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INTEGR. COMP. BIOL., 45:685695 (2005)

The Limits and Frontiers of Desiccation-Tolerant Life1

PETER ALPERT2

Biology Department, University of Massachusetts, Amherst, Massachusetts 01003-9297

SYNOPSIS

. Drying to equilibrium with the air is lethal to most species of animals and plants, makingdrought (i.e., low external water potential) a central problem for terrestrial life and a major cause ofagronomic failure and human famine. Surprisingly, a wide taxonomic variety of animals, microbes, andplants do tolerate complete desiccation, defined as water content below 0.1 g H 2O g

1 dry mass. Species infive phyla of animals and four divisions of plants contain desiccation-tolerant adults, juveniles, seeds, orspores. There seem to be few inherent limits on desiccation tolerance, since tolerant organisms can surviveextremely intense and prolonged desiccation. There seems to be little phylogenetic limitation of tolerance inplants but may be more in animals. Physical constraints may restrict tolerance of animals without rigidskeletons and to plants shorter than 3 m. Physiological constraints on tolerance in plants may include controlby hormones with multiple effects that could link tolerance to slow growth. Tolerance tends to be lower inorganisms from wetter habitats, and there may be selection against tolerance when water availability ishigh. Our current knowledge of limits to tolerance suggests that they pose few obstacles to engineeringtolerance in prokaryotes and in isolated cells and tissues, and there has already been much success on thisscientific frontier of desiccation tolerance. However, physical and physiological constraints and perhaps otherlimits may explain the lack of success in extending tolerance to whole, desiccation-sensitive, multicellularanimals and plants. Deeper understanding of the limits to desiccation tolerance in living things may be

needed to cross this next frontier.

INTRODUCTION

Drying to equilibrium with even moderately dry airis instantly lethal to most species of animals andplants, making water availability one of the most im-portant ecological factors and evolutionary pressureson terrestrial life. However, there are species of ani-mals, plants, and microbes that do tolerate completedesiccation. Among animals, desiccation tolerance iscommon in three phyla: nematodes (Wharton, 2003),rotifers (Ricci, 1998; Ricci and Carprioli 2005), and

tardigrades (Wright et al., 1992; Wright, 2001). Tol-erance in juveniles is known from two more phyla, inthe encysted embryos of one crustacean genus (Clegg,2005) and in the larva of one species of fly (Kikawadaet al., 2005). Among plants, desiccation tolerance iscommon in bryophytes (Proctor and Tuba, 2002; Oli-ver et al., 2005) and rare in adult pteridophytes andangiosperms (Porembski and Barthlott, 2000) yet com-mon in their spores, seeds, and pollen (Dickie andPrichard, 2002; Tweddle et al., 2003; Farmsworth,2004; Illing et al., 2005). The extent of desiccationtolerance remains less well known in prokaryotes andfungi, but many bacteria (Potts, 1994; Guerrero et al.,1999; Billi and Potts, 2002), terrestrial microalgae(Ong et al., 1992; Agrawal and Pal, 2003), and lichens(Palmqvist, 2000; Beckett et al., 2003; de la Torre etal., 2003; Kranner et al., 2003) and some yeasts (Sales

1 From the Symposium Drying Without Dying: The ComparativeMechanisms and Evolution of Desiccation Tolerance in Animals,Microbes, and Plants presented at the Annual Meeting of the Societyfor Integrative and Comparative Biology, 48 January 2005, at SanDiego, California. Contribution no. 2242 from the University of Cal-ifornia Bodega Marine Laboratory.

2 E-mail: [email protected]

et al., 2000) tolerate desiccation, as does at least oneintertidal macroalga (Abe et al., 2001).

The taxonomic diversity of tolerant species suggeststhat the potential to evolve tolerance is widespread,and the obvious advantages of tolerance for survivalon land suggest that there should be strong selectionfor tolerance. The main evolutionary puzzle about des-iccation tolerance is therefore why it is not more com-mon. A clue to this puzzle may lie in the morphologyand ecology of tolerance: desiccation-tolerant organ-

isms are either small or rare or both. No animals longerthan 5 cm tolerate desiccation. Tolerant floweringplants grow up to about 3 m tall but are largely con-fined to highly xeric habitats or microhabitats (Alpert,2000; Alpert and Oliver, 2002). Underlying these ap-parent morphological and ecological limits to desic-cation tolerance may be inherent or phylogenetic limitsto tolerance or physical or physiological constraints.

Understanding the limits to desiccation tolerance islikely to help us extend its frontiers. Perhaps the mostexciting scientific frontier of the study of desiccationtolerance is how to induce or engineer tolerance insensitive species (Crowe et al., 2005; Potts et al.,2005). Desiccation tolerance has already been induced

in human blood cells, greatly enhancing their medicaluse. Because no crops tolerate desiccation, drought re-mains a major cause of famine, and engineering des-iccation tolerance in crops might save many morelives.

The objective of this review is to consider whatmight limit the scope of desiccation tolerance in livingthings, whether these limits pose obstacles to extend-ing the frontiers of tolerance through human interven-tion, and, if so, how these obstacles might be over-come. The review offers a definition of desiccation


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tolerance, takes up different categories of possible lim-its, and ends by considering their relationship to suc-cess in confering tolerance upon new species. Therehave a been a number of previous reviews of desic-cation tolerance in individual taxa, and the reviewseeks to build upon these by citing them and morerecent research papers.

DEFINING DESICCATION TOLERANCE

Desiccation tolerance, as used here and in theother papers in this volume, is the ability to dry toequilibrium with air that is moderately to extremelydry and then regain normal function after rehydration.There are three key points to make about this defini-tion. First, desiccation tolerance is not the same thingas drought tolerance. Instead, desiccation tolerance isone mechanism of drought tolerance. Drought is lowwater availability in the environment of an organism,whereas desiccation, as used here, is low water contentin its cells. Many organisms tolerate drought by notdesiccating, through mechanisms such as water storage

in desert cacti or water synthesis in desert rodents.These organisms cannot desiccate without dying.

Second, desiccation tolerance here refers to com-plete desiccation, meaning complete air-dryness. In theliterature on marine algae and animals (e.g., Hoffmannand Harshman, 1999; Skene, 2004), desiccation tol-erance has sometimes been used to mean the ability tosurvive drying below full or optimal water content,that is, partial desiccation. An important functional dif-ference between complete and partial desiccation isthat complete desiccation seems always to be accom-panied by cessation of measurable metabolism, where-as studies of partial desiccation often focus on main-tenance of metabolism (e.g., Danks, 2000). To specifytolerance of complete desiccation, zoologists coinedthe term, anhydrobiosis (e.g., Crowe et al., 1992).This is a synonym for desiccation tolerance as gen-erally used in the literature on plants and as used here.

Third, a good quantitative definition of completedesiccation is probably drying to 0.1 g H2O g

1 drymass (10% water content [WC]) or less. This is rough-ly equivalent to air-dryness at 50% relative humidityand 20C and corresponds to a water potential of about100 MPa (Gaff, 1997; Haranczyk et al., 1998; Proc-tor, 2003). For example, desiccation-sensitive seeds diebefore they dry to 20% WC, whereas tolerant seedssurvive below 7% (Tweddle et al., 2003). Sensitiveprokaryotes fail to survive drying to 30% WC (Billiand Potts, 2002). The threshold of 10% WC appearsto have biological meaning, since it may correspondto the point at which there is no longer enough waterto form a monolayer around macromolecules, stoppingenzymatic reactions and thus metabolism (Billi andPotts, 2002).

It is not clear whether there is a continuum betweendesiccation tolerance and sensitivity. The best evi-dence for a continuum is in seeds (Sun and Liang,2001; Song et al., 2003; Berjak and Pammenter, 2004).For instance, Tweddle et al. (2003) classed about 2%

of some 8,000 species of plants as having seeds thatwere intermediate between being desiccation-tolerantand sensitive, based on their ability to survive dryingbelow 20% but not below 10% WC. Some intertidalalgae show intermediate tolerance, defined as surviv-ing a water potential of 15 MPa but not 150MPa (Abe et al., 2001; Burritt et al., 2002). Additional

evidence comes from quantitive biochemical differ-ences between tolerant and sensitive species, and fromgradations in tolerance during development. Some ofthe mechanisms involved in desiccation tolerance byadult seed plants, such as synthesis of LEA proteinsin response to dehydration, also occur to a lesser extentin sensitive species (Bartels and Salamini, 2001; Ra-manjulu and Bartels, 2002). Tolerance diminishes withage in some adult plants (Beckett, 2001; Vander Wil-ligen et al., 2003) and animals (Ricci and Pagani,1996). On the other hand, there appear to be no reportsof adult plants or animals that survive drying to equi-librium with 80% relative humidity but not 50%. Ifthere is not a complete gap between desiccation tol-

erance and sensitivity, there is certainly a strong bi-modality, with many sensitive, some tolerant, and fewintermediate species found so far.

THE LIMITS OF TOLERANCE

Inherent limits

A limit to a trait that appears to hold for all organ-isms might be called an inherent limit, especially ifthe limit is imposed by physics or some feature thatseems essential to life. For example, if a molecule thatall living things require and that none can synthesizede novo is destroyed below a certain water content,this could be an inherent limit to the minimum water

content that living things can survive. Neither evolu-tion nor engineering is likely to transgress such limits,and one might begin to look for them empirically bylooking for records of tolerance.

The minimum water content or water potential towhich desiccation-tolerance cells can survive drying isextremely low. Bacteria can recover from drying to2% WC (Potts, 1994), and plants and animals fromdrying to equilibrium with a relative humidity of1%(e.g., Pickup and Rothery, 1991). This seems likely topermit tolerant species to survive the most intense nat-ural drought, and so not to limit their tolerance ofdrought.

The maximum length of time that desiccation-tol-erance organisms can survive in the dry state is alsovery great. A seed of the sacred lotus, Nelumbo nu-cifera, was radiocarbon dated as being about 1,100years old and successfully germinated (Shen-Miller etal., 1995). This seed was retrieved from an ancientlake bed; other records of tolerance come from spec-imens stored indoors. Mosses and liverworts have re-covered after 2025 years at air-dryness, and adult an-giosperms and pteridophytes after 5 years (Alpert andOliver, 2002). The cyanobacterium Nostoc communeshows no significant DNA damage after 13 years of


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being dry and can resume growth after 55 years inherbarium storage (Shirkey et al., 2003). Egg cysts ofArtemia survive 15 years of desiccation (Clegg, 1967).Reports that nematodes can survive for 2040 yearsmay not be reliable, but rotifers and tardigrades cansurvive at least 9 years (Guidetti and Jonsson, 2002).It seems likely that some species from every major

group with tolerant species can survive at least severalyears of desiccation. If survival under natural condi-tions is similar to survival in storage, this is not a limitto tolerance of natural drought almost anywhere onEarth, nor an obstacle to engineering tolerance.

Since drought is often accompanied by heat or cold,desiccation tolerance might not enable organisms tosurvive drought if they are sensitive to extreme tem-peratures. However, at least some desiccation-tolerantplants and animals can tolerate, not only heat and cold,but also high doses of radiation and poisons while des-iccated (Alpert, 2000; Hoekstra, 2005). For example,desiccated tardigrades can survive treatments with x-rays and UV, and temperatures from near absolute zero

to over 100C (Jonsson and Bertolani, 2001). Nema-todes, rotifers, and tardigrades can all survive fumi-gation with methyl bromide (Jonsson and Guidetti,2001). High tolerance of radioactivity has led to thediscovery of new desiccation-tolerant bacteria in ra-dioactive work areas (Phillips et al., 2002b; Venkates-waran et al., 2003). The ability of many desiccation-tolerant animals to survive environmental extremes ap-pears to exceed the extremes they ever encounter innature, posing an interesting question as to the evo-lutionary origin of desiccation tolerance (Jonsson,2003). In sum, the records of tolerance of drought andtemperature by desiccation-tolerant organisms indicatethat inherent limits to tolerance are not likely to posean obstacle to the evolution or engineering of desic-cation tolerance.

Phylogenetic limits

Although desiccation tolerance is known from manydifferent clades, it is apparently absent from most phy-la of animals, including chordates. If tolerance is aderived trait, then its evolution in clades of sensitivespecies might be limited by availability of genetic var-iation. If tolerance is a basal trait, then its re-evolutionfollowing loss within a clade might be similarly lim-ited. Such phylogenetic limits to desiccation toler-ance need not pose an obstacle to engineering toler-ance in sensitive species, since it should be possibleto introduce genes from tolerant species. Identifyingphylogenetic limits is therefore a way of identifyingopportunities to extend the frontiers of tolerance.

Two lines of evidence suggest that desiccation tol-erance in plants is not phylogenetically limited. First,phylogenetic analyses indicate that tolerance in landplants is a basal trait (Oliver et al., 2000, 2005). Tol-erance may have been lost in vegetative tissues in as-sociation with the evolution of internal water transport,but conserved in the spores and seeds of vascularplants. Independent phylogenetic analysis suggests that

desiccation tolerance in seeds is a basal characteristic(Dickie and Prichard, 2002). Re-evolution of tolerancein adult plants, which appears to have occurred at leastnine times (Oliver et al., 2000), may depend mainlyon changes in gene expression since the genes neces-sary for tolerance in seeds or pollen are generally al-ready present (Bartels and Salamini, 2001). The grass

Eragrostis nindensis, whose seeds are tolerant, seed-lings are sensitive, and adults are tolerant (Vander Wil-ligen et al., 2003), might represent an intermediate inre-evolution of adult tolerance. One might thus be ableto engineer tolerance in sensitive plants by manipulat-ing regulatory switches for tolerance genes (Bartelsand Salamini, 2001), although multiple changes in reg-ulation are likely to be needed (Ramanjulu and Bartels,2002).

The second line of evidence comes from compari-sons of the genes associated with desiccation tolerancein different plants. Considerable homology betweenthe structural genes contrasts with differences betweenthe regulatory genes, suggesting that each re-evolution

of adult tolerance has involved novel changes in theregulation of a similar set of structural genes (D. Bar-tels, personal communication). For instance, at leastfour novel regulatory genes occur in the desiccation-tolerant angiosperm Craterostigma plantagineum(Bernacchia and Furini, 2004). Comparison of phos-pholipidases in C. plantagineum and Arabidopsis thal-iana, which is desiccation-sensitive, suggests that ho-mologous genes have been recruited to different pur-poses in the two species, to senescence and stomatalclosure in Arabidopsis and to desiccation tolerance inC. plantagineum (Bartels and Salamini, 2001); evolu-tion of tolerance may partly involve recruiting moreexisting genes to that purpose through regulation. Thisis not to suggest that the structural genes for toleranceare identical in all desiccation-tolerant plants. Thereare different routes to the synthesis of sucrose asso-ciated with desiccation tolerance in different species(Bartels and Mattar, 2002). Novel structural geneshave been identified in individual species, such asXvPer1, which encodes a stress-inducible antioxidantenzyme in Xerophyta viscosa (Mowla et al., 2002); thegene family CpPTP, which encodes proteins that mayreversibly restructure chloroplasts during desiccationin C. plantagineum (Phillips et al., 2002a); and CpEdi-9, which encodes a hydrophilic protein in mature seedsand in response to dehydration in leaf phloem in C.plantagineum (Rodrigo et al., 2004).

Further support for the idea that evolution of des-iccation tolerance in plants is not strongly limited byavailability of genetic variation comes from the bio-geography of tolerant angiosperms (Porembski andBarthlott, 2000). The majority are found largely onlarge rock outcrops, or inselbergs, in the tropics. Dif-ferent continents have very different sets of species,consistent with evolution of tolerance in differentgroups of angiosperms subject to similar selectivepressures. Biogeography does suggest that there maybe some phylogenetic limitation to tolerance, since


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rock pools on inselbergs in western and in southeasternAfrica both have tolerant bryophytes and lichens butonly those in southeastern Africa have tolerant angio-sperms (Krieger et al., 2000).

In contrast, it seems possible that the extent of des-iccation tolerance may be phylogenetically limited inmajor clades of animals. Direct evidence is very lim-

ited, since there appear to be no phylogenetic analysesof tolerance in animals apart from a survey suggestingthat tolerance is a basal characteristic in the class Bdel-loidea of the rotifers (Ricci, 1998). There is also someremarkable counterevidence in the apparent homologyof some genes associated with desiccation tolerance indifferent phyla of animals and even across animals,plants, and microbes. Named for their expression dur-ing induction of desiccation tolerance in maturingseeds, these Late Embryogenesis Abundant (LEA)genes encode a set of hydrophilic proteins that includedehydrins, which are produced in response to dryingin tolerant plants. In animals, the products of LEAgenes may act as molecular chaperones for DNA or

counter physical stress during desiccation (Wise,2003). In plants, they may increase the transition tem-perature and hydrogen bonding strength of sucroseglasses (Wolkers et al., 2001a), helping to inhibitmembrane fusion, protein denaturation, and effects offree radicals (Oliver et al., 2001). LEA homologueshave been found in nematodes and bacteria (Goyal etal., 2003; Browne et al., 2004; Wise and Tunnacliffe,2004), yeast (Garay-Arroyo et al., 2000; Sales et al.,2000), pollen (Wolkers et al., 2001a), bryophytes (Al-pert and Oliver, 2002), roots and shoots of dicotyle-donous plants (Schiller et al., 1997), and shoots ofmonocots (Bartels and Mattar, 2002).

Akin to phylogenetic limitation due to absence ofgenes for tolerance might be limits due to linkage be-tween genes for tolerance and other genes, such thatselection for traits that are not functionally linked totolerance nevertheless counters selection for tolerance(e.g., Feldgarden et al., 2003). For example, geneticlinkage between certain leaf traits and patterns of al-location in some plants appears to counter simulta-neous adaptation of leaves and allocation to climatechange (Etterson and Shaw, 2001). In Arabidopsis,correlation between water use efficiency and time re-quired to reach flowering might seem to indicate afunctional trade-off between drought tolerance and rateof reproduction but instead be at least partly due tofixation of genes that cause pleiotropy (Mckay et al.,2003). There seems to be no evidence so far for limitson tolerance due to genetic linkage in plants. However,lack of available genetic variation is one of severalplausible explanations for the apparent complete ab-sence of desiccation tolerance in most major clades ofanimals.

Physiological constraints

A limit to one trait that is imposed by the state ofanother trait is reasonably termed a constraint, giv-en the original sense of constrain, to force to do

something; and its literal sense, to tie together or com-press by tying (OED, 1989). A physiological con-straint on a biological unit may occur when it is con-trolled by the same physiological mechanism as an-other trait, since selection and regulation of the firsttrait may be limited by selection and regulation of theother. A prime example in animals is the regulation of

multiple traits by endocrine systems (Ricklefs and Wi-kelski, 2002; Zera and Zhao, 2004). In plants, controlof multiple traits by hormones can also impose phys-iological constraints (Farnsworth, 2004); tolerance ofdesiccation may be constrained by multiple effects ofthe hormone, abscisic acid or ABA.

ABA upregulates some aspects of desiccation tol-erance in bryophytes (Beckett et al., 2000; Guschinaet al., 2002; Zeng et al., 2002; Mayaba and Beckett,2003), seeds (Wakui and Takahata, 2002), vegetativetissues of angiosperms (Bartels and Salamini, 2001;Bernacchia and Furini, 2004; Vicre et al., 2004), andpossibly ferns (Pence, 2000). This has not been spe-cifically tied to other effects of the hormone in tolerant

species, but ABA tends in general to have effects thatlead to slower growth as well as higher tolerance ofstress by plants (Farnsworth, 2004) and selection forgrowth might counter selection for tolerance. For in-stance, a population of the desiccation-sensitive herbImpatiens capensis from a relatively wet habitatshowed less response to ABA than a population froma drier habitat; this could reflect selection in the firstpopulation for avoidance of reduction in reproductiveoutput due to ABA-mediated responses to cues fordrought that are inappropriate in a wetter habitat (Hes-chel and Hausmann, 2001).

Cytokinins are a second important example of planthormones with multiple effects (Farnsworth, 2004).No role has been established for cytokinins in desic-cation tolerance, but levels change in association withdesiccation in at least one tolerant species. As they dry,leaves ofCraterostigma wilmsii show a initial decreasein two cytokinins, zeatin and zeatin riboside, and thenan increase below 20% relative water content (RWC:WC/WC at full turgor; Vicre et al., 2004). Levels re-turn to normal after rehydration to 70% RWC. Under-standing the physiological constraints governed byplant hormones could identify opportunities to engi-neer tolerance of desiccation and other stresses (Farns-worth, 2004; Kim et al., 2004). For instance, if controlby ABA couples tolerance to slow growth, one mighttry to engineer release of tolerance from this control.Such release has already been engineered in transgeniccallus tissue of C. plantigineum through constitutiveexpression of the regulatory gene CDT-1 (Bartels andSalamini, 2001).

Another potential area of physiological constraint ontolerance may be regulation of senescence. Glutathioneis oxidized during desiccation in lichens, and desic-cation tolerance is correlated with the ability to reduceit again upon rehydration (Kranner, 2002). Correlationbetween levels of reduced glutathione and tolerancecould be related to prevention of oxidation damage


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during desiccation. For example, plants of an intertidalred algae collected from higher in the intertidal zoneand therefore subject to greater desiccation showedgreater upregulation of enzymes required to regenerateglutathione and ascorbate during dehydration down to40% WC and produced less hydrogen peroxide andlipid hydroperoxides (Burritt et al., 2002). However, a

low ratio of reduced to oxidized glutathione can alsocue apoptosis, suggesting that rapid reduction may alsobe needed to avoid prompting cell death (I. Kranner,personal communication).

Physical constraints

Physical traits that might constrain tolerance of des-iccation include rigidity and size. Tolerant animalsshrink as they desiccate, and it has been suggested thatactive contraction helps prevent mechanical damageduring desiccation (e.g., Ricci et al., 2003). For in-stance, rotifers and tardigrades adopt distinctive, high-ly compact forms as they dry (Jonsson, 2001; Ricci etal., 2003), and coiling has been used as a criterion for

non-lethal desiccation in nematodes (Treonis et al.,2000). Moreover, no desiccation-tolerant animals haverigid skeletons, at least not at the life stages wheretolerance is present.

Desiccation-tolerant plants do have relatively rigidcell walls and even wood. Special wall morphologiesmay promote folding as cells dry and reduce mechan-ical stress during desiccation in pteridophytes (Thom-son and Platt, 1997) and angiosperms (Farrant et al.,1999; Vander Willigen et al., 2004). Some angio-sperms change their cell wall composition during dry-ing (Vicre et al., 2004). In Craterostigma plantige-neum, an increase in the transcription of an alpha-ex-pansin cDNA during dehydration is associated with arise in activity of expansin and in cell wall flexibility(Jones and McQueen-Mason, 2004). Shrinkage ofplant cells during drying may be especially severe dueto the presence of large, water-filled vacuoles, andsome tolerant species appear to alleviate this by re-placing large vacuoles with numerous small ones andfilling them with non-aqueous compounds as cells dry(Thomson and Platt, 1997; Farrant, 2000).

Water transport through xylem imposes a second,related physical constraint on desiccation tolerance invascular plants (e.g., Sherwin et al., 1998). Undermost conditions and in all plants taller than about 3m, water is pulled up rather than pushed up throughfiles of dead cells in the xylem. When the negativepressure in the xylem exceeds that required to draw inan air bubble from a neighboring, air-filled conduit,cavitation results and interrupts water flow. In tolerantshrubs such as Myrothamnus flabellifolia, water col-umns must be regenerated after desiccation, and thisis variously thought to occur via root pressure (Schnei-der et al., 2000) or capillary action (Sherwin et al.,1998), possibly modified by an unusual lining of lipidson the inner surface of the cell wall (Wagner et al.,2000). Refilling may delay recovery from desiccation(Sherwin and Farrant, 1996) and is likely to be im-

possible at heights greater than 3 m. This may explainwhy there are no desiccation-tolerant trees, and a pre-ponderance of tree species or possibly greater diffi-culty in refilling their special form of xylem may ex-plain why there are no desiccation-tolerant adult gym-nosperms, despite the presence of tolerance in gym-nosperm pollen and seeds. Physical constraints on

rehydration have been less explored in animals, butneeds for water diffusion through cells might requireall tolerant organisms to be small or thin (Potts, 2001).

Differentiation and multicellularity do not imposeobvious constraints on desiccation tolerance in plantsor animals. Desiccation-tolerant plants have a widerange of specialized forms. For instance, the floatingand submerged leaves of Craterostigma intrepidus arespecialized as sun and shade leaves (Woitke et al.,2004). Tolerance in the larva of the fly Polypedilumvanderplanki shows that tolerance is compatible withhaving a central nervous system; excised tissue with-out nerves also survives desiccation in this species,showing that the nervous system is not essential for

tolerance (Watanabe et al., 2002).

Ecological limits

The likelihood that ability to tolerate intense or pro-longed drought does not effectively limit desiccationtolerance in living things is corroborated by their oc-currence in extreme habitats, such as in the hottest andcoldest deserts and on bare, non-porous rock (Alpert,2000; Alpert and Oliver, 2002). Tolerant cyanobacteriaare probably the main source of primary productivityin some sand crusts in the Negev Desert (Harel et al.,2004); they can recover 50% of their normal photo-system II activity in as little as five minutes after re-wetting. The Dry Valleys of Antartica may be largelypopulated by desiccation-tolerant species, includingnematodes in the soil (Treonis and Wall, 2005) andendolithic communities of lichens, cyanobacteria, andother bacteria (Hughes and Lawley, 2003). Graniticoutcrops in Brazil sport films composed mainly of cy-anobacteria plus cyanobacterial lichens (Buedel et al.,2002). Even a complete lack of liquid water may notexclude desiccation-tolerant species. For example, thealga Trentepolia odorata can rehydrate and photosyn-thesize with water vapor (Ong et al., 1992).

On a finer scale, desiccation-tolerant plant speciesmay nevertheless be excluded from the most xeric mi-crosites within a habitat by inability to maintain a pos-itive carbon balance over repeated cycles of desicca-tion and rehydration (Alpert, 1990, 2000). For in-stance, mosses at a site in the chaparral of southernCalifornia are much less abundant on the equator-fac-ing than on the pole-facing surfaces of granitic boul-ders (Alpert, 1985). This is probably because they areunable to recoup respiratory losses of carbon duringthe night and during recovery from desiccation beforedesiccation in the sun ends photosynthesis again (Al-pert and Oechel, 1985).

Although some aquatic plants and animals toleratedesiccation (e.g., Schiller et al., 1997; Ricci, 1998),


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desiccation tolerance is negatively associated with oc-currence in moist habitats. By far the most completeevidence for this is in seeds. Based on a survey ofover 8,000 species of angiosperms, Tweddle et al.(2003) found that the proportion of species with des-iccation-sensitive seeds tended to be higher in warmerand wetter habitats, and was highest (45%) among

non-pioneer species in evergreen tropical forests.Within the genus Coffea, degree of desiccation toler-ance in seeds decreases as number of dry months be-tween dispersal and the start of the wet season de-creases (Dussert et al., 2000)

Other comparisons between plants have mostly eachinvolved just a few species but generally also foundthat those from wetter habitats were less tolerant. Forinstance, lower ability to maintain photosynthesis dur-ing desiccation was associated with higher ambientwater availability in three species of Antarctic mosses(Robinson et al., 2000). Sensitivity to desiccation wasassociated with habitat wetness in other studies ofmosses by Franks and Bergstrom (2000) and Seel et

al. (1992), and in the common but not the rare mossesstudied by Cleavitt (2002). Schipperges and Rydin(1998) reported no relationship between recovery frompartial drying and microhabitat in six species of themoss genus Sphagnum, but none of these species tol-erated complete desiccation. Microdistributions ofeight epiphytic pteridophytes in a Mexican cloud for-est correlated more closely with tolerance of desicca-tion and high light than with maximum performance(Hietz and Briones, 2001). Exposure to high UV orPAR while desiccated did not affect photosystem II inthe xeric lichen Xanthorea parietina, but did have anegative effect on a lichen from more shaded habitats(Solhaug et al., 2003). Similarly, of three mosses, onlythe one from the most xeric habitats, Tortula rurali-formis, recovered photosythetic capacity if desiccatedin the light (Seel et al., 1992).

Comparisons between animals are fewer and lessconsistent. Solomon et al. (1999) reported an associ-ation between habitat dryness and desiccation toler-ance in three strains of the nematode Steinernema fel-tiae. Gal et al. (2001) found higher transcription ofglycogen synthase during dehydration of S. feltiaefrom a temperate than from a semi-arid habitat, sug-gesting a shift away from production of trehalose, asugar associated with tolerance, in the temperate strain.No subtidal marine tardigrades tolerate desiccation,whereas some of the intertidal (Clegg, 1967; Jonssonand Jaremo, 2003), semi-aquatic freshwater (Jonssonand Bertolani, 2001), and terrestrial species do. Of twocongeneric tardigrades from the upper intertidal, theone from exposed microsites was tolerant while theone from within barnacles was not (Grngaard et al.,1990; cf., Jonsson and Jaremo, 2003), and tardigradesin a population from a habitat with higher relative hu-midity and temperature were less tolerant than thosein a population from a less humid, cooler habitat (Hor-ikawa and Higashi, 2004). However, there was no dif-ference in tolerance between populations of tardi-

grades from the two sites sampled by Jonsson et al.,(2001), nor between populations of nematodes fromGreece and the UK (Menti et al., 1997). Torrenteraand Dodson (2004) noted differences in the phenologyof populations of Artemia in hypersaline pools andsalterns that differed in salinity, pH, and desiccationin Yucatan. In one of the most systematic comparisons

of tolerance in a group of animals, Ricci (1998) notedthat, of 15 rotifer species representing all four familesof the class Bbelloidea, all three desiccation-sensitivespecies were aquatic.

Two possible explanations for the scarcity of des-iccation-tolerant species in moist habitats are compet-itive exclusion by desiccation-sensitive species and se-lection against tolerance when water availability ishigh. Although there appear to be no experimentaltests for association between tolerance and competitiveability, it may be that desiccation-tolerant extremo-philes are actually competitively inferior normifu-ges. Work on mutants of Arabidopsis suggests thatloss of desiccation tolerance in seeds may require

changes in relatively few genes (Ooms et al., 1993).Derived desiccation sensitivity in seeds and rotifersfrom moist habitats could be due to lack of selectionto maintain tolerance or to selection against it, if thereis a trade off between tolerance and growth or repro-duction.

FRONTIERS OF TOLERANCE

The two main applications of research on desicca-tion tolerance in the past two decades have been at-tempts to induce tolerance in human cells for medicalpurposes and to engineer tolerance in crop plants tomake them less vulnerable to drought. Researchershave also tried to engineer or induce tolerance in ag-ronomic bacteria and in nematodes used for biologicalcontrol. Knowing what limits desiccation tolerance inprokaryotes and nematodes could also help controlpathenogenic species that are naturally desiccation-tol-erant (Breeuwer et al., 2003). There has been consid-erable success in conferring tolerance on isolatedmembrances and enzymes and on single cells (Billiand Potts, 2002; Crowe et al., 2005; Potts et al., 2005),which seems to accord with what we know about theinherent and phylogenetic limits of tolerance. Therehas been almost no success in crop plants, suggestingthe importance of physical or physiological constraintson tolerance in whole, multicellular organisms.

Making single cells tolerate desiccation

Desiccation tolerance in mammalian cells can be in-duced by treatment with trehalose, a sugar accumulat-ed during drying in many desiccation-tolerant animals.Whereas fresh human blood platelets have a shelf lifeof only about five days, platelets freeze-dried in a so-lution of trehalose can be stored much longer and thenrehydrated for use (Wolkers et al., 2001b, 2002). Thistechnique specifically preserves membrane microdo-mains (Crowe et al., 2003). Gordon et al. (2001) in-cubated human mesenchymal stem cells in 50 mmol


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trehalose and 3% glycerol, air-dried and stored themunder vacuum, and express mailed them from San Di-ego to Baltimore, where they recovered normal mor-phology, lability, and regeneration capacity after re-hydration. Incubation in a medium with a high treha-lose concentration can make corneal epithelial cellsdesiccation-tolerant (Matsuo, 2001). Chen et al. (2001)

introduced trehalose into mammalian cells with an en-gineered protein that formed pores in the plasma mem-brane; having about 1010 molecules of trehalose percell enabled cells to survive at 5% relative humidityand 20C for weeks. There are now techniques to loadhuman red blood cells with trehalose (Satpathy et al.,2004). Under optimal conditions, sugar alone may suf-fice to make mammalian cells tolerate desiccation(Crowe et al., 2002). Human cells can also toleratedesiccation in the absence of added sugars if driedslowly and stored under vacuum (Puhlev et al., 2001).

Desiccation tolerance can be induced in the bacteriaEscherichia coli and Pseudomonas putida with eithertrehalose or hydroectoine (de Castro et al., 2000; Tun-nacliffe et al., 2001; Manzanera et al., 2002, 2004).Loading the nitrogen-fixing mutualist bacterium Bra-dyrhizobium japonicum with trehalose by incubating itin the sugar during growth greatly improved its sub-sequent survival of desiccation, which is a major causeof failure of inoculation of leguminous crops with thebacterium in the field (Streeter, 2003).

There has also been some success in engineeringdesiccation tolerance in mammalian cells and bacteria.Guo et al. (2000) used a recombinant adenovirus vec-tor to express the otsA and otsB genes of Escherichiacoli, which encode enzymes that synthesize trehalose,in human primary fibroblasts and were able to main-

tain infected cells in the dry state for up to five days.However, engineering mouse cells to produce 80 mmoltrehalose did not make them fully desiccation-tolerant(de Castro and Tunnacliffe, 2000), even when extra-cellular trehalose was supplied (Tunnacliffe et al.,2001). Moving the spsA gene of a cyanobacterium intoE. coli resulted in production of sucrose-6-phosphatesynthetase and a 10,000-fold increase in survival ofdesiccation (Billi et al., 2000). It is also possible totransfer genes into naturally tolerant cyanobacteria(Billi et al., 2001), which might thereby become a des-iccation-tolerant source of useful metabolites. Genetransfer may even have been significant in some nat-ural origins of desiccation tolerance: the tolerant bac-

terium Dienococcus radiodurans appears to have ac-quired homologues of putative plant desiccation tol-erance genes by horizontal transfer (Makarova et al.,2001).

The success in making single cells tolerate desic-cation fits with knowledge of the limits of tolerance.There seem to be no significant inherent limits on des-iccation tolerance and no physical or physiologicalconstraints that apply to single, non-rigid prokaryoteor animal cells. If phylogeny limits tolerance in manyanimals via lack of available genetic variation for tol-

erance, then introducing genes for tolerance might beexpected to successfully overcome this limit.

Making whole organisms tolerate desiccation

It has so far been possible to increase tolerance ofpartial desiccation in multicellular animals and plants,but not to make them desiccation-tolerant. Treatment

with cold can increase production of trehalose and tol-erance in nematodes used for biological control (Gre-wal and Jagdale, 2002). Breeding can increase toler-ance of partial desiccation to some extent in the en-tomopathogenic nematode Heterorhabditis bacterio-phora (Strauch et al., 2004). Introduction of trehalosebiosynthetic genes into plants can increase their pro-duction of trehalose and their tolerance of variousstresses but has not resulted in desiccation tolerance(Penna, 2003). For example, regulated overexpressionof genes from E. coli in rice plants increased theirtrehalose concentration 310 times (Garg et al., 2002).The plants showed relatively low photooxidation andhigh ability to accumulate nutrients under salt,

drought, or cold stress but did not tolerate desiccation.Lack of success in making whole organisms tolerate

desiccation may suggest that the known limits that ap-ply to whole, multicellular organisms set major obsta-cles to engineering tolerance in them. For example,physical constraints in both animals and plants andphysiological constraints due to involvement of hor-nones in tolerance in plants are likely to require morethan just the engineering of synthesis of sugars to con-fer tolerance. To reduce mechanical stress, animalsmay need to contract during desiccation and plantsneed to have more flexible cell walls. Tolerance is un-likely to be engineered in plants taller than 3 m dueto the problem of refilling xylem. What we know about

the natural limits to desiccation tolerance in livingthings offers encouragement for further efforts to ar-tificially extend tolerance to single-celled organismsand to individual cells and tissues of multicellularones. However, we may need to know more about thelimits of desiccation tolerance before we can expect toextend it to desiccation-sensitive whole plants andmetazoans.

ACKNOWLEDGMENTS

I thank the participants in the symposium on des-iccation tolerance at the 2005 meeting of the Societyfor Integration and Comparative Biology for sharingtheir knowledge and insights.

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