Drying without dying: revealing the role of late embryogenesis abundant proteins during desiccation in Ramonda serbica

Abstract

Introduction: Resurrection plants (such as Ramonda serbica) can survive a long desiccation period andfully resume their metabolism upon watering. The hallmark of desiccation tolerance (DT) is the accumulation of protective, intrinsically disordered proteins(IDPs), called late embryogenesis abundant proteins (LEAPs). Although their high structural plasticity allows them to interact with various partners, nospecific cellular targets of LEAPs have been identified so far.Methods: To identify LEAPsinvolved in DT, differential transcriptome and proteome analyses of hydratedand desiccated R. serbica leaves were performed. The identified LEAPs were structurally characterisedand classified. To evaluate theirstructural propertiesin vitro and their potential functionsin vivo, the representative RsLEA proteins, were produced in Escherichia coli using recombinant DNA technology.Results: Members of the LEA4 protein family represent the majority of desiccation-inducible LEAPs. Even17 proteins belonging to the LEA4 protein family group were induced by desiccation. They show high disorder propensity (82 %), and at the same time, a high tendency to form α-helices (>80%). Although recombinant DNA technology has traditionally been used to overexpress and purify various globularproteins, the production of IDPsis challenging due to their high susceptibility to proteolytic cleavage andaggregation. Nevertheless, the representative LEAPs containing hexa-Histagsimmunoglobulin G-binding protein and a proteolytic TEV site were produced, purified and cleaved by TEV protease.Conclusion: The combination of in silico and in vitro results will be crucial for the identification of endogenous partners of LEAPs, providing further insight into their role in DT.