Date Published: April 25, 2019
Publisher: Public Library of Science
Author(s): Catherine Tesnière, Chloé Bessière, Martine Pradal, Isabelle Sanchez, Bruno Blondin, Frédéric Bigey, Alvaro Galli.
Nitrogen composition of the grape must has an impact on yeast growth and fermentation kinetics as well as on the organoleptic properties of the final product. In some technological processes, such as white wine/rosé winemaking, the yeast-assimilable nitrogen content is sometimes insufficient to cover yeast requirements, which can lead to slow or sluggish fermentations. Growth is nevertheless quickly restored upon relief from nutrient starvation, e.g. through the addition of ammonium nitrogen, allowing fermentation completion. The aim of this study was to determine how nitrogen repletion affected the transcriptional response of a Saccharomyces cerevisiae wine yeast strain, in particular within the first hour after nitrogen addition. We found almost 4800 genes induced or repressed, sometimes within minutes after nutrient changes. Some of these responses to nitrogen depended on the TOR pathway, which controls positively ribosomal protein genes, amino acid and purine biosynthesis or amino acid permease genes and negatively stress-response genes, and genes related to the retrograde response (RTG) specific to the tricarboxylic acid (TCA) cycle and nitrogen catabolite repression (NCR). Some unexpected transcriptional responses concerned all the glycolytic genes, carbohydrate metabolism and TCA cycle-related genes that were down-regulated, as well as genes from the lipid metabolism.
The yeast cell Saccharomyces cerevisiae is able to control its growth in response to changes in nutrient availability. Nitrogen limitation is one of the most frequent limitations observed during wine fermentation . The actual nitrogen content in must is dependent on many factors including rootstock, grape variety, climate, vine growing conditions, and grape processing. In enological conditions, musts are considered as nitrogen-limited when the yeast assimilable nitrogen (YAN) content is below 150 mg/L . YAN is a major factor influencing fermentation kinetics, the maximal fermentative rate being related to the nitrogen level in the must . In most cases of sluggish fermentations, nitrogen depletion quickly results in cells entering stationary phase. This phenomenon is not related to a decrease in viability, but could rather be related to a catabolic inactivation of the hexose transporters  or to lower protein synthesis and cell protein content . Other physiological changes such as autophagy, nitrogen recycling systems and the reorientation of the carbon flux to promote glycogen and trehalose storage have also been observed at the onset of nitrogen starvation . In addition, the transcriptional remodeling associated with the onset of starvation during wine alcoholic fermentations has been described , including the development of a general stress response. These transcriptional changes are mostly controlled by the TOR pathway, sensing cell nitrogen status and adapting nitrogen metabolism to nutrient availability [5, 6]. Nitrogen limitation stably arrests the cell cycle in G1/G0, whereas medium replenishment with the limiting nutrient quickly restores growth. Relief from nitrogen starvation is a way to increase the fermentation rate, while reducing its duration . In fact assimilable nitrogen addition to nitrogen-deficient must results in a reactivating protein synthesis and increasing sugar transport speed [7, 8]. Although this nitrogen addition is currently practiced using diammonium phosphate to reduce the risk of stuck fermentation in white and rosé wines, the molecular mechanisms triggered by nitrogen replenishment are still poorly understood.
The experimental design was mapped out on S1 Fig.
The addition of nitrogen to starved wine yeast cells thus contributed to the development of a favorable environment for wine yeast growth and also to limit the general stress response. During a very short time after the addition of nitrogen to the medium, we found thousands of genes induced or repressed, sometimes within minutes after nutrient changes. Some of these responses to nitrogen depended on the TOR pathway, which controls positively ribosomal protein genes, amino acid and purine biosynthesis or amino acid permease genes and negatively stress-response genes, and genes related to the retrograde response (RTG) specific to the tricarboxylic acid (TCA) cycle and nitrogen catabolite repression (NCR). Most of these responses are the opposite of the changes observed in yeasts deprived of nitrogen, when the cells reach the stage of the stationary phase . But we also detected unexpected transcriptional responses. These included all glycolytic genes, carbohydrate metabolism and TCA cycle genes that were downregulated, as well as genes derived from lipid metabolism.