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.

FIG. 1.

Anaerobic fermentation profiles under different nitrogen conditions. Cell density, viability, and fermentation rate of S. cerevisiae PYCC4072 grown in synthetic grape juice medium at 20°C under different nitrogen regimes were measured: cell density evaluated by turbidimetry (OD₆₄₀) (A); cell viability determined by counting CFU (B); concentration of glucose and CO_2, (C and D, respectively). Three main fermentation conditions with 200 g liter⁻¹ of glucose were established by manipulating the initial nitrogen concentration in the culture medium: •, 267 mg of N liter⁻¹, the nitrogen concentration required by the yeast strain to complete alcoholic fermentation (control fermentation); ▪, 67 mg of N liter⁻¹, the nitrogen concentration of the N-limiting fermentation; and □, the addition of 200 mg of N liter⁻¹ into the N-limiting fermentation, 72 h after inoculation (indicated by arrow), coinciding with 48 h of nitrogen depletion (refed fermentation). Values are presented as mean ± standard deviation from three independent experiments. Statistical significance (*, P < 0.05, between the control and N-starved fermentations) was determined by two-way ANOVA.

FIG. 2.

Nitrogen depletion induces ROS accumulation associated with a lower mitochondrial membrane potential. FACS measurements of intracellular ROS using MitoTracker Red CM-H₂XRos (MitoTracker) (A) and of superoxide anions using the superoxide-specific probe DHE (B) in S. cerevisiae cells under different fermentation conditions. Bar graphs indicate the percentage of cells exhibiting high levels of intracellular ROS detected by FACS measurements (20,000 cells per sample in three independent experiments) of MitoTracker- and DHE-positive cells. Values are mean ± standard deviations of all experiments. Statistical significance (*, P < 0.05) was determined by a Student's t test. (C) Overlay of FACS green fluorescence histograms (FL1 log) representative of S. cerevisiae cells obtained at time 96 h from the different fermentations and stained with rhodamine 123 (Rh123) as an indicator of mitochondrial membrane potential.

FIG. 3.

Sod2p and Cta1p activities are increased under nitrogen-limiting fermentations. Quantification of fold increases in the activity of peroxisomal catalase (Cta1p) (A), Cu/Zn-dependent superoxide dismutase (Sod1p) (B), and mitochondrial Mn-dependent superoxide dismutase (Sod2p) (C) of S. cerevisiae cells over time under the different indicated fermentation conditions. Cta1p, Sod1p, and Sod2p activity at each time point was normalized to the activity of cells from the control fermentation at time 24 h. Values are presented as mean ± standard deviation from three independent experiments. Statistical significance (*, P < 0.05) was determined by a Student's t test.

FIG. 4.

Nitrogen limitation induces cell membrane damage. Percentage of S. cerevisiae cells displaying affected plasma membrane integrity evaluated under the three different nitrogen regimes, as assessed by FACS quantification of cells after vital staining with propidium iodide.

FIG. 5.

Nitrogen limitation induces cell cycle arrest in G₀/G₁ phases. FACS analysis of the percentage of cells in each phase of the cell cycle using SYBR green staining of S. cerevisiae to measure DNA content. (A) Control fermentation. (B) Nitrogen-limiting fermentation. (C) Nitrogen refeeding after 72 h. Bar graphs correspond to a representative experiment performed out of three independent experiments.

FIG. 6.

Nitrogen limitation induces increased expression of ATG8, a marker of autophagy. Normalized fold expression levels of the ATG8 gene (actin, ACT1, was used as internal reference), evaluated by RT-PCR, in S. cerevisiae cells under the three different nitrogen regimes. Values indicate mean ± standard deviation from three independent experiments. Statistical significance (*, P < 0.05) was determined by a Student's t test.