Chloroplast biogenesis and regulation of photosynthesis
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 photosynthetic organisms 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).
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 Arabidopsis.
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 genes that are required for the expression of specific chloroplast genes. They 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 and translation
We are studying several nuclear mutants of Chlamydomonas that fail to accumulate specific chloroplast mRNAs. 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 often located within the mRNA 5'UTR (untranslated region). Cis-acting elements which are critical for stabilization of the RNA have been localized within their 5'UTRs. Because the nuclear transformation efficiency is high, we have used it for genomic complementation of the mutants and isolated the corresponding nuclear genes which are affected in these mutants. Some of these genes encode RNA-binding proteins derived from enzymes involved in RNA metabolism. These proteins appear to have been recruited for novel roles in chloroplast gene expression during evolution. Other genes involved in chloroplast gene expression encode proteins of the TPR (tetratricopeptide repeats), PPR (pentatrico peptide repeats) and OPR (octatrico peptide repeats) family containing degenerate repeats of 34 to 38 amino acids. Repeat-containing proteins are widespread in nature and are involved in a broad range of cellular activities. Some of these repeats appear to form RNA-binding domains.
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 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. Mab1 is required for the processing/stability of the same chloroplast mRNA. Tab3 belongs to the family of OPR proteins. 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 which have similar functions in RNA metabolism in higher plants.
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 (Cytb6f ) 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 Cytb6f 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 yield in state 1 and state 2, we have isolated a dozen of mutants deficient in state transitions. 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, Cytb6f and PSI and that its level changes 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. Using bioinformatics and reverse genetics approaches we have identified the PPH1 phosphatase in Arabidopsis which is able to dephosphorylate the LHCII proteins under state 1 conditions. The function of other chloroplast phosphatases is under investigation. The ultimate aim is to understand the regulation of this chloroplast-phosphatase thylakoid network.