Xanthophyll cycle induction by anaerobic conditions under low light in Chlamydomonas reinhardtii

The effect of anaerobiosis on the induction of the xanthophyll cycle was investigated in Chlamydomonas reinhardtii. The results showed that, anaerobiosis obtained by either sulfur starvation or by bubbling nitrogen in the culture grown in complete medium induced the xanthophyll cycle even when cultures were exposed to low light conditions. The zeaxanthin content reached 35 mmol mol^?1 Chl a, after 110 h in anaerobic sulfur-starved cultures, and 30 mmol mol^?1 Chl a within 24 h in sulfur replete cultures bubbled with nitrogen. Both starved and non-starved cultures grown under aerobic conditions, did not exhibit any sizeable increase in the zeaxanthin content. Chlorophyll fluorescence measurements revealed a decrease in the maximum photochemical quantum yield of PSII (F_v/F_m) by more than 50 %. The chlorophyll fluorescence kinetics (OJIP) analysis showed a strong rise at the J-step indicating a strong reduction of Q_A. Our findings demonstrated that anaerobiosis in low light exposed cultures induced the xanthophyll cycle through a strong increase of the level of plastoquinone pool reduction, which was associated to the formation of a trans-thylakoid membranes proton gradient, while in dark anaerobic cultures, no appreciable induction of xanthophyll cycle could be observed, despite the sizeable increase in non–photochemical quenching.     

Accumulation of starch in Chlamydomonas reinhardtii flagellar mutants.

Paralyzed flagellar mutants pf-1, pf-2, pf-7, and pf-18 of the green alga Chlamydomonas reinhardtii (Dangeard) were shown to store a significantly greater amount of starch than the motile wild type 137c+. The increase in starch storage was significant relative to protein, chlorophyll, and cell number. Analysis of average cell size revealed that the paralyzed mutants were larger than the wild type. This increase in storage molecule accumulation supports an inverse relationship between chemical energy storage and energy utilization for biomechanical/motile cellular functions. Chlamydomonas reinhardtii provides a useful model for studies of the role of cytoskeletal activity in the energy relationship and balance of organisms.     

Anionic modulation of the catalytic activity of hydrogenase from Chlamydomonas reinhardtii

Hydrogen production by cell-free extracts of Chlamydomonas reinhardtii is stimulated by anions when methyl viologen, reduced by dithionite, is used as the electron donor to hydrogenase. The increasing effectiveness of various anions closely follows their position in the Hofmeister chaotropic sequence. The most stimulatory anion tested, I^?, gives a six-fold increase in activity at a concentration of 0.5 n. The K_m of the enzyme for methyl viologen is not affected by anions, while the V is greatly increased. H₂ oxidation coupled to methyl viologen reduction is also greatly stimulated by anions. However, when reduced ferredoxin is used as the electron donor to hydrogenase, there is a very strong inhibition of H₂ production by salts. In this case, the V of the enzyme is unaffected, but there is a large increase in the K_m of the enzyme for ferredoxin. The most inhibitory salt tested, KI, decreases hydrogenase activity by 93% at a concentration of 0.2 n.     

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⁻.     

Pattern of CO2 fixation during vegetative development and gametic differentiation in Chlamydomonas reinhardtii

The rate of C¹⁴O₂ incorporation into amino acids and organic acids in C. reinhardtii is a function of particular stages of development in the life cycle of the alga. Gametic differentiation in nitrogen free medium is accompanied by a reduced rate of amino acid synthesis and a higher synthesis of organic acids than that found for the cells undergoing vegetative development. The addition of ammonium to differentiating gametes results in an increased synthesis of amino acids, particularly the basic ones, and a concomitant reduction in organic acid synthesis.     

Changes in C uptake in populations of Chlamydomonas reinhardtii selected at high CO2

Estimates of the effect of increased global atmospheric CO(2) levels on oceanic primary productivity depend on the physiological responses of contemporary phytoplankton populations. However, microalgal populations will possibly adapt to rising CO(2) levels in such a way that they become genetically different from contemporary populations. The unknown properties of these future populations introduce an undefined error into predictions of C pool dynamics, especially the presence and size of the biological C pump. To address the bias in predictions introduced by evolution, we measured the kinetics of CO(2) uptake in populations of Chlamydomonas reinhardtii that had been selected for growth at high CO(2) for 1000 generations. Following selection at high CO(2), the populations were unable to induce high-affinity CO(2) uptake, and one line had a lower rate of net CO(2) uptake. We attribute this to conditionally neutral mutations in genes affecting the C concentrating mechanism (CCM). Lower affinity CO(2) uptake, in addition to smaller population sizes, results in a significant reduction in net CO(2) uptake of about 38% relative to contemporary populations under the same conditions. This shows how predictions about the properties of communities in the future can be influenced by the effect of natural selection.     

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.     

Brownian dynamics and molecular dynamics study of the association between hydrogenase and ferredoxin from Chlamydomonas reinhardtii

The [FeFe] hydrogenase from the green alga Chlamydomonas reinhardtii can catalyze the reduction of protons to hydrogen gas using electrons supplied from photosystem I and transferred via ferredoxin. To better understand the association of the hydrogenase and the ferredoxin, we have simulated the process over multiple timescales. A Brownian dynamics simulation method gave an initial thorough sampling of the rigid-body translational and rotational phase spaces, and the resulting trajectories were used to compute the occupancy and free-energy landscapes. Several important hydrogenase-ferredoxin encounter complexes were identified from this analysis, which were then individually simulated using atomistic molecular dynamics to provide more details of the hydrogenase and ferredoxin interaction. The ferredoxin appeared to form reasonable complexes with the hydrogenase in multiple orientations, some of which were good candidates for inclusion in a transition state ensemble of configurations for electron transfer.     

The induction of the CO2-concentrating mechanism is correlated with the formation of the starch sheath around the pyrenoid of Chlamydomonas reinhardtii

The pyrenoid is a prominent proteinaceous structure found in the stroma of the chloroplast in unicellular eukaryotic algae, most multicellular algae, and some hornworts. The pyrenoid contains the enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase and is sometimes surrounded by a carbohydrate sheath. We have observed in the unicellular green alga Chlamydomonas reinhardtii Dangeard that the pyrenoid starch sheath is formed rapidly in response to a decrease in the CO₂ concentration in the environment. This formation of the starch sheath occurs coincidentally with the induction of the CO₂-concentrating mechanism. Pyrenoid starch-sheath formation is partly inhibited by the presence of acetate in the growth medium under light and low-CO₂ conditions. These growth conditions also partly inhibit the induction of the CO₂-concentrating mechanism. When cells are grown with acetate in the dark, the CO₂-concentrating mechanism is not induced and the pyrenoid starch sheath is not formed even though there is a large accumulation of starch in the chloroplast stroma. These observations indicate that pyrenoid starch-sheath formation correlates with induction of the CO₂-concentrating mechanism under low-CO₂ conditions. We suggest that this ultrastructural reorganization under lowCO₂ conditions plays a role in the CO₂-concentrating mechanism C. reinhardtii as well as in other eukaryotic algae.     

Carbon dioxide concentrating mechanism in Chlamydomonas reinhardtii: inorganic carbon transport and CO2 recapture

Many microalgae are capable of acclimating to CO(2) limited environments by operating a CO(2) concentrating mechanism (CCM), which is driven by various energy-coupled inorganic carbon (Ci; CO(2) and HCO(3)(-)) uptake systems. Chlamydomonas reinhardtii (hereafter, Chlamydomonas), a versatile genetic model organism, has been used for several decades to exemplify the active Ci transport in eukaryotic algae, but only recently have many molecular details behind these Ci uptake systems emerged. Recent advances in genetic and molecular approaches, combined with the genome sequencing of Chlamydomonas and several other eukaryotic algae have unraveled some unique characteristics associated with the Ci uptake mechanism and the Ci-recapture system in eukaryotic microalgae. Several good candidate genes for Ci transporters in Chlamydomonas have been identified, and a few specific gene products have been linked with the Ci uptake systems associated with the different acclimation states. This review will focus on the latest studies on characterization of functional components involved in the Ci uptake and the Ci-recapture in Chlamydomonas.     

The NIT1 promoter allows inducible and reversible silencing of centrin in Chlamydomonas reinhardtii

An inverted repeat corresponding to parts of the centrin gene of Chlamydomonas reinhardtii was placed downstream of the NIT1 promoter, which is induced by ammonium starvation. After induction, transformants developed centrin deficiency as assayed by immunofluorescence, Western blotting, and Northern blotting. The effect was reversible, demonstrating that the NIT1 promoter allowed controlled RNA interference in Chlamydomonas reinhardtii.     

Atomic resolution modeling of the ferredoxin:[FeFe] hydrogenase complex from Chlamydomonas reinhardtii

The [FeFe] hydrogenases HydA1 and HydA2 in the green alga Chlamydomonas reinhardtii catalyze the final reaction in a remarkable metabolic pathway allowing this photosynthetic organism to produce H(2) from water in the chloroplast. A [2Fe-2S] ferredoxin is a critical branch point in electron flow from Photosystem I toward a variety of metabolic fates, including proton reduction by hydrogenases. To better understand the binding determinants involved in ferredoxin:hydrogenase interactions, we have modeled Chlamydomonas PetF1 and HydA2 based on amino-acid sequence homology, and produced two promising electron-transfer model complexes by computational docking. To characterize these models, quantitative free energy calculations at atomic resolution were carried out, and detailed analysis of the interprotein interactions undertaken. The protein complex model we propose for ferredoxin:HydA2 interaction is energetically favored over the alternative candidate by 20 kcal/mol. This proposed model of the electron-transfer complex between PetF1 and HydA2 permits a more detailed view of the molecular events leading up to H(2) evolution, and suggests potential mutagenic strategies to modulate electron flow to HydA2.     

Effects of NaCl and Na2CO3 stresses on photosynthetic ability of Chlamydomonas reinhardtii

Chloride and carbonate salts are the main salts causing salinization and widely exist in aquatic environment, so algae may suffer from salinization stress for high water evaporation. In this study, in order to investigate and compare the toxic effects of the two salts on algal photosynthesis, we used NaCl and Na₂CO₃ to stress Chlamydomonas reinhardtii. Under the two salt stresses, the content of O ₂ ^?· and H₂O₂ in the cells was increased significantly, and it was much higher in Na₂CO₃ treatment than in NaCl treatment at the same Na⁺ concentration. The absorbance spectra and 4^th derivative spectra of photosynthetic pigments were declined under 300 mM NaCl and 25 mM Na₂CO₃ stresses, and remarkably changed under 50 mM and 100 mM Na₂CO₃ stresses. When the cells stressed by the two salts, the maximum quantum yield (Fv/Fm), electron transport rate (ETR) and photochemical quenching (qP) were reduced markedly, but the nonphotochemical dissipation (NPQ) was increased markedly. At the same Na⁺ concentration, Na₂CO₃ stress had stronger toxic effects on photosynthetic ability than NaCl stress.     

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.     

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.