Chlamydomonas reinhardtii is a unicellular green alga whose lineage diverged from land plants over 1 billion years ago. It is a model system for studying chloroplast-based photosynthesis, as well as the structure, assembly, and function of eukaryotic flagella (cilia), which were inherited from the common ancestor of plants and animals, but lost in land plants. We sequenced the~120-megabase nuclear genome of Chlamydomonas and performed comparative phylogenomic analyses, identifying genes encoding uncharacterized proteins that are likely associated with the function and biogenesis of chloroplasts or eukaryotic flagella. Analyses of the Chlamydomonas genome advance our understanding of the ancestral eukaryotic cell, reveal previously unknown genes associated with photosynthetic and flagellar functions, and establish links between ciliopathy and the composition and function of flagella.

The unicellular green alga Chlamydomonas reinhardtii is a widely used model organism for studies of oxygenic photosynthesis in eukaryotes. Here we describe the development of a resource for functional genomics of photosynthesis using insertional mutagenesis of the Chlamydomonas nuclear genome. Chlamydomonas cells were transformed with either of two plasmids conferring zeocin resistance, and insertional mutants were selected in the dark on acetate-containing medium to recover lightsensitive and nonphotosynthetic mutants. The population of insertional mutants was subjected to a battery of primary and secondary phenotypic screens to identify photosynthesis-related mutants that were pigment deficient, light sensitive, nonphotosynthetic, or hypersensitive to reactive oxygen species. Approximately 9% of the insertional mutants exhibited 1 or more of these phenotypes. Molecular analysis showed that each mutant line contains an average of 1.4 insertions, and genetic analysis indicated that approximately 50% of the mutations are tagged by the transforming DNA. Flanking DNA was isolated from the mutants, and sequence data for the insertion sites in 50 mutants are presented and discussed.As with other model organisms, the availability of genome sequence data is revolutionizing and revitalizing research into the biology of the unicellular green alga Chlamydomonas reinhardtii Ledford et al., 2005). Over the past four decades, many fundamental insights into the structure, function, assembly, and regulation of the photosynthetic apparatus have come from studies of Chlamydomonas, which offer several advantages for the genetic dissection of eukaryotic photosynthesis (for review, see Davies and Grossman, 1998; Hippler et al., 1998;Grossman, 2000;Rochaix, 2001). First and foremost, photosynthesis is fully dispensable in Chlamydomonas, as cells can grow heterotrophically in the dark using acetate as a sole carbon source. Cells grown in the dark, however, still synthesize and assemble a fully functional photosynthetic apparatus. This allows the isolation and analysis of mutants that are unable to perform photosynthesis, and lightsensitive mutants can be maintained in complete darkness. Because Chlamydomonas is predominantly maintained in a haploid form, it is not necessary to generate homozygous nuclear mutants, and mutants affecting photosynthesis can be screened immediately following mutagenesis. Chlamydomonas has an easily controlled and rapid sexual cycle (approximately 2 weeks) with the possibility of tetrad analysis, which facilitates genetic analysis. Its rapid cell-doubling time (approximately 10 h) and microbial lifestyle mean that it is easy to grow homogeneous cultures on any scale, simplifying physiological and biochemical characterization in comparison to multicellular land plants (Ledford et al., 2005). By way of example, the application of inhibitors and generators of various types of reactive oxygen species results in uniform uptake of the chemical by each cell. In land plants, multicellularity leads to differential ...

The maintenance of functional chloroplasts in photosynthetic eukaryotes requires real-time coordination of the nuclear and plastid genomes. Tetrapyrroles play a significant role in plastid-tonucleus retrograde signaling in plants to ensure that nuclear gene expression is attuned to the needs of the chloroplast. Well-known sites of synthesis of chlorophyll for photosynthesis, plant chloroplasts also export heme and heme-derived linear tetrapyrroles (bilins), two critical metabolites respectively required for essential cellular activities and for light sensing by phytochromes. Here we establish that Chlamydomonas reinhardtii, one of many chlorophyte species that lack phytochromes, can synthesize bilins in both plastid and cytosol compartments. Genetic analyses show that both pathways contribute to iron acquisition from extracellular heme, whereas the plastid-localized pathway is essential for light-dependent greening and phototrophic growth. Our discovery of a bilin-dependent nuclear gene network implicates a widespread use of bilins as retrograde signals in oxygenic photosynthetic species. Our studies also suggest that bilins trigger critical metabolic pathways to detoxify molecular oxygen produced by photosynthesis, thereby permitting survival and phototrophic growth during the light period.biliverdin | heme oxygenase | iron homeostasis | oxidative stress | RNA-Seq analysisT he daily light-dark cycle requires all oxygenic photosynthetic species to survive the repeated transition from prolonged darkness to phototrophic metabolism at dawn. Most plants are unable to synthesize chlorophyll in darkness and therefore accumulate photosensitizing chlorophyll precursors at night (1). Sunrise induces an oxidative burst as photosynthesis resumes, so the transition to daylight requires careful coordination of many lightdependent processes. Multiple photoreceptors perform such roles in plants, the most notable being the red-sensing, linear tetrapyrrole (bilin)-based phytochromes and the blue-sensing, flavin-based cryptochromes and phototropins (2-5). Bilins are well-established plant retrograde signals, synthesized in plastids but enabling light sensing by cytosolic phytochromes. Phytochrome photoconversion then triggers nuclear translocation to positively regulate photosynthesis-associated nuclear gene (PhANG) expression (6, 7).Genetic studies suggest that plastids also export negative retrograde signals, metabolites that suppress nuclear gene networks targeted by phytochromes (8-10). Among these metabolites are abscisic acid (ABA) (11), tetrapyrroles (12-14), 3′-phosphoadenosine 5′-phosphate (PAP) (15), β-cyclocitral (16), and methylerythritol cyclodiphosphate (MEcPP) (17). Although hypothetical export of a negative tetrapyrrole signal has received considerable support, biochemical evidence for such a retrograde signal remains equivocal in plants (18)(19)(20). Chlorophyte algae diverged from the streptophyte plant lineage over 500 million years ago but share a common chlorophyll a/b-based photosynthetic lightharvesting app...

The Chlamydomonas reinhardtii proton gradient regulation5 (Crpgr5) mutant shows phenotypic and functional traits similar to mutants in the Arabidopsis (Arabidopsis thaliana) ortholog, Atpgr5, providing strong evidence for conservation of PGR5-mediated cyclic electron flow (CEF). Comparing the Crpgr5 mutant with the wild type, we discriminate two pathways for CEF and determine their maximum electron flow rates. The PGR5/proton gradient regulation-like1 (PGRL1) ferredoxin (Fd) pathway, involved in recycling excess reductant to increase ATP synthesis, may be controlled by extreme photosystem I acceptor side limitation or ATP depletion. Here, we show that PGR5/PGRL1-Fd CEF functions in accordance with an ATP/redox control model. In the absence of Rubisco and PGR5, a sustained electron flow is maintained with molecular oxygen instead of carbon dioxide serving as the terminal electron acceptor. When photosynthetic control is decreased, compensatory alternative pathways can take the full load of linear electron flow. In the case of the ATP synthase pgr5 double mutant, a decrease in photosensitivity is observed compared with the single ATPase-less mutant that we assign to a decreased proton motive force. Altogether, our results suggest that PGR5/PGRL1-Fd CEF is most required under conditions when Fd becomes overreduced and photosystem I is subjected to photoinhibition. CEF is not a valve; it only recycles electrons, but in doing so, it generates a proton motive force that controls the rate of photosynthesis. The conditions where the PGR5 pathway is most required may vary in photosynthetic organisms like C. reinhardtii from anoxia to high light to limitations imposed at the level of carbon dioxide fixation.Photosynthesis is a highly regulated process that integrates different electron transfer pathways to convert light energy into ATP and NADPH and balance this production of chemical energy with its use in anabolic metabolism. Linear electron flow accounts for the major flux of electrons from the primary electron donor water to PSII and intersystem carriers to reduce NADP + , the terminal acceptor associated with PSI. Electron transfer is coupled to proton transfer through reactions involving plastoquinones/plastoquinols that are dependent on the activity of the cytochrome b 6 f complex (cyt f); the protons are transferred from the stroma into the thylakoid lumen. The proton motive force generated is used for ATP synthesis by the ATP synthase. The NADPH and ATP produced in the light serve as the energy/reductant that drives the fixation of carbon dioxide (CO 2 ) by Rubisco and the Calvin-Benson cycle and also supports other downstream metabolic reactions.The Calvin-Benson cycle has a stoichiometric requirement of 3 ATP and 2 NADPH per CO 2 molecule; this requirement is not fulfilled by linear electron flow, because it is slightly imbalanced in favor of NADPH production. Cyclic electron flow (CEF) pathways allow the cells to fulfill the energetic requirement for sustained CO 2 fixation through recycling or reoxidation of ...

Background: Photosystem II is an essential component of oxygenic photosynthesis.Results: Photosystem II is specifically decreased in rubredoxin mutants of the green alga Chlamydomonas reinhardtii, the cyanobacterium Synechocystis sp. PCC 6803, and the plant Arabidopsis thaliana.Conclusion: Rubredoxin is required for photosystem II, and not photosystem I, accumulation in these organisms.Significance: Rubredoxin was likely important in the evolution of oxygenic photosynthesis.

Singlet oxygen is a highly toxic and inevitable byproduct of oxygenic photosynthesis. The unicellular green alga Chlamydomonas reinhardtii is capable of acclimating specifically to singlet oxygen stress, but the retrograde signaling pathway from the chloroplast to the nucleus mediating this response is unknown. Here we describe a mutant, singlet oxygen acclimation knocked-out 1 (sak1), that lacks the acclimation response to singlet oxygen. Analysis of genome-wide changes in RNA abundance during acclimation to singlet oxygen revealed that SAK1 is a key regulator of the gene expression response during acclimation. The SAK1 gene encodes an uncharacterized protein with a domain conserved among chlorophytes and present in some bZIP transcription factors. The SAK1 protein is located in the cytosol, and it is induced and phosphorylated upon exposure to singlet oxygen, suggesting that it is a critical intermediate component of the retrograde signal transduction pathway leading to singlet oxygen acclimation.DOI: http://dx.doi.org/10.7554/eLife.02286.001

SUMMARYChlamydomonas reinhardtii is a unicellular green alga that is a key model organism in the study of photosynthesis and oxidative stress. Here we describe the large-scale generation of a population of insertional mutants that have been screened for phenotypes related to photosynthesis and the isolation of 459 flanking sequence tags from 439 mutants. Recent phylogenomic analysis has identified a core set of genes, named GreenCut2, that are conserved in green algae and plants. Many of these genes are likely to be central to the process of photosynthesis, and they are over-represented by sixfold among the screened insertional mutants, with insertion events isolated in or adjacent to 68 of 597 GreenCut2 genes. This enrichment thus provides experimental support for functional assignments based on previous bioinformatic analysis. To illustrate one of the uses of the population, a candidate gene approach based on genome position of the flanking sequence of the insertional mutant CAL027_01_20 was used to identify the molecular basis of the classical C. reinhardtii mutation ac17. These mutations were shown to affect the gene PDH2, which encodes a subunit of the plastid pyruvate dehydrogenase complex. The mutants and associated flanking sequence data described here are publicly available to the research community, and they represent one of the largest phenotyped collections of algal insertional mutants to date.

SUMMARYThe GENOMES UNCOUPLED 4 (GUN4) protein is found only in aerobic photosynthetic organisms. We investigated the role of GUN4 in metabolic activities of the Mg branch of the tetrapyrrole biosynthesis pathway and the plastid signal-mediated changes of nuclear gene expression in Chlamydomonas reinhardtii. In light, gun4 accumulates only 40% of the wild-type chlorophyll level. Light-or dark-grown gun4 mutant accumulates high levels of protoporphyrin IX (Proto), and displays increased sensitivity to moderate light intensities. Despite the photooxidative stress, gun4 fails to downregulate mRNA levels of the tetrapyrrole biosynthesis and the photosynthesis-associated nuclear genes (PhANGs). In contrast, upon illumination, the Proto-accumulating and light-sensitive chlD-1 mutant displays the expected downregulation of the same nuclear genes. Although chlD-1 and the wild type have similar GUN4 transcript levels, the GUN4 protein in chlD-1 is hardly detectable. Overexpression of GUN4 in chlD-1 modifies the downregulation of nuclear gene expression, but also increases light tolerance. Therefore, GUN4 is proposed to function in 'shielding' Proto, and most likely MgProto, by reducing reactivity with O 2 . Furthermore, GUN4 seems to be involved in sensing elevated levels of these photoreactive tetrapyrrole intermediates, and contributing to 1 O 2 -mediated retrograde signalling, originating from chlorophyll biosynthesis.