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Functional characterization of selected LEA proteins from Arabidopsis thaliana in yeast and in vitro

Planta volume 240, pages 325–336 (2014)Cite this article

Abstract

Main conclusion

Expression of eight LEA genes enhanced desiccation tolerance in yeast, including two LEA_2 genes encoding atypical, stably folded proteins. The recombinant proteins showed enzyme, but not membrane protection during drying.

To screen for possible functions of late embryogenesis abundant (LEA) proteins in cellular stress tolerance, 15 candidate genes from six Arabidopsis thaliana LEA protein families were expressed in Saccharomyces cerevisiae as a genetically amenable eukaryotic model organism. Desiccation stress experiments showed that eight of the 15 LEA proteins significantly enhanced yeast survival. While none of the proteins belonging to the LEA_1, LEA_5 or AtM families provided protection to yeast cells, two of three LEA_2 proteins, all three LEA_4 proteins and three of four dehydrins were effective. However, no significantly enhanced tolerance toward freezing, salt, osmotic or oxidative stress was observed. While most LEA proteins are highly hydrophilic and intrinsically disordered, LEA_2 proteins are “atypical”, since they are more hydrophobic and possess a stable folded structure in solution. Because nothing was known about the functional properties of LEA_2 proteins, we expressed the three Arabidopsis proteins LEA1, LEA26 and LEA27 in Escherichia coli. The bacteria expressed all three proteins in inclusion bodies from which they could be purified and refolded. Correct folding was ascertained by Fourier transform Infrared (FTIR) spectroscopy. None of the proteins was able to stabilize liposomes during freezing or drying, but they were all able to protect the enzyme lactate dehydrogenase (LDH) from inactivation during freezing. Significantly, only LEA1 and LEA27, which also protected yeast cells during drying, were able to stabilize LDH during desiccation and subsequent rehydration.

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Abbreviations

BSA:

Bovine serum albumin

CFU:

Colony-forming units

CD:

Circular dichroism

CF:

Carboxyfluorescein

EPC:

Egg phosphatidylcholine

FTIR:

Fourier transform infrared

GFP:

Green fluorescent protein

GOI:

Gene of interest

GRAVY:

Grand average of hydropathy

IDP:

Intrinsically disordered protein

LDH:

Lactate dehydrogenase

LEA:

Late embryogenesis abundant

POPC:

1-Palmitoyl-2-oleoyl-phosphatidylcholine

RH:

Relative humidity

TEN:

Tes, EDTA, NaCl buffer

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Author notes

  1. Michaela Hundertmark

    Present address: Vilmorin SA, Rotue du Manoir, 49250, La Ménitré, France

Affiliations

  1. Max-Planck-Institute of Molecular Plant Physiology, Am Mühlenberg 1, 14476, Potsdam, Germany

    Nghiem X. Dang, Antoneta V. Popova, Michaela Hundertmark & Dirk K. Hincha

  2. Faculty of Biotechnology, Hanoi University of Agriculture, Hanoi, Vietnam

    Nghiem X. Dang

  3. Institute of Biophysics and Biomedical Engineering, Bulgarian Academy of Sciences, 1113, Sofia, Bulgaria

    Antoneta V. Popova

Authors
  1. Nghiem X. Dang
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  2. Antoneta V. Popova
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  3. Michaela Hundertmark
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  4. Dirk K. Hincha
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Correspondence to Dirk K. Hincha.

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Dang, N.X., Popova, A.V., Hundertmark, M. et al. Functional characterization of selected LEA proteins from Arabidopsis thaliana in yeast and in vitro. Planta 240, 325–336 (2014). https://doi.org/10.1007/s00425-014-2089-z

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  • DOI: https://doi.org/10.1007/s00425-014-2089-z

Keywords

  • Arabidopsis
  • Desiccation
  • Enzyme
  • Freezing
  • LEA
  • Yeast