Accumulation of Non-Superoxide Anion Reactive Oxygen Species Mediates Nitrogen-Limited Alcoholic Fermentation by Saccharomyces cerevisiae

Abstract

Throughout alcoholic fermentation, nitrogen depletion is one of the most important environmental stresses that can negatively affect the yeast metabolic activity and ultimately leads to fermentation arrest. Thus, the identification of the underlying effects and biomarkers of nitrogen limitation is valuable for controlling, and therefore optimizing, alcoholic fermentation. In this study, reactive oxygen species (ROS), plasma membrane integrity, and cell cycle were evaluated in a wine strain of Saccharomyces cerevisiae during alcoholic fermentation in nitrogen-limiting medium under anaerobic conditions. The results indicated that nitrogen limitation leads to an increase in ROS and that the superoxide anion is a minor component of the ROS, but there is increased activity of both Sod2p and Cta1p. Associated with these effects was a decrease in plasma membrane integrity and a persistent cell cycle arrest at G 0 /G 1 phases. Moreover, under these conditions it appears that autophagy, evaluated by ATG8 expression, is induced, suggesting that this mechanism is essential for cell survival but does not prevent the cell cycle arrest observed in slow fermentation. Conversely, nitrogen refeeding allowed cells to reenter cell cycle by decreasing ROS generation and autophagy. Altogether, the results provide new insights on the understanding of wine fermentations under nitrogen-limiting conditions and further indicate that ROS accumulation, evaluated by the MitoTracker Red dye CM-H 2 XRos, and plasma membrane integrity could be useful as predictive markers of fermentation problems.

Guidelines for the use and interpretation of assays for monitoring autophagy

“…For example, autophagy induction is observed during the primary fermentation of synthetic grape must, 867 and during sparkling wine production (secondary fermentation). 868 A number of genome-wide studies have identified vacuolar functions and autophagy as relevant processes during primary wine fermentation or for ethanol tolerance, based on gene expression data or cell viability of knockout yeast strains; 867,[869][870][871][872][873] however, understanding the actual relevance of autophagy in yeast-driven food fermentation processes would require addressing this issue experimentally using some of the methods available for S. cerevisiae as described in these guidelines.…”

Polyhydroxyfullerene Binds Cadmium Ions and Alleviates Metal-Induced Oxidative Stress in Saccharomyces cerevisiae

“…This dye does not fluoresce in the reduced form, but when it enters an actively respiring cell, it is oxidized by ROS in the mitochondria to form a red fluorescent compound (32,37). Yeast cells, obtained as described above, were resuspended in 2 ml PBS containing 40 g ml Ϫ1 of the dye prepared in dimethyl sulfoxide (Ն99.9%; Sigma) and incubated for 15 min at 26°C in the dark.…”

“…The red fluorescence of PI or MitoTracker Red was detected on an FL3 log filter through a 590-nm long pass, a 620-nm band pass, and another 670-nm long pass. An acquisition protocol was defined to measure forward scatter (FS log), side scatter (SS log), and red fluorescence (FL3 log) on a 4-decade logarithmic scale (32,37). Each replicate was sampled three times, and 25,000 cells per sample were scanned.…”

“…Plasma membrane integrity was assessed by a membrane-impermeative dye, propidium iodide (PI; Molecular Probes, Eugene, OR), which enters the cells and binds to nucleic acids when plasma membrane disruption occurs; red fluorescence in cells indicates plasma membrane disruption (32). Nuclei were localized with 4=,6-diamidino-2-phenylindole dye (DAPI; Molecular Probes, Eugene, OR, USA), which forms a blue fluorescent complex with double-stranded DNA.…”

“…We selected the yeast Saccharomyces cerevisiae because (i) it has been used as a eukaryotic model system to study oxidative stress responses (30)(31)(32) and (ii) mounting evidence suggests that Cd can induce oxidative stress by accumulating ROS or free radicals (22,33,34). Because the uptake and toxicity of Cd in yeasts can change with environmental conditions (e.g., pH [35] and Cd concentration [36]), we assessed the effects of Cd and PHF alone and in mixtures on yeast growth, intracellular ROS accumulation, and plasma membrane integrity at different pHs and exposure concentrations.…”