Evidence for Inorganic Carbon Transport by Intact Chloroplasts of Chlamydomonas reinhardtii

Isolated intact chloroplasts from wall-less mutants of Chlamydomonas reinhardtii accumulate inorganic carbon (C_i) from the medium provided the cells had been adapted to low CO₂ photoautotrophic growth conditions. Chloroplasts from cultures grown on high (5%) CO₂ or photoheterotrophically with acetate did not accumulate inorganic carbon. Chloroplast C_i accumulation from low CO₂ grown cells was light dependent and was inhibited by uncouplers and inhibitors of electron transport. In a model for C_i accumulation by Chlamydomonas, it is proposed that CO₂ diffuses into the cell and C_i accumulation occurs in the chloroplast.     

Effect of Carbonic Anhydrase Inhibitors on Inorganic Carbon Accumulation by Chlamydomonas reinhardtii

Membrane-permeable and impermeable inhibitors of carbonic anhydrase have been used to assess the roles of extracellular and intracellular carbonic anhydrase on the inorganic carbon concentrating system in Chlamydomonas reinhardtii. Acetazolamide, ethoxzolamide, and a membrane-impermeable, dextran-bound sulfonamide were potent inhibitors of extracellular carbonic anhydrase measured with intact cells. At pH 5.1, where CO₂ is the predominant species of inorganic carbon, both acetazolamide and the dextran-bound sulfonamide had no effect on the concentration of CO₂ required for the half-maximal rate of photosynthetic O₂ evolution (K_0.5[CO₂]) or inorganic carbon accumulation. However, a more permeable inhibitor, ethoxzolamide, inhibited CO₂ fixation but increased the accumulation of inorganic carbon as compared with untreated cells. At pH 8, the K_0.5(CO₂) was increased from 0.6 micromolar to about 2 to 3 micromolar with both acetazolamide and the dextran-bound sulfonamide, but to a higher value of 60 micromolar with ethoxzolamide. These results are consistent with the hypothesis that CO₂ is the species of inorganic carbon which crosses the plasmalemma and that extracellular carbonic anhydrase is required to replenish CO₂ from HCO₃⁻ at high pH. These data also implicate a role for intracellular carbonic anhydrase in the inorganic carbon accumulating system, and indicate that both acetazolamide and the dextran-bound sulfonamide inhibit only the extracellular enzyme. It is suggested that HCO₃⁻ transport for internal accumulation might occur at the level of the chloroplast envelope.     

Form of Inorganic Carbon Utilized for Photosynthesis in Chlamydomonas reinhardtii1

The effect of carbonic anhydrase (CA) on time courses of photosynthetic¹⁴C incorporation in the presence of ¹⁴CO₂ or NaH¹⁴CO₃ was studiedwith cells of Chlamydomonas reinhardtii which had been grownunder ordinary air (low-CO₂ cells) or air enriched with 4% CO₂(high-CO₂ cells). Experimental data obtained at 20°C andpH 8.0 suggested that the major form of inorganic carbon utilizedby high-CO₂ cells was CO₂, while that utilized by low-CO₂ cellswas HCO₃^–. The cell suspension showed CA activity which was comparableto that observed in the sonicate of cells. Both activities werehigher in low-CO₂ cells than in high-CO₂ cells. The mechanism by which HCO₃^– is utilized by low-CO₂ cellsof C. reinhardtii is discussed. ³Present address: Department of Biology, Faculty of Science,University of Niigata, Niigata 950-21, Japan. (Received August 4, 1982; Accepted January 19, 1983)     

Carrier-mediated Uptake of Arginine and Urea by Chlamydomonas reinhardtii

Chlamydomonas reinhardtii possesses a high affinity, highly specific carrier involved in uptake of exogenous arginine. Carrier-mediated uptake of other amino acids cannot be detected, even in cultures maintained on amino acids as a nitrogen source or starved for nitrogen. This fact may contribute to the difficulty of isolating strains auxotrophic for amino acids other than arginine; conventional selection media may not supply adequate quantities of amino acids to permit growth of auxotrophs. A urea carrier is also present in C. reinhardtii but is readily distinguished from the arginine carrier on the basis of kinetic properties and sensitivity to a range of structural analogs. Ammonia appears to play a major role in regulating (depressing) activity of the arginine uptake system. Activity of the urea uptake system is elevated in nitrogen-starved cultures and elevated even further in the presence of urea or arginine. Extensive, independent fluctuations in the two uptake systems observed in semisynchronous cultures suggest that both are subject to modulation by a complex set of interacting endogenous and exogenous factors.     

Kinetic Characterization of Nitrite Uptake and Reduction by Chlamydomonas reinhardtii

Kinetics of nitrite uptake and reduction by Chlamydomonas reinhardtii cells growing phototrophically has been studied by means of progress curves and the Michaelis-Menten integrated equation. Both uptake and reduction processes exhibited hyperbolic saturation kinetics, the nitrite uptake system lacking a diffusion component. Nitrite uptake and reduction showed significant differences in K_s for nitrite at pH 7.5 (1.6 versus 20 micromolar, respectively), optimal pH, activation energy values, and sensitivity toward reagents of sulfhydryl groups. K_s values for nitrite uptake were halved in cells subjected to darkness or to nitrogen-starvation. Nitrate inhibited nitrite uptake by a partially competitive mechanism. The same inhibition pattern was found for nitrite uptake by C. reinhardtii mutant 305 cells incapable of nitrate assimilation. The results demonstrate that C. reinhardtii cells take up nitrite via a highly specific carrier, probably energy-dependent, kinetically responsive to environmental changes, distinguishable from the enzymic nitrite reduction and endowed with an active site for nitrite not usable for nitrate transport.     

Intracellular Carbon Partitioning in Chlamydomonas reinhardtii

Using enzymic and isotope techniques the intracellular partitioning of newly fixed carbon was studied in synchronized cells of Chlamydomonas reinhardtii. Starch and growth metabolism, i.e. the use of carbon in biosynthesis, were found to be the major sinks for photosynthetically fixed carbon in the alga. Sucrose does not accumulate in significant quantities. The amount of carbon partitioned either into starch or growth varies during the 12 hour light/12 hour dark cell cycle. Starch is accumulated at the beginning and at the end of the light period while a net breakdown is observed in the middle of the light period and in the dark. In contrast, nonsynchronized cells accumulate starch all the time in the light which suggests that carbon partitioning is controlled by the cell cycle. Labeled bicarbonate is incorporated into starch even at times when the total intracellular level of starch is decreasing. This indicates a turnover of the starch pool in the light with synthesis and degradation occurring simultaneously and at different rates.     

Internal Inorganic Carbon Pool of Chlamydomonas reinhardtii: EVIDENCE FOR A CARBON DIOXIDE-CONCENTRATING MECHANISM

The external inorganic carbon pool (CO₂ + HCO₃⁻) was measured in both high and low CO₂-grown cells of Chlamydomonas reinhardtii, using a silicone oil layer centrifugal filtering technique. The average internal pH values were measured for each cell type using [¹⁴C]dimethyloxazolidinedione, and the internal inorganic carbon pools were recalculated on a free CO₂ basis. These measurements indicated that low CO₂-grown cells were able to concentrate CO₂ up to 40-fold in relation to the external medium. Low and high CO₂-grown cells differed in their photosynthetic affinity for external CO₂. These differences could be most readily explained as being due to the relative CO₂-concentrating capacity of each cell type. This physiological adaptation appeared to be based on changes in the abilities of the cells actively to accumulate inorganic carbon using an energy-dependent transport system.     

Induction of Inorganic Carbon Accumulation in the Unicellular Green Algae Scenedesmus obliquus and Chlamydomonas reinhardtii

The induction of a dissolved inorganic carbon (DIC) accumulating mechanism in the two algal species Scenedesmus obliquus (WT) and Chlamydomonas reinhardtii (137 c+) was physiologically characterized by monitoring DIC uptake kinetics at a low and constant DIC concentration (120-140 micromolar), after transfer from high-DIC culturing conditions. A potentiometric titration method was used to measure and calculate algal DIC uptake. Full acclimation to low-DIC conditions was obtained within a period of 90 min, after which time the DIC uptake had been increased 7 to 10 times. Experiments were also conducted in the presence of inhibitors against DIC accumulation. The inhibitor of extracellular carbonic anhydrase (CA), acetazolamide (50 micromolar), inhibited the adaptation partly, while the inhibitor of both extra- and intracellular CA, ethoxyzolamide (50 micromolar) totally inhibited the acclimation. Cycloheximide (10 micrograms per milliliter), which inhibits protein synthesis on cytoplasmic ribosomes, and vanadate (180 micromolar), which inhibits the plasmamembrane bound ATPase, also inhibited the acclimation totally. These results taken together suggest that the algae are dependent on intracellular CA, plasmamembrane bound ATPase, and de novo protein synthesis for DIC accumulation. Also, these components are more important than extracellular CA for the overall function of the DIC-accumulating mechanism.     

Acclimation of Photosynthetic Light Reactions during Induction of Inorganic Carbon Accumulation in the Green Alga Chlamydomonas reinhardtii

Cells of the unicellular green algae Chlamydomonas reinhardtii were grown in high dissolved inorganic carbon (DIC) concentrations (supplied with 50 milliliters per liter CO₂[g]) and transferred to low DIC concentrations (supplied with ≤ 100 microliters per liter CO₂[g]). Immediately after transfer from high to low DIC the emission of photosystem II related chlorophyll a fluorescence was substantially quenched. It is hypothesized that the suddenly induced inorganic carbon limitation of photosynthesis resulted in a phosphorylation of LHCII, leading to the subsequent state 1 to state 2 transition. After 2 hours of low-DIC acclimation, 77 K fluorescence measurements revealed an increase in the fluorescence emitted from photosystem I, due to direct excitation, suggesting a change in photosystem II/photosystem I stoichiometry or an increased light harvesting capacity of photosystem I. After 5 to 6 hours of acclimation a considerable increase in spillover from photosystem II to photosystem I was observed. These adjustments of the photosynthetic light reactions reached steady-state after about 12 hours of low DIC treatment. The quencher of fluorescence could be removed by 5 minutes of dark treatment followed by 5 minutes of weak light treatment, of any of four different light qualities. It is hypothesized that this restoration of fluorescence was due to a state 2 to state 1 transition in low-DIC acclimated cells. A decreased ratio of violaxanthin to zeaxanthin was also observed in 12 hour low DIC treated cells, compared with high DIC grown cells. This ratio was not coupled to the level of fluorescence quenching. The role of different processes during the induction of a DIC accumulating mechanism is discussed.     

Bioflocculant produced by Chlamydomonas reinhardtii

Bioflocculants of Chlamydomonas reinhardtii were investigated under axenic conditions. C. reinhardtii was found to produce significant amounts of bioflocculants. Flocculating activity by C. reinhardtii began in the linear phase of growth and continued until the end of the stationary phase. The highest flocculating efficiency of the culture broth was 97.06%. The purified C. reinhardtii bioflocculant was composed of 42.1% (w/w) proteins, 48.3% carbohydrates, 8.7% lipids, and 0.01% nucleic acid. The optimum condition for bioflocculant production of C. reinhardtii was as follows: under temperature of 15°C to 25°C, pH 6–10 and illumination of 40–60 μmol photons m^?2 s^?1. The bioflocculants produced by C. reinhardtii showed maximum activity in pH ranges from 2 to 10. The flocculating activity was significantly enhanced by the addition of CaCl₂ as a co-flocculant at an optimal concentration of 4.5 mM.     

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.     

The Location of the Transporting System for Inorganic Carbon and the Nature of the Form Translocated in Chlamydomonas reinhardtii

The permeability of the plasmalemma of Chlamydomonas reinhardtiicells was increased by treatment with poly-L-lysine or dimethylsulphoxideas indicated by 3-phosphoglyceric acid dependent O₂ evolution.These treatments decreased the ability of the cells to accumulateinorganic carbon internally and hence their photosynthetic affinityfor inorganic carbon in the medium. With saturating light andinorganic carbon, the photosynthetic rate was less affectedby the poly-L-lysine and dimethylsulphoxide treatments. Thusthe poly-L-lysine and dimethylsulphoxide did not alter the activityof the chloroplasts but rather made the intracellular inorganiccarbon pool more freely exchangeable with the medium. It isconcluded that the transporting system for inorganic carbonis located at the plasmalemma. Treatment with Diamox, an inhibitor of carbonic anhydrase, didnot affect photosynthetic rate and accumulation of inorganiccarbon when CO₂ was supplied but strongly inhibited both parameterswhen HCO^–₃ was supplied. In a mutant of Chlamydomonasreinhardtii lacking a cell wall, carbonic anhydrase leaks tothe medium and uptake of inorganic carbon is much faster whenCO₂ is supplied than when HCO^–₃ is supplied. These resultssuggest that CO₂ rather than HCO^–₃ is the inorganic carbonspecies that is actively translocated across the plasmalemma. Key words: Chlamydomonas, Inorganic carbon uptake     

Uptake and Accumulation of Inorganic Carbon by a Freshwater Diatom

Colman, B. and Rotatore, C. 1988. Uptake and accumulation ofinorganic carbon by a freshwater diatom.—J. exp Bot 39:1025–1032. The mechanism of uptake of inorganic carbon and its accumulationhas been studied in the freshwater diatom Navicula pelliculosa.No external carbonic anhydrase could be detected, although itwas detected in cell extracts. The rate of photosynthetic O₂evolution, in media in the range pH 7.5–8.5, exceededthe calculated rate of CO₂ supply 2- to 5-fold, indicating thatHCO^–₃ was taken up by the cells. At an external pH of7.5, the internal pH, measured by ¹⁴C-dimethyloxazolidine-2,4-dione distribution between the cells and the medium, was pH7.6 in the light and pH 7.4 in the dark. Accumulation of inorganiccarbon was determined by the silicone oil centrifugation methodand inorganic carbon pools of 23.5 mol m^–3 were found,a concentration 21.6-fold that in the external medium. The resultsindicate an active accumulation of inorganic carbon againstpH and concentration gradients in this diatom, probably by activeHCO^–₃ uptake. Key words: Bicarbonate transport, carbon dioxide, carbonic anhydrase, CO₂ affinity, CO₂ concentrating mechanism, internal pH, Navicula pelliculosa     

Glycolate Metabolism and Excretion by Chlamydomonas reinhardtii

The flux of glycolate through the C₂ pathway in Chlamydomonas reinhardtii was estimated after inhibition of the pathway with aminooxyacetate (AOA) or aminoacetonitrile (AAN) by measurement of the accumulation of glycolate and glycine. Cells grown photoautotrophically in air excreted little glycolate except in the presence of 2 mm AOA when they excreted 5 micromoles glycolate per hour per milligram clorophyll. Cells grown on high CO₂ (1-5%) when transferred to air produced three times as much glycolate, with half of the glycolate metabolized and half excreted. The lower amount of glycolate produced by the air-grown cells reflects the presence of a CO₂ concentrating mechanism which raises the internal CO₂ level and decreases the ribulose-1,5-bisP oxygenase reaction for glycolate production. Despite the presence of the CO₂ concentrating mechanism, there was still a significant amount of glycolate produced and metabolized by air-grown Chlamydomonas. The capacity of these cells to metabolize between 5 and 10 micromoles of glycolate per hour per milligram chlorophyll was confirmed by measuring the biphasic uptake of added labeled glycolate. The initial rapid (<10 seconds) phase represented uptake of glycolate; the slow phase represented the metabolism of glycolate. The rates of glycolate metabolism were in agreement with those determined using the C₂-cycle inhibitors during CO₂ fixation.     

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.     

Uptake of polyamines by the unicellular green alga Chlamydomonas reinhardtii and their effect on ornithine decarboxylase activity

Uptake of exogenous polyamines by the unicellular green alga Chlamydomonas reinhardtii and their effects on polyamine metabolism were investigated. Our data show that, in contrast to mammalian cells, Chlamydomonas reinhardtii does not contain short-living, high-affinity polyamine transporters whose cellular level is dependent on the polyamine concentration. However, exogenous polyamines affect polyamine metabolism in Chlamydomonas cells. Exogenous putrescine caused a slow increase of both putrescine and spermidine and, vice versa, exogenous spermidine also led to an increase of the intracellular levels of both spermidine and putrescine. No intracellular spermine was detected under any conditions. Exogenous spermine was taken up by the cells and caused a decrease in their putrescine and spermidine levels. As in other organisms, exogenous polyamines led to a decrease in the activity of ornithine decarboxylase, a key enzyme of polyamine synthesis. In contrast to mammalian cells, this polyamine-induced decrease in ornithine decarboxylase activity is not mediated by a polyamine-dependent degradation or inactivation, but exclusively due to a decreased synthesis of ornithine decarboxylase. Translation of ornithine decarboxylase mRNA, but not overall protein biosynthesis is slowed by increased polyamine levels.     

Nitrate Reductase Regulates Expression of Nitrite Uptake and Nitrite Reductase Activities in Chlamydomonas reinhardtii

In Chlamydomonas reinhardtii mutants defective at the structural locus for nitrate reductase (nit-1) or at loci for biosynthesis of the molybdopterin cofactor (nit-3, nit-4, or nit-5 and nit-6), both nitrite uptake and nitrite reductase activities were repressed in ammonium-grown cells and expressed at high amounts in nitrogen-free media or in media containing nitrate or nitrite. In contrast, wild-type cells required nitrate induction for expression of high levels of both activities. In mutants defective at the regulatory locus for nitrate reductase (nit-2), very low levels of nitrite uptake and nitrite reductase activities were expressed even in the presence of nitrate or nitrite. Both restoration of nitrate reductase activity in mutants defective at nit-1, nit-3, and nit-4 by isolating diploid strains among them and transformation of a structural mutant upon integration of the wild-type nit-1 gene gave rise to the wild-type expression pattern for nitrite uptake and nitrite reductase activities. Conversely, inactivation of nitrate reductase by tungstate treatment in nitrate, nitrite, or nitrogen-free media made wild-type cells respond like nitrate reductase-deficient mutants with respect to the expression of nitrite uptake and nitrite reductase activities. Our results indicate that nit-2 is a regulatory locus for both the nitrite uptake system and nitrite reductase, and that the nitrate reductase enzyme plays an important role in the regulation of the expression of both enzyme activities.     

Carbon Supply and Photoacclimation Cross Talk in the Green Alga Chlamydomonas reinhardtii

Photosynthetic organisms are exposed to drastic changes in light conditions, which can affect their photosynthetic efficiency and induce photodamage. To face these changes, they have developed a series of acclimation mechanisms. In this work, we have studied the acclimation strategies of Chlamydomonas reinhardtii, a model green alga that can grow using various carbon sources and is thus an excellent system in which to study photosynthesis. Like other photosynthetic algae, it has evolved inducible mechanisms to adapt to conditions where carbon supply is limiting. We have analyzed how the carbon availability influences the composition and organization of the photosynthetic apparatus and the capacity of the cells to acclimate to different light conditions. Using electron microscopy, biochemical, and fluorescence measurements, we show that differences in CO₂ availability not only have a strong effect on the induction of the carbon-concentrating mechanisms but also change the acclimation strategy of the cells to light. For example, while cells in limiting CO₂ maintain a large antenna even in high light and switch on energy-dissipative mechanisms, cells in high CO₂ reduce the amount of pigments per cell and the antenna size. Our results show the high plasticity of the photosynthetic apparatus of C. reinhardtii. This alga is able to use various photoacclimation strategies, and the choice of which to activate strongly depends on the carbon availability.Light sustains virtually all life on Earth through the process of photosynthesis. However, light can be very harmful for oxygenic photosynthetic organisms, as excess absorption can lead to the production of reactive oxygen species. In order to survive and grow, these organisms have developed various photoacclimation mechanisms operating on different time scales that protect the cell from photodamage. In the green alga Chlamydomonas reinhardtii, these mechanisms vary from negative phototaxis and multicomponent nonphotochemical quenching (NPQ) to a number of physiological and biochemical changes (Erickson et al., 2015). C. reinhardtii cells are around 10 μm in diameter, and a large part of their total volume is occupied by a single horseshoe-shaped chloroplast (Sager and Palade, 1957). The photosynthetic machinery responsible for the light reactions is located in thylakoid membranes and contains four major components: PSII, cytochrome b₆f, PSI, and ATP synthase. Both photosystems bind chlorophyll (Chl) and carotenoid (Car) and are composed of a core and several outer antennae pigment-protein complexes, the main function of which is light harvesting and its conversion into chemical energy. The PSII core is composed of D1, D2, CP43, and CP47 pigment-protein complexes and several smaller subunits, the number of which varies between organisms (Shi et al., 2012). The outer antenna contains the light-harvesting complex II (LHCII), which in C. reinhardtii is encoded by nine LHCBM genes, and the minor antennae CP26 and CP29 (Nield et al., 2000; Teramoto et al., 2001; Natali and Croce, 2015). These complexes are assembled together to form PSII-LHCII supercomplexes (Tokutsu et al., 2012; Drop et al., 2014). The PSI core is composed of a PSAA-PSAB heterodimer and a number of smaller subunits (Jensen et al., 2007), and in C. reinhardtii the LHCI antenna consists of nine LHCA proteins (Mozzo et al., 2010) that are associated with the core to form the PSI-LHCI complex (Stauber et al., 2009; Drop et al., 2011).The composition and organization of the thylakoid membrane is light dependent. The gene expression of different LHCs has been reported to be affected by light acclimation (Teramoto et al., 2002; Durnford et al., 2003; Yamano et al., 2008) and to be NAB1 regulated (Mussgnug et al., 2005). It has been observed that long-term high-light exposure of C. reinhardtii cells leads to a 50% decrease of Chl content (Neale and Melis, 1986; Bonente et al., 2012) and to changes in Chl-to-Car ratio (Niyogi et al., 1997a; Baroli et al., 2003; Bonente et al., 2012), suggesting reduction of the antenna size (Neale and Melis, 1986), although, in a more recent report (Bonente et al., 2012), it was concluded that the antenna size is not modulated by light in this alga. Recently, a dependence of the antenna components on the carbon availability also was reported. It was shown that, when cells grown in acetate are shifted from high to low CO₂ concentration, the functional antenna size of PSII decreases and a down-regulation of LHCBM6/8 occurs (Berger et al., 2014).In the short term, the main response to high light is the dissipation of energy absorbed in excess heat in a process called qE, or energy-dependent quenching, which is the fastest component of NPQ. In land plants, the main player in this process is the protein PsbS (Li et al., 2002, 2004), while in C. reinhardtii, the process is centered around LHCSR1 and LHCSR3 (Peers et al., 2009; Dinc et al., 2016). LHCSR3, the most studied of the two, is a pigment-protein complex that is expressed within 1 h of high-light exposure (Allorent et al., 2013) in combination with CO₂ limitation (Yamano et al., 2008; Maruyama et al., 2014). The qE onset is triggered by lumen acidification sensed by LHCSR3/1 (Bonente et al., 2011; Liguori et al., 2013; Tokutsu and Minagawa, 2013; Dinc et al., 2016).Cars are well known to be involved in photoprotection. They quench triplet Chl and scavenge singlet oxygen (¹O₂; Frank and Cogdell, 1996). In C. reinhardtii, the antioxidant role of xanthophylls is well illustrated by the mutant npq1 lor1 lacking lutein and zeaxanthin (Niyogi et al., 1997b). This mutant is deficient in qE, but compared with other qE-deficient mutants like npq4 (Peers et al., 2009) and npq5 (Elrad et al., 2002), which are LHCSR3 and LHCBM1 knockouts, respectively, it is extremely light sensitive, due to the absence of quenching of triplet Chl and ¹O₂ by zeaxanthin and lutein.Aquatic oxygenic photosynthetic organisms meet several challenges in CO₂ fixation (Moroney and Ynalvez, 2007). First, the diffusion of CO₂ in water is 10,000 times slower than in air. Second, the CO₂-fixing enzyme Rubisco is not selective for CO₂ and also binds oxygen, resulting in the process of photorespiration. Third, the form of inorganic carbon depends on the pH (i.e. in alkaline pH, it is HCO₃⁻, while in acidic pH, it is CO₂; Beardall, 1981; Gehl et al., 1987). This diminishes even further the availability of CO₂ in the cell. In order to overcome these CO₂ fixation barriers, algae have developed carbon-concentrating mechanisms (CCMs; Moroney and Ynalvez, 2007). The essence of these processes lies in the active pumping of inorganic carbon in the cell via a number of transporters that concentrate it in the pyrenoid, a ball-like structure containing Rubisco, Rubisco activase, and intrapyrenoid thylakoids and surrounded by a starch sheath. In the pyrenoid, HCO₃⁻ is converted to CO₂ by CARBONIC ANHYDRASE3 (CAH3; Blanco-Rivero et al., 2012; Sinetova et al., 2012) and then fixed by Rubisco in the Calvin-Benson-Bassham cycle. CAH3 also is suggested to provide HCO₃⁻ in the proximity of the oxygen-evolving complex, where it may function as a proton carrier, removing H⁺ from water splitting to avoid photoinhibition (Villarejo et al., 2002; Shutova et al., 2008).C. reinhardtii also can grow mixotrophically using alternative organic carbon sources present in its environment. For example, it can take up acetate, which is then incorporated into the citric cycle, producing reducing equivalents and CO₂ (Johnson and Alric, 2012), and into the glyoxylate cycle, producing malate (Lauersen et al., 2016). In the presence of acetate, it has been reported that CO₂ uptake and oxygen evolution were decreased by half under saturating CO₂ and light intensities without affecting PSII efficiency, respiration, and cell growth (Heifetz et al., 2000). In addition, reactions of the oxidative pentose phosphate and glycolysis pathways, inactive under phototrophic conditions, show substantial flux under mixotrophic conditions (Chapman et al., 2015). Furthermore, acetate can replace PSII-associated HCO₃⁻, reducing ¹O₂ formation and, therefore, acting as a photoprotector during high-light acclimation (Roach et al., 2013).In short, high-light acclimation is a complex, multicomponent process that happens on different time scales. Furthermore, it is embedded in the overall metabolic network and is potentially influenced by different nutrients and metabolic states. A thorough understanding of this process and its regulation is crucial for fundamental research and applications. To determine if different carbon supply conditions trigger different light acclimation strategies and photoprotective responses, we systematically studied C. reinhardtii cells grown in mixotrophic, photoautotrophic, and high-CO₂ photoautotrophic conditions in different light intensities.We show that C. reinhardtii cells use different strategies to acclimate to high light depending on the carbon availability and trophic status. These results underline the strong connection between metabolism and light acclimation responses and reconcile the data from various reports. Furthermore, our study demonstrates how, in a dynamic system such as C. reinhardtii, a single change in growth conditions has large effects at multiple levels.     

Mating Type Determination of Chlamydomonas reinhardtii by PCR

We describe a quick and reliable protocol to determine the plus or minus mating type of haploid Chlamydomonas reinhardtii strains from very small amounts of cells. The method combines a fast DNA preparation adapted from forensic work of Walsh et al. (1991) with one for use with Chlamydomonas by Berthold et al. (1993). We used PCR to amplify the minus-specific mid gene (minus dominance) or the plus specific fus1 gene (fusion). Both primer pairs have the same optimum annealing temperature and could be used in the same PCR reaction. The fus1 and mid amplification products could be distinguished by agarose gel electrophoresis due to their different PCR product size. Diploid strains, which should have both mating type genes, could also be detected by the occurrence of both amplification products.