Chlororespiration and the process of carotenoid biosynthesis.

The plastoquinone pool during dark adaptation is reduced by endogenous reductants and oxidized at the expense of molecular oxygen. We report here on the redox state of plastoquinone in darkness, using as an indicator the chlorophyll fluorescence kinetics of whole cells of a Chlamydomonas reinhardtii mutant strain lacking the cytochrome b(6)f complex. When algae were equilibrated with a mixture of air and argon at 1.45% air, plastoquinol oxidation was inhibited whereas mitochondrial respiration was not. Consequently, mitochondrial oxidases cannot be responsible for the oxygen consumption linked to plastoquinol oxidation. Plastoquinol oxidation in darkness turned out to be sensitive to n-propyl gallate (PG) and insensitive to salicylhydroxamic acid (SHAM), whereas mitochondrial respiration was sensitive to SHAM and PG. Thus, both PG treatment and partial anaerobiosis allow to draw a distinction between an inhibition of plastoquinol oxidation and an inhibition of mitochondrial respiration, indicating the presence of a plastoquinol:oxygen oxidoreductase. The possible identification of this oxidase with an oxidase involved in carotenoid biosynthesis is discussed in view of various experimental data.

Chloroplast suppressors that act on a mitochondrial mutation in Chlamydomonas reinhardtii.

We report genetic evidence supporting the existence of suppressor genes in the chloroplast that act on a mitochondrial mutation that impairs heterotrophic growth in the green alga Chlamydomonas reinhardtii. One of these suppressors also acts on a point mutation in the rbcL gene in the chloroplast. These results are consistent with previous data showing that mitochondrial protein synthesis depends on chloroplast protein synthesis in C. reinhardtii. The nature of the interaction between chloroplasts and mitochondria is discussed in light of the requirement for import of tRNAs by plant mitochondria.

A new electrochemical gradient generator in thylakoid membranes of green algae.

Using a new method of delayed luminescence digital imaging, mutants of Chlorella sorokiniana lacking the chloroplast CF0CF1 ATP synthase were isolated for the first time. Biochemical characterization of these strains indicates a lack of detectable synthesis and accumulation of the ATP synthase subunits alpha-CF1 and beta-CF1. Functional characterization indicates the presence of a permanent electrochemical gradient (DeltaMu) across the thylakoid membrane in the dark-adapted state, which is not suppressed under anaerobic conditions. Contrary to what is observed in the presence of the CF0CF1 ATP synthase, this gradient is essentially due to an electric field component DeltaPsi with no detectable DeltapH component, under both aerobic and anaerobic conditions. Neither the CF0CF1 ATP synthase nor a respiratory process can thus be responsible for a permanent gradient detected under these conditions. The previous proposal of a new ATP-dependent electrogenic pump in thylakoid membranes is supported by these results that, in addition, indicate a specificity of this new pump for ions other than protons.

Limited photosynthetic electron flow but no CO2 fixation in Chlamydomonas mutants lacking photosystem I.

By measuring O2 and CO2 exchange in mutants of the green alga Chlamydomonas reinhardtii in which genes encoding the reaction center of photosystem I (psaA or psaB) have been deleted, we found that a photosystem II-dependent electron flow using O2 as the final acceptor can be sustained in the light. However, in contrast with recent reports using other Chlamydomonas mutants (B4 and F8), we show here that CO2 fixation does not occur in the absence of photosystem I. By deleting the psaA gene in both B4 and F8 strains, we conclude that the ability of these mutants to fix CO2 in the light is due to the presence of residual amounts of photosystem I.

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. We constructed chimeric genes consisting of the psaA and psaB 5' untranslated region (5' UTR) fused to the aminoglycoside adenyltransferase (aadA) coding sequence, which confers spectinomycin resistance. Insertion of these genes into the chloroplast genome through biolistic transformation and analysis of their expression in the TAB1 mutant nuclear background revealed that the psaB (but not the psaA) 5' UTR is the target of the wild-type TAB1 function. This suggests that TAB1 is required for the initiation of psaB mRNA translation. The dependence of PSAA synthesis or accumulation on PSAB synthesis is strongly suggested by the identification of a suppressor mutation within the psaB 5' UTR. The suppressor specifically restores the synthesis of both proteins in the presence of the tab1-F15 mutation. The location of the suppressor mutation within a putative base-paired region near the psaB initiation codon suggests a role for TAB1 in the activation of translation of the psaB mRNA.

Altered expression of nuclear genes encoding chloroplast polypeptides in non-photosynthetic mutants of Chlamydomonas reinhardtii: evidence for post-transcriptional regulation.

In photoautotrophic organisms it is well documented that the expression of nuclear genes encoding plastid proteins can be regulated at various levels. We present here the analysis of a non-photosynthetic strain (CC1051) of the green unicellular alga Chlamydomonas reinhardtii; this strain carries a mutation in the newly identified Cen gene involved in the co-regulated expression of several different nuclear genes encoding plastid proteins. We performed a differential screening strategy to isolate cDNAs corresponding to genes that are differentially expressed in mutant and wild-type strains. Extensive hybridization experiments revealed that the 15 cDNA clones isolated represent five different mRNAs that fail to accumulate in the non-photosynthetic mutant. Comparative analysis of DNA sequencing data showed that they all code for plastid proteins. In particular, we identified genes for the chlorophyll a/b binding protein of the light-harvesting complex II (LHCII), for subunits II and III of photosystem I (PsaD, PsaF), for pentose-5-phosphate 3-epimerase (PPE), an enzyme of the Calvin cycle, and for an unidentified 7 kDa protein with a suggested lumenal location. With the exception of the gene for LHCII, all proteins are encoded by single-copy genes. Evidence from run-on transcription experiments is presented showing that expression of the above mentioned plastid proteins is affected at the post-transcriptional level in the mutant strain CC1051 with a defect in the Cen gene. Our results suggest that the product of the Cen gene is involved in stabilization and/or processing of transcripts from nuclear genes encoding chloroplast proteins.

Etiolated cells of Chlamydomonas reinhardtii: choice material for characterization of mitochondrial membrane polypeptides.

We have investigated a light-conditional mutant of Chlamydomonas reinhardtii (J12) that is unable to synthesize chlorophyll in the dark with the aim of characterizing the mitochondrial membrane polypeptides of this alga. A crude membrane fraction derived from etiolated cells was analyzed by gel electrophoresis, immunoblot analysis, and pulse-labeling in the presence of specific protein synthesis inhibitors. This fraction contained both mitochondrial and etioplast membranes, and the latter contained appreciable amounts of subunits of the cytochrome b6f complex. The mitochondria-encoded subunit 1 of cytochrome-c oxidase called COX1 was identified, and its synthesis was detected in this membrane fraction. The redox-difference spectra of mitochondrial cytochromes were studied in whole cells and membrane fractions, in both respiratory-competent and -deficient strains. Mitochondrial membranes could be further purified after sucrose gradient centrifugation. The use of etiolated cells and their membrane extracts, in association with appropriate methodologies, opens ways to study the molecular genetics of mitochondria in C. reinhardtii and allows us to address the question of the cooperation established between the three genetic compartments of a plant cell.

New tools for mitochondrial genetics of Chlamydomonas reinhardtii: manganese mutagenesis and cytoduction.

A novel and efficient genetic procedure is described for generating mitochondrial mutants of the green alga Chlamydomonas reinhardtii. The development of a mutagenesis procedure using manganese cations and the application of cytoduction techniques resulted in a combined approach for the generation and analysis of mitochondrial mutants. Although mitochondrial mutations are inherited in sexual crosses from the minus mating type parent, the cytoduction technique can be used to transfer mitochondrial mutations into recipient strains with different genetic backgrounds, irrespective of their mating type. Cytoduction allows the transfer of mitochondrial markers from diploid to haploid cells also, which is of great benefit since diploid cells do not germinate in C. reinhardtii. We report here the isolation and characterisation of eight mutants, which are resistant to the antibiotics myxothiazol and mucidin. The mutants all have point mutations in the mitochondrial gene for apocytochrome b. Using in vitro-amplified cytb gene fragments as probes for direct DNA sequencing, three different types of single base pair substitutions were revealed in all mutants tested. In particular, amino acid substitutions in the mutant apocytochrome b polypeptide have been identified at residues 129, 132 and 137, which have been implicated in forming part of an antibiotic-binding niche. The amino acid substitution at position 132 has not been so far described for mutant apocytochrome b in any other organism, prokaryotic or eukaryotic. The genetic approach presented here confirms C. reinhardtii as a model system that is unique among plant cells.

Mitochondrial genetics of Chlamydomonas reinhardtii: resistance mutations marking the cytochrome b gene.

We describe the genetic and molecular analysis of the first non-Mendelian mutants of Chlamydomonas reinhardtii resistant to myxothiazol, an inhibitor of the respiratory cytochrome bc1 complex. Using a set of seven oligonucleotide probes, restriction fragments containing the mitochondrial cytochrome b (cyt b) gene from C. reinhardtii were isolated from a mitochondrial DNA library. This gene is located adjacent to the gene for subunit 4 of the mitochondrial NADH-dehydrogenase (ND4), near one end of the 15.8-kb linear mitochondrial genome of C. reinhardtii. The algal cytochrome b apoprotein contains 381 amino-acid residues and exhibits a sequence similarity of about 59% with other plant cytochrome b proteins. The cyt b gene from four myxothiazol resistant mutants of C. reinhardtii was amplified for DNA sequence analysis. In comparison to the wild-type strain, all mutants contain an identical point mutation in the cyt b gene, leading to a change of a phenylalanine codon to a leucine codon at amino acid position 129 of the cytochrome b protein. Segregation analysis in tetrads from reciprocal crosses of mutants with wild type shows a strict uniparental inheritance of this mutation from the mating type minus parent (UP-). However, mitochondrial markers from both parents are recovered in vegetative diploids in variable proportions from one experiment to the next for a given cross. On the average, a strong bias is seen for markers inherited from the mating type minus parent.

Translational accuracy and sexual differentiation in Chlamydomonas reinhardtii.

A renewal of ribosomes has been previously reported to occur during gametogenesis in C. reinhardtii. In order to further characterize these new ribosomes, we performed pulse-labelling experiments on whole cells of C. reinhardtii, during gametogenesis and in the presence of various aminoglycosides known to alter translational accuracy: Hygromycin and Paromomycin are assumed to increase the rate of translational errors at the level of 80S and 70S ribosomes whereas Kasugamycin is assumed to induce the opposite effect. Three lines of evidence support an increased inaccuracy in protein translation during gametogenesis: (1) gamete cells displayed a higher sensitivity than vegetative cells to Hygromycin and Paromomycin; 4 micrograms/ml Hygromycin cancelled cytoplasmic protein synthesis in gametes but not in vegetative cells; Paromomycin induced the synthesis of new polypeptides of high molecular weight and of nuclear origin in gametes but not in vegetative cells. In addition, chloroplast protein synthesis was more sensitive to Hygromycin and Paromomycin in gametes than in vegetative cells. (2) Kasugamycin-sensitive alterations of thylakoid membranes were detected during gametogenesis. (3) 35S-misincorporation in the OEE3 polypeptide, of nuclear origin and normally devoid of sulphur containing amino acids, was more than three times higher in gametes than in vegetative cells. This increase was prevented by Kasugamycin, suggesting that 80S translation in gametes was more inaccurate than in vegetative cells. The possible significance of these changes occurring during gametogenic differentiation is discussed in light of the importance of a modulation of translational accuracy at particular stages of the life cycle in other lower eukaryotes.

Nuclear and chloroplast mutations affect the synthesis or stability of the chloroplast psbC gene product in Chlamydomonas reinhardtii.

The psbC gene of Chlamydomonas reinhardtii encodes P6, the 43 kd photosystem II core polypeptide. The sequence of P6 is highly homologous to the corresponding protein in higher plants with the exception of the N-terminal region where the first 12 amino acids are missing. Translation of P6 is initiated at GUG in C. reinhardtii. The chloroplast mutant MA16 produces a highly unstable P6 protein. The mutation in this strain maps near the middle of the psbC gene and consists of a 6 bp duplication that creates a Ser-Leu repeat at the end of one transmembrane domain. Two nuclear mutants, F34 and F64, and one chloroplast mutant, FuD34, are unable to synthesize P6. All of these mutants accumulate wild-type levels of psbC mRNA. The FuD34 mutation has been localized near the middle of the 550 bp 5' untranslated region of psbC where the RNA can be folded into a stem-loop structure. A chloroplast suppressor of F34 has been isolated that partially restores synthesis of the 43 kd protein. The mutation of this suppressor is near that of FuD34, in the same stem-loop region. These chloroplast mutations appear to define the target site of a nuclear factor that is involved in P6 translation.

Mutant phenotypes support a trans-splicing mechanism for the expression of the tripartite psaA gene in the C. reinhardtii chloroplast.

The chloroplast psaA gene of the green unicellular alga Chlamydomonas reinhardtii consists of three exons that are transcribed from different strands. Analysis of numerous nuclear and chloroplast mutants that are deficient in photosystem I activity reveals that roughly one-quarter of them are specifically affected in psaA mRNA maturation. These mutants can be grouped into three phenotypic classes, based on their inability to perform either one or both splicing reactions. The data indicate that the three exons are transcribed independently as precursors which are normally assembled in trans and that the splicing reactions can occur in either order. While some chloroplast mutations could act in cis, the nuclear mutations that fall into several complementation groups probably affect factors specifically required for assembling psaA mRNA.

Restoration of phototrophic growth in a mutant of Chlamydomonas reinhardtii in which the chloroplast atpB gene of the ATP synthase has a deletion: an example of mitochondria-dependent photosynthesis.

The Chlamydomonas reinhardtii mutant FUD50 has a deletion in the atpB gene of the chloroplast ATP synthase [Woessner, J. P., Masson, A., Harris, E. H., Bennoun, P., Gillham, N. W., and Boynton, J. E. (1984) Plant Mol. Biol. 3, 177-190]. We have isolated a suppressed strain (FUD50su) that can grow under phototrophic conditions, although it still showed no synthesis of the beta subunit of coupling factor 1. Thylakoid membranes of the FUD50su strain were similar to those of the original FUD50 strain, in that they both lacked all the subunits making up the chloroplast ATP synthase complex. We show that photosynthesis in FUD50su is sensitive to inhibitors such as antimycin, specific for mitochondrial electron transport. This observation indicates that photosynthesis in the FUD50su strain is achieved through an unusual interaction between mitochondria and chloroplast. Exportation of light-induced reduced compounds from the chloroplast to the mitochondria elicits ATP formation in the latter, and ATP is subsequently imported to the chloroplast. The activation of such an ATP shuttle coupled to an NADPH shuttle would thus provide the reducing power and the free energy needed for carbon assimilation in a chloroplast that lacks chloroplast ATP synthase.

Structural and transcription analysis of two homologous genes for the P700 chlorophyll a-apoproteins in Chlamydomonas reinhardii: evidence for in vivo trans-splicing.

The two homologous genes for the P700 chlorophyll a-apoproteins (ps1A1 and ps1A2) are encoded by the plastom in the green alga Chlamydomonas reinhardii. The structure and organization of the two genes were determined by comparison with the homologous genes from maize using data from heterologous hybridizations as well as from DNA and RNA sequencing. While the ps1A2 (736 codons) gene shows a continuous gene organization, the ps1A1 (754 codons) gene possesses some unusual features. The discontinuous gene is split into three separate exons which are scattered around the circular chloroplast genome. Exon 1 (86 bp) is separated by approximately 50 kb from exon 2 (198 bp), which is located approximately 90 kb apart from exon 3 (1984 bp). All exons are flanked by intronic sequences of group II. Transcription analysis reveals that the ps1A2 gene hybridizes with a 2.8-kb transcript, while all exon regions of the ps1A1 gene are homologous to a mature mRNA of 2.7 kb. From our data we conclude that the three distantly separated exonic sequences of the ps1A1 gene constitute a functional gene which probably operates by a trans-splicing mechanism.

Expression of the nuclear encoded OEE1 protein is required for oxygen evolution and stability of photosystem II particles in Chlamydomonas reinhardtii.

In Chlamydomonas reinhardtii the oxygen evolving enhancer protein 1 (OEE1), which is part of the oxygen evolving complex of photosystem II (PS II), is coded for by a single nuclear gene (psb1). The nuclear mutant FuD44 specifically lacks the OEE1 polypeptide and is completely deficient in photosynthetic oxygen evolution. In this mutant a 5 kb DNA insertion into the 5' region of the psb1 gene results in the complete absence of OEE1 mRNA and protein. A revertant, FuD44-R 2, which is capable of 30% of the photosynthetic oxygen evolution of wild-type cells, has lost 4 kb of the 5 kb DNA insert, and accumulates both OEE1 mRNA and protein, although at levels somewhat less than those of wild-type cells. Absence of the OEE1 protein in the FuD44 mutant does not affect the accumulation of other nuclear encoded PS II peripheral polypeptides. OEE1 absence does, however, result in a more rapid turnover of the chloroplast encoded PS II core polypeptides, thus resulting in a substantial deficiency of PS II core polypeptides in FuD44 cells. These PS II core proteins again accumulate in revertant FuD44-R2 cells.

Lack of the D2 protein in a Chlamydomonas reinhardtii psbD mutant affects photosystem II stability and D1 expression.

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 reinhardtii and 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.

Characterization of photosystem II mutants of Chlamydomonas reinhardii lacking the psbA gene.

We have examined 78 chloroplast mutants of Chlamydomonas reinhardii lacking photosystem II activity. Most of them are unable to synthesize the 32 Kdalton protein. Analysis of 22 of these mutants reveals that they have deleted both copies of the psbA gene (which codes for the 32 Kdalton protein) in their chloroplast genome. Although these mutants are able to synthesize and to integrate the other photosystem II polypeptides in the thylakoid membranes, they are unable to assemble a stable functional photosystem II complex. The 32 Kprotein appears therefore to play an important role not only in photosystem II function, but also in stabilizing this complex.

Change in cytoplasmic ribosome properties during gametogenesis in the alga Chlamydomonas reinhardtii.

Cytoplasmic (80S) ribosomes isolated from gametes and vegetative cells of the unicellular alga Chlamydomonas reinhardtii have been compared. Ribosomes of these two cell types differ in their susceptibility towards aminoglycoside antibiotics while their response to other antibiotics are similar. Electrophoregrams of ribosomal proteins show clear-cut differences between the two kinds of ribosomes.

Herbicide resistance in Chlamydomonas reinhardtii results from a mutation in the chloroplast gene for the 32-kilodalton protein of photosystem II.

We report the isolation and characterization of a uniparental mutant of Chlamydomonas reinhardtii that 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[(14)C]atrazine at 0.1 muM. 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. reinhardtii mutant.