Differential gene expression under environmental stress in the freeze tolerant wood frog, Rana sylvatica
Abstract:
Freeze survival of wood frog, Rana sylvatica, involves adaptations including control over extracellular ice formation, production of glucose cryoprotectant, and resistance to freezing-caused intracellular dehydration and ischemia. Gene expression associated with stress survival was investigated in this freeze tolerant species. Freeze-inducible genes were found by differential screening of frog brain and liver cDNA libraries; these included mitochondrial genes [encoding ATPase subunit 6 & 8, 16S rRNA and NADH-ubiquinone oxidoreductase subunit 4 (ND4)], the phosphoglycerate kinase 1 (PGK1) gene, and genes whose products are involved in translational processes [acidic ribosomal phosphoprotein (P0), elongation factor 1 gamma subunit (EF-1 gamma)]. Another ribosome-associated gene, encoding ribosomal protein L7 (RPL7), was identified in skin via differential display of polymerase chain reaction (DD-PCR). This gene was up-regulated in skin of cold-acclimated frogs and brain of freeze-exposed frogs. Freezing stimulated the upregulation of the above genes in selected frog organs. Tissue-specific gene expression also occurred in frog brain and liver in response to anoxia or dehydration. Anoxia stimulated P0, PGK1, RPL7, 16S rRNA and ATPase 6 & 8 gene expression and modulated ND4and EF-l gamma expression, whereas dehydration enhanced the expression of genes such as PGK1.Upregulation of genes whose products are directly involved in energy generation (PGK1, ATPase subunit 6 & 8, ND4) and whose products are related to protein biosynthesis suggested that maintenance of minimal ATP levels and functional translation machinery may be critical for freezing survival. Freezing-induced ischemia may be a primary signal that triggers the upregulation of most of the isolated genes. However, low temperature seemed to play a role in the expression of ribosome associated protein genes, whereas freezing-related cell water stress may also regulate other selected genes (e.g. PGK1).Immunoblotting confirmed that elevated PGK1 transcripts resulted in increased enzyme protein and showed the potential physiological significance of up-regulated genes in response to stress. Immunoblotting also showed the elevation of Ca2+/ca1modulin-dependent protein kinase and phosphatase under freezing, anoxia and/or dehydration stresses which suggests that a Ca2~signaling pathway plays a role in stress-mediated gene expression. These cellular responses may play an important role in survival of environmental stress.