Final Report Summary - ECTOTOX (A toxico-genomic study of the model brown alga Ectocarpus siliculosus) Metals, due to their persistence, bioaccumulation and toxic effects, can pose immediate threats to the resident organisms of coastal waters and estuaries. Seaweeds are the pre-eminent primary producers of near-shore waters, providing habitats for a large diversity of other marine organisms. The lack of genomic information has been until now the main obstacle in the understanding of the molecular basis for metal (and other) stress responses in seaweeds. The recent sequencing of the brown alga Ectocarpus siliculosus genome has opened up the opportunity to address the above question. Phytochelatins (PCs) are the best characterized intracellular metal ligands in plants and their role is crucial to avoid or minimize the detrimental effects of essential and non-essential metals. PCs are small sulphur-rich oligopeptides synthesised through two enzymatic pathways that involve first the synthesis of glutathione (GSH) by γ-Glutamylcysteine Synthetase (ECS) and Glutathione Synthetase (GS) enzymes, then the conversion of GSH to PCs by Phytochelatin Synthetase (PCS). Glutathione is thus the primary pre-cursor for PC formation but also acts as antioxidant within the cell. Therefore, maintaining the equilibrium between synthesis and utilization of GSH in order to reduce oxidative damage and produce PCs may be critical to combating metal stress. Furthermore, the toxic effects of (some) metals are associated with reactive oxygen metabolism (ROM). Thus, tolerance to metal stress depends on defence systems that involve antioxidant enzymes, to prevent (or at least reduce) oxidative damage. The main aim of the project was to investigate oxidative stress responses, glutathione and phytochelatin production in several strains of Ectocarpus siliculosus, collected from areas with different pollution histories, exposed to a range of copper concentrations. In particular, the project aimed to assess the expression of the genes involved in GSH and PCs synthesis (ECS, GS and PCS) in these same strains and under the same copper conditions, thus allowing validation between phenotypic and genotypic responses.An extensive culture collection of 40 strains of E.siliculosus has been set up by the researcher at UoP, including both strains collected from a field trip done in 2010 along a copper polluted gradient site in Cornwall (UK) and axenic strains. Copper accumulation patterns under increasing copper concentrations have been assessed in four strains originally collected from areas with different copper pollution history. Sensitivity of spores to copper have also been analysed through measurement of growth and germination rate. Protocols for assessment of oxidative stress responses have been optimized for E. siliculosus and several components of the reactive oxygen metabolism (ROM) have been investigated (hydrogen peroxide production, lipid peroxidation). The cellular content of several specific antioxidants has also been measured (reduced and oxidized glutathione, phenols and chlorophylls content) and the activity of the antioxidant enzymes analysed (catalase, ascorbate peroxidase and superoxide dismutase). GSH and PCs content have also been measured in the same strains in response to copper exposure and a quantitative polymerase chain reaction assay (qPCR) has been developed in order to assess the expression profiles of the genes involved in the GSH and PCs synthesis pathway. In particular four genes were tested: one gene for PCS (Esil0399_0022), one gene for GS (Esil0066_0082) and two gene for γECS, γECS1 and γECS2 (Esil0250_0012 and Esil0184_0033 respectively).Our results showed, for the first time in the model brown alga E.siliculosus the presence of constitutive low levels of phytochelatins and that their synthesis can be induced in response to both high concentration of cadmium (500 µg L-1) and low concentrations of copper (50 µg L-1). Different PCs oligomers were detected (PC2, PC3 and PC4) and their molecular structure confirmed. Moreover, a marked strain specific response was observed. In fact copper induced PCs production was observed only in the copper tolerant strain (Ec524) compared to the strain collected from a pristine site (LIA4). Longer oligomers (PC3 and PC4) and thus higher metal binding capacity was also observed only in the copper tolerant strain Ec524. Results from the gene expression analysis in the copper tolerant strain Ec524 showed overexpression of all four genes tested. In particular, an abrupt increase of both phytochelatin synthase (PS) gene and reduced GSH content at the highest copper concentration tested (150 µg L-1) was observed suggesting that GSH and PCs play a significant role in copper homeostasis in this strain. In Ec524strain, copper binding to phytochelatins and activation of the antioxidant enzymes pool (ascorbate peroxidase and catalase) seem to be the combination of cellular mechanisms used to counteract copper toxicity and thus reducing ROS formation and lipid peroxidation. On the contrary, in LIA4 strain, collected from a pristine site, no enhanced PCs production was found and inhibition of the expression of the PS gene tested was observed above 50 µg L-1. Catalase was not significantly activated and APX only to a lesser extent than Ec524 strain. Total GSH pool increased at the highest copper concentration but to a much less extent than Ec524 strain. These results suggest that alternative chelators may play a role in metal homeostasis and also that 50 µg Cu L-1 may represent a threshold above which cell reactive oxygen metabolism is overwhelmed by copper toxicity (higher hydrogen peroxide and lipid peroxidation observed). Interestingly the axenic material of Ec524 strain showed a similar behaviour to the more copper sensitive strain LIA4 than to Ec524 non-axenic, both in terms of gene expression profiles and oxidative stress results. Nevertheless, a significantly higher total and intracellular copper content was observed at 100 and 150 µg Cu L-1 together with a higher concentration of phenols (both in control and copper exposed samples) in Ec524 axenic compared with other strains. Reduced GSH pool also increased above 25 µg Cu L-1.These results suggest that the absence of epiphytic bacteria can result in increased intracellular concentrations of copper resulting in greater toxicity in terms of ROS production and lipid peroxidation. Finally, REP10-11 strain collected in 2010 from a polluted (including Cu) site in UK, showed similar results to LIA4 although inhibition of the genes tested was observed already at concentrations above 25 µg Cu L-1, and total and intracellular copper content was the lowest of all strain at 100 and 150 µg Cu L-1. In REP10-11 an exclusion mechanism for Cu may be operating more effectively than intracellular metal chelation. Copper toxicity has also been evaluated on the unicellular stage (spores) of the life cycle. Measurements of germination and growth rates showed that this life stage is more copper sensitive than the adult (sporophyte) in the copper tolerant strain Ec524. Growth of spores is strongly affected and in particular, copper inhibits the development of round cells at a very early stage (few cells) of development. This leads to fewer upright filaments (UPRF) being formed and thus fewer reproductive organs as they are produced on UPRF.Implementation of the project resulted in novel and important information on copper toxicity mechanisms in the model brown alga Ectocarpus siliculosus. Results highlighted the importance of phytochelatins as intracellular metal chelators in brown algae in response to low copper levels. Phenotypic responses could be validated by gene expression profiles. Our results support the use of phytochelatins and related genes, especially phytochelatin synthase, as biomarkers of copper pollution. This can be of great interest in order to improve the monitoring tools available to environmental protection agencies. Moreover, the high variability between strains of E. siliculosus observed in response to copper exposure highlight the need to assess metal toxicity in a large number of strains (populations) and this supports the recent concept of E. siliculosus as a complex of cryptic species. Finally, our results also stress the importance of assessing metal toxicity in both axenic and non-axenic material as epiphytic bacteria play an important part in metal accumulation which can’t be ignored.
