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Chloroplasts rely on the translocon complexes in the outer and inner envelope membranes (the TOC and TIC complexes, respectively) to import thousands of different nuclear-encoded proteins from the cytosol1–4. Although previous studies indicated that the TOC and TIC complexes may assemble into larger supercomplexes5–7, the overall architectures of the TOC–TIC supercomplexes and the mechanism of preprotein translocation are unclear. Here we report the cryo-electron microscopy structure of the TOC–TIC supercomplex from Chlamydomonas reinhardtii. The major subunits of the TOC complex (Toc75, Toc90 and Toc34) and TIC complex (Tic214, Tic20, Tic100 and Tic56), three chloroplast translocon-associated proteins (Ctap3, Ctap4 and Ctap5) and three newly identified small inner-membrane proteins (Simp1–3) have been located in the supercomplex. As the largest protein, Tic214 traverses the inner membrane, the intermembrane space and the outer membrane, connecting the TOC complex with the TIC proteins. An inositol hexaphosphate molecule is located at the Tic214–Toc90 interface and stabilizes their assembly. Four lipid molecules are located within or above an inner-membrane funnel formed by Tic214, Tic20, Simp1 and Ctap5. Multiple potential pathways found in the TOC–TIC supercomplex may support translocation of different substrate preproteins into chloroplasts.

Although chloroplasts contain their own genetic system and are semi-autonomous cell organelles, plastid biogenesis and homeostasis are heavily dependent on the nucleo-cytosolic compartment. These two cellular compartments are closely co-ordinated through a complex signaling network comprising both anterograde and retrograde signaling chains. Developmental changes or any perturbation in the chloroplast system induced by a particular stress resulting from changes in environmental conditions such as excess light, elevated temperature, nutrient limitation, pathogen infection, give rise to specific signals. They migrate out of the chloroplast and are perceived by the nucleus where they elicit changes in expression of particular genes that allow for the maintenance of plastid homeostasis toward environmental cues. These genes mainly include those of photosynthesis-associated proteins, chaperones, proteases, nucleases and immune/defense proteins. Besides this transcriptional response, a chloroplast quality control system exists that is involved in the repair and turnover of damaged plastid proteins. This system degrades aggregated or damaged proteins and it can even remove entire chloroplasts when they have suffered heavy damage. This response comprises several processes such as plastid autophagy and ubiquitin–proteasome mediated proteolysis that occurs on the plastid envelope through the action of the ubiquitin–proteasome system.

Photosynthetic organisms are able to adapt to changes in light conditions by balancing the light excitation energy between the light-harvesting systems of photosystem (PS) II and photosystem I to optimize the photosynthetic yield. A key component in this process, called state transitions, is the chloroplast protein kinase Stt7/STN7, which senses the redox state of the plastoquinone pool. Upon preferential excitation of photosystem II, this kinase is activated through the cytochrome b6fcomplex and required for the phosphorylation of the light-harvesting system of photosystem II, a portion of which migrates to photosystem I (state 2). Preferential excitation of photosystem I leads to the inactivation of the kinase and to dephosphorylation of light-harvesting complex (LHC) II and its return to photosystem II (state 1). Here we compared the thylakoid phosphoproteome of the wild-type strain and the stt7mutant of Chlamydomonasunder state 1 and state 2 conditions. This analysis revealed that under state 2 conditions several Stt7-dependent phosphorylations of specific Thr residues occur in Lhcbm1/Lhcbm10, Lhcbm4/Lhcbm6/Lhcbm8/Lhcbm9, Lhcbm3, Lhcbm5, and CP29 located at the interface between PSII and its light-harvesting system. Among the two phosphorylation sites detected specifically in CP29 under state 2, one is Stt7-dependent. This phosphorylation may play a crucial role in the dissociation of CP29 from PSII and/or in its association to PSI where it serves as a docking site for LHCII in state 2. Moreover, Stt7 was required for the phosphorylation of the thylakoid protein kinase Stl1 under state 2 conditions, suggesting the existence of a thylakoid protein kinase cascade. Stt7 itself is phosphorylated at Ser533in state 2, but analysis of mutants with a S533A/D change indicated that this phosphorylation is not required for state transitions. Moreover, we also identified phosphorylation sites that are redox (state 2)-dependent but independent of Stt7 and additional phosphorylation sites that are redox-independent.

The psaA gene of the green alga Chlamydomonas reinhardtii consists of three exons that are widely separated on the chloroplast genome and transcribed independently. The exons are flanked by group II intron sequences. Maturation of the psaA mRNA requires two steps of splicing in trans between the transcripts of exons 1, 2, and 3. At least 14 nuclear loci and one chloroplast gene (tscA) are involved in this process. Recently the genes of three of these nuclear factors have been cloned. Raa3 is involved in the first trans-splicing reaction, and Raa1 and Raa2 are required for the second trans-splicing reaction. Here we show that Raa1 and Raa2 can be coimmunoprecipitated and that they are part of a high molecular weight complex of 400-500 kD. The size and integrity of the complex are affected by mutations in other complementation groups, suggesting that the corresponding proteins may also be components of this multiprotein complex or required for its assembly. Raa1 is also associated with a larger complex.

The gene encoding the chloroplast ribosomal protein S1 from Chlamydomonas reinhardtii, CreS1, was cloned and the RNA binding properties and the expression patterns were studied. Gel-shift analysis revealed that CreS1 binds AU-rich 5′-untranslated regions (5′-UTR) of chloroplast mRNAs with higher affinity than the corresponding sequence of a GC-rich nuclear transcript. The binding affinity of CreS1 for a mutant form of the psbD5′-UTR with a deletion of a U-rich stretch that is required for translation decreases 4-fold as compared to the wild-type 5′-UTR. Our results suggest that CreS1 protein interacts with U-rich sequences. Most of CreS1 is bound to high-molecular-weight complexes which co-migrate with the 30S small ribosomal subunit, and only a small fraction of CreS1 exists in its free form. CreS1 is localized mainly to the chloroplast stroma albeit a significant fraction is associated with chloroplast membranes. The results suggest that most of CreS1 is associated with the 30S ribosomal subunit throughout the translation process. Upon a shift of cells from the dark to the light, the mRNA levels of CreS1 and Psrp-7, both components of the 30S ribosomal subunit, increase transiently and return to the dark levels after 8 h. However, during this dark-to-light transition the levels of CreS1 and of other components of the 30S subunit remain the same suggesting that either protein synthesis or degradation is regulated. The possible implications of these findings are discussed.

Several membrane proteins were previously shown to bind to the 5′ leader of the chloroplast psbCmRNA in the unicellular eukaryotic alga Chlamydomonas reinhardtii. This study showed that these proteins have affinity for AU-rich RNAs, as determined by competition experiments. In addition, their binding activities are enhanced 13–15-fold by light, and a 46 kDa protein is activated within 1–10 min. This activation could be mediated by the modulation of ADP pools by the light-dependent reactions of photosynthesis and ATP synthase because (1) two inhibitors that block ATP synthesis also prevent this activation and (2) ADP inhibits the RNA-binding activity of this protein in vitro. An inhibitor of Photosystem II diminishes this induction, suggesting that reducing potential generated by the photosynthetic electron transport chain modulates this RNA-binding activity. The RNA-binding activities of two proteins (of 46 and 47 kDa) are inhibited by Mg-protoporphyrin IX methyl ester in vitrosuggesting they could be regulated by these intermediates in the chlorophyll biosynthetic pathway.

During the past 50 years, the green unicellular alga Chlamydomonas reinhardtiihas played a key role as model system for the study of photosynthesis and chloroplast biogenesis. This is due to its well-established nuclear and chloroplast genetics, its dispensable photosynthetic function in the presence of acetate, and its highly efficient nuclear and chloroplast transformation systems. Considerable progress has been achieved in our understanding of the structure, function, inheritance, and expression of nuclear, chloroplast, and mitochondrial genes and of the molecular cross-talk between the nuclear, chloroplast, and mitochondrial genetic systems.

Strains of Chlamydomonas reinhardtiilacking the PsaFgene or containing the mutation K23Q within the N-terminal part of PsaF are sensitive to high light (>400 μE m−2s−1) under aerobic conditions.In vitroexperiments indicate that the sensitivity to high light of the isolated photosystem I (PSI) complex from wild type and from PsaF mutants is similar. In vivomeasurements of photochemical quenching and oxygen evolution show that impairment of the donor side of PSI in the PsaF mutants leads to a diminished linear electron transfer and/or a decrease of photosystem II (PSII) activity in high light. Thermoluminescence measurements indicate that the PSII reaction center is directly affected under photo-oxidative stress when the rate of electron transfer becomes limiting in the PsaF-deficient strain and in the PsaF mutant K23Q. We have isolated a high light-resistant PsaF-deficient suppressor strain that has a high chlorophyll a/b ratio and is affected in the assembly of light-harvesting complex. These results indicate that under high light a functionally intact donor side of PSI is essential for protection ofC. reinhardtiiagainst photo-oxidative damage when the photosystems are properly connected to their light-harvesting antennae.

The two [4Fe-4S] clusters FAand FBare the terminal electron acceptors of photosystem I (PSI) that are bound by the stromal subunit PsaC. Soluble ferredoxin (Fd) binds to PSI via electrostatic interactions and is reduced by the outermost iron-sulfur cluster of PsaC. We have generated six site-directed mutants of the green alga Chlamydomonas reinhardtiiin which residues located close to the iron-sulfur clusters of PsaC are changed. The acidic residues Asp9and Glu46, which are located one residue upstream of the first cysteine liganding cluster FBand FA, respectively, were changed to a neutral or a basic amino acid. Although Fd reduction is not affected by the E46Q and E46K mutations, a slight increase of Fd affinity (from 1.3- to 2-fold) was observed by flash absorption spectroscopy for the D9N and D9K mutant PSI complexes. In the FA2triple mutant (V49I/K52T/R53Q), modification of residues located next to the FAcluster leads to partial destabilization of the PSI complex. The electron paramagnetic resonance properties of cluster FAare affected, and a 3-fold decrease of Fd affinity is observed. The introduction of positively charged residues close to the FBcluster in the FB1triple mutant (I12V/T15K/Q16R) results in a 60-fold increase of Fd affinity as measured by flash absorption spectroscopy and a larger amount of PsaC-Fd cross-linking product. The first-order kinetics are similar to wild type kinetics (two phases witht12of <1 and ≈4.5 μs) for all mutants except FB1, where Fd reduction is almost monophasic witht12< 1 μs. These data indicate that FBis the cluster interacting with Fd and therefore the outermost iron-sulfur cluster of PSI.

In photosynthetic cells of higher plants and algae, the distribution of light energy between photosystem I and photosystem II is controlled by light quality through a process called state transition. It involves a reorganization of the light-harvesting complex of photosystem II (LHCII) within the thylakoid membrane whereby light energy captured preferentially by photosystem II is redirected toward photosystem I or vice versa. State transition is correlated with the reversible phosphorylation of several LHCII proteins and requires the presence of functional cytochromeb6fcomplex. Most factors controlling state transition are still not identified. Here we describe the isolation of photoautotrophic mutants of the unicellular algaChlamydomonas reinhardtii, which are deficient in state transition. Mutant stt7is unable to undergo state transition and remains blocked in state I as assayed by fluorescence and photoacoustic measurements. Immunocytochemical studies indicate that the distribution of LHCII and of the cytochromeb6fcomplex between appressed and nonappressed thylakoid membranes does not change significantly during state transition in stt7,in contrast to the wild type. This mutant displays the same deficiency in LHCII phosphorylation as observed for mutants deficient in cytochromeb6fcomplex that are known to be unable to undergo state transition. The stt7mutant grows photoautotrophically, although at a slower rate than wild type, and does not appear to be more sensitive to photoinactivation than the wild-type strain. Mutant stt3-4bis partially deficient in state transition but is still able to phosphorylate LHCII. Potential factors affected in these mutant strains and the function of state transition in C. reinhardtiiare discussed.

D1 and D2, two chloroplast proteins with apparent mol. wt of 32 000‐34 000, play an important role in the photosynthetic reactions mediated by the membrane‐bound protein complex of photosystem II (PSII). We have isolated and characterized an uniparental, non‐photosynthetic mutant of Chlamydomonas reinhardtiiand show that the mutation is in the chloroplast gene psbD, coding for D2. A 46 bp direct DNA duplication in the coding region of the mutant gene causes a frame‐shift which results in a psbD transcript coding for 186 amino acid residues instead of the normal 352. The truncated D2 peptide is never seen, even after pulse‐labeling, suggesting that the mutant protein is very unstable. In addition, little or no D1 protein is detected in this mutant although the gene and normal levels of mRNA for D1 are present in mutant cells. All other core PSII proteins are synthesized and inserted into the membrane fraction, but never accumulate. These results suggest that D2 contributes not only to the stabilization of the PSII complex in the membrane, but also may play a specific role in the regulation of the D1 protein, either at the translational or post‐translational level.

The cleavable pre‐sequences of imported chloroplast and mitochondrial proteins have several features in common. This structural similarity prompted us to test whether a chloroplast pre‐sequence (‘transit peptide’) can also be decoded by the mitochondrial import machinery. In the green alga, Chlamydomonas reinhardtii, the small subunit of ribulose‐1,5‐bisphosphate carboxylase/oxygenase (Rubisco) (a chloroplast protein) is nuclear‐encoded and synthesized in the cytosol with a transient pre‐sequence of 45 residues. The 31 amino‐terminal residues of this chloroplast pre‐sequence were fused to mouse dihydrofolate reductase (a cytosolic protein) and to yeast cytochrome oxidase subunit IV (an imported mitochondrial protein) from which the authentic pre‐sequence had been removed. The chloroplast pre‐sequence transported both attached proteins into the yeast mitochondrial matrix or inner membrane, although it functioned less efficiently than an authentic mitochondrial pre‐sequence. We conclude that mitochondrial and chloroplast pre‐sequences perform their function by a similar mechanism.

The chloroplast psbA gene from the green unicellular alga Chlamydomonas reinhardiihas been localized, cloned and sequenced. This gene codes for the rapidly‐labeled 32‐kd protein of photosystem II, also identified as as herbicide‐binding protein. Unlike psbA in higher plants which is found in the large single copy region of the chloroplast genome and is uninterrupted, psbA in C. reinhardiiis located entirely within the inverted repeat, hence present in two identical copies per circular chloroplast genome, and contains four large introns. These introns range from 1.1 to 1.8 kb in size and fall into the category of Group I introns. Two of the introns contain open reading frames which are in‐frame with the preceding exon sequences. We present the nucleotide sequence for the C. reinhardii psbA 5′‐and 3′ ‐flanking sequences, the coding region contained in five exons and the deduced amino acid sequence. The algal gene codes for a protein of 352 amino acid residues which is 95% homologous, excluding the last eight amino acid residues, with the higher plant protein.

Using particle gun-mediated chloroplast transformation we have disrupted the psbK gene of Chlamydomonas reihardtii with an aadA expression cassette that confers resistance to spectinomycin. The transformants are unable to grow photoautotrophically, but they grow normally in acetate-containing medium. They are deficient in photosystem II activity as measured by fluorescence transients and O2 evolution and they accumulate less than 10% of wild-type levels of photosystem II as measured by immunochemical means. Pulse-labeling experiments indicate that the photosystem II complex is synthesized normally in the transformants. These results differ from those obtained previously with similar cyanobacterial psbK mutants that were still capable of photoautotrophic growth (Ikeuchi et al., J. Biol. Chem. 266 (1991) 1111–1115). In C. reinhardtii the psbK product is required for the stable assembly and/or stability of the photosystem II complex and essential for photoautotrophic growth. The data also suggest that the stability requirements of the photosynthetic complexes differ considerably between C. reinhardtii and cyanobacteria.

We report the rescue of an arginine-requiring mutant (arg7-8) of Chlamydomonas reinhardtii by complementation using total DNA from a genomic cosmid library. Using the glass-bead transformation method of Kindle [8] four putative transformants able to grow in the absence of exogenous arginine were obtained from 3×109 treated cells. Southern blot analysis reveals that at least three of the clones have acquired an additional copy of the gene (ARG7) encoding argininosuccinate lyase (ASL). The arginine-independent phenotype is stable in the absence of selective pressure and high levels of ASL activity are detected in all four clones. We conclude that these represent true transformants and that any stable nuclear mutant of Chlamydomonas could be rescued using this approach.

D1 and D2, two chloroplast proteins with apparent mol. wt of 32 000‐34 000, play an important role in the photosynthetic reactions mediated by the membrane‐bound protein complex of photosystem II (PSII). We have isolated and characterized an uniparental, non‐photosynthetic mutant of Chlamydomonas reinhardtiiand show that the mutation is in the chloroplast gene psbD, coding for D2. A 46 bp direct DNA duplication in the coding region of the mutant gene causes a frame‐shift which results in a psbD transcript coding for 186 amino acid residues instead of the normal 352. The truncated D2 peptide is never seen, even after pulse‐labeling, suggesting that the mutant protein is very unstable. In addition, little or no D1 protein is detected in this mutant although the gene and normal levels of mRNA for D1 are present in mutant cells. All other core PSII proteins are synthesized and inserted into the membrane fraction, but never accumulate. These results suggest that D2 contributes not only to the stabilization of the PSII complex in the membrane, but also may play a specific role in the regulation of the D1 protein, either at the translational or post‐translational level.

The chloroplast psbA gene from the green unicellular alga Chlamydomonas reinhardiihas been localized, cloned and sequenced. This gene codes for the rapidly‐labeled 32‐kd protein of photosystem II, also identified as as herbicide‐binding protein. Unlike psbA in higher plants which is found in the large single copy region of the chloroplast genome and is uninterrupted, psbA in C. reinhardiiis located entirely within the inverted repeat, hence present in two identical copies per circular chloroplast genome, and contains four large introns. These introns range from 1.1 to 1.8 kb in size and fall into the category of Group I introns. Two of the introns contain open reading frames which are in‐frame with the preceding exon sequences. We present the nucleotide sequence for the C. reinhardii psbA 5′‐and 3′ ‐flanking sequences, the coding region contained in five exons and the deduced amino acid sequence. The algal gene codes for a protein of 352 amino acid residues which is 95% homologous, excluding the last eight amino acid residues, with the higher plant protein.

The organization of the nuclear ribosomal units of five different species of Chlamydomonas has been examined by hybridization of their nuclear DNA fragments produced by several restriction endonucleases with a radioactively labelled probe consisting of the two cloned BamHI ribosomal fragments of C. reinhardii. The results indicate that a) the ribosomal units of these five species are structurally related, b) changes in the non transcribed spacer occur in C. eugametos and in C. globosa, c) the rDNA unit of C. intermedia contains either an enlarged internal transcribed spacer or a ribosomal intervening sequence, d) the rDNA units of C. reinhardii and C. callosa are indistinguishable.

Pulse labelled rDNA from cultured somatic cells of Xenopus laevis was examined by electron microscope autoradiography. The pattern of replication closely resembles that of bulk chromosomal DNA and differs considerably from rDNA synthesis during amplification in the oocyte. — About 0.15% of the rDNA molecules in the purified preparations were circular. The presence of interlocked circles of equal size indicates that the circles are not in vitro cyclization artefacts, but may represent free rRNA genes. A low frequency of circles was also seen in Xenopus blood rDNA. Their stability in high concentration of formamide suggests that they too did not arise after DNA extraction.

Chloroplast transit peptides from the green alga Chlamydomonas reinhardtiihave been analyzed and compared with chloroplast transit peptides from higher plants and mitochondrial targeting peptides from yeast, Neurospora and higher eukaryotes. In terms of length and amino acid composition, chloroplast transit peptides from C. reinhardtiiare more similar to mitochondrial targetting peptides than to chloroplast transit peptides from higher plants. They also contain the potential amphiphilic α‐helix characteristic of mitochondrial presequences. However, in similarity with chloroplast transit peptides from higher plants, they contain a C‐terminal region with the potential to form an amphiphilic β‐strand. As in higher plants, transit peptides that route proteins to the thylakoid lumen consist of an N‐tenninal domain similar to stroma‐targeting transit peptides attached to a C‐terminal apolar domain that share many characteristics with secretory signal peptides.

The cleavable pre‐sequences of imported chloroplast and mitochondrial proteins have several features in common. This structural similarity prompted us to test whether a chloroplast pre‐sequence (‘transit peptide’) can also be decoded by the mitochondrial import machinery. In the green alga, Chlamydomonas reinhardtii, the small subunit of ribulose‐1,5‐bisphosphate carboxylase/oxygenase (Rubisco) (a chloroplast protein) is nuclear‐encoded and synthesized in the cytosol with a transient pre‐sequence of 45 residues. The 31 amino‐terminal residues of this chloroplast pre‐sequence were fused to mouse dihydrofolate reductase (a cytosolic protein) and to yeast cytochrome oxidase subunit IV (an imported mitochondrial protein) from which the authentic pre‐sequence had been removed. The chloroplast pre‐sequence transported both attached proteins into the yeast mitochondrial matrix or inner membrane, although it functioned less efficiently than an authentic mitochondrial pre‐sequence. We conclude that mitochondrial and chloroplast pre‐sequences perform their function by a similar mechanism.

Summary We have isolated complementary DNA (cDNA) clones for apocytochrome c from the green algaChlamydomonas reinhardtii and shown that they are encoded by a single nuclear gene termedcyc.Cyc mRNA levels are found to depend primarily on the presence of acetate as a reduced carbon source in the culture medium. The deduced amino acid sequence shows that, apart from the probable removal of the initiating methionine,C. reinhardtii apocytochrome c is syntheszed in its mature form. Its structure is generally similar to that of cytochromes c from higher plants. Several punctual deviations from the general pattern of cytochrome c sequences that is found in other organisms have interesting structural and functional implications. These include, in particular, valines 19 and 39, asparagine 78, and alanine 83. A phylogenetic tree was constructed by the matrix method from cytochrome c data for a representative range of species. The results suggest thatC. reinhardtii diverged from higher plants approximately 700–750 million years ago; they also are not easy to reconcile with the current attribution ofChlamydomonas reinhardtii andEnteromorpha intestinalis to a unique phylum, because these two species probably diverged from one another at about the same time as they diverged from the line leading to higher plants.

We report the isolation and characterization of a uniparental mutant of Chlamydomonas reinhardtiithat is resistant to 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and 2-chloro-4-ethylamino-6-isopropylamino-s-triazine (atrazine). Such herbicides inhibit photosynthesis by preventing transfer of electrons in photosystem II from the primary stable electron acceptor Q to the secondary stable electron acceptor complex B, which is thought to contain a protein of 32 kDa and a bound quinone. It has been proposed that herbicide binding to the 32-kDa protein alters the B complex so that electron transfer from Q is prohibited. Both whole and broken-cell preparations of the mutant alga show a resistance to the effects of herbicide on electron transfer from Q to B, as measured by fluorescence-induction kinetics. In the absence of herbicide, mutant cells exhibit a slower rate of Q to B electron transfer than do wild-type cells. The 32-kDa protein from wild-type cells, but not mutant cells, binds azido[14C]atrazine at 0.1 μM. We have isolated psbA, the chloroplast gene for the 32-kDa protein, from both wild-type and herbicide-resistant algae and sequenced the coding regions of the gene that are contained in five exons. The only difference between the exon nucleotide sequences of the wild-type and mutant psbA is a single T-A to G-C transversion. This mutation results in a predicted amino acid change of serine in the wild-type protein to alanine in the mutant. We suggest that this alteration in the 32-kDa protein is the molecular basis for herbicide resistance in the C. reinhardtiimutant.

cDNA clones encoding three photosystem I subunits of Chlamydomonas reinhardtii with apparent molecular masses 13, 5 and 3 kDa (thylakoid polypeptides 28, 35 and 37; P28, P35 and P37, respectively) were isolated using gene specific oligonucleotides as probes. The sequences of these oligonucleotides were deduced from the N-terminal amino acid sequences of the proteins. The cDNAs were sequenced and used to probe Southern and Northern blots. The Southern blot analysis indicates that the proteins are encoded by single-copy genes. The mRNA sizes of the three components are 960 (P28), 1120 (P35) and 790 (P37) nucleotides. Comparison between the open reading frames of the cDNAs and the N-terminal amino acid sequences of the proteins indicates that the nascent polypeptides possess N-terminal transit sequences that are removed to give mature proteins of 11.0 (P28), 10.0 (P35) and 8.4 (P37) kDa. Analysis of the deduced protein sequences suggests that P28 and P35 are extrinsic membrane proteins and that P37 spans the thylakoid membrane. All three proteins have short transit peptides that probably route them to the stromal side of the thylakoid membrane.

Herbicide-resistant mutants of the eukaryotic green alga Chlamydomonas reinhardtii, that are altered in specific amino acids in their D-1 protein, show differential bicarbonate-reversible formate effects. These results suggest the involvement of D1 protein in the ‘bicarbonate effect’. A 25 mM formate treatment of mixotrophically or photoautotrophically grown wild type cells results in a slower rise of chlorophyll a fluorescence transient followed by a dramatically slowed decline during measurements in continuous light. These effects are fully reversed upon addition of 10 mM bicarbonate. The mutant BR-202 [L275F] is, however, highly insensitive to 25 mM formate suggesting that a significant change in formate (bicarbonate) binding has occurred in helix V of the D1 protein near histidine involved in Fe binding. With the exception of DCMU-4 [S264A], which is considerably more sensitive to formate than the wild type, five other different [V219I, A25IV, F255Y, G256D and cell-wall deficient CW-15] mutants display a relatively similar response to formate as wild type. Absence of formate effect on a PS II-lacking [FuD-7] mutant confirms the sole involvement of PS II in the ‘bicarbonate effect’.

Chloroplast transit peptides from the green alga Chlamydomonas reinhardtiihave been analyzed and compared with chloroplast transit peptides from higher plants and mitochondrial targeting peptides from yeast, Neurospora and higher eukaryotes. In terms of length and amino acid composition, chloroplast transit peptides from C. reinhardtiiare more similar to mitochondrial targetting peptides than to chloroplast transit peptides from higher plants. They also contain the potential amphiphilic α-helix characteristic of mitochondrial presequences. However, in similarity with chloroplast transit peptides from higher plants, they contain a C-terminal region with the potential to form an amphiphilic β-strand. As in higher plants, transit peptides that route proteins to the thylakoid lumen consist of an N-tenninal domain similar to stroma-targeting transit peptides attached to a C-terminal apolar domain that share many characteristics with secretory signal peptides.

The Chlamydomonas reinhardtiichloroplast mutants 18-5B and 18-7G lack both the chloroplast-encoded large subunit and nuclear-encoded small subunit of the chloroplast enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39). A chloroplast intergenic-suppression model has been postulated to account for the genetic instability of 18-5B revertants. Here, we have determined the molecular basis of the 18-5B and 18-7G mutants. They contain nonsense mutations close to the 3′ and 5′ ends of their large-subunit genes, respectively. Pulse-chase experiments revealed that the 18-5B mutant produces a truncated large subunit that is unstable. In connection with previous experiments, this work identifies nonsense suppression in the chloroplast. Small subunits are also synthesized but then degraded in the mutants. Thus, the coordinated absence of subunits is achieved through degradation of the small subunit in the specific absence of the large subunit.

Translation of the chloroplast psbCmRNA in Chlamydomonas reinhardtii has been shown previously to require interactions between its 5’ untranslated region (5’ UTR) and the functions encoded by two nuclear loci, which we name here TBC1and TBC2. We show that a 97-nucleotide (nt) region located in the middle of the psbC5’ UTR is required for translation initiation. Unlike most procaryotic cis-acting translational control elements, this region has a translational activation function and is located 236 nt upstream from the GUG translation initiation codon. In vivo pulse-labeling of chloroplast-encoded proteins and analyses of the expression of chimeric reporter genes in vivo reveal that a mutation of a newly described locus, TBC3, restores translation from the psbC5’ UTR in the absence of either this cis-acting element or the wild-type trans-acting TBC1function. These data demonstrate that sequences located in the middle of the psbC5’ UTR, TBC1, and TBC3functionally interact to control the translation of the psbCmRNA.

During oogenesis in many animals there is a massive extrachromosomal synthesis of the genes for ribosomal RNA. In Dytiscid beetles, as in the toad Xenopus, the amplified ribosomal DNA occurs as circular molecules of different sizes. The circles fall into size classes that are integral multiples of a unit circle. It is probable that the unit circle contains the coding sequence for one precursor ribosomal RNA molecule plus accompanying spacer sequences.

The genes coding for 4S RNA have been localized on the physical map of the chloroplast genome of Chlamydomonas reinhardii by hybridizing 32P-labelled 4S RNA to EcoRI, BamHI, BgIII and HindIII chloroplast DNA digests and to hybrid plasmids containing EcoRI and BamHI chloroplast DNA fragments. At least 10 EcoRI and 7 BamHI fragments carry sequences coding for 4S RNA. These genes are interspersed throughout the genome. The spacer between the 16S and 23S ribosomal RNA genes, which is repeated twice per chloroplast DNA molecule, codes for at least one 4S RNA, shown to be transcribed from the same strand as the ribosomal RNAs.

In Chlamydomonas reinhardtii the three exons of the psaA gene are widely scattered on the chloroplast genome: exons 1 and 2 are in opposite orientations and distant from each other and from exon 3. The mature mRNA, encoding a core polypeptide of photosystem I, is thus probably assembled from separate precursors by splicing in trans. We have isolated and characterized a set of mutants that are deficient in the maturation of psaA mRNA. The mutants belong to 14 nuclear complementation groups and one chloroplast locus that are required for the assembly of psaA mRNA. The chloroplast locus, tscA, is remote from any of the exons and must encode a factor required in trans. The mutants all show one of only three phenotypes that correspond to defects in one or other or both of the joining reactions. These phenotypes, and those of double mutants, are consistent with the existence of two alternative splicing pathways.

The chloroplast ribosomal intron of Chlamydomonas reinhardtii encodes a sequence-specific DNA endonuclease (I-CreI), which is most probably involved in the mobility of this intron. Here we show that I-CreI generates a 4 by staggered cleavage just downstream of the intron insertion site. The I-CreI recognition sequence is 19–24 by in size and is located asymmetrically around the intron insertion site. Screening of natural variants of the I-CreI recognition sequence indicates that the I-CreI endonuclease tolerates single and even multiple base changes within its recognition sequence.

We have isolated and characterized a nuclear mutant, 222E, in Chlamydomonas reinhardtii, which is defective in photosystem II (PSII). Polypeptide P5, the product of psbB, is not produced in this mutant, leading to a destabilization of other PSII components. The mutant specifically fails to accumulate psbB transcripts and displays an altered transcription pattern downstream of psbB. Pulse-labelling experiments suggest that mRNA stability and/or processing are affected by the alteration of a nuclear gene product in this mutant. We show that the C. reinhardtii psbB gene is co-transcribed with a small open reading frame that is highly conserved in location and amino acid sequence in land plants. The 5' and 3' termini of the psbB transcript have been mapped to 35 bases upstream of the initiation codon and approximately 600 bases downstream of the stop codon. The 3' flanking region contains two potential stem-loops, of which the larger (with an estimated free energy of -46 kcal) is near the 3' terminus of the transcript.

The sequences of the nuclear genes of the 33 kDa (OEE1) and the 16 kDa (OEE3) polypeptides of the oxygen evolving complex of Chlamydomonas reinhardtii have been established. Comparison between the OEE1 protein sequences of C. reinhardtii and higher plants and cyanobacteria reveals 67 and 47% homology. In contrast, C. reinhardtii and higher plants have only 28% overall homology for OEE3 which is mostly limited to the central portion of the protein. The transit peptides of the C. reinhardtii proteins consist of 52 (OEE1) and, most likely, 51 (OEE1) amino acids. They have a basic amino terminal region and, at least in the case of OEE1, a hydrophobic segment at their carboxy terminal end typical of thylakoid lumen proteins. Comparison of the genomic and cDNA clones indicates that the OEE1 and OEE3 genes contain five and four introns, respectively, some of which are located within the coding sequences of the transit peptides.

cDNA clones encoding two Photosystem I subunits of Chlamydomonas reinhardtii with apparent molecular masses of 18 and 11 kDa (thylakoid polypeptides 21 and 30; P21 and P30 respectively) were isolated using oligonucleotides, the sequences of which were deduced from the N-terminal amino acid sequences of the proteins. The cDNAs were sequenced and used to probe Southern and Northern blots. The Southern blot analysis indicates that both proteins are encoded by single-copy genes. The mRNA sizes of the two components are 1400 and 740 nucleotides, respectively. Comparison between the open reading frames of the cDNAs and the N-terminal amino acid sequences of the proteins indicates that the molecular masses of the mature proteins are 17.9 (P21) and 8.1 kDa (P30). Analysis of the deduced protein sequences predicts that both subunits are extrinsic membrane proteins with net positive charges. The amino acid sequences of the transit peptides suggest that P21 and P30 are routed towards the lumenal and stromal sides of the thylakoid membranes, respectively.

The introduction of exogenous DNA into the nuclear genome of Chlamydomonas reinhardtii occurs predominantly via non-homologous (illegitimate) recombination and results in integration at apparently-random loci. Using truncated and modified versions of the C. reinhardtii ARG7 gene in a series of transformation experiments, we demonstrate that homologous recombination between introduced DNA molecules occurs readily in C. reinhardtii, requires a region of homology of no more than 230 bp, and gives rise to intact copies of ARG7 in the nuclear genome. Evidence is presented for homologous recombination between introduced ARG7 DNA and the resident copy of the gene, and for the de-novo synthesis of the ARG7 sequence during transformation.

In the secondary structure model that has been proposed for the trans-spliced intron 1 in the Chlamydomonas reinhardtii psaA gene, a third RNA species (tscA RNA) interacts with the 5' and 3' intron parts flanking the exons to reconstitute a composite structure with several features of group-II introns. To test the validity of this model, we undertook the sequencing and modelling of equivalent introns in the psaA gene from other unicellular green algae belonging to the highly diversified genus Chlamydomonas. Our comparative analysis supports the model reported for the C. reinhardtii psaA intron 1, and also indicates that the 5' end of the tscA RNA and the region downstream from the psaA exon 1 cannot be folded into a structure typical of domain I as described for most group-II introns. It is possible that a fourth RNA species, yet to be discovered, provides the parts of domain I which are apparently missing.

The Chlamydomonas reinhardtii gene ARG7 encoding the enzyme argininosuccinate lyase is a popular marker for the nuclear transformation of this alga. We present the complete nucleotide sequence of ARG7. The gene spans 7 kilobases and is split by 12 introns containing highly repetitive microsatellite elements. Molecular analysis of eight different arg7 mutants indicates that none of the mutations is the result of gross rearrangements at the ARG7 locus. We describe our analysis of factors influencing transformation efficiency and the construction of a cosmid library of the Chlamydomonas genome in a customised vector carrying the ARG7 marker. This library should greatly facilitate the cloning of Chlamydomonas genes by complementation.

Four cDNAs (cDNAs 1–4),162,338,321 and 167 by in size, that contain repetitive DNA sequences, were isolated from C. reinhardtii. cDNAs 1, 2 and 3 hybridized to multiple transcripts in poly A+ RNA. Each of the four repeat families is comprised of an extremely heterogeneous population of interspersed nuclear DNA sequences most of which are less than 0.5 kbp in size. A large number of restriction fragment length polymorphisms were uncovered by using cDNAs 1 and 2 as hybridization probes. cDNA2 was compared to two different genomic DNA sequences: the first sequence was complementary to a central 136 bases of cDNA2, which is bordered by a 15-bp imperfect direct repeat; the second sequence lacks a poly-dA tail, but is otherwise colinear along its entire length with cDNA2. This suggests that some members of the cDNA2 repeat family contain signals for polyadenylation. The majority of accumulated transcripts that hybridize to cDNA2 have the same 5'–3' orientation as cDNA2.

A 5.3 kb chloroplast restriction fragment of Chlamydomonas reinhardii containing an origin of DNA replication and a sequence capable of promoting autonomous replication in C. reinhardii (ARC sequence) also carries an ARS sequence (autonomous replication in yeast). The ARC and ARS elements have been physically mapped and shown to be distinct from the origin of DNA replication. Similarly, restriction fragments containing the origin of chloroplast DNA replication from Euglena gracilis are unable to promote autonomous replication in yeast.

The ribulosebisphosphate carboxylase/oxygenase-defective Chlamydomonas mutant, 10-6C, was the first mutant to be physically defined in chloroplast DNA. In this report, a photosynthesis-competent revertant of the 10-6C mutant has been found to result from true reversion within the chloroplast large-subunit gene. This result supports the original assignment of the 10-6C mutation within the large-subunit gene.

Chloroplast genomes contain a single ClpP1 gene encoding one of the catalytic subunits of the evolutionarily conserved ATP-dependent Clp protease. Efforts to inactivate this protease in the chloroplast through targeted disruption of the clpP1gene have failed, suggesting that it is essential for cell survival in plants. To circumvent this problem, a repressible chloroplast gene expression system was developed in the green unicellular alga Chlamydomonas reinhardtii. This system takes advantage of the nuclear Nac2gene fused to the MetEpromoter and Thi4riboswitch, which can be repressed by adding vitamin B12 and thiamine to the growth medium. Nac2encodes a chloroplast protein that interacts specifically with the 5′UTR of the psbDmRNA and is involved in processing/translation of this transcript. Loss of Nac2 leads to the specific degradation of psbDmRNA. Because the psbD5′UTR is necessary and sufficient for the Nac2-dependent stability of psbDmRNA, this dependence can be transferred to any chloroplast gene by linking its coding sequence to the psbD5 ‘UTR. In this way it was possible to repress the clpP1gene in a reversible way with vitamins.

A unique feature of the ATP-dependent ClpP protease of eukaryotic photosynthetic organisms is that its catalytic subunit ClpP1 is encoded by the chloroplast genome. Attempts to inactivate this subunit through chloroplast transformation have failed because it is essential for cell survival. To study the function of ClpP we have developed a repressible chloroplast gene expression system in Chlamydomonas reinhardtii. This system is based on the use of a chimeric nuclear gene in which the vitamin-repressible MetEpromoter and Thi4riboswitch have been fused to the coding sequence of Nac2. Upon entry into the chloroplast the Nac2 protein specifically interacts with the psbD5’UTR and is required for the proper processing/translation of the psbDmRNA. This property can be conveyed to any chloroplast mRNA by replacing its 5’UTR with that of psbD. In this study we have chosen clpP1as plastid target gene and examined the cellular events induced upon depletion of ClpP through transcriptomic, proteomic, biochemical and electron microscope analysis. Among the most striking features, a massive increase in protein abundance occurs for plastid chaperones, proteases and proteins involved in membrane assembly/disassembly strongly suggesting the existence of a chloroplast unfolded protein response.

Three small RNAs of the cytoplasmic 80S ribosomes of the green unicellular alga Chlamydomonas reinhardii have been sequenced. They include two species of ribosomal 5S RNA, a major and a minor one of 122 and 121 nucleotides respectively, which differ from each other by 17 bases, and also the ribosomal 5.8S RNA of 156 nucleotides. Novel structural features can be recognized in the 5S RNAs of C. reinhardii by a comparison with published 5S RNA sequences. In addition the secondary structure of these small RNA molecules has been examined using a newly developed method based on differential nuclease susceptibility.

TOC1, a transposable element from Chlamydomonas reinhardtii, is 5662 bases long. The 217 and 237 base long terminal repeat sequences of TOC1 are unusually arranged around the 4600 and 123 base unique regions: [217]-4600-[237][217]-123-[237] Although TOC1 contains long terminal repeats and most TOC1 elements are complete, features shared with virus-like retroposons, its unique 4600 base reglon is more similar to the structure of the Li family of non-virus retroposons: first, 11 3/4 tandemly repeated copies of a 76 base repeat are found 813 bases from the left end of TOC1, and second using the universal genetic code large open reading frames were not found in TOC1. The relationship between TOC1, virus-like retroposons and the Li family of non-virus retroposons is unclear and may be very distant since only poor similarity was found between the TOC1 encoded OAFs and retrovirus polypeptides. The length of the tandem array of 76 base repeat sequences was conserved in most TOC1 elements and solo 76 base repeat sequences were not found outside TOC1 elements in the C.reinhardtii genome. Nucleotide substitutions allow all copies of the 76 base repeat to be distinguished from one another.