The intrinsically disordered protein LEA7 from Arabidopsis thaliana protects the isolated enzyme lactate dehydrogenase and enzymes in a soluble leaf proteome during freezing and drying

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Highlights

  • LEA7 interacts with membranes in the dry state, but does not stabilize liposomes during drying.

  • LEA7 stabilizes enzymes during drying and freezing.

  • Protection of lactate dehydrogenase is not based on an anti-aggregation activity.

  • There is a mutual structural influence of LEA7 and the Arabidopsis soluble proteome.

  • Effects of LEA7 on the Arabidopsis soluble proteome are enhanced by the presence of sugars.

Abstract

The accumulation of Late Embryogenesis Abundant (LEA) proteins in plants is associated with tolerance against stresses such as freezing and desiccation. Two main functions have been attributed to LEA proteins: membrane stabilization and enzyme protection. We have hypothesized previously that LEA7 from Arabidopsis thaliana may stabilize membranes because it interacts with liposomes in the dry state. Here we show that LEA7, contrary to this expectation, did not stabilize liposomes during drying and rehydration. Instead, it partially preserved the activity of the enzyme lactate dehydrogenase (LDH) during drying and freezing. Fourier-transform infrared (FTIR) spectroscopy showed no evidence of aggregation of LDH in the dry or rehydrated state under conditions that lead to complete loss of activity. To approximate the complex influence of intracellular conditions on the protective effects of a LEA protein in a convenient in-vitro assay, we measured the activity of two Arabidopsis enzymes (glucose-6-P dehydrogenase and ADP-glucose pyrophosphorylase) in total soluble leaf protein extract (Arabidopsis soluble proteome, ASP) after drying and rehydration or freezing and thawing. LEA7 partially preserved the activity of both enzymes under these conditions, suggesting its role as an enzyme protectant in vivo. Further FTIR analyses indicated the partial reversibility of protein aggregation in the dry ASP during rehydration. Similarly, aggregation in the dry ASP was strongly reduced by LEA7. In addition, mixtures of LEA7 with sucrose or verbascose reduced aggregation more than the single additives, presumably through the effects of the protein on the H-bonding network of the sugar glasses.

Introduction

Anhydrobiosis or “life without water” is a phenomenon that has received much attention and although mechanisms responsible for cellular desiccation tolerance have been proposed (e.g. [1], [2]), many functional aspects are still unresolved [3]. There is, however, widespread consensus that sugars and Late Embryogenesis Abundant (LEA) proteins can be major contributors to cell stability in the dry state, even in cells that naturally do not contain LEA proteins [4]. In addition, some organisms can achieve desiccation tolerance without the accumulation of sugars [5], [6]. LEA proteins have been first identified in plant seeds during maturation, when the seeds attain desiccation tolerance [7], but were later also found in vegetative plant organs, in bacteria and various anhydrobiotic invertebrates [8], [9].

The precise in vivo function of most LEA proteins remains unresolved, which may at least in part be due to their unstructured nature in solution, which has made functional predictions impossible. However, many of these proteins fold mainly into α-helices during drying [10]. Results from various in vitro assays suggest that some LEA proteins are involved in the stabilization of cellular constituents such as proteins and membranes, but other functions have also been proposed [8], [9]. Only for the cold induced Arabidopsis thaliana LEA proteins COR15A and COR15B, membrane stabilization during freezing could be clearly established as their in vivo function, while enzyme stabilization could be excluded [11]. However, many in vitro investigations have shown that LEA proteins can effectively prevent inactivation of sensitive enzymes such as lactate dehydrogenase (LDH) during freezing or drying [9], [12]. Under the appropriate drying conditions such enzymes aggregate, which may contribute to their inactivation. Aggregation can be prevented by many LEA proteins that are thought to function as “molecular shields” by preventing direct contact between enzyme molecules [13], [14], [15].

In addition, as an adaptive response to water loss, most organisms accumulate compatible solutes, such as sugars [16]. During drying, most sugars do not crystallize, but rather form a glass. Due to the low molecular mobility, the glassy state immobilizes macromolecules, thus providing protection e.g. to cells in dry plant seeds [17], [18]. LEA proteins can be embedded in such a glassy matrix, increasing the glass transition temperature [19]. It has been proposed that H-bonding interactions between sugars and proteins may enhance the stability of cytoplasmic glasses in seeds and pollen, thereby contributing to the exceptional stability of these structures in the dry state [2], [18], [20].

The aim of the present study was to investigate the functional activity of the structurally disordered protein LEA7 from A. thaliana with respect to dehydration and freezing stress. LEA7 is located in the cytosolic compartment of plant cells [21] and increases the desiccation tolerance of transgenic yeast cells [22]. In a previous study we presented evidence that LEA7 is able to interact with lipid membranes in the fully hydrated and in the dry state [23], suggesting a function of the protein in membrane stabilization. Here, we show that LEA7 is not able to protect liposomes during dehydration, but rather has protective activity for the labile enzyme LDH and enzymes found in the total soluble proteome of Arabidopsis leaves. We present evidence from Fourier-transform infrared (FTIR) spectroscopy for interactions between LEA7 and LDH and also between LEA7 and the Arabidopsis proteome, thus decreasing the degree of protein aggregation and preservation of enzyme activity. In the presence of LEA7 the average strength of H-bonding interactions in dry sugar (sucrose and verbascose) glasses was increased, indicating that protein and sugars interact to form a more tightly packed matrix in comparison to pure sugars.

Section snippets

Materials

Lactate dehydrogenase from rabbit muscle (LDH), β-lactoglobulin (LG) from bovine milk and sucrose were obtained from Sigma (St. Louis, MO), verbascose from Megazyme (Wicklow, Ireland). RNaseA from bovine pancreas (RNaseA) was from Roche (Basel, Switzerland). D2O (99.98%) was purchased from Deutero GmbH (Kastellaun, Germany) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) was obtained from Avanti Polar Lipids (Alabaster, AL).

Expression and purification of recombinant LEA7

The LEA7 gene (At1g52690) was cloned into the pDEST17

Liposomes are not stabilized by LEA7 during drying

Our previous investigation indicated that LEA7 interacts with liposomes in the dry state [23]. Based on these data we hypothesized that LEA7 may stabilize membranes against the stresses associated with drying and rehydration. Here, we tested this hypothesis by monitoring leakage of the fluorescent dye CF from liposomes (Fig. 1). The results indicate that LEA7 only had a marginal effect on liposome stability. CF leakage was reduced by about 7%, from 98% in the absence of additives to 91% in the

Discussion

Two main functions have been attributed to LEA proteins, namely membrane and enzyme protection during freezing and drying [9], [49]. Although our knowledge about the structural requirements for LEA proteins to perform either function is not sufficient yet to propose that they are mutually exclusive, evidence in favor of such a hypothesis is accumulating. Examples include two LEA proteins from a desiccation-tolerant rotifer, of which one shows enzyme protection and one membrane interaction [50],

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    1

    Permanent address: Institute of Biophysics and Biomedical Engineering, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria.

    2

    Present address: Siemens AG, Siemensdamm 50, D-13629 Berlin, Germany.

    3

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

    4

    Present address: UMR 1332, Biologie du Fruit et Pathologie, INRA Bordeaux-Aquitaine, 33882 Villeneuve d'Ornon Cedex, France.

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