The Circadian Clock in Chlamydomonas reinhardtii. What Is It For? What Is It Similar To?

The physiology of the circadian (daily) clock has been well studied in the unicellular eukaryote Chlamydomonas reinhardtii. Circadian rhythms of phototaxis, chemotaxis, cell division, UV sensitivity, and adherence to glass have been characterized in this green alga. Circadian phototaxis was even shown to operate in outer space! The related phenomenon of photoperiodic time measurement of germination has been demonstrated. The C. reinhardtii system now offers genetic and proteomic opportunities that make it an excellent unicellular eukaryotic model organism to study the circadian clock at all levels of organization. Several clock-controlled genes have been identified as well as a clock-controlled RNA-binding protein that acts on circadian output. A computer-based search in C. reinhardtii for components of the circadian system that are similar to those from other model species has shown that some phototransduction components and especially kinases and phosphatases are well conserved in this green alga, while their target proteins appear to be different. The first functional proteomic approaches have discovered novel components of the circadian system, including a protein disulfide isomerase and a tetratricopeptide repeat protein. Circadian (daily) rhythms are endogenous biological programs that control metabolic, physiological, and/or behavioral events to occur at optimal phases of the daily cycle. In addition to exhibiting a self-sustained oscillation in constant conditions that can be entrained to environmental cycles, circadian rhythms run at essentially the same rate at different ambient temperatures (i.e. they are “temperature compensated”). C. reinhardtii has long been a preferred model system for the analysis of circadian rhythms because of the genetic and molecular techniques that have been developed for C. reinhardtii and are described elsewhere in this issue (Harris, 1989, 2001; Johnson et al., 1992; Fuhrmann, 2002). The availability of its complete nuclear, chloroplast, and mitochondrial genome sequences and of more than 200,000 expressed sequence tags (ESTs) that have been assembled into approximately 10,000 unique cDNAs (Grossman et al., 2003) render it a highly attractive model system. The circadian clock in C. reinhardtii is known to modulate a number of processes. As is the case for other flagellates, C. reinhardtii is able to orientate itself toward the light, a process known as photoaccumulation or phototaxis. This process is known to be rhythmically modulated in some species; Victor Bruce demonstrated circadian rhythms of photoaccumulation in C. reinhardtii more than 30 years ago (Bruce, 1970). The algae swim toward a supplied light source with maximum accumulation during the day phase. Computerized apparatuses can automatically monitor this rhythm (Mergenhagen, 1984; Kondo et al., 1991; Johnson et al., 1992). Mergenhagen and Mergenhagen (1987) used an automated apparatus aboard a spacecraft to confirm that the photoaccumulation rhythm persisted in outer space when the cells orbited the earth every 90 min! The rhythms continued for at least 6 d in microgravity without any terrestrial cue of the time of day. Therefore, these studies of C. reinhardtii showed conclusively that the circadian clock can run independently from daily cycles of gravity, magnetism, cosmic ray irradiation, and so on. There are also circadian rhythms in C. reinhardtii that peak during the night phase, for example, chemotaxis to ammonium (Byrne et al., 1992). The cells swim maximally toward this nitrogen source during the middle of the night phase, even though the light-dependent uptake of ammonium does not occur until dawn. The uptake of nitrite also peaks at dawn, as does the activity of nitrite reductase (Pajuelo et al., 1995). Chemotaxis versus phototaxis (as well as the uptake and further metabolism of N sources) is an example of temporal separation of processes in algae. In other words, the daily biological clock organizes a temporal program that triggers biological events to occur at specific phases throughout the daily environmental cycle; phototaxis during daytime optimizes photosynthesis, and chemotaxis (to ammonium) in the nighttime allows C. reinhardtii to find and store nutrients when solar energy is not available. Another circadian rhythm in C. reinhardtii that peaks during the night is the ability of the cells to adhere to a glass surface (Straley and Bruce, 1979). Yet another important rhythmic property is the temporal control of cell division, as first studied by Bruce (1970) and later by Goto and Johnson (1995). Several clock mutants that have an altered period were isolated by Victor Bruce using the phototaxis rhythm as a screen (Bruce, 1972; Bruce and Bruce, 1978). These mutants were called period (per), a name that was also given to a famous clock gene in Drosophila (but they are almost certainly unrelated since there are no putative homologs of the Drosophila per gene in the C. reinhardtii genome; see below). Victor Bruce demonstrated with one of the long-period mutants (per4) that three independent rhythms (phototaxis, adhesion to glass, and cell division) were affected by the mutation, implying that a component of the central oscillator may be defective in the per4 mutant (Straley and Bruce, 1979). The question of why organisms have endogenous temporal programs is closely linked with identifying the selective forces that encouraged the original evolution of these timers. Perhaps an initial driving force for the early evolution of circadian clocks could have been to phase cellular events that are inhibited by sunlight to occur in the night. This idea has been called the “escape from light” hypothesis (Pittendrigh, 1993). That speculation seems plausible when one considers the numerous examples of microorganisms with 24-h cell division cycles in which DNA replication and cell division occur during the night (Edmunds, 1988). Some of the events of the cell division cycle in these microorganisms might be sensitive to sunlight, e.g. replication during S phase. Survival of Chlamydomonas cells after irradiation by UV light as a function of the time in a light/dark cycle. Chlamydomonas cultures were plated onto agar medium and treated with equal amounts of UV light at different phases of a 12-h-light/12-h-dark cycle. Survival was measured as the colony-forming ability of cells following treatment as compared to that of cells that were not irradiated with UV light (modified from Nikaido and Johnson, 2000). Plants and animals sense the season of the year by measuring the duration of the day and/or night in the natural environment and respond appropriately so as to adapt to seasonal changes in their environment (Thomas and Vince-Prue, 1997). This phenomenon is called photoperiodic time measurement (PTM). PTM is of fundamental importance to the biological adaptations of organisms to their environment, especially in the cases of reproduction and development. The model for PTM that has become generally accepted is that a circadian (daily) clock is the timer that somehow measures the length of the night and triggers the developmental events controlled by photoperiodism, as first proposed by Bünning (1936) and now supported by multiple lines of evidence (Thomas and Vince-Prue, 1997). Germination in different photoperiods in Chlamydomonas. A, Photoperiodic response curve. The germination efficiencies 10 d after mating are shown as a function of the light duration in light/dark cycles of 24 h total duration (white squares). Constant light (LL, filled square) represents cells continuously exposed to light for 24 h a day. B, Germination efficiencies in two different photoperiods (LD 12:12 versus LD 9:15) at different light fluences. Data are plotted as the total light fluence in a day, calculated as irradiance (light intensity, μE m−2 s−1) × (duration of light treatment in seconds). A and B, Germinated cells were counted at 10 d after mating, all data are shown with SEM (modified from Suzuki and Johnson, 2002). So far, most of the studies on the molecular basis of the circadian clock in C. reinhardtii have focused on clock-controlled genes. In particular, circadian control of transcriptional rate has been found for a number of genes, resulting in rhythms of RNA abundance over the circadian cycle. These data have been described in a recent review (Mittag and Wagner, 2003) and are summarized in Table I Circadian changes in mRNA abundance and/or transcription rate in C. reinhardtii References: Jacobshagen and Johnson (1994); Memon et al. (1995); Fujiwara et al. (1996); Hwang et al. (1996); Jacobshagen et al. (1996, 2001); Savard et al. (1996); and Lemaire et al. (1999). Circadian changes in mRNA abundance and/or transcription rate in C. reinhardtii References: Jacobshagen and Johnson (1994); Memon et al. (1995); Fujiwara et al. (1996); Hwang et al. (1996); Jacobshagen et al. (1996, 2001); Savard et al. (1996); and Lemaire et al. (1999). Other molecular studies concern the circadian binding activity of an RNA-binding protein called CHLAMY 1 (Mittag, 1996). This protein is an analog of CCTR (clock controlled translational regulator) from the dinoflagellate alga Lingulodinium polyedra (for review, see Mittag, 2003). Its binding activity increases at the end of the subjective day and stays at a high level until the middle of the subjective night. CHLAMY 1 interacts with mRNAs that have an UG repeat of at least seven units (Waltenberger et al., 2001), and we assume that it represses translation (Mittag, 2003). The UG-containing mRNAs encode mainly proteins of the nitrogen metabolic pathways (NRT2;3, a nitrite/nitrate transporter; nitrite reductase; Gln synthetase; arginino-succinate lyase) and carbon metabolic pathways (small subunit of Rubisco; LIP36-G1, a CO2 shuffling protein in the outer chloroplast membrane; CCM, a CO2 key regulator). CHLAMY 1 represents a novel type of heteromeric RNA-binding protein that consists of subunits comprising three RNA-recognition motifs and three Lys-homology motifs (Zhao et al., 2004). CHLAMY 1's circadian binding activity appears to be controlled at the posttranslational level by time-dependent formation of protein complexes. One of the subunits of CHLAMY 1, named C3, is conserved in mammals, where it appears to be involved in myotonic dystrophy (Zhao et al., 2004). Interestingly, patients suffering from myotonic dystrophy have disturbances in their circadian system (Okumura et al., 2002). The entire nuclear genome (version 2) of C. reinhardtii has been sequenced by the U.S. Department of Energy, and the information is available at the Joint Genome Institute (JGI) Web site (http://genome.jgi-psf.org/chlre2/chlre2.home.html). We searched the C. reinhardtii nuclear genome for potential homologs to genes that are known to encode components of the circadian system in other organisms (Table II Clock-related proteins from model organisms that are conserved in C. reinhardtii Sequence analysis was carried out as explained in the first paragraph of the section “Exploring the C. reinhardtii Genome with Regard to Components of the Circadian System.” Briefly, the JGI BLAST page was used in combination with tBLASTn in addition to a NCBI conserved domain search and NCBI protein BLAST. Clock proteins that do not show any significant similarity (labeled as “no hits” in the output file of the JGI blast) to proteins of C. reinhardtii are: S. elongatus KAI A, KAI B, KAI C, LDPA, PEX, RPOD2, and SAS A; N. crassa FRQ; Arabidopsis ELF3, GI, PHYB, PHYE, PIF3, and TEJ; D. melanogaster CLK, CYC, LARK, PDP1, PER, TIM, and VRI; and mammal BMAL1, BMAL2, m-CLOCK, CREB, DBP, and Other proteins that show a similarity to proteins of C. reinhardtii are: N. crassa and and Arabidopsis and was as those cases when the of including functional was over the total length of the protein. in the number of from the of similarity is a that the number of one can to see by when a of a the or the it is to the more significant is the total number of of the protein from the organism used for the similarity are in the domain is also in the putative in the C. reinhardtii genome and is the of and the domain is also in the putative in the C. reinhardtii genome is of the of model of that show similarity to proteins of C. reinhardtii have been to functional Clock-related proteins from model organisms that are conserved in C. reinhardtii Sequence analysis was carried out as explained in the first paragraph of the section “Exploring the C. reinhardtii Genome with Regard to Components of the Circadian System.” Briefly, the JGI BLAST page was used in combination with tBLASTn in addition to a NCBI conserved domain search and NCBI protein BLAST. Clock proteins that do not show any significant similarity (labeled as “no hits” in the output file of the JGI blast) to proteins of C. reinhardtii are: S. elongatus KAI A, KAI B, KAI C, LDPA, PEX, RPOD2, and SAS A; N. crassa FRQ; Arabidopsis ELF3, GI, PHYB, PHYE, PIF3, and TEJ; D. melanogaster CLK, CYC, LARK, PDP1, PER, TIM, and VRI; and mammal BMAL1, BMAL2, m-CLOCK, CREB, DBP, and Other proteins that show a similarity to proteins of C. reinhardtii are: N. crassa and and Arabidopsis and was as those cases when the of including functional was over the total length of the protein. in the number of from the of similarity is a that the number of one can to see by when a of a the or the it is to the more significant is the total number of of the protein from the organism used for the similarity are in the domain is also in the putative in the C. reinhardtii genome and is the of and the domain is also in the putative in the C. reinhardtii genome is of the of model of that show similarity to proteins of C. reinhardtii have been to functional In we have searched the C. reinhardtii chloroplast and mitochondrial for potential homologs to the genes of S. elongatus in Table this the chloroplast and mitochondrial sequences of C. reinhardtii were in all and compared by the BLAST Sequence with S. elongatus protein. no significant were found to any of the proteins of S. In all circadian model studied so far, and have been proposed to be key (for review, see et al., 2001; et al., and Johnson, 2004). these some molecular components are conserved organisms with to their protein domain and their and as transcription of clock components in and kinases components of the endogenous system in N. and mammals, the target proteins are different. In some cases the protein domain is conserved a of its function is as in the case of acts as in Arabidopsis and is a component of the in The similarity search can a C. reinhardtii its function the circadian system have to be for temporal of central oscillator proteins is to be to the 24-h In all eukaryotic examples D. and kinases and to the of kinases are involved et al., 2001; et al., et al., et al., and 2004). the target components are different on the organism in N. in in D. melanogaster and mammals, their temporal appears to be for circadian In C. and are conserved (Table the which has been shown to the of and in D. is also conserved in C. reinhardtii. it has been demonstrated that the protein subunits and are also of the in D. melanogaster by of the that they in a circadian et al., 2004). subunits are well conserved in C. reinhardtii. for the subunit of protein 1 and for a subunit of have been found in the circadian clock et al., 2004). can on the central clock protein is also highly conserved in C. reinhardtii (Table and have been to specific the Chlamydomonas is of the central that and appears to be to the et al., 2000). and are for clock of circadian in C. reinhardtii. Therefore, kinases and phosphatases that could be involved in circadian are the C. reinhardtii In there are potential homologs in the C. reinhardtii nuclear genome to all known eukaryotic kinases and the other the is different with to the clock components that are of these there were no significant found in the C. reinhardtii genome to the famous clock proteins PER, or might be that the of for C. reinhardtii be its and by S. elongatus and one genes from the there are central clock components that from the example, there are no putative homologs in C. reinhardtii to the central clock genes or of S. The homologs that do appear are to and The gene is a gene that has a domain that is similar to that of The function of is and its similarity to is to no sequence is a for RNA and is to be a gene of in does not have a clock with clock genes those of in phototransduction and no other specific clock homologs were and potential homologs to phototransduction genes be in the The to putative central clock components of Arabidopsis are putative homologs in the C. reinhardtii genome for the Arabidopsis clock components or and 2003). similarity can be found to or the and There are also of similarity to the central clock genes and (Table these are to found these proteins (i.e. DNA binding in and or in than to the of these clock genes that the In there is a section of similarity the RNA of the output gene of Arabidopsis et al., and Therefore, the green algae Chlamydomonas and the putative clock genes are in and genes not in the genes that are to be central components of the Several years from cells that as well as light could the phase of the circadian clock in C. reinhardtii et al., 1991; Kondo et al., were at and to in the and at and to in the was as a since was not The data available at that time that could be A potential has not been The C. reinhardtii genome some of that have been characterized in other organisms were found in the C. reinhardtii One is the et al., which can as circadian in Drosophila or or as a component of the circadian in et al., 2001), as The similarity search identified two genes that encode potential in C. reinhardtii. Arabidopsis or 2) were used for the one gene model was Table the other when D. melanogaster or or 2) were used for the a different gene model was the Table gene proteins are that all the of we that C. reinhardtii has two one of which is more closely related to Arabidopsis and the other to and have sequenced a that the protein to their et al., and 2004). this sequence is and the C. reinhardtii an of similarity is found gene model the protein of this gene model is not with the protein sequence by et al. resulting in some the the the potential from gene model has not been described and to to be in where genes be since these could The other is an of the of was discovered in Arabidopsis and et al., and was identified in C. reinhardtii et al., 2002). In C. multiple in its including changes in chemotaxis and et al., 2004). with those can be found in the of C. reinhardtii et al., 2004). The circadian clock in Arabidopsis the of et al., 2000). and a in the circadian system of C. for an the there have been for in C. until now data for a in C. reinhardtii. the A, B, C, and proteins from Arabidopsis were used for the could be C. reinhardtii proteins with A, C, and and essentially to or we have used the protein sequence from the green alga and the sequence from for the similarity there were no when the and sequences were the C. reinhardtii nuclear genome These that there is no in C. reinhardtii or that these have C. reinhardtii and other another protein from Arabidopsis is which similarity in its to by the an important the circadian system reproduction in its is not to as it can be also found in and in et al., and et al., 2003). In the two in the and In C. there are two sequences that show similarity to those and Table In the case of the of similarity could be than the in Table II because there is a of sequence to the of the the that C. reinhardtii that could to a C. reinhardtii offers excellent to the of functional chloroplast, and mitochondrial genome sequences as well as sequences are available (Grossman et al., 2003). some proteins can now be identified at the to with proteins a are not to be identified by Therefore, of proteins is a for and C. reinhardtii can be and in so as to proteomic studies have been carried out in C. reinhardtii with to the the chloroplast and the circadian system et al., et al., et al., 2004). In a first to functional to proteins from C. proteins were by et al., 2004). that were in constant light were throughout the subjective day and night. were carried out using and a proteins a of for a daily rhythm of These two proteins were with and identified by et al., 2004). One of the proteins was a protein disulfide isomerase Its was during the middle of the subjective night and at the of the subjective day. is known to be an that in the of proteins in the can function as a molecular et al., the and of disulfide during protein Interestingly, a chloroplast been described in C. reinhardtii that is a component of the protein the protein of is in the of translation in C. reinhardtii in a and and et al., The other protein identified in the proteomic search was a protein et al., 2004). Its also in the middle of the subjective its is in the middle of the subjective day. proteins have been proposed to with of a central and 1992). the molecular of the circadian clocks in D. melanogaster and N. crassa show a of proteins in the of the circadian oscillator et al., et al., 2003). There are also proteins described in C. reinhardtii that are to of chloroplast and that are of including RNA et al., et al., 2000). The of a of physiological, and techniques with the available genome and sequences C. reinhardtii to be an attractive eukaryotic model organism for studies of the circadian The of the of the circadian clock in and is of and have been as potential on the of the similarity search and their the Circadian that control might also be to especially when one considers that proteins from the apparatus and the of C. reinhardtii are well conserved in in the apparatus and the of changes of of as well as a with and et al., et al., et al., We can make on the of molecular components of the circadian oscillator from model organisms with potential from C. reinhardtii. The clock kinases and phosphatases from and are well conserved in this green alga, and it is that they may be also involved in its circadian system. this does not out the that these kinases and phosphatases may also in other cellular processes. The same for the sequences found in C. reinhardtii. In this it is that there appears to be two one of which is more closely related to while the other is more similar to a of that interacts with in Arabidopsis and its et al., 2001), appears to occur in C. reinhardtii. The central clock components that are of and are in most cases to a of organisms PER, are not in the C. reinhardtii this this green alga have its central clock The evolution of specific proteins that are key components of the central oscillator seems to have independently in different We for on the We are for information supplied by the C. reinhardtii genome of the U.S. Department of