Degree

Doctor of Philosophy (PhD)

Department

Biological Sciences

Document Type

Dissertation

Abstract

This dissertation focuses on two major topics germane to mechanisms by which animals tolerate extreme water stress. First, the impact of transgenic expression of late embryogenesis abundant proteins (LEA) on water stress tolerance of the fruit fly Drosophila melanogaster was investigated. The overall aim was to extend current understanding of the protective properties of LEA proteins documented with isolated cells to a desiccation-sensitive organism during exposure to drying and hyperosmotic stress. Four lines of D. melanogaster were created that expressed transgenes encoding selected LEA proteins originally identified in embryos of the extremophile Artemia franciscana. After 80% tissue water loss, embryos that expressed AfrLEA2 or AfrLEA3m eclosed two days earlier than embryos without AfrLEA proteins. When results for all Afrlea lines were combined for third instar larvae, Kaplan-Meier survival curves indicated the percent water lost at the LT50 (lethal time for 50% mortality) was significantly greater for the AfrLEA lines (78%) versus Gal4GFP controls (52%). When adults were challenged with hyperosmotic stress and progeny forced to develop under these conditions, offspring of fly lines that expressed AfrLEA2, AfrLEA3m, or AfrLEA6 exhibited significantly greater success in reaching pupation, compared to wild type flies.

The goal of the second topic was to gather information to ultimately evaluate whether lipid compositions of the outer and inner mitochondrial membranes (OMM and IMM, respectively) from embryos of A. franciscana might directly enhance membrane stability in the dried state, or perhaps improve it indirectly by increasing the binding of LEA proteins. After mitochondrial purification, the IMM and OMM were separated by hypoosmotic swelling of mitochondria and purified by sucrose gradient centrifugation. Results showed low cross-contamination between the IMM and OMM. The most abundant lipids in OMM and IMM were phosphatidylcholine (PC) and phosphatidylinositol (PI), respectively. As compared to OMM isolated from rat liver, the OMM from A. franciscana embryos contained more PI and phosphatidylglycerol (PG) and less PC. Approximately 75% of the acyl chains of phospholipids in both membranes was unsaturated. Importantly, triacylglycerol was preferentially associated with the OMM, which may stabilize this bilayer during drying. These studies highlight the complexity of desiccation tolerance in whole animals.

Committee Chair

Hand, Steven C.

Available for download on Sunday, July 07, 2024

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