Hand 2022 Abstract Bioblast
|2.3. «10+5» https://doi.org/10.26124/bec:2022-0001|
Event: Bioblast 2022
Metabolic depression in animals is positively correlated with survival under environmental stress. Selected invertebrates and certain fish enter diapause — a developmentally programed dormancy characterized by suppression of development and metabolism. In crustacean embryos the metabolic arrest of OXPHOS is profound, and accordingly these embryos survive years of anoxia and severe desiccation . Mechanisms by which mitochondria and other cellular components tolerate such insults are multifaceted but include targeting of Late Embryogenesis Abundant (LEA) proteins to cellular compartments. LEA proteins are family of intrinsically disordered proteins (IDPs) reported to improve cellular tolerance to water stress. Open questions are whether gain of secondary structure by LEA proteins during drying is a prerequisite for this stabilizing function and whether their protective abilities as documented with isolated cells can be extended to a desiccation-sensitive, whole organism during water stress.
Proteins were characterized by catalytic activity, Western blotting and circular dichroism spectroscopy (CD). Recombinant AfrLEA2, a Group 3 LEA protein from Artemia franciscana, was expressed in bacterial cells and purified by HisTrap affinity chromatography and by anion exchange. Phosphofructokinase (PFK) was extracted from rabbit muscle and purified by ultracentrifugation, isopropanol precipitation, DEAE chromatography and heat treatment. We used incremental drying (equilibration to a series of relative humidities, RH) to test the ability of AfrLEA2 to protect desiccation-sensitive PFK. Fly lines of Drosophila melanogaster that expressed the PhiC31 integrase were injected with expression vectors containing the desired LEA transgenes. The non-parametric Kaplan–Meier method was used for analysis of survivorship curves.
After quantifying the ability of AfrLEA2 to protect PFK activity during incremental drying, parallel experiments used CD to measure gain of secondary structure in AfrLEA2. Protection of PFK by AfrLEA2 coincided with incremental gain of α-helix in AfrLEA2 as RH decreased . To evaluate the impact of LEA proteins in whole organisms during water stress, embryos of D. melanogaster were dried to 80 % tissue water and then rehydrated . Embryos from fly lines that expressed AfrLEA2 or AfrLEA3m eclosed 2 days earlier than wild-type embryos or embryos expressing green fluorescent protein (Gal4GFP control). With third instar larval stages, Kaplan–Meier survival curves indicated a significant improvement in survivorship in fly lines expressing AfrLEA proteins compared with Gal4GFP controls. The percent water lost at the LT50 (lethal time for 50 % mortality) for the AfrLEA lines was 78 % versus 52 % for Gal4GFP controls. Finally, offspring of fly lines that expressed AfrLEA2, AfrLEA3m or AfrLEA6 exhibited significantly greater success in reaching pupation, compared with wild-type flies, when adults were challenged with hyperosmotic stress (NaCl-fortified medium) and progeny forced to develop under these conditions .
In conclusion, metabolic shutdown is a prerequisite for successful tolerance of severe stress in many species. Our experimental evidence links the acquisition of α-helix in a LEA protein with stabilization of a target protein (PFK) across a graded series of hydration states. Additionally, our gain of function studies with D. melanogaster show that LEA proteins improve tolerance to water stress in a desiccation sensitive-species that normally lacks these proteins.
- Hand SC, Moore DS, Patil Y (2018) Challenges during diapause and anhydrobiosis: mitochondrial bioenergetics and desiccation. tolerance. https://doi.org/10.1002/iub.1953
- LeBlanc BM, Hand SC (2021) Target enzymes are stabilized by AfrLEA6 and a gain of α-helix coincides with protection by a group 3 LEA protein during incremental drying. https://doi.org/10.1016/j.bbapap.2021.140642
- Anderson JM, Hand SC (2021) Transgenic expression of late embryogenesis abundant proteins improves tolerance to water stress in Drosophila melanogaster. https://doi.org/10.1242/jeb.238204
• Keywords: Metabolic arrest, Diapause, Water stress, Late embryogenesis abundant proteins, Intrinsically disordered proteins • Bioblast editor: Plangger M
Affiliation and support
- Dept Biological Sciences, Louisiana State Univ, Baton Rouge, USA – shand@LSU.edu
- Supported by NSF grant IOS-1457061/IOS-1456809.
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