Proteomic Analysis of Rana sylvatica Reveals Differentially Expressed Proteins in Liver in Response to Anoxia, Dehydration or Freezing Stress

Ectothermic animals that live in seasonally cold regions are adapted to seasonal variation and specific environmental conditions. During the winter, some amphibians hibernate terrestrially and encounter a limited amount of environmental water, deficient oxygen, and extremely low temperatures that can cause whole body freezing. These stresses trigger physiological and biochemical adaptations in amphibians that allow them to survive. Rana sylvatica, commonly known as the wood frog, shows excellent freeze tolerance. They can slow their metabolic activity to a near halt and endure freezing of 65-70% of their total body water as extracellular ice during hibernation, returning to normal when the temperatures rise again. To investigate the molecular adaptations of freeze-tolerant wood frogs, a comprehensive proteomic analysis was performed on frog liver tissue after anoxia, dehydration, or freezing exposures using a label-free LC-MS/MS proteomic approach. Quantitative proteomic analysis revealed that 87, 118, and 86 proteins were significantly upregulated in dehydrated, anoxic, and frozen groups, respectively, suggesting potential protective functions. The presence of three upregulated enzymes, glutathione S-transferase (GST), aldolase (ALDOA), and sorbitol dehydrogenase (SORD), were validated. For all enzymes, the specific enzymatic activity was significantly higher in liver of frozen and anoxic groups compared to controls. This study reveals that GST, ALDOA, and SORD might participate in the freeze tolerance mechanism by contributing to regulating cellular detoxification and energy metabolism.