Effect of CUP1 copy number and pH on copper resistance of Saccharomyces cerevisiae enological strains.

The widespread use of copper-based pesticides in winemaking can affect wine fermentation. Therefore, it is crucial to assess the resistance levels of Saccharomyces cerevisiae wine strains in enological growth conditions. In the context of winemaking, grape juice is a complex environment capable of chelating copper and is characterized by a distinctly acidic pH. In this work, the effects of copper concentration on the growth of 10 S. cerevisiae strains, isolated from an enological environment, and one commercial starter were tested in YNB minimal medium and synthetic must, mimicking enological conditions. In minimal medium, resistance to copper varied among yeasts (50-600 µM), revealing the presence of three resistance levels (high, intermediate, and low). Representative strains of the three groups were tested at a pH range from 5.2 to 3.0 at the copper concentration that showed a 20-25 % growth reduction. At pH range 5.2-4.5, a growth reduction was observed, while, conversely, a strain-specific recovery was observed at pH range 3.2-3.0. In synthetic must, the strains showed higher copper resistance levels than in minimal medium (50-4000 µM). In both synthetic must and minimal medium, a significant logarithmic correlation was found between copper resistance and CUP1 gene copy number. The copy number tended to better explain resistance in minimal medium compared to synthetic must. The results shed light on the role of CUP1 copy number within an enological environment.

Zn deficiency disrupts Cu and S homeostasis in Chlamydomonas resulting in over accumulation of Cu and Cysteine.

Growth of Chlamydomonas reinhardtii in zinc (Zn) limited medium leads to disruption of copper (Cu) homeostasis, resulting in up to 40-fold Cu over-accumulation relative to its typical Cu quota. We show that Chlamydomonas controls its Cu quota by balancing Cu import and export, which is disrupted in a Zn deficient cell, thus establishing a mechanistic connection between Cu and Zn homeostasis. Transcriptomics, proteomics and elemental profiling revealed that Zn-limited Chlamydomonas cells up-regulate a subset of genes encoding "first responder" proteins involved in sulfur (S) assimilation and consequently accumulate more intracellular S, which is incorporated into L-cysteine, γ-glutamylcysteine, and homocysteine. Most prominently, in the absence of Zn, free L-cysteine is increased ∼80-fold, corresponding to ∼2.8 × 109 molecules/cell. Interestingly, classic S-containing metal binding ligands like glutathione and phytochelatins do not increase. X-ray fluorescence microscopy showed foci of S accumulation in Zn-limited cells that co-localize with Cu, phosphorus and calcium, consistent with Cu-thiol complexes in the acidocalcisome, the site of Cu(I) accumulation. Notably, cells that have been previously starved for Cu do not accumulate S or Cys, causally connecting cysteine synthesis with Cu accumulation. We suggest that cysteine is an in vivo Cu(I) ligand, perhaps ancestral, that buffers cytosolic Cu.

Cu accumulation, detoxification and tolerance in the red swamp crayfish Procambarus clarkii.

Copper is an essential metal but potentially toxic to aquatic animals at high levels. The present study investigated physiologically adaptive responses to waterborne Cu2+ exposure (0, 0.03, 0.30, 3.00 mg/L) in a representative species of crustaceans, the red swamp crayfish (Procambarus clarkii) for 7 d, followed by a 7-d depuration period. The tissue-specific distribution of Cu showed that crayfish hepatopancreas was the primary accumulating site among internal tissues. During Cu2+ exposure, crayfish repressed the expression level of Cu homeostasis genes (Ctr1, Atox1, copper-transporting ATPase 2, MTF-1/2, and MT) in hepatopancreas to inhibit intracellular Cu transporting. Cu2+-exposed crayfish increased activities of GPx and GST, GSH contents, and mRNA expression of antioxidative enzyme genes (Cu/Zn-sod, cat, gpx, gst) to cope with the Cu2+-induced oxidative stress which accompanied by an increased MDA content. Additionally, after a 7-d depuration, crayfish effectively eliminated excess Cu from hepatopancreas by up-regulating expression level of Cu homeostasis genes, and recovered from oxidative damage by enhancing antioxidative enzyme gene expression (Cu/Zn-sod, cat, gpx, gst) and consuming more GSH, which thereby caused a return of the MDA level to the control value. Overall, our study provided new insights into the regulatory mechanisms of cellular Cu homeostasis system and antioxidative system, contributing to Cu detoxification and tolerance ability exhibited by crayfish under Cu2+ stress and after withdrawal of Cu2+ stress.