Novel insights into the function of LHCSR3 in Chlamydomonas reinhardtii

Light is essential for photosynthesis but excess light is hazardous as it may lead to the formation of reactive oxygen species. Photosynthetic organisms struggle to optimize light utilization and photosynthesis while minimizing photo-oxidative damage. Hereby light to heat dissipation via specialized proteins is a potent mechanism to acclimate toward excess light. In the green alga Chlamydomonas reinhardtii the expression of an ancient light-harvesting protein LHCSR3 enables cells to dissipate harmful excess energy. Herein we summarize newest insights into the function of LHCSR3 from C. reinhardtii.     

UV-A/B radiation rapidly activates photoprotective mechanisms in Chlamydomonas reinhardtii

Conversion of light energy into chemical energy through photosynthesis in the chloroplasts of photosynthetic organisms is essential for photoautotrophic growth, and non-photochemical quenching (NPQ) of excess light energy prevents the generation of reactive oxygen species and maintains efficient photosynthesis under high light. In the unicellular green alga Chlamydomonas reinhardtii, NPQ is activated as a photoprotective mechanism through wavelength-specific light signaling pathways mediated by the phototropin (blue light) and ultra-violet (UV) light photoreceptors, but the biological significance of photoprotection activation by light with different qualities remains poorly understood. Here, we demonstrate that NPQ-dependent photoprotection is activated more rapidly by UV than by visible light. We found that induction of gene expression and protein accumulation related to photoprotection was significantly faster and greater in magnitude under UV treatment compared with that under blue- or red-light treatment. Furthermore, the action spectrum of UV-dependent induction of photoprotective factors implied that C. reinhardtii senses relatively long-wavelength UV (including UV-A/B), whereas the model dicot plant Arabidopsis (Arabidopsis thaliana) preferentially senses relatively short-wavelength UV (mainly UV-B/C) for induction of photoprotective responses. Therefore, we hypothesize that C. reinhardtii developed a UV response distinct from that of land plants.

In contrast to land plants, which sense short-wave UV light, the unicellular green alga Chlamydomonas reinhardtii senses long-wavelength UV light for photoprotective responses.     

Chloroplast suppressors that act on a mitochondrial mutation in Chlamydomonas reinhardtii

We report genetic evidence supporting the existence of suppressor genes in the chloroplast that act on a mitochondrial mutation that impairs heterotrophic growth in the green alga Chlamydomonas reinhardtii. One of these suppressors also acts on a point mutation in the rbcL gene in the chloroplast. These results are consistent with previous data showing that mitochondrial protein synthesis depends on chloroplast protein synthesis in C. reinhardtii. The nature of the interaction between chloroplasts and mitochondria is discussed in light of the requirement for import of tRNAs by plant mitochondria.     

Expression by Chlamydomonas reinhardtii of a chloroplast ATP synthase with polyhistidine-tagged beta subunits

The green alga Chlamydomonas reinhardtii is a model organism for the study of photosynthesis. The chloroplast ATP synthase is responsible for the synthesis of ATP during photosynthesis. Using genetic engineering and biolistic transformation, a string of eight histidine residues has been inserted into the amino-terminal end of the β subunit of this enzyme in C. reinhardtii. The incorporation of these amino acids did not impact the function of the ATP synthase either in vivo or in vitro and the resulting strain of C. reinhardtii showed normal growth. The addition of these amino acids can be seen through altered gel mobility of the β subunit and the binding of a polyhistidine-specific dye to the subunit. The purified his-tagged CF1 has normal Mg²⁺-ATPase activity, which can be stimulated by alcohol and detergents and the enzyme remains active while bound to a nickel-coated surface. Potential uses for this tagged enzyme as a biochemical tool are discussed.     

The role of respiration in the activation of photosynthesis upon illumination of dark adapted Chlamydomonas reinhardtii

It is reported that O₂ is required for the activation of photosynthesis in dark adapted Chlamydomonas reinhardtii in State 1, under low light intensity. The concentration of dissolved O₂ of ca. 9 µM is sufficient to saturate the requirement. When the concentration of O₂ is 3 μM or below, the activation of photosynthesis is strongly inhibited by myxothiazol, a specific inhibitor of the mitochondrial cytochrome bc₁. The effect of this inhibitor decreases as the O₂ concentration is raised, to disappear completely above 50 μM. Low concentrations of uncouplers delay the activation of photosynthesis, but do not inhibit it when steady state is reached. It is concluded that in State 1 C. reinhardtii mitochondrial respiration is required for the activation of photosynthesis upon illumination of dark adapted cells only when the concentration of O₂ is too low (less than 5 μM) to allow an appreciable activity of the Mehler reaction. The role of respiration does not seem to be due to the synthesis of ATP by oxidative phosphorylation, because photosynthesis activation is not sensitive to oligomycin.     

Cytochrome and alternative pathway respiration in green algae : measurements using inhibitors and o(2) discrimination

Inhibitor titration curves and discrimination against ¹⁸O₂ by mitochondrial respiration in three strains of green algae (Selenastrum minutum [Naeg.] Collins, and two strains of Chlamydomonas reinhardtii Dangeard) with differing respiratory capabilities were determined. Discrimination for cytochrome pathway respiration ranged from 19.89 to 20.43%. Discrimination for alternative pathway respiration by wild-type C. reinhardtii (measured in the presence of KCN) was 25.46%, while discrimination values for a cytochrome oxidase deficient mutant of C. reinhardtii ranged from 24.24 to 24.96%. In the absence of KCN, the alternative pathway was not engaged in wild-type C. reinhardtii, the only algal strain that possessed both cytochrome and alternative pathway capacities.     

Method for assembling and expressing multiple genes in the nucleus of microalgae

The green alga, Chlamydomonas reinhardtii, is a model organism used in the study of photosynthesis and biotechnological research. Despite its importance, a complete set of genetic tools has yet to be developed. Here, we report the development of a new method for constructing a multi-gene pathway in Saccharomyces cerevisiae and integrating the assembled pathway into the nuclear genome of C. reinhardtii. To demonstrate the use of this method, we assembled and functionally expressed up to three reporter proteins (Ble, AphVIII, and GFP) simultaneously in the nucleus of C. reinhardtii. This new molecular tool should aid efforts to engineer microalgae for biofuel and biopharmaceutical production.     

The three genomes of Chlamydomonas

During the past 50 years, the green unicellular alga Chlamydomonas reinhardtii has played a key role as model system for the study of photosynthesis and chloroplast biogenesis. This is due to its well-established nuclear and chloroplast genetics, its dispensable photosynthetic function in the presence of acetate, and its highly efficient nuclear and chloroplast transformation systems. Considerable progress has been achieved in our understanding of the structure, function, inheritance, and expression of nuclear, chloroplast, and mitochondrial genes and of the molecular cross-talk between the nuclear, chloroplast, and mitochondrial genetic systems. This revised version was published online in June 2006 with corrections to the Cover Date.     

Regulation of alternative oxidase 1 in Chlamydomonas reinhardtii during sulfur starvation

The mitochondrial respiratory chain in plants, some protists and many fungi consists of the ATP-coupling cyanide-sensitive cytochrome pathway and the cyanide-resistant alternative respiratory pathway. The alternative pathway is mediated by alternative oxidase (AOX). Although AOX has been proposed to play essential roles in nutrient stress tolerance of plants and protists, the effects of sulfur (S) deprivation, on AOX are largely unknown. The unicellular green alga Chlamydomonas reinhardtii reacts to S limitation conditions with the induced expression of many genes. In this work, we demonstrated that exposure of C. reinhardtii to S deprivation results in the up-regulation of AOX1 expression and an increased AOX1 protein. Furthermore, S-deprived C. reinhardtii cells display the enhanced AOX1 capacity. Moreover, nitrate assimilation regulatory protein (NIT2) is involved in the control of the AOX1 gene expression in the absence of S. Together, the results clearly indicate that AOX1 relates to S limitation stress responses and is regulated in a NIT2-dependent manner, probably together with yet-unknown regulatory factor(s).     

Recycling of the major thylakoid lipid MGDG and its role in lipid homeostasis in Chlamydomonas reinhardtii

Monogalactosyldiacylglycerol (MGDG), the most abundant lipid in thylakoid membranes, is involved in photosynthesis and chloroplast development. MGDG lipase has an important role in lipid remodeling in Chlamydomonas reinhardtii. However, the process related to turnover of the lysogalactolipid that results from MGDG degradation, monogalactosylmonoacylglycerol (MGMG), remains to be clarified. Here we identified a homolog of Arabidopsis thaliana lysophosphatidylcholine acyltransferase (LPCAT) and characterized two independent knockdown (KD) alleles in C. reinhardtii. The enzyme designated as C. reinhardtiiLysolipid Acyltransferase 1 (CrLAT1) has a conserved membrane-bound O-acyl transferase domain. LPCAT from Arabidopsis has a key role in deacylation of phosphatidylcholine (PC). Chlamydomonas reinhardtii, however, lacks PC, and thus we hypothesized that CrLAT1 has some other important function in major lipid flow in this organism. In the CrLAT1 KD mutants, the amount of MGMG was increased, but triacylglycerols (TAGs) were decreased. The proportion of more saturated 18:1 (9) MGDG was lower in the KD mutants than in their parental strain, CC-4533. In contrast, the proportion of MGMG has decreased in the CrLAT1 overexpression (OE) mutants, and the proportion of 18:1 (9) MGDG was higher in the OE mutants than in the empty vector control cells. Thus, CrLAT1 is involved in the recycling of MGDG in the chloroplast and maintains lipid homeostasis in C. reinhardtii.

A recycling system of the major thylakoid lipid monogalactosyldiacylglycerol in the Chlamydomonas reinhardtii chloroplast contributes to lipid homeostasis.     

microRNAs in a multicellular green alga Volvox carteri

microRNAs(miRNAs)have emerged as key components in the eukaryotic gene regulatory network.We and others have previously identified many miRNAs in a unicellular green alga,Chlamydomonas reinhardtii.To investigate whether miRNA-mediated gene regulation is a general mechanism in green algae and how miRNAs have been evolved in the green algal lineage,we examined small RNAs in Volvox carteri,a multicellular species in the same family with Chlamydomonas reinhardtii.We identified 174 miRNAs in Volvox,with many of them being highly enriched in gonidia or somatic cells.The targets of the miRNAs were predicted and many of them were subjected to miRNA-mediated cleavage in vivo,suggesting that miRNAs play regulatory roles in the biology of green algae.Our catalog of miRNAs and their targets provides a resource for further studies on the evolution,biological functions,and genomic properties of miRNAs in green algae.     

High-Throughput Genotyping of Green Algal Mutants Reveals Random Distribution of Mutagenic Insertion Sites and Endonucleolytic Cleavage of Transforming DNA

A high-throughput genetic screening platform in a single-celled photosynthetic eukaryote would be a transformative addition to the plant biology toolbox. Here, we present ChlaMmeSeq (Chlamydomonas MmeI-based insertion site Sequencing), a tool for simultaneous mapping of tens of thousands of mutagenic insertion sites in the eukaryotic unicellular green alga Chlamydomonas reinhardtii. We first validated ChlaMmeSeq by in-depth characterization of individual insertion sites. We then applied ChlaMmeSeq to a mutant pool and mapped 11,478 insertions, covering 39% of annotated protein coding genes. We observe that insertions are distributed in a manner largely indistinguishable from random, indicating that mutants in nearly all genes can be obtained efficiently. The data reveal that sequence-specific endonucleolytic activities cleave the transforming DNA and allow us to propose a simple model to explain the origin of the poorly understood exogenous sequences that sometimes surround insertion sites. ChlaMmeSeq is quantitatively reproducible, enabling its use for pooled enrichment screens and for the generation of indexed mutant libraries. Additionally, ChlaMmeSeq allows genotyping of hits from Chlamydomonas screens on an unprecedented scale, opening the door to comprehensive identification of genes with roles in photosynthesis, algal lipid metabolism, the algal carbon-concentrating mechanism, phototaxis, the biogenesis and function of cilia, and other processes for which C. reinhardtii is a leading model system.     

Biochemical and physiological characterization of the pyruvate formate-lyase Pfl1 of Chlamydomonas reinhardtii, a typically bacterial enzyme in a eukaryotic alga

The unicellular green alga Chlamydomonas reinhardtii has a special type of anaerobic metabolism that is quite unusual for eukaryotes. It has two oxygen-sensitive [Fe-Fe] hydrogenases (EC 1.12.7.2) that are coupled to photosynthesis and, in addition, a formate- and ethanol-producing fermentative metabolism, which was proposed to be initiated by pyruvate formate-lyase (Pfl; EC 2.3.1.54). Pfl enzymes are commonly found in prokaryotes but only rarely in eukaryotes. Both the hydrogen- and the formate/ethanol-producing pathways are involved in a sustained anaerobic metabolism of the alga, which can be induced by sulfur depletion in illuminated cultures. Before now, the presence of a Pfl protein in C. reinhardtii was predicted from formate secretion and the homology of the deduced protein of the PFL1 gene model to known Pfl enzymes. In this study, we proved the formate-producing activity of the putative Pfl1 enzyme by heterologous expression of the C. reinhardtii PFL1 cDNA in Escherichia coli and subsequent in vitro activity tests of the purified protein. Furthermore, a Pfl-deficient E. coli strain secretes formate when expressing the PFL1 cDNA of C. reinhardtii. We also examined the Pfl1 fermentation pathway of C. reinhardtii under the physiological condition of sulfur depletion. Genetic and biochemical analyses show that sulfur-depleted algae express genes encoding enzymes acting downstream of Pfl1 and also potentially ethanol-producing enzymes, such as pyruvate decarboxylase (EC 4.1.1.1) or pyruvate ferredoxin oxidoreductase (EC 1.2.7.1). The latter enzymes might substitute for Pfl1 activity when Pfl1 is specifically inhibited by hypophosphite.