New insights in copper handling strategies in the green alga Chlamydomonas reinhardtii under low-iron condition.

Copper (Cu) is a redox-active transition element critical to various metabolic processes. These functions are accomplished in tandem with Cu-binding ligands, mainly proteins. The main goal of this work was to understand the mechanisms that govern the intracellular fate of Cu in the freshwater green alga, Chlamydomonas reinhardtii, and more specifically to understand the mechanisms underlying Cu detoxification by algal cells in low-Fe conditions. We show that Cu accumulation was up to 51-fold greater for algae exposed to Cu in low-Fe medium as compared to the replete-Fe growth medium. Using the stable isotope 65Cu as a tracer, we studied the subcellular distribution of Cu within the various cell compartments of C. reinhardtii. These data were coupled with metallomic and proteomic approaches to identify potential Cu-binding ligands in the heat-stable proteins and peptides fraction of the cytosol. Cu was mostly found in the organelles (78%), and in the heat-stable proteins and peptides (21%) fractions. The organelle fraction appeared to also be the main target compartment of Cu accumulation in Fe-depleted cells. As Fe levels in the medium were shown to influence Cu homeostasis, we found that C. reinhardtii can cope with this additional stress by utilizing different Cu-binding ligands. Indeed, in addition to expected Cu-binding ligands such as glutathione and phytochelatins, 25 proteins were detected that may also play a role in the Cu-detoxification processes in C. reinhardtii. Our results shed new light on the coping mechanisms of C. reinhardtii when exposed to environmental conditions that induce high rates of Cu accumulation.

Role of iron in gene expression and in the modulation of copper uptake in a freshwater alga: Insights on Cu and Fe assimilation pathways.

Metal uptake and toxicity can generally be related to its aqueous speciation and to the presence of competitive ions as described by the biotic ligand model. Beyond these simple chemical interactions at the surface of aquatic organisms, several internal biological feedback mechanisms can also modulate metal uptake. This is particularly important for essential elements for which specific transport systems were developed over the course of evolution. Based on the results of short-term Cu2+ uptake experiments and on the analysis of the expression of certain genes involved in Cu and Fe homeostasis, we studied the effects of Fe3+ on Cu2+ uptake by the freshwater green alga Chlamydomonas reinhardtii. We observed a significant increase in Cu2+ uptake rate in algal cells acclimated to a low Fe3+ medium up to 4.7 times greater compared to non-acclimated algal cells. The overexpression of the ferroxidase FOX1 and permease FTR1 genes suggests an activation of the high affinity Fe3+ assimilation system, which could constitute a plausible explanation for the increase in Cu2+ uptake rate in acclimatized algae. We show that Fe availability can have a significant impact on Cu uptake. Our observations reinforce the importance of considering physiological factors to better predict metal bioavailability.

Iron Modulation of Copper Uptake and Toxicity in a Green Alga ( Chlamydomonas reinhardtii).

Little attention has been paid to the role of essential trace elements on the toxicity of another element. In this work, we examined if low concentrations of essential elements (Co, Mn, Zn, and Fe) modified the response of a freshwater green alga ( Chlamydomonas reinhardtii) to copper. To do so, we followed cell growth over 72 h in exposure media where the essential element concentrations were manipulated. Among these elements, iron proved to have a strong impact on the cells' response to copper. The free Cu2+ concentrations required to inhibit cellular growth by 50% (EC50) over 72 h decreased from 2 nM in regular Fe medium (10-17.6 M Fe3+) to 4 pM in low iron medium (10-19.0 M Fe3+); a 500-fold increase in toxicity. Moreover, at low Cu2+ concentrations (10-13.0 to 10-10.5 M), Cu uptake increased under low iron conditions but remain relatively stable under regular iron conditions. These results show clearly that iron plays a protective role against copper uptake and toxicity to C. reinhardtii. In freshwaters, iron is always abundant but the expected free iron concentrations in surface waters can vary between 10-14.0 to 10-20.0 M, depending on pH (e.g., when pH increases from 6 to 8). We conclude that copper toxicity in natural waters can be modulated by iron and that, in some conditions, the Biotic Ligand Model may need to be further developed to account for the influence of iron.