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To better understand the physiological and molecular response of S. cerevisiae to hyperosmotic stress, chitin and glucan contents in the cell wall and sensitivity of the cell wall to calcofluor white (CFW) stain and cell walldegrading enzymes were assayed. Further analysis of the cell membrane integrity of S. cerevisiae was performed by staining with dual dyes (PI and Hoechst 33342). Finally, gene expression profiles of S. cerevisiae under extreme high glucose pressure were conducted by RNA sequencing (RNAseq). The alterations in membrane integrity and wall integrity in S. cerevisiae were found after high sugar stress. Hyperosmotic stresstreated cells increased sensitivity to CFW stain and cell walldegrading enzymes demonstrated that hyperosmotic stress disrupted cell wall properties. It was also found that hyperosmotic stresstreated cells increased sensitivity to PI stain, indicating that the membrane was damaged. Global transcript measurements confirmed the membrane damage observations by showing downregulation of membrane components and changes in fatty acid biosynthesis. High sugar stress did cause a compensatory response to cell wall damage through downregulation of several genes (ROM1, RLM1, PIR3, YGP1 and CWP1) involved with the cell wall integrity (CWI) signaling pathway. In addition, the previous studies were combined to discuss the CWI signaling pathway of yeast response to different stresses. The research results provided a reference for understanding hyperosmotic stress mechanism.