Spontaneous dominant mutations in chlamydomonas highlight ongoing evolution by gene diversification.

We characterized two spontaneous and dominant nuclear mutations in the unicellular alga Chlamydomonas reinhardtii, ncc1 and ncc2 (for nuclear control of chloroplast gene expression), which affect two octotricopeptide repeat (OPR) proteins encoded in a cluster of paralogous genes on chromosome 15. Both mutations cause a single amino acid substitution in one OPR repeat. As a result, the mutated NCC1 and NCC2 proteins now recognize new targets that we identified in the coding sequences of the chloroplast atpA and petA genes, respectively.

Thylakoid FtsH protease contributes to photosystem II and cytochrome b6f remodeling in Chlamydomonas reinhardtii under stress conditions.

FtsH is the major thylakoid membrane protease found in organisms performing oxygenic photosynthesis. Here, we show that FtsH from Chlamydomonas reinhardtii forms heterooligomers comprising two subunits, FtsH1 and FtsH2. We characterized this protease using FtsH mutants that we identified through a genetic suppressor approach that restored phototrophic growth of mutants originally defective for cytochrome b6f accumulation.

A dual strategy to cope with high light in Chlamydomonas reinhardtii.

Absorption of light in excess of the capacity for photosynthetic electron transport is damaging to photosynthetic organisms. Several mechanisms exist to avoid photodamage, which are collectively referred to as nonphotochemical quenching. This term comprises at least two major processes.

Dual functions of the nucleus-encoded factor TDA1 in trapping and translation activation of atpA transcripts in Chlamydomonas reinhardtii chloroplasts.

After endosymbiosis, organelles lost most of their initial genome. Moreover, expression of the few remaining genes became tightly controlled by the nucleus through trans-acting protein factors that are required for post-transcriptional expression (maturation/stability or translation) of a single (or a few) specific organelle target mRNA(s). Here, we characterize the nucleus-encoded TDA1 factor, which is specifically required for translation of the chloroplast atpA transcript that encodes subunit α of ATP synthase in Chlamydomonas reinhardtii.

Impaired respiration discloses the physiological significance of state transitions in Chlamydomonas.

State transitions correspond to a major regulation process for photosynthesis, whereby chlorophyll protein complexes responsible for light harvesting migrate between photosystem II and photosystem I in response to changes in the redox poise of the intersystem electron carriers. Here we disclose their physiological significance in Chlamydomonas reinhardtii using a genetic approach. Using single and double mutants defective for state transitions and/or mitochondrial respiration, we show that photosynthetic growth, and therefore biomass production, critically depends on state transitions in respiratory-defective conditions.

Molecular identification and function of cis- and trans-acting determinants for petA transcript stability in Chlamydomonas reinhardtii chloroplasts.

In organelles, the posttranscriptional steps of gene expression are tightly controlled by nucleus-encoded factors, most often acting in a gene-specific manner. Despite the molecular identification of a growing number of factors, their mode of action remains largely unknown. In the green alga Chlamydomonas reinhardtii, expression of the chloroplast petA gene, which codes for cytochrome f, depends on two specific nucleus-encoded factors.

Evidence for regulatory function of nucleus-encoded factors on mRNA stabilization and translation in the chloroplast.

A salient feature of organelle gene expression is the requirement for nucleus-encoded factors that act posttranscriptionally in a gene-specific manner. A central issue is to understand whether these factors are merely constitutive or have a regulatory function. In the unicellular alga Chlamydomonas reinhardtii, expression of the chloroplast petA gene-encoding cytochrome f, a major subunit of the cytochrome b(6)f complex, depends on two specific nucleus-encoded factors: MCA1, required for stable accumulation of the petA transcript, and TCA1, required for its translation.

Relationship between mRNA levels and protein accumulation in a chloroplast promoter-mutant of Chlamydomonas reinhardtii.

The photosynthetic chloroplast mutant G64 of Chlamydomonas reinhardtii was shown to contain a single point mutation within the 5' region of the psbD gene encoding the D2 protein of the photosystem II reaction center. The mutation affects the sequence element TATAATAT which has previously been hypothesized to function as the psbD promoter. Run-on analysis confirmed that transcription of psbD in the mutant was reduced to approximately 10% of the wild-type level.

Biogenesis of PSI involves a cascade of translational autoregulation in the chloroplast of Chlamydomonas.

Photosystem I comprises 13 subunits in Chlamydomonas reinhardtii, four of which-the major reaction center I subunits PsaA and PsaB, PsaC and PsaJ-are chloroplast genome-encoded. We demonstrate that PSI biogenesis involves an assembly-governed regulation of synthesis of the major chloroplast-encoded subunits where the presence of PsaB is required to observe significant rates of PsaA synthesis and the presence of PsaA is required to observe significant rates of PsaC synthesis. Using chimeric genes expressed in the chloroplast, we show that these regulatory processes correspond to autoregulation of translation for PsaA and PsaC.

A single mutation that causes phosphatidylglycerol deficiency impairs synthesis of photosystem II cores in Chlamydomonas reinhardtii.

Two mutants of Chlamydomonas reinhardtii, mf1 and mf2, characterized by a marked reduction in their phosphatidylglycerol content together with a complete loss in its Delta3-trans hexadecenoic acid-containing form, also lost photosystem II (PSII) activity. Genetic analysis of crosses between mf2 and wild-type strains shows a strict cosegregation of the PSII and lipid deficiencies, while phenotypic analysis of phototrophic revertant strains suggests that one single nuclear mutation is responsible for the pleiotropic phenotype of the mutants. The nearly complete absence of PSII core is due to a severely decreased synthesis of two subunits, D1 and apoCP47, which is not due to a decrease in translation initiation.

Tab2 is a novel conserved RNA binding protein required for translation of the chloroplast psaB mRNA.

The chloroplast psaB mRNA encodes one of the reaction centre polypeptides of photosystem I. Protein pulse-labelling profiles indicate that the mutant strain of Chlamydomonas reinhardtii, F14, affected at the nuclear locus TAB2, is deficient in the translation of psaB mRNA and thus deficient in photosystem I activity. Genetic studies reveal that the target site for Tab2 is situated within the psaB 5'UTR.

A dominant nuclear mutation in Chlamydomonas identifies a factor controlling chloroplast mRNA stability by acting on the coding region of the atpA transcript.

We have characterized a nuclear mutation, mda1-ncc1, that affects mRNA stability for the atpA gene cluster in the chloroplast of Chlamydomonas. Unlike all nuclear mutations altering chloroplast gene expression described to date, mda1-ncc1 is a dominant mutation that still allows accumulation of detectable amounts of atpA mRNAs. At variance with the subset of these mutations that affect mRNA stability through the 5' UTR of a single chloroplast transcript, the mutated version of MDA1 acts on the coding region of the atpA message.

Characterization of Tbc2, a nucleus-encoded factor specifically required for translation of the chloroplast psbC mRNA in Chlamydomonas reinhardtii.

Genetic analysis has revealed that the three nucleus-encoded factors Tbc1, Tbc2, and Tbc3 are involved in the translation of the chloroplast psbC mRNA of the eukaryotic green alga Chlamydomonas reinhardtii. In this study we report the isolation and phenotypic characterization of two new tbc2 mutant alleles and their use for cloning and characterizing the Tbc2 gene by genomic complementation. TBC2 encodes a protein of 1,115 residues containing nine copies of a novel degenerate 38-40 amino acid repeat with a quasiconserved PPPEW motif near its COOH-terminal end.

TCA1, a single nuclear-encoded translational activator specific for petA mRNA in Chlamydomonas reinhardtii chloroplast.

We isolated seven allelic nuclear mutants of Chlamydomonas reinhardtii specifically blocked in the translation of cytochrome f, a major chloroplast-encoded subunit of the photosynthetic electron transport chain encoded by the petA gene. We recovered one chloroplast suppressor in which the coding region of petA was now expressed under the control of a duplicated 5' untranslated region from another open reading frame of presently unknown function. Since we also recovered 14 nuclear intragenic suppressors, we ended up with 21 alleles of a single nuclear gene we called TCA1 for translation of cytochrome b(6)f complex petA mRNA.

Assembly-controlled regulation of chloroplast gene translation.

Studies of the biogenesis of the photosynthetic protein complexes in the unicellular green alga Chlamydomonas reinhardtii have pointed to the importance of the concerted expression of nuclear and chloroplast genomes. The accumulation of chloroplast- and nuclear-encoded subunits is concerted, most often as a result of the rapid proteolytic disposal of unassembled subunits, but the rate of synthesis of some chloroplast-encoded subunits from photosynthetic protein complexes, designed as CES proteins (Controlled by Epistasy of Synthesis), is regulated by the availability of their assembly partners from the same complex. Cytochrome f, a major subunit of the cytochrome b(6)f complex is a model protein for the study of the CES process.

A nucleus-encoded suppressor defines a new factor which can promote petD mRNA stability in the chloroplast of Chlamydomonas reinhardtii.

Mutations in the Chlamydomonas reinhardtii nuclear gene MCD1 specifically destabilize the chloroplast petD mRNA, which encodes subunit IV of the cytochrome b6/f complex. The MCD1 gene product is thought to interact with the mRNA 5' end to protect it from degradation by a 5' --> 3' exoribonuclease and may also have a role in translation initiation. Here we report the isolation and characterization of a semidominant, allele-specific, nucleus-encoded suppressor of the mcd1-2 mutation.

In vivo evidence for 5'-->3' exoribonuclease degradation of an unstable chloroplast mRNA.

The acetate-requiring Chlamydomonas reinhardtii nuclear mutant F16 harbors the mutation mcd1-1 and fails to accumulate the cytochrome b6/f complex. The primary defect of mcd1-1 was determined to be the instability of petD mRNA, which encodes subunit IV of the complex. Chimeric reporter genes introduced by chloroplast transformation demonstrated that the determinant of petD mRNA instability in the mcd1-1 background is located in the 5' untranslated region (UTR).

Translation of cytochrome f is autoregulated through the 5' untranslated region of petA mRNA in Chlamydomonas chloroplasts.

A process that we refer to as control by epistasy of synthesis (CES process) occurs during chloroplast protein biogenesis in Chlamydomonas reinhardtii: the synthesis of some chloroplast-encoded subunits, the CES subunits, is strongly attenuated when some other subunits from the same complex, the dominant subunits, are missing. Herein we investigate the molecular basis of the CES process for the biogenesis of the cytochrome b6f complex and show that negative autoregulation of cytochrome f translation occurs in the absence of other complex subunits. This autoregulation is mediated by an interaction, either direct or indirect, between the 5' untranslated region of petA mRNA, which encodes cytochrome f, and the C-terminal domain of the unassembled protein.

Genetic analysis of chloroplast c-type cytochrome assembly in Chlamydomonas reinhardtii: One chloroplast locus and at least four nuclear loci are required for heme attachment.

Chloroplasts contain up to two c-type cytochromes, membrane-anchored cytochrome f and soluble cytochrome c6. To elucidate the post-translational events required for their assembly, acetate-requiring mutants of Chlamydomonas reinhardtii that have combined deficiencies in both plastid-encoded cytochrome f and nucleus-encoded cytochrome c6 have been identified and analyzed. For strains ct34 and ct59, where the phenotype displays uniparental inheritance, the mutations were localized to the chloroplast ccsA gene, which was shown previously to be required for heme attachment to chloroplast apocytochromes.

Molecular genetic identification of a pathway for heme binding to cytochrome b6.

Heme binding to cytochrome b6 is resistant, in part, to denaturing conditions that typically destroy the noncovalent interactions between the b hemes and their apoproteins, suggesting that one of two b hemes of holocytochrome b6 is tightly bound to the polypeptide. We exploited this property to define a pathway for the conversion of apo- to holocytochrome b6, and to identify mutants that are blocked at one step of this pathway. Chlamydomonas reinhardtii strains carrying substitutions in either one of the four histidines that coordinate the bh or bl hemes to the apoprotein were created.

Translation of the chloroplast psbC mRNA is controlled by interactions between its 5' leader and the nuclear loci TBC1 and TBC3 in Chlamydomonas reinhardtii.

Translation of the chloroplast psbC mRNA 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 TBC1 and TBC2. We show that a 97-nucleotide (nt) region located in the middle of the psbC 5' 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.

A nuclear-encoded function essential for translation of the chloroplast psaB mRNA in chlamydomonas.

We report the analysis of a photosystem I-deficient mutant of Chlamydomonas reinhardtii, F15, that contains a mutation at the TAB1 (for translation of psaB mRNA) nuclear locus. Pulse labeling of chloroplast proteins revealed that the synthesis of the two photosystem I reaction center polypeptides PSAA and PSAB was undetectable in this mutant. The mRNA levels of these proteins were only moderately reduced, suggesting that the primary defect occurs at a step during or after translation.

Molecular genetic analysis of plastocyanin biosynthesis in Chlamydomonas reinhardtii.

Five plastocyanin-deficient mutants were identified from a population of UV-mutagenized Chlamydomonas reinhardtii cells. Genetic complementation experiments indicated that four mutants represented alleles at the PCY1 locus (pcy1-2, pcy1-3, pcy1-4, and pcy1-5). Sequence analysis confirmed that two strains, pcy1-2 and pcy1-3, carry a frameshift (-1) and a nonsense mutation, respectively, while strains pcy1-4 and pcy1-5 synthesize an extended protein as a result of read-through mutations at the stop codon.

Nuclear mutants of Chlamydomonas reinhardtii defective in the biogenesis of the cytochrome b6f complex.

The random integration of transforming DNA into the nuclear genome of Chlamydomonas has been employed as an insertional mutagen to generate a collection of photosynthetic mutants that display abnormal steady-state fluorescence levels and an acetate-requiring phenotype. Electron paramagnetic resonance spectroscopy was then used to identify those mutants that specifically lack a functional cytochrome b6f complex. Our analysis of RNA and protein synthesis in five of these mutants reveals four separate phenotypes.

Chloroplasts can accommodate inclusion bodies. Evidence from a mutant of Chlamydomonas reinhardtii defective in the assembly of the chloroplast ATP synthase.

We identified two neighboring missense mutations in the chloroplast atpA gene which are responsible for the defect of ATP synthase assembly in the FUD16 mutant from Chlamydomonas reinhardtii. The two corresponding amino acid substitutions, Ile184-->Asn and Asn186-->Tyr, occurred at strictly conserved sites among the alpha and beta subunits of (C)F1 complexes from bacteria, mitochondria, and chloroplasts. The altered region in the alpha polypeptide chain is located 7 amino acids downstream of the P-loop, which forms most of the conserved nucleotide binding site.