Singlet oxygen-induced signalling depends on the metabolic status of the Chlamydomonas reinhardtii cell.

Using a mutant screen, we identified trehalose 6-phosphate phosphatase 1 (TSPP1) as a functional enzyme dephosphorylating trehalose 6-phosphate (Tre6P) to trehalose in Chlamydomonas reinhardtii. The tspp1 knock-out results in reprogramming of the cell metabolism via altered transcriptome. As a secondary effect, tspp1 also shows impairment in 1O2-induced chloroplast retrograde signalling. From transcriptomic analysis and metabolite profiling, we conclude that accumulation or deficiency of certain metabolites directly affect 1O2-signalling. 1O2-inducible GLUTATHIONE PEROXIDASE 5 (GPX5) gene expression is suppressed by increased content of fumarate and 2-oxoglutarate, intermediates in the tricarboxylic acid cycle (TCA cycle) in mitochondria and dicarboxylate metabolism in the cytosol, but also myo-inositol, involved in inositol phosphate metabolism and phosphatidylinositol signalling system. Application of another TCA cycle intermediate, aconitate, recovers 1O2-signalling and GPX5 expression in otherwise aconitate-deficient tspp1. Genes encoding known essential components of chloroplast-to-nucleus 1O2-signalling, PSBP2, MBS, and SAK1, show decreased transcript levels in tspp1, which also can be rescued by exogenous application of aconitate. We demonstrate that chloroplast retrograde signalling involving 1O2 depends on mitochondrial and cytosolic processes and that the metabolic status of the cell determines the response to 1O2.

Interactions Between Carbon Metabolism and Photosynthetic Electron Transport in a Chlamydomonas reinhardtii Mutant Without CO2 Fixation by RuBisCO.

A Chlamydomonas reinhardtii RuBisCO-less mutant, ΔrbcL, was used to study carbohydrate metabolism without fixation of atmospheric carbon. The regulatory mechanism(s) that control linear electron flow, known as "photosynthetic control," are amplified in ΔrbcL at the onset of illumination. With the aim to understand the metabolites that control this regulatory response, we have correlated the kinetics of primary carbon metabolites to chlorophyll fluorescence induction curves. We identify that ΔrbcL in the absence of acetate generates adenosine triphosphate (ATP) via photosynthetic electron transfer reactions. Also, metabolites of the Calvin Benson Bassham (CBB) cycle are responsive to the light. Indeed, ribulose 1,5-bisphosphate (RuBP), the last intermediate before carboxylation by Ribulose-1,5-bisphosphate carboxylase-oxygenase, accumulates significantly with time, and CBB cycle intermediates for RuBP regeneration, dihydroxyacetone phosphate (DHAP), pentose phosphates and ribose-5-phosphate (R5P) are rapidly accumulated in the first seconds of illumination, then consumed, showing that although the CBB is blocked, these enzymes are still transiently active. In opposition, in the presence of acetate, consumption of CBB cycle intermediates is strongly diminished, suggesting that the link between light and primary carbon metabolism is almost lost. Phosphorylated hexoses and starch accumulate significantly. We show that acetate uptake results in heterotrophic metabolism dominating phototrophic metabolism, with glyoxylate and tricarboxylic acid (TCA) cycle intermediates being the most highly represented metabolites, specifically succinate and malate. These findings allow us to hypothesize which metabolites and metabolic pathways are relevant to the upregulation of processes like cyclic electron flow that are implicated in photosynthetic control mechanisms.

The function of PROTOPORPHYRINOGEN IX OXIDASE in chlorophyll biosynthesis requires oxidised plastoquinone in Chlamydomonas reinhardtii.

In the last common enzymatic step of tetrapyrrole biosynthesis, prior to the branching point leading to the biosynthesis of heme and chlorophyll, protoporphyrinogen IX (Protogen) is oxidised to protoporphyrin IX (Proto) by protoporphyrinogen IX oxidase (PPX). The absence of thylakoid-localised plastid terminal oxidase 2 (PTOX2) and cytochrome b6f complex in the ptox2 petB mutant, results in almost complete reduction of the plastoquinone pool (PQ pool) in light. Here we show that the lack of oxidised PQ impairs PPX function, leading to accumulation and subsequently uncontrolled oxidation of Protogen to non-metabolised Proto. Addition of 3(3,4-Dichlorophenyl)-1,1-dimethylurea (DCMU) prevents the over-reduction of the PQ pool in ptox2 petB and decreases Proto accumulation. This observation strongly indicates the need of oxidised PQ as the electron acceptor for the PPX reaction in Chlamydomonas reinhardtii. The PPX-PQ pool interaction is proposed to function as a feedback loop between photosynthetic electron transport and chlorophyll biosynthesis.

Mg chelatase in chlorophyll synthesis and retrograde signaling in Chlamydomonas reinhardtii: CHLI2 cannot substitute for CHLI1.

The oligomeric Mg chelatase (MgCh), consisting of the subunits CHLH, CHLI, and CHLD, is located at the central site of chlorophyll synthesis, but is also thought to have an additional function in regulatory feedback control of the tetrapyrrole biosynthesis pathway and in chloroplast retrograde signaling. In Arabidopsis thaliana and Chlamydomonas reinhardtii, two genes have been proposed to encode the CHLI subunit of MgCh. While the role of CHLI1 in A. thaliana MgCh has been substantially elucidated, different reports provide inconsistent results with regard to the function of CHLI2 in Mg chelation and retrograde signaling. In the present report, the possible functions of both isoforms were analyzed in C. reinhardtii Knockout of the CHLI1 gene resulted in complete loss of MgCh activity, absence of chlorophyll, acute light sensitivity, and, as a consequence, down-regulation of tetrapyrrole biosynthesis and photosynthesis-associated nuclear genes. These observations indicate a phenotypical resemblance of chli1 to the chlh and chld C. reinhardtii mutants previously reported. The key role of CHLI1 for MgCh reaction in comparison with the second isoform was confirmed by the rescue of chli1 with genomic CHLI1 Because CHLI2 in C. reinhardtii shows lower expression than CHLI1, strains overexpressing CHLI2 were produced in the chli1 background. However, no complementation of the chli1 phenotype was observed. Silencing of CHLI2 in the wild-type background did not result in any changes in the accumulation of tetrapyrrole intermediates or of chlorophyll. The results suggest that, unlike in A. thaliana, changes in CHLI2 content observed in the present studies do not affect formation and activity of MgCh in C. reinhardtii.

Regulation and function of tetrapyrrole biosynthesis in plants and algae.

Tetrapyrroles are macrocyclic molecules with various structural variants and multiple functions in Prokaryotes and Eukaryotes. Present knowledge about the metabolism of tetrapyrroles reflects the complex evolution of the pathway in different kingdoms of organisms, the complexity of structural and enzymatic variations of enzymatic steps, as well as a wide range of regulatory mechanisms, which ensure adequate synthesis of tetrapyrrole end-products at any time of development and environmental condition. This review intends to highlight new findings of research on tetrapyrrole biosynthesis in plants and algae. In the course of the heme and chlorophyll synthesis in these photosynthetic organisms, glutamate, one of the central and abundant metabolites, is converted into highly photoreactive tetrapyrrole intermediates. Thereby, several mechanisms of posttranslational control are thought to be essential for a tight regulation of each enzymatic step. Finally, we wish to discuss the potential role of tetrapyrroles in retrograde signaling and point out perspectives of the formation of macromolecular protein complexes in tetrapyrrole biosynthesis as an efficient mechanism to ensure a fine-tuned metabolic flow in the pathway. This article is part of a Special Issue entitled: Chloroplast Biogenesis.

The GUN4 protein plays a regulatory role in tetrapyrrole biosynthesis and chloroplast-to-nucleus signalling in Chlamydomonas reinhardtii.

The GENOMES UNCOUPLED 4 (GUN4) protein is found only in aerobic photosynthetic organisms. We investigated the role of GUN4 in metabolic activities of the Mg branch of the tetrapyrrole biosynthesis pathway and the plastid signal-mediated changes of nuclear gene expression in Chlamydomonas reinhardtii. In light, gun4 accumulates only 40% of the wild-type chlorophyll level. Light- or dark-grown gun4 mutant accumulates high levels of protoporphyrin IX (Proto), and displays increased sensitivity to moderate light intensities. Despite the photooxidative stress, gun4 fails to downregulate mRNA levels of the tetrapyrrole biosynthesis and the photosynthesis-associated nuclear genes (PhANGs). In contrast, upon illumination, the Proto-accumulating and light-sensitive chlD-1 mutant displays the expected downregulation of the same nuclear genes. Although chlD-1 and the wild type have similar GUN4 transcript levels, the GUN4 protein in chlD-1 is hardly detectable. Overexpression of GUN4 in chlD-1 modifies the downregulation of nuclear gene expression, but also increases light tolerance. Therefore, GUN4 is proposed to function in 'shielding' Proto, and most likely MgProto, by reducing reactivity with O2 . Furthermore, GUN4 seems to be involved in sensing elevated levels of these photoreactive tetrapyrrole intermediates, and contributing to (1) O2 -mediated retrograde signalling, originating from chlorophyll biosynthesis.

The PSBP2 protein of Chlamydomonas reinhardtii is required for singlet oxygen-dependent signaling.

In the green alga Chlamydomonas reinhardtii, the cytosolic Glutathione Peroxidase 5 gene (GPX5) is known to be transcriptionally up-regulated in response to singlet oxygen ((1)O(2)). As demonstrated by previous studies, fusion of the promoter region of GPX5 to the Arylsulfatase 2 gene (ARS2) creates an effective reporter system that can be used to monitor (1)O(2)-driven GPX5 expression. This system was also used in this study to generate a stably transformed C. reinhardtii strain which expresses ARS2 in a (1)O(2)-dependent manner, resulting in the synthesis of a functional protein with detectable activity. Using the strain of C. reinhardtii harboring a (1)O(2)-sensitive reporter construct, a secondary mutagenic screen was performed. This allowed identification of mutant cell lines that were unable to up-regulate expression of the GPX5-ARS2 fusion in response to (1)O(2). In one of these lines, the mutation was subsequently localized to the first exon of the PSBP-like gene (PSBP2). The PSBP2 gene is part of a small protein family in C. reinhardtii, also present in all angiosperms studied thus far. While each member of the PSBP protein family contains a similar domain to the PSBP1 protein, which is a member of the oxygen evolving complex of photosystem II (PSII), the PSBP2 protein does not appear to be involved in PSII function, but may function as a sensor and/or signal mediating molecule of the (1)O(2) generated in the chloroplast.

Inactivation of the STT7 gene protects PsaF-deficient Chlamydomonas reinhardtii cells from oxidative stress under high light.

Photosystem I (PSI) utilizes light energy to excite electrons for the reduction of NADP(+) , and like photosystem II, it is sensitive to excess light. When PSI is excited and unable to be reduced by the electron transport chain, the special pair of chlorophyll molecules, P700(+) , will take electrons from neighboring sources leading to cellular damage. A Chlamydomonas reinhardtii mutant, which is defective in the production of the PsaF subunit of PSI, provides an ideal platform for studying the processes involved in protecting PSI from excess light. This strain dies following the exposure to high light (HL) because of photo-oxidative damage. We used a second-site suppressor screen to identify genes involved in protecting PsaF-deficient PSI from excess light. In doing so, we demonstrated that the absence of the STT7 protein, which is required for LHCII phosphorylation and the process of state transitions suppresses the psaF HL-lethal phenotype. On the basis of chlorophyll fluorescence measurements, the psaF mutant has a more reduced plastoquinone pool at a given photosynthetic photon flux density than the wild-type cells. Under these conditions the process of state transitions will become active, resulting in the transfer of phosphorylated LHCII proteins to PSI, further increasing the excitation of PSI. However, in the psaF stt7 double mutant, the LHCII proteins will not be transferred to PSI, and thus the level of PSI excitation will remain lower. This study provides clear genetic evidence that the HL-lethal phenotype of the psaF mutant is because of PSI overexciation.

Mitochondrial and chloroplastic targeting signals of NADP+-dependent isocitrate dehydrogenase.

Many mitochondrial and chloroplast proteins are encoded in the nucleus and subsequently imported into the organelles via active protein transport systems. While usually highly specific, some proteins are dual-targeted to both organelles. In tobacco (Nicotiana tabacum L.), the cDNA encoding the mitochondrial isoform of NADP+-dependent isocitrate dehydrogenase (NADP+-ICDH) contains two translational ATG start sites, suggesting the possibility of tandem targeting signals. In this work, the putative mitochondrial and chloroplastic targeting signals from NADP+-ICDH were fused to a yellow fluorescent protein (YFP) reporter to generate a series of constructs and introduced into tobacco leaves by Agrobacterium-mediated transient transformation. The subsequent sub-cellular locations of the ICDH:YFP fusion proteins were then examined using confocal microscopy. Constructs predicted to be targeted to the chloroplast all localized to the chloroplast. However, this was not the case for all of the constructs that were predicted to be mitochondrial targeted. Although some constructs localized to mitochondria as expected, others appeared to be chloroplast localized. This was attributed to an additional 50 amino acid residues of the mature NADP+-ICDH protein that were present in those constructs, generated from either 'Xanthi' or 'Petit Havana' cultivars of tobacco. The results of this study raise interesting questions regarding the targeting and processing of organellar isoforms of NADP+-ICDH.