The Diurnal Logic of the Expression of the Chloroplast Genome in Chlamydomonas reinhardtii

Bock R (2014) The Diurnal Logic of the Expression of the Chloroplast Genome in Chlamydomonas reinhardtii. PLoS ONE 9(10): e108760. doi:10.1371/journal.pone.0108760 The Diurnal Logic of the Expression of the Chloroplast Genome in Chlamydomonas reinhardtii Frederik Bo rnke, Leibniz-Institute for Vegetable and Ornamental Plants, Germany 0 Max-Planck-Institut fu r Molekulare Pflanzenphysiologie , Potsdam, Golm , Germany Chloroplasts are derived from cyanobacteria and have retained a bacterial-type genome and gene expression machinery. The chloroplast genome encodes many of the core components of the photosynthetic apparatus in the thylakoid membranes. To avoid photooxidative damage and production of harmful reactive oxygen species (ROS) by incompletely assembled thylakoid protein complexes, chloroplast gene expression must be tightly regulated and co-ordinated with gene expression in the nucleus. Little is known about the control of chloroplast gene expression at the genome-wide level in response to internal rhythms and external cues. To obtain a comprehensive picture of organelle transcript levels in the unicellular model alga Chlamydomonas reinhardtii in diurnal conditions, a qRT-PCR platform was developed and used to quantify 68 chloroplast, 21 mitochondrial as well as 71 nuclear transcripts in cells grown in highly controlled 12 h light/12 h dark cycles. Interestingly, in anticipation of dusk, chloroplast transcripts from genes involved in transcription reached peak levels first, followed by transcripts from genes involved in translation, and finally photosynthesis gene transcripts. This pattern matches perfectly the theoretical demands of a cell ''waking up'' from the night. A similar trend was observed in the nuclear transcripts. These results suggest a striking internal logic in the expression of the chloroplast genome and a previously unappreciated complexity in the regulation of chloroplast genes. - Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its Supporting Information files. Funding: This work was supported by grants from the BMBF (Systems Biology Initiative FORSYS: GoFORSYS) and the Deutsche Forschungsgemeinschaft (FOR2092, BO 1482/17-1) to RB. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. The unicellular green alga Chlamydomonas reinhardtii has become a model organism for many research areas, including chloroplast biology and photosynthesis research [1], [2], [3], [4], [5]. Moreover, its capacity to produce molecular hydrogen and to synthesize and accumulate considerable amounts of oil also have made C. reinhardtii an attractive target organism of many efforts to develop algae as an efficient and sustainable production platform for biofuels [6], [7], [8], [9]. Since all algal biofuels and renewable green chemical feedstocks are directly or indirectly derived from photosynthesis and many of the core components of the photosynthetic apparatus are encoded in the chloroplast (plastid) DNA, a comprehensive understanding of the mechanisms that govern expression of the chloroplast genome is of great importance to all future attempts to rationally engineer primary metabolism. However, our knowledge about the regulatory mechanisms operating in the control of chloroplast gene expression in response to endogenous cues and environmental stimuli and the signaling pathways co-ordinating expression of the chloroplast genome with that of the nuclear genome is still far from complete. The chloroplast genome of C. reinhardtii is 204 kb in size and contains 99 genes [10]. The vast majority of these genes belong to the two main gene classes found in plastid genomes of algae and embryophyte plants: photosynthesis genes and genetic system genes [11]. The photosynthesis-related genes comprise genes for subunits of the major multiprotein complexes in the thylakoid membrane that participate in photosynthetic electron transfer and ATP generation (photosystem II, cytochrome b6f complex, photosystem I, ATP synthase). The genetic system genes encode components of the transcriptional apparatus (subunits of the plastid-encoded RNA polymerase, PEP) and the translational machinery (rRNAs, tRNAs, ribosomal proteins). Genes in the chloroplast genome of C. reinhardtii are transcribed by a multisubunit RNA polymerase that resembles bacterial RNA polymerases of the a2bb type. All of its core subunits are encoded in the chloroplast DNA and, for this reason, the enzyme has been dubbed PEP (for plastid-encoded RNA polymerase). In Chlamydomonas, there appears to be no nucleusencoded plastid RNA polymerase that has been shown to be additionally present in plastids of embryophyte plants [12], [13]. The PEP core complex interacts with the single sigma factor found in the nuclear genome of C. reinhardtii (RPOD) to confer sequence specificity in promoter recognition [14], [15]. TATA-like -10 box sequences (and, in some cases, the extended -10 box motif TATAATAT) are found upstream of many reading frames in the C. reinhardtii plastid DNA and are thought to act as key promoter elements. However, several other types of promoters have been identified, and a consensus is yet to emerge [16], [17], [18]. Natural conditions for C. reinhardtii involve several environmental variables which vary reliably over a day. The strongest signal over a day is light, which is often used in laboratory conditions to simulate a diurnal cycle. Many cellular processes respond to light-dark cycles, such as regulation of the cell cycle, as well as various steps of chloroplast gene expression. As examples, the structure of the entire plastid genome is most accessible early in the light period, and the half-life of several mRNAs is much shorter in the light period than in the dark [19], [20]. So far, nine chloroplast transcripts have been quantified over a diurnal cycle in at least one study (Figure S1 in File S1). The majority of these attain their highest levels in the light period. However, the majority of the mRNAs which have been studied so far are involved in photosynthesis. Neither RNA polymerase subunits nor any subunits of the ribosome have been analyzed. Interestingly, when the diurnal regulation of chloroplast transcription in C. reinhardtii was studied, it was found to be accompanied by little or no fluctuation in RNA polymerase activity or in the level of RPOD, the only sigma factor in chloroplasts of Chlamydomonas [21]. Here, we have undertaken a systematic study of the expression of the chloroplast genome in Chlamydomonas under diurnal conditions. Our work has uncovered a surprising internal logic in the expression of the chloroplast genome in that transcripts of the transcriptional apparatus peak first, followed by transcripts of t (...truncated)