Rehydrated

liquid crystalline dehydrated with trehalose Q

liquid crystalline dehydrated with trehalose Q

leakage gel

*CKy liquid crystalline no leakage -►

liquid crystalline no leakage -►

liquid crystalline tigure 3.5 The water replacement hypothesis for the protective action of trehalose on membranes during desiccation and rehydration. Dehydration causes membranes to change from a liquid crystalline state to a gel state. Reversal of this change upon rehydration causes membranes to become transiently leaky, resulting in the fatal loss of cell contents. Trehalose replaces the water molecules associated with the membrane and prevents these harmful changes. Redrawn from a figure in Crowe et al. (1992).

liquid crystal to a gel. When water is added, the molecules move apart again and revert back to the liquid crystalline state. This change causes the membrane to become leaky, for a while, which results in the loss of substances from within the cell. This leakage of materials from the cell could be fatal for an anhydrobiotic organism. Trehalose attaches to the membrane and prevents it from changing from the liquid crystalline to the gel state when it loses water. When water returns, there is thus no change of state and the leakage of materials from the cell is prevented (Figure 3.5). This proposed mechanism is called 'the water replacement hypothesis' since trehalose replaces the water molecules in the membrane and prevents it from undergoing changes in state which would prove fatal during desiccation and rehydration.

Trehalose appears to play a number of other important roles in anhydrobiosis, in addition to preventing transitions in membranes which could result in the leakage of cell contents. Most sugars are susceptible to oxidation when in contact with air and form reaction products with proteins (the Maillard or browning reaction), but this is not the case with trehalose which is a non-reducing sugar. As water is lost from cells, the contents pack together and membranes which were previously separated could come into contact and fuse together. Such membrane fusion would violate the integrity of cellular compartments which would fall apart when rehydrated. Trehalose is thought to prevent membrane fusion from occurring. When you gently heat a sugar, it melts and then, on cooling, resolidifies into a glassy state. This is how boiled sweets, lollies and mints are made. Sugar glasses are also formed as a result of desiccation. As well as preventing membrane fusion, the formation of a sugar glass may trap the tissues of an anhy-drobiotic organism in a sticky, immobile and stable medium which would prevent any deterioration from occurring.

Some proteins are susceptible to damage (denaturation) during desiccation; trehalose stabilises and protects these proteins. How it does so is not entirely clear. The properties of a protein depend on it maintaining its correct shape or conformation. The conformation is at least partly dependent on the coating of water molecules which bond to the surface of the protein. Between one- and three-quarters of a gram of water bind to each gram of protein in solution. This makes up most the bound water in the cell, although perhaps the term 'bound water' is misleading since it can exchange rapidly with the free water in the cell. Loss of this water, however, could affect the conformation of a protein and result in the loss of its vital biological properties (denaturation). Trehalose prevents protein denaturation during desiccation, perhaps by taking the place of the water molecules associated with its surface and thus preserving its conformation.

Is trehalose the answer?

In 1971, John Crowe published a paper in the American Naturalist entitled 'Anhydrobiosis: an unsolved problem'. Some 20 years later, and after extensive studies on the stabilisation of membranes by sugars and other compounds, he was led to declare that 'a single perturbation - synthesis of a disaccharide such as trehalose or sucrose -is sufficient to achieve survival'. In other words, the problem of anhydrobiosis had been solved and the answer was trehalose (or sucrose in plants). The evidence that trehalose stabilises membranes and proteins during desiccation is impressive. It is the most efficient compound of all those which have been tested in this respect. It must be said, however, that these studies have largely been conducted on isolated proteins, artificial membranes or on membranes which have been isolated from organisms which are clearly not anhydrobiotic (such as membranes from the muscles of lobsters). This is understandable, since it is difficult to demonstrate the activity of trehalose in an intact organism. However, the attachment of trehalose to membranes in an anhydrobiotic animal has yet to be demonstrated.

Trehalose is also produced by a number of animals which are not capable of anhydrobiosis. Ascaris lumbricoides is a large (10 centimetre) nematode which is parasitic in the intestine of humans. It contains trehalose within its body fluids and throughout many of its tissues, and yet is never exposed to desiccation. A number of other parasitic, as well as free-living, nematodes have been shown to produce trehalose. The main functions of trehalose in parasitic nematodes appear to be as a store of carbohydrates and as a blood sugar which is transported to provide the tissues with carbohydrates. Glucose is the sugar which is the main source of energy in organisms. Trehalose consists of two glucose units and can thus be used to store and transport glucose. The main carbohydrate store in animals is, however, glyco-gen. This is a much more efficient glucose store than trehalose, since it does not cause osmotic problems to the animal and it yields more energy than does trehalose. Parasitic nematodes produce both trehalose and glycogen, so what is the role of trehalose in this situation? Nematode parasites living in the intestine of their host are bathed in a solution of easily absorbed nutrients, including glucose. The trehalose may well assist in the uptake of glucose across the intestine of the nem-atode. Since trehalose does not cross cell membranes, the glucose can be locked up in the form of trehalose, which dissolves in the body fluids and can be transported to the tissues. This prevents glucose from leaking back across the wall of the intestine and being lost to the parasite. The nematode can thus maintain favourable conditions for the uptake of glucose from its host.

The ability of nematodes to synthesise trehalose may explain the widespread occurrence of anhydrobiosis in this group. Insects also produce trehalose, in which it is the major blood sugar. Despite the presence of trehalose, anhydrobiosis is rare in insects. Insects are certainly found in many situations where an ability to survive anhydrobi-otically would be useful to them. If trehalose is the only adaptation needed, why is anhydrobiosis not more common in insects? Some recent studies on nematodes have cast further doubt on the proposition that trehalose is the sole adaptation necessary for anhydrobiotic survival. Some nematodes require slow drying even after they have completed the production of trehalose to concentrations found in the desiccated state. Other nematodes will not survive anhydrobiotically even though they produce trehalose in response to desiccation stress. It is clear that other, as yet unknown, adaptations are necessary for anhy-drobiosis.

It is not to be denied that trehalose plays an important role in anhy-drobiosis. It does not, however, appear to be the only adaptation which is necessary. Rather than considering the problem to have been solved, we need to explore what those other adaptations might be.

Recovering from anhydrobiosis

Surviving desiccation is only half the story. When water returns, the organism must absorb the water and resume active life. This could be as stressful as was the loss of water during desiccation, with the potential for material to be lost from cells and for disruption to occur. You might expect a dry nematode, for example, to take up water very quickly like a piece of dry cotton. Although the initial rate of water

figure 3.6 The appearance under the light microscope of the anhydrobiotic nematode Ditylenchus dipsaci. In (a), the nematode has not been exposed to desiccation; in (b) the nematode is completely desiccated (specimen mounted in a non-aqueous medium); and in (c) the nematode has recovered activity after a period of anhydrobiosis and then immersion in water for two hours. During rehydration, a hyaline layer (HL - comprising the muscle cells and epidermis) appears between the intestinal cells and the cuticle. Large globules within the intestinal cells also emerge, due to the fusion of smaller droplets of lipid (LD). From Wharton et al. (1985), reproduced with the permission of the Journal of Zoology.

figure 3.6 The appearance under the light microscope of the anhydrobiotic nematode Ditylenchus dipsaci. In (a), the nematode has not been exposed to desiccation; in (b) the nematode is completely desiccated (specimen mounted in a non-aqueous medium); and in (c) the nematode has recovered activity after a period of anhydrobiosis and then immersion in water for two hours. During rehydration, a hyaline layer (HL - comprising the muscle cells and epidermis) appears between the intestinal cells and the cuticle. Large globules within the intestinal cells also emerge, due to the fusion of smaller droplets of lipid (LD). From Wharton et al. (1985), reproduced with the permission of the Journal of Zoology.

uptake is indeed rapid, returning to a water content half that of normal within a few minutes, the rate of uptake slows and it takes several hours for the normal water content to be reached. Just as a slow rate of water loss was important to survive desiccation, a controlled process of recovery is necessary. In some cases, recovery is enhanced if the dry animals are exposed to moist air before immersion in water.

Anhydrobiotic nematodes do not start moving for several hours after they are reimmersed in water. This apparent period of inactivity is called 'the lag phase'. Although the nematodes do not move during the lag phase, they are certainly not inert. If you measure their metabolism, it commences immediately on immersion in water and rapidly rises to normal levels. During the lag phase, the nematode is metabolising like crazy but not moving - so what is it doing? In some nematodes, an orderly series of changes in their body structure has been observed. Most noticeable is the formation of large droplets in the intestinal cells and the appearance of a clear layer in between the intestine and the cuticle (Figure 3.6). The large droplets are due to small droplets of lipid fusing together to form larger ones, while the clear layer is due mainly to an increase in the thickness of the muscle cells of the animal. The structural changes are broadly the reverse of those observed during desiccation. The nematode appears to be undergoing some sort of process of repair, or restoration of a normal physiological state, during the lag phase that has to be completed before it can start moving again. If repair is occurring during the lag phase, you would expect its length to increase with more severe desiccation and hence with more damage to repair. This is indeed what happens, with the plant-parasitic nematode Ditylenchus dipsaci taking three times as long to recover after desiccation at 0 per cent relative humidity than after drying at 98 per cent relative humidity. It is the severity of the stress imposed during drying that determines the length of the lag phase, rather than the final relative humidity to which the nematode is exposed. A lag phase, the length of which appears to depend on the severity of desiccation, has also been observed in tardigrades.

There is more direct evidence for repair during the lag phase. The nematode cuticle acts a barrier to the exchange of materials with the outside world. This permeability barrier is partly destroyed by desiccation and then repaired during rehydration. The cells of some anhydro-biotes leak substances from within the cell when first immersed in water. The termination of this leakage could be due to the repair of membrane damage or to a physical change in membranes.

As well as repairing or restoring membranes and other structural elements, anhydrobiotic organisms will have to restore other aspects of the functioning of their cells to normal. The removal and then return of water will produce profound disturbances to the cell's internal environment. The water balance of the cell is obviously disrupted. After desiccation, the cell is shrunken. Sufficient water must be absorbed during rehydration to restore its normal water balance, but not too much or the cell will burst. The cell needs to restore its normal volume. All cells actively maintain a difference in the concentrations of various substances within the cell, compared with those outside the cell. As water is removed, the concentration of dissolved substances will increase and then decrease again when water returns. As well as restoring its water balance, the cell will also need to restore the normal concentrations of substances important to its functioning. This is of particular importance for muscle and nerve cells which depend on there being differences in the concentrations of particular ions (electrically charged atoms or molecules dissolved in water) between the inside and outside of the cell in order to function. Nerve impulses involve movements of sodium and potassium ions across the membranes of nerve cells, while muscle contraction depends on the movement of calcium ions. Restoring the concentration gradients of these substances is important for the nerves and muscles to work and this may explain the lack of movement by anhydrobiotes during the lag phase. Other features of the cell that may require restoration after anhydrobiosis include: pH, osmotic balance and the concentrations of oxygen and other dissolved gases.

The processes of repair and restoration may need to proceed to a certain level before the activity of anhydrobiotic animals will resume. Further repair may be necessary, however, before they will survive a further episode of desiccation. For some anhydrobiotes, survival has been shown to decline with repeated cycles of desiccation and rehydra-tion. This may have been simply a result of the organism not being given sufficient time to recover before exposure to another bout of desiccation. The ability of animals to survive anhydrobiotically involves a suite of mechanisms, or potential mechanisms. These are summarised in Figure 3.7.

the mechanisms of anhydrobiosis in plants Although the seeds, spores and pollen of many plants are capable of anhydrobiosis, it is difficult to distinguish the mechanisms which enable them to survive anhydrobiotically from those associated with their formation, germination and development. The tissues of resurrection plants and of desiccation-tolerant mosses, algae and lichens are, however, mature and provide systems in which the mechanisms of anhydrobiosis can be investigated (Figure 3.8). We would like many

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