INTEGR.COMP.BIOL., 45:685±695 (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, making drought (i.e., low external water potential) a central problem for terrestrial life and a major cause of agronomic failure and human famine. Surprisingly, a wide taxonomic variety of animals, microbes, and Ϫ1 plants do tolerate complete desiccation, de®ned as water content below 0.1 g H2Og dry mass. Species in ®ve phyla of animals and four divisions of plants contain desiccation-tolerant adults, juveniles, seeds, or spores. There seem to be few inherent limits on desiccation tolerance, since tolerant organisms can survive extremely intense and prolonged desiccation. There seems to be little phylogenetic limitation of tolerance in plants but may be more in animals. Physical constraints may restrict tolerance of animals without rigid skeletons and to plants shorter than 3 m. Physiological constraints on tolerance in plants may include control by hormones with multiple effects that could link tolerance to slow growth. Tolerance tends to be lower in organisms from wetter habitats, and there may be selection against tolerance when water availability is high. Our current knowledge of limits to tolerance suggests that they pose few obstacles to engineering tolerance in prokaryotes and in isolated cells and tissues, and there has already been much success on this scienti®c frontier of desiccation tolerance. However, physical and physiological constraints and perhaps other limits may explain the lack of success in extending tolerance to whole, desiccation-sensitive, multicellular animals and plants. Deeper understanding of the limits to desiccation tolerance in living things may be needed to cross this next frontier.

INTRODUCTION et al., 2000) tolerate desiccation, as does at least one Drying to equilibrium with even moderately dry air intertidal macroalga (Abe et al., 2001). is instantly lethal to most species of animals and The taxonomic diversity of tolerant species suggests plants, making water availability one of the most im- that the potential to evolve tolerance is widespread, portant ecological factors and evolutionary pressures and the obvious advantages of tolerance for survival on terrestrial life. However, there are species of ani- on land suggest that there should be strong selection mals, plants, and microbes that do tolerate complete for tolerance. The main evolutionary puzzle about des- desiccation. Among animals, desiccation tolerance is iccation tolerance is therefore why it is not more com- common in three phyla: nematodes (Wharton, 2003), mon. A clue to this puzzle may lie in the morphology rotifers (Ricci, 1998; Ricci and Carprioli 2005), and and ecology of tolerance: desiccation-tolerant organ- tardigrades (Wright et al., 1992; Wright, 2001). Tol- isms are either small or rare or both. No animals longer erance in juveniles is known from two more phyla, in than 5 cm tolerate desiccation. Tolerant ¯owering the encysted embryos of one crustacean genus (Clegg, plants grow up to about 3 m tall but are largely con- 2005) and in the larva of one species of ¯y (Kikawada ®ned to highly xeric habitats or microhabitats (Alpert, et al., 2005). Among plants, desiccation tolerance is 2000; Alpert and Oliver, 2002). Underlying these ap- common in bryophytes (Proctor and Tuba, 2002; Oli- parent morphological and ecological limits to desic- ver et al., 2005) and rare in adult pteridophytes and cation tolerance may be inherent or phylogenetic limits angiosperms (Porembski and Barthlott, 2000) yet com- to tolerance or physical or physiological constraints. mon in their spores, seeds, and pollen (Dickie and Understanding the limits to desiccation tolerance is Prichard, 2002; Tweddle et al., 2003; Farmsworth, likely to help us extend its frontiers. Perhaps the most 2004; Illing et al., 2005). The extent of desiccation exciting scienti®c frontier of the study of desiccation tolerance remains less well known in prokaryotes and tolerance is how to induce or engineer tolerance in fungi, but many bacteria (Potts, 1994; Guerrero et al., sensitive species (Crowe et al., 2005; Potts et al., 1999; Billi and Potts, 2002), terrestrial microalgae 2005). Desiccation tolerance has already been induced (Ong et al., 1992; Agrawal and Pal, 2003), and lichens in human blood cells, greatly enhancing their medical (Palmqvist, 2000; Beckett et al., 2003; de la Torre et use. Because no crops tolerate desiccation, drought re- al., 2003; Kranner et al., 2003) and some yeasts (Sales mains a major cause of famine, and engineering des- iccation tolerance in crops might save many more lives. 1 From the Symposium Drying Without Dying: The Comparative The objective of this review is to consider what Mechanisms and Evolution of Desiccation Tolerance in Animals, might limit the scope of desiccation tolerance in living Microbes, and Plants presented at the Annual Meeting of the Society things, whether these limits pose obstacles to extend- for Integrative and Comparative Biology, 4±8 January 2005, at San ing the frontiers of tolerance through human interven- Diego, California. Contribution no. 2242 from the University of Cal- ifornia Bodega Marine Laboratory. tion, and, if so, how these obstacles might be over- 2 E-mail: [email protected] come. The review offers a de®nition of desiccation

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tolerance, takes up different categories of possible lim- of some 8,000 species of plants as having seeds that its, and ends by considering their relationship to suc- were intermediate between being desiccation-tolerant cess in confering tolerance upon new species. There and sensitive, based on their ability to survive drying have a been a number of previous reviews of desic- below 20% but not below 10% WC. Some intertidal cation tolerance in individual taxa, and the review algae show intermediate tolerance, de®ned as surviv- seeks to build upon these by citing them and more ing a water potential of ϽϪ15 MPa but not ϽϪ150 recent research papers. MPa (Abe et al., 2001; Burritt et al., 2002). Additional evidence comes from quantitive biochemical differ- DEFINING DESICCATION TOLERANCE ences between tolerant and sensitive species, and from ``Desiccation tolerance,'' as used here and in the gradations in tolerance during development. Some of other papers in this volume, is the ability to dry to the mechanisms involved in desiccation tolerance by equilibrium with air that is moderately to extremely adult seed plants, such as synthesis of LEA proteins dry and then regain normal function after rehydration. in response to dehydration, also occur to a lesser extent There are three key points to make about this de®ni- in sensitive species (Bartels and Salamini, 2001; Ra- tion. First, desiccation tolerance is not the same thing manjulu and Bartels, 2002). Tolerance diminishes with as drought tolerance. Instead, desiccation tolerance is age in some adult plants (Beckett, 2001; Vander Wil- one mechanism of drought tolerance. Drought is low ligen et al., 2003) and animals (Ricci and Pagani, water availability in the environment of an organism, 1996). On the other hand, there appear to be no reports whereas desiccation, as used here, is low water content of adult plants or animals that survive drying to equi- in its cells. Many organisms tolerate drought by not librium with 80% relative humidity but not 50%. If desiccating, through mechanisms such as water storage there is not a complete gap between desiccation tol- in desert cacti or water synthesis in desert rodents. erance and sensitivity, there is certainly a strong bi- These organisms cannot desiccate without dying. modality, with many sensitive, some tolerant, and few Second, desiccation tolerance here refers to com- intermediate species found so far. plete desiccation, meaning complete air-dryness. In the literature on marine algae and animals (e.g., Hoffmann THE LIMITS OF TOLERANCE and Harshman, 1999; Skene, 2004), desiccation tol- Inherent limits erance has sometimes been used to mean the ability to survive drying below full or optimal water content, A limit to a trait that appears to hold for all organ- that is, partial desiccation. An important functional dif- isms might be called an ``inherent limit,'' especially if ference between complete and partial desiccation is the limit is imposed by physics or some feature that that complete desiccation seems always to be accom- seems essential to life. For example, if a molecule that panied by cessation of measurable metabolism, where- all living things require and that none can synthesize as studies of partial desiccation often focus on main- de novo is destroyed below a certain water content, tenance of metabolism (e.g., Danks, 2000). To specify this could be an inherent limit to the minimum water tolerance of complete desiccation, zoologists coined content that living things can survive. Neither evolu- the term, ``anhydrobiosis'' (e.g., Crowe et al., 1992). tion nor engineering is likely to transgress such limits, This is a synonym for ``desiccation tolerance'' as gen- and one might begin to look for them empirically by erally used in the literature on plants and as used here. looking for records of tolerance. Third, a good quantitative de®nition of complete The minimum water content or water potential to Ͻ Ϫ1 which desiccation-tolerance cells can survive drying is desiccation is probably drying to 0.1gH2Og dry mass (10% water content [WC]) or less. This is rough- extremely low. Bacteria can recover from drying to ly equivalent to air-dryness at 50% relative humidity 2% WC (Potts, 1994), and plants and animals from and 20ЊC and corresponds to a water potential of about drying to equilibrium with a relative humidity of Ͻ1% Ϫ100 MPa (Gaff, 1997; Haranczyk et al., 1998; Proc- (e.g., Pickup and Rothery, 1991). This seems likely to tor, 2003). For example, desiccation-sensitive seeds die permit tolerant species to survive the most intense nat- before they dry to 20% WC, whereas tolerant seeds ural drought, and so not to limit their tolerance of survive below 7% (Tweddle et al., 2003). Sensitive drought. prokaryotes fail to survive drying to 30% WC (Billi The maximum length of time that desiccation-tol- and Potts, 2002). The threshold of 10% WC appears erance organisms can survive in the dry state is also to have biological meaning, since it may correspond very great. A seed of the sacred lotus, Nelumbo nu- to the point at which there is no longer enough water cifera, was radiocarbon dated as being about 1,100 to form a monolayer around macromolecules, stopping years old and successfully germinated (Shen-Miller et enzymatic reactions and thus metabolism (Billi and al., 1995). This seed was retrieved from an ancient Potts, 2002). lake bed; other records of tolerance come from spec- It is not clear whether there is a continuum between imens stored indoors. Mosses and liverworts have re- desiccation tolerance and sensitivity. The best evi- covered after 20±25 years at air-dryness, and adult an- dence for a continuum is in seeds (Sun and Liang, giosperms and pteridophytes after 5 years (Alpert and 2001; Song et al., 2003; Berjak and Pammenter, 2004). Oliver, 2002). The cyanobacterium Nostoc commune For instance, Tweddle et al. (2003) classed about 2% shows no signi®cant DNA damage after 13 years of INTRODUCTION 687 being dry and can resume growth after 55 years in desiccation tolerance in seeds is a basal characteristic herbarium storage (Shirkey et al., 2003). Egg cysts of (Dickie and Prichard, 2002). Re-evolution of tolerance Artemia survive 15 years of desiccation (Clegg, 1967). in adult plants, which appears to have occurred at least Reports that nematodes can survive for 20±40 years nine times (Oliver et al., 2000), may depend mainly may not be reliable, but rotifers and tardigrades can on changes in gene expression since the genes neces- survive at least 9 years (Guidetti and Jonsson, 2002). sary for tolerance in seeds or pollen are generally al- It seems likely that some species from every major ready present (Bartels and Salamini, 2001). The grass group with tolerant species can survive at least several Eragrostis nindensis, whose seeds are tolerant, seed- years of desiccation. If survival under natural condi- lings are sensitive, and adults are tolerant (Vander Wil- tions is similar to survival in storage, this is not a limit ligen et al., 2003), might represent an intermediate in to tolerance of natural drought almost anywhere on re-evolution of adult tolerance. One might thus be able Earth, nor an obstacle to engineering tolerance. to engineer tolerance in sensitive plants by manipulat- Since drought is often accompanied by heat or cold, ing regulatory switches for tolerance genes (Bartels desiccation tolerance might not enable organisms to and Salamini, 2001), although multiple changes in reg- survive drought if they are sensitive to extreme tem- ulation are likely to be needed (Ramanjulu and Bartels, peratures. However, at least some desiccation-tolerant 2002). plants and animals can tolerate, not only heat and cold, The second line of evidence comes from compari- but also high doses of radiation and poisons while des- sons of the genes associated with desiccation tolerance iccated (Alpert, 2000; Hoekstra, 2005). For example, in different plants. Considerable homology between desiccated tardigrades can survive treatments with x- the structural genes contrasts with differences between rays and UV, and temperatures from near absolute zero the regulatory genes, suggesting that each re-evolution to over 100ЊC (JoÈnsson and Bertolani, 2001). Nema- of adult tolerance has involved novel changes in the todes, rotifers, and tardigrades can all survive fumi- regulation of a similar set of structural genes (D. Bar- gation with methyl bromide (JoÈnsson and Guidetti, tels, personal communication). For instance, at least 2001). High tolerance of radioactivity has led to the four novel regulatory genes occur in the desiccation- discovery of new desiccation-tolerant bacteria in ra- tolerant angiosperm Craterostigma plantagineum dioactive work areas (Phillips et al., 2002b; Venkates- (Bernacchia and Furini, 2004). Comparison of phos- waran et al., 2003). The ability of many desiccation- pholipidases in C. plantagineum and Arabidopsis thal- tolerant animals to survive environmental extremes ap- iana, which is desiccation-sensitive, suggests that ho- pears to exceed the extremes they ever encounter in mologous genes have been recruited to different pur- nature, posing an interesting question as to the evo- poses in the two species, to senescence and stomatal lutionary origin of desiccation tolerance (JoÈnsson, closure in Arabidopsis and to desiccation tolerance in 2003). In sum, the records of tolerance of drought and C. plantagineum (Bartels and Salamini, 2001); evolu- temperature by desiccation-tolerant organisms indicate tion of tolerance may partly involve recruiting more that inherent limits to tolerance are not likely to pose existing genes to that purpose through regulation. This an obstacle to the evolution or engineering of desic- is not to suggest that the structural genes for tolerance cation tolerance. are identical in all desiccation-tolerant plants. There are different routes to the synthesis of sucrose asso- Phylogenetic limits ciated with desiccation tolerance in different species Although desiccation tolerance is known from many (Bartels and Mattar, 2002). Novel structural genes different clades, it is apparently absent from most phy- have been identi®ed in individual species, such as la of animals, including chordates. If tolerance is a XvPer1, which encodes a stress-inducible antioxidant derived trait, then its evolution in clades of sensitive enzyme in Xerophyta viscosa (Mowla et al., 2002); the species might be limited by availability of genetic var- gene family CpPTP, which encodes proteins that may iation. If tolerance is a basal trait, then its re-evolution reversibly restructure chloroplasts during desiccation following loss within a clade might be similarly lim- in C. plantagineum (Phillips et al., 2002a); and CpEdi- ited. Such ``phylogenetic limits'' to desiccation toler- 9, which encodes a hydrophilic protein in mature seeds ance need not pose an obstacle to engineering toler- and in response to dehydration in leaf phloem in C. ance in sensitive species, since it should be possible plantagineum (Rodrigo et al., 2004). to introduce genes from tolerant species. Identifying Further support for the idea that evolution of des- phylogenetic limits is therefore a way of identifying iccation tolerance in plants is not strongly limited by opportunities to extend the frontiers of tolerance. availability of genetic variation comes from the bio- Two lines of evidence suggest that desiccation tol- geography of tolerant angiosperms (Porembski and erance in plants is not phylogenetically limited. First, Barthlott, 2000). The majority are found largely on phylogenetic analyses indicate that tolerance in land large rock outcrops, or inselbergs, in the tropics. Dif- plants is a basal trait (Oliver et al., 2000, 2005). Tol- ferent continents have very different sets of species, erance may have been lost in vegetative tissues in as- consistent with evolution of tolerance in different sociation with the evolution of internal water transport, groups of angiosperms subject to similar selective but conserved in the spores and seeds of vascular pressures. Biogeography does suggest that there may plants. Independent phylogenetic analysis suggests that be some phylogenetic limitation to tolerance, since 688 PETER ALPERT

rock pools on inselbergs in western and in southeastern something; and its literal sense, to tie together or com- Africa both have tolerant bryophytes and lichens but press by tying (OED, 1989). A physiological con- only those in southeastern Africa have tolerant angio- straint on a biological unit may occur when it is con- sperms (Krieger et al., 2000). trolled by the same physiological mechanism as an- In contrast, it seems possible that the extent of des- other trait, since selection and regulation of the ®rst iccation tolerance may be phylogenetically limited in trait may be limited by selection and regulation of the major clades of animals. Direct evidence is very lim- other. A prime example in animals is the regulation of ited, since there appear to be no phylogenetic analyses multiple traits by endocrine systems (Ricklefs and Wi- of tolerance in animals apart from a survey suggesting kelski, 2002; Zera and Zhao, 2004). In plants, control that tolerance is a basal characteristic in the class Bdel- of multiple traits by hormones can also impose phys- loidea of the rotifers (Ricci, 1998). There is also some iological constraints (Farnsworth, 2004); tolerance of remarkable counterevidence in the apparent homology desiccation may be constrained by multiple effects of of some genes associated with desiccation tolerance in the hormone, abscisic acid or ABA. different phyla of animals and even across animals, ABA upregulates some aspects of desiccation tol- plants, and microbes. Named for their expression dur- erance in bryophytes (Beckett et al., 2000; Guschina ing induction of desiccation tolerance in maturing et al., 2002; Zeng et al., 2002; Mayaba and Beckett, seeds, these Late Embryogenesis Abundant (LEA) 2003), seeds (Wakui and Takahata, 2002), vegetative genes encode a set of hydrophilic proteins that include tissues of angiosperms (Bartels and Salamini, 2001; dehydrins, which are produced in response to drying Bernacchia and Furini, 2004; Vicre et al., 2004), and in tolerant plants. In animals, the products of LEA possibly ferns (Pence, 2000). This has not been spe- genes may act as molecular chaperones for DNA or ci®cally tied to other effects of the hormone in tolerant counter physical stress during desiccation (Wise, species, but ABA tends in general to have effects that 2003). In plants, they may increase the transition tem- lead to slower growth as well as higher tolerance of perature and hydrogen bonding strength of sucrose stress by plants (Farnsworth, 2004) and selection for glasses (Wolkers et al., 2001a), helping to inhibit growth might counter selection for tolerance. For in- membrane fusion, protein denaturation, and effects of stance, a population of the desiccation-sensitive herb free radicals (Oliver et al., 2001). LEA homologues Impatiens capensis from a relatively wet habitat have been found in nematodes and bacteria (Goyal et showed less response to ABA than a population from al., 2003; Browne et al., 2004; Wise and Tunnacliffe, a drier habitat; this could re¯ect selection in the ®rst 2004), yeast (Garay-Arroyo et al., 2000; Sales et al., population for avoidance of reduction in reproductive 2000), pollen (Wolkers et al., 2001a), bryophytes (Al- output due to ABA-mediated responses to cues for pert and Oliver, 2002), roots and shoots of dicotyle- drought that are inappropriate in a wetter habitat (Hes- donous plants (Schiller et al., 1997), and shoots of chel and Hausmann, 2001). monocots (Bartels and Mattar, 2002). Cytokinins are a second important example of plant Akin to phylogenetic limitation due to absence of hormones with multiple effects (Farnsworth, 2004). genes for tolerance might be limits due to linkage be- No role has been established for cytokinins in desic- tween genes for tolerance and other genes, such that cation tolerance, but levels change in association with selection for traits that are not functionally linked to desiccation in at least one tolerant species. As they dry, tolerance nevertheless counters selection for tolerance leaves of Craterostigma wilmsii show a initial decrease (e.g., Feldgarden et al., 2003). For example, genetic in two cytokinins, zeatin and zeatin riboside, and then linkage between certain leaf traits and patterns of al- an increase below 20% relative water content (RWC: location in some plants appears to counter simulta- WC/WC at full turgor; Vicre et al., 2004). Levels re- neous adaptation of leaves and allocation to climate turn to normal after rehydration to 70% RWC. Under- change (Etterson and Shaw, 2001). In Arabidopsis, standing the physiological constraints governed by correlation between water use ef®ciency and time re- plant hormones could identify opportunities to engi- quired to reach ¯owering might seem to indicate a neer tolerance of desiccation and other stresses (Farns- functional trade-off between drought tolerance and rate worth, 2004; Kim et al., 2004). For instance, if control of reproduction but instead be at least partly due to by ABA couples tolerance to slow growth, one might ®xation of genes that cause pleiotropy (Mckay et al., try to engineer release of tolerance from this control. 2003). There seems to be no evidence so far for limits Such release has already been engineered in transgenic on tolerance due to genetic linkage in plants. However, callus tissue of C. plantigineum through constitutive lack of available genetic variation is one of several expression of the regulatory gene CDT-1 (Bartels and plausible explanations for the apparent complete ab- Salamini, 2001). sence of desiccation tolerance in most major clades of Another potential area of physiological constraint on animals. tolerance may be regulation of senescence. Glutathione is oxidized during desiccation in lichens, and desic- Physiological constraints cation tolerance is correlated with the ability to reduce A limit to one trait that is imposed by the state of it again upon rehydration (Kranner, 2002). Correlation another trait is reasonably termed a ``constraint,'' giv- between levels of reduced glutathione and tolerance en the original sense of ``constrain,'' to force to do could be related to prevention of oxidation damage INTRODUCTION 689 during desiccation. For example, plants of an intertidal possible at heights greater than 3 m. This may explain red algae collected from higher in the intertidal zone why there are no desiccation-tolerant trees, and a pre- and therefore subject to greater desiccation showed ponderance of tree species or possibly greater dif®- greater upregulation of enzymes required to regenerate culty in re®lling their special form of xylem may ex- glutathione and ascorbate during dehydration down to plain why there are no desiccation-tolerant adult gym- 40% WC and produced less hydrogen peroxide and nosperms, despite the presence of tolerance in gym- lipid hydroperoxides (Burritt et al., 2002). However, a nosperm pollen and seeds. Physical constraints on low ratio of reduced to oxidized glutathione can also rehydration have been less explored in animals, but cue apoptosis, suggesting that rapid reduction may also needs for water diffusion through cells might require be needed to avoid prompting cell death (I. Kranner, all tolerant organisms to be small or thin (Potts, 2001). personal communication). Differentiation and multicellularity do not impose obvious constraints on desiccation tolerance in plants Physical constraints or animals. Desiccation-tolerant plants have a wide Physical traits that might constrain tolerance of des- range of specialized forms. For instance, the ¯oating iccation include rigidity and size. Tolerant animals and submerged leaves of Craterostigma intrepidus are shrink as they desiccate, and it has been suggested that specialized as sun and shade leaves (Woitke et al., active contraction helps prevent mechanical damage 2004). Tolerance in the larva of the ¯y Polypedilum during desiccation (e.g., Ricci et al., 2003). For in- vanderplanki shows that tolerance is compatible with stance, rotifers and tardigrades adopt distinctive, high- having a central nervous system; excised tissue with- ly compact forms as they dry (JoÈnsson, 2001; Ricci et out nerves also survives desiccation in this species, al., 2003), and coiling has been used as a criterion for showing that the nervous system is not essential for non-lethal desiccation in nematodes (Treonis et al., tolerance (Watanabe et al., 2002). 2000). Moreover, no desiccation-tolerant animals have rigid skeletons, at least not at the life stages where Ecological limits tolerance is present. The likelihood that ability to tolerate intense or pro- Desiccation-tolerant plants do have relatively rigid longed drought does not effectively limit desiccation cell walls and even wood. Special wall morphologies tolerance in living things is corroborated by their oc- may promote folding as cells dry and reduce mechan- currence in extreme habitats, such as in the hottest and ical stress during desiccation in pteridophytes (Thom- coldest deserts and on bare, non-porous rock (Alpert, son and Platt, 1997) and angiosperms (Farrant et al., 2000; Alpert and Oliver, 2002). Tolerant cyanobacteria 1999; Vander Willigen et al., 2004). Some angio- are probably the main source of primary productivity sperms change their cell wall composition during dry- in some sand crusts in the Negev Desert (Harel et al., ing (Vicre et al., 2004). In Craterostigma plantige- 2004); they can recover 50% of their normal photo- neum, an increase in the transcription of an alpha-ex- system II activity in as little as ®ve minutes after re- pansin cDNA during dehydration is associated with a wetting. The Dry Valleys of Antartica may be largely rise in activity of expansin and in cell wall ¯exibility populated by desiccation-tolerant species, including (Jones and McQueen-Mason, 2004). Shrinkage of nematodes in the soil (Treonis and Wall, 2005) and plant cells during drying may be especially severe due endolithic communities of lichens, cyanobacteria, and to the presence of large, water-®lled vacuoles, and other bacteria (Hughes and Lawley, 2003). Granitic some tolerant species appear to alleviate this by re- outcrops in Brazil sport ®lms composed mainly of cy- placing large vacuoles with numerous small ones and anobacteria plus cyanobacterial lichens (Buedel et al., ®lling them with non-aqueous compounds as cells dry 2002). Even a complete lack of liquid water may not (Thomson and Platt, 1997; Farrant, 2000). exclude desiccation-tolerant species. For example, the Water transport through xylem imposes a second, alga Trentepolia odorata can rehydrate and photosyn- related physical constraint on desiccation tolerance in thesize with water vapor (Ong et al., 1992). vascular plants (e.g., Sherwin et al., 1998). Under On a ®ner scale, desiccation-tolerant plant species most conditions and in all plants taller than about 3 may nevertheless be excluded from the most xeric mi- m, water is pulled up rather than pushed up through crosites within a habitat by inability to maintain a pos- ®les of dead cells in the xylem. When the negative itive carbon balance over repeated cycles of desicca- pressure in the xylem exceeds that required to draw in tion and rehydration (Alpert, 1990, 2000). For in- an air bubble from a neighboring, air-®lled conduit, stance, mosses at a site in the chaparral of southern cavitation results and interrupts water ¯ow. In tolerant California are much less abundant on the equator-fac- shrubs such as Myrothamnus ¯abellifolia, water col- ing than on the pole-facing surfaces of granitic boul- umns must be regenerated after desiccation, and this ders (Alpert, 1985). This is probably because they are is variously thought to occur via root pressure (Schnei- unable to recoup respiratory losses of carbon during der et al., 2000) or capillary action (Sherwin et al., the night and during recovery from desiccation before 1998), possibly modi®ed by an unusual lining of lipids desiccation in the sun ends photosynthesis again (Al- on the inner surface of the cell wall (Wagner et al., pert and Oechel, 1985). 2000). Re®lling may delay recovery from desiccation Although some aquatic plants and animals tolerate (Sherwin and Farrant, 1996) and is likely to be im- desiccation (e.g., Schiller et al., 1997; Ricci, 1998), 690 PETER ALPERT

desiccation tolerance is negatively associated with oc- grades from the two sites sampled by JoÈnsson et al., currence in moist habitats. By far the most complete (2001), nor between populations of nematodes from evidence for this is in seeds. Based on a survey of Greece and the UK (Menti et al., 1997). Torrentera over 8,000 species of angiosperms, Tweddle et al. and Dodson (2004) noted differences in the phenology (2003) found that the proportion of species with des- of populations of Artemia in hypersaline pools and iccation-sensitive seeds tended to be higher in warmer salterns that differed in salinity, pH, and desiccation and wetter habitats, and was highest (45%) among in Yucatan. In one of the most systematic comparisons non-pioneer species in evergreen tropical forests. of tolerance in a group of animals, Ricci (1998) noted Within the genus Coffea, degree of desiccation toler- that, of 15 rotifer species representing all four familes ance in seeds decreases as number of dry months be- of the class Bbelloidea, all three desiccation-sensitive tween dispersal and the start of the wet season de- species were aquatic. creases (Dussert et al., 2000) Two possible explanations for the scarcity of des- Other comparisons between plants have mostly each iccation-tolerant species in moist habitats are compet- involved just a few species but generally also found itive exclusion by desiccation-sensitive species and se- that those from wetter habitats were less tolerant. For lection against tolerance when water availability is instance, lower ability to maintain photosynthesis dur- high. Although there appear to be no experimental ing desiccation was associated with higher ambient tests for association between tolerance and competitive water availability in three species of Antarctic mosses ability, it may be that desiccation-tolerant ``extremo- (Robinson et al., 2000). Sensitivity to desiccation was philes'' are actually competitively inferior ``normifu- associated with habitat wetness in other studies of ges.'' Work on mutants of Arabidopsis suggests that mosses by Franks and Bergstrom (2000) and Seel et loss of desiccation tolerance in seeds may require al. (1992), and in the common but not the rare mosses changes in relatively few genes (Ooms et al., 1993). studied by Cleavitt (2002). Schipperges and Rydin Derived desiccation sensitivity in seeds and rotifers (1998) reported no relationship between recovery from from moist habitats could be due to lack of selection partial drying and microhabitat in six species of the to maintain tolerance or to selection against it, if there moss genus Sphagnum, but none of these species tol- is a trade off between tolerance and growth or repro- erated complete desiccation. Microdistributions of duction. eight epiphytic pteridophytes in a Mexican cloud for- est correlated more closely with tolerance of desicca- FRONTIERS OF TOLERANCE tion and high light than with maximum performance The two main applications of research on desicca- (Hietz and Briones, 2001). Exposure to high UV or tion tolerance in the past two decades have been at- PAR while desiccated did not affect photosystem II in tempts to induce tolerance in human cells for medical the xeric lichen Xanthorea parietina, but did have a purposes and to engineer tolerance in crop plants to negative effect on a lichen from more shaded habitats make them less vulnerable to drought. Researchers (Solhaug et al., 2003). Similarly, of three mosses, only have also tried to engineer or induce tolerance in ag- the one from the most xeric habitats, Tortula rurali- ronomic bacteria and in nematodes used for biological formis, recovered photosythetic capacity if desiccated control. Knowing what limits desiccation tolerance in in the light (Seel et al., 1992). prokaryotes and nematodes could also help control Comparisons between animals are fewer and less pathenogenic species that are naturally desiccation-tol- consistent. Solomon et al. (1999) reported an associ- erant (Breeuwer et al., 2003). There has been consid- ation between habitat dryness and desiccation toler- erable success in conferring tolerance on isolated ance in three strains of the nematode Steinernema fel- membrances and enzymes and on single cells (Billi tiae. Gal et al. (2001) found higher transcription of and Potts, 2002; Crowe et al., 2005; Potts et al., 2005), glycogen synthase during dehydration of S. feltiae which seems to accord with what we know about the from a temperate than from a semi-arid habitat, sug- inherent and phylogenetic limits of tolerance. There gesting a shift away from production of trehalose, a has been almost no success in crop plants, suggesting sugar associated with tolerance, in the temperate strain. the importance of physical or physiological constraints No subtidal marine tardigrades tolerate desiccation, on tolerance in whole, multicellular organisms. whereas some of the intertidal (Clegg, 1967; JoÈnsson and JaÈremo, 2003), semi-aquatic freshwater (JoÈnsson Making single cells tolerate desiccation and Bertolani, 2001), and terrestrial species do. Of two Desiccation tolerance in mammalian cells can be in- congeneric tardigrades from the upper intertidal, the duced by treatment with trehalose, a sugar accumulat- one from exposed microsites was tolerant while the ed during drying in many desiccation-tolerant animals. one from within barnacles was not (Grùngaard et al., Whereas fresh human blood platelets have a shelf life 1990; cf.,JoÈnsson and JaÈremo, 2003), and tardigrades of only about ®ve days, platelets freeze-dried in a so- in a population from a habitat with higher relative hu- lution of trehalose can be stored much longer and then midity and temperature were less tolerant than those rehydrated for use (Wolkers et al., 2001b, 2002). This in a population from a less humid, cooler habitat (Hor- technique speci®cally preserves membrane microdo- ikawa and Higashi, 2004). However, there was no dif- mains (Crowe et al., 2003). Gordon et al. (2001) in- ference in tolerance between populations of tardi- cubated human mesenchymal stem cells in 50 mmol INTRODUCTION 691 trehalose and 3% glycerol, air-dried and stored them erance, then introducing genes for tolerance might be under vacuum, and express mailed them from San Di- expected to successfully overcome this limit. ego to Baltimore, where they recovered normal mor- phology, lability, and regeneration capacity after re- Making whole organisms tolerate desiccation hydration. Incubation in a medium with a high treha- It has so far been possible to increase tolerance of lose concentration can make corneal epithelial cells partial desiccation in multicellular animals and plants, desiccation-tolerant (Matsuo, 2001). Chen et al. (2001) but not to make them desiccation-tolerant. Treatment introduced trehalose into mammalian cells with an en- with cold can increase production of trehalose and tol- gineered protein that formed pores in the plasma mem- erance in nematodes used for biological control (Gre- brane; having about 1010 molecules of trehalose per wal and Jagdale, 2002). Breeding can increase toler- cell enabled cells to survive at 5% relative humidity ance of partial desiccation to some extent in the en- and 20ЊC for weeks. There are now techniques to load tomopathogenic nematode Heterorhabditis bacterio- human red blood cells with trehalose (Satpathy et al., phora (Strauch et al., 2004). Introduction of trehalose 2004). Under optimal conditions, sugar alone may suf- biosynthetic genes into plants can increase their pro- ®ce to make mammalian cells tolerate desiccation duction of trehalose and their tolerance of various (Crowe et al., 2002). Human cells can also tolerate stresses but has not resulted in desiccation tolerance desiccation in the absence of added sugars if dried (Penna, 2003). For example, regulated overexpression slowly and stored under vacuum (Puhlev et al., 2001). of genes from E. coli in rice plants increased their Desiccation tolerance can be induced in the bacteria trehalose concentration 3±10 times (Garg et al., 2002). Escherichia coli and Pseudomonas putida with either The plants showed relatively low photooxidation and trehalose or hydroectoine (de Castro et al., 2000; Tun- high ability to accumulate nutrients under salt, nacliffe et al., 2001; Manzanera et al., 2002, 2004). drought, or cold stress but did not tolerate desiccation. Loading the nitrogen-®xing mutualist bacterium Bra- Lack of success in making whole organisms tolerate dyrhizobium japonicum with trehalose by incubating it desiccation may suggest that the known limits that ap- in the sugar during growth greatly improved its sub- ply to whole, multicellular organisms set major obsta- sequent survival of desiccation, which is a major cause cles to engineering tolerance in them. For example, physical constraints in both animals and plants and of failure of inoculation of leguminous crops with the physiological constraints due to involvement of hor- bacterium in the ®eld (Streeter, 2003). nones in tolerance in plants are likely to require more There has also been some success in engineering than just the engineering of synthesis of sugars to con- desiccation tolerance in mammalian cells and bacteria. fer tolerance. To reduce mechanical stress, animals Guo et al. (2000) used a recombinant adenovirus vec- may need to contract during desiccation and plants tor to express the otsA and otsB genes of Escherichia need to have more ¯exible cell walls. Tolerance is un- coli, which encode enzymes that synthesize trehalose, likely to be engineered in plants taller than 3 m due in human primary ®broblasts and were able to main- to the problem of re®lling xylem. What we know about tain infected cells in the dry state for up to ®ve days. the natural limits to desiccation tolerance in living However, engineering mouse cells to produce 80 mmol things offers encouragement for further efforts to ar- trehalose did not make them fully desiccation-tolerant ti®cially extend tolerance to single-celled organisms (de Castro and Tunnacliffe, 2000), even when extra- and to individual cells and tissues of multicellular cellular trehalose was supplied (Tunnacliffe et al., ones. However, we may need to know more about the 2001). Moving the spsA gene of a cyanobacterium into limits of desiccation tolerance before we can expect to E. coli resulted in production of sucrose-6-phosphate extend it to desiccation-sensitive whole plants and synthetase and a 10,000-fold increase in survival of metazoans. desiccation (Billi et al., 2000). It is also possible to transfer genes into naturally tolerant cyanobacteria ACKNOWLEDGMENTS (Billi et al., 2001), which might thereby become a des- I thank the participants in the symposium on des- iccation-tolerant source of useful metabolites. Gene iccation tolerance at the 2005 meeting of the Society transfer may even have been signi®cant in some nat- for Integration and Comparative Biology for sharing ural origins of desiccation tolerance: the tolerant bac- their knowledge and insights. terium Dienococcus radiodurans appears to have ac- quired homologues of putative plant desiccation tol- REFERENCES erance genes by horizontal transfer (Makarova et al., Abe, S., A. Kurashima, Y. 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