Chlamydomonas reinhardtii J-D Rochaix, University of Geneva, Geneva, Switzerland © 2013 Elsevier Inc. All rights reserved.
Glossary 5′UTR, 3′UTR 5′-untranslated, 3′-untranslated region of mRNA. These regions are at the ends of the mRNA and do not encode amino acids. EST Expressed sequence tags generated by rcverse transcription and cloning of short cDNAs of mRNAs. LHCII Light harvesting system of photosystem II consisting of chlorophyll-binding proteins which absorb the light energy. MicroRNA Small 21–22 nucleotide RNAs that are involved in silencing specific mRNAs either by degradation of the RNA or by translational arrest. NPQ Nonphotochemical quenching of chlorophyll fluorescence. Upon light absorption by the chlorophyll antenna, the light excitation energy can be used for photosynthesis, a process which is accompanied by a
The Organism Chlamydomonas reinhardtii cells are oval shaped, c. 10 μm in length and 3 μm in width, with two flagellae at their anterior end (Figure 1). The cells contain a single chloroplast occupying 40% of the cell volume and several mitochondria. These cells exist as mating-type (+) or mating-type (–). The haploid vege tative cells multiply through mitotic divisions. However, upon nitrogen starvation the vegetative cells differentiate into gametes and cells of opposite mating-type fuse to give rise to a zygote, which will undergo meiosis under appropriate light– dark conditions and produce four haploid daughter cells that can resume vegetative growth. It is also possible to recover mitotically dividing diploid cells after the mating reaction. In the presence of acetate in the growth medium, the photo synthetic function of C. reinhardtii cells is dispensable. This feature has been exploited extensively for isolating and main taining mutants deficient in photosynthetic activity. Cells can thus be grown under three different conditions: in minimal medium with light and CO2 as the sole carbon source (photo trophic growth), in acetate-containing medium with light (mixotrophic growth) or without light (heterotrophic growth). Importantly, C. reinhardtii cells grown in the dark maintain a functional photosynthetic apparatus because of their ability to synthesize chlorophyll in the absence of light. The growth of the cells can easily be synchronized by light–dark cycles.
Three Genetic Systems Like plants, C. reinhardtii contains three genetic systems located in the nucleus, chloroplast, and mitochondria. Mutations in the genomes of these three compartments can be recognized by their unique segregation patterns during crosses. Whereas nuclear mutations segregate according to the classical
Brenner’s Encyclopedia of Genetics, 2nd edition, Volume 1
decrease of fluorescence (photochemical quenching). If the excitation energy exceeds the absorption capacity of the photosynthetic machinery, the excess excitation energy can be dissipated as heat. This process is also accompanied by a lowering of chlorophyll fluorescence (nonphotochemical quenching). A further decrease in fluorescence can be caused by state transitions in which part of the LHCII antenna moves from photosystem II to photosystem I in a reversible way. Stroma Soluble part of the chloroplast. Thylakoids Internal chloroplast membranes where the primary reactions of photosynthesis occur. Xanthophylls Carotenoids containing either hydroxyl groups and/or pairs of hydrogen atoms that are substituted by oxygen atoms.
Mendelian rules, chloroplast and mitochondrial mutations are normally transmitted uniparentally from the mt+ and mt– parents, respectively (Table 1). Analysis of the sequence of the 121-mb nuclear genome of C. reinhardtii has revealed 15′143 gene models of which only 56% are so far supported by expressed sequence tag (EST) coverage. This analysis has provided insights into the last com mon ancestor of plants and animals and identified novel plastid- and cilia-related genes. Currently, the genetic map includes 148 loci distributed over 17 linkage groups. In addi tion, crosses between a field isolate and the standard laboratory strain revealed 8775 polymorphisms giving rise to a marker density of 1 per 13 kb. The nuclear transformation yield is sufficiently high to allow for genomic complementation of nuclear mutants. Moreover, because nuclear transformation of this alga occurs mainly through nonhomologous recombina tion, the transforming DNA integrates randomly into the nuclear genome. Thus, transformation of C. reinhardtii can be used as a mutagen for tagging genes. The recent findings of microRNAs, small 21–22 nucleotide RNAs, together with the presence of Dicer and Argonaute nuclease proteins in this alga, indicate that RNA-silencing systems involved in modulating the stability and translation of mRNAs were a feature of primi tive eukaryotic cells and that they evolved before multicellularity. This system can also be used for nuclear reverse genetics in C. reinhardtii by specifically downregulating the expression of a gene of interest. The 204'210 bp chloroplast genome of C. reinhardtii con tains 34 genes involved in photosynthesis, 31 genes involved in chloroplast transcription and translation, 1 protease gene, 29 tRNA genes, and 9 genes of unknown function. Chloroplast transformation can be achieved routinely in C. reinhardtii by bombarding cells with DNA-coated gold or tungsten particles. In contrast to nuclear transformation, chloroplast transforma tion occurs exclusively through homologous recombination.
Chlamydomonas reinhardtii consisting of chloroplast regulatory elements fused to reporter genes for identifying the target sites of specific nucleus-encoded factors required for chloroplast gene expression. This analysis has revealed that some of these factors act on the 5'-UTR of specific mRNAs and that they are required for mRNA stability, splicing, or translation. The 15.8-kb linear mitochondrial genome of C. reinhardtii encodes seven proteins involved in respiration, one protein resembling a reverse transcriptase, two ribosomal RNA genes that are fragmented and interspersed with other coding sequences, and three tRNA genes. The other tRNAs have to be imported from the chloroplast or cytosol. Mutants with dele tions in the mitochondrial apocytochrome b gene require light for growth and are unable to grow on acetate medium in the dark, indicating that mitochondrial respiration is dispensable in the light.
Biogenesis of the Photosynthetic Apparatus c
Figure 1 Section through a cell of Chlamydomonas reinhardtii, showing cup-shaped chloroplast (c) with thylakoid membranes, prominent nucleus (n), and mitochondria (m). Courtesy of U. Goodenough. Reproduced from Rocheix J-D (1996) Chalmydomonas. Encyclopedia of Molecular Biology and Molecular Medicine , vol. 1, pp. 347–360. Weinjeim: VHC Verlagsgesellschaft, with permission from VHC Verlagsgesellschaft.
Because foreign selectable marker genes are available conferring resistance to specific antibiotics in the chloroplast, it is possible to inactivate specific genes or to perform site-directed mutagen esis on any plastid gene of interest. This reverse genetics approach has been rather successful for elucidating the func tion of conserved genes of unknown function present in the plastid genomes of several plants, algae, and cyanobacteria. Chloroplast transformation has also been very useful for study ing chloroplast gene expression, especially when it is combined with classical genetic analysis. It has been possible to dissect regulatory elements such as promoters and 5'- and 3'-UTRs (untranslated region of RNA), and to use chimeric genes
The primary reactions of photosynthesis occur in the thylakoid membranes, where light energy is collected and converted into chemical energy through charge separations across the membrane followed by a series of complex oxidoreduction reactions. Ultimately, this process leads to the formation of an electrochemical gradient across this membrane and the production of adenosine triphosphate (ATP) and nicotinamide adenine dinucleotide phosphate (NADPH), both of which are required to drive the Calvin cycle, which results in CO2 fixation and the synthesis of carbohydrates (Figure 2). Four multimo lecular complexes are involved in these primary reactions: photosystem II and photosystem I and their associated lightharvesting systems, the cytochrome b6f complex, and the ATP synthase. Because the photosynthetic apparatus of C.reinhardtii is similar to its homolog in higher plants, this alga is appropriate for studying the biogenesis and function of the photosynthetic complexes and for investigating more generally the genetic interactions between the nuclear, chloroplast, and mitochon drial compartments. The biosynthesis of the photosynthetic apparatus of C. reinhardtii occurs though the concerted action of two genetic systems located in the nucleus and chloroplast, respectively. Several subunits of the photosynthetic complexes are encoded by the chloroplast genome and translated on 70S chloroplast ribosomes. The remaining subunits are encoded by nuclear genes, translated on cytosolic 80S ribosomes as pre cursors with a transit peptide at the N-terminal end, which targets the protein to the chloroplast compartment (Figure 3). In the final steps, chloroplast and nucleus-encoded subunits
Three genetic systems in Chlamydomonas
Complexity of genome (kb)
Number of linkage groups
Nucleus Chloroplast Mitochondria
1.2 × 105 204 15.8
17 1 1
me up+ up−
Abbreviations: me, Mendelian inheritance; up+, up−, uniparentally inherited from the mating-type+ and mating-type− parent, respectively.
1 + – 2O2 + 2H
Figure 2 Photosynthetic electron transport chain with the four major complexes photosystem II (PSII) and photosystem I (PSI) with their associated chlorophyll antenna, the cytochrome b6f complex (cytb6/f), and the ATP synthase. Electron pathways are indicated by broken lines. CEF, cyclic electron flow; Fd, ferredoxin; FNR, ferredoxin–NADP reductase; PQ, plastoquinone; PQH2, plastoquinol; PC, plastocyanin; chl, chlorophyll.
Adaptation of the Photosynthetic Apparatus
Nuclear DNA mRNA 80S
Assembly Translation and integration Stability RNA Processing Splicing
Figure 3 Biosynthesis of the photosynthetic apparatus. The nucleus and chloroplast are shown in the upper and lower parts, respectively. Chloroplast (70S) and cytosolic (80S) ribosomes are shown. The pathways for synthesis of proteins of the photosynthetic complexes are indicated by continuous lines. The role of the nucleus-encoded factors in the different posttranscriptional steps of chloroplast gene expression are shown by dashed lines.
are assembled either in the stroma or in the thylakoid mem branes to form functional protein complexes. An extensive analysis of nuclear mutants deficient in photosynthesis has revealed that besides the mutations that directly affect the genes of the components of the photosynthetic apparatus, the vast majority of the muta tions are in genes encoding factors that are required for several chloroplast posttransciptional steps including RNA stability, RNA processing, translation and the assembly of photosynthetic complexes. The number of these genes is surprisingly high and their products act mostly in a genespecific manner.
Plants and algae have the remarkable ability to adapt and to modulate the operation of the photosynthetic apparatus in response to changes in light quality and quantity. Too much light can be harmful and excess light energy can be dissipated as fluorescence or heat (nonphotochemical quenching, NPQ). At least part of this nonradiative energy dissipation occurs through reversible covalent modifications of the thylakoid xanthophylls and involves the reductive de-epoxidation of vio laxanthin to zeaxanthin (xanthophyll cycle) that is triggered by the pH gradient produced by photosynthetic electron flow. A genetic analysis of NPQ-deficient mutants provided direct genetic evidence for the importance of zeaxanthin in NPQ and also revealed that the pigments of the xanthophyll cycle derived from β-carotene, and lutein derived from α-carotene are required both for NPQ and for protection against oxidative damage in high light. Photosystem II and photosystem I act in series in the photo synthetic electron transport chain and they are connected to two distinct antennae systems with different light absorption properties. Upon changes in light quality and intensity, a reor ganization of the antennae occurs to ensure a balanced excitation of the two photosystems and hence an optimal photosynthetic quantum yield. This process is called state tran sition and involves the reversible displacement of the antenna of photosystem II to photosystem I under conditions in which photosystem II is overstimulated relative to photosystem I. A key step in state transitions is the activation of a kinase that specifically phosphorylates the LHCII proteins. The activa tion is triggered through a signal transduction chain that involves the redox state of the plastquinone pool and a func tional cytochrome b6f complex. A genetic approach has identified several mutants deficient in state transitions and in the phosphorylation of LHCII. The gene affected in one of these mutants encodes a thylakoid-associated protein kinase, called Stt7 which is conserved in land plants. Mutants blocked in state 1 are unable to switch to cyclic electron flow under state 2 conditions. Thus, state transitions in C. reinhardtii serve not
only as a light-adaptation mechanism, but also for rerouting photosynthetic electron flow from linear to cyclic electron flow, thereby allowing the organism to adapt to changes in cellular demand for ATP. Mutants affected in state transitions offer promising possibilities to identify all the factors involved in this process. Under conditions of excess reducing power generated by photosystem I in an anaerobic environment, C. reinhardtii cells can evolve hydrogen. Although this property was known since many years, it is only recently that this alga has been intensively studied and manipulated for improved hydrogen production. In this respect microalgae offer promising possibi lities for the environmentally friendly generation of biofuels.
Function and Assembly of the Flagellar Apparatus C. reinhardtii has also proved to be useful for studies on flagellar function and assembly, especially for understanding the com plex functional interrelationships between the various flagellar subcomplexes in vivo. This alga possesses two flagella, located at the anterior end of the cell, that are assembled on basal bodies. During cell division, basal bodies migrate to the interior of the cell and function as centrioles by organizing the spindle appara tus. The flagellar system of Chlamydomonas has proved to be particularly well suited for studying microtubule assembly, func tion, and motility. This is because flagellar biosynthesis can be readily synchronized and numerous mutants affected in the function and assembly of the flagellar apparatus have been isolated. These mutants can be separated into two major classes: those with abnormal or no motility, usually called paralyzed mutants, and those defective in flagellar assembly. Because flagellar structure has been conserved throughout evolution, results obtained with Chlamydomonas are relevant for understanding human diseases associated with flagellar or ciliary dysfunction. They include primary ciliary dyskinesis which affect the motility of cilia, polycystic kidney disease which involves in some cases a defect in assembly of the
primary cilia, and retinitis pigmentosa which causes retinal degeneration through a defect in transport of proteins through the connecting cilium of the photoreceptor cells and leads thereby to blindness. Several of the Chlamydomonas flagellar proteins are remarkably similar to human proteins associated with some of these diseases.
See also: Chloroplasts; Culture Collections; Flagella; Chloroplasts, Genetics of; Kinases (Protein Kinases); Mendelian Genetics; Mitochondria; Organelles; Photosynthesis, Genetics of; Uniparental Inheritance
Further Reading Harris EB, Stern D, and Witman G (eds.) (2009) The Chlamydomonas Sourcebook, 2nd edn., 3 vols. Amsterdam: Academic Press, Elsevier. Eberhard S, Finazzi G, and Wollman FA (2008) The dynamics of photosynthesis. Annual Review of Genetics 42: 463–515. Hemschemeier A, Melis A, and Happe T (2009) Analytical approaches to photobiological hydrogen production in unicellular green algae. Photosynthesis Research 102: 523–540. Li Z, Wakao S, Fischer BB, and Niyogi KK (2009) Sensing and responding to excess light. Annual Review of Plant Biology 60: 239–260. Merchant S, Prochnik SE, Vallon O, et al. (2007) The Chlamydomonas genome reveals the evolution of key animal and plant functions. Science 318: 245–250. Molnar A, Schwach F, Studholme DJ, Thuenemann EC, and Baulcombe D (2007) miRNAs control gene expression in the single-cell alga Chlamydomonas reinhardtii. Nature 447: 1126–1129. Rocheix J-D (1996) Chalmydomonas. Encyclopedia of Molecular Biology and Molecular Medicine, vol. 1, pp. 347–360. Weinjeim: VHC Verlagsgesellschaft. Rochaix JD (2007) Role of thylakoid protein kinases in photosynthetic acclimation. FEBS Letters 581: 2768–2775.
Relevant Websites http://www.chlamy.org – Chlamy Center.
http://genome.jgi-psf.org – JGI Genome Blast; Chlamydomonas reinhardtii v3.0.