Long-term acclimation to cadmium exposure reveals extensive phenotypic plasticity in Chlamydomonas.

Increasing industrial and anthropogenic activities are producing and releasing more and more pollutants in the environment. Among them, toxic metals are one of the major threats for human health and natural ecosystems. Because photosynthetic organisms play a critical role in primary productivity and pollution management, investigating their response to metal toxicity is of major interest. Here, the green microalga Chlamydomonas (Chlamydomonas reinhardtii) was subjected to short (3 d) or chronic (6 months) exposure to 50 µM cadmium (Cd), and the recovery from chronic exposure was also examined. An extensive phenotypic characterization and transcriptomic analysis showed that the impact of Cd on biomass production of short-term (ST) exposed cells was almost entirely abolished by long-term (LT) acclimation. The underlying mechanisms were initiated at ST and further amplified after LT exposure resulting in a reversible equilibrium allowing biomass production similar to control condition. This included modification of cell wall-related gene expression and biofilm-like structure formation, dynamics of metal ion uptake and homeostasis, photosynthesis efficiency recovery and Cd acclimation through metal homeostasis adjustment. The contribution of the identified coordination of phosphorus and iron homeostasis (partly) mediated by the main phosphorus homeostasis regulator, Phosphate Starvation Response 1, and a basic Helix-Loop-Helix transcription factor (Cre05.g241636) was further investigated. The study reveals the highly dynamic physiological plasticity enabling algal cell growth in an extreme environment.

Toxicity, Physiological, and Ultrastructural Effects of Arsenic and Cadmium on the Extremophilic Microalga Chlamydomonas acidophila.

The cytotoxicity of cadmium (Cd), arsenate (As(V)), and arsenite (As(III)) on a strain of Chlamydomonas acidophila, isolated from the Rio Tinto, an acidic environment containing high metal(l)oid concentrations, was analyzed. We used a broad array of methods to produce complementary information: cell viability and reactive oxygen species (ROS) generation measures, ultrastructural observations, transmission electron microscopy energy dispersive x-ray microanalysis (TEM-XEDS), and gene expression. This acidophilic microorganism was affected differently by the tested metal/metalloid: It showed high resistance to arsenic while Cd was the most toxic heavy metal, showing an LC50 = 1.94 µM. Arsenite was almost four-fold more toxic (LC50= 10.91 mM) than arsenate (LC50 = 41.63 mM). Assessment of ROS generation indicated that both arsenic oxidation states generate superoxide anions. Ultrastructural analysis of exposed cells revealed that stigma, chloroplast, nucleus, and mitochondria were the main toxicity targets. Intense vacuolization and accumulation of energy reserves (starch deposits and lipid droplets) were observed after treatments. Electron-dense intracellular nanoparticle-like formation appeared in two cellular locations: inside cytoplasmic vacuoles and entrapped into the capsule, around each cell. The chemical nature (Cd or As) of these intracellular deposits was confirmed by TEM-XEDS. Additionally, they also contained an unexpected high content in phosphorous, which might support an essential role of poly-phosphates in metal resistance.

Manganese co-localizes with calcium and phosphorus in Chlamydomonas acidocalcisomes and is mobilized in manganese-deficient conditions.

Exposing cells to excess metal concentrations well beyond the cellular quota is a powerful tool for understanding the molecular mechanisms of metal homeostasis. Such improved understanding may enable bioengineering of organisms with improved nutrition and bioremediation capacity. We report here that Chlamydomonas reinhardtii can accumulate manganese (Mn) in proportion to extracellular supply, up to 30-fold greater than its typical quota and with remarkable tolerance. As visualized by X-ray fluorescence microscopy and nanoscale secondary ion MS (nanoSIMS), Mn largely co-localizes with phosphorus (P) and calcium (Ca), consistent with the Mn-accumulating site being an acidic vacuole, known as the acidocalcisome. Vacuolar Mn stores are accessible reserves that can be mobilized in Mn-deficient conditions to support algal growth. We noted that Mn accumulation depends on cellular polyphosphate (polyP) content, indicated by 1) a consistent failure of C. reinhardtii vtc1 mutant strains, which are deficient in polyphosphate synthesis, to accumulate Mn and 2) a drastic reduction of the Mn storage capacity in P-deficient cells. Rather surprisingly, X-ray absorption spectroscopy, EPR, and electron nuclear double resonance revealed that only little Mn2+ is stably complexed with polyP, indicating that polyP is not the final Mn ligand. We propose that polyPs are a critical component of Mn accumulation in Chlamydomonas by driving Mn relocation from the cytosol to acidocalcisomes. Within these structures, polyP may, in turn, escort vacuolar Mn to a number of storage ligands, including phosphate and phytate, and other, yet unidentified, compounds.

A highly efficient sulfadiazine selection system for the generation of transgenic plants and algae.

The genetic transformation of plant cells is critically dependent on the availability of efficient selectable marker gene. Sulfonamides are herbicides that, by inhibiting the folic acid biosynthetic pathway, suppress the growth of untransformed cells. Sulfonamide resistance genes that were previously developed as selectable markers for plant transformation were based on the assumption that, in plants, the folic acid biosynthetic pathway resides in the chloroplast compartment. Consequently, the Sul resistance protein, a herbicide-insensitive dihydropteroate synthase, was targeted to the chloroplast. Although these vectors produce transgenic plants, the transformation efficiencies are low compared to other markers. Here, we show that this inefficiency is due to the erroneous assumption that the folic acid pathway is located in chloroplasts. When the RbcS transit peptide was replaced by a transit peptide for protein import into mitochondria, the compartment where folic acid biosynthesis takes place in yeast, much higher resistance to sulfonamide and much higher transformation efficiencies are obtained, suggesting that current sul vectors are likely to function due to low-level mistargeting of the resistance protein to mitochondria. We constructed a series of optimized transformation vectors and demonstrate that they produce transgenic events at very high frequency in both the seed plant tobacco and the green alga Chlamydomonas reinhardtii. Co-transformation experiments in tobacco revealed that sul is even superior to nptII, the currently most efficient selectable marker gene, and thus provides an attractive marker for the high-throughput genetic transformation of plants and algae.

Proteome evolution under non-substitutable resource limitation.

Resource limitation is a major driver of the ecological and evolutionary dynamics of organisms. Short-term responses to resource limitation include plastic changes in molecular phenotypes including protein expression. Yet little is known about the evolution of the molecular phenotype under longer-term resource limitation. Here, we combine experimental evolution of the green alga Chlamydomonas reinhardtii under multiple different non-substitutable resource limitation regimes with proteomic measurements to investigate evolutionary adaptation of the molecular phenotype. We demonstrate convergent proteomic evolution of core metabolic functions, including the Calvin-Benson cycle and gluconeogenesis, across different resource limitation environments. We do not observe proteomic changes consistent with optimized uptake of particular limiting resources. Instead, we report that adaptation proceeds in similar directions under different types of non-substitutable resource limitation. This largely convergent evolution of the expression of core metabolic proteins is associated with an improvement in the resource assimilation efficiency of nitrogen and phosphorus into biomass.

Decreased phosphorus incorporation explains the negative effect of high iron concentrations in the green microalga Chlamydomonas acidophila.

The green microalga Chlamydomonas acidophila is an important primary producer in very acidic lakes (pH 2.0-3.5), characterized by high concentrations of ferric iron (up to 1 g total Fe L-1) and low rates of primary production. It was previously suggested that these high iron concentrations result in high iron accumulation and inhibit photosynthesis in C. acidophila. To test this, the alga was grown in sterilized lake water and in medium with varying total iron concentrations under limiting and sufficient inorganic phosphorus (Pi) supply, because Pi is an important growth limiting nutrient in acidic waters. Photosynthesis and growth of C. acidophila as measured over 5 days were largely unaffected by high total iron concentrations and only decreased if free ionic Fe3+ concentrations exceeded 100 mg Fe3+ L-1. Although C. acidophila was relatively rich in iron (up to 5 mmol Fe: mol C), we found no evidence of iron toxicity. In contrast, a concentration of 260 mg total Fe L-1 (i.e. 15 mg free ionic Fe3+ L-1), which is common in many acidic lakes, reduced Pi-incorporation by 50% and will result in Pi-limited photosynthesis. The resulting Pi-limitation present at high iron and Pi concentrations was illustrated by elevated maximum Pi-uptake rates. No direct toxic effects of high iron were found, but unfavourable chemical Pi-speciation reduced growth of the acidophile alga.

PtDRG1, a Desiccation Response Gene from Pyropia tenera (Rhodophyta), Exhibits Chaperone Function and Enhances Abiotic Stress Tolerance.

Pyropia are commercially valuable marine red algae that grow in the intertidal zone. They are extremely tolerant to desiccation stress. We have previously identified and reported desiccation response genes (DRGs) based on transcriptome analysis of P. tenera. Among them, PtDRG1 encodes a polypeptide of 22.6 kDa that is located in the chloroplast. PtDRG1 does not share sequence homology with any known gene deposited in public database. Transcription of PtDRG1 gene was upregulated by osmotic stress induced by mannitol or H2O2 as well as desiccation stress, but not by heat. When PtDRG1 was overexpressed in Escherichia coli or Chlamydomonas, transformed cells grew much better than control cells under high temperature as well as osmotic stress induced by mannitol and NaCl. In addition, PtDRG1 significantly reduced thermal aggregation of substrate protein under heat stress condition. These results demonstrate that PtDRG1 has a chaperone function and plays a role in tolerance mechanism for abiotic stress. This study shows that red algae have unknown stress proteins such as PtDRG1 that contributes to stress tolerance.

Basis of genetic adaptation to heavy metal stress in the acidophilic green alga Chlamydomonas acidophila.

To better understand heavy metal tolerance in Chlamydomonas acidophila, an extremophilic green alga, we assembled its transcriptome and measured transcriptomic expression before and after Cd exposure in this and the neutrophilic model microalga Chlamydomonas reinhardtii. Genes possibly related to heavy metal tolerance and detoxification were identified and analyzed as potential key innovations that enable this species to live in an extremely acid habitat with high levels of heavy metals. In addition we provide a data set of single orthologous genes from eight green algal species as a valuable resource for comparative studies including eukaryotic extremophiles. Our results based on differential gene expression, detection of unique genes and analyses of codon usage all indicate that there are important genetic differences in C. acidophila compared to C. reinhardtii. Several efflux family proteins were identified as candidate key genes for adaptation to acid environments. This study suggests for the first time that exposure to cadmium strongly increases transposon expression in green algae, and that oil biosynthesis genes are induced in Chlamydomonas under heavy metal stress. Finally, the comparison of the transcriptomes of several acidophilic and non-acidophilic algae showed that the Chlamydomonas genus is polyphyletic and that acidophilic algae have distinctive aminoacid usage patterns.

Biological effects of four iron-containing nanoremediation materials on the green alga Chlamydomonas sp.

As nanoremediation strategies for in-situ groundwater treatment extend beyond nanoiron-based applications to adsorption and oxidation, ecotoxicological evaluations of newly developed materials are required. The biological effects of four new materials with different iron (Fe) speciations ([i] FerMEG12 - pristine flake-like milled Fe(0) nanoparticles (nZVI), [ii] Carbo-Iron® - Fe(0)-nanoclusters containing activated carbon (AC) composite, [iii] Trap-Ox® Fe-BEA35 (Fe-zeolite) - Fe-doped zeolite, and [iv] Nano-Goethite - 'pure' FeOOH) were studied using the unicellular green alga Chlamydomonas sp. as a model test system. Algal growth rate, chlorophyll fluorescence, efficiency of photosystem II, membrane integrity and reactive oxygen species (ROS) generation were assessed following exposure to 10, 50 and 500 mg L-1 of the particles for 2 h and 24 h. The particles had a concentration-, material- and time-dependent effect on Chlamydomonas sp., with increased algal growth rate after 24 h. Conversely, significant intracellular ROS levels were detected after 2 h, with much lower levels after 24 h. All Fe-nanomaterials displayed similar Z-average sizes and zeta-potentials at 2 h and 24 h. Effects on Chlamydomonas sp. decreased in the order FerMEG12 > Carbo-Iron® > Fe-zeolite > Nano-Goethite. Ecotoxicological studies were challenged due to some particle properties, i.e. dark colour, effect of constituents and a tendency to agglomerate, especially at high concentrations. All particles exhibited potential to induce significant toxicity at high concentrations (500 mg L-1), though such concentrations would rapidly decrease to mg or µg L-1 in aquatic environments, levels harmless to Chlamydomonas sp. The presented findings contribute to the practical usage of particle-based nanoremediation in environmental restoration.

Influence of sulphate on the reduction of cadmium toxicity in the microalga Chlamydomonas moewusii.

Cadmium is considered as one of the most hazardous metals for living organism and ecosystems. Environmental factors play an important role since they alter the toxicity of metals by varying the bioavailability of these elements for the organisms. The aim of the present study was to investigate, using the freshwater microalga Chlamydomonas moewusii, the existence of an interaction between cadmium and sulphate as a factor that varied the toxicity of this metal. Different cell parameters such as cell growth, content of chlorophylls and biosynthesis of phytochelatins (PCs) were determined. A two-way ANOVA showed that the interaction had a significant effect size of 21% (p<0.001) for the growth of this microalga and around of a 6% on the content of chlorophylls/cell. The effect of this inhibition was that when the concentration of sulphate increased, a lower toxic effect of cadmium on the growth and on the content of chlorophylls was observed. In addition, the increase of sulphate concentration allowed the biosynthesis of a higher amount of PCs and/or PCs with higher chain length. This higher biosynthesis was responsible for the reduction of the toxic effect of cadmium and explained the interaction.

Isobolographic analysis of the interaction between cadmium (II) and sodium sulphate: toxicological consequences.

Sulphate is an essential nutrient for autotrophic organisms and has been shown to have important implications in certain processes of tolerance to cadmium toxicity. Sodium sulphate is the main salt of sulphate in the natural environments. The concentration of this salt is increasing in the aquatic environments due to environmental pollution. The aim of this study was to investigate, using an analysis of isobolograms, the type and the degree of the interaction between Cd(II) and sodium sulphate in the freshwater microalga Chlamydomonas moewusii. Two blocks of experiments were performed, one at sub-optimal sodium sulphate concentrations (<14.2 mg/L) and the other at supra-optimal concentrations (>14.2 mg/L). Three fixed ratios (2:1, 1:1, and 1:2) of the individual EC50 for cadmium and sodium sulphate were used within each block. The isobolographic analysis of interaction at sub-optimal concentrations showed a stronger antagonistic effect with values of interaction index (γ) between 1.46 and 3.4. However, the isobologram with sodium sulphate at supra-optimal concentrations revealed a slight but significant synergistic effect between both chemicals with an interaction index between 0.54 and 0.64. This synergic effect resulted in the potentiation of the toxic effects of cadmium, synergy that was related to the increase of the ionic strength and of two species of cadmium, CdSO4 (aq), and Cd(SO4)2(2-) , in the medium. Results of the current study suggest that sodium sulphate is able to perform a dual antagonist/synergist effect on cadmium toxicity. This role was concentration dependent.

Towards autotrophic tissue engineering: Photosynthetic gene therapy for regeneration.

The use of artificial tissues in regenerative medicine is limited due to hypoxia. As a strategy to overcome this drawback, we have shown that photosynthetic biomaterials can produce and provide oxygen independently of blood perfusion by generating chimeric animal-plant tissues during dermal regeneration. In this work, we demonstrate the safety and efficacy of photosynthetic biomaterials in vivo after engraftment in a fully immunocompetent mouse skin defect model. Further, we show that it is also possible to genetically engineer such photosynthetic scaffolds to deliver other key molecules in addition to oxygen. As a proof-of-concept, biomaterials were loaded with gene modified microalgae expressing the angiogenic recombinant protein VEGF. Survival of the algae, growth factor delivery and regenerative potential were evaluated in vitro and in vivo. This work proposes the use of photosynthetic gene therapy in regenerative medicine and provides scientific evidence for the use of engineered microalgae as an alternative to deliver recombinant molecules for gene therapy.

The Antarctic Psychrophile Chlamydomonas sp. UWO 241 Preferentially Phosphorylates a Photosystem I-Cytochrome b6/f Supercomplex.

Chlamydomonas sp. UWO 241 (UWO 241) is a psychrophilic green alga isolated from Antarctica. A unique characteristic of this algal strain is its inability to undergo state transitions coupled with the absence of photosystem II (PSII) light-harvesting complex protein phosphorylation. We show that UWO 241 preferentially phosphorylates specific polypeptides associated with an approximately 1,000-kD pigment-protein supercomplex that contains components of both photosystem I (PSI) and the cytochrome b6/f (Cyt b6/f) complex. Liquid chromatography nano-tandem mass spectrometry was used to identify three major phosphorylated proteins associated with this PSI-Cyt b6/f supercomplex, two 17-kD PSII subunit P-like proteins and a 70-kD ATP-dependent zinc metalloprotease, FtsH. The PSII subunit P-like protein sequence exhibited 70.6% similarity to the authentic PSII subunit P protein associated with the oxygen-evolving complex of PSII in Chlamydomonas reinhardtii. Tyrosine-146 was identified as a unique phosphorylation site on the UWO 241 PSII subunit P-like polypeptide. Assessment of PSI cyclic electron transport by in vivo P700 photooxidation and the dark relaxation kinetics of P700(+) indicated that UWO 241 exhibited PSI cyclic electron transport rates that were 3 times faster and more sensitive to antimycin A than the mesophile control, Chlamydomonas raudensis SAG 49.72. The stability of the PSI-Cyt b6/f supercomplex was dependent upon the phosphorylation status of the PsbP-like protein and the zinc metalloprotease FtsH as well as the presence of high salt. We suggest that adaptation of UWO 241 to its unique low-temperature and high-salt environment favors the phosphorylation of a PSI-Cyt b6/f supercomplex to regulate PSI cyclic electron transport rather than the regulation of state transitions through the phosphorylation of PSII light-harvesting complex proteins.

Unraveling vitamin B12-responsive gene regulation in algae.

Photosynthetic microalgae play a vital role in primary productivity and biogeochemical cycling in both marine and freshwater systems across the globe. However, the growth of these cosmopolitan organisms depends on the bioavailability of nutrients such as vitamins. Approximately one-half of all microalgal species requires vitamin B12 as a growth supplement. The major determinant of algal B12 requirements is defined by the isoform of methionine synthase possessed by an alga, such that the presence of the B12-independent methionine synthase (METE) enables growth without this vitamin. Moreover, the widespread but phylogenetically unrelated distribution of B12 auxotrophy across the algal lineages suggests that the METE gene has been lost multiple times in evolution. Given that METE expression is repressed by the presence of B12, prolonged repression by a reliable source of the vitamin could lead to the accumulation of mutations and eventually gene loss. Here, we probe METE gene regulation by B12 and methionine/folate cycle metabolites in both marine and freshwater microalgal species. In addition, we identify a B12-responsive element of Chlamydomonas reinhardtii METE using a reporter gene approach. We show that complete repression of the reporter occurs via a region spanning -574 to -90 bp upstream of the METE start codon. A proteomics study reveals that two other genes (S-Adenosylhomocysteine hydrolase and Serine hydroxymethyltransferase2) involved in the methionine-folate cycle are also repressed by B12 in C. reinhardtii. The strong repressible nature and high sensitivity of the B12-responsive element has promising biotechnological applications as a cost-effective regulatory gene expression tool.

Sulphate, more than a nutrient, protects the microalga Chlamydomonas moewusii from cadmium toxicity.

Sulphur is an essential macroelement that plays important roles in living organisms. The thiol rich sulphur compounds, such as cysteine, γ-Glu-Cys, glutathione and phytochelatins participate in the tolerance mechanisms against cadmium toxicity. Plants, algae, yeasts and most prokaryotes cover their demand for reduced sulphur by reduction of inorganic sulphate. The aim of this study was to investigate, using a bifactorial experimental design, the effect of different sulphate concentrations in the nutrient solution on cadmium toxicity in the freshwater microalga Chlamydomonas moewusii. Cell growth, kinetic parameters of sulphate utilization and intracellular concentrations of low-molecular mass thiol compounds were determined. A mathematical model to describe the growth of this microalga based on the effects of sulphate and cadmium was obtained. An ANOVA revealed an interaction between them, 16% of the effect sizes was explained by this interaction. A higher amount of sulphate in the culture medium allowed a higher cadmium tolerance due to an increase in the thiol compound biosynthesis. The amount of low-molecular mass thiol compounds, mainly phytochelatins, synthesized by this microalga was significantly dependent on the sulphate and cadmium concentrations; the higher phytochelatin content was obtained in cultures with 4 mg Cd/L and 1mM sulphate. The maximum EC50 value (based on nominal cadmium concentration) reached for this microalga was 4.46 ± 0.42 mg Cd/L when the sulphate concentration added to the culture medium was also 1mM. An increase in the sulphate concentration, in deficient environments, could alleviate the toxic effect of this metal; however, a relative excess is also negative. The results obtained showed a substrate inhibition for this nutrient. An uncompetitive model for sulphate was chosen to establish the mathematical model that links both factors.

The salt stress-induced LPA response in Chlamydomonas is produced via PLA2 hydrolysis of DGK-generated phosphatidic acid.

The unicellular green alga Chlamydomonas has frequently been used as a eukaryotic model system to study intracellular phospholipid signaling pathways in response to environmental stresses. Earlier, we found that hypersalinity induced a rapid increase in the putative lipid second messenger, phosphatidic acid (PA), which was suggested to be generated via activation of a phospholipase D (PLD) pathway and the combined action of a phospholipase C/diacylglycerol kinase (PLC/DGK) pathway. Lysophosphatidic acid (LPA) was also increased and was suggested to reflect a phospholipase A2 (PLA2) activity based on pharmacological evidence. The question of PA's and LPA's origin is, however, more complicated, especially as both function as precursors in the biosynthesis of phospho- and galactolipids. To address this complexity, a combination of fatty acid-molecular species analysis and in vivo ³²P-radiolabeling was performed. Evidence is provided that LPA is formed from a distinct pool of PA characterized by a high α-linolenic acid (18:3n-3) content. This molecular species was highly enriched in the polyphosphoinositide fraction, which is the substrate for PLC to form diacylglycerol. Together with differential ³²P-radiolabeling studies and earlier PLD-transphosphatidylation and PLA2-inhibitor assays, the data were consistent with the hypothesis that the salt-induced LPA response is primarily generated through PLA2-mediated hydrolysis of DGK-generated PA and that PLD or de novo synthesis [via endoplasmic reticulum - or plastid-localized routes] is not a major contributor.

The expression of a carbon concentrating mechanism in Chlamydomonas acidophila under variable phosphorus, iron, and CO2 concentrations.

The CO(2) acquisition was analyzed in Chlamydomonas acidophila at pH 2.4 in a range of medium P and Fe concentrations and at high and low CO(2) condition. The inorganic carbon concentrating factor (CCF) was related to cellular P quota (Q(p)), maximum CO(2)-uptake rate by photosynthesis (V(max,O2)), half saturation constant for CO(2) uptake (K(0.5)), and medium Fe concentration. There was no effect of the medium Fe concentration on the CCF. The CCF increased with increasing Q(p) in both high and low CO(2) grown algae, but maximum Q(p) was 6-fold higher in the low CO(2) cells. In high CO(2) conditions, the CCF was low, ranging between 0.8 and 3.5. High CCF values up to 9.1 were only observed in CO(2)-limited cells, but P- and CO(2)-colimited cells had a low CCF. High CCF did not relate with a low K(0.5) as all CO(2)-limited cells had a low K(0.5) (<4 µM CO(2)). High C(i)-pools in cells with high Q(p) suggested the presence of an active CO(2)-uptake mechanism. The CCF also increased with increasing V(max,O2) which reflect an adaptation to the nutrient in highest demand (CO(2)) under balanced growth conditions. It is proposed that the size of the CCF in C. acidophila is more strongly related to porter density for CO(2) uptake (reflected in V(max,O2)) and less- to high-affinity CO(2) uptake (low K(0.5)) at balanced growth. In addition, high CCF can only be realized with high Q(p).

Nickel and low CO2-controlled motility in Chlamydomonas through complementation of a paralyzed flagella mutant with chemically regulated promoters.

BACKGROUND: Chlamydomonas reinhardtii is a model system for the biology of unicellular green algae. Chemically regulated promoters, such as the nickel-inducible CYC6 or the low CO2-inducible CAH1 promoter, may prove useful for expressing, at precise times during its cell cycle, proteins with relevant biological functions, or complementing mutants in genes encoding such proteins. To this date, this has not been reported for the above promoters. RESULTS: We fused the CYC6 and CAH1 promoters to an HA-tagged RSP3 gene, encoding a protein of the flagellar radial spoke complex. The constructs were used for chemically regulated complementation of the pf14 mutant, carrying an ochre mutation in the RSP3 gene. 7 to 8% of the transformants showed cells with restored motility after induction with nickel or transfer to low CO2 conditions, but not in non-inducing conditions. Maximum complementation (5% motile cells) was reached with very different kinetics (5-6 hours for CAH1, 48 hours for CYC6). The two inducible promoters drive much lower levels of RSP3 protein expression than the constitutive PSAD promoter, which shows almost complete rescue of motility. CONCLUSIONS: To our knowledge, this is the first example of the use of the CYC6 or CAH1 promoters to perform a chemically regulated complementation of a Chlamydomonas mutant. Based on our data, the CYC6 and CAH1 promoters should be capable of fully complementing mutants in genes whose products exert their biological activity at low concentrations.

Effect of aluminum on cellular division and photosynthetic electron transport in Euglena gracilis and Chlamydomonas acidophila.

The present study investigated aluminum's effect on cellular division and the photosynthetic processes in Euglena gracilis and Chlamydomonas acidophila at pH 3.0, at which Al is present mostly as Al(3+), AlSO(4) (+), and Al(SO(4))(2) (-). These algal species were exposed to 100, 188, and 740 microM Al, and after 24 h cell-bound Al was significantly different from control only for the highest concentration tested. However, very different effects of Al on algal cellular division, biomass per cell, and photosynthetic activity were found. Aluminum stimulated cell division but decreased at some level biomass per cell in C. acidophila. Primary photochemistry of photosynthesis, as Photosystem II quantum yield, and energy dissipation via nonphotochemical activity were slightly affected. However, for E. gracilis, under the same conditions, Al did not show a stimulating effect on cellular division or photosynthetic activity. Primary photochemical activity was diminished, and energy dissipation via nonphotochemical pathways was strongly increased. Therefore, when Al is highly available in aquatic ecosystems, these effects may indicate very different response mechanisms that are dependent on algal species.

Cadmium toxicity on the freshwater microalga Chlamydomonas moewusii Gerloff: Biosynthesis of thiol compounds.

Cadmium (Cd) toxicity and production of different thiols (phytochelatins, glutathione, gamma-Glu-Cys and cysteine) were studied in the microalga Chlamydomonas moewusii exposed to different concentrations of this metal (1, 2, 4, 6, 8, and 10 mg/L) for 96 h. The inhibitory effect of Cd on growth was demonstrated. The value of EC50 (metal concentration which reduces the population growth to 50% of the control) obtained for this microalga was estimated at 4.1 +/- 0.8 mg/L of Cd after 96 h of exposure. The amount of thiol compounds synthesized by C. moewusii changed with Cd concentration. Cysteine concentrations were significantly higher compared to those of gamma-Glu-Cys and glutathione in all the Cd concentrations assayed. The amino acid cysteine reached its higher levels in those cultures in which a decrease in the concentration of phytochelatins (PCs) was observed. Both cysteine and glutathione concentrations showed significant differences along the Cd concentrations assayed, while the amount of gamma-Glu-Cys detected remained stable. The PCs detected were of two, three, and four subunits. The level of PC(2) was higher than that of PC(3) and PC(4). PC(4) was detected only in the cultures exposed to the Cd concentrations of 1 and 2 mg/L, in which the synthesis of phytochelatins was higher. A rapid increase in the production of PC(2) and PC(3) was observed up to a Cd concentration of 2 mg/L, after which their levels began to decrease. Phytochelatins were not detected in cultures without Cd (controls) and in those exposed to the maximum Cd concentration (10 mg/L), in which cell growth was completely inhibited.