Advances in the study of cylindrospermopsin: detection of potentially cylindrospermopsin-producing cyanobacteria, and prospect for the initial steps of the toxin synthesis

Tesis doctoral inédita, leída en Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología. Fecha de lectura: 19-12-201

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

Autophagy constitutes an essential process triggered upon 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 repor

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 networksThis work was funded by a grant from the Spanish Ministry of Economy and Competitiveness ( AGL2014–53771-R ). Alfonso Blázquez-Castro acknowledges funding under the Marie Skłodowska-Curie Action COFUND 2015 (EU project 713366 – InterTalentum

Attenuation of mercury phytotoxicity with a high nutritional level of nitrate in alfalfa plants grown hydroponically

Mercury (Hg) is one of the most dangerous pollutant heavy metals to the environment, which causes several toxic effects in plants upon accumulation, such as induction of oxidative stress. Nitrate (NO3 – ) is the prevalent form to incorporate nitrogen (N) in higher plants, through its reduction to nitrite (NO2 – ) by the enzyme nitrate reductase (NR). We studied the physiological alterations caused by Hg (0, 6 and 30 µM) in alfalfa plants grown at two different levels of NO3 – : low, (2 mM; LN), and high (12 mM; HN) for one week using a semi-hydroponic culture system. Several parameters of oxidative stress such as lipid peroxidation, chlorophyll content, biothiol concen tration, and ascorbate peroxidase (APX) and glutathione reductase (GR) activities showed that HN plants were less affected by Hg. Nitrate reductase activity and NO3 – concentration were also altered under Hg stress, with lower impact in plants nourished with high NO3 – . Our results highlight the importance of the NO3 – nutritional status to improve tolerance to toxic metals like H

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 decontaminationWork supported by the Spanish State Research Agency (AEI) (Spain) (projects AGL2014–53771-R and AGL2017–87591-R). The FTIR experiments were performed at MIRAS beamline at ALBA Synchrotron with the collaboration of ALBA staff and supported by travel grants for experiments No 2016091860 and 201702211

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 transpor

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 organismsThis work was funded by a grant from the Spanish Ministry of Economy and Competitiveness (AGL2014-53771-R

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

Glutathione metabolism in plants under metal and metalloid stress and its impact on the cellular redox homoeostasis

Efficient phytoremediation of soils polluted with toxic elements greatly depends on the ability of selected plants to withstand the damages induced by these contaminants. Among other metabolites, glutathione (GSH) plays a fundamental dual role in tolerance as an antioxidant required for the attenuation of reactive oxygen species (ROS), such as superoxide (O-) and hydrogen peroxide (H2O2) and as a precursor of phytochelatins (PCs). Understanding the regulatory mechanisms involved in sulphur assimilation and biothiols’ metabolism under metal and metalloid stress will provide the tools to select and obtain more tolerant plants with improved performance, where the cellular redox status and stress-related phytohormones are key players. Metal uptake and distribution depend greatly on the biothiol metabolism, and advanced metallomic analytical techniques offer the tools to characterize in detail functional aspects of metal(loid)-biothiol interaction. Therefore, we present in this chapter an insight in the impact of GSH on the cellular redox balance under metal stress, and how biothiols affect the dynamics of these contaminants in plants with possible implications for future phytoremediation approache