Minireview

Biochemical and molecular responses to water stress in

resurrection plants

Giovanni Bernacchiaa,* and Antonella Furinib

aUniversita di Ferrara, Dip. Biologia, Via Borsari 46, 44100 Ferrara, ItalybUniversita di Verona, Dip. Scientifico e Tecnologico, Strada Le Grazie 15, 37134 Verona, Italy*Corresponding author, e-mail: bhg@unife.it

Received 13 October 2003; revised 19 November 2003

A small group of angiosperms, known as resurrection plants,

can tolerate extreme dehydration. They survive in arid envir-

onments because they are able to dehydrate, remain quiescentduring long periods of drought, and then resurrect upon rehy-

dration. Dehydration induces the expression of a large number

of transcripts in resurrection plants. Gene products with a

putative protective function such as LEA proteins have beenidentified; they are expressed at high levels in the cytoplasm or

in chloroplasts upon dehydration and/or ABA treatment of

vegetative tissue. An increase in sugar concentration is usually

observed at the onset of desiccation in vegetative tissue of

resurrection plants. These sugars may be effective in osmotic

adjustment or they may stabilize membrane structures andproteins. Regulatory genes such as a protein translation initia-

tion factor, homeodomain-leucine zipper genes and a gene

probably working as a regulatory RNA have been isolated

and characterized. The knowledge of the biochemical andmolecular responses that occur during the onset of drought

may help to improve water stress tolerance in plants of agro-

nomic importance.

Introduction

Since agriculture began, drought, or more generallyinadequate water availability, has been one of themajor crop production-limiting factor, causing famineand death. It continues to be a permanent constraint toagricultural production in many developing countriesand causes yield reduction in developed ones (Ceccarelliand Grando 1996). Thus, in environments where waterdeficit occurs, understanding the mechanisms that allowplants to tolerate drought is an urgent need for agriculture.

Some angiosperms are able to cope with arid environ-ments by mechanisms that mitigate drought stress, such asstomatal closure, partial senescence of tissues, reductionof leaf growth, development of water storage organs, andincreased root length and density, in order to use watermore efficiently. Water flux through the plant can bereduced or water uptake can be increased by several phy-siological adaptations. These mechanisms allow plants tosurvive in arid environments by lessening the severity ofdrought stress, but they do not make these plants tolerantto desiccation. In fact, with long periods of drought theseplants will dehydrate and die (Scott 2000).

The modern scientific study of drought tolerance beganin 1702 when Anthony von Leeuwenhoek discovered thatrotifers could survive without water for months. Now it isknown that a few groups of animals and a wide variety ofplants can tolerate desiccation in the adult stages of theirlife cycle. Many lichens, bryophytes, and a few ferns cansurvive in a dried state. Among vascular plants a smallgroup of angiosperms known as poikilohydric or resurrec-tion plants (Gaff 1987) can tolerate extreme dehydration.Desiccation tolerance allows a plant to survive equilibriumwith 0% air humidity and it is preserved until waterbecomes available. Upon watering, dried resurrectionplants rapidly revive and become fully photosyntheticallyactive within 24h (Bernacchia et al. 1996). In addition, theprocesses of drying and rehydration cause only limiteddamage to plant tissues. Thus, resurrection plants havethe advantage over other species in arid environments inthat they can remain quiescent during long periods ofdrought. They can resurrect upon rehydration, grow andreproduce long before non-resurrecting plants have theopportunity to do so (Scott 2000).

PHYSIOLOGIA PLANTARUM 121: 175–181. 2004 DOI: 10.1111/j.1399-3054.2004.00321.x

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Physiol. Plant. 121, 2004 175

Desiccation tolerance is common in non-vascularplants, but is rare in vascular plants, even though driedstructures (i.e. seeds and pollen grains) are frequentlypresent in most plant species. This would suggest thatthe genetic properties required for tolerance are presentin flowering plants and that only some tightly regulatedprogrammes of gene expression in plant development arerequired to induce the revival of a dried plant body(Hartung et al. 1998). The study of desiccation-tolerantplants started at the beginning of the last century(Irmscher 1912) with the botanical description of severalspecies, and has continued until the present day, focusingon the physiological, biochemical and molecular aspectsof this phenomenon.

The importance of drought resistance in determiningcrop productivity is well known. In arid lands or envir-onments where drought occurs, the mechanisms of desic-cation tolerance can play a role in plant survival untilwater becomes available. It is justifiable that a largenumber of scientists are interested in plant responses towater deficit at physiological, biochemical and molecularlevels because this knowledge may be applied in thedevelopment of drought-tolerant agricultural species.The purpose of this minireview is to show what is cur-rently known about resurrection plants with a specialfocus on the biochemical and molecular events thatlead to desiccation tolerance.

Resurrection plants: definition and distribution

Angiosperms that possess the unique ability to withstanddesiccation of their vegetative tissues and to revive froman air-dried state are called resurrection plants (Gaff1987, Fig. 1). These poikilohydrous plants can experiencedifferent rates of desiccation, depending upon the waterstatus of the environment, and they can recover unin-jured from complete dryness within 24 h of contact withwater. Some resurrection plants lose chlorophyll anddismantle their photosynthetic apparatus during theonset of drought and they are then defined as poikilo-chlorophyllous. Others retain chlorophyll and the photo-synthetic machinery during dehydration and aretherefore called homoiochlorophyllous (Tuba et al.1998). In earlier studies, Bewley (1979) suggested thatto survive in a dehydrated state, plants must be able tolimit the damage, they must maintain the physiological

integrity in the desiccated state, and they must possessrepair mechanisms to recover from the damage uponrehydration. More recent studies (Bewley and Oliver1992) confirmed that interpretation and suggested thatdesiccation-tolerant plants can also be classified into twogroups based on their resistance mechanism: protectionof cellular integrity or cellular repair of desiccation-induced damage.

Resurrection plants are widely distributed, they arefound in all continents except Antarctica. According toGaff (1987), they are mainly concentrated in arid cli-mates such as southern Africa, eastern South Americaand western Australia, while only a few species have beenfound in Europe in the Balkan mountains (Stefanov et al.1992). About 330 species of angiosperms have beenfound to survive desiccation (Gaff 1987; Porembskiand Barthlott 2000), but no resurrection gymnospermsare known (Hartung et al. 1998). There are bothmonocotyledonous plants such as Xerophyta viscosa andSporobolus stapfianus and dicotyledonous species such asMyrothamnus flabellifolia, Craterostigma plantagineumand Chamaegigas intrepidus. The latter is the uniqueexample known of aquatic resurrection plants (Hartunget al. 1998). Despite their broad geographical distribu-tion, the ecological range for resurrection plants is nar-row. Usually they are found in habitats subjected tolengthy periods of drought, where rainfall is extremelysporadic, particularly on rock outcrops below 2000 m intropical and subtropical areas, and to a lesser extent intemperate zones (Porembski and Barthlott 2000).

Resurrection plants have evolved structures andmechanisms to allow survival under extreme conditions.For instance, a recently described and very interestingaspect of the physiology of resurrection plants is theresponse of the xylem to desiccation. In fact, when theplant is in the air-dried state the xylem vessels are com-pletely embolized. During rehydration, the xylem mustbe able to refill, avoiding the formation of air bubbles, inorder to provide a supply of water to the leaves. Vulner-ability of xylem to the formation of air bubbles dependson wood anatomy, particularly the size of pores in inter-vessel pits. Larger pores are more susceptible to air entrythan small pores. In M. flabellifolia narrow reticulatexylem vessels have been observed, which cavitate upondesiccation but refill from root pressure and capillaritywhen water becomes available (Sherwin et al. 1998).

Fig. 1. An example of resurrection plant: Craterostigma plantagineum.

176 Physiol. Plant. 121, 2004

Although the mechanisms that allow these plants tosurvive a severe water stress have potential applicationsin agriculture, molecular and biochemical studies areto date restricted to a few species: C. plantagineum(reviewed in Ingram and Bartels 1996; Bartels andSalamini 2001), S. stapfianus (Neale et al. 2000),X. viscosa (Mundree et al. 2000) and the moss Tortularuralis (Bewley and Oliver 1992; Oliver and Bewley 1997;Oliver et al. 2000).

Molecular and biochemical studies

LEA proteins

Many transcripts and proteins accumulate during dryingin resurrection plants and some of them have beencloned and/or sequenced. They include, for instance,gene products that may play a role in protecting cyto-plasmic structures during dehydration such as LateEmbryogenesis Abundant (LEA) proteins. LEA proteinsinclude a large group of proteins that accumulate inmature embryos during the onset of desiccation (Galauet al. 1986), and they can be induced in immatureembryos by ABA treatment. Generally their expressionis found in osmotically stressed or ABA-treated tissue(Mundy and Chua 1988) in desiccation-tolerant as wellas in desiccation-sensitive plants (Skriver and Mundy1990; Campbell and Close 1997; Svensson et al. 2002).

LEA proteins, even if quite heterogeneous, are usuallyrich in hydrophilic amino acids, are water soluble andcan be divided into different groups based on sequencesimilarities and properties. For example group 1 LEAproteins are characterized by a 20-amino-acid-motif thatwas first found in the wheat Em protein (Litts et al.1987). The most widely distributed and best-studiedLEA genes belong to group 2, which includes a set ofgenes rapidly induced by ABA (RAB, King et al. 1992)or by water stress (dehydrins in barley and maize, Close1997) in embryos and vegetative tissues of different plantspecies. LEA2 proteins are hydrophilic; they share aconserved serine and lysine-rich motif and remain solu-ble after boiling. Group 3 LEA proteins are character-ized by a motif of 11 amino acids, which is predicted toform an amphipathic a-helix probably involved in struc-tural interactions (Dure et al. 1989). Group 4 and 5 showrelatively less conserved structures and are probablyinvolved in protecting membranes and binding water,respectively (Ingram and Bartels 1996). Sequence homo-logies to LEA genes from all five groups have beenisolated and characterized in the resurrection plantC. plantagineum (Bartels and Salamini 2001). They areexpressed at high levels in the cytoplasm or in the chloro-plast upon dehydration and/or ABA treatment of vege-tative or callus tissue. The accumulation of LEA proteinsin desiccated vegetative tissues of C. plantagineumpointed out the similarity between seeds and resurrectionplants: both systems survive in an air-dried state andrevive during rehydration. In addition, the high cellularconcentration of different LEA proteins in desiccated

C. plantagineum tissues and their high hydrophilic struc-ture suggest that they may play a role in protectingcellular structures during drought stress.

A cellular protection mechanism is also present inthe moss T. ruralis, whose cellular integrity is maintainedduring drying, allowing a rapid recovery of the photo-synthetic activity upon rehydration (Proctor andSmirnoff 2000). Interestingly, it has been reported thatdehydrin-type LEA proteins accumulate constitutivelyalso in the hydrated state of vegetative tissue of T. ruralis(Bewley et al. 1993). By using purified antibodies raisedagainst corn seedling dehydrin, it was shown thatT. ruralis produces two dehydrins of 90 and 35 kDa.They are present constitutively and do not appear toincrease during rapid or slow drying (Bewley et al.1993). This fact may indicate that different mechanismsof drought tolerance are present in Bryophytes and invascular plants. Nevertheless, T. ruralis gametophytesrecover at a much slower rate if the water loss occursrapidly and this suggests that this moss can activatesystem(s) during the desiccation phase that can decreasethe time necessary to recover upon rehydration.

Carbohydrate metabolism

In addition to the synthesis of proteins, an increasedconcentration of sugars is usually observed in seeds ofmany species at the onset of desiccation (Leprince et al.1993) and also in vegetative tissues of resurrection plantssuch as C. plantagineum (Bianchi et al. 1991), Ramondamyconi, Haberlea rhodopensis (Muller et al. 1997),S. stapfianus (Albini et al. 1994) and others. Studyingthe mechanisms by which diverse organisms such asbacteria, fungi and yeast can withstand desiccation,Crowe and Crowe (1986) reported that in Saccharomycescerevisiae trehalose was effective in preserving the integ-rity of membranes during desiccation and rehydration.This sugar is extremely rare in plants where sucrose andother sugars may play a similar role. Sugars may beeffective in osmotic adjustment during water loss, butthey may protect the cells by causing, during severedesiccation, glass formation with the mechanical proper-ties of a solid. This phenomenon has been observed, forinstance, in maize seeds and has been correlated withtheir viability (Williams and Leopold 1989). In addition,sucrose may preserve the integrity of the lipid bilayerduring dehydration (Crowe and Crowe 1986).

It has been observed that in many desiccation-tolerantplants, independently of the species of soluble sugaraccumulated in fully hydrated tissues, sucrose is invari-ably present at high levels in the dried state (Leprinceet al. 1993). A particular case is represented by the leavesof C. plantagineum. During desiccation, the unusualeight-carbon sugar 2-octulose, representing approxi-mately 90% of the total sugar in fully hydrated leaves,is converted in sucrose, which then represents about 40%of the DW (Bianchi et al. 1991).

2-Octulose is the main photosynthetic storage sugarthat C. plantagineum leaves build up during the day.

Physiol. Plant. 121, 2004 177

During dehydration, 2-octulose is rapidly converted intosucrose without any significant alteration in the overallsugar content. Little is known about the mechanism ofthis conversion. Nevertheless, it has been shown to cor-relate with an increased synthesis of sucrose-metabolizingenzymes. Specific isoenzymes of sucrose synthase andsucrose phosphate synthase are in fact differentiallyexpressed during this phase, along with an upregulationof glyceraldehyde dehydrogenase (reviewed in Ingram andBartels 1996; Bartels and Salamini 2001). Interestingly,fully hydrated roots of C. plantagineum accumulate theoligosaccharide stachyose and its level does not appearto change significantly during dehydration. Because thereis no additional information on the sugar composition ofC. plantagineum dehydrated roots, it is not known if thesealso accumulate sucrose during dehydration or rely on thepresence of this oligosaccharide for their desiccationtolerance.

Sucrose is also the only available sugar for cellularprotection in T. ruralis. The amount of this sugar ingametophytic cells is usually 10% of the DW. Wet-dryepisodes do not induce changes in sucrose concentration,thus indicating that this moss needs to maintain suffi-cient amount of this sugar in the event of desiccation(Oliver and Bewley 1997). Other resurrection plants relyon different carbohydrate sources for their survival: inX. viscosa for instance, glucose and fructose accumulateas leaves dry, while in Ramonda it was reported thatstarch content declines as sucrose accumulates duringdrying (Muller et al. 1997).

In C. plantagineum plants, the rehydration process hasbeen studied in terms of metabolic activity and geneexpression profiles (Bernacchia et al. 1995, 1996). Afast recovery of full photosynthetic activity and ofrespiration was observed upon rewatering, along with arestart of the synthesis of related proteins (Bartels andSalamini 2001). Differential screenings also revealed aparticular behaviour of transketolase-encoding genes inrehydrating C. plantagineum leaves. In fact, two genescoding for putatively cytosolic transketolases are upre-gulated during recovery, while the plastid-localized oneappears to be expressed independently of the water cont-ent of the plant (Bernacchia et al. 1995). These resultssuggest a possible role of transketolases in the sugarconversions taking place during the dehydration-rehydration cycle, even if questions about the nature ofthe metabolic pathway are still open.

Role of ABA

The involvement of ABA in the desiccation tolerance ofresurrection plants is well known. The ABA content hasbeen measured in fully hydrated and dehydrated leavesof several resurrection plants (Gaff and Loveys 1984;Schiller et al. 1997; Bartels and Salamini 2001). In gen-eral a 3- to 7-fold increase of leaf ABA content wasobserved upon desiccation; moreover, when leaves ofM. flabellifolia and Borya nitida were too rapidly dehy-drated, the increase in ABA was not observed and leaves

did not survive after rehydration (Gaff and Loveys1984). As reported, leaves of C. plantagineum do notrequire ABA treatment to establish or to increasedrought tolerance (Bartels and Salamini 2001); however,a feature of this species is that a pretreatment with ABAprior to dehydration is essential to induce desiccationtolerance in dediferrentiated callus tissue.

Most of the proteins studied so far showing anincreased accumulation during drought in resurrectionplants are also induced by an exogenous ABA treatment.Genetic studies with Arabidopsis thaliana mutants (Parcyet al. 1994) defective in their sensitivity to ABA or withan altered ABA biosynthetic pathway also confirmed therole of ABA in inducing gene expression in response towater stress. Furthermore, different types of cis-actingelements relevant to ABA and/or drought have beenextensively analysed (Skriver et al. 1991; Yamaguchi-Shinozaki and Shinozaki 1994). In the resurrectionplant C. plantagineum, the promoters studied are derivedfrom LEA-like genes induced by dehydration and ABA(Furini et al. 1996; Ingram and Bartels 1996). Thesepromoters were analysed in transgenic Arabidopsis andtobacco plants and resulted expressed in seeds and pollenin the dry state. This observation suggests that in vege-tative tissues of C. plantagineum the signal transductionpathway from water stress to gene expression requiresthe activation of specific genes, which in angiosperms areexpressed only in dried organs.Arabidopsis abscisic acid-insensitive abi3 mutant seeds

do not acquire desiccation tolerance and remain non-dormant (Ooms et al. 1993). Further studies (Parcyet al. 1994) indicated that the ABI3 encodes a transcrip-tional factor that regulates the expression of severalgenes during seed development. The ABI3 gene is specif-ically expressed in seeds but its ectopic expression leadsto the accumulation of seed-specific transcripts in trans-genic Arabidopsis plantlets in response to ABA. In add-ition, it has been observed (Furini et al. 1996) that theectopic expression of ABI3 also conferred the ability toinduce C. plantagineum promoter activity of LEA-likegenes in vegetative tissues of transgenic Arabidopsisafter ABA treatment. These results led to the hypoth-esis that a gene product necessary for the induction ofABA-and/or desiccation-inducible genes accumulates invegetative tissue of C. plantagineum while its analogouscounterpart accumulates in seeds of angiosperms. AnABI3 homologue was isolated from C. plantagineum toverify if this gene may be responsible for the activation ofdehydration- and/or ABA-inducible genes. The encodedproduct was, indeed, able to induce LEA-like genes intransient expression assays, but its expression was notdetected in vegetative tissue of C. plantagineum (Bartelsand Salamini 2001), indicating that another factor maybe involved in the induction of drought-tolerance genes.

Genes induced at very low levels during the earlystages of desiccation have been isolated from the resur-rection grass Sporobolus stapfianus. The SDG134c gene,encoding an eIF1 protein translation initiation factor,was detected at very low levels in hydrated leaves and

178 Physiol. Plant. 121, 2004

the transcript level increased and persisted duringdrought (Neale et al. 2000). In desiccation-tolerantplants, many components essential for re-initiation ofmetabolic activity are present in the dried tissue. It wassuggested (Neale et al. 2000) that SDG134c is requiredduring the rehydration process that leads to full meta-bolic activity within several hours.

Homeodomain-leucine zipper (HD-ZIP) genes arespecific for plants and are involved in the regulation ofdevelopmental processes and induced by biotic and abio-tic stresses. From C. plantagineum two HD-ZIP family IIgenes were isolated and their products were shown tointeract in a yeast (S. cerevisiae) two-hybrid system(Frank et al. 1998). These genes, CPHB-1 andCPHB-2,are of particular interest because both are induced bydehydration but show different responses to ABA:CPHB-1 was not induced by the phytohormone, whilethe transcript level of CPHB-2 increased upon ABAtreatment. This suggests that ABA-dependent andABA-independent pathways are responsible for adapta-tion to dehydration in C. plantagineum (Table 1). In fact,previous studies on Arabidopsis ABA mutants haverevealed the existence of different water-stress signal-transduction pathways. ABA regulates one mechanism,while the other works independently of the phytohor-mone (Shinozaki and Yamaguchi-Shinozaki 2000).These observations strengthen the hypothesis that similarmechanisms underlying water-stress responses exist inresurrection and in non-resurrection plants.

Other genes that act as putative regulators during thedehydration stress have been isolated from C. plantagi-neum. For example, Myb-related genes and a heat-shocktranscriptional factor (for a review see Phillips et al.2002) are induced by dehydration. A further gene iso-lated via T-DNA activation tagging is implicated in the

process of desiccation tolerance: CDT-1 is a member of arepeated gene family that, when transcribed at high levelin callus tissue, leads to the constitutive activation ofdesiccation-related genes. This gene upregulation enablescallus cells to survive desiccation without an ABA pre-treatment (Furini et al. 1997). CDT-1 mRNA does notcarry a long ORF starting with ATG and no proteinproduct was detected by in vitro translation. Therefore,further experiments were designed to verify whetherCDT-1 might be involved in the activation of the signalpathway that leads to desiccation tolerance via regula-tory RNA or via a small polypeptide. Preliminary resultssuggest (Furini et al., unpublished data) that a putativepeptide encoded by CDT-1 is not required for conferringdesiccation tolerance in C. plantagineum callus tissue,therefore strengthening the hypothesis that the CDT-1transcript may be responsible for the observed phenotype.

Conclusions

The importance of drought resistance in determiningcrop productivity is well known. In arid lands or envir-onments where drought occurs, the mechanisms of desic-cation tolerance may promote plant survival until soilmoisture levels improve. Thus, studies of these mechan-isms have potential applications in the development ofdesiccation-tolerant crops. Although more work is stillnecessary, many genes that play a possible role in desic-cation tolerance have been isolated from resurrectionplants and the function of the encoded proteins havebeen established for several of them. Molecularresponses to dehydration such as the expression of LEAgenes and changes in sugar metabolism and regulatorypathways have been extensively studied.

It is known from molecular marker analysis (Quarrie1996) that tolerance to water stress is a quantitative trait;nevertheless all the knowledge so far achieved with theavailable gene technology opens up new opportunitiesfor improving stress tolerance in crop plants. Forinstance, in transgenic Arabidopsis plants carrying thetranscriptional activator DREB1a driven by an abioticstress-inducible promoter, induction of LEA genes wasobserved and these plants were more tolerant to waterstress (Kasuga et al. 1999).

The use of cDNA microarrays to identify drought-and cold-inducible genes in Arabidopsis allowed the isol-ation of stress-induced genes that were not previouslyidentified (Seki et al. 2001). This technology may beapplied for the isolation of new genes involved in thesignal transduction pathway that leads resurrectionplants to survive extreme water stress.

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Table 1. The different responses observed in resurrection plantsupon water stress.

Resurrection plantscan exploit

damage repair mechanismcell protection mechanism

jt

Signal transductionpathways can be

ABA-dependentABA-independent

jt

Regulatory factorsinvolved

ABI3CDT1CPHB-1 and CPHB-2 (HD-ZIPs)Myb factorsTranslation factor eIF1. . .j

t

Molecularresponses

Protective proteins j tLEA, structural,hydrophilic.

Protective molecules j t Sucrose:osmoprotection,glass formation.

Metabolic enzymes j t Sugars conversions:Sucrose synthaseSucrose-P-synthaseTransketolases (?)

jt

Morphologicalresponses

chlorophyll loss, poikilochlorophyllousno chlorophyll loss, homoiochlorophyllous

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