title: 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.
authors: Department of Biological Science, Eastern Illinois University, Charleston, IL 61920, USA. mrmarunde@eiu.edu
authors: Matthew R
authors: MR
authors: Marunde
authors: Dilini A
authors: DA
authors: Samarajeewa
authors: John
authors: J
authors: Anderson
authors: Shumin
authors: S
authors: Li
authors: Steven C
authors: SC
authors: Hand
authors: Michael A
authors: MA
authors: Menze
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001156:Artemia / Q000737:chemistry / Q000196:embryology / Q000235:genetics / Q000502:physiology*; D060829:Arthropod Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D015153:Blotting, Western; D019069:Cell Respiration; D002478:Cells, Cultured; D003890:Desiccation; D004331:Drosophila melanogaster / Q000737:chemistry / Q000196:embryology / Q000235:genetics / Q000502:physiology*; D004591:Electrophoresis, Polyacrylamide Gel; D004625:Embryo, Nonmammalian / Q000737:chemistry / Q000196:embryology / Q000502:physiology; D008928:Mitochondria / Q000378:metabolism; D017385:Sequence Homology; D014162:Transfection
chemical_list: D060829:Arthropod Proteins