Antarctic intertidal macroalgae under predicted increased temperatures mediated by global climate change: Would they cope?

The Antarctic Peninsula is one of the regions to be most affected by increase in sea surface temperatures (SSTs) mediated by Global Climate Change; indeed, most negative predictions imply an up to 6 °C increment by the end of the XXI century. Temperature is one of the most important factors mediating diversity and distribution of macroalgae, although there is still no consensus as to the likely effects of higher SSTs, especially for polar seaweeds. Some available information suggests that potential strategies to withstand future increases in SSTs will be founded upon the glutathione-ascorbate cycle and the induction of chaperone-functioning heat shock proteins (HSPs); however, their eventual role, even for general stress responses, is unclear. The intertidal green, brown and red macroalgae species Monostroma hariotii, Adenocystis utricularis and Pyropia endiviifolia, respectively, from King George Island, Antarctic Peninsula, were exposed to 2 °C (control) and 8 °C (climate change scenario) for up to 5 days (d). Photosynthetic activity (αETR and ETRmax, and EkETR), photoinhibition (Fv/Fm) and photoprotection processes (αNPQ, NPQmax, and EkNPQ) provided no evidence of negative ecophysiological effects. There were moderate increases in H2O2 production and levels of lipid peroxidation with temperature, results supported by stable levels of total glutathione and ascorbate pools, with mostly higher levels of reduced ascorbate and glutathione than oxidized forms in all species. Transcripts of P. endiviifolia indicated a general upregulation of all antioxidant enzymes and HSPs genes studied under warmer temperature, although with different levels of activation with time. This pioneering investigation exploring different levels of biological organization, suggested that Antarctic intertidal macroalgae may be able to withstand future rise in SSTs, probably slightly altering their latitudinal distribution and/or range of thermal tolerance, by exhibiting robust glutathione-ascorbate production and recycling, as well as the induction of associated antioxidant enzymatic machinery and the syntheses of HSPs.

MAPK Pathway under Chronic Copper Excess in Green Macroalgae (Chlorophyta): Involvement in the Regulation of Detoxification Mechanisms.

Following the physiological complementary/parallel Celis-Plá et al., by inhibiting extracellular signal regulated kinases (ERK), c-Jun N-terminal kinases (JNK), and cytokinin specific binding protein (p38), we assessed the role of the mitogen-activated protein kinases (MAPK) pathway in detoxification responses mediated by chronic copper (10 µM) in U. compressa. Parameters were taken at 6, 24, and 48 h, and 6 days (d). H2O2 and lipid peroxidation under copper and inhibition of ERK, JNK, or p38 alone increased but recovered by the sixth day. By blocking two or more MAPKs under copper, H2O2 and lipid peroxidation decayed even below controls. Inhibition of more than one MAPK (at 6 d) caused a decrease in total glutathione (reduced glutathione (GSH) + oxidised glutathione (GSSG)) and ascorbate (reduced ascorbate (ASC) + dehydroascorbate (DHA)), although in the latter it did not occur when the whole MAPK was blocked. Catalase (CAT), superoxide dismutase (SOD), thioredoxin (TRX) ascorbate peroxidase (APX), dehydroascorbate reductase (DHAR), and glutathione synthase (GS), were downregulated when blocking more than one MAPK pathway. When one MAPK pathway was blocked under copper, a recovery and even enhancement of detoxification mechanisms was observed, likely due to crosstalk within the MAPKs and/or other signalling processes. In contrast, when more than one MAPK pathway were blocked under copper, impairment of detoxification defences occurred, demonstrating that MAPKs were key signalling mechanisms for detoxification in macroalgae.

MAPK Pathway under Chronic Copper Excess in Green Macroalgae (Chlorophyta): Influence on Metal Exclusion/Extrusion Mechanisms and Photosynthesis.

There is currently no information regarding the role that whole mitogen activated protein kinase (MAPK) pathways play in counteracting environmental stress in photosynthetic organisms. To address this gap, we exposed Ulva compressa to chronic levels of copper (10 µM) specific inhibitors of Extracellular Signal Regulated Kinases (ERK), c-Jun N-terminal Kinases (JNK), and Cytokinin Specific Binding Protein (p38) MAPKs alone or in combination. Intracellular copper accumulation and photosynthetic activity (in vivo chlorophyll a fluorescence) were measured after 6 h, 24 h, 48 h, and 6 days of exposure. By day 6, when one (except JNK) or more of the MAPK pathways were inhibited under copper stress, there was a decrease in copper accumulation compared with algae exposed to copper alone. When at least two MAPKs were blocked, there was a decrease in photosynthetic activity expressed in lower productivity (ETRmax), efficiency (αETR), and saturation of irradiance (EkETR), accompanied by higher non-photochemical quenching (NPQmax), compared to both the control and copper-only treatments. In terms of accumulation, once the MAPK pathways were partially or completely blocked under copper, there was crosstalk between these and other signaling mechanisms to enhance metal extrusion/exclusion from cells. Crosstalk occurred among MAPK pathways to maintain photosynthesis homeostasis, demonstrating the importance of the signaling pathways for physiological performance. This study is complemented by a parallel/complementary article Rodríguez-Rojas et al. on the role of MAPKs in copper-detoxification.

Copper excess detoxification is mediated by a coordinated and complementary induction of glutathione, phytochelatins and metallothioneins in the green seaweed Ulva compressa.

In order to analyze the involvement of intracellular thiol-chelators in the accumulation and detoxification of copper, the marine alga Ulva compressa was cultivated with increasing concentrations of copper such as 2.5, 5, 7.5 and 10 µM for up to 12 d, and the amount of intracellular copper, glutathione (GSH), phytochelatins (PCs) and transcripts encoding three metallothioneins (MTs) were determined. Over this exposure period and concentration range there was a linear correlation between intracellular copper and the copper concentration in the culture medium. Increases in GSH concentrations occurred mainly between days 1 and 3 and at lower concentrations of copper (2.5 and 5 µM). The level of PCs, and particularly PC2, increased from day 1 of exposure mainly at higher concentrations of copper (7.5 and 10 µM). The levels of transcripts encoding MT7 increased at day 3, whereas those of MT3 and MT6 increased between days 9-12, mainly at higher concentrations of copper. Thus in U. compressa, the initial responses to increasing intracellular copper concentrations are increases in GSH and PCs that are followed by higher levels of MTs expression, suggesting that thiol-containing peptides and proteins may participate in copper accumulation and detoxification responding in a coordinated and complementary manner. In addition, the alga was cultivated with 10 µM copper for 5 d and transferred to synthetic seawater with no copper and cultivated for 3 d. The release of copper from cells to culture medium was observed and accompanied by a similar nanomolar amount of GSH; no PCs or small proteins were detected. These results could suggest that a component of the detoxification mechanism also involves the release of copper and GSH to the extracellular medium.

Phaeoviral Infections Are Present in Macrocystis, Ecklonia and Undaria (Laminariales) and Are Influenced by Wave Exposure in Ectocarpales.

Two sister orders of the brown macroalgae (class Phaeophyceae), the morphologically complex Laminariales (commonly referred to as kelp) and the morphologically simple Ectocarpales are natural hosts for the dsDNA phaeoviruses (family Phycodnaviridae) that persist as proviruses in the genomes of their hosts. We have previously shown that the major capsid protein (MCP) and DNA polymerase concatenated gene phylogeny splits phaeoviruses into two subgroups, A and B (both infecting Ectocarpales), while MCP-based phylogeny suggests that the kelp phaeoviruses form a distinct third subgroup C. Here we used MCP to better understand the host range of phaeoviruses by screening a further 96 and 909 samples representing 11 and 3 species of kelp and Ectocarpales, respectively. Sporophyte kelp samples were collected from their various natural coastal habitats spanning five continents: Africa, Asia, Australia, Europe, and South America. Our phylogenetic analyses showed that while most of the kelp phaeoviruses, including one from Macrocystispyrifera, belonged to the previously designated subgroup C, new lineages of Phaeovirus in 3 kelp species, Ecklonia maxima, Ecklonia radiata, Undaria pinnatifida, grouped instead with subgroup A. In addition, we observed a prevalence of 26% and 63% in kelp and Ectocarpales, respectively. Although not common, multiple phaeoviral infections per individual were observed, with the Ectocarpales having both intra- and inter-subgroup phaeoviral infections. Only intra-subgroup phaeoviral infections were observed in kelp. Furthermore, prevalence of phaeoviral infections within the Ectocarpales is also linked to their exposure to waves. We conclude that phaeoviral infection is a widely occurring phenomenon in both lineages, and that phaeoviruses have diversified with their hosts at least since the divergence of the Laminariales and Ectocarpales.

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.

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.

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.

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.