Pyrenoidal sequestration of cadmium impairs carbon dioxide fixation in a microalga.

Mixotrophic microorganisms are able to use organic carbon as well as inorganic carbon sources and thus, play an essential role in the biogeochemical carbon cycle. In aquatic ecosystems, the alteration of carbon dioxide (CO2 ) fixation by toxic metals such as cadmium - classified as a priority pollutant - could contribute to the unbalance of the carbon cycle. In consequence, the investigation of cadmium impact on carbon assimilation in mixotrophic microorganisms is of high interest. We exposed the mixotrophic microalga Chlamydomonas reinhardtii to cadmium in a growth medium containing both CO2 and labelled 13 C-[1,2] acetate as carbon sources. We showed that the accumulation of cadmium in the pyrenoid, where it was predominantly bound to sulphur ligands, impaired CO2 fixation to the benefit of acetate assimilation. Transmission electron microscopy (TEM)/X-ray energy dispersive spectroscopy (X-EDS) and micro X-ray fluorescence (µXRF)/micro X-ray absorption near-edge structure (µXANES) at Cd LIII- edge indicated the localization and the speciation of cadmium in the cellular structure. In addition, nanoscale secondary ion mass spectrometry (NanoSIMS) analysis of the 13 C/12 C ratio in pyrenoid and starch granules revealed the origin of carbon sources. The fraction of carbon in starch originating from CO2 decreased from 73 to 39% during cadmium stress. For the first time, the complementary use of high-resolution elemental and isotopic imaging techniques allowed relating the impact of cadmium at the subcellular level with carbon assimilation in a mixotrophic microalga.

Algicidal properties of extracts from Cinnamomum camphora fresh leaves and their main compounds.

Plant allelochemicals are considered as the source of effective, economic and friendly-environmental algaecides. To uncover the anti-algal activities of Cinnamomum camphora fresh leaves and their main algicidal agents, we investigated the inhibitory effects of water and methanol extracts from C. camphora fresh leaves on Microcystis aeruginosa and Chlamydomonas reinhardtii cell growth, analyzed the composition of the water and methanol extracts, and determined the main compounds in extracts on the growth of the two algae and their anti-algal mechanism from photosynthetic abilities. Water and methanol extracts from C. camphora fresh leaves can inhibit M. aeruginosa and C. reinhardtii cell growth, and methanol extracts showed stronger inhibitory effects, due to their more compounds and higher molar concentration. There were 23 compounds in the water extracts, mainly including terpenoids, esters, alcohols, and ketones. Compared to the water extracts, 9 new compounds were detected in the methanol extracts, and the molar concentration of total compounds in methanol extracts increased by 1.3 folds. Camphor, α-terpineol and linalool were 3 main compounds in the water and methanol extracts. Their mixture (1: 3: 6) and individual compound showed remarkable inhibition on M. aeruginosa and C. reinhardtii cell growth. The degradation of photosynthetic pigments and the reduction of maximum quantum yield of photosystem II (PSII) photochemistry, coefficient of photochemical quenching as well as apparent electron transport rate in C. reinhardtii cells aggravated gradually with increasing the concentration of the mixture and individual compound, while the non-photochemical dissipation of absorbed light energy increased gradually, which led to the decline of photosynthetic abilities. This indicated that camphor, α-terpineol and linalool were 3 main algicidal agents in C. camphora fresh leaf extracts, and they inhibited algal growth by inducing photosynthetic pigment degradation and declining PSII efficiency. Therefore, C. camphora fresh leaf extracts and their main components have potential utilization values as algaecides.

Inhibitory effects of extracts from Cinnamomum camphora fallen leaves on algae.

Natural allelochemicals are considered as a source of algaecides. To uncover the anti-algal activity of Cinnamomum camphora fallen leaves and promote their usage as algaecides, the composition of their water and methanol extracts was analyzed, and the inhibitory effects of extracts on the growth of Microcystis aeruginosa and Chlamydomonas reinhardtii, and chlorophyll (Chl) content and photosynthetic abilities in C. reinhardtii were investigated. Twenty-five compounds were detected in the water extracts, mainly including terpenoids, esters, alcohols, and ketones. Compared to water extracts, there were more compounds and higher concentration in methanol extracts. Both water and methanol extracts inhibited the growth of the two algae, and 15 mg·ml-1 methanol extracts killed the algal cells after 48 h. The levels of Chl a and Chl b, as well as maximum quantum yield of photosystem II photochemistry (Fv/Fm) in C. reinhardtii cells reduced gradually with increasing the concentration of extracts, while the maximum quantum yield of non-photochemical de-excitation (φDO) increased gradually. At the same concentration, methanol extracts showed stronger inhibitory effects than water extracts, due to their higher number of compounds and higher concentration. Therefore, C. camphora fallen leaves have a potential value as an algaecide.

Response of energy microalgae Chlamydomonas reinhardtii to nitrogen and phosphorus stress.

Microalgae can effectively absorb nitrogen (N) and phosphorus (P) in wastewater, while growth characteristics can be affected by such nutrients. The influences of the N and P concentration on growth, biomass yield, protein yield, and cell ultrastructure of Chlamydomonas reinhardtii (C. reinhardtii) were investigated in this study. The results showed that, in the optimum conditions (24-72 mg/L for N and 4.5-13.5 mg/L for P), the final biomass and protein content of C. reinhardtii could reach maximum value, and the cell organelles (chloroplast, mitochondria,etc.) showed good structures with larger chloroplasts, and more and neater thylakoids. However, if the concentration of nutrients was much higher or lower than the optimal value, it would cause adverse effects on the growth of C. reinhardtii, especially in high nitrogen (1000 mg/L) and low phosphorus (0.5 mg/L) conditions. Under these extreme conditions, the ultrastructure of the cells was also damaged significantly as follows: the majority of the organelles were deformed, the chloroplast membrane became shrunken, and the mitochondria became swollen, even partial disintegrated (differing slightly under high-N and low-P conditions); furthermore, it is found that C. reinhardtii was more sensitive to low-P stress. On the basis of these results, our findings have general implications in the application of wastewater treatment.

Effects of phosphorus sources on volatile organic compound emissions from Microcystis flos-aquae and their toxic effects on Chlamydomonas reinhardtii.

There is diverse phosphorus (P) in eutrophicated waters, but it is considered as a crucial nutrient for cyanobacteria growth due to its easy precipitation as insoluble salts. To uncover the effects of complex P nutrients on the emission of volatile organic compounds (VOCs) from cyanobacteria and their toxic effects on other algae, the VOCs from Microcystis flos-aquae supplied with different types and amount of P nutrients were analyzed, and the effects of VOCs and their two main compounds on Chlamydomonas reinhardtii growth were investigated. When M. flos-aquae cells were supplied with K2HPO4, sodium pyrophosphate and sodium hexametaphosphate as the sole P source, 27, 23 and 29 compounds were found, respectively, including furans, sulfocompounds, terpenoids, benzenes, aldehydes, hydrocarbons and esters. With K2HPO4 as the sole P source, the VOC emission increased with reducing P amount, and the maximum emission was found under Non-P condition. In the treatments of M. flos-aquae VOCs under Non-P condition and two main terpenoids (eucalyptol and limonene) in the VOCs, remarkable decreases were found in C. reinhardtii cell growth, photosynthetic pigment content and photosynthetic abilities. Therefore, we deduce that multiple P nutrients in eutrophicated waters induce different VOC emissions from cyanobacteria, and P amount reduction caused by natural precipitation and algal massive growth results in more VOC emissions. These VOCs play toxic roles in cyanobacteria becoming dominant species, and eucalyptol and limonene are two toxic agents.

Effect of tetracycline on the growth and nutrient removal capacity of Chlamydomonas reinhardtii in simulated effluent from wastewater treatment plants.

The aim of this work was to study the effect of tetracycline, which is on the growth, physiological characteristics, and contaminants removal by Chlamydomonas reinhardtii. The results showed that the biomass and photosynthetic pigment concentration of C. reinhardtii exposed to tetracycline were lower than those of the control, while the superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) activities, and the malondialdehyde (MDA) content, were higher than those of the control. Additionally, when the tetracycline concentration reached 0.25mg/L, the removal of total nitrogen (TN), total phosphorus (TP), and chemical oxygen demand (COD) decreased from 80.8 to 55.0%, 100 to 92.5%, and 36.5 to 11.5%, respectively. Thus, tetracycline concentrations of 0-0.25mg/L are expected to have a significant effect on the growth and nutrient removal of C. reinhardtii in recycled water from wastewater treatment plants.

The effect of rapamycin on biodiesel-producing protist Euglena gracilis.

Rapamycin induces autophagy with lipid remodeling in yeast and mammalian cells. To investigate the lipid biosynthesis of Euglena gracilis, rapamycin was supplemented in comparison with two model algae, Chlamydomonas reinhardtii and Cyanidioschyzon merolae. In Euglena, rapamycin induced the reduction of chlorophylls and the accumulation of neutral lipids without deterring its cell proliferation. Its lipidomic profile revealed that the fatty acid composition did not alter by supplementing rapamycin. In Chlamydomonas, however, rapamycin induced serious growth inhibition as reported elsewhere. With a lower concentration of rapamycin, the alga accumulated neutral lipids without reducing chlorophylls. In Cyanidioschyzon, rapamycin did not increase neutral lipids but reduced its chlorophyll content. We also tested fatty acid elongase inhibitors such as pyroxasulfone or flufenacet in Euglena with no significant change in its neutral lipid contents. In summary, controlled supplementation of rapamycin can increase the yield of neutral lipids while the scheme is not always applicable for other algal species.

Phosphite cannot be used as a phosphorus source but is non-toxic for microalgae.

Phosphorous (P) plays a critical role for all living organisms as a structural component of RNA, DNA and phospholipids. Microalgae are autotrophs organisms that have been reported to only assimilate the fully oxidized phosphate (Pi) as P source. However, there are microorganisms capable of utilizing P reduced compounds (i.e. phosphite (Phi) and hypophosphite) as a sole P source, such as bacteria and cyanobacteria. In this study, we evaluated whether microalgae, such as Chlamydomonas reinhardtii, Botryococcus braunii and Ettlia oleoabundans, are capable of using Phi as a sole P source. Our studies revealed that these three microalgae are unable to use Phi as a sole P source. We also found that when Phi is present at concentrations equal or higher than that of Pi, Phi has an inhibitory effect on C. reinhardtii growth. However, since C. reinhardtii was able to survive for a long period of cultivation in the presence of high concentrations of Phi and to recover cell division capacity after transfer to media containing Pi, we noticed that Phi is not toxic for this microalga. We propose that the inhibitory effect of Phi on C. reinhardtii growth might be caused, at least in part, by a competition between the transport of Pi and Phi.

Effects of nitrogen and phosphorus on arsenite accumulation, oxidation, and toxicity in Chlamydomonas reinhardtii.

We studied arsenite (iAs(III)) accumulation, oxidation, and toxicity in the freshwater green alga Chlamydomonas reinhardtii under nutrient-enriched (+NP), phosphorus-limited (-P), and nitrogen-limited (-N) conditions. The -P alga (55.1 µM) had a Michaelis constant (Kd) for uptake approximately one tenth of the +NP (419 µM) and -N (501 µM) cells, indicating iAs(III) uptake inhibition by extracellular phosphate. This conclusion was supported by the hyperbolic reduction in iAs(III) uptake rate (V) from 9.2 to 0.8 µmol/g-dw/h when the extracellular phosphate concentration went up from 0 to 250 µM. The maximal iAs(III) uptake rate (Vmax) of the -N alga (24.3 µmol/g-dw/h) was twice as much as that of the +NP (12 µmol/g-dw/h) and -P (8.1 µmol/g-dw/h) cells. It implies that more arsenic transporters were synthesized under the -N condition. Once accumulated, iAs(III) was oxidized and a higher proportion of arsenate (iAs(V)) was observed at lower [As]dis or under nutrient-limited conditions. Nevertheless, iAs(III) oxidation mainly occurred outside the cells with the extent of oxidation reciprocal to [As]dis. Based on the logistic modeling of the concentration-response curves in the +NP, -P, and -N toxicity tests, iAs(III) had an [As]dis-based EC50 of 1763, 13.1, and 1208 µM and an intracellular arsenic concentration based EC50 of 35.6, 28.8, and 195 µmol/g-dw, respectively. Higher iAs(III) toxicity to the -P cells occured because of their increased iAs(III) accumulation, whereas the underlying mechanisms why the -N alga was more tolerant need to be further revealed. Overall, both N and P had remarkable effects on the behavior and effects of iAs(III), which cannot be disregarded in the biogeochemical cycling research of arsenic.

Uranium accumulation and toxicity in the green alga Chlamydomonas reinhardtii is modulated by pH.

The effects of pH on metal uptake and toxicity in aquatic organisms are currently poorly understood and remain an evolving topic in studies about the biotic ligand model (BLM). In the present study, the authors investigated how pH may influence long-term (4 d) uranium (U) accumulation and chronic toxicity in batch cultures of the freshwater green alga Chlamydomonas reinhardtii. The toxicity expressed as a function of the free uranyl ion was much greater at pH 7 (effective concentration, 50% [EC50] = 1.8 × 10(-9) M UO2 (2+) ) than at pH 5 (EC50 = 1.2 × 10(-7) M UO2 (2+) ). The net accumulation rate of U in algal cells was much higher at pH 7 than at pH 5 for the same free [UO2 (2+) ], but the cells exposed at pH 5 were also more sensitive to intracellular U than the cells at pH 7 with EC50s of 4.0 × 10(-15) and 7.1 × 10(-13) mol of internalized U cell(-1) , respectively. The higher cellular sensitivity to U at pH 5 than at pH 7 could be explained partly by the increase in cytosolic U binding to algal soluble proteins or enzymes at pH 5 as observed by subcellular fractionation. To predict U accumulation and toxicity in algae accurately, the important modulating effects of pH on U accumulation and U cellular sensitivity should be considered in the BLM.

Sensitivity evaluation of the green alga Chlamydomonas reinhardtii to uranium by pulse amplitude modulated (PAM) fluorometry.

Although ecotoxicological studies tend to address the toxicity thresholds of uranium in freshwaters, there is a lack of information on the effects of the metal on physiological processes, particularly in aquatic plants. Knowing that uranium alters photosynthesis via impairment of the water photo-oxidation process, we determined whether pulse amplitude modulated (PAM) fluorometry was a relevant tool for assessing the impact of uranium on the green alga Chlamydomonas reinhardtii and investigated how and to what extent uranium hampered photosynthetic performance. Photosynthetic activity and quenching were assessed from fluorescence induction curves generated by PAM fluorometry, after 1 and 5h of uranium exposure in controlled conditions. The oxygen-evolving complex (OEC) of PSII was identified as the primary action site of uranium, through alteration of the water photo-oxidation process as revealed by F0/Fv. Limiting re-oxidation of the plastoquinone pool, uranium impaired the electron flux between the photosystems until almost complete inhibition of the PSII quantum efficiency ( [Formula: see text] , EC50=303 ± 64 µg UL(-1) after 5h of exposure) was observed. Non-photochemical quenching (qN) was identified as the most sensitive fluorescence parameter (EC50=142 ± 98 µg UL(-1) after 5h of exposure), indicating that light energy not used in photochemistry was dissipated in non-radiative processes. It was shown that parameters which stemmed from fluorescence induction kinetics are valuable indicators for evaluating the impact of uranium on PSII in green algae. PAM fluorometry provided a rapid and reasonably sensitive method for assessing stress response to uranium in microalgae.

Transfer modelling and toxicity evaluation of the effluent from an installation of cleansing and uranium recovery using a battery of bioassays.

On July 7, 2008, a leak of effluent from an Installation of Cleansing and Uranium Recovery (Tricastin, France) led to the spillage of uranium in a stream. The acute toxicity of the effluent was evaluated, and compared to the toxicity of uranium nitrate in bioassays using several organisms: Chlamydomonas reinhardtii, Daphnia magna, Chironomus riparius and Danio rerio. A sediment bioassay was also performed on C. riparius using water and sediment sampled along the river. Results showed that effluent EC(50) 72 h was 0.65 mg U/l for algae and LC(50) 48 h was 1.67 mg U/l for daphnia, while values obtained for uranium nitrate were higher. The LC(50) 96 h of effluent to C. riparius was 22.7 mg U/l, similar to value for uranium nitrate; the sediment collected was not toxic to C. riparius larvae. The LOEC of effluent and uranium nitrate on HT(50) of D. rerio were similar (0.03 mg U/l), but larvae were more sensitive to uranium nitrate than to effluent. Our results suggest that other substances contained in the effluent could potentially be toxic to wildlife in association with uranium. In parallel, the modelling of the transfers based on uranium measurements in the surface water was used to fill data gaps and assess the impact along the river. These results provided an estimate of exposure conditions that occurred along the river. This approach allowed us to see that the risk to ecosystem during this incident was certainly low and concerned a short period of time, but it could have existed at least for some species.

Extract of Lillium candidum L. can modulate the genotoxicity of the antibiotic zeocin.

Lilium candidum L. extract (LE) is well known in folk medicine for the treatment of burns, ulcers, inflammations and for healing wounds. This work aims to clarify whether the genotoxic potential of the radiomimetic antibiotic zeocin (Zeo) could be modulated by LE. Our results indicate that LE exerts no cytotoxic, DNA-damaging and clastogenic activity in in Chlamydomonas reinhardtii, Pisum sativum L. and Hordeum vulgare L. test systems over a broad concentration range. Weak but statistically significant clastogenic effects due to the induction of micronuclei and chromosome aberrations have been observed in H. vulgare L. after treatment with 200 and 300 µg/mL LE. To discriminate protective from adverse action of LE different experimental designs have been used. Our results demonstrate that the treatment with mixtures of LE and Zeo causes an increase in the level of DNA damage, micronuclei and "metaphases with chromatid aberrations" (MwA). Clear evidence has been also obtained indicating that pretreatment with LE given 4 h before the treatment with Zeo accelerates the rejoining kinetics of Zeo-induced DNA damage in P. sativum L. and C. reinhardtii, and can decrease clastogenic effect of Zeo measured as frequencies of micronuclei and MwA in H. vulgare L. Here, we show for the first time that LE can modulate the genotoxic effects of zeocin. The molecular mode of action strongly depends on the experimental design and varies from synergistic to protective effect (adaptive response-AR). Our results also revealed that LE-induced AR to zeocin involves up-regulation of DSB rejoining in C. reinhardtii and P. sativum L. cells.

Using FTIR spectroscopy for rapid determination of lipid accumulation in response to nitrogen limitation in freshwater microalgae.

In this study Fourier transform infrared micro-spectroscopy (FTIR) was used to determine lipid and carbohydrate content over time in the freshwater microalgae Chlamydomonas reinhardtii and Scenedesmus subspicatus grown in batch culture in limiting concentrations of nitrogen (N). Both algae exhibited restricted cell division and increased cell size following N-limitation. FTIR spectra of cells in N-limited media showed increasing lipid:amide I and carbohydrate:amide I ratios over time. The use of lipid- and starch-staining dyes confirmed that the observed ratio changes were due to increased lipid and carbohydrate synthesis. These results demonstrate rapid metabolic responses of C. reinhardtii and S. subspicatus to changing nutrient availability, and indicate the efficiency of FTIR as a reliable method for high-throughput determination of lipid induction.

Culture of microalgae Chlamydomonas reinhardtii in wastewater for biomass feedstock production.

The objective of this research was to develop large-scale technologies to produce oil-rich algal biomass from wastewater. The experiments were conducted using Erlenmeyer flasks and biocoil photobioreactor. Chlamydomonas reinhardtii was grown in artificial media and wastewaters taken from three different stages of the treatment process, namely, influent, effluent, and centrate. Each of wastewaters contained different levels of nutrients. The specific growth rate of C. reinhardtii in different cultures was monitored over a period of 10 days. The biomass yield of microalgae and associated nitrogen and phosphorous removal were evaluated. Effects of CO(2) and pH on the growth were also studied. The level of nutrients greatly influenced algae growth. High levels of nutrients seem to inhibit algae growth in the beginning, but provided sustained growth to a high degree. The studies have shown that the optimal pH for C. reinhardtii is in the range of 7.5. An injection of air and a moderate amount of CO(2) promoted algae growth. However, too much CO(2) inhibited algae growth due to a significant decrease in pH. The experimental results showed that algal dry biomass yield reached a maximum of 2.0 g L(-1) day(-1) in the biocoil. The oil content of microalgae of C. reinhardtii was 25.25% (w/w) in dry biomass weight. In the biocoil, 55.8 mg nitrogen and 17.4 mg phosphorus per liter per day were effectively removed from the centrate wastewater. Ferric chloride was found to be an effective flocculent that helps the algae settle for easy harvest and separation from the culture media.

Genotoxicity and antigenotoxicity evaluation of non-photoactivated hypericin.

The potential genotoxicity and antigenotoxicity of non-photoactivated hypericin was investigated in five experimental models. Hypericin was non-mutagenic in the Ames assay, with and without metabolic activation. It did not exert a protective effect against mutagenicity induced by 9-aminoacridine. In a yeast (Saccharomyces cerevisiae) assay, hypericin did not increase the frequency of mitotic crossovers or total aberrants at the ade(2) locus, the number of convertants at the trp5 locus, or the number of revertants at the ilv1 locus. In combined application with 4-nitroquinoline-1-oxide, it significantly enhanced the number of revertants at the ilv1 locus at the highest concentration used. Hypericin was not mutagenic in the alga Chlamydomonas reinhardtii. However, in combined application with methyl methane sulfonate, toxicity and mutagenicity were slightly reduced. In a chromosome aberration assay using three mammalian cell lines, hypericin did not alter the frequency of structural chromosome aberrations, and in the DPPH radical scavenging assay, it did not exert any antioxidant effects.

Metal-phytoplankton interactions: modeling the effect of competing ions (H+, Ca2+, and Mg2+) on uranium uptake.

The influence of pH and hardness cation concentrations on uranium uptake by a green alga, Chlamydomonas reinhardtii, was investigated through short-term exposure experiments. Uranium uptake at pH 5 and at pH 7 was measured over a large concentration range (0.020-2.0 microM 233U), and the effects of hardness cations were studied over environmentally pertinent concentration ranges (approximately 0.05-2 mM) at a constant uranium concentration (0.25 microM). Calcium and magnesium inhibited uranyl uptake, but the influence of pH was more complex than anticipated. The equilibrium biotic ligand paradigm of metal bioavailability predicts that two distinct phenomena of antipathetic effect will influence uranium availability as pH is varied. Increasing pH reduces the concentration of protons, thus reducing competition for the physiologically active sites, whereas the concomitant complexation by carbonates and hydroxides reduces the free uranyl activity. Maximum uranium uptake rates observed at pH 7, however, were far greater than those observed at pH 5, suggesting a noncompetitive inhibition of metal transport by protons. Modeling on the basis of our results strongly suggests that cells grown and exposed at pH 7 have either a greater internalization rate of uranyl or a higher number of transport sites compared with cells grown and exposed at pH 5. We thus conclude that the simple proton-metal competition described by the biotic ligand model cannot successfully depict uranium-algae interactions. The development of an appropriate model incorporating the influence of protons to predict metal uptake and toxicity will be more challenging than anticipated.

Effect of selenate on growth and photosynthesis of Chlamydomonas reinhardtii.

Algal communities play a crucial role in aquatic food webs by facilitating the transfer of dissolved inorganic selenium (both an essential trace element and a toxic compound for a wide variety of organisms) to higher trophic levels. The dominant inorganic chemical species of selenium in freshwaters are selenite (SeO(3)(2-)) and selenate (SeO(4)(2-)). At environmental concentrations, selenite is not likely to have direct toxic effects on phytoplankton growth [Morlon, H., Fortin, C., Floriani, M., Adam, C., Garnier-Laplace, J., Boudou, A., 2005a. Toxicity of selenite in the unicellular green alga Chlamydomonas reinharditii: comparison between effects at the population and sub-cellular level. Aquat. Toxicol. 73(1), 65-78]. The effects of selenate, on the other hand, are poorly documented. We studied the effects of selenate on Chlamydomonas reinhardtii growth (a common parameter in phytotoxicity tests). Growth inhibition (96-h IC(50)) was observed at 4.5+/-0.2 microM selenate (p<0.001), an effective concentration which is low compared to environmental concentrations. Growth inhibition at high selenium concentrations may result from impaired photosynthesis. This is why we also studied the effects of selenate on the photosynthetic process (not previously assessed in this species to our knowledge) as well as selenate's effects on cell ultrastructure. The observed ultrastructural damage (chloroplast alterations, loss of appressed domains) confirmed that chloroplasts are important targets in the mechanism of selenium toxicity. Furthermore, the inhibition of photosynthetic electron transport evaluated by chlorophyll fluorescence induction confirmed this hypothesis and demonstrated that selenate disrupts the photosynthetic electron chain. Compared to the classical 'growth inhibition' parameter used in phytotoxicity tests, cell diameter and operational photosynthetic yield were more sensitive and may be convenient tools for selenate toxicity assessment in non-target plants.

Manganese deficiency in Chlamydomonas results in loss of photosystem II and MnSOD function, sensitivity to peroxides, and secondary phosphorus and iron deficiency.

For photoheterotrophic growth, a Chlamydomonas reinhardtii cell requires at least 1.7 x 10(7) manganese ions in the medium. At lower manganese ion concentrations (typically <0.5 microm), cells divide more slowly, accumulate less chlorophyll, and the culture reaches stationary phase at lower cell density. Below 0.1 microm supplemental manganese ion in the medium, the cells are photosynthetically defective. This is accompanied by decreased abundance of D1, which binds the Mn(4)Ca cluster, and release of the OEE proteins from the membrane. Assay of Mn superoxide dismutase (MnSOD) indicates loss of activity of two isozymes in proportion to the Mn deficiency. The expression of MSD3 through MSD5, encoding various isoforms of the MnSODs, is up-regulated severalfold in Mn-deficient cells, but neither expression nor activity of the plastid Fe-containing superoxide dismutase is changed, which contrasts with the dramatically increased MSD3 expression and plastid MnSOD activity in Fe-deficient cells. Mn-deficient cells are selectively sensitive to peroxide but not methyl viologen or Rose Bengal, and GPXs, APX, and MSRA2 genes (encoding glutathione peroxidase, ascorbate peroxidase, and methionine sulfoxide reductase 2) are slightly up-regulated. Elemental analysis indicates that the Mn, Fe, and P contents of cells in the Mn-deficient cultures were reduced in proportion to the deficiency. A natural resistance-associated macrophage protein homolog and one of five metal tolerance proteins were induced in Mn-deficient cells but not in Fe-deficient cells, suggesting that the corresponding gene products may be components of a Mn(2+)-selective assimilation pathway.

Selenite transport and its inhibition in the unicellular green alga Chlamydomonas reinhardtii.

The influence of time, ambient concentration, and medium composition on selenite (Se(IV)) uptake by the unicellular green alga Chlamydomonas reinhardtii has been investigated. The aims of the performed experiments were to describe the kinetics of accumulation, to characterize transport capacities, to identify key nutrients influencing absorption, and to establish links between speciation and bioavailability. Our results suggested that the adsorbed fraction was negligible compared to the absorbed one. Over the short time scale considered, the absorption was linear with time, with an estimated conductance of approximately 0.2 nmol/m2/h/nM. Uptake was proportional to ambient levels in a broad range of intermediate concentrations (from nM to microM). However, conductances were higher at low concentrations (< nM) and then decreased with increasing concentrations (> microM). These results suggested that a specific but rapidly saturated transport system was involved at low concentrations, coupled with a nonspecific one that was only saturated at high ambient concentrations (approximately mM). The latter could involve transporters used by anionic macronutrients, which is supported by the fact that increasing sulfate and nitrate concentrations induced significant inhibition of Se(IV) uptake. Finally, Se(IV) speciation changes caused by varying pH did not significantly affect bioavailability.