Claudio A. Sáez

Copper-induced intra-specific oxidative damage and antioxidant responses in strains of the brown alga Ectocarpus siliculosus with different pollution histories.

Inter- and intra-specific variation in metal resistance has been observed in the ecologically and economically important marine brown macroalgae (Phaeophyceae), but the mechanisms of cellular tolerance are not well elucidated. To investigate inter-population responses of brown seaweeds to copper (Cu) pollution, the extent of oxidative damage and antioxidant responses were compared in three strains of the filamentous brown seaweed Ectocarpus siliculosus, the model organism for the algal class Phaeophyceae that diverged from other major eukaryotic groups over a billion year ago. Strains isolated from locations with different pollution histories (i.e. LIA, from a pristine site in Scotland; REP and Es524 from Cu-contaminated sites in England and Chile, respectively) were exposed to total dissolved Cu concentrations (CuT) of up to 2.4 μM (equivalent to 128 nM Cu(2+)) for 10 d. LIA exhibited oxidative stress, with increases in hydrogen peroxide (H2O2) and lipid peroxidation (measured as TBARS levels), and decreased concentrations of photosynthetic pigments. Es524 presented no apparent oxidative damage whereas in REP, TBARS increased, revealing some level of oxidative damage. Adjustments to activities of enzymes and antioxidant compounds concentrations in Es524 and REP were strain and treatment dependent. Mitigation of oxidative stress in Es524 was by increased activities of superoxide dismutases (SOD) at low CuT, and catalase (CAT) and ascorbate peroxidase (APX) at all CuT, accompanied by higher levels of antioxidants (ascorbate, glutathione, phenolics) at higher CuT. In REP, only APX activity increased, as did the antioxidants. For the first time evidence is presented for distinctive oxidative stress defences under excess Cu in two populations of a species of brown seaweed from environments contaminated by Cu.

A Simple and Effective Method for High Quality Co-Extraction of Genomic DNA and Total RNA from Low Biomass Ectocarpus siliculosus, the Model Brown Alga

The brown seaweed Ectocarpus siliculosus is an emerging model species distributed worldwide in temperate coastal ecosystems. Over 1500 strains of E. siliculosus are available in culture from a broad range of geographic locations and ecological niches. To elucidate the molecular mechanisms underlying its capacity to cope with different environmental and biotic stressors, genomic and transcriptomic studies are necessary; this requires the co-isolation of genomic DNA and total RNA. In brown algae, extraction of nucleic acids is hindered by high concentrations of secondary metabolites that co-precipitate with nucleic acids. Here, we propose a reliable, rapid and cost-effective procedure for the co-isolation of high-quality nucleic acids using small quantities of biomass (25-, 50- and 100 mg) from strains of E. siliculosus (RHO12; LIA4A; EC524 and REP10–11) isolated from sites with different environmental conditions. The procedure employs a high pH extraction buffer (pH 9.5) which contains 100 mM Tris-HCl and 150 mM NaCl, with the addition of 5 mM DTT and 1% sarkosyl to ensure maximum solubility of nucleic acids, effective inhibition of nuclease activity and removal of interfering contaminants (e.g. polysaccharides, polyphenols). The use of sodium acetate together with isopropanol shortened precipitation time and enhanced the yields of DNA/RNA. A phenol:chlorophorm:isoamyl alcohol step was subsequently used to purify the nucleic acids. The present protocol produces high yields of nucleic acids from only 25 mg of fresh algal biomass (0.195 and 0.284 µg mg−1 fresh weigh of RNA and DNA, respectively) and the high quality of the extracted nucleic acids was confirmed through spectrophotometric and electrophoretic analyses. The isolated RNA can be used directly in downstream applications such as RT-PCR and the genomic DNA was suitable for PCR, producing reliable restriction enzyme digestion patterns. Co-isolation of DNA/RNA from different strains indicates that this method is likely to have wider applications for intra- and inter-specific studies on other brown algae.

Variation in patterns of metal accumulation in thallus parts of Lessonia trabeculata (Laminariales; Phaeophyceae): implications for biomonitoring.

Seaweeds are well known to concentrate metals from seawater and have been employed as monitors of metal pollution in coastal waters and estuaries. However, research showing that various intrinsic and extrinsic factors can influence metal accumulation, raises doubts about the basis for using seaweeds in biomonitoring programmes. The thallus of brown seaweeds of the order Laminariales (kelps) is morphologically complex but there is limited information about the variation in metal accumulation between the different parts, which might result in erroneous conclusions being drawn if not accounted for in the biomonitoring protocol. To assess patterns of individual metals in the differentiated parts of the thallus (blade, stipe, holdfast), concentrations of a wide range of essential and non-essential metals (Fe, Cr, Cu, Zn, Mn, Pb, Cd, Ni and Al) were measured in the kelp Lessonia trabeculata. Seaweeds were collected from three sampling stations located at 5, 30 and 60 m from an illegal sewage outfall close to Ventanas, Chile and from a pristine location at Faro Curaumilla. For the majority of metals the highest concentrations in bottom sediment and seaweed samples were found at the site closest to the outfall, with concentrations decreasing with distance from the outfall and at control stations; the exception was Cd, concentrations of which were higher at control stations. The patterns of metal concentrations in different thallus parts were metal specific and independent of sampling station. These results and the available literature suggest that biomonitoring of metals using seaweeds must take account of differences in the accumulation of metals in thallus parts of complex seaweeds.

Response differences between Ectocarpus siliculosus populations to copper stress involve cellular exclusion and induction of the phytochelatin biosynthetic pathway

Some populations of brown seaweed species inhabit metal-polluted environments and can develop tolerance to metal stress, but the mechanisms by which this is accomplished are still to be elucidated. To address this, the responses of two strains of the model brown alga Ectocarpus siliculosus isolated from sites with different histories of metal contamination exposed to total copper (CuT) concentrations ranging between 0 and 2.4 μM for 10 days were investigated. The synthesis of the metal-chelator phytochelatin (PCs) and relative levels of transcripts encoding the enzymes γ-glutamylcysteine synthetase (γ-GCS), glutathione synthase (GS) and phytochelatin synthase (PCS) that participate in the PC biosynthetic pathway were measured, along with the effects on growth, and adsorption and uptake of Cu. Growth of strain LIA, from a pristine site in Scotland, was inhibited to a greater extent, and at lower concentrations, than that of Es524, isolated from a Cu-contaminated site in Chile. Concentrations of intra-cellular Cu were higher and the exchangeable fraction was lower in LIA than Es524, especially at the highest exposure levels. Total glutathione concentrations increased in both strains with Cu exposure, whereas total PCs levels were higher in Es524 than LIA; PC2 and PC3 were detected in Es524 but PC2 only was found in LIA. The greater production and levels of polymerisation of PCs in Es524 can be explained by the up-regulation of genes encoding for key enzymes involved in the synthesis of PCs. In Es524 there was an increase in the transcripts of γ-GCS, GS and PCS, particularly under high Cu exposure, whereas in LIA4 transcripts of γ-GCS1 increased only slightly, γ-GCS2 and GS decreased and PCS did not change. The consequences of higher intra-cellular concentrations of Cu, lower production of PCs, and lower expression of enzymes involved in GSH-PCs synthesis may be contributing to an induced oxidative stress condition in LIA, which explains, at least in part, the observed sensitivity of LIA to Cu. Therefore, responses to Cu exposure in E. siliculosus relate to the contamination histories of the locations from where the strains were isolated and differences in Cu exclusion and PCs production are in part responsible for the development of intra-specific resistance.

Mechanisms of metal tolerance in marine macroalgae, with emphasis on copper tolerance in Chlorophyta and Rhodophyta

Green and red macroalgae are closely related organisms, and with terrestrial plants, and constitute the base of marine food webs in coastal ecosystems. Green and red seaweeds, as all living organisms, require essential metals, such as copper, iron, zinc, which can act as co-factors for several proteins and enzymes; however, these metals in excess can induce stress and impair cell viability. Most important negative effects of metal excess are related to the induction of an oxidative stress condition, characterized by the over-accumulation of Reactive Oxygen Species (ROS). In this respect, copper, abundant in wastewaters disposed to coastal environments from domestic and industrial activities, has been one of the most studied metals. Different investigations have provided evidence that green and red macroalgae display several defenses against copper excess to prevent, or at least reduce, stress and damage, among which are cellular exclusion mechanisms, synthesis of metal-chelating compounds, and the activation of the antioxidant system. Most important defense mechanisms identified in green and red seaweed involve: metal-binding to cell wall and epibionts; syntheses of metallothioneins and phytochelatins that accumulate in the cytoplasm; and the increase in the activity of antioxidant enzymes such as superoxide dismutase, ascorbate peroxidase, glutathione peroxidase and catalase, and greater production of antioxidant metabolites as glutathione and ascorbate in organelles and the cytoplasm. In this review, we go through historical records, latest advances, and pending tasks aiming to expand our current knowledge on defense mechanisms to copper excess in green and red macroalgae, with emphasis on biochemical and molecular aspects.

Copper-induced activation of TRP channels promotes extracellular calcium entry, activation of CaMs and CDPKs, copper entry and membrane depolarization in Ulva compressa

In order to identify channels involved in membrane depolarization, Ulva compressa was incubated with agonists of TRP channels C5, A1 and V1, and the level of intracellular calcium was detected. Agonists of TRPC5, A1 and V1 induced increases in intracellular calcium at 4, 9, and 11 min of exposure, respectively, and antagonists of TRPC5, A1, and V1 corresponding to SKF-96365 (SKF), HC-030031 (HC), and capsazepin (CPZ), respectively, inhibited calcium increases indicating that functional TRPs exist in U. compressa. In addition, copper excess induced increases in intracellular calcium at 4, 9, and 12 min which were inhibited by SKF, HC, and CPZ, respectively, indicating that copper activate TRPC5, A1, and V1 channels. Moreover, copper-induced calcium increases were inhibited by EGTA, a non-permeable calcium chelating agent, but not by thapsigargin, an inhibitor of endoplasmic reticulum (ER) calcium ATPase, indicating that activation of TRPs leads to extracellular calcium entry. Furthermore, copper-induced calcium increases were not inhibited by W-7, an inhibitor of CaMs, and staurosporine, an inhibitor of CDPKs, indicating that extracellular calcium entry did not require activation of CaMs and CDPKs. In addition, copper induced membrane depolarization events at 4, 8, and 11 min and these events were inhibited by SKF, HC, CPZ, and bathocuproine, a specific copper chelating agent, indicating that copper entry through TRP channels leads to membrane depolarization. Moreover, membrane depolarization events were inhibited by W-7 and staurosporine, indicating that activation of CaMs and CDPKs is required to allow copper entry through TRPs. Interestingly, copper-induced calcium increases and depolarization events were light-dependent and were inhibited by DCMU, an inhibitor of photosystem II, and ATP-γ-S, a non-hydrolizable analog of ATP, suggesting that ATP derived from photosynthesis is required to activate TRPs. Thus, light-dependent copper-induced activation TRPC5, A1 and V1 promotes extracellular calcium entry leading to activation of CaMs and CDPKs which, in turn, promotes copper entry through TRP channels and membrane depolarization.

Oligo-carrageenan kappa increases NADPH, ascorbate and glutathione syntheses and TRR/TRX activities enhancing photosynthesis, basal metabolism, and growth in Eucalyptus trees

In order to analyze the effect of OC kappa in redox status, photosynthesis, basal metabolism and growth in Eucalyptus globulus, trees were treated with water (control), with OC kappa at 1 mg mL−1, or treated with inhibitors of NAD(P)H, ascorbate (ASC), and glutathione (GSH) syntheses and thioredoxin reductase (TRR) activity, CHS-828, lycorine, buthionine sulfoximine (BSO), and auranofin, respectively, and with OC kappa, and cultivated for 4 months. Treatment with OC kappa induced an increase in NADPH, ASC, and GSH syntheses, TRR and thioredoxin (TRX) activities, photosynthesis, growth and activities of basal metabolism enzymes such as rubisco, glutamine synthetase (GlnS), adenosine 5′-phosphosulfate reductase (APR), involved in C, N, and S assimilation, respectively, Krebs cycle and purine/pyrimidine synthesis enzymes. Treatment with inhibitors and OC kappa showed that increases in ASC, GSH, and TRR/TRX enhanced NADPH synthesis, increases in NADPH and TRR/TRX enhanced ASC and GSH syntheses, and only the increase in NADPH enhanced TRR/TRX activities. In addition, the increase in NADPH, ASC, GSH, and TRR/TRX enhanced photosynthesis and growth. Moreover, the increase in NADPH, ASC and TRR/TRX enhanced activities of rubisco, Krebs cycle, and purine/pyrimidine synthesis enzymes, the increase in GSH, NADPH, and TRR/TRX enhanced APR activity, and the increase in NADPH and TRR/TRX enhanced GlnS activity. Thus, OC kappa increases NADPH, ASC, and GSH syntheses leading to a more reducing redox status, the increase in NADPH, ASC, GSH syntheses, and TRR/TRX activities are cross-talking events leading to activation of photosynthesis, basal metabolism, and growth in Eucalyptus trees.

A novel field transplantation technique reveals intra-specific metal-induced oxidative responses in strains of Ectocarpus siliculosus with different pollution histories.

A novel field transplantation technique, in which seaweed material is incorporated into dialysis tubing, was used to investigate intra-specific responses to metals in the model brown alga Ectocarpus siliculosus. Metal accumulation in the two strains was similar, with higher concentrations in material deployed to the metal-contaminated site (Ventanas, Chile) than the pristine site (Quintay, Chile). However, the oxidative responses differed. At Ventanas, strain Es147 (from low-polluted site) underwent oxidative damage whereas Es524 (from highly polluted site) was not affected. Concentrations of reduced ascorbate (ASC) and reduced glutathione (GSH) were significantly higher in Es524. Activities of the antioxidant enzymes superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT), and glutathione reductase (GR) all increased in Es524, whereas only SOD increased in Es147. For the first time, employing a field transplantation technique, we provide unambiguous evidence of inter-population variation of metal-tolerance in brown algae and establish that antioxidant defences are, in part, responsible.

Enzymatic antioxidant defences are transcriptionally regulated in Es524, a copper- tolerant strain of Ectocarpus siliculosus (Ectocarpales, Phaeophyceae)

Abstract: Previous investigations have revealed that Ectocarpus siliculosus strain Es524, isolated from a highly copper (Cu)-polluted location in Chile, displays higher activities of the antioxidant enzymes superoxide dismutase (SOD), ascorbate peroxidase (APX) and catalase (CAT) under Cu excess. This partly explains its enhanced tolerance compared with other E. siliculosus strains. However, the transcriptomic basis behind the activity of these antioxidant enzymes is still unknown. We have conducted laboratory experiments with Es524 under control conditions and exposure to 2.4 μM total Cu. Under Cu exposure, Es524 showed a decrease in cell length and cell plasmolysis, revealing a certain level of cytophysiological stress. The genes encoding for iron-SOD, APX and CAT were overexpressed under Cu excess, demonstrating that higher activities of these enzymes are transcriptionally regulated. It is now more apparent that the induction of SOD, APX and CAT are conserved molecular and biochemical mechanisms developed after a long history of Cu exposure which is responsible, at least in part, for intraspecific Cu tolerance in E. siliculosus.

Changes in invertebrate assemblages inhabiting Lessonia spicata (Phaeophyceae) holdfasts after the 2010 earthquake-mediated coastal uplift in Chile

On February 27th 2010, a mega-earthquake struck Chile causing coastal coseismic uplift. In this study we compared the invertebrate fauna from uplifted (UH) a...