Proteomic analysis of the Cyanophora paradoxa muroplast provides clues on early events in plastid endosymbiosis.

Glaucophytes represent the first lineage of photosynthetic eukaryotes of primary endosymbiotic origin that diverged after plastid establishment. The muroplast of Cyanophora paradoxa represents a primitive plastid that resembles its cyanobacterial ancestor in pigment composition and the presence of a peptidoglycan wall. To attain insights into the evolutionary history of cyanobiont integration and plastid development, it would thus be highly desirable to obtain knowledge on the composition of the glaucophyte plastid proteome. Here, we provide the first proteomic analysis of the muroplast of C. paradoxa. Mass spectrometric analysis of the muroplast proteome identified 510 proteins with high confidence. The protein repertoire of the muroplast revealed novel paths for reduced carbon flow and export to the cytosol through a sugar phosphate transporter of chlamydial origin. We propose that C. paradoxa possesses a primordial plastid mirroring the situation in the early protoalga.

An intermediate membrane subfraction in cyanobacteria is involved in an assembly network for Photosystem II biogenesis.

Early steps in the biogenesis of Photosystem II (PSII) in the cyanobacterium Synechocystis sp. PCC 6803 are thought to occur in a specialized membrane fraction that is characterized by the specific accumulation of the PSII assembly factor PratA and its interaction partner pD1, the precursor of the D1 protein of PSII. Here, we report the molecular characterization of this membrane fraction, called the PratA-defined membrane (PDM), with regard to its lipid and pigment composition and its association with PSII assembly factors, including YCF48, Slr1471, Sll0933, and Pitt. We demonstrate that YCF48 and Slr1471 are present and that the chlorophyll precursor chlorophyllide a accumulates in the PDM. Analysis of PDMs from various mutant lines suggests a central role for PratA in the spatial organization of PSII biogenesis. Moreover, quantitative immunoblot analyses revealed a network of interdependences between several PSII assembly factors and chlorophyll synthesis. In addition, formation of complexes containing both YCF48 and Sll0933 was substantiated by co-immunoprecipitation experiments. The findings are integrated into a refined model for PSII biogenesis in Synechocystis 6803.

In-depth analysis of the distinctive effects of norflurazon implies that tetrapyrrole biosynthesis, organellar gene expression and ABA cooperate in the GUN-type of plastid signalling.

Application of norflurazon (NF) damages plastids, induces photobleaching and represses expression of the nuclear LHCB1.2 gene encoding a light-harvesting protein. In genomes uncoupled (gun) mutants, LHCB1.2 expression is maintained in the presence of NF. The mutants gun2, gun4 and gun5 exhibit perturbations in tetrapyrrole biosynthesis, but gun1 is defective in organellar gene expression (OGE). How gun mutations affect nuclear gene expression (NGE) and why the signals elicited by the two types evoke the same response remains unknown. Here we show that the carotenoid biosynthesis inhibitors amitrole and flurochloridone can replace NF in gun assays, whereas novel tetrapyrrole pathway mutations do not provoke a gun phenotype. Changes in haem levels also do not account for LHCB1.2 derepression in NF-treated gun mutants. Pigment measurements indicated that gun mutants are not resistant to NF, but gun2, gun4 and gun5 retain low levels of lutein, as well as of neoxanthin and violaxanthin, the precursors of abscisic acid (ABA). This might explain the enhanced ABA sensitivity of gun4 and gun5 plants found in germination assays. Metabolite profiling and analyses of reactive oxygen species and cellular redox state failed to suggest a link between gun mutations and altered LHCB1.2 expression. However, in contrast to NF-treated wild-type plants, gun mutants retain to a marked extent the capability to express the plastome-encoded proteins AtpB and RbcL. This, together with the finding that application of ABA can partially restore LHCB1.2 expression in NF-treated wild-type plants, supports the view that tetrapyrrole, OGE and ABA signalling are interconnected.

Pitt, a novel tetratricopeptide repeat protein involved in light-dependent chlorophyll biosynthesis and thylakoid membrane biogenesis in Synechocystis sp. PCC 6803.

Biogenesis of photosynthetic pigment/protein complexes is a highly regulated process that requires various assisting factors. Here, we report on the molecular analysis of the Pitt gene (slr1644) from the cyanobacterium Synechocystis sp. PCC 6803 (Synechocystis 6803) that encodes a membrane-bound tetratricopeptide repeat (TPR) protein of formerly unknown function. Targeted inactivation of Pitt affected photosynthetic performance and light-dependent chlorophyll synthesis. Yeast two-hybrid analyses and native PAGE strongly suggest a complex formation between Pitt and the light-dependent protochlorophyllide oxidoreductase (POR). Consistently, POR levels are approximately threefold reduced in the pitt insertion mutant. The membrane sublocalization of Pitt was found to be dependent on the presence of the periplasmic photosystem II (PSII) biogenesis factor PratA, supporting the idea that Pitt is involved in the early steps of photosynthetic pigment/protein complex formation.

Chloroplast biogenesis: the use of mutants to study the etioplast-chloroplast transition.

In angiosperm plants, the etioplast-chloroplast transition is light-dependent. A key factor in this process is the protochlorophyllide oxidoreductase A (PORA), which catalyzes the light-induced reduction of protochlorophyllide to chlorophyllide. The import pathway of the precursor protein prePORA into chloroplasts was analyzed in vivo and in vitro by using homozygous loss-of-function mutants in genes coding for chlorophyllide a oxygenase (CAO) or for members of the outer-envelope solute-channel protein family of 16 kDa (OEP16), both of which have been implied to be key factors for the import of prePORA. Our in vivo analyses show that cao or oep16 mutants contain a normally structured prolamellar body that contains the protochlorophyllide holochrome. Furthermore, etioplasts from cao and oep16 mutants contain PORA protein as found by mass spectrometry. Our data demonstrate that both CAO and OEP16 are dispensable for chloroplast biogenesis and play no central role in the import of prePORA in vivo and in vitro as further indicated by protein import studies.

Inhibition of lycopene cyclase results in accumulation of chlorophyll precursors.

Free porphyrins and their magnesium complexes, including chlorophylls, are potent photo-sensitizers. Plants usually accumulate these compounds bound to proteins together with protective compounds like carotenoids. Besides their protective role, carotenoids can play a structural role in these complexes. To analyze the effect of impaired carotenogenesis on plastid membranes we applied to barley seedlings the bleaching herbicide 2-(4-chlorophenylthio)triethylamine (CPTA) as a specific inhibitor for the cyclization of lycopene. To avoid interference with photo-oxidation, the essential experiments were performed on seedlings grown in darkness. While the amount of total carotenoids decreased, we found accumulation of more 6-carotene than lycopene in darkness clearly showing that CPTA inhibits the lycopene beta-cyclase more effectively than the lycopene epsilon-cyclase. The CPTA treatment resulted in accumulation of non-photoactive protochlorophyllide a; the amount of photoactive protochlorophyllide and NADPH:protochlorophyllide oxidoreductase remained constant. Further, the level of Mg protophorphyrin and its monomethyl ester increased to an extent similar to that obtained by application of 5-aminolevulinic acid (ALA). The perturbation of the ultrastructure of etioplast inner membranes, observed after CPTA-treatment, was not found after ALA-treatment; this excluded the accumulated tetrapyrroles as responsible for the perturbation. By contrast, the down-regulation of Lhcb and RbcS genes found after CPTA-treatment was compatible with the presumed role of Mg protophorphyrin as "plastid signal" for regulation of nuclear gene expression. Possible mechanisms for enhancement of tetrapyrrole accumulation by non-cyclic carotenoids are discussed.

Identification of a plastid response element that acts as an enhancer within the Chlamydomonas HSP70A promoter.

Chloroplast-derived signals control a subset of nuclear genes in higher plants and eukaryotic algae. Among the types of signals identified are intermediates of chlorophyll biosynthesis such as Mg-protoporphyrin IX (MgProto). In Chlamydomonas reinhardtii, it was suggested that this tetrapyrrole mediates the light induction of chaperone gene HSP70A. Here we have analyzed cis elements involved in the regulation of HSP70A by MgProto and light. We identified two promoters and between their transcription start sites two regulatory regions that each may confer inducibility by MgProto and light to both HSP70A promoters. These regulatory regions, when cloned in front of basal non-light inducible heterologous promoters, conferred inducibility by MgProto and light. The orientation and distance independent function of these cis-regulatory sequences qualifies them as enhancers that mediate the response of nuclear genes to a chloroplast signal. Mutational analysis of one of these regulatory regions and an alignment with promoters of other MgProto-inducible genes revealed the sequence motif (G/C)CGA(C/T)N(A/G)N15 (T/C/A)(A/T/G) which, as shown for HSP70A, may confer MgProto responsiveness. This cis-acting sequence element is employed for induction of HSP70A by both MgProto and light, lending support to the model that light induction of this gene is mediated via MgProto.

Mg-protoporphyrin IX and heme control HEMA, the gene encoding the first specific step of tetrapyrrole biosynthesis, in Chlamydomonas reinhardtii.

HEMA encodes glutamyl-tRNA reductase (GluTR), which catalyzes the first step specific for tetrapyrrole biosynthesis in plants, archaea, and most eubacteria. In higher plants, GluTR is feedback inhibited by heme and intermediates of chlorophyll biosynthesis. It plays a key role in controlling flux through the tetrapyrrole biosynthetic pathway. This enzyme, which in Chlamydomonas reinhardtii is encoded by a single gene (HEMA), exhibits homology to GluTRs of higher plants and cyanobacteria. HEMA mRNA accumulation was inducible not only by light but also by treatment of dark-adapted cells with Mg-protoporphyrin IX (MgProto) or hemin. The specificity of these tetrapyrroles as inducers was demonstrated by the absence of induction observed upon the feeding of protoporphyrin IX, the precursor of both heme and MgProto, or chlorophyllide. The HEMA mRNA accumulation following treatment of cells with light and hemin was accompanied by increased amounts of GluTR. However, the feeding of MgProto did not suggest a role for Mg-tetrapyrroles in posttranscriptional regulation. The induction by light but not that by the tetrapyrroles was prevented by inhibition of cytoplasmic protein synthesis. Since MgProto is synthesized exclusively in plastids and heme is synthesized in plastids and mitochondria, the data suggest a role of these compounds as organellar signals that control expression of the nuclear HEMA gene.

Involvement of tetrapyrroles in inter-organellar signaling in plants and algae.

For the assembly of a functional chloroplast, the coordinated expression of genes distributed between nucleus and chloroplasts is a prerequisite. While the nucleus plays an undisputed dominant role in controling biogenesis and functioning of chloroplasts, plastidic signals appear to control the expression of a subset of nuclear genes; the majority of which encodes chloroplast constituents. Tetrapyrrole biosynthesis intermediates are attractive candidates for one type of plastidic signal ever since an involvement of Mg-porphyrins in signaling from chloroplast to nucleus was first demonstrated in Chlamydomonas reinhardtii. Since then, Mg-protoporphyrin IX has been shown to exert a regulatory function on nuclear genes in higher plants as well. Here we review evidence for the role played by tetrapyrroles in inter-organellar communication. We also report on a screening for nuclear genes that may be subject to regulation by tetrapyrroles. This revealed that (i) >HEMA, the gene encoding the first enzyme specific for porphyrin biosynthesis is induced by Mg-protoporphyrin IX, (ii) several nuclear HSP70 genes are regulated by tetrapyrroles. Members of the gene family induced by the feeding of Mg-rotoporphyrin IX encode chaperones located in either the chloroplast or the cytosol. These results point to an important role of Mg-tetrapyrroles as plastidic signal in controling the initial step of porphyrin biosynthesis, and the synthesis of chaperones involved in protein folding in cytosol/stroma, protein transport into organelles, and the stress response.

Chlorophylls of the c family: absolute configuration and inhibition of NADPH:protochlorophyllide oxidoreductase.

Using circular dichroism (CD) spectroscopy, the stereochemistry at C-13(2) of members of the chlorophyll (Chl) c family, namely Chls c(1), c(2), c(3) and [8-vinyl]-protochlorophyllide a (Pchlide a) was determined. By comparison with spectra of known enantiomers, all Chl c members turned out to have the (R) configuration, which is in agreement with considerations drawn from chlorophyll biosynthesis. Except for a double bond in the side chain at C-17, the chemical structure of Chl c(1) is identical with Pchlide a, the natural substrate of the light-dependent NADPH:protochlorophyllide oxidoreductase (POR). Thus, lack of binding to the active site due to the wrong configuration at C-13(2), which had been proposed previously, cannot be an explanation for inactivity of Chl c in this enzymic reaction. Our results show rather that Chl c(1) is a competitive inhibitor for this enzyme, tested with Pchlide a and Zn-protopheophorbide a (Zn-Ppheide a) as substrates.

Pre-loading of chlorophyll synthase with tetraprenyl diphosphate is an obligatory step in chlorophyll biosynthesis.

The reaction of recombinant chlorophyll synthase from Avena sativa, expressed in Escherichia coli, was investigated. To verify the identity of the recombinant and native enzymes, reaction rates were determined for both enzyme preparations with several chlorophyllide analogs. The rates of esterification of these modified substrates ranged from 0 to 100% of the rate with the natural substrate, and were nearly identical for both enzyme preparations. The Lineweaver-Burk plot for variation of both chlorophyllide a and phytyl diphosphate concentration showed parallel lines, indicative of a 'ping-pong' mechanism. Pre-incubation with phytyl diphosphate exhibited an initial rapid reaction phase, which did not occur after pre-incubation with chlorophyllide. We conclude that the tetraprenyl diphosphate must bind to the enzyme as the first substrate and esterification occurs when this pre-loaded enzyme meets the second substrate, chlorophyllide. Approximately 10-17% of the recombinant enzyme were pre-loaded with phytyl diphosphate under the experimental conditions. The rapid reaction phase is also observed for the chlorophyll synthase reaction in etiolated barley leaves in addition to the well-known slow phase. This indicates that pre-loading of the enzyme with tetraprenyl diphosphate is also the basis for the reaction in vivo.