figure 3.7 The changes that occur during desiccation and rehydration of an anhydrobiotic animal, such as a nematode.

agriculturally important plants to be more drought tolerant. If the mechanisms of survival in anhydrobiotic plants can be understood, it may be possible to transfer these abilities to crop plants, using the techniques of genetic engineering, to allow them to be grown in more drought-prone areas.

Anhydrobiotic plants can be divided into two groups on the basis of the rate of desiccation which they can survive. The ability to survive fast rates of water loss is restricted to some species within the less complex groups of plants: mosses, liverworts, algae and lichens. These have little ability to conserve water and their water content rapidly comes into balance with that of their environment. Desiccation-tolerant higher plants (resurrection plants) require slow drying in order to survive anhydrobiotically and they have morphological and physio-

figure 3.8 Changes in shoots of the African resurrection plant, Myrothamnus flabellifola, during rehydration. The branch on the left was air dry, the remainder (from left to right) were following four, eight, 20 and 24 hours of immersion of the stem in water. The leaves expand and rotate downwards as they rehydrate. The leaves of the fully hydrated plant are up to 8-10 millimetres long. Photo: Don Gaff.

figure 3.8 Changes in shoots of the African resurrection plant, Myrothamnus flabellifola, during rehydration. The branch on the left was air dry, the remainder (from left to right) were following four, eight, 20 and 24 hours of immersion of the stem in water. The leaves expand and rotate downwards as they rehydrate. The leaves of the fully hydrated plant are up to 8-10 millimetres long. Photo: Don Gaff.

logical mechanisms which ensure the slow rate of water loss necessary for their survival. These two groups of anhydrobiotic plants have been referred to as fully desiccation-tolerant plants and modified desiccation-tolerant plants respectively. Fully desiccation-tolerant plants must always be prepared for periods of anhydrobiosis, while the mechanisms which enable modified desiccation-tolerant plants to survive are mobilised only when they are exposed to desiccation. For the higher plants, the costs involved in diverting resources from growth and reproduction to survival are thus only incurred when they are faced with a life-threatening exposure to desiccation.

Structural preservation in the dry state varies in different species of anhydrobiotic plants. In some, their structure is well preserved and they retain chlorophyll, while in others there is extensive degradation of the structures within their cells and chlorophyll is lost. During rehy-dration, the normal cell structure takes several days to be restored and chlorophyll is resynthesised. There are thus two different strategies for


surviving desiccation. Some species prevent damage during desiccation, while others repair the damage when water returns. For many species, some mixture of these two strategies is likely to be important.

A number of different mechanisms have been suggested to be important in the anhydrobiotic survival of resurrection plants. Trehalose is rare in plants, although its presence has been reported in a few species, but other sugars (such as sucrose, raffinose and maltose), polyols (such as glycerol, sorbitol and mannitol) or amino acids (such as proline) may play a similar role to trehalose in plants, protecting membranes and proteins during desiccation. The sugars may also be involved in the formation of glasses which stabilise the cells during anhydrobiosis. Glass formation has been demonstrated in plant seeds.

Biological reactions with oxygen can generate highly reactive charged molecules, particularly the superoxide radical (O~) and the hydroxyl radical (OH-). These radicals react destructively with membranes and biological molecules. Organisms have various mechanisms for protecting themselves from, and preventing the accumulation of, these reactive radicals. In particular, they have enzymes, such as superoxide dismutase, catalase and peroxidase, which facilitate their removal. These protective reactions may be lost during desiccation, since metabolism ceases, but resurrection plants may have an enhanced ability for the functioning of these enzymes to continue during water loss and rehydration. The loss of chlorophyll during the desiccation of some anhydrobiotic plants is interesting in this respect since photosynthesis involves reactions with oxygen which can generate these reactive radicals.

One approach to determining the mechanisms which might be important in stress resistance is to compare the pattern of protein production in the stressed and the unstressed organism. This approach, called proteomics, aims to look at the total pattern of proteins produced (the proteome). The proteome differs from the genome (the complement of genes) since not all proteins encoded by genes are produced at any one time. Any shift in the pattern of protein production, or gene expression, in response to a stress such as desiccation may indicate the mechanisms that are important in the response to that stress. This approach has yet to be applied to anhydrobiosis in animals, but there have been some promising results with the response of plants to drought and desiccation, including that of seeds and of resurrection plants.

Although the overall rate of protein synthesis eventually declines during exposure to drought, there are proteins synthesised which are either not produced by the plants when not under stress or which are produced in greater quantities. One group of these desiccation-induced proteins has been called 'dehydrins'. Dehydrins have been identified in seeds, in resurrection plants and in desiccation-tolerant mosses and liverworts. They have even been reported from several species of cya-nobacteria and are thus found in a wide range of photosynthetic organisms. They are present in the nucleus, the chloroplasts and the cytoplasm of cells. In addition to plant tissues which are anhydrobiotic, dehydrins are found in a variety of plants during drought which, although drought resistant, will not survive anhydrobiotically. Many of the proteins produced by mature plants during drought are also produced by seeds in the later stages of their development (late embryogenesis-abundant proteins). Seeds dehydrate during the final stages of their development. The production of related proteins by seeds and by mature tissues during drought suggests that similar mechanisms are involved in surviving desiccation in both of these situations.

The synthesis of dehydrins by higher plants is triggered by desiccation and by exposure to abscisic acid, a plant hormone which is associated with plant responses to a variety of environmental stresses. Some of the changes observed in resurrection plants are, however, independent of the action of abscisic acid. The responses of plants to drought, cold and osmotic stress are similar since all these stresses involve problems of water availability. Exposure to low temperature induces a physiological drought since the increased resistance of roots to water movement means that the water lost by transpiration through the leaves exceeds that taken up by the roots. Many plants have been shown to synthesise dehydrins in response to cold stress. In mosses, which will survive rapid desiccation, dehydrins and sugars are present in fully hydrated tissue and protein synthesis is not triggered by desiccation. Thus, they have the compounds which protect them during desiccation permanently present in their tissues.

The function of many of these desiccation-induced proteins has yet to be deciphered. Some are proteases which may break down redundant proteins, thus releasing a source of amino acids which can be used for making new proteins. Others are involved in the synthetic pathways responsible for producing sugars and other compounds which are thought to act as protective substances during desiccation. Membrane proteins responsible for the transport of water and ions are likely to be important in controlling water flux during desiccation and rehydra-tion. Some proteins may be involved in structural rearrangements, repair after rehydration and the removal of toxins (such as superoxide and hydroxyl radicals). Proteins that are also found in the final stages of seed formation are good at absorbing and holding on to water. They may thus enable the seed to maintain a minimum water content in the face of desiccation. They may also be involved in the protection of other proteins and, together with sugars, the formation of a glassy state which stabilises the structure of the tissue. Dehydrins have been found which are associated with membranes, suggesting a role in their stabilisation. They may interact with the surface of macromolecules and membranes, together with sugars, preserving their structural integrity during desiccation.

As well as mechanisms that protect cells during dehydration and in the dried state, repair following rehydration also appears to be important. Some anhydrobiotic plants show extensive structural disruption which is repaired on rehydration. Even where structural disruption is not visible, the leakage of cell contents into the surrounding water indicates that some membrane damage has occurred. In some resurrection plants, the production of several specific proteins associated with the rehydration phase has been demonstrated. In desiccation-tolerant mosses, there is no protein synthesis during desiccation but there is during rehydration. The metabolism of mosses rapidly recovers after rehydration, but the time taken to recover depends on the rate of water loss during desiccation. The more severe the desiccation stress, the longer it takes the moss to recover. This parallels observations on the lag phase of anhydrobiotic nematodes. The proteins which are synthe-sised during rehydration have been called 'rehydrins'. Their functions are unknown but they are thought to be involved in the process of repair of the damage that results from desiccation.

There are thus a variety of mechanisms that appear to be involved in anhydrobiosis in plants. Plants vary in their relative reliance on protective mechanisms during desiccation and repair mechanisms during rehydration. Protection involves both low molecular weight substances, such as sucrose, and proteins like dehydrins. In higher plants (resurrection plants), protective mechanisms are induced by desiccation stress, while in lower plants (mosses, liverworts, algae and lichens) the protective mechanisms are perpetually present. Although the mechanisms of anhydrobiosis and drought tolerance in plants are still not fully understood, it is clear that the application of the techniques of molecular biology to the problem is making good progress towards that goal. Such an approach has yet to be applied to the study of anhydrobiosis in animals, but, no doubt, some surprises await us when it is.

anhydrobiosis in microorganisms

Bacteria are exposed to desiccation if they are carried into the air, are living on the surface of (or within) the soil when it dries, and if they are on or inside rocks and on the surface of plants and the skins of animals. Most bacteria have some degree of tolerance to desiccation. Some will survive after rapid drying, while others need a slow rate of water loss to survive. In general, they survive for longer after slow rather than fast drying. Spores, cysts and other sorts of resting stages survive better than do normal vegetative cells. These resting stages have some sort of structure outside the cell that forms a wall which protects it against environmental hazards, including desiccation. Viable bacteria have been isolated from sediments between 10000 and 13000 years old from ice cores at Vostok station in Antarctica. Under these conditions, the bacteria had been freeze dried for this period of time. Only spore-forming bacteria were found in the deeper, and therefore older, sediments.

As with animals, anhydrobiotic microorganisms accumulate treha-lose. This is thought to stabilise membranes and proteins by replacing the water which plays a part in maintaining their structure and/or by forming a glass-like state which immobilises the cell's internal organisation. Other mechanisms may be involved in anhydrobiosis, such as preventing the accumulation of reactive oxygen radicals, repair mechanisms during rehydration and proteins which protect and assist in the folding of other proteins (molecular chaperones). Microorganisms may have less ability to synthesise specific proteins involved in the response to desiccation (like the dehydrins in plants), since (being single cells) their dehydration is likely to be much more rapid than that of a plant or animal. Many bacteria secrete large amounts of material to the outside of their cells (polysaccharides - large molecules consisting of repeating sugar subunits), which form a matrix or sheath surrounding them. These extracellular polysaccharides are hygroscopic (they absorb water). Thus, they may reduce the rate of water loss during desiccation and assist with water uptake during rehydration. They may also have protective properties, such as the formation of a glass, or some other stable state, during desiccation.

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