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 media 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 G0/G1 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 re-feeding allowed cells to re-enter 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 MitoTracker Red CM-H2XRos, and plasma membrane integrity could be useful as predictive markers of fermentation problems.
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