Lipidomic Analysis of Chlamydomonas reinhardtii under Nitrogen and Sulfur Deprivation

Chlamydomonas reinhardtii accumulates lipids under complete nutrient starvation conditions while overall growth in biomass stops. In order to better understand biochemical changes under nutrient deprivation that maintain production of algal biomass, we used a lipidomic assay for analyzing the temporal regulation of the composition of complex lipids in C. reinhardtii in response to nitrogen and sulfur deprivation. Using a chip-based nanoelectrospray direct infusion into an ion trap mass spectrometer, we measured a diversity of lipid species reported for C. reinhardtii, including PG phosphatidylglycerols, PI Phosphatidylinositols, MGDG monogalactosyldiacylglycerols, DGDG digalactosyldiacylglycerols, SQDG sulfoquinovosyldiacylglycerols, DGTS homoserine ether lipids and TAG triacylglycerols. Individual lipid species were annotated by matching mass precursors and MS/MS fragmentations to the in-house LipidBlast mass spectral database and MS2Analyzer. Multivariate statistics showed a clear impact on overall lipidomic phenotypes on both the temporal and the nutrition stress level. Homoserine-lipids were found up-regulated at late growth time points and higher cell density, while triacyclglycerols showed opposite regulation of unsaturated and saturated fatty acyl chains under nutritional deprivation.     

The Requirement for Carotenoids in the Assembly and Function of the Photosynthetic Complexes in Chlamydomonas reinhardtii

We have investigated the importance of carotenoids on the accumulation and function of the photosynthetic apparatus using a mutant of the green alga Chlamydomonas reinhardtii lacking carotenoids. The FN68 mutant is deficient in phytoene synthase, the first enzyme of the carotenoid biosynthesis pathway, and therefore is unable to synthesize any carotenes and xanthophylls. We find that FN68 is unable to accumulate the light-harvesting complexes associated with both photosystems as well as the RC subunits of photosystem II. The accumulation of the cytochrome b₆f complex is also strongly reduced to a level approximately 10% that of the wild type. However, the residual fraction of assembled cytochrome b₆f complexes exhibits single-turnover electron transfer kinetics comparable to those observed in the wild-type strain. Surprisingly, photosystem I is assembled to significant levels in the absence of carotenoids in FN68 and possesses functional properties that are very similar to those of the wild-type complex.Carotenoids (Cars) are fundamental components of the photosynthetic apparatus (Young and Britton, 1993, and refs. therein). The vast majority of Cars are noncovalently bound to either the core or the antenna subunits of PSI or PSII (Siefermann-Harms, 1985; Bassi et al., 1993). The most abundant Car bound to the core subunits of both photosystems is β-carotene, which is found in the vast majority of oxygenic organisms (Siefermann-Harms, 1985; Bassi et al., 1993). The light-harvesting complexes (LHCs) that act as the outer antenna in plants and green algae bind a wider range of oxygenated Cars, known as xanthophylls, the most abundant of which is lutein (Siefermann-Harms, 1985; Bassi et al., 1993; Jennings et al., 1996). The stoichiometry of xanthophylls binding to LHC complexes depends on the particular complexes and often on the illumination conditions during the organism’s growth (Siefermann-Harms, 1985; Demmig-Adams, 1990; Horton et al., 1996). Intriguingly, a molecule of β-carotene (as well as a molecule of chlorophyll [Chl] a) is found also in the cytochrome (Cyt) b₆f complex (Kurisu et al., 2003; Stroebel et al., 2003).Cars have multiple functions in the photosynthetic process; they act as light-harvesting pigments (Frank and Cogdell, 1993), enlarging the optical cross section to radiation that is poorly absorbed by Chl. Moreover, Cars play a crucial role in processes such as nonphotochemical quenching that control the efficiency of light harvesting in response to the intensity of the incident radiation (for review, see Demmig-Adams, 1990; Horton et al., 1996; Niyogi, 1999). Probably the most important role of Cars in photosynthesis is the quenching of the excited triplet state of Chl (for review, see Frank and Cogdell, 1993; Giacometti et al., 2007), preventing the formation of highly reactive singlet oxygen, which represents the principal species active under high light stress (Hideg et al., 1994; Krieger-Liszkay, 2005). The importance of Cars is demonstrated by the observation that disruption of their biosynthesis through mutation, or by inhibition of a key enzyme in the pathway, leads to either lethal phenotypes or to rapid photobleaching of the photosynthetic tissue (Claes, 1957; Faludi-Dániel et al., 1968, 1970; Bolychevtseva et al., 1995; Trebst and Depka, 1997).Moreover, it has been shown that the presence of xanthophylls is absolutely necessary for refolding in vitro of LHC I and LHC II antenna complexes (Plumley and Schmidt, 1987; Paulsen et al., 1993; Sandonà et al., 1998). Such Cars, therefore, have a structural role, as well as their involvement in light harvesting, nonphotochemical quenching regulation, and the quenching of the Chl triplet state. Whether Cars also play a key structural role in the formation and stability of the core complexes of both PSI and PSII has not been systematically explored, since assembly of these complexes in vitro is not feasible. Studies in vivo using higher plants are complicated by the fact that Car deficiency is lethal and can be studied only during the early stages of greening and leaf development (Faludi-Dániel et al., 1968, 1970; Inwood et al., 2008). In these studies, it was shown that the accumulation of PSII complexes was greatly impaired in mutants of maize (Zea mays; Faludi-Dániel et al., 1968, 1970; Inwood et al., 2008), while the assembly of PSI appeared to be less sensitive to Car availability. In mutants of the cyanobacterium Synechocystis sp. PCC 6803 lacking the genes for phytoene desaturase or ζ-carotene desaturase, there was a complete loss of PSII assembly, while functional PSI complexes were assembled, albeit with slightly altered electron transfer kinetics with respect to the wild-type complex (Bautista et al., 2005). In agreement with the higher sensitivity of PSII assembly to Car availability, Trebst and Depka (1997) reported a specific effect on the synthesis of the D1 subunit of PSII RC upon treatment with phytoene desaturase inhibitors. On the other hand, it has recently been reported that in lycopene-β-cyclase mutants of Arabidopsis (Arabidopsis thaliana) that have a decreased amount of β-carotene (bound to the RC) with respect to most of the xanthophyll pool pigments (bound to the LHCs), the level of accumulation of PSI complexes, particularly that of the LHC I complement, was more affected that that of PSII, probably also because of an increased sensitivity to photodamage of mutated PSI RC (Cazzaniga et al., 2012; Fiore et al., 2012).In this investigation, we have studied the accumulation and functionality of the major chromophore-binding complexes of the photosynthetic apparatus, PSI, PSII, and Cyt b₆f, in a Car-less mutant of the green alga Chlamydomonas reinhardtii (FN68) that is blocked at the first committed step of Car biosynthesis, namely, phytoene synthesis (McCarthy et al., 2004). Although the mutant is incapable of growing under phototrophic or photomixotrophic conditions, it can grow in complete darkness on a medium supplemented with a carbon source. Here, we show that the PSII core and antenna complexes fail to accumulate in the mutant and that the Cyt b₆f complex accumulates to approximately one-tenth of the wild-type level. On the other hand, the PSI reaction center accumulates in FN68 and possesses electron transfer properties that are remarkably similar to those of wild-type PSI. Interestingly, we find that the level of PSI accumulation differs in other phytoene synthase null mutants, suggesting that additional mutations in one or other of these strains affect PSI stability. Nevertheless, our findings demonstrate that Cars are not required for either the assembly or the functionality of PSI in vivo.     

The Regulation of Photosynthetic Electron Transport during Nutrient Deprivation in Chlamydomonas reinhardtii

The light-saturated rate of photosynthetic O₂ evolution in Chlamydomonas reinhardtii declined by approximately 75% on a per-cell basis after 4 d of P starvation or 1 d of S starvation. Quantitation of the partial reactions of photosynthetic electron transport demonstrated that the light-saturated rate of photosystem (PS) I activity was unaffected by P or S limitation, whereas light-saturated PSII activity was reduced by more than 50%. This decline in PSII activity correlated with a decline in both the maximal quantum efficiency of PSII and the accumulation of the secondary quinone electron acceptor of PSII nonreducing centers (PSII centers capable of performing a charge separation but unable to reduce the plastoquinone pool). In addition to a decline in the light-saturated rate of O₂ evolution, there was reduced efficiency of excitation energy transfer to the reaction centers of PSII (because of dissipation of absorbed light energy as heat and because of a transition to state 2). These findings establish a common suite of alterations in photosynthetic electron transport that results in decreased linear electron flow when C. reinhardtii is limited for either P or S. It was interesting that the decline in the maximum quantum efficiency of PSII and the accumulation of the secondary quinone electron acceptor of PSII nonreducing centers were regulated specifically during S-limited growth by the SacI gene product, which was previously shown to be critical for the acclimation of C. reinhardtii to S limitation (J.P. Davies, F.H. Yildiz, and A.R. Grossman [1996] EMBO J 15: 2150–2159).     

A Galactoglycerolipid Lipase Is Required for Triacylglycerol Accumulation and Survival Following Nitrogen Deprivation in Chlamydomonas reinhardtii

Following N deprivation, microalgae accumulate triacylglycerols (TAGs). To gain mechanistic insights into this phenomenon, we identified mutants with reduced TAG content following N deprivation in the model alga Chlamydomonas reinhardtii. In one of the mutants, the disruption of a galactoglycerolipid lipase-encoding gene, designated PLASTID GALACTOGLYCEROLIPID DEGRADATION1 (PGD1), was responsible for the primary phenotype: reduced TAG content, altered TAG composition, and reduced galactoglycerolipid turnover. The recombinant PGD1 protein, which was purified from Escherichia coli extracts, hydrolyzed monogalactosyldiacylglycerol into its lyso-lipid derivative. In vivo pulse-chase labeling identified galactoglycerolipid pools as a major source of fatty acids esterified in TAGs following N deprivation. Moreover, the fatty acid flux from plastid lipids to TAG was decreased in the pgd1 mutant. Apparently, de novo–synthesized fatty acids in Chlamydomonas reinhardtii are, at least partially, first incorporated into plastid lipids before they enter TAG synthesis. As a secondary effect, the pgd1 mutant exhibited a loss of viability following N deprivation, which could be avoided by blocking photosynthetic electron transport. Thus, the pgd1 mutant provides evidence for an important biological function of TAG synthesis following N deprivation, namely, relieving a detrimental overreduction of the photosynthetic electron transport chain.     

Photosynthetic efficiency of Chlamydomonas reinhardtii in flashing light

Efficient light to biomass conversion in photobioreactors is crucial for economically feasible microalgae production processes. It has been suggested that photosynthesis is enhanced in short light path photobioreactors by mixing‐induced flashing light regimes. In this study, photosynthetic efficiency and growth of the green microalga Chlamydomonas reinhardtii were measured using LED light to simulate light/dark cycles ranging from 5 to 100 Hz at a light‐dark ratio of 0.1 and a flash intensity of 1000 µmol m⁻² s⁻¹. Light flashing at 100 Hz yielded the same photosynthetic efficiency and specific growth rate as cultivation under continuous illumination with the same time‐averaged light intensity (i.e., 100 µmol m⁻² s⁻¹). The efficiency and growth rate decreased with decreasing flash frequency. Even at 5 Hz flashing, the rate of linear electron transport during the flash was still 2.5 times higher than during maximal growth under continuous light, suggesting storage of reducing equivalents during the flash which are available during the dark period. In this way the dark reaction of photosynthesis can continue during the dark time of a light/dark cycle. Understanding photosynthetic growth in dynamic light regimes is crucial for model development to predict microalgal photobioreactor productivities. Biotechnol. Bioeng. 2011;108: 2905–2913. © 2011 Wiley Periodicals, Inc.     

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).     

The Type II NADPH Dehydrogenase Facilitates Cyclic Electron Flow,Energy-Dependent Quenching,and Chlororespiratory Metabolism during Acclimation of Chlamydomonas reinhardtii to Nitrogen Deprivation

When photosynthetic organisms are deprived of nitrogen (N), the capacity to grow and assimilate carbon becomes limited, causing a decrease in the productive use of absorbed light energy and likely a rise in the cellular reduction state. Although there is a scarcity of N in many terrestrial and aquatic environments, a mechanistic understanding of how photosynthesis adjusts to low-N conditions and the enzymes/activities integral to these adjustments have not been described. In this work, we use biochemical and biophysical analyses of photoautotrophically grown wild-type and mutant strains of Chlamydomonas reinhardtii to determine the integration of electron transport pathways critical for maintaining active photosynthetic complexes even after exposure of cells to N deprivation for 3 d. Key to acclimation is the type II NADPH dehydrogenase, NDA2, which drives cyclic electron flow (CEF), chlororespiration, and the generation of an H⁺ gradient across the thylakoid membranes. N deprivation elicited a doubling of the rate of NDA2-dependent CEF, with little contribution from PGR5/PGRL1-dependent CEF. The H⁺ gradient generated by CEF is essential to sustain nonphotochemical quenching, while an increase in the level of reduced plastoquinone would promote a state transition; both are necessary to down-regulate photosystem II activity. Moreover, stimulation of NDA2-dependent chlororespiration affords additional relief from the elevated reduction state associated with N deprivation through plastid terminal oxidase-dependent water synthesis. Overall, rerouting electrons through the NDA2 catalytic hub in response to photoautotrophic N deprivation sustains cell viability while promoting the dissipation of excess excitation energy through quenching and chlororespiratory processes.Oxygenic photosynthesis involves the conversion of light energy into chemical bond energy by plants, green algae, and cyanobacteria and the use of that energy to fix CO₂. The photosynthetic electron transport system, located in thylakoid membranes, involves several major protein complexes: PSII (water-plastoquinone oxidoreductase), cytochrome b₆f (cyt b6f; plastoquinone-plastocyanin oxidoreductase), PSI (plastocyanin-ferredoxin oxidoreductase), and the ATP synthase (CF_oCF₁). Light energy absorbed by the photosynthetic apparatus is used to establish both linear electron flow (LEF) and cyclic electron flow (CEF), which drive the production of ATP and NADPH, the chemical products of the light reactions needed for CO₂ fixation in the Calvin-Benson-Bassham (CBB) cycle.With the absorption of light energy by pigment-protein complexes associated with PSII, energy is funneled into unique chlorophyll (Chl) molecules located in the PSII reaction center (RC), where it can elicit a charge separation that generates a large enough oxidizing potential to extract electrons from water. In LEF, electrons from PSII RCs are transferred sequentially along a set of electron carriers, initially reducing the plastoquinone (PQ) pool, then the cyt b₆f complex, and subsequently the lumenal electron carrier plastocyanin (PC). Light energy absorbed by PSI excites a special pair of Chl molecules (P700), causing a charge separation that generates the most negative redox potential in nature (Nelson and Yocum, 2006). The energized electron, which is replaced by electrons from PC, is sequentially transferred to ferredoxin and ferredoxin NADP⁺ reductase, generating reductant in the form of NADPH.Electron transport from water to NADPH in LEF is accompanied by the transport of H⁺ into the thylakoid lumen. For each water molecule oxidized, two H⁺ are released in the thylakoid lumen. In addition, H⁺ are moved into the lumen by the transfer of electrons through cyt b₆f (Q cycle). H⁺ accumulation in the thylakoid lumen dramatically alters the lumenal pH, and the transmembrane H⁺ gradient (ΔpH) together with the transmembrane ion gradient constitute the proton motive force (pmf), which drives ATP formation by ATP synthase (Mitchell, 1961, 1966, 2011). This pmf also promotes other cellular processes, including the dissipation of excess absorbed excitation energy as heat in a photoprotective process (see below; Li et al., 2009; Erickson et al., 2015). The NADPH and ATP molecules generated by LEF and CEF fuel the synthesis of reduced carbon backbones (in the CBB cycle) used in the production of many cellular metabolites and fixed carbon storage polymers.A basic role for CEF is to increase the ATP-NADPH ratio, which can satisfy the energy requirements of the cell and augment the synthesis of ATP by LEF, which is required to sustain CO₂ fixation by the CBB cycle (Allen, 2003; Kramer et al., 2004; Iwai et al., 2010; Alric, 2014). There are two distinct CEF pathways identified in plants and algae. In both pathways, electrons flow from the PQ pool through cyt b₆f to reduce the oxidized form of P700 (P700⁺). In one CEF pathway, electrons are transferred back to the PQ pool prior to the formation of NADPH. This route involves the proteins PGR5 and PGRL1 (DalCorso et al., 2008; Tolleter et al., 2011; Hertle et al., 2013) and is termed PGR5/L1-dependent CEF. A second route for CEF includes an NADPH dehydrogenase that oxidizes NADPH (product of LEF) to NADP⁺, simultaneously reducing the PQ (Allen, 2003; Kramer et al., 2004; Rumeau et al., 2007). The reduced PQ pool is then oxidized by cyt b₆f, causing H⁺ translocation into the thylakoid lumen, followed by the transfer of electrons to P700⁺ via PC. In the green alga Chlamydomonas reinhardtii, this second route for CEF involves a type II NADPH dehydrogenase (NDA2; Jans et al., 2008; Desplats et al., 2009).Oxygenic photosynthetic organisms have inhabited the planet for approximately 3 billion years and have developed numerous strategies to acclimate to environmental fluctuations. These acclimation processes confer flexibility to the photosynthetic machinery, allowing it to adjust to changes in conditions that impact the metabolic/energetic state of the organism and, most importantly, the formation of reactive oxygen species that may damage the photosynthetic apparatus and other cellular components (Li et al., 2009). Several ways in which the photosynthetic apparatus adjusts to environmental fluctuations have been established. A well-studied acclimation process, nonphotochemical quenching (NPQ), reduces the excitation pressure on PSII when oxidized downstream electron acceptors are not available (Eberhard et al., 2008; Li et al., 2009; Erickson et al., 2015). Several processes constitute NPQ, as follows. (1) qT, which involves the physical movement of light-harvesting complexes (LHCs) from one photosystem to another (this is also designated state transitions; Rochaix, 2014). (2) qE, which involves thermal dissipation of the excitation energy. This energy-dependent process requires an elevated ΔpH and involves an LHC-like protein, LHCSR3 (in C. reinhardtii) or PSBS (in plants), as well as the accumulation of specific xanthophylls (mainly lutein in C. reinhardtii and zeaxanthin in plants; Niyogi et al., 1997b; Li et al., 2000, 2004; Peers et al., 2009). (3) qZ, which is energy independent and involves the accumulation of zeaxanthin (Dall’Osto et al., 2005; Nilkens et al., 2010). (4) qI, which promotes quenching following physical damage to PSII core subunits (Aro et al., 1993). Additional mechanisms that can impact LEF and CEF are the synthesis and degradation of pigment molecules, changes in levels of RC and antenna complexes, and the control of electron distribution between LEF and CEF as the energetic demands of the cell change (Allen, 2003; Kramer et al., 2004). In addition, electrons can be consumed by mitochondrial and chlororespiratory activities (Bennoun, 1982; Peltier and Cournac, 2002; Johnson et al., 2014; Bailleul et al., 2015). The latter mainly involves the plastid terminal oxidase PTOX2, which catalyzes the oxidation of the PQ pool and the reduction of oxygen and H⁺ to form water molecules (Houille-Vernes et al., 2011; Nawrocki et al., 2015).Photosynthetic processes also must be modulated as organisms experience changes in the levels of available nutrients (Grossman and Takahashi, 2001). The macronutrient nitrogen (N), which represents 3% to 5% of the dry weight of photosynthetic organisms, is required to synthesize many biological molecules (e.g. amino acids, nucleic acids, and various metabolites) and also participates in posttranslational modifications of proteins (e.g. S-nitrosylation; Romero-Puertas et al., 2013). Importantly, N is highly abundant in chloroplasts in the form of DNA, ribosomes, Chl, and polypeptides (e.g. Rubisco and LHCs; Evans, 1989; Raven, 2013). Furthermore, there is a strong integration between N and carbon assimilation. During N limitation under photoautotrophic conditions, the inability of the organism to synthesize amino acids and other N-containing molecules necessary for cell growth and division can feed back to inhibit both carbon fixation by the CBB cycle and electron transport processes and also can negatively impact the expression of genes encoding key CBB cycle enzymes (Terashima and Evans, 1988; Huppe and Turpin, 1994; Nunes-Nesi et al., 2010).C. reinhardtii is a well-established model organism in which to study photosynthesis and acclimation processes, including acclimation to nutrient limitation (Wykoff et al., 1998; Grossman and Takahashi, 2001; Moseley et al., 2006; Grossman et al., 2009; Terauchi et al., 2010; Aksoy et al., 2013). This unicellular alga grows rapidly as a photoheterotroph (on fixed carbon in the light) or as a heterotroph (on fixed carbon in the dark), has completely sequenced nuclear, chloroplast, and mitochondrial genomes, can be used for classical genetic analyses, and is haploid, which makes some aspects of molecular manipulation (e.g. the generation of knockout mutants) easier (Merchant et al., 2007; Blaby et al., 2014). In the past few years, there have been many studies on the ways in which C. reinhardtii responds to N deprivation (Bulté and Wollman, 1992; Blaby et al., 2013; Goodenough et al., 2014; Schmollinger et al., 2014; Wei et al., 2015; Juergens et al., 2015). Cells deprived of N under photoheterotrophic conditions (i.e. acetate as an external carbon source) minimize the use of N (referred to as N sparing) and induce mechanisms associated with scavenging N from both external and internal pools, all of which eventually lead to proteome modifications and an elevated carbon-N ratio (Schmollinger et al., 2014). Acclimation under photoheterotrophic conditions also causes dramatic modifications of cellular metabolism and energetics: photosynthesis is down-regulated at multiple levels, with a portion of its N content recycled (mainly Chl and polypeptides of the photosynthetic apparatus), while there is enhanced accumulation of mitochondrial complexes leading to increased respiratory activity (Schmollinger et al., 2014; Juergens et al., 2015). Additionally, while fixed carbon cannot be used for growth in the absence of N, it may be stored as starch and triacylglycerol (Work et al., 2010; Siaut et al., 2011; Davey et al., 2014; Goodenough et al., 2014).In contrast to the acclimation of photoheterotrophically grown C. reinhardtii to N deprivation, little is known about how the photosynthetic machinery in this alga adjusts in response to N deprivation under photoautotrophic conditions, when the cells absolutely require photosynthetic energy generation for maintenance. Specifically, we sought to understand how photosynthesis adjusts to metabolic restrictions that slow down the CBB cycle, which in turn could cause the accumulation of photoreductant, particularly NADPH, as the demand for electrons declines (Peltier and Schmidt, 1991; Rumeau et al., 2007). Based on analyses of mutants and the use of spectroscopic and fluorescence measurements, we established a critical role for NDA2 in the acclimation of C. reinhardtii to N deprivation under photoautotrophic conditions, including (1) an augmented capacity for alternative routes of electron utilization (which decrease the NADPH-NADP⁺ ratio) based on increased NDA2-dependent CEF and chlororespiration, and (2) elevated qE, which relies on the H⁺ gradient generated by NDA2-dependent CEF.     

Effect of Nitrogen Supply on the Kinetics and Regulation of Nitrate Assimilation in Chlamydomonas reinhardtii Dangeard

Concentration-dependent NO     

Photosynthetic efficiency of Chlamydomonas reinhardtii in attenuated, flashing light

As a result of mixing and light attenuation, algae in a photobioreactor (PBR) alternate between light and dark zones and, therefore, experience variations in photon flux density (PFD). These variations in PFD are called light/dark (L/D) cycles. The objective of this study was to determine how these L/D cycles affect biomass yield on light energy in microalgae cultivation. For our work, we used controlled, short light path, laboratory, turbidostat‐operated PBRs equipped with a LED light source for square‐wave L/D cycles with frequencies from 1 to 100 Hz. Biomass density was adjusted that the PFD leaving the PBR was equal to the compensation point of photosynthesis. Algae were acclimated to a sub‐saturating incident PFD of 220 µmol m^?2 s^?1 for continuous light. Using a duty cycle of 0.5, we observed that L/D cycles of 1 and 10 Hz resulted on average in a 10% lower biomass yield, but L/D cycles of 100 Hz resulted on average in a 35% higher biomass yield than the yield obtained in continuous light. Our results show that interaction of L/D cycle frequency, culture density and incident PFD play a role in overall PBR productivity. Hence, appropriate L/D cycle setting by mixing strategy appears as a possible way to reduce the effect that dark zone exposure impinges on biomass yield in microalgae cultivation. The results may find application in optimization of outdoor PBR design to maximize biomass yields. Biotechnol. Bioeng. 2012; 109: 2567–2574. © 2012 Wiley Periodicals, Inc.     

Regulation of Photosynthetic Capacity in Chlamydomonas mundana

A regulatory system has been described in the obligately phototrophic green alga Chlamydomonas mundana. Cells grown in acetate media are unable to fix carbon dioxide in the light but carry out a photoassimilation of acetate to carbohydrate: cells cultured with carbon dioxide as the sole source of cellular carbon carry out typical green plant photosynthesis. The control appears to take place at the level of the reductive pentose phosphate cycle. The presence of sodium acetate in the medium strongly inhibits formation of ribulose-1.5-diphosphate carboxylase, ribulose-5-phosphate kinase, and one of the 2 fructose-1,6-diphosphate aldolase activities of the cell. Ribose-5-phosphate isomerase is present in higher activity in autotrophic cells. Changes in the levels of triose phosphate dehydrogenase were also noted. The total pigment content of the cell and the photosynthetic electron transport reactions are not altered under different conditions of growth.     

Blue Light Regulation of Cell Division in Chlamydomonas reinhardtii

A delay in cell division was observed when synchronized cultures of the unicellular green alga Chlamydomonas reinhardtii growing under heterotrophic conditions were exposed to white light during the second half of the growth period. This effect was also observed when photosynthesis was blocked by addition of the photosystem II inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea. Light pulses of 10 minutes were sufficient to induce a delay in cell division in the presence or absence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea. A delay in cell division was induced by blue light but not by illumination with red or far-red light. The equal intensity action spectrum revealed two peaks at 400 and 500 nm.     

Heavy-Metal Regulation of Thioredoxin Gene Expression in Chlamydomonas reinhardtii

Heavy metals are highly toxic compounds for cells. In this report we demonstrate that the expression of Chlamydomonas reinhardtii thioredoxins (TRX) m and h is induced by heavy metals. Upon exposure of the cells to Cd and Hg, a strong accumulation of both messengers was observed. Western-blot experiments revealed that among these two TRXs, only TRX h polypeptides accumulated in response to the toxic cations. A biochemical analysis indicated that heavy metals inhibit TRX activity, presumably by binding at the level of their active site. Sequence analysis of the C. reinhardtii TRX h promoter revealed the presence of cis-acting elements related to cadmium induction. The origins and purposes of this regulation are discussed. Our data suggest, for the first time to our knowledge, a possible implication of TRXs in defense mechanisms against heavy metals.     

Dark Anaerobic Inactivation of Photosynthetic Oxygen Evolution by Chlamydomonas reinhardtii

When intact cells of Chlamydomonas reinhardtii were anaerobicallyincubated in the dark, rapid inactivation of oxygen evolutionwith benzoquinone as the Hill oxidant occurred. Measurementsof electron transport using thylakoids isolated after anaerobictreatment showed that the inactivation occurred at, or before,the secondary electron acceptor of PS II, whereas PS I activitywas largely unaffected. In addition, after anaerobic treatmentfluorescence transients measured with no addition or with dibromomethylisopropylbenzoquinonepresent were virtually the same as those obtained with DCMUpresent. When 10 mM NaHCO₃ was added to inactivated cells, partof the oxygen evolution capacity was restored rapidly. However,almost complete recovery (within 20 to 25 min) required theaddition of oxygen as well. This recovery was not light-dependentand was faster in the presence of 1 mM KCN. We suggest thatthe in activation of benzoquinone-dependent oxygen evolutionwas due to both bicarbonate depletion and reduction of the plastoquinonepool. ¹Present address: Institute of Molecular Biophysics, FloridaState University, Tallahassee, Florida 32306, U.S.A. (Received January 17, 1984; Accepted February 25, 1984)     

Function and dynamics of PKD2 in Chlamydomonas reinhardtii flagella

To analyze the function of ciliary polycystic kidney disease 2 (PKD2) and its relationship to intraflagellar transport (IFT), we cloned the gene encoding Chlamydomonas reinhardtii PKD2 (CrPKD2), a protein with the characteristics of PKD2 family members. Three forms of this protein (210, 120, and 90 kD) were detected in whole cells; the two smaller forms are cleavage products of the 210-kD protein and were the predominant forms in flagella. In cells expressing CrPKD2-GFP, about 10% of flagellar CrPKD2-GFP was observed moving in the flagellar membrane. When IFT was blocked, fluorescence recovery after photobleaching of flagellar CrPKD2-GFP was attenuated and CrPKD2 accumulated in the flagella. Flagellar CrPKD2 increased fourfold during gametogenesis, and several CrPKD2 RNA interference strains showed defects in flagella-dependent mating. These results suggest that the CrPKD2 cation channel is involved in coupling flagellar adhesion at the beginning of mating to the increase in flagellar calcium required for subsequent steps in mating.     

Regulation by ammonium of nitrate and nitrite assimilation in Chlamydomonas reinhardtii

The inhibitor of mRNA synthesis, 6-methylpurine, inhibited nitrate reductase derepression in either ammonium-grown or methylammonium-treated wild-type cells of Chlamydomonas reinhardtii, but not in nitrogen-starved cells. In contrast, 6-methylpurine did not inhibit nitrate reductase synthesis in the methylammonium-resistant mutant 2170 (ma-1) either grown on ammonium, treated with methylammonium or nitrogen starved, but did inhibit the continuous synthesis of nitrate reductase, which required the presence of nitrate in the media. In both wild-type and mutant 2170 grown on ammonium and transferred to nitrate media, cycloheximide immediately prevented nitrate reductase derepression when added either at the beginning or at different times of induction treatment. Unlike wild-type cells, mutant 2170 was able to take up either nitrate or nitrite simultaneously with ammonium in whose presence nitrate and nitrite reductases were synthesized. However, synthesis of nitrate reductase was progressively inhibited in the mutant cells when the intracellular ammonium levels were raised as a result of an increase in either the external pH or the extracellular ammonium concentrations. The results rule out the existence of maturase-like proteins in Chlamydomonas and indicate that ammonium has a double effect on the regulation of nitrate reductase synthesis: (a) it prevents nitrate reductase mRNA production; and (b) it controls negatively the expression of this mRNA.     

Regulation by light of ammonium transport systems in Chlamydomonas reinhardtii

The unicellular green alga Chlamydomonas reinhardtii is able to take up methylammonium/ammonium from the medium at different stages of its sexual life cycle. Vegetative cells and pre‐gametes mostly used a low‐affinity system (LATS) component, but gametes obtained after light treatment of N‐deprived pre‐gametes expressed both LATS and high‐affinity system (HATS) components for the uptake of methylammonium/ammonium. The activity of the LATS component was stimulated by light in only 5 min in a process independent of protein synthesis. By using the lrg6 mutant that produces sexually competent gametes in the dark, light effects on ammonium transport and gamete differentiation have been separately analysed. We have found light regulation of four Amt1 genes: Amt1; 1, Amt1; 2, Amt1; 4 and Amt1; 5. Whereas light‐dependent expression of Amt1; 1, Amt1; 2 and Amt1; 4 was independent of gametogenesis, and that of Amt1; 5 was activated in the lrg6 mutant, suggesting a connection between this transporter and the subsequent events taking place during gametogenesis.     

Regulation of the low-CO2-inducible polypeptides in Chlamydomonas reinhardtii

Polypeptides of 21, 36 and 37 kDa are induced in the unicellular green alga Chlamydomonas reinhardtii Dang. when cells are transferred from high (2%) to low (0.03%) CO₂ concentrations. The synthesis of these polypeptides is correlated with the induction of the CO₂-concentrating mechanism. In this work we studied the effect of the growth conditions on the synthesis of these polypeptides with the aim of clarifying whether the induction of all three of these low-CO₂-inducible polypeptides requires the same environmental factor. Our results showed that induction of the 21- and 36-kDa polypeptides under low-CO₂ conditions occurred only in the light, while the 37-kDa periplasmic carbonic anhydrase (EC 4.2.1.1) was induced in light, darkness, and in both synchronous and asynchronous cultures. In addition, induction of these polypeptides appeared to be determined more by the O₂/CO₂ ratio than by the CO₂ concentrations. None of these polypeptides could be induced in either of two different mutants of C. reinhardtii, one lacking ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) and the other with inactive enzyme. Our results indicate that the 21- and 36-kDa polypeptides are regulated by a mechanism different from that controlling the 37-kDa polypeptide.Abbreviations pCA (periplasmic) carbonic anhydrase - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - TAP Trisacetate phosphate medium The authors thank Prof. M. Spalding (Iowa State University, USA) for providing antisera to LIP-21 and LIP-36. We thank Prof. S. Bartlett and Dr. J. Moroney (Louisiana State University, USA) for providing antibodies to C. reinhardtii, Rubisco and 37-kDa pCA, respectively. This work was supported by the Instituto Tecnologico de Canarias.