Improved tolerance to salt and water stress in Drosophila melanogaster cells conferred by late embryogenesis abundant protein

Abstract

Mechanisms that govern anhydrobiosis involve the accumulation of highly hydrophilic macromolecules, such as late embryogenesis abundant (LEA) proteins. Group 1 LEA proteins comprised of 181 (AfLEA1.1) and 197 (AfLEA1.3) amino acids were cloned from embryos of Artemia franciscana and expressed in Drosophila melanogaster cells (Kc167). Confocal microscopy revealed a construct composed of green fluorescent protein (GFP) and AfLEA1.3 accumulates in the mitochondria (AfLEA1.3-GFP), while AfLEA1.1-GFP was found in the cytoplasm. In the presence of mixed substrates, oxygen consumption was statistically identical for permeabilized Kc167 control and Kc167-AfLEA1.3 cells. Acute titrations of permeabilized cells with NaCl up to 500 mM led to successive drops in oxygen flux, which were significantly ameliorated by 18% in Kc167-AfLEA1.3 cells compared to Kc167 controls. Mitochondria were isolated from both cell types and resuspended in a sucrose-based buffer solution. The purified mitochondria from Kc167 control cells showed significantly larger reductions in respiratory capacities after one freeze-thaw cycle (-80°C) compared to mitochondria isolated from Kc167-AfLEA1.3 cells. When cultured in the presence of a non-permeant osmolyte (50-200 mM sucrose) cells expressing AfLEA1.3 showed significantly improved viability (10-15%) during this hyperosmotic challenge as compared to Kc167 controls. Furthermore, Kc167-AfLEA1.3 cells survived desiccation by convective air drying in presence of 200 mM extracellular trehalose to lower final moisture contents than did control Kc167 cells (0.36 g H2O/g DW vs.1.02 g H2O/g DW). Thus, AfLEA1.3 exerts a protective influence on mitochondrial function and increases viability of Kc167 cells during water stress.