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 500mM 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 (50-200mM 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 200mM extracellular trehalose to lower final moisture contents than did control Kc167 cells (0.36g H2O/g DW vs.1.02g H2O/g DW). Thus, AfLEA1.3 exerts a protective influence on mitochondrial function and increases viability of Kc167 cells during water stress. © 2013 Elsevier Ltd.
Publication Source (Journal or Book title)
Journal of Insect Physiology
Marunde, M., Samarajeewa, D., Anderson, J., Li, S., Hand, S., & Menze, M. (2013). Improved tolerance to salt and water stress in Drosophila melanogaster cells conferred by late embryogenesis abundant protein. Journal of Insect Physiology, 59 (4), 377-386. https://doi.org/10.1016/j.jinsphys.2013.01.004