Jean-David Rochaix

Michel Goldschmidt-Clermont

A unique feature of plant and algal cells is that they contain three distinct genetic systems located in the nucleus, chloroplast and mitochondria. These systems comprise their own genome and protein synthesizing machineries. Our long-range goal is to understand the molecular cross-talk between these systems. This coordination is required for the biogenesis and function of the organelles. Using the combined approaches of molecular genetics and biochemistry, we study the interactions between the nucleo-cytosolic and chloroplast systems that are involved in the assembly of the photosynthetic apparatus. We also study the remarkable ability of plants to adapt to changes in both light quality and quantity, and in particular the dynamic acclimation processes which occur in the thylakoid membranes (the photosynthetic membranes of the chloroplast).

Introduction

The biogenesis of the photosynthetic apparatus and its regulation play a key role in plant development. We study this process in the green unicellular alga Chlamydomonas reinhardtii which has emerged as a powerful model system. Photosynthetic function is dispensable when this alga is grown in the presence of acetate as a carbon source, a property that greatly facilitates the isolation of mutants and their genetic analysis. Nuclear, chloroplast and mitochondrial transformation can be easily achieved. Furthermore the sequences of the nuclear, chloroplast and mitochondrial genomes of this alga have been determined (see the website of the Chlamydomonas Center: http://www.chlamy.org/). We also use the model plant Arabidopsis thaliana in some of our studies. It is of particular interest to compare a unicellular, motile alga like Chlamydomonas with a multicellular, sessile plant like Arabidosis.

Chloroplast biogenesis and function depend on the concerted action of the nuclear and organellar genetic systems. While the majority of chloroplast proteins are nucleus-encoded, the chloroplast genome contains a relatively small number of genes, required mainly for photosynthesis and chloroplast gene expression. Genetic analysis of mutants deficient in photosynthetic activity has revealed a surprisingly large number of nuclear loci that are required for the expression of specific chloroplast genes. These loci appear to be involved in several post-transcriptional steps including RNA stability, RNA processing, splicing, translation and assembly of photosynthetic complexes.

The role of the nucleus in chloroplast biogenesis: Chloroplast RNA metabolism

We are studying several nuclear mutants of Chlamydomonas that fail to accumulate specific chloroplast mRNAs such as psbD, psbB or psaB. Because chloroplast RNA stability, processing and translation are closely coupled, any of these steps could be affected in the mutants. We have shown that the target site of the nucleus-encoded function affected in these mutants is located within the mRNA 5'UTR (untranslated region). Cis-acting elements which are critical for stabilization of the RNA have been localized within the psbD and psbB 5'UTRs. Because the nuclear transformation efficiency is high, we have used it for genomic complementation of the mutants and isolated the nuclear Nac2 gene, which is required specifically for the stable accumulation of psbD mRNA and Mbb1, required for the accumulation of the mRNAs from the psbB/ T /H gene cluster. The proteins encoded by these two genes both contain 9 or 10 tetratrico-peptide repeats (TPR) and are part of high molecular weight complexes. A single amino acid change within one of the Nac2 TPRs abrogates Nac2 function, indicating that the TPRs are important for mRNA stability, processing and/or translation. In the case of Nac2, the TPR region appears to act as RNA binding domain. It is interesting to note that Mbb1 has an orthologue in Arabidopsis, HCF107, which is also involved in RNA processing of the psbH mRNA.

The chloroplast psaA gene, encoding one of the apoproteins of photosystem I, has an unusual structure in Chlamydomonas reinhardtii. It consists of three exons that are scattered at widely separate loci of the plastid genome and are flanked by group II intron sequences. The mature mRNA is assembled from three separate precursors in two steps of splicing in trans. We have obtained numerous nuclear mutants that fail to assemble mature psaA RNA. They belong to three phenotypic classes: some are defective for the trans-splicing of the first split intron of psaA (class C mutants), some for the trans-splicing of the second split intron (class A), and others for both trans-splicing steps (class B). A complementation analysis has shown that in each phenotypic class there are several nuclear genes belonging to a total of at least fourteen loci. A remarkable feature is that the first psaA intron consists of at least three independently transcribed parts. The chloroplast locus, tscA, encodes a small RNA which is thought to assemble with the precursors of exon 1 and exon 2 to form the characteristic structure of group II introns. This tripartite psaA intron could represent an intermediate in the evolution of group II introns to nuclear introns. Our recent efforts to understand psaA trans-splicing have focused on the cloning and characterization of three of the nuclear genes, Raa1, Raa2 and Raa3, and the polypeptides they encode. Raa1 is a very large protein that contains five OPR repeats (see below) and is required for trans-splicing of both introns. Raa2, which is involved in splicing of the second intron, is related to pseudo-uridine synthases and is associated together with Raa1 in a 500kDa complex. The size and integrity of this complex is affected by mutations mapped in other complementation groups suggesting that other proteins are also present in this multiprotein complex. Alternatively, these other factors could be required for the assembly of the complex. Raa3, required for trans-splicing the first intron, forms a 1800 kDa complex with tscA RNA and the psaA exon1 precursor. Thus, at least two different RNA-protein complexes have been identified which could represent plastid spliceosomal complexes of group II introns.

Tab2 and Tab3 are two nuclear genes that control translation of the chloroplast psaB mRNA via cis-acting sequences in its 5' UTR. We have also isolated and begun to characterize the Mab1 gene, which is required for the stability of the same chloroplast mRNA. The Tab2 gene encodes a polypeptide of 358 amino acids with a N-terminal transit peptide responsible for targeting the protein into the chloroplast. An interesting feature is that this protein contains orthologs in Arabidopsis and also in brown algae and cyanobacteria. It thus represents the first mRNA-specific translation factor that is conserved in both prokaryotic and eukaryotic photosynthetic organisms. Tab3 belongs to the family of OPR proteins (octatrico peptide repeats). These 38 amino acid repeats are also found in a family of chloroplast proteins such as the trans-splicing factor Raa1 and the translation factors Tbc2 and Tda1. Thus the OPR proteins of Chlamydomonas may be the functional counterparts of the large family of PPR proteins (pentatrico peptide repeat) which have similar functions in RNA metabolism in higher plants.

Chloroplast-nuclear signaling in Chlamydomonas

Coordination between the activities of organelles and the nucleus requires the exchange of signals. In recent years it has become clear that nuclear-chloroplast interactions operate bidirectionally from the nucleus to the chloroplast and vice-versa. Although the chloroplast signals involved in this response are still enigmatic, several processes are known to give rise to these signals: accumulation of certain tetrapyrrole intermediates, changes in redox state of the plastoquinone pool, arrest of chloroplast protein synthesis, singlet oxygen production and the activity of the cytochrome b6f complex.

In photosynthetic organisms the accumulation of harmful photodynamic chlorophyll precursors is prevented because of the tight regulation of the tetrapyrrole pathway. FLU is one of the regulatory factors involved in this process in land plants. We have examined the function of a Flu-like gene (FLP) from Chlamydomonas which gives rise to two FLP transcripts through alternative splicing. These transcripts are translated into a short and a long protein that differ by only 12 amino acids but which interact differently with glutamyl tRNA reductase, an enzyme involved in an early step of the chlorophyll biosynthetic pathway. Expression of FLPs is light-regulated at the level of RNA accumulation and splicing, and is altered by mutations affecting the pathway. The relative levels of the long and short forms of FLP can be correlated with the accumulation of specific porphyrin intermediates some of which have been implicated in a signalling chain from the chloroplast to the nucleus. Reciprocally, reduction of the FLP proteins by RNA interference leads to the accumulation of several porphyrin intermediates and to photobleaching when cells are transferred from the dark to the light. Thus the FLP proteins act as regulators of chlorophyll synthesis and their expression is controlled by light and plastid signals. Using biochemical and genetic tools we are trying to elucidate the molecular basis of this phenomenon.

Adaptation to a changing light environment through state transitions

Plants and algae have the remarkable ability to adapt to changes in light quality and quantity. They balance energy input and consumption in the short term through non-photochemical dissipation of excess energy and through state transitions. This adjustment occurs at the level of the primary reactions of photosynthesis catalyzed by photosystem II (PSII) and photosystem I (PSI) which are linked in series through the plastoquinone pool, the cytochrome b₆f complex and plastocyanin. Because the antenna systems of PSII and PSI have different light absorption properties, changes in light conditions lead to unequal excitation of the photosystems and to changes in the redox state of the plastoquinone pool. Reduction of the plastoquinone pool by overexcitation of PSII relative to PSI leads to the activation of a kinase through the cytochrome b₆f complex and to phosphorylation of the light-harvesting system of PSII (LHCII) (state 2), a fraction of which is then displaced from PSII to PSI. Overexcitation of PSI relative to PSII leads to the oxidation of the plastoquinone pool, inactivation of the kinase and dephosphorylation of the mobile LHCII and its return to PSII (state 1).This reversible redistribution of excitation energy between the two photosystems leads to an overall increase in photosynthetic quantum yield. It also triggers a change from linear to cyclic electron flow (or vice-versa) and plays a key role in ATP homeostasis.

Using a fluorescence video-imaging screen based on the differences in fluorescence in state 1 and state 2, we have isolated a dozen of mutants deficient in state transitions which are currently characterized. Amongst these mutants, stt7 was found to be deficient in the thylakoid serine-threonine protein kinase Stt7. The Stt7 kinase is required for LHCII phosphorylation and state transitions. It is conserved in Arabidopsis and belongs to a small family of thylakoid protein kinases including Stt7 and Stl1 in Chlamydomonas and STN7 and STN8, respectively, in Arabidopsis. We have found that Stt7 is associated with LHCII, the cytochrome b6f complex and PSI and that it levels change during state transitions. Although we have been able to identify several of its substrates (collaboration with Alex Vener, Linkoping University), the mechanism of regulation of the kinase remains unknown and is under investigation.

Plant Growth in a changing Environment

SystemsX.ch RTD project

This new interdisciplinary project aims to use the approaches of systems biology to study and model plant growth at the cellular, tissue and whole plant levels. We will be particularly involved in a subproject that will investigate how the developmental processes that promote growth are integrated with the underlying metabolic fluxes in Arabidopsis. In a collaboration with the ETH Zürich and the University of Neuchâtel, we will analyze a series of mutants that show defects in photosynthetic performance, as an effort to elucidate regulatory pathways and identify novel factors that control photosynthetic capacity and growth. Data from molecular profiling of transcripts and of proteins, from metabolic flux analysis and from measurements of photosynthetic parameters will be analyzed to understand correlations and causal factors linking them. These data will be used to evaluate and refine models of regulatory networks, and together with those of the other modules in the project, to establish models of Arabidopsis growth.

SOLAR-H2: European Solar-Fuel Initiative. Renewable Hydrogen from Sun and Water. Science Linking Molecular Biomimetics and Genetics

Seventh Framework Programme FP7 project.

This project involves 12 European research teams and integrates two frontline research topics: artificial photosynthesis in man-made biomimetic systems, and photobiological H2 production in living organisms. C. reinhardtii is able to evolve hydrogen under anaerobic conditions in the light. However this occurs only transiently because the hydrogenase is quickly inactivated by the oxygen produced through photosynthesis. To circumvent this problem we have developed an inducible chloroplast gene expression system in C. reinhardtii which allows us to turn off PSII. Under these conditions oxygen is consumed by respiration and hydrogen production is induced until all the reduced carbon sources are exhausted. We have taken advantage of the properties of the copper-sensitive cytochrome c6 promoter and of the nucleus-encoded Nac2 chloroplast protein. This protein is specifically required for the stable accumulation of the chloroplast psbD RNA and acts on its 5'UTR. We have constructed a strain containing the Nac2 coding sequence driven by the cytochrome c6 promoter so that psbD is expressed in copper-depleted but not in copper replete medium. Because psbD encodes the D2 reaction center polypeptide of photosystem II (PSII), the repression of psbD leads to the loss of PSII. We have tested this system for hydrogen production. Upon addition of copper to cells pre-grown in copper deficient medium, PSII levels declined to a level at which oxygen consumption by respiration exceeded oxygen evolution by PSII. The resulting anaerobic conditions led to the induction of hydrogenase activity. Because the Cyc6 promoter is also induced under anaerobic conditions, this system opens possibilities for sustained cycling hydrogen production. Moreover, this inducible gene expression system is applicable to any chloroplast gene by replacing its 5'UTR with the psbD 5'UTR in the same genetic background.

We have developed a second inducible system in which the Nac2 gene is driven by the vitamin B12-responsive promoter and a thiamine pyrophosphate (TPP) riboswitch within the Nac2 5'UTR (collaboration with Martin Croft and Allison Smith), University of Cambridge). With this system psbD expression and PSII activity can be repressed by adding B12 and TPP to the growth medium. Besides hydrogen production this system can be used for transient expression of foreign proteins in the chloroplast and for elucidating the function of essential chloroplast genes.

Green algae as oral vaccines for fish

Project homepage.

National Research Programme NRP 59.

For the expression of foreign proteins, the chloroplast compartment offers many advantages (precise and predictable gene insertion by homologous recombination, high expression levels, stable gene expression). In the chloroplast of Chlamydomonas, we are trying to express bacterial antigens from Aeromonas salmonicida, a pathogenic agent that causes furunculosis in fish. In a collaboration with the groups of Profs J. Frey and H. Segner at the Veterinary Faculty of the University of Bern, we will then test whether such Chlamydomonas lines can be used for oral vaccination of rainbow trout. Development of such vaccines for fish is highly desirable to reduce the widespread use of antibiotics in aquaculture.