Functional genomics of eukaryotic photosynthesis using insertional mutagenesis of Chlamydomonas reinhardtii

The unicellular green alga Chlamydomonas reinhardtii is a widely used model organism for studies of oxygenic photosynthesis in eukaryotes. Here we describe the development of a resource for functional genomics of photosynthesis using insertional mutagenesis of the Chlamydomonas nuclear genome. Chlamydomonas cells were transformed with either of two plasmids conferring zeocin resistance, and insertional mutants were selected in the dark on acetate-containing medium to recover light-sensitive and nonphotosynthetic mutants. The population of insertional mutants was subjected to a battery of primary and secondary phenotypic screens to identify photosynthesis-related mutants that were pigment deficient, light sensitive, nonphotosynthetic, or hypersensitive to reactive oxygen species. Approximately 9% of the insertional mutants exhibited 1 or more of these phenotypes. Molecular analysis showed that each mutant line contains an average of 1.4 insertions, and genetic analysis indicated that approximately 50% of the mutations are tagged by the transforming DNA. Flanking DNA was isolated from the mutants, and sequence data for the insertion sites in 50 mutants are presented and discussed.     

Functional genomics of plant photosynthesis in the fast lane using Chlamydomonas reinhardtii

Oxygenic photosynthesis by algae and plants supports much of life on Earth. Several model organisms are used to study this vital process, but the unicellular green alga Chlamydomonas reinhardtii offers significant advantages for the genetic dissection of photosynthesis. Recent experiments with Chlamydomonas have substantially advanced our understanding of several aspects of photosynthesis, including chloroplast biogenesis, structure-function relationships in photosynthetic complexes, and environmental regulation. Chlamydomonas is therefore the organism of choice for elucidating detailed functions of the hundreds of genes involved in plant photosynthesis.     

Chlamydomonas reinhardtii: biological rationale for genomics

Chlamydomonas reinhardtii has been the subject of genetic, biochemical, cytological, and molecular analyses for over 50 years. It is an ideal model system for the study of flagella and basal bodies as well as the study of photosynthesis and chloroplast biogenesis, cell-cell recognition and fusion, phototaxis, and secretion. It is clear that many of the genes identified in Chlamydomonas have homologs in land plants as well as animals. Thus, a genomic approach in Chlamydomonas will provide another important avenue for the understanding of important biological processes.     

Chlamydomonas reinhardtii

Recombinant proteins have become more and more important for the pharmaceutical and chemical industry. Although various systems for protein expression have been developed, there is an increasing demand for inexpensive methods of large-scale production. Eukaryotic algae could serve as a novel option for the manufacturing of recombinant proteins, as they can be cultivated in a cheap and easy manner and grown to high cell densities. Being a model organism, the unicellular green alga Chlamydomonas reinhardtii has been studied intensively over the last decades and offers now a complete toolset for genetic manipulation. Recently, the successful expression of several proteins with pharmaceutical relevance has been reported from the nuclear and the chloroplastic genome of this alga, demonstrating its ability for biotechnological applications.     

Effect of methylmercury on primary photosynthesis processes in green microalgae Chlamydomonas reinhardtii

The sensitivity of green microalgae Chlamydomonas reinhardtii to methylmercury chloride (MeHg) and chloride mercury (HgCl2) was evaluated by measuring chlorophyll fluorescence parameters by the pulse-amplitude-modulation (PAM) fluorometry. It was shown that MeHg at concentrations above 1 microM decreased the Fv/Fm ratio, which characterizes the maximal efficiency of energy utilization in photosystem II. The degree of inhibition depended on the time of treatment and was always higher under illumination conditions (50 microE.m-2.s-1) than under dark conditions. A similar regularity was observed for the delta F/Fm' ratio, which characterizes the real efficiency of energy storage at the given intensity of the photosynthesis-exciting light. Incubation with 5 microM HgCl2 for 5 h did not affect both ratios. The decrease in Fm at constant F0 as well as changes in the fast fluorescence kinetics after MeHg treatment of algae cells indicated the damage on the donor side of photosystem II and the damage of the electron transfer from QA to QB. The reduction of photochemical fluorescence quenching (qN) under MeHg treatment is also evidence of the increase in the fraction of closed reaction centers (QA-). At the same time, increase in the steady-state level of P700 photooxidation indicated a disturbance of electron transfer between photosystems. The present study demonstrates that methylmercury treatment damaged the photosynthetic electron transfer chain at several sites. The inhibitory effect of methylmercury is much stronger than the effect of mercury chloride on photosynthetic processes.     

In vivo interactions between photosynthesis,mitorespiration, and chlororespiration in Chlamydomonas reinhardtii

Interactions between photosynthesis, mitochondrial respiration (mitorespiration), and chlororespiration have been investigated in the green alga Chlamydomonas reinhardtii using flash illumination and a bare platinum electrode. Depending on the physiological status of algae, flash illumination was found to induce either a fast (t(1/2) approximately 300 ms) or slow (t(1/2) approximately 3 s) transient inhibition of oxygen uptake. Based on the effects of the mitorespiratory inhibitors myxothiazol and salicyl hydroxamic acid (SHAM), and of propyl gallate, an inhibitor of the chlororespiratory oxidase, we conclude that the fast transient is due to the flash-induced inhibition of chlororespiration and that the slow transient is due to the flash-induced inhibition of mitorespiration. By measuring blue-green fluorescence changes, related to the redox status of the pyridine nucleotide pool, and chlorophyll fluorescence, related to the redox status of plastoquinones (PQs) in C. reinhardtii wild type and in a photosystem I-deficient mutant, we show that interactions between photosynthesis and chlororespiration are favored when PQ and pyridine nucleotide pools are reduced, whereas interactions between photosynthesis and mitorespiration are favored at more oxidized states. We conclude that the plastid oxidase, similar to the mitochondrial alternative oxidase, becomes significantly engaged when the PQ pool becomes highly reduced, and thereby prevents its over-reduction.     

A phenotypic screening platform to identify small molecule modulators of Chlamydomonas reinhardtii growth,motility and photosynthesis

Chemical biology, the interfacial discipline of using small molecules as probes to investigate biology, is a powerful approach of developing specific, rapidly acting tools that can be applied across organisms. The single-celled alga Chlamydomonas reinhardtii is an excellent model system because of its photosynthetic ability, cilia-related motility and simple genetics. We report the results of an automated fitness screen of 5,445 small molecules and subsequent assays on motility/phototaxis and photosynthesis. Cheminformatic analysis revealed active core structures and was used to construct a naïve Bayes model that successfully predicts algal bioactive compounds.     

Discovery of photosynthesis genes through whole-genome sequencing of acetate-requiring mutants of Chlamydomonas reinhardtii

Large-scale mutant libraries have been indispensable for genetic studies, and the development of next-generation genome sequencing technologies has greatly advanced efforts to analyze mutants. In this work, we sequenced the genomes of 660 Chlamydomonas reinhardtii acetate-requiring mutants, part of a larger photosynthesis mutant collection previously generated by insertional mutagenesis with a linearized plasmid. We identified 554 insertion events from 509 mutants by mapping the plasmid insertion sites through paired-end sequences, in which one end aligned to the plasmid and the other to a chromosomal location. Nearly all (96%) of the events were associated with deletions, duplications, or more complex rearrangements of genomic DNA at the sites of plasmid insertion, and together with deletions that were unassociated with a plasmid insertion, 1470 genes were identified to be affected. Functional annotations of these genes were enriched in those related to photosynthesis, signaling, and tetrapyrrole synthesis as would be expected from a library enriched for photosynthesis mutants. Systematic manual analysis of the disrupted genes for each mutant generated a list of 253 higher-confidence candidate photosynthesis genes, and we experimentally validated two genes that are essential for photoautotrophic growth, CrLPA3 and CrPSBP4. The inventory of candidate genes includes 53 genes from a phylogenomically defined set of conserved genes in green algae and plants. Altogether, 70 candidate genes encode proteins with previously characterized functions in photosynthesis in Chlamydomonas, land plants, and/or cyanobacteria; 14 genes encode proteins previously shown to have functions unrelated to photosynthesis. Among the remaining 169 uncharacterized genes, 38 genes encode proteins without any functional annotation, signifying that our results connect a function related to photosynthesis to these previously unknown proteins. This mutant library, with genome sequences that reveal the molecular extent of the chromosomal lesions and resulting higher-confidence candidate genes, will aid in advancing gene discovery and protein functional analysis in photosynthesis.     

Chemoresponses of Chlamydomonas reinhardtii

Cells of Chlamydomonas reinhardtii have been found to respond to chemicals in two ways: chemokinesis and chemotaxis. Several amino acids, fatty acids, and inorganic salts can stimulate these responses.     

Recovery of photosynthesis and phtosystem II fluorescence in Chlamydomonas reinhardtii after exposure to three levels of high light

Recovery from 60 min of photoinhibitory treatment at photosynthetic photon flux densities of 500, 1400 and 2200 μMmol m^?2 s^? was followed in cells of the green alga Chlamydomonas reinhardtii grown at 125 μMmol m^?2 s^?1. These light treatments represent photoregulation, moderate photoinhibition and strong photoinhibition, respectively. Treatment in photoregulatory light resulted in an increased maximal rate of oxygen evolution (P_max) and an increased quantum yield (Φ), but a 15% decrease in F_v/F_M. Treatment at moderately photoinhibitory light resulted in a 30% decrease in F_v/F_M and an approximately equal decrease in Φ. Recovery in dim light restored F_v/F_M within 15 and 45 min after high light treatment at 500 and 1400 μMmol m^?2 s^?1, respectively. Convexity (Θ), a measure of the extent of co-limitation between PS II turnover and whole-chain electron transport, and Φ approached, but did not reach the control level during recovery after exposure to 1400 μMmol m^?2 s^?1, whereas P_max increased above the control. Treatment at 2200 μMmol m^?2 s^?1 resulted in a strong reduction of the modeled parameters Φ, Θ and P_max. Subsequent recovery was initially rapid but the rate decreased, and a complete recovery was not reached within 120 min. Based on the results, it is hypothesized that exposure to high light results in two phenomena. The first, expressed at all three light intensities, involves redistribution within the different aspects of PS II heterogeneity rather than a photoinhibitory destruction of PS II reaction centers. The second, most strongly expressed at 2200 μmol m^?2 s^?1, is a physical damage to PS II shown as an almost total loss of PS II_α and PS II Q_B-reducing centers. Thus recovery displayed two phase, the first was rapid and the only visible phase in algae exposed to 500 and 1400 μmol m^?2 s^?1. The second phase was slow and visible only in the later part of recovery in cells exposed to 2200 μmol m^?2 s^?1.     

Crop evolution: from genetics to genomics

The advent of the genomics age has greatly facilitated the study of crop evolution. While full-scale genome sequencing projects are underway for just a handful of crop plants, recent years have witnessed a tremendous increase in the availability of DNA sequence data for virtually all major crops. Such resources have bolstered 'traditional' genetic approaches such as QTL mapping and candidate gene-based association studies. They have also allowed us to undertake genome-wide analyses in which we simultaneously consider the importance of a large and essentially random collection of genes. These sorts of analyses promise a more or less unbiased view of the genetic basis of crop evolution and will probably result in the identification of agronomically important genes that would have otherwise been overlooked.     

The effect of triacontanol on growth, photosynthesis and photorespiration in Chlamydomonas reinhardtii and Anacystis nidulans

Chlamydomonas reinhardtii Dangerad 11–32(90) (−), which exhibits C3 properties, and Anacystis nidulans (Strain no. UTEX 625), which exhibits C4 properties, were used to study the effects of triacontanol on growth, photosynthesis and photorespiration. Photosynthetic rate was measured as CO2 uptake and the O2 inhibition of photosynthesis was used as a measure of photorespiration. Triacontanol dissolved in chloroform and dispersed in Tween-20 and triacontanol colloidally dispersed in an aqueous solution of sodium tallow alkyl sulfate were tested. Chlamydomonas cultures increased significantly in cell number after 4 days, and in chlorophyll content after 3 days of treatment with 2.3 × 10⁻⁸ M TRIA in chloroform/Tween-20. In cultures of Anacystis the chlorophyll content became significantly higher 3 days after treatment with 2.3 × 10⁻⁹ M TRIA and the cell number was noticeably higher than the controls.
CO₂ uptake by triacontanol-treated Chlamydomonas cultures was about the same in both 2 and 21% O2, and the O2 inhibition was significantly reduced as compared with the controls. Photosynthesis in Anacystis was O2-insensitive under the experimental condition used. When Anacystis was treated with triacontanol there was no change in the rate of CO2 uptake and no change in the O2 sensitivity of its CO2 uptake. It appears that triacontanol affects some process which regulated the balance between photosynthesis and photorespiration, but other processes which result in increased growth are probably also affected.     

Reactivation of photosynthesis in the photoinhibited green alga Chlamydomonas reinhardtii: Role of dark respiration and of light

Effect of quality, quantity and minimum duration of light on the process of recovery was investigated in the photoinhibited cells of the green alga Chlamydomonas reinhardtii. Complete and rapid reactivation of photosynthesis took place in diffuse white light of 25 mol m^–2 s^–1. The recovery was partial (< 10%) in the dark. Far red (725 nm), red (660 nm) and blue light (480 nm) in the range of 10 to 75 mol m^–2 s^–1 did not enhance the process of reactivation. Photoinhibited cells incubated in dark for 15 min when exposed for 5 min to diffuse light (25 mol m^–2 s^–1) showed complete reactivation. Even exposure of 15 min dark incubated photoinhibited cells to photoinhibitory light (2500 mol m^–2 s^–1) for 5 s fully regained the photosynthesis. The study indicated a very precise and triggering effect of light in the process of reactivation. The dark respiratory inhibitor KCN and uncouplers FCCP and CCCP increased the susceptibility of C. reinhardtii to photoinhibition and also prevented photoinhibited cells to reactivate fully even after longer period of incubation under suitable reactivating conditions. Of the various possibilities envisaged to assign the role of dark respiration in recovery process, supply of ATP by mitochondrial respiration appeared sound and pertinent.Abbreviations CCCP- carbonyl cyanide m-chlorophenylhydrazone - D1- 32 kDa protein of PS II reaction center - FCCP- carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone - KCN- potassium cyanide - PBQ- phenyl-p-benzoquinone - PFD- photon flux density - SHAM- salicylhydroxamic acid NBRI Research Publication No. 431.     

Posttranslational events leading to the assembly of photosystem II protein complex: a study using photosynthesis mutants from Chlamydomonas reinhardtii

We studied the assembly of photosystem II (PSII) in several mutants from Chlamydomonas reinhardtii which were unable to synthesize either one PSII core subunit (P6 [43 kD], D1, or D2) or one oxygen-evolving enhancer (OEE1 or OEE2) subunit. Synthesis of the PSII subunits was analyzed on electrophoretograms of cells pulse labeled with [14C]acetate. Their accumulation in thylakoid membranes was studied on immunoblots, their chlorophyll-binding ability on nondenaturating gels, their assembly by detergent fractionation, their stability by pulse-chase experiments and determination of in vitro protease sensitivity, and their localization by immunocytochemistry. In Chlamydomonas, the PSII core subunits P5 (47 kD), D1, and D2 are synthesized in a concerted manner while P6 synthesis is independent. P5 and P6 accumulate independently of each other in the stacked membranes. They bind chlorophyll soon after, or concomitantly with, their synthesis and independently of the presence of the other PSII subunits. Resistance to degradation increases step by step: beginning with assembly of P5, D1, and D2, then with binding of P6, and, finally, with binding of the OEE subunits on two independent high affinity sites (one for OEE1 and another for OEE2 to which OEE3 binds). In the absence of PSII cores, the OEE subunits accumulate independently in the thylakoid lumen and bind loosely to the membranes; OEE1 was found on stacked membranes, but OEE2 was found on either stacked or unstacked membranes depending on whether or not P6 was synthesized.     

Portrait of a species: Chlamydomonas reinhardtii

Chlamydomonas reinhardtii, the first alga subject to a genome project, has been the object of numerous morphological, physiological, and genetic studies. The organism has two genetically determined mating types (plus and minus) and all stages of the simple life cycle can be evoked in culture. In the nearly 60 years since the first standard laboratory strains were isolated, numerous crosses and exchanges among laboratories have led to some confusion concerning strain genealogy. Here we use analyses of the nuclear internal transcribed spacer regions and other genetic traits to resolve these issues, correctly identify strains currently available, and analyze phylogenetic relationships with all other available similar chlamydomonad types. The presence of a 10-bp indel in ITS2 in some but not all copies of the nuclear ribosomal cistrons of an individual organism, and the changing ratios of these in crosses, provide a tool to investigate mechanisms of concerted evolution. The standard C. reinhardtii strains, plus C. smithii +, plus the new eastern North American C. reinhardtii isolates, comprise one morphological species, one biological species of high sexual intercompatibility, and essentially identical ITS sequences (except the tip of helix I of ITS2). However, variant RFLP patterns characterize strains from each geographic site.     

Mechanistic modeling of sulfur‐deprived photosynthesis and hydrogen production in suspensions of Chlamydomonas reinhardtii

The ability of unicellular green algal species such as Chlamydomonas reinhardtii to produce hydrogen gas via iron‐hydrogenase is well known. However, the oxygen‐sensitive hydrogenase is closely linked to the photosynthetic chain in such a way that hydrogen and oxygen production need to be separated temporally for sustained photo‐production. Under illumination, sulfur‐deprivation has been shown to accommodate the production of hydrogen gas by partially‐deactivating O₂ evolution activity, leading to anaerobiosis in a sealed culture. As these facets are coupled, and the system complex, mathematical approaches potentially are of significant value since they may reveal improved or even optimal schemes for maximizing hydrogen production. Here, a mechanistic model of the system is constructed from consideration of the essential pathways and processes. The role of sulfur in photosynthesis (via PSII) and the storage and catabolism of endogenous substrate, and thus growth and decay of culture density, are explicitly modeled in order to describe and explore the complex interactions that lead to H₂ production during sulfur‐deprivation. As far as possible, functional forms and parameter values are determined or estimated from experimental data. The model is compared with published experimental studies and, encouragingly, qualitative agreement for trends in hydrogen yield and initiation time are found. It is then employed to probe optimal external sulfur and illumination conditions for hydrogen production, which are found to differ depending on whether a maximum yield of gas or initial production rate is required. The model constitutes a powerful theoretical tool for investigating novel sulfur cycling regimes that may ultimately be used to improve the commercial viability of hydrogen gas production from microorganisms. Biotechnol. Bioeng. 2014;111: 320–335. © 2013 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.     

Chlamydomonas: Fast tracking from genomics

Elucidating biological processes has relied on the establishment of model organisms, many of which offer advantageous features such as rapid axenic growth, extensive knowledge of their physiological features and gene content, and the ease with which they can be genetically manipulated. The unicellular green alga Chlamydomonas reinhardtii has been an exemplary model that has enabled many scientific breakthroughs over the decades, especially in the fields of photosynthesis, cilia function and biogenesis, and the acclimation of photosynthetic organisms to their environment. Here, we discuss recent molecular/technological advances that have been applied to C. reinhardtii and how they have further fostered its development as a “flagship” algal system. We also explore the future promise of this alga in leveraging advances in the fields of genomics, proteomics, imaging, and synthetic biology for addressing critical future biological issues.     

Glycolate: Dichlorophenolindophenol Oxidoreductase in Chlamydomonas reinhardtii

Glycolate: dichlorophenolindophenol oxidoreductase levels in Chlamydomonas reinhardtii are not under the immediate control of the CO₂ tension to which the cells are exposed. The enzyme is synthesized initially in 5% CO₂ in air at a similar rate to that in air. It disappears from the cell under nitrogen-limitation.