Late Embryogenesis Abundant (LEA) proteins confer water stress tolerance to mammalian somatic cells

M Czernik,A Fidanza,F.P Luongo,L Valbonetti,P.A Scapolo,P Patrizio,P Loi

doi:10.1016/j.cryobiol.2020.01.009

Abstract

Late Embryogenesis Abundant (LEA) proteins are commonly found in plants and other organisms capable of undergoing severe and reversible dehydration, a phenomenon termed “anhydrobiosis”. Here, we have produced a tagged version for three different LEA proteins: pTag-RAB17-GFP-N, Zea mays dehydrin-1dhn, expressed in the nucleo-cytoplasm; pTag-WCOR410-RFP, Tricum aestivum cold acclimation protein WCOR410, binds to cellular membranes, and pTag-LEA-BFP, Artemia franciscana LEA protein group 3 that targets the mitochondria. Sheep fibroblasts transfected with single or all three LEA proteins were subjected to air drying under controlled conditions. After rehydration, cell viability and functionality of the membrane/mitochondria were assessed. After 4 h of air drying, cells from the un-transfected control group were almost completely nonviable (1% cell alive), while cells expressing LEA proteins showed high viability (more than 30%), with the highest viability (58%) observed in fibroblasts expressing all three LEA proteins. Growth rate was markedly compromised in control cells, while LEA-expressing cells proliferated at a rate comparable to non-air-dried cells. Plasmalemma, cytoskeleton and mitochondria appeared unaffected in LEA-expressing cells, confirming the protection conferred by LEA proteins on these organelles during dehydration stress. This is likely to be an effective strategy when aiming to confer desiccation tolerance to mammalian cells.

Highlights

Water is essential for life (Hand et al, 2007; Menze et al, 2009) yet many organisms are able to survive almost completely dehydrated (>99% of their body water is removed)(Crow et al, 1992)
The results showed that LateEmbryogenesis Abundant proteins (LEAp), one expressed in the cytoplasm, AfrLEA2, and the other AfrLEA3m in the mitochondria, together with trehalose, effectively protected the cells from the desiccation stress
To confirm the proper localization of the Late Embryogenesis Abundant (LEA) proteins, sheep fibroblasts were transfected with empty vectors EV-GFP, EV-RFP and EV-BFP as a control

Summary

Introduction

Water is essential for life (Hand et al, 2007; Menze et al, 2009) yet many organisms are able to survive almost completely dehydrated (>99% of their body water is removed). In nature, this phenomenon is known as “anhydrobiosis”, and is conserved across vegetal and animal phyla (Hincha et al, 1992). Embryogenesis Abundant proteins (LEAp) are the best characterized and perhaps the most interesting (Marunde et al, 2013). LEA proteins were first discovered in cotton seeds more than 30 years ago (Dure et al, 1981) and were later found in seeds and vegetative tissues of several other plants (Shih et al, 2008). A relatively recent survey, probably not updated, contains 769 LEAp entries from 196 organisms (Hunault and Jaspard, 2009)

Methods

Results

Conclusion