Desiccation-tolerant Organisms Research Articles

Antioxidant defences in hydrated and desiccated states of the tardigrade Paramacrobiotus richtersi

Reactive oxygen species (ROS) are formed in all aerobic organisms, potentially leading to oxidative damage of all biological molecules. A number of defence mechanisms have developed to protect the organism from attack by ROS. Desiccation tolerance is correlated with an increase in the antioxidant potential in several organisms, but the regulation of the antioxidant defence system is complex and its role in desiccation-tolerant organisms is not yet firmly established. To determine if anhydrobiotic tardigrades have an antioxidant defence system, capable of counteracting ROS, we compared the activity of several antioxidant enzymes, the fatty acid composition and Heat shock protein expression in two physiological states (desiccated vs. hydrated) of the tardigrade Paramacrobiotus richtersi. In hydrated tardigrades, superoxide dismutase and catalase show comparable activities, while in desiccated specimens the activity of superoxide dismutase increases. Both glutathione peroxidase and glutathione were induced by desiccation. The percentage of fatty acid composition of polyunsaturated fatty acids and the amount of thiobarbituric acid reactive substances are higher in desiccated animals than in hydrated ones. Lastly, desiccated tardigrades did not differ significantly from the hydrated ones in the relative levels of Hsp70 and Hsp90. These results indicate that the possession of antioxidant metabolism could represent a crucial strategy to avoid damages during desiccation in anhydrobiotic tardigrades.

Analyse post-génomique de la tolérance à la dessiccation

Desiccation tolerance is the capacity to survive complete drying. It is an ancient trait that can be found in prokaryotes, fungi, primitive animals (often at the larval stages), whole plants, pollens and seeds. In the dry state, metabolism is suspended and the duration that anhydrobiotes can survive ranges from years to centuries. Whereas genes induced by drought stress have been successfully enumerated in tissues that are sensitive to cellular desiccation, we have little knowledge as to the adaptive role of these genes in establishing desiccation tolerance at the cellular level. This paper reviews postgenomic approaches in a variety of desiccation tolerant organisms in which the genetic responses have been investigated when they acquire the capacity of tolerating extremes of dehydration or when they are dry. Accumulation of non-reducing sugars, LEA proteins and a coordinated repression of metabolism appear to be the essential and universal attributes that can confer desiccation tolerance. The protective mechanisms of these attributes are described. Furthermore, it is most likely that other mechanisms have evolved since the function of about 30% of the genes involved in desiccation tolerance remains to be elucidated. The question of the overlap between desiccation tolerance and drought tolerance is briefly addressed.

Hydrophilic protein associated with desiccation tolerance exhibits broad protein stabilization function.

The ability of certain plants, invertebrates, and microorganisms to survive almost complete loss of water has long been recognized, but the molecular mechanisms of this phenomenon remain to be defined. One phylogenetically widespread adaptation is the presence of abundant, highly hydrophilic proteins in desiccation-tolerant organisms. The best characterized of these polypeptides are the late embryogenesis abundant (LEA) proteins, first described in plant seeds >20 years ago but recently identified in invertebrates and bacteria. The function of these largely unstructured proteins has been unclear, but we now show that a group 3 LEA protein from the desiccation-tolerant nematode Aphelenchus avenae is able to prevent aggregation of a wide range of other proteins both in vitro and in vivo. The presence of water is essential for maintenance of the structure of many proteins, and therefore desiccation stress induces unfolding and aggregation. The nematode LEA protein is able to abrogate desiccation-induced aggregation of the water-soluble proteomes from nematodes and mammalian cells and affords protection during both dehydration and rehydration. Furthermore, when coexpressed in a human cell line, the LEA protein reduces the propensity of polyglutamine and polyalanine expansion proteins associated with neurodegenerative diseases to form aggregates, demonstrating in vivo function of an LEA protein as an antiaggregant. Finally, human cells expressing LEA protein exhibit increased survival of dehydration imposed by osmotic upshift, consistent with a broad protein stabilization function of LEA proteins under conditions of water stress.

Structure and function of a mitochondrial late embryogenesis abundant protein are revealed by desiccation.

Few organisms are able to withstand desiccation stress; however, desiccation tolerance is widespread among plant seeds. Survival without water relies on an array of mechanisms, including the accumulation of stress proteins such as the late embryogenesis abundant (LEA) proteins. These hydrophilic proteins are prominent in plant seeds but also found in desiccation-tolerant organisms. In spite of many theories and observations, LEA protein function remains unclear. Here, we show that LEAM, a mitochondrial LEA protein expressed in seeds, is a natively unfolded protein, which reversibly folds into alpha-helices upon desiccation. Structural modeling revealed an analogy with class A amphipathic helices of apolipoproteins that coat low-density lipoprotein particles in mammals. LEAM appears spontaneously modified by deamidation and oxidation of several residues that contribute to its structural features. LEAM interacts with membranes in the dry state and protects liposomes subjected to drying. The overall results provide strong evidence that LEAM protects the inner mitochondrial membrane during desiccation. According to sequence analyses of several homologous proteins from various desiccation-tolerant organisms, a similar protection mechanism likely acts with other types of cellular membranes.

Constraints of tolerance: why are desiccation-tolerant organisms so small or rare?

Drying to equilibrium with the air kills nearly all animals and flowering plants, including livestock and crops. This makes drought a key ecological problem for terrestrial life and a major cause of human famine. However, the ability to tolerate complete desiccation is widespread in organisms that are either <5 mm long or found mainly where desiccation-sensitive organisms are scarce. This suggests that there is a trade-off between desiccation tolerance and growth. Recent molecular and biochemical research shows that organisms tolerate desiccation through a set of mechanisms, including sugars that replace water and form glasses, proteins that stabilize macromolecules and membranes, and anti-oxidants that counter damage by reactive oxygen species. These protections are often induced by drying, and some of the genes involved may be homologous in microbes, plants and animals. Understanding how mechanisms of desiccation tolerance may constrain growth might show how to undo the constraint in some economically important macroorganisms and elucidate the much-studied but elusive relationship between tolerance of stress and productivity.

Unifying perspectives of some mechanisms basic to desiccation tolerance across life forms

Desiccation-tolerant organisms, or life-cycle stages of particular taxa, occur among animals, higher and lower plants, terrestrial micro-algae, lichens and bacteria. Recent investigations have revealed that a number of mechanisms conferring desiccation tolerance appear to be common to the diversity of life forms able to survive extreme dehydration. In particular, parallel processes involving late embryogenic abundant (LEA) or LEA-like proteins, accumulation of sugars, aspects of active oxygen species (AOS) and non-enzymic and enzymic antioxidants have been the recent focus of attention, some across a diversity of organisms. The present contribution considers advances made from the study of these processes, particularly in enhancing current understanding of the composition of, and protection afforded by, the glassy state in desiccated organisms. Strong evidence that proteins, particularly LEAs, are implicated in glass formation is reviewed, and behaviour of such proteins upon dehydration is discussed in this context. The question of the ability of cells to survive complete water removal is considered in the context that it is unlikely, and that the basis of the deterioration of very dry seeds results from abstraction of water necessary to maintain the integrity of the intracellular glassy state. Finally, the revelation that desiccated seeds deteriorate with time, even under extremely good genebanking conditions, is discussed.

Sharing the Secrets of Life Without Water

Water and life are inseparable. No known living thing can function without water, and there is life wherever there is water on Earth (Rothschild and Mancinelli, 2001). One of the greatest problems of living on land is thus that the air is almost always deadly dry. For example, at equilbrium with air of 50% relative humidity at 200C, cells have a water content of about 0.1 g H20 g-' dry mass. This is probably not enough water to surround the proteins and membranes in a cell (Billi and Potts, 2002) and so stops metabolism and kills almost all animals and plants. However, a very few animals, a few plants, and an unknown proportion of microbes can be separated from water for a time. They can dry without dying, survive for hours to decades in a desiccated, ametabolic state, and then recover full function after rewetting. They are either small or found mainly where few other organisms can survive. Though still little-known even among biologists, this general taxonomic and ecological scope of desiccation tolerance was well-established a half-century ago (Alpert, 2000). What has awaited answer with more modern techniques are the twin puzzles raised by the prodigious survival yet modest distribution of desiccation-tolerant organisms: How do they tolerate desiccation? and Why are they not more common?

Dehydration-regulated processing of late embryogenesis abundant protein in a desiccation-tolerant nematode

Late embryogenesis abundant (LEA) proteins occur in desiccation-tolerant organisms, including the nematode Aphelenchus avenae, and are thought to protect other proteins from aggregation. Surprisingly, expression of the LEA protein AavLEA1 in A. avenae is partially discordant with that of its gene: protein is present in hydrated animals despite low cognate mRNA levels. Moreover, on desiccation, when its gene is upregulated, AavLEA1 is specifically cleaved to discrete, smaller polypeptides. A processing activity was found in protein extracts of dehydrated, but not hydrated, nematodes, and main cleavage sites were mapped to 11-mer repeated motifs in the AavLEA1 sequence. Processed polypeptides retain function as protein anti-aggregants and we hypothesise that the expression pattern and cleavage of LEA protein allow rapid, maximal availability of active molecules to the dehydrating animal.

Glass formation in plant anhydrobiotes: survival in the dry state*1

Anhydrobiotes can resist complete dehydration and survive the dry state for extended periods of time. During drying, cytoplasmic viscosity increases dramatically and in the dry state, the cytoplasm transforms into a glassy state. Plant anhydrobiotes possess large amounts of soluble non-reducing sugars and their state diagrams resemble those of simple sugar mixtures. However, more detailed in vivo measurements using techniques such as Fourier transform infrared spectroscopy and electron paramagnetic resonance spectroscopy reveal that these intracellular glasses are complex systems with properties quite different from those of simple sugar glasses. Intracellular glasses exhibit a high molecular packing and slow molecular mobility, resembling glasses made of mixtures of proteins and sugars, which potentially interact with additional cytoplasmic components such as salts, organic acids, and amino acids. Above the glass transition temperature, the cytoplasm of biological systems still exhibits a high stability and low molecular mobility, which could serve as an ecological advantage. All desiccation-tolerant organisms form glasses upon drying, but desiccation-sensitive organisms generally lose their viability during drying at water contents at which the glassy state has not yet been formed, suggesting that other factors are necessary for desiccation tolerance. Nevertheless, the formation of intracellular glasses is indispensable to survive the dry state. Storage stability of seeds and pollens is related to the molecular mobility and packing density of the intracellular glass, suggesting that the characteristic properties of intracellular glasses provide stability for long-term survival.

Membrane behavior as influenced by partitioning of amphiphiles during drying: a comparative study in anhydrobiotic plant systems

During cellular desiccation, reduction in volume can in principle cause amphiphilic compounds to partition from the cytoplasm into membranes, with structural perturbance as the result. Here, we studied the effect of partitioning of endogenous amphiphiles on membrane surface dynamics in desiccation-tolerant and -intolerant, higher and lower plant systems, using electron paramagnetic resonance (EPR) spin probe techniques. Labeling cells with the amphiphilic spin probe perdeuterated TEMPONE (PDT) enabled partitioning into the various phases to be followed. During drying, PDT molecules preferentially partitioned from the aqueous cytoplasm into the membrane surface and, at advanced stages of water loss, also into oil bodies. There was no specific partition behavior that could be correlated with lower/higher plants or with desiccation-tolerance. In vivo labeling with 5-doxylstearate (5-DS) enabled membrane surface fluidity to be characterized. In hydrated plants, the 5-DS spectra contained an immobile and a fluid component. The characteristics of the immobile component could not be specifically correlated with either lower or higher plants, or with desiccation tolerance. The relative contribution of the fluid component to the 5-DS spectra was higher in lower plants than in higher plants, but considerably decreased with drying in all desiccation-tolerant organisms. In contrast, the proportion of the fluid component in desiccation-sensitive wheat seedling root was higher than that in desiccation-tolerant wheat axis and considerably increased at the onset of water loss. We suggest that partitioning of amphipaths fluidize the membrane surface, but that in desiccation-tolerant systems the membranes are protected from excessive fluidization.

Membrane behaviour in seeds and other systems at low water content: the various effects of solutes

A common feature of desiccation-tolerant organisms, such as orthodox seeds, is the presence of large quantities of sugars, especially di- and oligosaccharides. These sugars may be one component of the suite of adaptations that allow anhydrobiotes to survive the loss of most of their cellular water. This paper describes the physical effects of dehydration on cellular ultrastructure, with particular emphasis on membranes, and explains quantitatively how sugars and other solutes can influence these physical effects. As a result of dehydration, the surfaces of membranes are brought into close approach, which causes physical stresses that can lead to a variety of effects, including demixing of membrane components and fluid-to-gel phase transitions of membrane lipids. The presence of small solutes, such as sugars, between membranes can limit their close approach and, thereby, diminish the physical stresses that cause lipid fluid-to-gel phase transitions to occur during dehydration. Thus, in the presence of intermembrane sugars, the lipid fluid-to-gel phase transition temperature (Tm) does not increase as much as it does in the absence of sugars. Vitrification of the intermembrane sugar solution has the additional effect of adding a mechanical resistance to the lipid phase transition; therefore, when sugars vitrify between fluid phase bilayers, Tm is depressed below its fully hydrated value (To). These effects occur only for solutes small enough to remain in very narrow spaces between membranes at low hydration. Large solutes, such as polymers, may be excluded from such regions and, therefore, do not diminish the physical forces that lead to membrane changes at low hydration.

Methods for dehydration-tolerance: Depression of the phase transition temperature in dry membranes and carbohydrate vitrification

AbstractAnhydrobiosis, or life without water, is the remarkable ability of certain types of plants and animals to survive almost total dehydration. This phenomenon requires a coordinated series of events within the cells of anhydrobiotes that protect their cellular components, particularly proteins and lipid membranes, from damage caused by the removal of water. Much of what is now understood about preserving biological samples during drying was learned by studying naturally desiccation-tolerant organisms and extended using model systems such as phospholipid vesicles. Most anhydrobiotic organisms accumulate disaccharides in their cells and tissues during the dehydration process. These carbohydrates, usually sucrose or trehalose, satisfy two criteria that appear to be necessary for protecting membranes during desiccation and during storage in the dry state. These requirements include: (1) depression of the gel-to-liquid crystalline phase transition temperature (Tm) in the dehydrated lipid to a temperature at or near that of the hydrated lipid, a process that appears to require a direct interaction between the carbohydrates and the lipid molecules of the membrane; and (2) formation of a carbohydrate glass with a relatively high glass transition temperature, leading to inhibition of fusion between the vesicles.

Arbutin and sucrose in the leaves of the resurrection plant Myrothamnus flabellifolia

Forty-two per cent of the desiccated leaves of Myrothamnus flabellifolia was extracted in methanol, and contained two major components, sucrose (30%) and arbutin (65%). Its arbutin content can probably explain the purported medicinal properties of this plant. By analogy with other desiccation-tolerant organisms, sucrose and arbutin may be partially responsible for the extreme resistance of Myrothamnus to environmental stress.