Water availability has pronounced influences on animal activity and distribution patterns. Drying due to evaporative water loss is the most common mechanism for dehydration, and it is a universal problem confronting terrestrial animals. The majority of animal species do not tolerate severe water stress. This research will address precise mechanisms that protect animals during water stress at levels from the molecular to the organismal. Understanding these protective mechanisms may have translational application for broad scientific communities. For example, the ability for scientists working with important genetic models to prolong the storage of young animal stages by imposing desiccation would be of practical benefit for maintaining these animals more economically. The advantage would extend to national stock centers and teaching laboratories. Engineering desiccation tolerance in dehydration-sensitive organisms is complex, but could provide possibilities for dry storage of valuable live organisms that may be difficult and expensive to maintain in laboratory culture. Further, the biomedical implications of some findings could be significant. Storage of dried cells would increase the availability of replacement cells for many clinical applications and in regenerative medicine. Educationally, the project will facilitate the training of graduate students and undergraduates from a wide diversity of backgrounds. Outreach activities planned as part of the project will contribute to the general education of international scientists (including researchers in biotechnology from the private sector, National Laboratory scientists, and academic faculty wishing to acquire new skills).
This collaborative research will evaluate the impact on desiccation tolerance of multiple Late Embryogenesis Abundant (LEA) proteins originating from anhydrobiotic embryos of the brine shrimp Artemia franciscana. The project will refine and expand the understanding of how LEA proteins promote stabilization of targeted biological structures at various hydration states along a continuum from full hydration to water contents of 2% or less. Hypotheses will be tested with isolated macromolecules, liposomes, insect cells lines, and larvae of Drosophila melanogaster. One novel hypothesis is that the function displayed by individual LEA proteins is multifaceted depending upon the severity of desiccation. Such functional plasticity for individual LEA proteins would represent a new paradigm for protection of biological structures as hydration state varies. A new LEA protein from A. franciscana (AfrLEA6), with high sequence homology to a plant LEA protein recently linked to long-term protection against desiccation damage, has been cloned, sequenced and expressed. AfrLEA6 will be stably transfected into insect cell lines to explore whether this protein will extend long-term desiccation tolerance, in the presence and absence of other co-transfected LEA proteins and stabilizing sugars. Finally, fly lines of Drosophila melanogaster will be created that transgenically express multiple LEA proteins. Because larval stages of D. melanogaster naturally contain high endogenous levels of trehalose (a sugar known to provide protection synergistically with LEA proteins), larvae will be used to test for improved tolerance to drying in this species that is generally desiccation-sensitive and does not contain LEA genes in its genome.
