (Sicher and Jensen, 1979;Salvucci et al., 1986;Arrivault et al., 2009). In    Table S1. RT 4 reactions were performed using ReverTra Ace (TOYOBO, Japan) with each primer set 5 (Table S1)

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For immunoblot analysis, 2 µg of total proteins extracted from the whole cells 16 and 20 µg of total protein extracted from isolated chloroplasts ( Figure S2 The 90 bp of DNA fragment for the 32 P-labeled probe was generated by PCR 7 amplification using the primers, uprbcL90f and uprbcL90r (Table S1). Reactions (20 µl) 8 contained 1 nM 32 P-labeled probe, 0.1 ng poly(dI-dC)(dI-dC), binding buffer A for 9 Figure  before the incubation ( Figure 4C). The gel was dried and analyzed using a BAS1000 1 (Fujix, Japan) or Personal Molecular Imager FX (bio-rad, U.S.A.).         3 rbcS, small subunit of Rubisco; cbbX; AAA + family protein; ilvH, small subunit of 4 acetolactate synthase; psbY, photosystem II subunit Y; psbY, photosystem II subunit Y; 5 ycf30, LysR-type transcription factor Ycf30; cysW, sulfate transport system permease 6 protein CysW; crtR, beta-carotene hydroxylase. Arrows (a-c) indicate the transcripts 7 transcribed from the rbcL promoter. The sizes and positions of PCR products in B are