Adaptation to desiccation tolerance or anhydrobiosis has puzzled scientists for more than 300 years. Over the last few decades, considerable emphasis has been placed on understanding the role of two key molecules involved in anhydrobiosis: a peculiar disaccharide named trehalose and the hydrophilic LEA (Late Embryogenesis Abundant) proteins. In an article published in the Biochemical Journal in 2005, Alan Tunnacliffe and colleagues found that LEA proteins (alone, or more so in combination with trehalose) can protect stress-sensitive enzymes, such as citrate synthase and lactate dehydrogenase, from aggregation due to desiccation and freezing. Upon heat-stress, however, LEA proteins alone cannot prevent these enzymes from aggregating unless trehalose is present. This is the first report that LEA proteins can act as ‘molecular shields’ to prevent aggregation-induced cell damage due to water loss.

Many organisms are exposed to a stressful environment, either continuously or at least during certain periods of their lives. To thrive in unfavourable and extreme conditions, such as heat excess, salinity, freezing or desiccation, they possess several mechanisms. An amazing adaptation is anhydrobiosis (‘life without water’), which is the ability to survive in a completely dehydrated state and revive as soon as water is available. Although this was described by van Leeuwenhoek over 300 years ago, the biochemistry on which it is based is only now beginning to be elucidated.

A common mechanism to counteract abiotic stress is the synthesis of osmotically active compounds compatible with metabolism. Among the many osmoprotectants found in Nature, the non-reducing disaccharide trehalose is probably the most widespread and ancient one. This remarkable carbohydrate is present in several species of anhydrobiotic bacteria, fungi and invertebrates, as well as in the so-called ‘resurrection plants’ [1]. It basically functions by interacting with cell membranes to prevent their disruption or fusion, and thus leakage of cytosol components. In addition, trehalose protects proteins from desiccation-induced denaturation, possibly by replacing water molecules at their surface. These sugar ‘glasses’ apparently trap macromolecules in an immobile and stable medium, hence preventing cell deterioration.

Given these properties of trehalose, its biosynthetic genes have been used to generate transgenic plants and micro-organisms that are tolerant to several abiotic stress conditions. Tunnacliffe and co-workers [2] have coined the term ‘anhydrobiotic engineering’ to describe the process whereby a desiccation-sensitive mammalian cell is converted into a desiccation-tolerant one by enabling it to synthesize trehalose. They have genetically engineered a mouse cell line to accumulate trehalose, and this leads to an increased tolerance to osmotic stress [3]. However, trehalose alone was not sufficient to confer a state of anhydrobiosis, suggesting that further adaptations are required. This view is supported by bdelloid rotifers, animals that exhibit excellent desiccation tolerance even though trehalose and other disaccharides are absent from their systems [4].

In addition to osmoprotectants, a group of proteins have been reckoned to play a major role in stress tolerance. LEA (Late Embryogenesis Abundant) proteins are an evolutionarily conserved group of proteins, common among different plant taxa and cyanobacteria, as well as in some non-photosynthetic organisms, like bacteria, yeast and several invertebrates [5]. These proteins were initially identified in mature embryos prior to desiccation of seeds, and have been studied most extensively in relation to drought and cold stress, as well as in response to exogenous abscisic acid. They are highly hydrophilic and thermostable proteins, composed largely of the amino acids glycine, alanine and glutamine, and lacking cysteine and tryptophan. LEA proteins may be localized in the nucleus, cytoplasm, mitochondria, chloroplast and plasma membrane, and have been classified in at least six groups that are defined on the basis of their expression pattern and sequence. The most common LEA proteins are referred to as groups 1, 2 and 3, which are found unfolded in their native states. The first two groups are present only in plants, whereas group 3 LEA proteins are found in a wide variety of organisms. Although little is known about their mechanism of action, they are proposed to function as hydration buffers and ion chelators to protect macromolecules or stabilize membranes at the onset of dehydration.

The role of LEA proteins in stress tolerance has been partially revealed by performing gain-of-function experiments and the use of mutants. When overexpressed in transgenic rice and wheat, barley HVA1 group 3 LEA protein confers tolerance to both water deficiency and saline stress. Similar results were obtained in yeast cells overexpressing plant LEA proteins [5]. However, transgenic plants or cells are not desiccation-tolerant or anhydrobiotic in any of these examples. Recently, plants having two Arabidopsis-knockout mutations in genes belonging to the group 1 LEA protein family display premature seed dehydration and maturation, which demonstrates for the first time that an LEA protein is required for normal seed development [6]. LEA proteins are also present in animals, such as the anhydrobiotic nematode Aphelenchus avenae, which has a gene that is up-regulated in response to desiccation stress and whose encoded protein shares sequence similarity with a group 3 LEA protein [7].

Novel bioinformatic tools, such as POPP (Protein or Oligonucleotide Probability Profile), have predicted DNA binding activity for group 1 and group 2 LEA proteins and desiccation-induced formation of intracellular filaments by group 3 LEA proteins, possibly to increase mechanical strength [8]. In an article published in the Biochemical Journal in 2005, Tunnacliffe and co-workers [9] tested the hypothesis of their molecular chaperone activity on the basis of this bioinformatics information, and made an important advance in the field. They subjected two sensitive enzymes, CS (citrate synthase) and LDH (lactate dehydrogenase), to stress conditions in vitro in the presence or absence of recombinant forms of a group 3 LEA protein from the anhydrobiotic nematode A. avenae and a group 1 LEA protein from wheat seeds. CS is inactivated and forms aggregates at 40 °C or above, and this aggregation is decreased by molecular chaperones, such as the small heat-shock protein p26 from brine shrimp. However, after adding either of the LEA proteins to CS at molar ratios up to 100:1 and subjecting the mixture to heat stress, they found that the aggregates still formed, suggesting that LEA proteins alone are incapable of preventing substrate unfolding due to heat stress. In the presence of 400 mM trehalose and no LEA proteins, however, heat-induced CS aggregation was inhibited. On the other hand, at only a few degrees higher (43 °C) and in the presence of less trehalose (100 mM), partial aggregation of CS did occur.

Interestingly, when nematode or wheat LEA protein was added to this solution, a decrease in CS aggregation was noted, suggesting that LEA proteins do indeed enhance the protective effect from trehalose against heat-induced CS aggregation.

Since LEA proteins are not thought to be involved in the heat-shock response, Tunnacliffe and co-workers [9] also explored the ability of LEA proteins to attenuate CS aggregation induced by freezing or drying. They found that repeated rounds of vacuum-drying and rehydration provoked CS aggregation and loss of activity, whereas addition of either LEA protein almost completely inhibited aggregation. To support these observations, the same experiments were performed with LDH, which is also inactivated by the loss of water. However, when nematode or wheat LEA proteins were present, enzyme function was maintained. Trehalose could also prevent LDH aggregation due to drying. As before, in combination, trehalose and LEA proteins demonstrated a greater-than-additive effect. Similar results were observed when inducing aggregation by freezing.

Overall, these results led the authors to propose a model for the function of LEA proteins [9]. In this model, the role of LEA proteins as molecular chaperones was abandoned because of major differences with chaperones. Chaperones have defined secondary and tertiary structures and bind unfolded proteins to allow them to refold correctly, thereby preventing irreversible aggregation. In contrast, LEA proteins are unfolded in their native state and suppress protein aggregation under water-stress conditions. In this regard, it is their unordered and flexible structures that may allow LEA proteins to act as molecular shields by forming a physical barrier between neighbouring CS molecules and preventing contact between them [9].

Recently, the same research group has reported that the nematode group 3 LEA protein can also function in vivo [10]. When this LEA protein was expressed in a human cell line, it protected the water-soluble proteome from desiccation-induced aggregation and increased cell survival upon osmotic stress. Moreover, the LEA protein prevented protein aggregation in human cells that co-expressed proteins associated with neurodegenerative disorders, such as Huntington's disease. Interestingly, trehalose was shown also to have an anti-aggregation effect on huntingtin in vitro, as well as in transgenic mouse models of Huntington's disease, thus making trehalose a potential therapeutic agent [11]. This protective effect was previously thought to be caused by trehalose binding to expanded huntingtin, thereby stabilizing the partially unfolded mutant protein, but Tunnacliffe and colleagues recently demonstrated that trehalose induces autophagy, thereby enhancing clearance of the mutant protein [12]. Autophagy is a process that allows bulk degradation of cytoplasmic contents.

If trehalose and LEA proteins alone are insufficient to explain desiccation tolerance, could they be combined to convert a stress-sensitive organism into an anhydrobiotic one? A revision of the original concept of anhydrobiotic engineering could now include both trehalose and LEA proteins that may therefore act in concert to promote formation of anhydrobiotic cells and organisms. Mammalian cells could then be stored in a dried state rather than being frozen. This would reduce storage costs for products based, for example, on cell technology, hybridoma cell libraries and biosensors, and could also be of great use in tissue engineering, drug delivery and preservation of cell-producing high-value biomolecules. So far, in vivo studies of combined expression of trehalose and LEA proteins in transgenic cells or organisms have not been reported, but surely this would be a major advance towards engineering desiccation tolerance.

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