Exogenous Trehalose Assists Zygosaccharomyces rouxii in Resisting High-Temperature Stress Mainly by Activating Genes Rather than Entering Metabolism

Abstract

Zygosaccharomyces rouxii is a typical aroma-producing yeast in food brewing, but it has low heat resistance and poor proliferation ability at high temperature. Trehalose is generally considered to be a protective agent that helps stable yeast cells resist heat shock stress, but its functional mechanism for yeast cells in the adaptation period under heat stress is unclear. In this study, the physiological metabolism changes, specific gene transcription expression characteristics, and transcriptome differences of Z. rouxii under different carbon sources under high-temperature stress (40 °C) were compared to explore the mechanism of trehalose inducing Z. rouxii to recover and proliferate under high-temperature stress during the adaptation period. The results showed that high concentration of trehalose (20% Tre) could not be used as the main carbon source for the proliferation of Z. rouxii under long-term high-temperature stress, but it helped to maintain the stability of the cell population. The intracellular trehalose of Z. rouxii was mainly derived from the synthesis and metabolism of intracellular glucose, and the extracellular acetic acid concentration showed an upward trend with the improvement of yeast growth. A high concentration of trehalose (20% Tre) can promote the expression of high glucose receptor gene GRT2 (12.0-fold) and induce the up-regulation of HSF1 (27.1-fold), MSN4 (58.9-fold), HXK1 (8.3-fold), and other signal transduction protein genes, and the increase of trehalose concentration will maintain the temporal up-regulation of these genes. Transcriptome analysis showed that trehalose concentration and the presence of glucose had a significant effect on the gene expression of Z. rouxii under high-temperature stress. In summary, trehalose assists Z. rouxii in adapting to high temperature by changing gene expression levels, and assists Z. rouxii in absorbing glucose to achieve cell proliferation.

Keywords: Zygosaccharomyces rouxii; high-temperature adversity; proliferation; transcriptome; trehalose.

Figure 1

Effects of carbon source concentration and ratio on high-temperature (40 °C) growth of Z. rouxii: (A) the growth of Z. rouxii with 2% Glc and different Tre concentrations; (B) the growth of Z. rouxii under different concentrations of Glc and 20% Tre; (C) the growth of Z. rouxii under different osmotic conditions; (D) analysis of growth period of Z. rouxii. Abbreviations: Tre, trehalose; Srt, sorbitol; Xyl, xylitol; Gly, glycerol. Note: significance test: * p < 0.05; ** p < 0.01.

Figure 2

Changes of intracellular and extracellular trehalose and glucose concentrations in Z. rouxii grown at 40 °C. Note: The hollow point is the extracellular data point, and the solid point is the intracellular data point.

Figure 3

Detection of intracellular and extracellular stress-resistant carbon metabolites in Z. rouxii at 40 °C. Note: The hollow point is the extracellular data point, and the solid point is the intracellular data point.

Figure 4

(A) Changes of intracellular and extracellular acetic acid in Z. rouxii; (B) changes of intracellular malic acid in Z. rouxii. Note: The hollow point is the extracellular data point, and the solid point is the intracellular data point.

Figure 5

Relative transcriptional expression changes of carbohydrate metabolite synthesis gene mRNA. Note: The solid line arrow indicates one-step reaction, and the dotted line arrow indicates multi-step reaction. (A) 2% Tre; (B) 20% Tre; (C) 20% Tre + 2% Glc. Significance test: * p < 0.05; ** p < 0.01; *** p < 0.001.

Figure 6

Relative transcriptional expression changes of transport regulatory protein gene mRNA. Note: (A) 2% Tre; (B) 20% Tre; (C) 20% Tre + 2% Glc. Significance test: ** p < 0.01; *** p < 0.001.

Figure 7

Relative transcriptional expression changes of stress-resistant global regulatory protein gene mRNA. Note: (A) 2% Tre; (B) 20% Tre; (C) 20% Tre + 2% Glc. Significance test: * p < 0.05; ** p < 0.01; *** p < 0.001.

Figure 8

Volcanic map of expression difference of different samples. Note: (A) 2% Tre, 24 h; (B) 2% Tre, 48 h; (C) 20% Tre, 24 h; (D) 20% Tre, 48 h; (E) 20% Tre + 2% Glc, 24 h; (F) 20% Tre + 2% Glc, 48 h. The abscissa is the multiple of gene/transcript expression difference between the two samples, that is, the expression of the treated sample is divided by the expression of the control sample, and the ordinate is the statistical test value of the difference in gene expression change, that is, the p value. The larger the −log10 (Pvalue), the more significant the difference in expression, and the values of the horizontal and vertical coordinates are logarithmically processed. Each point in the figure represents a specific gene. The red point represents a significantly up-regulated gene, the blue point represents a significantly down-regulated gene, and the gray point is a non-significant difference gene.

Figure 9

The model of trehalose assisting Z. rouxii to resist heat shock stress. Note: The black solid line arrow represents a one-step reaction; the black dotted arrow represents a multi-step reaction; the black dotted line arrow represents the anti-stress function of the substance; the blue solid arrow represents the NADPH production pathway; the blue dotted arrow represents the transport of acetic acid; the green arrow represents the absorption of Tre; the red arrow represents the absorption of Glc.