Based on our proteomic data, inhibitors-responsive proteins could be divided into two main categories. One category of proteins is related to the stress response involved in the UPR, while the other is to adjust cells’ metabolism to overcome the deleterious effects of PFA. It means that the responses of yeast to PFA have two aspects (). Firstly, the respond of yeast cells to the stress of inhibitors is to up-regulate the proteins in cells’ rescues and defense mechanism through UPR. Secondly, the defense mechanism of yeast cells against PFA is to regulate the carbon and nitrogen metabolism and preserve the derived energy to increase its tolerance.
Schematic depicting the effects of PFA on the physiology of S. cerevisiae.
It is indicated by this study that PFA causes severe oxidative stress on yeast cells, which subsequently respond to such stress by up-regulating the UPR. Many researches indicated that furfural induced the oxidative stress in yeast 
. Acetic acid caused the accumulation of anions, thereby resulting in the oxidative stress 
. Phenol induced the accumulation of reactive oxygen species 
. It was also found in our previous study that the metabolites in response to oxidative stress (including inositol and phenylethylamine) were increased dramatically in the presence of PFA 
. The proteins related to cell rescue and defense pathways were up-regulated to protect yeast cells from the toxic effects of PFA in the tolerant yeast strain. The UPR is an intracellular signaling pathway that is activated by the accumulation of unfolded proteins in the endoplasmic reticulum (ER) 
. The UPR not only regulates the genes related to the secretory pathway, but also the cell fate, protein metabolism, amino acid and lipid metabolism 
. Some external factors, such as oxidative stress and treatment of chemicals, would lead to the accumulation of endoplasmic misfolded proteins. Several signaling pathways, collectively known as UPR, have evolved to detect the accumulation of misfolded proteins in the ER and activate cellular responses to maintain homeostasis and a normal flux of proteins in the ER 
. Our present results indicate that PFA induced oxidative stress in yeast, which causes the accumulation of misfolded proteins in the UPR, including the enhancment of protein folding, arrest of the translation of most proteins, and acceleration of the degradation of proteins 
Yeast also defends against PFA by regulating the nitrogen metabolism. It has been found that PFA affects the normal metabolism of amino acids and nucleotides. Lower levels of amino acids and nucleotides are important for yeast to conserve energy consumption, which would benefit for the tolerance of yeast to inhibitors. Regulation of amino acids and nucleotides metabolism related enzymes would increase the tolerance of yeast. This study also indicates that yeast cells need to produce more energy and decrease the metabolism rate to reduce the energy consumption to defense against the stress of PFA. However, it may also reduce the fermentation rate which would lead to low ethanol production efficiency in the absence of PFA as concluded in our previous study 
. It was found in the metabolomics study that the consumption rate of glucose and the production rate of ethanol were slightly lower in the tolerant yeast strain 
. But, in the presence of PFA, the fermentation rate of the tolerant strain was much higher than the parental yeast due to its strongly enhanced tolerance to the toxicity of PFA. Amino acids are important primary metabolites, which are involved in many cellular physiological functions. It has been reported that supplementation of arginine, serine, histidine and aromatic amino acids significantly enhance the tolerance of cells to furfural 
. Acetic acid is proved to cause the upregulation of many genes related to amino acids biosynthesis, including arginine, histidine, and tryptophan 
. Supplementation of arginine and lysine has been proved to significantly enhance the acid tolerance of Salmonella typhimurium
. Acetic acid causes high expression level of genes encoding arginine and lysine decarboxylases 
. Our previous study found that the genes in the amino acids metabolism (especially arginine, histidine, and tryptophan) were up-regulated by acetic acid 
. Supplementation of methionine could increase the tolerance of yeast cells to thermal and oxidative stress, shortening the lag phase of cells 
. These results were in accordance with our study that the PFA caused the up-regulation of proteins in the amino acids (methionine, asparagines, glutamate) metabolism. It suggests that these amino acids are of importance for the survival of yeast cells in the presence of inhibitors. In a metabolomic study, the levels of most amino acids increased significantly in the parental yeast, whereas decreased slightly in the tolerant yeast 
. It indicates that the changes of amino acids are not only due to the regulation proteins in the amino acids biosynthesis, but also the protein degradation discussed above.
It is found that S needed to produce more energy derived from glycolysis to defend against the stress and repair the damage caused by PFA. Proteomic researches on the responses of yeast to furfural revealed that furfural induced the stress response and many aspects of metabolism were interrupted, including glucose fermentation, tricarboxylic acid cycle, and glycerol metabolism 
. Metabolic flux analysis also showed that furfural affected glycolytic and TCA fluxes, which were involved in energy metabolism 
. Undissociated weak acids are liposoluble and can diffuse across the plasma membrane into the cytosol, which were proposed to be an important reason inhibiting the growth of microorganisms 
. With the increase of intracellular acetic acid, more membrane ATPase is needed to pump protons out of the cell at the expense of ATP hydrolysis 
. At high acetic acid concentrations, the proton pumping capacity of cells is exhausted, resulting in the depletion of ATP content, dissipation of the proton motive force, and acidification of cytoplasm 
. Thus, much more energy is needed to defend against the stress of PFA, preventing the depletion of cellular energy. The increment of energy for N was significantly less than S, due to the higher tolerance of N to PFA. In the present study, most detected proteins related to energy were involved in glycolysis and gluconeogenesis. Alcohol dehydrogenases (ADH) are NADH-dependent enzymes that play a key role in the reduction of furfural to less toxic furfuryl alcohol 
. In this study, Adh1p and Adh6p were both up-regulated by PFA in S and N, suggesting the two enzymes were crucial for the cellular detoxification of furfural. Glycerol 3-phosphate dehydrogenase (Gpd1p), the most important enzyme in the biosynthesis of glycerol, was up-regulated only in S, but not in N. It was reported that glycerol was formed to regenerate excess NADH to maintain the intracellular redox balance 
. Our results further indicate that furfural acts as an alternative redox sink that can oxidize the excessive NADH. The reduction of furfural is dependent upon ADH 
, which would improve the tolerance of yeast to PFA.
In all, two main strategies are found for yeast to defense against the stress of PFA. One strategy is for yeast to have higher capability in its detoxification and toleration of oxidative stresses. The other strategy is to decrease the metabolic rate of nitrogen to reduce the consumption of energy.