Synchrotron Radiation-Fourier Transformed Infrared microspectroscopy (µSR-FTIR) reveals multiple metabolism alterations in microalgae induced by cadmium and mercury.

Toxic metals such as cadmium (Cd) and mercury (Hg) represent a threat to photosynthetic organisms of polluted aquatic ecosystems, and knowledge about mechanisms of toxicity is essential for appropriate assessment of environmental risks. We used Synchrotron Radiation-Fourier Transformed Infrared microspectroscopy (µSR-FTIR) to characterise major changes of biomolecules caused by Cd and Hg in the model green microalga Chlamydomonas reinhardtii. µSR-FTIR showed several metabolic alterations in different biochemical groups such as carbohydrates, proteins, and lipids in a time-dose dependent manner, with the strongest changes occurring at concentrations above 10 µM Cd and 15 µM Hg after short-term (24 h) treatments. This occurred in a context where metals triggered intracellular oxidative stress and chloroplast damage, along with autophagy induction by overexpressing AUTOPHAGY-RELATED PROTEIN 8 (ATG8). Thin layer chromatography analysis confirmed that toxic metals promoted remarkable changes in lipid profile, with higher degree of esterified fatty acid unsaturation as detected by gas chromatography coupled with mass spectrometry. Under Cd stress, there was specifically higher unsaturation of free fatty acids, while Hg led to stronger unsaturation in monogalactosyldiacylglycerol. µSR-FTIR spectroscopy proved as a valuable tool to identify biochemical alterations in microalgae, information that could be exploited to optimise approaches for metal decontamination.

Physiological and Molecular Alterations of Phycobionts of Genus Trebouxia and Coccomyxa Exposed to Cadmium.

Several studies on aeroterrestrial microalgae are unravelling their resistance mechanisms to different abiotic stressors, including hazardous metals, pointing to their future role as bioremediation microorganisms. In the present study, physiological and molecular alterations of four phycobionts of genus Trebouxia (T. TR1 and T. TR9) and Coccomyxa (C. subellipsoidea and C. simplex) exposed to Cd were studied. Cd accumulation and subcellular distribution, cell wall structure, production of biothiols (GSH and phytochelatins), reactive oxygen species (ROS) formation, expression of key antioxidant genes and ROS-related enzymes were evaluated to determine the physiological differences among the four microalgae, with the aim to identify the most suitable microorganism for further biotechnological applications. After 7 days of Cd exposure, Coccomyxa algae showed higher capacity of Cd intake than Trebouxia species, with C. subellipsoidea being the highest Cd accumulator at both intracellular and, especially, cell wall level. Cd induced ROS formation in the four microalgae, but to a greater extent in both Coccomyxa algae. Trebouxia TR9 showed the lowest Cd-dependent oxidative stress probably due to glutathione reductase induction. All microalgae synthetized phytochelatins in response to Cd but in a species-specific and a dose-dependent manner. Results from this study agree with the notion that each microalga has evolved a distinct strategy to detoxify hazardous metals like Cd and to cope with oxidative stress associated with them. Coccomyxa subellipsoidea and Trebouxia TR9 appear as the most interesting candidates for further applications.

Characterization of Oxidative Lipidomics and Autophagy Induction in Chlamydomonas reinhardtii Under Abiotic Stress.

Autophagy constitutes an essential process triggered by oxidative stress that enables cells to recycle damaged biomolecules and organelles, which is eventually traced by immunodetection with anti-ATG8. In parallel with autophagy induction, carbon metabolism in Chlamydomonas reinhardtii under abiotic stress is diverged toward lipid biosynthesis and lipid droplet accumulation, which can be analyzed by a simple thin-layer chromatography and in vivo staining with the fluorescent probe BODIPY 493/503. We show the responses in Chlamydomonas cells exposed to mercury or cadmium (0-50 µM doses), as examples of oxidative stress-mediated changes in autophagy and lipid metabolism, monitored with the procedures described in this report.

Fibers spreading worldwide: Microplastics and other anthropogenic litter in an Arctic freshwater lake.

We investigated the presence of microplastics and other anthropogenic litter in the sediments adhered to rocks of an Arctic freshwater lake at Ny-Ålesund (Svalbard Archipelago, 78°N; 11°E). Most of the sampled microparticles were fibers (>90%). The identification of polymer types and additives was performed by combining three spectroscopic techniques, namely Raman Microscopy, Fourier-Transform Infrared microspectroscopy (µFTIR) and Synchrotron Radiation µFTIR (SR-FTIR). SR-FTIR confirmed the presence of poly(ethylene terephthalate) fibers, while RAMAN spectroscopy provided evidence of fibers containing industrial additives. Our results estimated an average concentration of 400 microparticles/m2 of rocks identified as anthropogenic litter, which included an estimation of 90 microplastics/m2 identified as polyester fibers; the rest are mostly natural fibers with evidence of anthropogenic origin. Taken together, the results proved the occurrence of anthropogenic pollutants in remote polar areas. Their probable origin is the long range atmospheric transport.

Aluminium triggers oxidative stress and antioxidant response in the microalgae Scenedesmus sp.

Aluminium (Al) water pollution is an increasing environmental problem and comprehensive analysis of toxic responses of aquatic primary producer organisms is imperative. We characterized the antioxidant response of Scenedesmus sp. microalga to Al-induced oxidative stress. After 72 h of exposure to Al (0, 10, and 100 µM) in a modified Bold Basal Medium (pH 5.0), we observed cell aggregation and alterations in the subcellular structure, strong lipid peroxidation and oxidative stress induction (detected with the fluorescent probe 2',7'-dichlorodihydrofluorescein diacetate) in parallel with Al accumulation in cells. At the same time, Al toxicity caused depletion of important macronutrients like Ca, which is important for cell-wall structure. Analysis of antioxidant enzymatic activities in Al-treated Scenedesmus cells revealed that catalase, ascorbate peroxidase, as well as different isoforms of superoxide dismutase were inhibited especially at the highest Al dose (100 µM), cells that accumulated the highest concentration of Al. On the other hand, glutathione reductase activity increased at that Al concentration. Immunodetection after Western-blotting confirmed that only ascorbate peroxidase inhibition was apparently due to a decrease in enzyme levels. However, the inhibition of catalase and activation of glutathione reductase activities seemed related with post-translational modifications in protein function as protein expression decreased or increased, respectively under Al stress. Our results may help to understand toxic mechanisms triggered by Al in freshwater microalgae, which in turn could aid to select suitable biomarkers of Al contamination in aquatic ecosystems.

Whole-Genome Sequence of Toxic Freshwater Cyanobacterium Chrysosporum ovalisporum Strain UAM-MAO.

Here, we report the complete nucleotide sequence of Chrysosporum ovalisporum UAM-MAO, a filamentous, cylindrospermopsin-producing cyanobacterium involved in bloom forming in freshwater systems worldwide. It was isolated from an artificial pond in Madrid, Spain. The genome sequence contains 336 contigs, consisting of 7,478,035 bp and 2,851 putative protein-coding genes.

Fluorescent in vivo imaging of reactive oxygen species and redox potential in plants.

Reactive oxygen species (ROS) are by-products of aerobic metabolism, and excessive production can result in oxidative stress and cell damage. In addition, ROS function as cellular messengers, working as redox regulators in a multitude of biological processes. Understanding ROS signalling and stress responses requires methods for precise imaging and quantification to monitor local, subcellular and global ROS dynamics with high selectivity, sensitivity and spatiotemporal resolution. In this review, we summarize the present knowledge for in vivo plant ROS imaging and detection, using both chemical probes and fluorescent protein-based biosensors. Certain characteristics of plant tissues, for example high background autofluorescence in photosynthetic organs and the multitude of endogenous antioxidants, can interfere with ROS and redox potential detection, making imaging extra challenging. Novel methods and techniques to measure in vivo plant ROS and redox changes with better selectivity, accuracy, and spatiotemporal resolution are therefore desirable to fully acknowledge the remarkably complex plant ROS signalling networks.

Influence of Glycine and Arginine on Cylindrospermopsin Production and aoa Gene Expression in Aphanizomenon ovalisporum.

Arginine (Arg) and glycine (Gly) seem to be the only substrates accepted by the amidinotransferase that catalyze the first step of the synthesis pathway of the cyanotoxin cylindrospermopsin (CYN), leading to guanidinoacetate (GAA). Here, the effect of these amino acids on the production of CYN in cultures of the cylindrospermopsin-producing strain, Aphanizomenon ovalisporum UAM-MAO, has been studied. Arg clearly increased CYN content, the increment appearing triphasic along the culture. On the contrary, Gly caused a decrease of CYN, observable from the first day on. Interestingly, the transcript of the gene ntcA, key in nitrogen metabolism control, was also enhanced in the presence of Arg and/or Gly, the trend of the transcript oscillations being like that of aoa/cyr. The inhibitory effect of Gly in CYN production seems not to result from diminishing the activity of genes considered involved in CYN synthesis, since Gly, as Arg, enhance the transcription of genes aoaA-C and cyrJ. On the other hand, culture growth is affected by Arg and Gly in a similar way to CYN production, with Arg stimulating and Gly impairing it. Taken together, our data show that the influence of both Arg and Gly on CYN changes seems not to be due to a specific effect on the first step of CYN synthesis; it rather appears to be the result of changes in the physiological cell status.

In vivo ROS and redox potential fluorescent detection in plants: Present approaches and future perspectives.

Reactive oxygen species (ROS) are metabolic by-products in aerobic organisms including plants. Endogenously produced ROS act as cellular messengers and redox regulators involved in several plant biological processes, but excessive accumulation of ROS cause oxidative stress and cell damage. Understanding ROS signalling and stress responses requires precise imaging and quantification of local, subcellular and global ROS dynamics with high selectivity, sensitivity, and spatiotemporal resolution. Several fluorescent vital dyes have been tested so far, which helped to provide relevant spatially resolved information of oxidative stress dynamics in plants subjected to harmful environmental conditions. However, certain plant characteristics, such as high background fluorescence of plant tissues in vivo and antioxidant mechanisms, can interfere with ROS detection. The development of improved small-molecule fluorescent dyes and protein-based ROS sensors targeted to subcellular compartments will enable in vivo monitoring of ROS and redox changes in photosynthetic organisms.

Genome Engineering in Cyanobacteria: Where We Are and Where We Need To Go.

Genome engineering of cyanobacteria is a promising area of development in order to produce fuels, feedstocks, and value-added chemicals in a sustainable way. Unfortunately, the current state of genome engineering tools for cyanobacteria lags far behind those of model organisms such as Escherichia coli and Saccharomyces cerevisiae. In this review, we present the current state of synthetic biology tools for genome engineering efforts in the most widely used cyanobacteria strains and areas that need concerted research efforts to improve tool development. Cyanobacteria pose unique challenges to genome engineering efforts because their cellular biology differs significantly from other eubacteria; therefore, tools developed for other genera are not directly transferrable. Standardized parts, such as promoters and ribosome binding sites, which control gene expression, require characterization in cyanobacteria in order to have fully predictable results. The application of these tools to genome engineering efforts is also discussed; the ability to do genome-wide searching and to introduce multiple mutations simultaneously is an area that needs additional research in order to enable fast and efficient strain engineering.

First evidence of accumulation in cyanobacteria of guanidinoacetate, a precursor of the toxin cylindrospermopsin.

Guanidinoacetate (GAA) is one of the most extensively studied toxic guanidine compounds. Changes in GAA can affect the nervous system and induce hyperhomocysteinemia, representing a risk factor for cardiovascular diseases. In cyanobacteria, GAA is thought to be an intermediate in the synthesis of the toxin cylindrospermopsin (CYN), one of the most common known cyanotoxins that affects multiple organs and functions in animals and plants. In spite of the evidence supporting GAA toxicity and its role in CYN synthesis, no data have been reported on the accumulation of GAA in any cyanobacterium. We have analyzed and compared the content of GAA in cultures of diverse cyanobacteria types, both cylindrospermopsin producing (CYN(+)) and not producing (CYN(-)). The results obtained show that GAA accumulates in the majority of the strains tested, although the highest content was found in one of the CYN(+) strain, Aphanizomenon ovalisporum UAM-MAO. In this strain, both GAA and CYN can be located within and out the cells. In conclusion, GAA appears to be a general cyanobacterial metabolite that due to its proven toxic should be considered when studying and managing cyanobacteria toxicity.

Characterization of Aphanizomenon ovalisporum amidinotransferase involved in cylindrospermopsin synthesis.

An increasing abundance of Aphanizomenon ovalisporum in water bodies from diverse world regions has been reported in the last few years, with the majority of the isolated strains producing the toxin cylindrospermopsin (CYN), leading to a rise in ecological and health risks. The understanding of CYN synthesis is crucial in the control of CYN production. An amidinotransferase (AMDT) seems to be the first enzyme involved in the synthesis of CYN. In this study, we have cloned and overexpressed the aoaA gene from the constitutive CYN producer A. ovalisporum UAM-MAO. The recombinant purified AoaA was characterized, confirming that it is an l-arginine:glycine AMDT. It shows an optimal activity between 32 and 37°C, at pH from 8 to 9. The activity exhibits a mixed (ping-pong/sequential) kinetic mechanism, and is inhibited by the reaction product guanidine acetate (GAA) in a noncompetitive manner. Mg(2+) stimulates AoaA activity while Co(2+) and Mn(2+) inhibit it. AoaA conserves the critical residues of the catalytic site and substrate specificity of AMDTs, as the previously reported AMDT from Cylindrospermopsis raciborskii Cyr. Both proteins can be included in a new group of prokaryotic AMDTs involved in CYN production.

Detection of potentially producing cylindrospermopsin and microcystin strains in mixed populations of cyanobacteria by simultaneous amplification of cylindrospermopsin and microcystin gene regions.

Cyanobacterial blooms are frequently formed by heterogeneous populations of toxin-producing and non-producing strains. Microcystins (MC) and cylindrospermopsin (CYN) are the most representative cyanobacterial toxins. We have developed a multiplex PCR assay that allows simultaneous detection of MC(+) and/or CYN(+) strains in mixed populations of cyanobacteria. Various primer sets were designed using mcy and aoa gene sequences related with MC and CYN synthesis respectively, to amplify at the same time aoa and mcy sequences. Purified DNA, cultured cell mixtures and field samples with MC and CYN producing strains were used as DNA template. The results show: (i) the expected amplicons were only observed with toxic strains; (ii) cells were suitable as a source of purified DNA for the multiplex PCR; (iii) the assay could detect simultaneously 3 aoa and 3 mcy gene regions with mixed CYN(+) and MC(+) cyanobacteria cells. The method could be applied to environmental samples, allowing in a rapid, economical and easy way to detect simultaneously the presence of CYN(+) and MC(+) cyanobacteria in sestonic fractions of water samples.

Assessment of microcystins in lake water and fish (Mugilidae, Liza sp.) in the largest Spanish coastal lake.

Cyanobacteria dominance and cyanotoxin production can become major threats to humans and aquatic life, especially in warm shallow lakes, which are often dominated by cyanobacteria. This study investigates the occurrence and distribution of microcystins (MCYST) in water, cell-bound and in the tissues of the commercial mugilid Liza sp. in the largest, coastal, Spanish Mediterranean lake (Albufera of Valencia). This is the first report concerning microcystin accumulation in tissues of mugilid fish species. Considerable amounts of microcystins were found in the water and seston, which correlated with development of Microcystis aeruginosa populations in the lake. The MCYST concentrations found in Lake Albufera (mean 1.7 and 17 µg/L and maximum 16 and 120 µg/L in water and seston, respectively) exceeded by one to two orders of magnitude the guideline levels proposed by the World Health Organization and were higher than that reported in other lakes of the Mediterranean zone. The presence of MCYST was found in all the fishes studied and accumulated differently among tissues of the commercial species Liza sp. Toxin accumulation in fish tissues showed that although the target organ for MCYST was the liver, high concentrations of microcystins were also found in other analysed tissues (liver>intestine>gills>muscle). Human tolerable daily intake for microcystins is assessed relative to the WHO guidelines, and potential toxicological risks for humans, wildlife and related ecosystems of the lake are discussed.