Mercury (Hg) is a global pollutant that affects numerous marine aquatic ecosystems. We isolated Chlorococcum dorsiventrale Ch-UB5 microalga from coastal areas of Tunisia suffering from metal pollution and analyzed its tolerance to Hg. This strain accumulated substantial amounts of Hg and was able to remove up to 95% of added metal after 24 and 72 h in axenic cultures. Mercury led to lesser biomass growth, higher cell aggregation, significant inhibition of photochemical activity, and appearance of oxidative stress and altered redox enzymatic activities, with proliferation of starch granules and neutral lipids vesicles. Such changes matched the biomolecular profile observed using Fourier Transformed Infrared spectroscopy, with remarkable spectral changes corresponding to lipids, proteins and carbohydrates. C. dorsiventrale accumulated the chloroplastic heat shock protein HSP70B and the autophagy-related ATG8 protein, probably to counteract the toxic effects of Hg. However, long-term treatments (72 h) usually resulted in poorer physiological and metabolic responses, associated with acute stress. C. dorsiventrale has potential use for Hg phycoremediation in marine ecosystems, with the ability to accumulating energetic reserves that could be used for biofuel production, supporting the notion of using of C. dorsiventrale for sustainable green chemistry in parallel to metal removal.

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.

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 mu M doses), as examples of oxidative stress-mediated changes in autophagy and lipid metabolism, monitored with the procedures described in this report.

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 mu M) 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 mu M), 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.

There is a little clinical data to the use of statins and new androgen receptor inhibitors (ARIs) in patients with mCRPC. Statins can enhance the effect of new ARIs in patients with mCRPC through a competitive interactions, decreasing the recruitment as an androgenic precursor. The aim of the study is to determine whether the use of statins with new ARIs has a clinical impact on survival in patients with mCRPC. Patients diagnosed of mCRPC and treated with new ARIs without previous chemotherapy were included. The patients were stratified by statins or non-statins. We described categorical and continuous variables in each group. Comparisons of survival rates were performed using log rank statistics. Multivariate analysis was performed with Cox regression modelling. Value of p < .05 was set as significant. The variables studied were: hemoglobin, lymphocytes, alkaline phosphatase, pain, use of opioids, type of ARIs, radiotherapy, androgen deprivation (AD), prostate-specific-antigen (PSA) at diagnosis, nadir PSA and statins and PSA response at 4, 8, 12, and16 weeks. Between April 2009 and August 2018, 64 patients (42 Abidaterone and 22 Enzalutamide) were included. The median survival of the entire cohort of patients was 40.8 months (95% CI 33.64 – 48.08). Overall survival (OS) was 43.2 months for the statin group (95% CI 36.98 – 49.41, p = 0.035) and 29.7 months for the non-statin group (95% CI 23.45 – 36.01). Progression-free survival (PFS) was 18 months for the statin group (95% CI 8.64 – 27.35, p = 0.004) and 7 months for the non-statin group (95% CI 4.60 – 9.39). No relationship was observed between statins and the lowering of PSA at 4, 8, 12 and 16 weeks compared to the non-statin group (P = ns). On multivariate analysis, factors associated to survival were LDH (HR 1,007, CI 95%, 1,002 – 1,013, p = 0.01), Neutrophils (HR 1,001, CI 95% 1,000 – 1,001, p = 0.049), and statins (HR 2,790, CI 95% 1,011 – 7,698, p = 0.045). In this retrospective cohort, the use of statins increases overall and progression-free survival compared to the use of ARIs without statins in mCRPC. Factors associated to survival were LDH, neutrophils, and statins. Statins do not modify the PSA response in the first 4 months of treatment.Abstract 2642; Table 1Patient characteristicsSTATINS (n = 32 ptes)Not STATINS (n = 32 ptes)pMedian age at diagnosis (range)69,8 years (46 – 85)67,9 years (43 – 86)0.415KPS <80 >8010 (31%) 22 (69%)10 (31%) 22 (69%)0.6Gleason score <7 8 - 1012 (37,5%) 20 (62,5%)15 (46,9%) 17 (53,1%)0.133Initial clinical stage Localized Metastatic13 (40,6%) 19 (59,4%)15 (46,9%) 17 (53,1%)0.614Time to CRPC months (range)62,5m (6 – 144m)59,09m (3 – 192m)0.755KPS at CRPC >80 <8015 (46,9%) 17 (53,1%)11 (34,4%) 21 (65,6%)0.309Alkaline phosphate (U/L)280 (58 – 452)365 (98 – 784)0.127LDH (U/L)338 (54 – 1074)552 (102 – 1990)0.019PSA (ng/ml)72,8 (1,14 – 368)96,7 (0,97 – 1006)0.503Type of AD Abiraterone Enzalutamide21 (65,6%) 11 (34,4%)21 (65,6%) 11 (34,4%)0.604 Open table in a new tab

Traditional agricultural practices are based mainly on maintaining sufficient soil fertility to obtain appropriate crop yield. However, soils are not only a source of nutrients for plants, but also a potential source of metals, which decrease crop productivity and may pose a risk to human health. Over the last decades, recurrent cases of soil pollution by metals are appearing in diverse geographical locations throughout the world, mostly associated with inefficient management of industrial wastewater effluents, poor containment of mining leachates and residues, and as a consequence of natural geological sources. Among the eco-friendly cleanup technologies, in situ contaminant stabilization ("inactivation") and plant-based approaches ("phytoremediation") are proposed to cope with the above-mentioned contamination challenge. In this chapter, the authors discuss the main plant species suitable for phytoremediating metal-polluted soils, and the most appropriate agronomic techniques to increase phytoremediation efficiency (e.g., pruning, plant density, soil pH, fertilization).

•Description of ROS origin, homeostasis and in vivo detection in plants.•Small molecules and fluorescent dyes used for ROS and redox imaging in plants.•Protein-based fluorescent probes to study plant redox changes and ROS production.•Techniques and future strategies to study ROS signalling and redox changes.

Chlamydomonas reinhardtii is a green unicellular eukaryotic model organism for studying relevant biological and biotechnological questions. The availability of genomic resources and the growing interest in C. reinhardtii as an emerging cell factory for the industrial production of biopharmaceuticals require an in-depth analysis of protein N-glycosylation in this organism. Accordingly, we used a comprehensive approach including genomic, glycomic, and glycoproteomic techniques to unravel the N-glycosylation pathway of C. reinhardtii. Using mass-spectrometry-based approaches, we found that both endogenous soluble and membrane-bound proteins carry predominantly oligomannosides ranging from Man-2 to Man-5. In addition, minor complex N-linked glycans were identified as being composed of partially 6-O-methylated Man-3 to Man-5 carrying one or two xylose residues. These findings were supported by results from a glycoproteomic approach that led to the identification of 86 glycoproteins. Here, a combination of in-source collision-induced dissodiation (CID) for glycan fragmentation followed by mass tag-triggered CID for peptide sequencing and PNGase F treatment of glycopeptides in the presence of (18)O-labeled water in conjunction with CID mass spectrometric analyses were employed. In conclusion, our data support the notion that the biosynthesis and maturation of N-linked glycans in the endoplasmic reticulum and Golgi apparatus occur via a GnT I-independent pathway yielding novel complex N-linked glycans that maturate differently from their counterparts in land plants.

BACKGROUND: Cah3 is the only carbonic anhydrase (CA) isoform located in the thylakoid lumen of Chlamydomonas reinhardtii. Previous studies demonstrated its association with the donor side of the photosystem II (PSII) where it is required for the optimal function of the water oxidizing complex. However this enzyme has also been frequently proposed to perform a critical function in inorganic carbon acquisition and CO(2) fixation and all mutants lacking Cah3 exhibit very poor growth after transfer to low CO(2) conditions. RESULTS/CONCLUSIONS: In the present work we demonstrate that after transfer to low CO(2), Cah3 is phosphorylated and that phosphorylation is correlated to changes in its localization and its increase in activity. When C. reinhardtii wild-type cells were acclimated to limiting CO(2) conditions, the Cah3 activity increased about 5-6 fold. Under these conditions, there were no detectable changes in the level of the Cah3 polypeptide. The increase in activity was specifically inhibited in the presence of Staurosporine, a protein kinase inhibitor, suggesting that the Cah3 protein was post-translationally regulated via phosphorylation. Immunoprecipitation and in vitro dephosphorylation experiments confirm this hypothesis. In vivo phosphorylation analysis of thylakoid polypeptides indicates that there was a 3-fold increase in the phosphorylation signal of the Cah3 polypeptide within the first two hours after transfer to low CO(2) conditions. The increase in the phosphorylation signal was correlated with changes in the intracellular localization of the Cah3 protein. Under high CO(2) conditions, the Cah3 protein was only associated with the donor side of PSII in the stroma thylakoids. In contrast, in cells grown at limiting CO(2) the protein was partly concentrated in the thylakoids crossing the pyrenoid, which did not contain PSII and were surrounded by Rubisco molecules. SIGNIFICANCE: This is the first report of a CA being post-translationally regulated and describing phosphorylation events in the thylakoid lumen.

Polypeptides of 21, 36 and 37 kDa are induced in the unicellular green alga Chlamydomonas reinhardtii when cells are transferred from high (5 %) to low (0.03 %) CO2 concentrations. The synthesis of these polypeptides is correlated with the induction of the CO2- concentrating mechanism Interaction between the induction of low-CO2-inducible polypeptides. the CO2-concentrating mechanism and photorespiration has been studied in wild-type C. reinhardtii with the aim of clarifying whether the glycolate pathway is involved in algal acclimation to limiting CO2 conditions. Our results showed that the induction of the 37 kDa periplasmic carbonic anhydrase and 21 kDa polypeptide under low-CO2 conditions was not observed in the presence of aminooxyacetate, an inhibitor which completely blocks glycolate metabolism. However, the induction of the 36 kDa polypeptide was not affected by this inhibitor. The presence of aminooxyacetate during the acclimation to low CO2 conditions also inhibited the increase in the photosynthetic affinity for inorganic carbon shown by low-CO2-acclimated Chlamydomonas cells without the inhibitor. Our results suggest that the induction of the CO2 concentrating mechanism may require the function of the glycolate pathway. In addition our results also indicate that there is differential regulation of the induction of these three low-CO2-inducible polypeptides in Chlamylomonas reinhardtii.

Using mass-spectrometric measurements of 18O exchange from 13C18O2 we determined the activity of carbonic anhydrase (CA; EC 4.2.1.1) in chloroplast envelope membranes isolated from Chlamydomonas reinhardtii cw-15. Our results show an enrichment of CA activity in these fractions relative to the activity in the crude chloroplast. The envelope CA activity increased about 8-fold during the acclimation to low-CO2 conditions and was completely induced within the first 4 h after the transfer to air levels of CO2. The CA-activity was not dissociated from envelope membranes after salt treatment. In addition, no cross-reactivity with other CA isoenzymes of Chlamydomonas was observed in our chloroplast envelope membranes. All these observations indicated that the protein responsible for this activity was a new CA isoenzyme, which was an integral component of the chloroplast envelopes from Chlamydomonas. The catalytic properties of the envelope CA activity were completely different from those of the thylakoid isoenzyme, showing a high requirement for Mg2+ and a high sensitivity to ethoxyzolamide. Analysis of the integral envelope proteins showed that there were no detectable differences between high- and low-inorganic carbon (Ci) cells, suggesting that the new CA activity was constitutively expressed in both high- and low-Ci cells. Two different high-Ci-requiring mutants of C. reinhardtii, cia-3 and pmp-1, had a reduced envelope CA activity. We propose that this activity could play a role in the uptake of inorganic carbon at the chloroplast envelope membranes.

In the green alga Chlorella vulgaris UAM 101, a CO2-concentrating mechanism (CCM) is induced when cells are transferred from high (5%) to low (0.03%) CO2 concentrations. The induction of the CCM is correlated with de-novo synthesis of several polypeptides that remain to be identified. The internal carbonic anhydrase (CA; EC 4.2.1.1) activity increased 6- to 7-fold within 6 h of acclimation to air. When crude homogenates were further separated into soluble and insoluble fractions, nearly all of the CA activity was associated with the membrane fraction. Immunoblot analysis of cell homogenates probed with antibodies raised against the 37-kDa subunit of periplasmic CA of Chlamydomonas reinhardtii showed a cross-reaction with a single 38-kDa polypeptide in both high- and low-CO2-grown cells. The up-regulation of the expression of the 38-kDa polypeptide was closely correlated with the increase in internal CA activity. Furthermore, its subcellular location was also correlated with the distribution of the activity. Immunoblot analysis of pyrenoid fractions showed that the 38-kDa polypeptide was concentrated in the pyrenoids from low-CO2-grown cells but was not present in pyrenoids from high-CO2-grown cells. In addition, immunogold labeling experiments showed that the protein was mainly associated with membranes crossing the pyrenoid, while it was absent from the pyrenoid matrix. These studies have identified a putative intracellular CA polypeptide associated with the pyrenoid in Chlorella vulgaris, suggesting that this structure may play an important role in the operation of the CCM and the acclimation to low CO2 conditions.

In the green alga Chlorella vulgaris UAM 101, a CO2‐concentrating mechanism is induced when the cells are growing under low CO2 conditions. We have investigated the effect of glucose on the induction of this mechanism. Cells adapted to low CO2 in the presence of glucose showed a reduced ability to transport and fix external inorganic carbon. This reduction was correlated with a decrease in internal carbonic anhydrase activity. 3‐O‐methyl‐glucose, a nonmetabolizable analog of glucose, caused a more dramatic repression of these phenomena. Immunoblot analyses of total cell protein of Chlorella vulgaris UAM 101 against large subunit of ribulose‐1.5‐bisphosphate carboxylase/oxygenase and ribulose 1.5‐bisphosphate‐carboxylase/oxygenase activase polyclonal antibodies showed that the expression of these two polypeptides was affected by neither CO2 level, nor glucose or 3‐O‐methyl‐glucose. Ultrastructure studies showed that the low CO2‐induced development of the pyrenoid was also affected by glucose. Immunocytochemical data demonstrated that ribulose‐1.5‐bisphosphate carboxylase/oxygenase was exclusively located in the pyrenoid matrix. This localization and the density of labeling of the pyrenoid region were affected by neither CO2 level nor the presence of glucose.

In Chlamydomonas reinhardtii the formation of a starch sheath surrounding the pyrenoid is observed when cells grown under high CO 2 (5% CO 2 in air) are transferred to low CO 2 (0.03%) conditions. Formation of the starch sheath occurs coincidentally with induction of the CO 2 concentrating mechanism and with de novo synthesis of 5 polypeptides with molecular masses of 21, 36, 37, 42–44 kDa. We studied the effect of CO 2 concentrations on photosynthesis, ultrastructure and protein synthesis in Chlamydomonas reinhardtii cw‐15 (wild phenotype for photosynthesis) and in the starch‐less mutant BAFJ ‐6, with the aim to clarify the role of the pyrenoid starch sheath in the operation of the CO 2 concentrating mechanism and whether these low CO 2 ‐inducible polypeptides are involved in the formation of starch sheath. When wild type and starch‐less mutant cells were transferred from high to low CO 2 , the CO 2 requirement for half‐maximal rates of photosynthesis decreased from 40 μM to 2 μM CO 2 . 35 SO 4 2‐ labeling analyses showed that the starch‐less mutant induced the 5 low CO 2 ‐inducible polypeptides. These observations suggest that the starch‐less mutant was able to induce a fully active CO 2 concentrating mechanism. Since the starch‐less mutant did not form a pyrenoid starch sheath, we suggest that the starch sheath is not involved in the operation of the CO 2 concentrating mechanism and that none of these 5 low CO 2 ‐inducible proteins is involved in the formation of the starch sheath in Chlamydomonas .

Immature inclusions representing three progressive steps of carboxysome biogenesis have been identified in Synechococcus during the period of adaptation to low-CO2 conditions: (a) ring-shaped structures, (b) electron-translucent inclusions with the shape of a carboxysome and the internal orderly arrangement of ribulose-1,5-bisphosphate carboxylase oxygenase (Rubisco) molecules, and (c) carboxysomes with an internal electron-translucent area, which seem to be the penultimate stage of carboxysome maturation. The ability to build up normal carboxysomes is impaired in three (M3, EK6, and D4) of four high-carbon-requiring mutants studied in this work. M3 and EK6 exhibit abundant immature electron-translucent carboxysomes but no mature ones. This finding supports the contention that an open reading frame located 7.5 kb upstream of the gene encoding the large subunit of Rubisco (altered in M3) is involved in the carboxysome composition and confirms the structural role of the small subunit of Rubisco (slightly modified in EK6) in the assembly of these structures. D4 shows few typical carboxysomes and frequent immature types, its genetic lesion affecting the apparently unrelated gene encoding a subunit of phosphoribosyl aminoamidazole carboxylase of the purine biosynthesis pathway. Revertants EK20 (EK6) and RK13 (D4) have normal carboxysomes, which means that the restoration of the ability to grow under low CO2 coincides with the proper assembling of these structures. N5, a transport mutant due to the alteration of the gene encoding subunit 2 of NADH dehydrogenase, shows an increase in the number and size of carboxysomes and frequent bar-shaped ones.

In Chlorella vulgaris UAM 101, the presence of glucose altered the photosynthetic and respiratory metabolism in the light. When glucose was added to the growth medium, an increase in the cellular level of enzymes involved in glucose oxidation, namely glucose‐6‐P dehydrogenase (EC 1.1.1.49) and NAD+‐glyceraldehyde‐3‐P dehydrogenase (EC 1.2.1.12), was observed. Glucose also enhanced respiratory O2 consumption. In addition, CO2 released by glucose oxidation was refixed in photosynthesis. The presence of glucose also affected photosynthesis. Phosphoribulokinase (EC 2.7.1.19) and NADP+‐dependent glyceraldehyde‐3‐P dehydrogenase (EC 1.2.1.13), two regulatory enzymes of the reductive pentose phosphate cycle, were increased by glucose. However, Rubisco (EC 4.1.1.39) activity of these cells was lower than that of autotrophic cells. Despite these alterations, the photosynthetic O2 evolution was not significantly inhibited by glucose. On the other hand, an increase in the cytosolic NADP+‐glyceraldehyde‐3‐P dehydrogenase (EC 1.2.1.9) that is involved in obtaining reducing power for anabolic processes was observed. The CO2 levels in the growth medium did not significantly affect the cellular level of enzymes measured in this work, except those involved in biosynthetic pathways. These data suggest that the effect of glucose on photosynthesis and respiration can be explained by alteration of the cellular level of photosynthetic enzymes and respiratory substrates, respectively.