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.

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.

Proteomic analysis and identification of copper stress-regulated proteins in the marine alga Scytosiphon gracilis (Phaeophyceae).

A proteomic analysis combining peptide de novo sequencing and BLAST analysis was used to identify novel proteins involved in copper tolerance in the marine alga Scytosiphon gracilis (Phaeophyceae). Algal material was cultivated in seawater without copper (control) or supplemented with 100 microg L(-1) for 4 days, and protein extracts were separated by two-dimensional gel electrophoresis (2-DE). From the proteins obtained in the copper treatment, 25 over-expressed, 5 under-expressed and 5 proteins with no changes as compared with the control, were selected for sequencing. Tryptic-peptides obtained from 35 spots were analyzed by capillary liquid chromatography and tandem mass spectroscopy (capLC/MS/MS), and protein identity was determined by BLASTP. We identified 19 over-expressed proteins, including a chloroplast peroxiredoxin, a cytosolic phosphomannomutase, a cytosolic glyceraldehyde-3-phosphate dehydrogenase, 3 ABC transporters, a chaperonine, a subunit of the proteasome and a tRNA synthase, among others. The possible involvement of these over-expressed proteins in buffering oxidative stress and avoiding metal uptake in S. gracilis exposed to copper excess is discussed taking into consideration the information available for other plant models.

Differential responses to copper-induced oxidative stress in the marine macroalgae Lessonia nigrescens and Scytosiphon lomentaria (Phaeophyceae).

In order to help explain the absence of the brown kelp Lessonia nigrescens from a coastal environment chronically enriched with copper, we characterized the biochemical responses induced by copper stress in this kelp and compared them with those displayed by the copper tolerant brown alga Scytosiphon lomentaria. These algae were cultivated with increasing concentrations of copper (20, 40 and 100microgL(-1)) for 96h and the temporal production of hydrogen peroxide, superoxide anions and lipoperoxides as well as the activities of antioxidant enzymes catalase (CAT), glutathione peroxidase (GP), ascorbate peroxidase (AP), dehydroascorbate reductase (DHAR) and glutathione reductase (GR) and the activity of the defense enzyme lipoxygenase (LOX) were determined. In L. nigrescens and S. lomentaria, a single peak of hydrogen peroxide was detected, with similar maxima after 3h of copper exposure, although in L. nigrescens buffering took longer. Superoxide anions, on the other hand, were only detected in L. nigrescens. The production of lipoperoxides in L. nigrescens increased steadily at higher copper levels, in a pattern clearly different to their rapid stabilization in S. lomentaria. We suggest that the accumulation of lipoperoxides might be related to LOX, whose activity also increases with exposure time. Furthermore, activities of the antioxidant enzymes CAT, GP, AP and DHAR were lower in L. nigrescens than in S. lomentaria, and GP and DHAR were completely inhibited at higher copper concentrations. Since these enzymes also detoxify fatty acid hydroperoxides, their inhibition, together with the activation of LOX, may explain the persistent and copper-dependent levels of lipoperoxides in L. nigrescens. Based on terrestrial plant models demonstrating toxic effects of lipoperoxides, and on our results on organellar ultrastructural changes, we suggest that copper toxicity induced an uncontrolled lipoperoxide accumulation which may lead to cell damage and dysfunction in L. nigrescens, explaining at least partially, the absence of this kelp in a copper-enriched coastal environment.

LAMINARIOCOLAX SP. (PHAEOPHYCEAE) ASSOCIATED WITH GALL DEVELOPMENTS IN LESSONIA NIGRESCENS (PHAEOPHYCEAE)(1).

As part of a long-term study on the biology and ecology of the intertidal kelp Lessonia nigrescens Bory, we report on the occurrence of gall development on this alga, identified the possible causal agent, and assessed the extent of the phenomenon in two wild stands of the host. Our results showed that galls affecting natural populations of L. nigrescens were associated with the infection by a filamentous brown algal endophyte of the genus Laminariocolax. Assignment to Laminariocolax of the endophytes isolated from cultured gall tissue was based on the (i) high internal transcribed spacer 1 (ITS1) sequence similarity and phylogenetic relationship between the Chilean isolates and several species of the genus Laminariocolax endophytic in other kelps, (ii) reproductive and vegetative features of the endophyte in culture, and (iii) anatomical agreement of fully developed galls of Lessonia with those described for other kelp galls caused by endophytic members of Laminariocolax. Unequivocal identification at the species level of the endophytes infecting Lessonia, however, awaits further studies.

TWO-DIMENSIONAL GEL ELECTROPHORESIS ANALYSIS OF BROWN ALGAL PROTEIN EXTRACTS(1).

High-quality protein extracts are required for proteomic studies, a field that is poorly developed for marine macroalgae. A reliable phenol extraction protocol using Scytosiphon gracilis Kogame and Ectocarpus siliculosus (Dillwyn) Lyngb. (Phaeophyceae) as algal models resulted in high-quality protein extracts. The performance of the new protocol was tested against four methods available for vascular plants and a seaweed. The protocol, which includes an initial step to remove salts from the algal tissues, allowed the use of highly resolving two-dimensional gel electrophoresis (2-DE) protein analyses, providing the opportunity to unravel potentially novel physiological processes unique to this group of marine organisms.