Role of abscisic acid (ABA) in activating antioxidant tolerance responses to desiccation stress in intertidal seaweed species.

MAIN CONCLUSION: The hormone ABA regulates the oxidative stress state under desiccation in seaweed species; an environmental condition generated during daily tidal changes. Desiccation is one of the most important factors that determine the distribution pattern of intertidal seaweeds. Among most tolerant seaweed is Pyropia orbicularis, which colonizes upper intertidal zones along the Chilean coast. P. orbicularis employs diverse mechanisms of desiccation tolerance (DT) (among others, e.g., antioxidant activation, photoinhibition, and osmo-compatible solute overproduction) such as those used by resurrection plants and bryophytes. In these organisms, the hormone abscisic acid (ABA) plays an important role in regulating responses to water deficit, including gene expression and the activity of antioxidant enzymes. The present study determined the effect of ABA on the activation of antioxidant responses during desiccation in P. orbicularis and in the sensitive species Mazzaella laminarioides and Lessonia spicata. Changes in endogenous free and conjugated ABA, water content during the hydration-desiccation cycle, enzymatic antioxidant activities [ascorbate peroxidase (AP), catalase (CAT) and peroxiredoxine (PRX)], and levels of lipid peroxidation and cell viability were evaluated. The results showed that P. orbicularis had free ABA levels 4-7 times higher than sensitive species, which was overproduced during water deficit. Using two ABA inhibitors (sodium tungstate and ancymidol), ABA was found to regulate the activation of the antioxidant enzymes activities during desiccation. In individuals exposed to exogenous ABA the enzyme activity increased, concomitant with low lipid peroxidation and high cell viability. These results demonstrate the participation of ABA in the regulation of DT in seaweeds, and suggest that regulatory mechanisms with ABA signaling could be of great importance for the adaptation of these organisms to dehydration.

Identification of proteins involved in desiccation tolerance in the red seaweed Pyropia orbicularis (Rhodophyta, Bangiales).

Extreme reduction in cellular water content leads to desiccation, which, if persistent, affects the physiology of organisms, mainly through oxidative stress. Some organisms are highly tolerant to desiccation, including resurrection plants and certain intertidal seaweeds. One such species is Pyropia orbicularis, a rhodophycean that colonizes upper intertidal zones along the Chilean coast. Despite long, daily periods of air exposure due to tides, this alga is highly tolerant to desiccation. The present study examined the proteome of P. orbicularis by 2DE and LC-MS/MS analyses to determine the proteins associated with desiccation tolerance (DT). The results showed that, under natural conditions, there were significant changes in the protein profile during low tide as compared to naturally hydrated plants at high tide. These changes were mainly in newly appeared proteins spots such as chaperones, monodehydroascorbate reductase, and manganese superoxide dismutase, among others. Previously undescribed proteins under desiccation conditions included phycobiliproteins, glyoxalase I, and phosphomannomutase. These changes evidenced that several physiological responses involved in DT are activated during low tide, including decreased photosynthetic activity, increased antioxidant capacity, and the preservation of cell physiology by regulating water content, cell wall structure, and cell volume. Similar responses have been observed in resurrection plants and bryophytes exposed to desiccation. Therefore, the coordinated activation of different desiccation tolerance pathways in P. orbicularis could explain the successful biological performance of this seaweed in the upper intertidal rocky zones.

Transcriptomic and metabolomic analysis of copper stress acclimation in Ectocarpus siliculosus highlights signaling and tolerance mechanisms in brown algae.

BACKGROUND: Brown algae are sessile macro-organisms of great ecological relevance in coastal ecosystems. They evolved independently from land plants and other multicellular lineages, and therefore hold several original ontogenic and metabolic features. Most brown algae grow along the coastal zone where they face frequent environmental changes, including exposure to toxic levels of heavy metals such as copper (Cu). RESULTS: We carried out large-scale transcriptomic and metabolomic analyses to decipher the short-term acclimation of the brown algal model E. siliculosus to Cu stress, and compared these data to results known for other abiotic stressors. This comparison demonstrates that Cu induces oxidative stress in E. siliculosus as illustrated by the transcriptomic overlap between Cu and H2O2 treatments. The common response to Cu and H2O2 consisted in the activation of the oxylipin and the repression of inositol signaling pathways, together with the regulation of genes coding for several transcription-associated proteins. Concomitantly, Cu stress specifically activated a set of genes coding for orthologs of ABC transporters, a P1B-type ATPase, ROS detoxification systems such as a vanadium-dependent bromoperoxidase, and induced an increase of free fatty acid contents. Finally we observed, as a common abiotic stress mechanism, the activation of autophagic processes on one hand and the repression of genes involved in nitrogen assimilation on the other hand. CONCLUSIONS: Comparisons with data from green plants indicate that some processes involved in Cu and oxidative stress response are conserved across these two distant lineages. At the same time the high number of yet uncharacterized brown alga-specific genes induced in response to copper stress underlines the potential to discover new components and molecular interactions unique to these organisms. Of particular interest for future research is the potential cross-talk between reactive oxygen species (ROS)-, myo-inositol-, and oxylipin signaling.

Comparative analysis of peroxiredoxin activation in the brown macroalgae Scytosiphon gracilis and Lessonia nigrescens (Phaeophyceae) under copper stress.

Among thiol-dependent peroxidases (TDPs) peroxiredoxins (PRXs) standout, since they are enzymes capable of reducing hydrogen peroxide, alkylhydroperoxides and peroxynitrite, and have been detected in a proteomic study of the copper-tolerant species Scytosiphon gracilis. In order to determine the importance of these enzymes in copper-stress tolerance, TDP activity and type II peroxiredoxin (II PRX) protein expression were compared between the opportunistic S. gracilis and the brown kelp Lessonia nigrescens, a species absent from copper-impacted sites due to insufficient copper-tolerance mechanisms. Individuals of both species were cultured with increasing copper concentrations (0-300 µg l(-1) Cu) for 96 h and TDP activity and lipoperoxides (LPXs) were determined together with II PRX expression by immunofluorescence and Western blot analysis. The results showed that TDP activity was higher in S. gracilis than L. nigrescens in all copper concentrations, independent of the reducing agent used (dithiothreitol, thioredoxin or glutaredoxin). This activity was copper inhibited in L. nigrescens at lower copper concentrations (20 µg l(-1) Cu) compared to S. gracilis (100 µg l(-1) Cu). The loss of activity coincided in both species with an increase in LPX, which suggests that TDP may control LPX production. Moreover, II PRX protein levels increased under copper stress only in S. gracilis. These results suggest that in S. gracilis TDP, particularly type II peroxiredoxin (II PRX), acts as an active antioxidant barrier attenuating the LPX levels generated by copper, which is not the case in L. nigrescens. Thus, from an ecological point of view these results help explaining the inability of L. nigrescens to flourish in copper-enriched environments.

Seaweeds early development: detrimental effects of desiccation and attenuation by algal extracts.

The effects of desiccation on the early development stages of Mazzaella laminarioides, Scytosiphon lomentaria and Lessonia nigrescens, algal species with different patterns of distribution across the intertidal zone, were examined in the laboratory. In addition, the protective effect against desiccation was evaluated using algal extracts, including those from Porphyra columbina, a macroalga tolerant to desiccation that lives in the uppermost part of the intertidal zone. Our results showed that M. laminarioides displayed the highest resistance to daily desiccation, followed by S. lomentaria, whereas L. nigrescens was the most susceptible. Spores from L. nigrescens exposed to desiccation, although being able to germinate, ceased further post-germination development. In addition, our results showed that all species exposed to extracts from desiccated P. columbina successfully completed their development and strongly suggest the occurrence of compounds with protective properties that help in attenuating the stress caused by desiccation. Finally, our results indicate that the magnitude of the effects generated by desiccation on the early algal development is related to the position of the species in the intertidal zone, and that the protective effects of P. columbina extracts reveal an exceptional metabolism of this species under desiccation stress.

Tolerance to oxidative stress induced by desiccation in Porphyra columbina (Bangiales, Rhodophyta).

Unravelling the mechanisms underlying desiccation tolerance is crucial in order to understand the position of algal species in the intertidal zone. The alga Porphyra columbina lives in the uppermost part of the rocky intertidal zones around the world and was selected as a model for this study. Naturally desiccated plants were collected during low tide and studied for morphological changes, oxidative burst induction, biomolecule oxidation, antioxidant responses, and photosynthetic status. Naturally hydrated plants collected during high tides were used for comparative purposes. In addition, changes induced by desiccation were assessed in vitro and the capacity to recover from desiccation was determined by rehydrating the fronds in seawater. The global results show that desiccation induces morphological and cellular alterations accompanied by a loss of ∼96% of the water content. Overproduction of reactive oxygen species (ROS) was induced by desiccation and two peaks of H(2)O(2) were detected at 1 and 3 h of desiccation. However, during in vitro rehydration post-desiccation, the ROS quickly returned to the basal levels. At the biomolecular level, only a low production of oxidized proteins was recorded during desiccation, whereas the activity of diverse antioxidant enzymes increased. However, this activity diminished to near basal levels during rehydration. The photosynthetic efficiency (F(v)/F(m)) during desiccation declined by 94-96% of the values recorded in hydrated plants. This reduction was generated by the low levels of trapped energy flux per cross-section (TRo/CS), electron transport flux per CS (ETo/CS), and density of reaction centres (RC/SCo) as well as the chlorophyll content. The inverse pattern was observed for the levels of phycocyanin and phycoerythrin content. F(v)/F(m) and the photosynthetic indicators were restored to normal levels after only 5 min of rehydration. The results indicate that desiccation in P. columbina causes overproduction of ROS that is efficiently attenuated. The morphological and photosynthetic changes could be operating as tolerance mechanisms due to the fact that these responses principally prevent biomolecular alteration and cellular collapse. Thus, the activation of different physiological mechanisms helps to explain the high tolerance to desiccation of P. columbina and, at least in part, the position of this species at the highest level in the intertidal zone.

Copper stress proteomics highlights local adaptation of two strains of the model brown alga Ectocarpus siliculosus.

Ectocarpus siliculosus is a cosmopolitan brown alga with capacity to thrive in copper enriched environments. Analysis of copper toxicity was conducted in two strains of E. siliculosus isolated from (i) an uncontaminated coast in southern Peru (Es32) and (ii) a copper polluted rocky beach in northern Chile (Es524). Es32 was more sensitive than Es524, with toxicity detected at 50 microg/L Cu, whereas Es524 displayed negative effects only when exposed to 250 microg/L Cu. Differential soluble proteome profiling for each strain exposed to sub-lethal copper levels allowed to identify the induction of proteins related to processes such as energy production, glutathione metabolism as well as accumulation of HSPs. In addition, the inter-strain comparison of stress-related proteomes led to identify features related to copper tolerance in Es524, such as striking expression of a PSII Mn-stabilizing protein and a Fucoxanthine chlorophyll a-c binding protein. Es524 also expressed specific stress-related enzymes such as RNA helicases from the DEAD box families and a vanadium-dependent bromoperoxidase. These observations were supported by RT-qPCR for some of the identified genes and an enzyme activity assay for vanadium-dependent bromoperoxidase. Therefore, the occurrence of two different phenotypes within two distinct E. siliculosus strains studied at the physiological and proteomic levels strongly suggest that persistent copper stress may represent a selective force leading to the development of strains genetically adapted to copper contaminated sites.

Genetic diversity of Ectocarpus (Ectocarpales, Phaeophyceae) in Peru and northern Chile, the area of origin of the genome-sequenced strain.

• The origin of the Ectocarpus strain used for genome sequencing (the 'genome strain') was Peru, where no Ectocarpus had been reported previously. To study the genetic diversity in the region and to increase the number of individuals from this area available for genetic experiments, 119 new Ectocarpus strains were isolated from eight localities along the 3000 km of coastline from central Peru to central Chile. • Internal transcribed spacer 1 (ITS1) genotyping revealed nine different genotypes, five of which were endemic to the area studied and three of which were previously unknown. • Individuals of the same genotype as the genome strain occurred from Peru to northernmost Chile, representing 61% of the samples in this area, from which five more genotypes were isolated. Further south, down to central Chile, most individuals belonged to Ectocarpus siliculosus, Ectocarpus fasciculatus and Ectocarpus crouaniorum. In sexual crosses, the genome strain and the new isolates of the same genotype were fully compatible. • Sequences from four nuclear and cytoplasmic genetic markers (ITS1, ITS2, Rubisco spacer and Cytochrome-c oxidase subunit 3 (cox3)) separated the genome strain from the known species of Ectocarpus. It may in future be recognized as a separate species.

Co-occurring increases of calcium and organellar reactive oxygen species determine differential activation of antioxidant and defense enzymes in Ulva compressa (Chlorophyta) exposed to copper excess.

In order to analyse copper-induced calcium release and (reactive oxygen species) ROS accumulation and their role in antioxidant and defense enzymes activation, the marine alga Ulva compressa was exposed to 10 µM copper for 7 d. The level of calcium, extracellular hydrogen peroxide (eHP), intracellular hydrogen peroxide (iHP) and superoxide anions (SA) as well as the activities of ascorbate peroxidase (AP), glutathione reductase (GR), glutathione-S-transferase (GST), phenylalanine ammonia lyase (PAL) and lipoxygenase (LOX) were determined. Calcium release showed a triphasic pattern with peaks at 2, 3 and 12 h. The second peak was coincident with increases in eHP and iHP and the third peak with the second increase of iHP. A delayed wave of SA occurred after day 3 and was not accompanied by calcium release. The accumulation of iHP and SA was mainly inhibited by organellar electron transport chains inhibitors (OETCI), whereas calcium release was inhibited by ryanodine. AP activation ceased almost completely after the use of OETCI. On the other hand, GR and GST activities were partially inhibited, whereas defense enzymes were not inhibited. In contrast, PAL and LOX were inhibited by ryanodine, whereas AP was not inhibited. Thus, copper stress induces calcium release and organellar ROS accumulation that determine the differential activation of antioxidant and defense enzymes.

Changes in epiphytic bacterial communities of intertidal seaweeds modulated by host, temporality, and copper enrichment.

This study reports on the factors involved in regulating the composition and structure of bacterial communities epiphytic on intertidal macroalgae, exploring their temporal variability and the role of copper pollution. Culture-independent, molecular approaches were chosen for this purpose and three host species were used as models: the ephemeral Ulva spp. (Chlorophyceae) and Scytosiphon lomentaria (Phaeophyceae) and the long-living Lessonia nigrescens (Phaeophyceae). The algae were collected from two coastal areas in Northern Chile, where the main contrast was the concentration of copper in the seawater column resulting from copper-mine waste disposals. We found a clear and strong effect in the structure of the bacterial communities associated with the algal species serving as host. The structure of the bacterial communities also varied through time. The effect of copper on the structure of the epiphytic bacterial communities was significant in Ulva spp., but not on L. nigrescens. The use of 16S rRNA gene library analysis to compare bacterial communities in Ulva revealed that they were composed of five phyla and six classes, with approximately 35 bacterial species, dominated by members of Bacteroidetes (Cytophaga-Flavobacteria-Bacteroides) and α-Proteobacteria, in both non-polluted and polluted sites. Less common groups, such as the Verrucomicrobiae, were exclusively found in polluted sites. This work shows that the structure of bacterial communities epiphytic on macroalgae is hierarchically determined by algal species > temporal changes > copper levels.

Phylogeographic analyses of the 30 degrees S south-east Pacific biogeographic transition zone establish the occurrence of a sharp genetic discontinuity in the kelp Lessonia nigrescens: vicariance or parapatry?

Phylogeographic studies are lacking in the Southern Hemisphere, and in particular in the south-eastern Pacific. To infer the possible scenario for the debated biogeographic transition zone located at 30-33 degrees S along the Chilean coast, we investigated whether there is a concordance between the phylogeographic pattern and the biogeographic transition in the intertidal kelp Lessonia nigrescens whose distribution is continuous across this transition zone. Using a combination of four makers located in the three genomic compartments (chloroplast, mitochondria and nucleus), we showed the presence of two main divergent lineages, possibly cryptic species. There was an exact match of the phylogeographic break with the 30 degrees S biogeographic transition zone, suggesting a common origin. The combined information given by the multilocus approach and by the population analysis suggested the occurrence of a budding speciation, with a northward range expansion.

Genetic variation in wild and cultivated populations of the haploid-diploid red alga Gracilaria chilensis: how farming practices favor asexual reproduction and heterozygosity.

The extent of changes in genetic diversity and life-history traits associated with farming was investigated in the haploid-diploid red alga, Gracilaria chilensis, cultivated in Chile. This alga belongs to one of the most frequently cultivated seaweed genera around the world. Fifteen farmed populations, 11 wild populations, and two subspontaneous populations were sampled along the Chilean coast. The frequency of reproductive versus vegetative individuals and of haploid versus diploid individuals was checked in each population. In addition, the distribution of genetic variation in wild and cultivated populations was analyzed using six microsatellite markers. Our results first demonstrated that farmed populations are maintained almost exclusively by vegetative propagation. Moreover, the predominance of diploid individuals in farms showed that farming practices had significantly modified life-history traits as compared to wild populations. Second, the expected reduction in genetic diversity due to a cultivation bottleneck and subsequent clonal propagation was detected in farms. Finally, our study suggested that cultural practices in the southern part of the country contributed to the spread of selected genotypes at a local scale. Altogether, these results document for the first time that involuntary selection could operate during the first step of domestication in a marine plant.

Copper stress induces biosynthesis of octadecanoid and eicosanoid oxygenated derivatives in the brown algal kelp Laminaria digitata.

To better understand the toxicity and the orchestration of antioxidant defenses of marine brown algae in response to copper-induced stress, lipid peroxidation processes were investigated in the brown alga Laminaria digitata. The expression of genes involved in cell protection and anti-oxidant responses were monitored by semi-quantitative reverse transcriptase polymerase chain reaction and the lipid peroxidation products were further characterized by profiling oxylipin signatures using high-pressure liquid chromatography-mass spectrometry. Exposure to copper excess triggers lipoperoxide accumulation and upregulates the expression of stress related genes. It also increases the release of free polyunsaturated fatty acids, leading to an oxidative cascade through at least two distinct mechanisms. Incubations in presence of inhibitors of lipoxygenases and cycloxygenases showed that in addition to the reactive oxygen species-mediated processes, copper stress induces the synthesis of oxylipins through enzymatic mechanisms. Among complex oxylipins, cyclopentenones from C18 and C20 fatty acids such as 12-oxo-PDA and prostaglandins were detected for the first time in brown algae, as well as unique compounds such as the 18-hydroxy-17-oxo-eicosatetraenoic acid. These results suggest that lipid peroxidation participates in the toxic effects of copper and that lipid peroxidation derivatives may regulate protective mechanisms by employing plant-like octadecanoid signals but also eicosanoid oxylipins which are absent in vascular plants.

Changes in bacterial community structure associated with coastal copper enrichment.

Marine bacterial communities isolated from the water column, sediment, the rock surface, and the green seaweed Ulva compressa were studied in an intertidal ecosystem. The study area included a coastal zone chronically affected by copper mine waste disposals. Bacterial community composition was analyzed by terminal restriction fragment length polymorphism (T-RFLP) of 16S rRNA genes, and multivariate analyses of T-RFLP data sets were used for comparisons. Results showed that diversity and richness indexes were not able to detect differences among compartments. However, comparisons within the same compartment clearly showed that copper enrichment was associated with changes in the composition of the bacterial communities and revealed that the magnitude of the effect depends on the compartment being considered. In this context, communities from sediments appeared as the most affected by copper enrichment. The present study also demonstrated that intertidal bacterial communities were dominated by Gammaproteobacteria, Firmicutes, and Actinobacteria and the changes in these communities were mainly due to changes in their relative abundances.

Effects of copper on early developmental stages of Lessonia nigrescens Bory (Phaeophyceae).

Copper effects on the early developmental gametophytic and sporophytic stages of the kelp Lessonia nigrescens were tested in gradients of increasing concentrations of ASV-labile copper. The results demonstrated a high sensitivity to copper of all life-history stages of the alga, where even the lowest tested concentration affected spore release as well as their subsequent settlement. More significant, concentrations higher than 7.87 microg L(-1) totally interrupted the development of the spores after they settle. This effect led to a failure in the formation of male and female gametophytes and, as a consequence, to a complete disruption of the normal life cycle of the kelp. Thus, we suggest that the absence of L. nigrescens from copper-enriched environments results from the high sensitivity of its early life cycle stages, which limits growth and maturation of the gametophytic microscopic phase and, as a consequence, prevents development of the macroscopic sporophytic phase.

Micro-evolution due to pollution: possible consequences for ecosystem responses to toxic stress.

Polluting events can change community structure and ecosystem functioning. Selection of genetically inherited tolerance on exposed populations, here referred as micro-evolution due to pollution, has been recognized as one of the causes of these changes. However, there is a gap between studies addressing this process and those assessing effects at higher levels of biological organization. In this review we attempt to address these evolutionary considerations into the ecological risk assessment (ERA) of polluting events and to trigger the discussion about the consequences of this process for the ecosystem response to toxic stress. We provide clear evidence that pollution drives micro-evolutionary processes in several species. When this process occurs, populations inhabiting environments that become polluted may persist. However, due to the existence of ecological costs derived from the loss of genetic variability, negative pleiotropy with fitness traits and/or from physiological alterations, micro-evolution due to pollution may alter different properties of the affected populations. Despite the existence of empirical evidence showing that safety margins currently applied in the ERA process may account for pollution-induced genetic changes in tolerance, information regarding long-term ecological consequences at higher levels of biological organization due to ecological costs is not explicitly considered in these procedures. In relation to this, we present four testable hypotheses considering that micro-evolution due to pollution acts upon the variability of functional response traits of the exposed populations and generates changes on their functional effect traits, therefore, modifying the way species exploit their ecological niches and participate in the overall ecosystem functioning.

An assessment of the impact of copper mine tailings disposal on meiofaunal assemblages using microcosm bioassays.

Microcosms were used to assess the impact of copper mine tailings disposal on the littoral meiofaunal assemblages of the Atacama region of northern Chile. The specific purpose was to establish a cause and effect relationship between the elevated copper concentrations and altered meiofaunal assemblages observed at the study sites. Meiofaunal assemblages were exposed to a series of copper concentrations to assess general toxicity, both densities and taxa diversities decreased with increasing copper. Natural coarse sediments were mixed with a tailings substitute to assess the physical impact of the tailings dumping on meiofaunal assemblages. Meiofaunal assemblage densities increased with increasing amounts of tailings substitute, entirely due to an increase in surface utilising foraminiferans. However, taxa diversities decreased as the interstitial spaces became blocked. Finally, the microcosms were used to conduct bioassays of sediments and seawaters from the impacted sites. The sediments from the impacted sites proved to be toxic resulting in reduced meiofaunal densities and taxa diversities. Seawater samples did not prove to be significantly toxic. The use of microcosms has allowed the effects of the physical and chemical components of tailings to be assessed individually, which was not possible in the field. Additionally, it allowed a cause and effect relationship to be established between elevated concentrations of porewater copper observed in the field and the reduced densities and taxa diversities of the meiofaunal assemblages observed at the same sites.

Modulatory effect of the exudates released by the brown kelp Lessonia spicata on the toxicity of copper in early developmental stages of ecologically related organisms.

Lessonia spicata is a key dominant species along the Pacific coast of South America, providing a habitat for many organisms. However, this role can be affected by abiotic stress, such as metals. To counteract the toxic effect, L. spicata, among other seaweeds, releases exudates that bind metals. In this study, tolerances to copper of organisms related to the kelp forest (spores of Ulva lactuca (Chlorophyceae) and L. spicata (Phaeophyceae) and Zoea I of Taliepus dentatus (Milne-Edwards, Crustacea)) were studied; then, exudates are assessed by their protective effect. Exudates increase the 48-h 50% effective concentration (EC50) of the germination of spores from 8 to 23 µg Cu L-1 for U. lactuca and from 119 to 213 µg Cu L-1 for L. spicata and the survival of the larvae Zoea I 48-h 50% of lethal concentration (LC50) from 144 to 249 µg Cu L-1. Results indicated that exudates had a protective effect. Each species is specifically sensitive to copper. Crab larvae Zoea I were able to support higher doses, and exposure before hatching increased their tolerance.

Herbivore-induced chemical and molecular responses of the kelps Laminaria digitata and Lessonia spicata.

Kelps are founding species of temperate marine ecosystems, living in intertidal coastal areas where they are often challenged by generalist and specialist herbivores. As most sessile organisms, kelps develop defensive strategies to restrain grazing damage and preserve their own fitness during interactions with herbivores. To decipher some inducible defense and signaling mechanisms, we carried out metabolome and transcriptome analyses in two emblematic kelp species, Lessonia spicata from South Pacific coasts and Laminaria digitata from North Atlantic, when challenged with their main specialist herbivores. Mass spectrometry based metabolomics revealed large metabolic changes induced in these two brown algae following challenges with their own specialist herbivores. Targeted metabolic profiling of L. spicata further showed that free fatty acid (FFA) and amino acid (AA) metabolisms were particularly regulated under grazing. An early stress response was illustrated by the accumulation of Sulphur containing amino acids in the first twelve hours of herbivory pressure. At latter time periods (after 24 hours), we observed FFA liberation and eicosanoid oxylipins synthesis likely representing metabolites related to stress. Global transcriptomic analysis identified sets of candidate genes specifically induced by grazing in both kelps. qPCR analysis of the top candidate genes during a 48-hours time course validated the results. Most of these genes were particularly activated by herbivore challenge after 24 hours, suggesting that transcriptional reprogramming could be operated at this time period. We demonstrated the potential utility of these genes as molecular markers for herbivory by measuring their inductions in grazed individuals of field harvested L. digitata and L. spicata. By unravelling the regulation of some metabolites and genes following grazing pressure in two kelps representative of the two hemispheres, this work contributes to provide a set of herbivore-induced chemical and molecular responses in kelp species, showing similar inducible responses upon specialist herbivores in their respective ecosystems.

Kinetics of copper accumulation in Lessonia nigrescens (Phaeophyceae) under conditions of environmental oxidative stress.

Juvenile individuals of the brown kelp Lessonia nigrescens were exposed to a coastal environment chronically impacted by copper mine wastes and currently displaying more than 250 nM of total dissolved copper. The kinetic of copper accumulation in the intra and extracellular compartments was determined and correlated to the oxidative burst resulting from copper-mediated oxidative stress. Accumulation involved an initial adsorption onto the outer cell wall followed by a slower uptake into the cells. A linear pattern of copper uptake over time was found during the first 52 h of exposure, and a steady state was reached at 76 h. The resulting oxidative stress was found to be inefficiently attenuated, and the intracellular level of copper remained sufficiently high to determine a persistently higher than normal level of reactive oxygen species (ROS). Thus, our results strongly suggest that, in L. nigrescens, copper needs to reach an intracellular threshold before oxidative burst develops. Furthermore, it was found that the high ROS levels generated by copper accumulation within the cells persists after the oxidative burst has ceased, suggesting a limited capacity of the algal tissue to buffer the increases of ROS caused by the environmental copper levels.