# Proteomics Analysis of _Zygosaccharomyces mellis_ in Response to Sugar Stress
## 4 Discussion
Intracellular glycerol and trehalose, which can be used as osmotic protectants, will accumulate in yeast cells in response to external osmotic stress. It was reported that trehalose and glycerol can protect the cell membranes in response to sugar, NaCl and metal ions [7; 39; 40]. iTRAQ data revealed that the proteins involved in the degradation of glycerol and trehalose were downregulated in the assayed yeast. Among them, glycerol kinase, dihydroxyacetone kinase 1 and glycerol-3-phosphate/dihydroxyacetone phosphate acyltransferase (38) are involved in the degradation of glycerol [41; 42; 43], and phosphoglucomutase-2 is involved in the degradation of trehalose [40]. In yeasts, there are two well-established pathways to produce glycerol, including the Gcy-Dak pathway and the Gpd-Gpp pathway [7]. The proteins related to glycerol generation, including _TPS and otsB_, had been identified in response to high sugar stress in _Z. mellis_[6], but in this study, no proteins involved in these biosynthetic pathways were significantly differentially expressed. These results indicated that glycerol and trehalose also accumulated via decreased degradation, which is consistent with previous studies showing that glycerol accumulated under high osmotic pressure by retention [44]. As a result, this study revealed that osmotic protectants accumulated rapidly in the early stages of high-sugar fermentation to resist external hypertonic pressure in _Z. mellis_. Many studies have shown that antioxidant enzymes, such as SOD, CAT and POD, can play an important role against reactive oxygen species (ROS) to protect organisms from oxidative damage [45; 46; 47]. When yeast is under stress, the intracellular ROS content will rapidly increase, and the enzyme activities and the expression level of the encoding genes will be upregulated to regulate the intracellular ROS balance [32; 48]. SOD catalyzes two molecules of superoxide anion (O\({}_{2}-\)) to generate hydrogen peroxide (H\({}_{2}\)O\({}_{2}\)) and oxygen (O\({}_{2}\)), and CAT and POD then decompose H2O2 into H2O and O2 [49]. The improved antioxidant enzyme activities could enhance the high salt tolerance of _Z. rouxii_[32]. In addition, the expression level of SOD was upregulated, which was consistent with the result of transcriptomics analysis [6]. In _S. cerevisiae_, SOD could protect RNR from inactivation, and sod1\(\Delta\) and sod2\(\Delta\) mutants showed decreased growth under aerobic conditions [50]. In this study, the enzyme activities and protein expression of SOD, CAT and POD were increased during fermentation to eliminate the damaging effect of H\({}_{2}\)O\({}_{2}\) on cells and avoid oxidative damage in a high-sugar environment. Osmotic stress can increase the activities of MDH and SDH, which are the key enzymes of glucose metabolism and the TCA cycle and are involved in energy metabolism processes and respiration [51]. In fungi, the inhibition of enzyme activities involved in energy metabolism and respiration, especially SDH and MDH, resulted in the inhibition of mycelial growth [52]. Mitochondrial defects lead to the hyperaccumulation of ROS in response to salt stress [53]. In the proteome experiment, 45 proteins were identified that are related to energy metabolism, 29 of which were upregulated, while the other 16 were downregulated (Table S4). Among them, cytochrome c oxidase is the terminal enzyme of the mitochondrial membrane respiratory chain, and its function is directly related to ATP synthesis and associated with programmed cell death in yeast [54]. In addition, 7 of 10 proteins involved in the TCA cycle were upregulated (Table 2). Among these proteins, the expression levels of the SDH and MDH proteins were upregulated, which was consistent with the observed changes in enzyme activity. It has been shown that the expression of energy-related genes, especially those related to respiration and energy metabolism, increases with the duration of hyperosmalor stress in _S. cerevisiae_[55].