a study of tolerance
Why trehalose synthesis is induced?
LEA proteins and cryptobiosis
Identification of a trehalose transporter
Radiation tolerance in the sleeping chironomid
The rapid synthesis of trehalose, which plays an important role during anhydrobiosis, is enhanced when the water content of the body falls under 75%. So, what are the factors triggering this synthesis of trehalose?
As water is getting out of the larva, the relative concentration of sugars and ions increases in the body and the osmotic pressure rises consequently. We focused on this variation of osmotic pressure and made experiments that mimic the increase of osmotic pressure in the body of larvae. Larvae were immersed for 24 hrs into solutions containing sodium chloride, mannitol, glycerol or DMSO (dimethyl sulfoxide) at various concentrations and the trehalose content of the body was then measured. As a result, a peak of trehalose accumulation was observed for sodium chloride at approximately 350 milliosmole (around a concentration of 1%).

The observed trehalose accumulation was similar to the amount accumulated during anhydrobiosis. In comparison, the other treatments increased osmotic pressure but did not induce in trehalose accumulation. From those results, it is clear that the induction of trehalose synthesis is not dependent on a simple physical stimulus such as osmotic pressure. Since other types of salt solutions presented an inducing effect similar to that of sodium chloride, we suggested the existence of a sensor capable of detecting changes of ionic concentrations in the sleeping chironomid.
As exemplified by the lotus seeds found in an archeological site of the Jomon period in Japan, which germinated after 2000 years of quiescence, plant seeds are in general remarkably resistant to desiccation and this is phenomenon is called dormancy. Some proteins, which are produced in a very large amount during the preparation of dormancy in plant seeds, were discovered 30 years ago and those proteins were named LEA (Late Embryo Abundant) proteins. Since LEA proteins are also strongly accumulated in some kind of pollen, which present also resistance to desiccation, those proteins were consequently suggested to be involved in the resistance to desiccation. Thereafter, LEA proteins were shown to accumulate in response to desiccation or salt (changes in ion concentration) stress in other parts of the plants such as leaves or roots, as well.

On the basis of their primary structure and expression profile, LEA proteins were classified in groups 1 to 6, but all of them have in common a high hydrophilicity (they are easily solvated by water). In vitro, they can prevent the aggregation of proteins, which is generally observed along with desiccation or salt stress. In contrast with other proteins, whose structure is generally altered under conditions of desiccation stress, LEA proteins present the interesting particularity of having normally a random structure, which turns to a coiled -helix structure when exposed to desiccation stress.
LEA proteins were thought to be plant-specific proteins, but the team of Tunnacliffe demonstrated in 2002 that LEA proteins were also present in a nematode worm undergoing cryptobiosis. In other words, LEA proteins were discovered in animals for the first time. Thereafter, our group identified also 3 cDNA (PvLea1, PvLea2, PvLea3) encoding group 3 LEA proteins in larvae of the sleeping chironomid. The corresponding LEA proteins were expressed in baculovirus and even after boiling for 15 min, proteins did not precipitate by aggregation. This demonstrates the high hydrophilic character of those 3 LEA proteins from P. vanderplanki. The expression of the 3 genes was induced by both desiccation and salt stress.

These results suggest that the expression of LEA proteins in the sleeping chironomid is triggered by the rise of ion concentration in the hemolymph during desiccation, similarly to trehalose synthesis.

We made hypotheses concerning the role of LEA proteins during the process of desiccation in larvae of the sleeping chironomid. Two possible functions were distinguished. First, during the process of desiccation in the larvae, the structure of group 3 LEA proteins undergoes a transition from a random state to an organized coiled -helix structure. Since this the hydrophilic part of the -helix structure becomes polarized, it may bind to intracellular ions and thus prevent the salt precipitation (denaturation and aggregation of proteins due to high salt concentration) of the other proteins. Because they are amphiphilic, LEA proteins may also bind to the cellular membrane and to the hydrophobic regions of other proteins, thus avoiding the irreversible denaturation of biological membranes and proteins.

Second, the hydrophobic regions of group 3 LEA proteins bind together and form a 3-dimensional structure. During desiccation, LEA proteins form fibre-like structures, which may act as cytoskeleton. Together with the vitrification of trehalose, LEA proteins may thus act as the steel bars in reinforced concrete. We propose that such a structure would avoid excessive shrinking of the cells during desiccation.

To summarize, LEA proteins are strongly induced during the desiccation of P. vanderplanki larvae and acting together with trehalose, they protect biomolecules from denaturation during cryptobiosis.
Living cells are surrounded by a phospholipid bilayer membrane. This phospholipid bilayer membrane does not allow the exchanges of most solutes (sugars, aminoacids or ions) and even water cannot pass through the membrane. The only way for water and hydrophilic molecules to enter in or go out from the cell consist in passing through membrane proteins, called transporters (or channels), which are embedded in the phospholipid bilayer.

There are many types of transporters, from those with high specificity, which allow the permeability of only one kind of molecule, to transporters with a broad range of substrates. In humans, for example, whatever the quantity of food is ingested, if there was no glucose transporter, glucose in the blood would never arrive into the cells of peripheral tissues.

In the sleeping chironomid, the existence of a trehalose transporter was expected, since the trehalose that is synthesized in large quantity in the fat body should be redistributed to the cells of other tissues. However, at that time there was no trehalose transporter identified from any multicellular organism. As the sleeping chironomid was expected to express a large amount of trehalose transporter, we decided to identify the corresponding gene. As a result, the full-length cDNA sequence was characterized. This cDNA was artificially expressed in African clawed frog (Xenopus laevis) oocytes, which lack trehalose, and then oocytes were placed into a buffer containing trehalose. Trehalose was finally detected inside the oocyte cells, showing that trehalose was transported selectively through the cell membrane. This result comfirmed that our cDNA was actually encoding a trehalose transporter and consequently, we named the corresponding gene Tret1.

We investigated the properties of the TRET1 protein, obtained from the Tret1 gene and showed that this protein presented a very high affinity for trehalose and that it was a facilitated diffusion type transporter (meaning that it needs no energy from the cell for transporting trehalose). Moreover, TRET1 presented a very high capacity for trehalose transport and was able to diffuse trehalose with sufficient velocity even at concentrations over 100mM. This concentration corresponds to the amount of trehalose found in the body of the sleeping chironomid during the induction of cryptobiosis. Tre1 is only expressed in the fat body and its expression is strongly induced during the process of desiccation. Consequently, we showed that TRET1 is responsible for the transport to the hemolymph of the large amount of trehalose, which is synthesized in the fat body.

As we explained previously ("The surprising resistance of the Sleeping Chironomid"), the larvae of the sleeping chironomid present a high tolerance towards radiations. By studying further this tolerance phenomenon, we can consider the phenomenon of tolerance from a different point of view.

In the sleeping chironomid, desiccated larvae can survive to higher doses of radiation than larvae, which are not desiccated (called active larvae hereunder). This survival means that larvae are still alive 48 hrs after exposure to radiations. However, interesting facts are emerging when we look at the subsequent development and reproduction. If larvae exposed to a 100 Gy dose of g rays are grown further, active larvae will produce next generation, but desiccated larvae will not. Females actually lay eggs, but they do not hatch. When comparing radiation doses, which do not affect sexual glands (30 Gy for active larvae and 10 Gy for desiccated larvae), it appears that the effects of radiations are stronger on desiccated larvae. For living organisms, producing the next generation, which ultimately means surviving, is a major goal. Desiccation was thus suggested to have a negative effect on the protection of the sexual glands.

Organisms, which are tolerant towards radiations, are generally also resistant to desiccation. However, this “tolerance” includes quantities of radiations that do not occur in the nature. It is hard to believe that organisms evolved to acquire a tolerance towards conditions they have no chance to face. It is probable that the capacity to resist to desiccation, which organisms developed, overlaps with the mechanism of radiation tolerance.

Precisely, what are the effects of irradiation? Radiations differ on the basis of the energy they bring to tissues and this defines the quality of the radiations. The quality of radiations is roughly divided in low-LET (linear energy transfer) radiations and high-LET radiations. Low-LET radiations comprise X rays and Gamma Rays, whereas proton beam and ion beam, which are used in the latest medical treatments, are classified in high-LET radiations. Those radiations are absorbed in tissues and the corresponding energy ionizes the molecules and atoms in the cells. In case of direct action, the target itself is ionized, but in case of indirect action, water undergoes the action of radiations and generates instable elements, such as ions or radicals, which will in turn attack the target molecules. Low-LET radiations are responsible for indirect action, whereas high-LET radiations have a direct action on target molecules. DNA is the most sensible molecule to these actions of radiations, which cause single strand break or double strand break of the DNA double helix.

In the case of the sleeping chironomid, radicals are generated during the process of desiccation and provoke damages to DNA and cell membrane. However, trehalose and LEA proteins are thought to have a buffer effect towards this phenomenon. Thus they would protect the cell against the harmful effects of low-LET radiations by removing the resulting radicals. Furthermore, desiccated larvae were suggested to present a high capacity of DNA repair, since they tolerate as well g rays and ion beam. As an indicator of DNA repair capacity, we investigated the subcritical radiation dose, Dq (defining the capacity to recover from damages due to radiations). Desiccated and active larvae of the sleeping chironomid and two other normal chironomid species (Chironomus yoshimatsui & Polypedilum nubifer) were compared and the Dq value of the sleeping chironomid was higher than the value of the two other species, thus indicating that its constitutive capacity of DNA repair was very high. We also observed that the Dq value of desiccated larvae was even higher.
We understand now that the expression of DNA repair enzymes increases during the induction of the desiccation process. We also noticed that larvae in a physiological state between the active and the desiccated forms, when exposed to ion beam irradiation, did survive longer as the exposure was performed closer to the desiccated state. On the basis of these results, we suggested that the DNA repair function was enhanced gradually during the process of desiccation.

We would like to stress here the fact, that the character of anhydrobiosis has evolved in order to protect the body against desiccation, and that this character is required to withstand the desiccated state until recovery. In other words, this suggests that this character is needed because cells are damaged during desiccation. We investigated the level of damage to DNA in the cells just after recovery, using a technique called Comet Assay, and the DNA of almost every cell was actually broken. Until recently, we believed that cells were fully protected against damages during anhydrobiosis, since the sleeping chironomid was able to survive, even in a totally desiccated state. However, dehydration represents a severe stress and DNA was damaged in cells. Nevertheless, we suggest that those damages do not exceed the recovering capacity of the sleeping chironomid, which prepared DNA repair enzymes in advance.