Michel Goldschmidt-Clermont

The molecular biology of chloroplasts and mitochondria in Chlamydomonas

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Participation of nuclear genes in chloroplast gene expression.

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Sequence, evolution and differential expression of the two genes encoding variant small subunits of ribulose bisphosphate carboxylase/oxygenase in Chlamydomonas reinhardtii

The expression of the plastid genome is dependent on a large number of nucleus-encoded factors. Some of these factors have been identified through biochemical assays, and many others by genetic screens in Arabidopsis, Chlamydomonas and maize. Nucleus-encoded factors function in each step in plastid gene expression, including transcription, RNA editing, RNA splicing, RNA processing, RNA degradation, and translation. Many of the factors discovered via biochemical approaches play general roles as components of the basic gene expression machinery, whereas the majority of those identified by genetic approaches are specifically required for the expression of small subsets of chloroplast genes and are involved in post-transcriptional steps. Some of the nucleus-encoded factors may play regulatory roles and modulate chloroplast gene expression in response to developmental or environmental cues. They may also serve to couple chloroplast gene expression with the assembly of the protein products into the large complexes of the photosynthetic apparatus. The convergence of biochemical approaches with those of classical and reverse genetics, and the contributions from large scale genomic sequencing should result in rapid advances in our understanding of the regulatory interactions that govern plastid gene expression.

Coordination of Nuclear and Chloroplast Gene Expression in Plant Cells

We have sequenced the two genes for the small subunit of ribulose bisphosphate carboxylase/oxygenase (Rubisco) in Chlamydomonas reinhardtii and analyzed their expression. The two genes encode variant small subunits that differ by four amino acid residues. Both genes are expressed and each is transcribed into an RNA of distinct size. The accumulation of the two RNAs changes depending on the growth conditions, so the small subunit composition of Rubisco may be expected to differ in response to the environment. The C. reinhardtii small subunit sequence is homologous to those of vascular plants or cyanobacteria, but is longer at the amino terminus and in internal positions. The number and location of the intervening sequences in the genes from C. reinhardtii and from other plants differ. In several cases, internal length differences in the polypeptide coincide with the positions of introns in the coding sequence. Thus, changes in the exon structure of the genes during evolution may have been accompanied by substantial changes in the encoded protein. The translation and splicing signals in C. reinhardtii are similar to those of other eukaryotes, but the transcription signals are less conserved and the highly biased codon usage is very unusual.

The PPH1 phosphatase is specifically involved in LHCII dephosphorylation and state transitions in Arabidopsis

Plastid proteins are encoded in two genomes, one in the nucleus and the other in the organelle. The expression of genes in these two compartments in coordinated during development and in response to environmental parameters such as light. Two converging approaches reveal features of this coordination: the biochemical analysis of proteins involved in gene expression, and the genetic analysis of mutants affected in plastid function or development. Because the majority of proteins implicated in plastid gene expression are encoded in the nucleus, regulatory processes in the nucleus and in the cytoplasm control plastid gene expression, in particular during development. Many nucleus-encoded factors involved in transcriptional and posttranscriptional steps of plastid gene expression have been characterized. We are also beginning to understand whether and how certain developmental or environmental signals perceived in one compartment may be transduced to the other.

Directed chloroplast transformation in Chlamydomonas reinhardtii: insertional inactivation of the psaC gene encoding the iron sulfur protein destabilizes photosystem I.

The ability of plants to adapt to changing light conditions depends on a protein kinase network in the chloroplast that leads to the reversible phosphorylation of key proteins in the photosynthetic membrane. Phosphorylation regulates, in a process called state transition, a profound reorganization of the electron transfer chain and remodeling of the thylakoid membranes. Phosphorylation governs the association of the mobile part of the light-harvesting antenna LHCII with either photosystem I or photosystem II. Recent work has identified the redox-regulated protein kinase STN7 as a major actor in state transitions, but the nature of the corresponding phosphatases remained unknown. Here we identify a phosphatase of Arabidopsis thaliana, called PPH1, which is specifically required for the dephosphorylation of light-harvesting complex II (LHCII). We show that this single phosphatase is largely responsible for the dephosphorylation of Lhcb1 and Lhcb2 but not of the photosystem II core proteins. PPH1, which belongs to the family of monomeric PP2C type phosphatases, is a chloroplast protein and is mainly associated with the stroma lamellae of the thylakoid membranes. We demonstrate that loss of PPH1 leads to an increase in the antenna size of photosystem I and to a strong impairment of state transitions. Thus phosphorylation and dephosphorylation of LHCII appear to be specifically mediated by the kinase/phosphatase pair STN7 and PPH1. These two proteins emerge as key players in the adaptation of the photosynthetic apparatus to changes in light quality and quantity.

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

The chloroplast gene psaC encoding the iron sulfur protein of photosystem I (PSI) from the green alga Chlamydomonas reinhardtii has been cloned and characterized. The deduced amino acid sequence is highly related to that of higher plants and cyanobacteria. Using a particle gun, wild type C. reinhardtii cells have been transformed with a plasmid carrying the psaC gene disrupted by an aadA gene cassette designed to express spectinomycin/streptomycin resistance in the chloroplast. Transformants selected on plates containing acetate as a reduced carbon source and spectinomycin are unable to grow on minimal medium lacking acetate and are deficient in PSI activity. Southern blot analysis of total cell DNA of the transformants shows that the wild type psaC gene has been replaced by the interrupted psaC gene through homologous recombination. While authentic transcripts of the psaC gene are no longer detected, aadA gives rise to a few transcripts in the transformants. Biochemical analysis indicates that neither PSI reaction center subunits nor the seven small subunits belonging to PSI accumulate stably in the thylakoid membranes of the transformants. Pulse‐chase labeling of cell proteins shows that the PSI reaction center subunits are synthesized normally but turn over rapidly in the transformants. We conclude that the iron sulfur binding protein encoded by the psaC gene is an essential component, both for photochemical activity and for stable assembly of PSI. The present study suggests that any chloroplast gene encoding a component of the photosynthetic apparatus can be disrupted in C. reinhardtii using the strategy described.

Mutant Phenotypes Support a Trans-Splicing Mechanism for the Expression of the Tripartite psaA Gene in the C. reinhardtii Chloroplast

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.

The Effect of Heat Shock on Gene Expression in Drosophila melanogaster

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.

Mutation at the chlamydomonas nuclear NAC2 locus specifically affects stability of the chloroplast psbD transcript encoding polypeptide D2 of PS II

Publisher Summary This chapter elaborates the effect of heat shock on gene expression in drosophila melanogaster. Drosophila melanogaster tissue culture cells, normally grown at 25°C, were labeled with 35S-methionine for 2h at this temperature, or for 2h at 37°C, starting 1h after the temperature shift. Heat shocked cells contain a new class of larger polysomes as compared to normal cells at 25°C and a major fraction of polyribosomes with 20 to 30 ribosomes, and a minor fraction of smaller polyribosomes. The de novo synthesized poly(A) + RNA found in these polyribosomes sediments as two main component which includes a major fraction of 20 S RNA predominant in the large polyribosomes, and a minor 12 S fraction found mostly in small polyribosomes. The 20 S and 12 S RNA fractions were found to code for the large, and small heat shock polypeptides respectively. The poly(A) + labeled at 25°C, in contrast, sediments quite heterogeneously, and codes for a whole spectrum of proteins normally synthesized in vivo at 25°C.