Changes in Photorespiratory Enzyme Activity in Response to Limiting CO(2) in Chlamydomonas reinhardtii

The activity of two photorespiratory enzymes, phosphoglycolate phosphatase (PGPase) and glycolate dehydrogenase (glycolate DH), changes when CO₂-enriched wild-type (WT) Chlamydomonas reinhardtii cells are transferred to air levels of CO₂. Adaptation to air levels of CO₂ by Chlamydomonas involves induction of a CO₂-concentrating mechanism (CCM) which increases the internal inorganic carbon concentration and suppresses oxygenase activity of ribulose-1,5-bisphosphate carboxylase/oxygenase. PGPase in cell extracts shows a transient increase in activity that reaches a maximum 3 to 5 hours after transfer and then declines to the original level within 48 hours. The decline in PGPase activity begins at about the time that physiological evidence indicates the CCM is approaching maximal activity. Glycolate DH activity in 24 hour air-adapted WT cells is double that seen in CO₂-enriched cells. Unlike WT, the high-CO₂-requiring mutant, cia-5, does not respond to limiting CO₂ conditions: it does not induce any known aspects of the CCM and it does not show changes in PGPase or glycolate DH activities. Other known mutants of the CCM show patterns of PGPase and glycolate DH activity after transfer to limiting CO₂ which are different from WT and cia-5 but which are consistent with changes in activity being initiated by the same factor that induces the CCM, although secondary regulation must also be involved.     

Short-Flagella Mutants of Chlamydomonas reinhardtii

Six short-flagella mutants were isolated by screening clones of mutagenized Chlamydomonas for slow swimmers. The six mutants identify three unlinked Mendelian genes, with three mutations in gene shf-1, two in shf-2 and one in shf-3. shf-1 and shf-2 have been mapped to chromosomes VI and I, respectively. Two of the shf-1 mutations have temperature-sensitive flagellar-assembly phenotypes, and one shf-2 mutant has a cold-sensitive phenotype. shf shf double mutants were constructed; depending on the alleles present they showed either flagellaless or short-flagella phenotypes. Phenotypic revertants of shf-1 and shf-2 mutants were isolated, and certain of them were found to carry extragenic suppressors, some dominant and some recessive. We suspect that the shf mutations affect components of a specific flagellar size-control system, the existence of which has been suggested by a variety of physiological experiments.     

Reduced Inorganic Carbon Transport in a CO(2)-Requiring Mutant of Chlamydomonas reinhardii

A mendelian mutant of the unicellular green alga Chlamydomonas reinhardii has been isolated that is deficient in inorganic carbon transport. This mutant strain, designated pmp-1-16-5K (gene locus pmp-1), was selected on the basis of a requirement of elevated CO₂ concentration for photoautrophic growth. Inorganic carbon accumulation in the mutant was considerably reduced in comparison to wild type, and the CO₂ response of photosynthesis indicated a reduced affinity for CO₂ in the mutant. At air levels of CO₂ (0.03-0.04%), O₂ inhibited photosynthesis and stimulated the synthesis of photorespiratory intermediates in the mutant but not in wild type. Neither strain was significantly affected by O₂ at saturating CO₂ concentration. Thus, the primary consequence of inorganic carbon transport deficiency in the mutant was a much lower internal CO₂ concentration compared to wild type. From these observations, we conclude that enzyme-mediated transport of inorganic carbon is an essential component of the CO₂ concentrating system in C. reinhardii photosynthesis.     

A Metabolomic Approach to Study Major Metabolite Changes during Acclimation to Limiting CO2 in Chlamydomonas reinhardtii

Using a gas chromatography-mass spectrometry-time of flight technique, we determined major metabolite changes during induction of the carbon-concentrating mechanism in the unicellular green alga Chlamydomonas reinhardtii. In total, 128 metabolites with significant differences between high- and low-CO₂-grown cells were detected, of which 82 were wholly or partially identified, including amino acids, lipids, and carbohydrates. In a 24-h time course experiment, we show that the amino acids serine and phenylalanine increase transiently while aspartate and glutamate decrease after transfer to low CO₂. The biggest differences were typically observed 3 h after transfer to low-CO₂ conditions. Therefore, we made a careful metabolomic examination at the 3-h time point, comparing low-CO₂ treatment to high-CO₂ control. Five metabolites involved in photorespiration, 11 amino acids, and one lipid were increased, while six amino acids and, interestingly, 21 lipids were significantly lower. Our conclusion is that the metabolic pattern during early induction of the carbon-concentrating mechanism fit a model where photorespiration is increasing.Most microalgae express a carbon-concentrating mechanism (CCM; Raven et al., 2005). The green alga Chlamydomonas reinhardtii is one of the species with a CCM (Giordano et al., 2005). The CCM in C. reinhardtii is typically induced when the concentration of CO₂ in the air bubbled through the cultures is decreased to around 0.5% or lower (Vance and Spalding, 2005). Within the first few hours after starting the induction, numerous genes are either up- or down-regulated (Miura et al., 2004; Yamano et al., 2008; Yamano and Fukuzawa, 2009). However, the change in gene expression is only manifested as rather limited detectable changes in the abundance of proteins (Manuel and Moroney, 1988; Spalding and Jeffrey, 1989). Even though the response to decreased concentrations of inorganic carbon (C_i) is fast (Eriksson et al., 1998), the algal cells go through a transient phase before the CCM is fully operational.Many genes coding for enzymes of the photorespiratory pathway are up-regulated (Marek and Spalding, 1991; Miura et al., 2002, 2004) within 20 min and show a transient expression pattern (Tural and Moroney, 2005; Yamano et al., 2008). A decline in starch content is also detectable within 30 min after transfer to low CO₂ (Kuchitsu et al., 1988). This decline in starch is followed, after about 2 h, by a net synthesis of starch (Thyssen et al., 2001) that is mainly deposited around the pyrenoid, rather than as the starch grains distributed in the stroma normally found in high-CO₂-grown cells. When the CCM is fully induced, the alga can concentrate C_i inside the cell/chloroplast against a free-energy gradient. Accumulation of C_i increases the CO₂-oxygen ratio at the site of Rubisco (Giordano et al., 2005), with a corresponding increase in photosynthesis, a decrease in photorespiration, and a greater capacity for net organic carbon production at low external C_i (Giordano et al., 2003).Induction of the CCM is thus known to affect genes of many different pathways, and especially the photorespiratory pathway has been studied extensively (Moroney et al., 1986; Tural and Moroney, 2005). The aim of this work was to screen for metabolic changes in order to find key metabolites that could trigger the expression of genes that regulate the CCM. We have extended the analyses by using metabolomics to detect changes in major metabolites, particularly in the beginning of the induction period but also in preliminary experiments, over a 24-h time period. The resulting metabolic changes have enabled us to propose a working model for the coordinated regulation of cellular metabolism during the induction of the CCM in C. reinhardtii.     

Molecular and Structural Changes in Chlamydomonas under Limiting CO2 (A Possible Mitochondrial Role in Adaptation)

When Chlamydomonas reinhardtii cells are transferred to limiting CO2, one response is the induction of a CO2-concentrating mechanism (CCM) with components that remain to be identified. Characterization of membrane-associated proteins induced by this transfer revealed that synthesis of the 21-kD protein (LIP-21) was regulated at the level of translatable message abundance and correlated well with the induction of CCM activity. Phase partitioning of LIP-21 and the previously characterized LIP-36 showed that both appeared to be peripherally associated with membranes, which limits their potential to function as transporters of inorganic carbon. Ultrastructural changes that occur when cells are transferred to limiting CO2 were also examined to help form a model for the CCM or other aspects of adaptation to limiting CO2. Changes were observed in vacuolization, starch distribution, and mitochondrial location. The mitochondria relocated from within the cup of the chloroplast to between the chloroplast envelope and the plasma membrane. In addition, immunogold labeling demonstrated that LIP-21 was localized specifically to the peripheral mitochondria. These data suggest that mitochondria, although not previously incorporated into models for the CCM, may play an important role in the cell's adaptation to limiting CO2.     

Active CO(2) Transport by the Green Alga Chlamydomonas reinhardtii

Mass spectrometric measurements of dissolved free ¹³CO₂ were used to monitor CO₂ uptake by air grown (low CO₂) cells and protoplasts from the green alga Chlamydomonas reinhardtii. In the presence of 50 micromolar dissolved inorganic carbon and light, protoplasts which had been washed free of external carbonic anhydrase reduced the ¹³CO₂ concentration in the medium to close to zero. Similar results were obtained with low CO₂ cells treated with 50 micromolar acetazolamide. Addition of carbonic anhydrase to protoplasts after the period of rapid CO₂ uptake revealed that the removal of CO₂ from the medium in the light was due to selective and active CO₂ transport rather than uptake of total dissolved inorganic carbon. In the light, low CO₂ cells and protoplasts incubated with carbonic anhydrase took up CO₂ at an apparently low rate which reflected the uptake of total dissolved inorganic carbon. No net CO₂ uptake occurred in the dark. Measurement of chlorophyll a fluorescence yield with low CO₂ cells and washed protoplasts showed that variable fluorescence was mainly influenced by energy quenching which was reciprocally related to photosynthetic activity with its highest value at the CO₂ compensation point. During the linear uptake of CO₂, low CO₂ cells and protoplasts incubated with carbonic anhydrase showed similar rates of net O₂ evolution (102 and 108 micromoles per milligram of chlorophyll per hour, respectively). The rate of net O₂ evolution (83 micromoles per milligram of chlorophyll per hour) with washed protoplasts was 20 to 30% lower during the period of rapid CO₂ uptake and decreased to a still lower value of 46 micromoles per milligram of chlorophyll per hour when most of the free CO₂ had been removed from the medium. The addition of carbonic anhydrase at this point resulted in more than a doubling of the rate of O₂ evolution. These results show low CO₂ cells of Chlamydomonas are able to transport both CO₂ and HCO₃⁻ but CO₂ is preferentially removed from the medium. The external carbonic anhydrase is important in the supply to the cells of free CO₂ from the dehydration of HCO₃⁻.     

Adaptation to CO(2) Level and Changes in the Phosphorylation of Thylakoid Proteins during the Cell Cycle of Chlamydomonas reinhardtii

The photosynthetic performance of synchronously grown Chlamydomonas reinhardtii alternated rhythmically during the cell cycle. The activity of the “CO₂ concentrating mechanism” including the ability to accumulate CO₂ internally and the activity of carbonic anhydrase peaked after 6 to 9 hours of light and reached minimum after 6 to 9 hours of dark. Consequently, the apparent photosynthetic affinity to extracellular CO₂ alternated rhythmically. At the end of the dark period the cells behaved as if they were adapted to high CO₂ even though they were continuously aerated with air. Results from experiments in which the light or dark periods were extended bear on the interaction between the internal (cell cycle or biological clock) and the external (light) signal. The observed rhythmical alterations in photosynthetic V_max may result from changes in PSII activity. The latter may be partly explained by the capacity for phosphorylation of thylakoid proteins, which reached maximum after 9 hours of light and decreased toward the dark period.     

Membrane-Associated Polypeptides Induced in Chlamydomonas by Limiting CO(2) Concentrations

Chlamydomonas reinhardtii and other unicellular green algae have a high apparent affinity for CO₂, little O₂ inhibition of photosynthesis, and reduced photorespiration. These characteristics result from operation of a CO₂-concentrating system. The CO₂-concentrating system involves active inorganic carbon transport and is under environmental control. Cells grown at limiting CO₂ concentrations have inorganic carbon transport activity, but cells grown at 5% CO₂ do not. Four membrane-associated polypeptides (M_r 19, 21, 35, and 36 kilodaltons) have been identified which either appear or increase in abundance during adaptation to limiting CO₂ concentrations. The appearance of two of the polypeptides occurs over roughly the same time course as the appearance of the CO₂-concentrating system activity in response to CO₂ limitation.     

Carbonic Anhydrase Activity in Isolated Chloroplasts of Wild-Type and High-CO2-Dependent Mutants of Chlamydomonas reinhardtii as Studied by a New Assay

In an assay of carbonic anhydrase (CA), NAH14CO3 soltution at the bottom of a sealed vessel releases 14CO2, which diffuses to the top of the vessel to be assimilated by photosynthesizing Chlamydomonas reinhardtii cells that have been adapted to a low-CO2 environment. The assay is initiated by illuminating the cells and is stopped by turning the light off and killing the cells with acid. Enzyme activity was estimated from acid-stable radioactivity. With bovine CA, 1.5 Wilbur-Anderson units (WAU) was consistently measured at 5- to 6-fold above background. Sonicated whole cells of air-adapted wild-type C. reinhardtii had 740 [plus or minus] 12.4 WAU/mg chlorophyll (Chl). Sonicated chloroplasts from a mixotrophically grown wall-less strain, cw-15, had 35.5 [plus or minus] 2.6 WAU/mg Chl, whereas chloroplasts from wall-less external CA mutant strain cia5/cw-15 had 33.8 [plus or minus] 1.9 WAU/mg Chl. Sonicated chloroplasts from the wall-less mutant strain cia-3/cw-15, believed to lack an internal CA, had 2.8 [plus or minus] 3.2 WAU/mg Chl. Sonicated whole cells from cia3/cw-15 had 2.8 [plus or minus] 7.8 WAU/mg Chl. Acetazolamide, ethoxyzolamide, and p-aminomethylbenzene sulfonamide (Mafenide) at 100 [mu]M inhibited CA in sonicated chloroplasts from cia-5/cw-15. Treatment at 80[deg]C for 10 min inhibited this CA activity by 90.8 [plus or minus] 3.6%. Thus, a sensitive 14C assay has confirmed the presence of a CA in cw-15 and cia-5/cw-15 chloroplasts and the lack of a CA in cia-3/cw-15 chloroplasts. Our results indicate that HCO3- is the inorganic carbon species that is accumulated by chloroplasts of Chlamydomonas and that chloroplastic CA is responsible for the majority of internal CA activity.     

Control of CO(2) Fixation by Cell-free Extracts of Chlamydomonas reinhardtii

Contrary to earlier reports, CO₂ fixation by extracts of Chlamydomonas is inhibited by glutamate and aspartate. These amino acids and some organic acids are shown to be inhibitors of phosphoenolpyruvate carboxylase. Inorganic phosphate is shown to activate CO₂ fixation, but there is a time lag before inorganic phosphate exerts its full activating effect.     

Temperature-Sensitive, Photosynthesis-Deficient Mutants of Chlamydomonas reinhardtii

Mutants of the unicellular, green alga Chlamydomonas reinhardtii were recovered by screening for the absence of photoautotrophic growth at 35°C. Whereas nonconditional mutants required acetate for growth at both 25 and 35°C, the conditional mutants have normal photoautotrophic growth at 25°C. The conditional mutants consisted of two classes: (a) Temperature-sensitive mutants died under all growth conditions at 35°C, but (b) temperature-sensitive, acetate-requiring mutants were capable of heterotrophic growth at 35°C when supplied with acetate in the dark. The majority of mutants within the latter of these two classes had defects in photosynthetic functions. These defects included altered pigmentation, reduced whole-chain electron-transport activity, reduced ribulosebis-phosphate carboxylase activity, or pleiotropic alterations in a number of these photosynthetic components. Both nuclear and chloroplast mutants were identified, and a correlation between light-sensitive and photosynthesis-deficient phenotypes was observed.     

H(2) and CO(2) Evolution by Anaerobically Adapted Chlamydomonas reinhardtii F-60

Using manometric and enzymic techniques, H₂ and CO₂ evolution in darkness and light has been studied in the green alga Chlamydomonas reinhardtii F-60. F-60 is a mutant strain characterized by an incomplete photosynthetic carbon reduction cycle but an intact electron transport chain.     

Insertional mutants of Chlamydomonas reinhardtii that require elevated CO(2) for survival

Aquatic photosynthetic organisms live in quite variable conditions of CO(2) availability. To survive in limiting CO(2) conditions, Chlamydomonas reinhardtii and other microalgae show adaptive changes, such as induction of a CO(2)-concentrating mechanism, changes in cell organization, increased photorespiratory enzyme activity, induction of periplasmic carbonic anhydrase and specific polypeptides (mitochondrial carbonic anhydrases and putative chloroplast carrier proteins), and transient down-regulation in the synthesis of Rubisco. The signal for acclimation to limiting CO(2) in C. reinhardtii is unidentified, and it is not known how they sense a change of CO(2) level. The limiting CO(2) signals must be transduced into the changes in gene expression observed during acclimation, so mutational analyses should be helpful for investigating the signal transduction pathway for low CO(2) acclimation. Eight independently isolated mutants of C. reinhardtii that require high CO(2) for photoautotrophic growth were tested by complementation group analysis. These mutants are likely to be defective in some aspects of the acclimation to low CO(2) because they differ from wild type in their growth and in the expression patterns of five low CO(2)-inducible genes (Cah1, Mca1, Mca2, Ccp1, and Ccp2). Two of the new mutants formed a single complementation group along with the previously described mutant cia-5, which appears to be defective in the signal transduction pathway for low CO(2) acclimation. The other mutations represent six additional, independent complementation groups.     

Complement-Mediated Cytolysis of Cell-Wall Mutants of Chlamydomonas reinhardtii

Treatment of the cell wall-less mutant CW 15 of Chlamydomonasreinhardtii with human serum leads to a marked increase of thecell volume, followed by an irreversible cytolysis. Heat-inactivatedserum as a control reveals no cytotoxic effects on CW 15. Experimentswith C4-, properdin-, C3-, and factor H-depleted sera indicatethe alternative pathway of complement as being responsible forthe serum-mediated lysis. After immunofluorescence marking aswell as electromicroscopically after negative staining the membraneattacking complex of complement, C5b-9, could be demonstratedon the surface of CW 15. These results together with the observationthat cells of the wild-type strain 11-32c of C. reinhardtiiare not lysed by active serum suggest that only protoplastsof Chlamydomonas carry surface structures capable to activatethe alternative pathway of complement. In order to find out whether other cell wall mutants of C. reinhardtii,besides CW 15, can also activate the human complement system,we tested three strains each of the three known mutant categories.Strains CW 4, CW 9, and CW 19, representing category A, andstrains CW 3, CW 10, and CW 92, representing category C, andCW 8 and CW 18, accounting for category B, were cytolysed bynormal human serum. Only one type used in our experiments, CW20 of category B, resisted serum treatment, suggesting the needto redefine this category. ¹This paper is dedicated to Professor Dr. Andr? Pirson on theoccasion of his 80th birthday (Received December 1, 1989; Accepted April 5, 1990)     

Isolation and Characterization of Chemotaxis Mutants of Chlamydomonas reinhardtii

Zoospores of Chlamydomonas reinhardtii exhibit chemotaxis towards maltose, sucrose, xylose, mannitol, and ammonium. Ten independent mutants defective in chemotaxis towards sugars have been isolated. These mutants form five phenotypic classes. Genetic analysis of two mutant strains defective in chemotaxis to maltose (CHE1, CHE3) and two mutant strains defective in chemotaxis to sucrose (CHE2, CHE4) indicated that the defect in them depended on single nuclear recessive mutant alleles. Mutations mal1, mal2, suc1, and suc2 represent four chemotactic loci that are unlinked to the marker mt located on the linkage group VI. Four loci are unlinked to each other. These observations suggest that the mal and the suc loci do not constitute a spatially single functional group.     

Flagellar Morphology in Stumpy-Flagella Mutants of Chlamydomonas reinhardtii1

Sixteen new mutants of the biflagellate green alga Chlamydomonas reinhardtii with either stumpy-flagella or no flagella at all were examined by electron microscopy. Four of the mutants were found to carry short bulbous flagella containing amorphous electron-dense material which may represent unassembled flagellar protein. Basal bodies of normal ultrastructure were present in all mutants. Dikaryon dominance tests indicated that the stumpy mutations were recessive to wild-type in all cases tested. Stumpy mutations also conferred a measure of detergent resistance to Chlamydomonas, apparently by affecting the detergent-solubility of the flagellar membrane.     

Salt-Sensitive Mutants of Chlamydomonas reinhardtii Isolated after Insertional Tagging

We describe the isolation of salt-sensitive Chlamydomonas reinhardtii mutants by insertional mutagenesis using the nitrate reductase (Nit1) gene. The plasmid pMN24, containing Nit1, was used for transformation of 305CW15 (nit1 cw15 mt+), and transformants were selected for complementation of the nit- phenotype. From 6875 nit+ colonies, four transformants (S4, S18, S46, and S66) were isolated that exhibited both Na+ and Li+ sensitivity (sod-), and another transformant (S33) was selected that exhibited sensitivity to Li+ but not Na+ (lit-) based on relative growth comparisons with the wild-type strain. S33, S46, and S66 were no more growth inhibited by sorbitol than was 305CW15. In comparison, S4 and S18 exhibited substantial growth inhibition in medium supplemented with sorbitol. Genetic analyses indicated that the salt-sensitive mutants were each defective in a single recessive gene. The mutant genes in S4 (sod1), S33 (lit1), and S66 (sod3) are linked to a functional copy of Nit1 and are presumably tagged with a pMN24 insertion.     

Hydrogen Production by CO2 Deprived Photoautotrophic Chlamydomonas reinhardtii Cultures

Biochemistry (Moscow) - Light-dependent hydrogen production by microalgae attracts attention of researchers because of the potential practical application. It is generally recognized that...