Insights on Structure and Function of a Late Embryogenesis Abundant Protein from Amaranthus cruentus: An Intrinsically Disordered Protein Involved in Protection against Desiccation, Oxidant Conditions, and Osmotic Stress.

Alma L Saucedo,Abraham Escobedo-Moratilla,Daniel A Fernández-Velasco,Federico Del Río-Portilla,Angel A Guevara-García,Ana P Barba De La Rosa,Luis A De Luna-Valdez,Esaú Bojorquéz-Velázquez,Eric E Hernández-Domínguez

doi:10.3389/fpls.2017.00497

Abstract

Late embryogenesis abundant (LEA) proteins are part of a large protein family that protect other proteins from aggregation due to desiccation or osmotic stresses. Recently, the Amaranthus cruentus seed proteome was characterized by 2D-PAGE and one highly accumulated protein spot was identified as a LEA protein and was named AcLEA. In this work, AcLEA cDNA was cloned into an expression vector and the recombinant protein was purified and characterized. AcLEA encodes a 172 amino acid polypeptide with a predicted molecular mass of 18.34 kDa and estimated pI of 8.58. Phylogenetic analysis revealed that AcLEA is evolutionarily close to the LEA3 group. Structural characteristics were revealed by nuclear magnetic resonance and circular dichroism methods. We have shown that recombinant AcLEA is an intrinsically disordered protein in solution even at high salinity and osmotic pressures, but it has a strong tendency to take a secondary structure, mainly folded as α-helix, when an inductive additive is present. Recombinant AcLEA function was evaluated using Escherichia coli as in vivo model showing the important protection role against desiccation, oxidant conditions, and osmotic stress. AcLEA recombinant protein was localized in cytoplasm of Nicotiana benthamiana protoplasts and orthologs were detected in seeds of wild and domesticated amaranth species. Interestingly AcLEA was detected in leaves, stems, and roots but only in plants subjected to salt stress. This fact could indicate the important role of AcLEA protection during plant stress in all amaranth species studied.

Highlights

Seeds can withstand the loss of cellular water during the maturation phase of their development by the accumulation of high levels of ubiquitous proteins named late embryogenesis abundant (LEA) proteins (Ali-Benali et al, 2005; Dalal et al, 2009; Liu et al, 2013; Avelange-Macherel et al, 2015)
AcLEA cDNA contains an ORF of 516 bp that codifies for a 172 amino acids protein with a molecular mass calculated of 18.34 kDa and a theoretical pI of 8.58, values that corresponded to experimental data previously reported (Maldonado-Cervantes et al, 2014)
Group 3 LEA proteins are characterized by a repetitive motif of 11 amino acids TAQAAKDKTSE in the middle of the sequence that is preceded or followed by ATEAAKQKASE; in the N-terminal region is usually conserved the SYKAGETKGRKT; GGVLQQTGEQV and AADAVKHTLGM are frequently observed in the C-terminal

Summary

Introduction

Seeds can withstand the loss of cellular water during the maturation phase of their development by the accumulation of high levels of ubiquitous proteins named late embryogenesis abundant (LEA) proteins (Ali-Benali et al, 2005; Dalal et al, 2009; Liu et al, 2013; Avelange-Macherel et al, 2015). LEA proteins are reported in response to water limitation, which suggests that they have an important role in desiccation tolerance (Tunnacliffe and Wise, 2007; Tunnacliffe et al, 2010; Hand et al, 2011). In spite of their widely recognized importance for desiccation tolerance, the molecular function of LEA proteins is only starting to emerge, with a variety of functions in agreement with their diversity (Battaglia and Covarrubias, 2013). LEA proteins are intrinsically disordered proteins (IDP) in aqueous solutions (Wolkers et al, 2001; Goyal et al, 2003; Boucher et al, 2010; Tompa and Kovacs, 2010; Popova et al, 2011), they may acquire some structure folding into α-helical conformations during partial or complete dehydration (Shih et al, 2004; Tolleter et al, 2007; Hincha and Thalhammer, 2012)

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