Comparative transcriptomic analysis reveals similarities and dissimilarities in Saccharomyces cerevisiae wine strains response to nitrogen availability

Abstract

Nitrogen levels in grape-juices are of major importance in winemaking ensuring adequate yeast growth and fermentation performance. Here we used a comparative transcriptome analysis to uncover wine yeasts responses to nitrogen availability during fermentation. Gene expression was assessed in three genetically and phenotypically divergent commercial wine strains (CEG, VL1 and QA23), under low (67 mg/L) and high nitrogen (670 mg/L) regimes, at three time points during fermentation (12 h, 24 h and 96 h). Two-way ANOVA analysis of each fermentation condition led to the identification of genes whose expression was dependent on strain, fermentation stage and on the interaction of both factors. The high fermenter yeast strain QA23 was more clearly distinct from the other two strains, by differential expression of genes involved in flocculation, mitochondrial functions, energy generation and protein folding and stabilization. For all strains, higher transcriptional variability due to fermentation stage was seen in the high nitrogen fermentations. A positive correlation between maximum fermentation rate and the expression of genes involved in stress response was observed. The finding of common genes correlated with both fermentation activity and nitrogen up-take underlies the role of nitrogen on yeast fermentative fitness. The comparative analysis of genes differentially expressed between both fermentation conditions at 12 h, where the main difference was the level of nitrogen available, showed the highest variability amongst strains revealing strain-specific responses. Nevertheless, we were able to identify a small set of genes whose expression profiles can quantitatively assess the common response of the yeast strains to varying nitrogen conditions. The use of three contrasting yeast strains in gene expression analysis prompts the identification of more reliable, accurate and reproducible biomarkers that will facilitate the diagnosis of deficiency of this nutrient in the grape-musts and the development of strategies to optimize yeast performance in industrial fermentations.

Fig 1. Genomic variability of VL1, CEG and QA23 yeast strains.

Dendrograms obtained by composite hierarchical analysis of PCR interdelta and M13 patterns using Pearson's correlation coefficient and the UPGMA clustering method for 20 S. cerevisiae commercial selected strains. The strains grouped according to their genomic similarity. The three strains used in this study were selected on the basis of their phenotypic differences [8] and their different inter-delta and M13 PCR profiles, as shown.

Fig 2. Genotype, environment, and genotype–environment interaction effects in wine yeast strains.

Differentially expressed genes at each nitrogen regime studied (LN and HN), identified using two-way ANOVA. The number of genes significantly affected by genotype (strain), by environment (fermentation stage) and by the interaction between both factors is represented with bars. The selection of genes showing differential expression (with a significant effect of the different factors) was defined at FDR<0.05 using the Benjamini and Hochberg correction.

Fig 3. Hierarchical clustering of genes identified as exhibiting significant genotype (G), environmental (E), and gene-by-environment (GEI) effects in each of the fermentations (LN and HN).

The diagrams show the log2 expression differences in the indicated strains compared to the mean expression of that gene in all strains in each fermentation. Each row represents a given gene and each column represents a different strain within which expression is ordered by fermentation stage (12, 24 and 96h). Red and green correspond to higher and lower expression, respectively. Clusters are annotated at the right with characteristic GO category functional enrichments (S2 Table).

Fig 4. Venn diagrams for genes whose expression was significantly altered in the three wine yeast strains in LN and HN fermentations.

Red indicated genes whose expression was altered in CEG, green in QA23 and violet in VL1.