Jacqueline Girard-Bascou

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.

A small chloroplast RNA may be required for trans-splicing in chlamydomonas reinhardtii

In C. reinhardtii, the mature psaA mRNA is assembled by a process involving trans-splicing of three separate transcripts encoded at three widely scattered loci of the chloroplast genome. At least one additional chloroplast locus (tscA) is required for trans-splicing of exons 1 and 2. We have mapped this gene by transformation of a deletion mutant with a particle gun. The 0.7 kb region of the chloroplast genome that is sufficient to rescue tscA function has been subjected to insertion mutagenesis, showing that it does not contain significant open reading frames. We suggest from these experiments that the product of the tscA gene may be a small chloroplast RNA that acts in trans in the first trans-splicing reaction of psaA. A model for the mode of action of this RNA is presented, in which the characteristic structure of group II introns is assembled from three separate transcripts.

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.

Trans-splicing mutants of Chlamydomonas reinhardtii

SummaryIn 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.

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.

A dual strategy to cope with high light in Chlamydomonas reinhardtii

To protect photosynthetic organisms from photodamage, excess energy in photosystem II must be dissipated. In C. reinhardtii, two alternative mechanisms (energy thermal dissipation [qE] and kinase-mediated migration of light harvesting proteins [qT]) synergistically modulate photoprotection via the reversible migration of LHCSR3, a key qE effector in this alga, between photosystems II and I during qT. 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. State transitions (qT) represent changes in the relative antenna sizes of photosystems II and I. High energy quenching (qE) is the increased thermal dissipation of light energy triggered by lumen acidification. To investigate the respective roles of qE and qT in photoprotection, a mutant (npq4 stt7-9) was generated in Chlamydomonas reinhardtii by crossing the state transition–deficient mutant (stt7-9) with a strain having a largely reduced qE capacity (npq4). The comparative phenotypic analysis of the wild type, single mutants, and double mutants reveals that both state transitions and qE are induced by high light. Moreover, the double mutant exhibits an increased photosensitivity with respect to the single mutants and the wild type. Therefore, we suggest that besides qE, state transitions also play a photoprotective role during high light acclimation of the cells, most likely by decreasing hydrogen peroxide production. These results are discussed in terms of the relative photoprotective benefit related to thermal dissipation of excess light and/or to the physical displacement of antennas from photosystem II.

Evidence for Nuclear Control of the Expression of the atpA and atpB Chloroplast Genes in Chlamydomonas.

We analyzed three nuclear mutants of Chlamydomonas reinhardtii altered in the expression of the chloroplast genes atpA or atpB coding for the [alpha] or [beta] subunit of the chloroplast ATP synthase. These mutants revealed the existence of three nuclear products controlling the expression of the two chloroplast genes: the first one acts on the translation of the atpA transcript, and the two others act specifically on the stability of either the atpB or the atpA mRNAs. The nuclear mutation responsible for the decreased stability of the atpB mRNA prevented translation of the corresponding polypeptide. In contrast, the mutation responsible for the decreased stability of the atpA mRNA had limited effect on the translation of the [alpha] subunit, thereby allowing its accumulation and assembly in an active ATP synthase. Although acting originally on the expression of only one of the two main coupling factor 1 subunits, the three mutations caused a change in the translation rate of the other subunit, as viewed in 5-min pulse labeling experiments. This is indicative of a concerted expression of the [alpha] and [beta] subunits at an early post-translational step, or during translation, that may be critical for the assembly of the chloroplast ATP synthase.

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

SummaryWe 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.

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. The downregulation of translation occurs at some early stage since it arises from the interaction between unassembled PsaA and PsaC polypeptides and 5′ untranslated regions of psaA and psaC mRNAs, respectively. These assembly‐dependent autoregulations of translation represent two new instances of a control by epistasy of synthesis process that turns out to be a general feature of protein expression in the chloroplast of C. reinhardtii.

Molecular Genetic Identification of a Pathway for Heme Binding to Cytochrome b 6

Heme binding to cytochromeb 6 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 b 6 is tightly bound to the polypeptide. We exploited this property to define a pathway for the conversion of apo- to holocytochrome b 6, 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. These mutations resulted in the appearance of distinct immunoreactive species of cytochrome b 6, which allowed us to specifically identify cytochrome b 6 with altered bh or bl ligation. In gabaculine-treated (i.e. heme-depleted) wild type and site-directed mutant strains, we established that (i) the single immunoreactive band, observed in strains carrying the bl site-directed mutations, corresponds to apocytochrome b 6 and (ii) the additional band present in strains carrying bhsite-directed mutations corresponds to a bl-heme-dependent intermediate in the formation of holocytochrome b 6. Five nuclear mutants (ccb strains) that are defective in holocytochromeb 6 formation display a phenotype that is indistinguishable from that of strains carrying site-directed bh ligand mutants. The defect is specific for cytochromeb 6 assembly, because the ccbstrains can synthesize other b cytochromes and allc-type cytochromes. The ccb strains, which define four nuclear loci (CCB1, CCB2,CCB3, and CCB4), provide the first evidence that a b-type cytochrome requires trans-acting factors for its heme association.