Trehalose and plant stress responses:friend or foe?Olivier Fernandez1, Linda Bethencourt2, Anthony Quero3, Rajbir S. Sangwan2 andChristophe Clement1

1 Universite de Reims Champagne Ardenne, Unite de Recherche Vignes et Vins de Champagne – Stress et Environnement (EA 2069),

UFR Sciences Exactes et Naturelles, BP 1039, 51687 Reims Cedex 2, France2 Universite de Picardie Jules Verne, Unite de Recherche EA3900 Biologie des Plantes et Controle Insectes Ravageurs,

Laboratoire Androgenese et Biotechnologie, Faculte des Sciences, 33 Rue Saint-Leu, 80039 Amiens, France3 Universite de Picardie Jules Verne, Laboratoire des Polysaccharides Microbiens et Vegetaux, IUT d’Amiens, Departement de

Genie Biologique, Avenue des Facultes, Le Bailly, 80025 Amiens Cedex, France

Review

The disaccharide trehalose is involved in stress responsein many organisms. However, in plants, its precise roleremains unclear, although some data indicate that tre-halose has a protective role during abiotic stresses. Bycontrast, some trehalose metabolism mutants exhibitgrowth aberrations, revealing potential negative effectson plant physiology. Contradictory effects also appearunder biotic stress conditions. Specifically, trehalose isessential for the infectivity of several pathogens but atthe same time elicits plant defense. Here, we argue thattrehalose should not be regarded only as a protectivesugar but rather like a double-faced molecule and thatfurther investigation is required to elucidate its exactrole in stress tolerance in plants.

Trehalose: a ‘‘minor’’ sugar involved in plant responsesto stressTrehalose is a nonreducing disaccharide composed of twoglucose residues bound by an a-a-(1!1) linkage. Trehalosewas first reported in 1832 [1,2] in rye following ergotinfection; it has since been detected in a wide range oforganisms, including bacteria, fungi, invertebrates andplants [2], and is often associated with stress-resistantphenotypes [3–6]. For years, trehalose was thought to berestricted to resurrection plants, such as Myrothamnusflabellifolius or Selaginella lepidophylla [7,8] because, inthese plants, trehalose reaches easily detectable levels [upto 10 mg g�1 fresh weight (FW)]. Later, trehalose wasdetected in themodel plantArabidopsis (Arabidopsis thali-ana) using validamycin A, an inhibitor of the trehalose-degrading enzyme, trehalase [9]. More recently, trehalosehas been detected in crops, such as rice (Oryza sativa) [10]and tobacco (Nicotiana tabacum) [11], at concentrations ofapproximately 10 mg g�1 FW.

Among disaccharides, trehalose has particular proper-ties. Both reducing ends of the molecule are involved informing the glycosidic bond. Therefore, trehalose is resist-ant to acidic hydrolysis and is stable in solution at hightemperatures, even under acidic conditions [1]. Otherproperties related to the stability of the a-a-(1!1) linkagehave been extensively reviewed in [1]. Two additional

Corresponding author: Clement, C. (christophe.clement@univ-reims.fr).

1360-1385/$ – see front matter � 2010 Published by Elsevier Ltd. doi:10.1016/j.tplants.2010.04.

mechanisms indicate that trehalose is an appropriatemembrane and molecule stabilizer. These are the waterreplacement and the glass formation mechanisms. In thewater replacement mechanism, trehalose replaces waterby establishing hydrogen bonds with membranes and/ormacromolecules during dehydration or freezing [12]. In theglass formation mechanism, trehalose not only crystallizesbut can also solidifies into a glassy state and is the onlysugar that can remain in glass-like state when completelydehydrated [1,2]. It is thought that the glassy state oftrehalose can prevent biomolecules denaturing duringdehydration, thereby enabling their functional activity tobe retained when rehydrated.

Throughout their life cycle, plants have to cope withmultiple stresses that alter normal plant physiology, plantgrowth and development [13–15]. Stresses are usuallycategorized as abiotic or biotic. Abiotic stresses are causedby physical or chemical environmental factors, such ascold, heat, salinity, drought, wind, chemicals, oxidationor radiation. Biotic stresses are brought about by biologicalagents, such as bacteria, fungi, insects or herbivores [13].Although stresses do not necessarily threaten plant survi-val, they can disrupt physiological processes to varyingdegrees, ranging from the disruption of standard vitalfunctions to complete tissue collapse [14,15]. To counteractthis damage, plants are equipped with a large set ofdefense mechanisms, some of which are constitutive andothers that are only activated when a stress-specific signalis perceived. The cascade of events that occurs in responseto both categories of stress consists of: (i) mobilizing anetwork of signal transduction pathways [16–18]; (ii) indu-cing the expression of sets of downstream genes [16]; (iii)synthesizing specific proteins, known as the pathogenesis-related (PR) proteins [19]; and (iv) accumulating compa-tible metabolites, such as specific sugars, proline [20] oranti-microbial molecules, such as phytoalexins [21].

Because of its original chemical and physical properties,as well as its demonstrated role in stress management inyeast, fungi and bacteria [3–5], there is growing interest inunderstanding whether trehalose (and/or its precursortrehalose-6-phosphate T6P) is involved in plant stressresponses. Recently, the sweetie mutant of A. thalianawas characterized as accumulating abnormal amounts of

004 Trends in Plant Science 15 (2010) 409–417 409

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trehalose (up to four times more than the wild type) [22].This mutant exhibits higher constitutive expression ofgenes involved in response to abiotic stress. It also exhibitssimilar developmental aberrations to those described inother plants that overexpress genes responsible for treha-lose biosynthesis.

In this review, we discuss how trehalose, T6P and theirmetabolism are involved in plant responses to both abioticand biotic stresses. We also examine current hypothesesabout the putative mechanisms by which trehalose canhelp plants dealing with environmental fluctuations.Finally, we discuss the apparent contradiction betweensome deleterious effects of trehalose on plant developmentand the positive role of trehalose in response to stress.

Trehalose metabolism in plantsAlthough five different trehalose synthesis pathways existin bacteria, fungi, yeast and algae [7,8,23], trehalose bio-synthesis in higher plants only occurs in the trehalose

Figure 1. Identified trehalose metabolic pathways. The sole trehalose synthesis pathway (

in various eukaryotic and prokaryotic organisms. Alternate trehalose synthesis pathways

pathways have been only described in prokaryotic organisms. TreP is restricted to fungi a

Abbreviations: TPS, Trehalose-6-phosphate Synthase; TPP, Trehalose-6-phosphate Phosp

trehalose trehalohydrolase; TS, Trehalose Synthase; TreT, Trehalose Glycosyltransferring

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phosphate synthase (TPS)–trehalose phosphate phospha-tase (TPP) pathway (also known as OtsA–OtsB pathway)(Figure 1). The first step, catalyzed by TPS, involves thebinding of a glucose-6-P to a UDP-glucose to produce T6P,which is cleaved into trehalose by TPP [7]. Trehalasebreaks down trehalose to form two glucose residues. Thisprocess has been found in all organisms that synthesizetrehalose [1,2,7], even when distinct forms of trehalasecoexist, for example in yeast [24].

Trehalose related genes in plants

The description of trehalose biosynthesis genes in plantsbegan in the late 1990 s with the characterization of the A.thaliana TPS (AtTPS1) and TPP encoding genes (AtTPPAand AtTPPB) [25,26]. These three genes encode functionalTPS and TPP proteins because they complement tps1 andtps2 yeast mutants, which are deficient in TPS and TPPactivities, respectively. Systematic sequencing of A. thali-ana has allowed in-depth investigations of trehalose

TPS–TPP) as described in plants (blue box). The TPS–TPP pathway can also be found

present in other organisms are shown below the blue box. TreT, TreY–TreZ and TS

nd bacteria. The trehalose degradation pathway in plants is shown in the yellow box.

hatase; TreH, Trehalase; TreY, Maltooligosyl-trehalose Synthase; TreZ, Maltooligosyl-

Synthase; TreP, Trehalose Phosphorylase; UDP, Uridine DiPhosphate.

Table 1. Effect of stress on trehalose metabolism

Stress Plant–pathogen Fluctuations of trehalose

metabolism

Organ Refs

Abiotic Drought Myrothamnus flabellifolius Trehalose accumulation Leaves [7,8,30]

Selaginella tamariscina Trehalose accumulation Leaves [29]

Cotton (Gossypium hirsutum) Induction of TPS gene expression Leaves/roots [32]

Maize (Zea maize) Induction of TPS1 gene expression Ears/tassels [33]

Repression of TPP gene expression Tassels

Rice (Oryza sativa) Induction of OsTPP1and OsTPP2 Shoots/roots [34,35]

Wheat (Triticum aestivum) Trehalose accumulation Shoots/roots [66]

Heat Arabidopsis thaliana Trehalose accumulation Shoots [31]

Chilling Arabidopsis thaliana Trehalose accumulation Shoots [31]

Rice (Oryza sativa) Trehalose accumulation Shoots/roots [34,35]

Induction of OsTPP1and OsTPP2 Shoots/roots

Salinity Rice (Oryza sativa) Induction of OsTPP1and OsTPP2 Shoots/roots

Medicago trunculata Trehalose accumulation Nodules [36]

Repression of MtTRE gene expression Nodules

Wheat (Triticum aestivum) Trehalose accumulation Shoots/roots [66]

Biotic Symbiosis Soybean (Glycine max)�Bradyrhizobium japonicum

Trehalose accumulation Nodules [49]

Common bean (Phaseolus

vulgaris)�Rhizobium tropici

Trehalose accumulation Nodules [50]

Popular tremula�Amanita

muscaria

Trehalose accumulation Hartig net hyphae [54]

Pathogen Arabidopsis thaliana�Plasmodiophora brassicae

Trehalose accumulation Infected roots/hypocotyls/

stem/leaves

[57]

Induction of AtTRE1 gene expression

and increased trehalase activity

Infected roots/hypocotyls

Pine (Pinus sylvestris)�Armillaria ostoyae

Trehalose accumulation Infected roots [58]

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biosynthesis genes in plants [27]. A large number of TPSgenes have been found in A. thaliana. Using in silicoanalysis, 10 homologs of AtTPS1 have been identified;these can be divided into two classes. Class I genes(AtTPS1–AtTPS4) encode proteins that have a TPSdomain closely related to Saccharomyces cerevisiaeScTPS1. Among them, the AtTPS1 gene encodes a proteinwith a unique N-terminal region. It has been proposed thatthis N-terminal extension can take over the regulatoryfunction exerted by the ScTSL1 protein, in the yeasttrehalose synthesis complex [27]. Class II genes(AtTPS5–AtTPS11) encode proteins with a TPP domain,exhibiting a strong homology to AtTPPA and AtTPPB, butonly 30% homology with class I proteins (AtTPS1–

AtTPS4). The function of these class II genes remainslargely unknown. Ten homologs of TPP have been foundin A. thaliana (AtTPPA–AtTPPJ) [23,27]. In this plant, aswell as in others, TPP contains a consensus sequence,characteristic of the haloacid dehalogenase or phosphataseboxes superfamily [23]. Interestingly, TPS and TPP formmultigenic families in other plants. For example, rice(Oryza Sativa) contains at least nine OsTPS and nineOsTPP genes [23]. According to most authors, this redun-dancy is an indicator that either trehalose or T6P hasan important metabolic role. By contrast, trehalase isencoded by a unique gene in A. thaliana, rice and soybean(Glycine max) [28]. The complete trehalose biosynthesisgene expression pattern has been extensively reviewedin [7].

Trehalose and the plant response to abiotic stressesTrehalose metabolism under abiotic stress conditions

The role of trehalose in abiotic stress tolerance (Table 1)was first demonstrated in resurrection plants, such as

Myrothamnus flabellifolius, Selaginella tamariscina orSelaginella lepidophylla. These desiccation tolerant plantscan withstand almost complete dehydration and, uponrehydration, regain complete viability [29,30]. Interest-ingly, in these three species, trehalose is the main solublesugar, with levels reaching 3 mg g–1 FW inM. flabellifoliusand 12 mg g�1 FW in S. lepidophylla. During dehydration,trehalose concentration only slightly increases and it actsas a protector for both proteins and membranes [8,29].

Trehalose accumulation under abiotic stresses is notrestricted to resurrection plants. In A. thaliana, the tre-halose level doubled within 4 h of heat stress (40 8C) andincreased eightfold 4 days after cold exposure (4 8C) [31].Depending on the organ examined, the accumulation oftrehalose during stress is related to the transcriptionalactivation of the genes involved in trehalose biosynthesis.For example, expression of the TPS1 gene is droughtinducible in cotton (Gossypium hirsutum) leaves and roots[32]. In maize (Zea mays), this gene is overexpressed inears under drought conditions, whereas the TPP gene isrepressed in tassels [33]. In rice, two TPP genes (OsTPP1andOsTPP2) have been found to be transiently induced bychilling stress (4 8C), drought and exogenous abscisic acid(ABA) application (50 mM), in both seedling roots andshoots [34]. As expected, the TPP activity and trehalosecontent in roots has been found to increase after chillingstress [34,35]. Salinity stress, caused by a 150-mM NaCltreatment in shoots, resulted in the expression of OsTPP1but not OsTTP2 [35]. These data indicate an involvementofOsTTP1 andOsTPP2 expression in stress responses andsuggest that ABA plays a role in their regulation [34,35].

Under stress conditions, the level of trehalose in plantorgans could also be regulated by its degradation. Forexample, under salt stress, trehalose accumulates in

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nodules of Medicago truncatula and the trehalase gene(MtTRE1) is repressed, suggesting that the increase in thetrehalose level results, at least in part, from MtTRE1transcriptional repression [36]. Furthermore, amicroarrayanalysis has revealed that most of the genes involved intrehalose metabolism in A. thaliana respond to a widerange of abiotic stresses, such as cold, salt and UV [37].This finding indicates that trehalose and/or T6P areinvolved in the response to abiotic environmental fluctu-ations.

Plant stress tolerance modulation by trehalose

treatment

In rice, exogenous application of trehalose significantlyreduces damage caused by salt stress. Its action resultsin: (i) preservation of root integrity; (ii) reduction of bothNa+ accumulation and chlorophyll loss in leaf blades; (iii)growth inhibition; and (iv) moderation of the expression ofthe osmotically responsive salT gene [38]. Furthermore,adding exogenous trehalose (30 mM) to the culture mediaof liquid-grown seeds of A. thaliana provokes a transientincrease in trehalose concentration (up to 300 mg g–1 FWafter 12 h, below 20 mg g–1 FW after 24 h) and induces theexpression of genes involved in detoxification and stressresponse within 6–12 h, as well as the synthesis of relatedproteins [39,40]. These findings suggest that exogenoustrehalose acts as an elicitor of genes involved in abioticstress responses [39,40].

Plant abiotic stress tolerance modulation by engineering

the trehalose synthesis pathway

Different research groups have attempted to create stress-tolerant plants by introducing trehalose biosynthetic genesinto crops. Drought tolerance was one of the first traitsobtained by constitutive overexpression (promoter 35S) ofthe yeast ScTPS1 gene in tobacco (Nicotiana tabaccum)[41] and the AtTPS1 gene in A. thaliana [42]. Resultanttransgenic lines accumulate trehalose up to levels of170 mg g�1 FW and 25 mg g�1 FW, respectively;A. thalianatransformants also showed an increase in T6P content(3 mg g�1 FW versus 0.75 mg g�1 FW in the wild type)[42]. Improved drought tolerance was also achieved withstress inducible and chloroplast-targeted expression of theplastid TPS1 gene in tobacco [11] and by expression ofbifunctional fusion genes, OtsA–OtsB and ScTPS–ScTPP,in rice and tobacco, respectively [10,11]. Both transgeniclines also accumulate trehalose (50 mg g�1 FW and200 mg g�1 FW, respectively).

Other resistance characters have been achieved usingtransgenic strategies related to trehalose metabolism.Transgenic tomatoes (Solanum lycopersicum) overexpres-sing the ScTPS1 gene are more resistant to salt, droughtand oxidative stresses [43]. Improved freezing and heatstress tolerance have been obtained in A. thaliana byconstitutive or stress-inducible expression of a bifunctionalyeast ScTPS1–ScTPS2 gene, leading to weak but signifi-cant accumulation of trehalose (up to 40 mg g�1 FW) [44].Rice overexpressing the Escherichia coli trehalose syn-thesis genes (OtsA and OtsB) becomes tolerant to saltand low-temperature stresses. These plants are character-ized by trehalose accumulation (increased threefold to

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tenfold, when compared with the non-transgenic controls),stronger photosynthetic activity and global accumulationof carbohydrates [10]. Modification of soluble carbohydratecontent suggests that the stress tolerance phenotype intrehalose genetically engineered plants could be partly dueto modulation of sugar sensing and carbohydrate metab-olism. In this regard, the A. thaliana sweetie mutant is ofspecial interest [22]. Indeed, sweetie plants share physio-logical traits with trehalose genetically engineered plantsbecause they show (i) overexpression of stress-relatedgenes; (ii) hyperaccumulation of carbohydrates; and (iii)hyperaccumulation of trehalose and T6P. Nevertheless,the SWEETIE protein does not show any homology withtrehalose biosynthetic enzymes, supporting the idea thattrehalose and/or T6P could be responsible for the stress-tolerant phenotype of this mutant. Another characteristicshared by sweetie and some trehalose genetically engin-eered plants is the alteration of plant development, forexample severe dwarfism [22]. Constitutive overexpres-sion of the yeast ScTPS1 gene in tobacco and tomato[41,43] causes growth aberrations in these plants. Thistakes the form of stunted growth in tobacco and of abnor-mal root development in tomato. Such developmentalalterations might be correlated with interactions betweentrehalose and general carbohydrate metabolism [7].Indeed, expression of ApL3, a gene involved in starchsynthesis, is induced in A. thaliana when grown in treha-lose-containing medium [45]. Furthermore, T6P accumu-lation provokes growth inhibition in A. thaliana [46]. Theclose correlation between primary carbon metabolismindispensable to normal plant development and trehalosestress-related metabolism might explain why manipulat-ing stress tolerance by engineering trehalose biosynthesisis a delicate process.

Trehalose and plant responses to biotic stressesBecause there are common mechanisms implicated inabiotic stresses [47], it might be expected that trehaloseplays a role in the plant response to biotic interactions(Table 1). However, little information is available althoughreports indicate trehalose could have contradictory roles.

Trehalose and plant–microorganism symbiosis

By definition, symbiotic plant–microorganism interactionsbenefit the host. The formation of rhizobia nodules onlegumes or mycorrhizal associations often result inincreased host biomass or improved resistance to stress.However, the onset of the plant–microorganism interactionhas some similarity to biotic stress, in so far as plantsactivate defense responses that are later overcome by thesymbiont [48]. Interestingly, trehalose is involved in theseinteractions. The presence of trehalose was reported in theearly 1980 s in soybean (Glycine max) nodules [49] and wasassayed in several legumes at concentrations between0.1 mg g�1 dry weight (DW) and 14 mg g�1 DW [50]. Eventhough trehalose synthesis occurs in bacteria, most treha-lose is located in the cytoplasm of host plant cells [50].

Trehalose could be involved at two levels in these inter-actions. First, the presence of trehalose in nodules couldimprove host tolerance to stress, as revealed by theincrease in trehalose synthesis in nodules during drought

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stress [50]. A further demonstration of this trehalose actionhas been achieved using ReOtsA, a Rhizobium etli strainthat overexpresses otsA, a trehalose synthesis gene in E.coli [51]. Common bean (Phaseolus vulgaris) plants inocu-lated with ReOtsA developed more nodules (by 27%),greater nitrogenase activity (by 38%) and increased bio-mass (by 25%), when compared with host plants inoculatedwith the wild-type strain. The opposite phenotype wasobserved with beans inoculated with the R. etli TPS KOmutant, as revealed by a decrease of nodule number (by26%), nitrogenase activity (by 45%) and biomass (by 40%)[51]. Furthermore, plants inoculated with ReOtsA fullyrecovered after a 3-week period of drought stress, whereasplants inoculated with the wild-type strain or the R. etliTPS KO mutant did not [51].

Second, trehalose accumulation in the symbiont couldhelp it to resist host defense responses induced at the startof the association. Indeed, Sinorhizobium meliloti andSinorhizobium medicae thuB mutants, affected in treha-lose degradation, accumulate higher levels of the disac-charide and are able to form more nodules than the wild-type strains [52].

Trehalose is also involved in ectomycorrhizal symbiosis[53]. A study of the interaction between the fly agaricfungus (Amanita muscaria) and poplar (Populus tremu-la�tremuloides) found that the transcript level of genesencoding fungal trehalose synthesis enzymes increased[54]. Subsequently, trehalose accumulated by a factor ofthree in the Hartig net, a complex network of fungalhyphae which is the site of intense nutrient exchangebetween the fungus and the host plant [54]. It has beensuggested that trehalose could create a carbon sink for thesymbiont, resulting in attraction of photoassimilates.

Trehalose: elicitor of the plant defense response

Trehalose acts as an elicitor of plant defense mechanisms.Transcriptomic analysis performed on A. thaliana grownon 30-mM trehalose-containing media has revealed theinduction of the expression of genes known for their rolein plant defense. For example, the expression of WRKY6and b-1,3-glucanase genes, which encode a defense-relatedtranscription factor and a PR protein, respectively, isinduced [46]. Furthermore, it has been reported that tre-halose treatment on wheat induced partial protectionagainst the necrotrophic agent Blumeria graminis [55].Protection was correlated with a significant increase inphenylalanine ammonia–lyase activity or the accumu-lation of reactive oxygen species (ROS) at the infectionpoint. Interestingly, the addition of trehalose has littleeffect on the pathogen itself. It neither modifies pathogenmembrane lipid composition [56] nor conidia germination[55], which supports the elicitor hypothesis. However, atleast some of these results were obtained by sprayingtrehalose at a concentration of 15 g l–1, far higher thanphysiological levels. This raises the question of whetherdefenses were triggered following osmotic stress or by trueelicitation.

Trehalose: an aspect of pathogen attack

To date, there has been little investigation of trehaloseaccumulation during pathogenic infection. In the compa-

tible interaction between A. thaliana and Plasmodiophorabrassicae, trehalose accumulates in infected roots andhypocotyls up to concentrations of 10 mg g–1 DW [57].Trehalose accumulation has also recently been describedin roots of Scots pine (Pinus sylvestris) following infectionby Armillaria ostoyae [58]. The presence of the disacchar-ide in both plants and pathogenic fungi makes it difficult toidentify their respective contribution to trehalose accumu-lation in infected organs. This appears to be anotherexample of the contradictory effects of trehalose; it is botha plant stress protector and a multifunctional sugar infungi [59] that plays a part in their infectivity. The role oftrehalose in infectivity is illustrated by the decreasedpathogenicity of the deletion tps1 mutant of Magnaporthegrisea [60,61].

A study of both host and pathogen trehalosemetabolismgenes in the A. thaliana–Plasmodiophora brassicae patho-system suggests that trehalose is produced by thepathogen as an element of its suite of virulence tools[57]. Indeed, it has been demonstrated that a gene relatedto trehalose synthesis in the pathogen, PbTPS, is upregu-lated at the onset of the infection, thus resulting in treha-lose accumulation. In themeantime,AtTRE is upregulatedbefore trehalose accumulation in infected tissue, whichsuggests that trehalase might be part of a plant defensesystem to counteract trehalose accumulation. In thisexample, trehalose overaccumulation in plant tissue is athreat to plant survival. Thus, in precise locations and atparticular concentrations, trehalose can have a negativeimpact on plants.

Trehalose and plant stress response: potentialmechanisms of actionTrehalose: a compatible solute in higher plants?

Compatible solutes are non-toxic molecules able toaccumulate at high concentrations in the cytoplasm. Theyparticipate in turgor maintenance and/or the protection ofmacromolecular structures against the destabilizing effectof anhydrobiotic conditions [62]. This definition covers abroad range of molecules, including saccharides such astrehalose [63]. In many organisms, trehalose has beenreported as a better stabilizer than other sugars for pro-tecting membranes and biomolecules [2,3,12], and a com-patible solute [23].

The notion of trehalose as a compatible solute iscontroversial in plants and needs further investigation.If trehalose concentration in resurrection plants reacheslevels consistent with the compatible solutes theory, theconcentration of trehalose in other plants is low, whichsuggests that trehalose is not a compatible solute[42,64,65]. Furthermore, trehalose genetically engin-eered plants exhibit altered morphology, possibly causedby toxicity of high trehalose concentrations, indicatingthat trehalose is a non-compatible solute [41,43,64].However, many plants accumulate trehalose in specificorgans when stressed [10,66]. In addition, when trans-formed with the chloroplast-targeted ScTPS1 gene, A.thaliana becomes drought-tolerant, without growthaberrations [11]. These results suggest that trehalosemight act as a compatible solute in specific organs, cellsor organelles.

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Trehalose and antioxidant response

Production of ROS under both abiotic and biotic stresses isa common plant response to adverse environmental con-ditions [67]. Overproduction of ROS can oxidize plant cellmembranes and macromolecules [67,68], resulting in thetriggering of antioxidant reactions that trehalose synthesiscould be a part of.

In wheat (Triticum aestivum) exposed to heat stress,trehalose has been shown to scavenge ROS in a concen-tration-dependent manner, reaching the maximumneutralization effect at 50 mM [69]. A similar reactionwas reported when yeast cells became more tolerant toH2O2 after treatment with 10% trehalose [70]. Neverthe-less, trehalose accumulation might reduce ROS signalingand affect the subsequent plant defense response.

Trehalose and programmed cell death

In plants, programmed cell death (PCD) can occur follow-ing both pathogen attacks and environmental fluctuations[71]. For example, PCD is activated in tobacco when it isinfected with Pseudomonas syringae pv. phaseolicola [72]and in A. thaliana following UV-C radiation [73]. Antia-poptotic effects of trehalose have been widely documentedin animals. The ability of trehalose to improve the con-servation of stem cells and platelets during freezing hasbeen correlated with an antiapoptotic effect [74,75]. Sur-prisingly, the effect of trehalose on plant PCD has onlybeen documented once. Florets conserved in water supple-mented with trehalose exhibited delayed wilting anddecreased PCD [76]. Furthermore, trehalose degradationseems to be important during senescence, which is closelyrelated to PCD [71]. Based on the Genevestigator V3 tool[77], we conclude that the trehalase encoding gene (AtTRE)is strongly upregulated during senescence. This couldindicate that trehalose degradation is required for thedevelopment of the PCD process and strongly suggeststhat trehalose has an antiapoptotic effect in plants.

Table 2. Demonstrated and suspected effects (protective or adver

Context Status Protective effect

Abiotic stress Demonstrated Trehalose gene overexpression

confers abiotic stress resistance

against drought, salt and cold

stress in different plants

Biotic stress Demonstrated Trehalose elicits plant defense

Demonstrated Trehalose is a key metabolite in

plant–rhizobacteria interactions

Compatible solute Suspected Trehalose could participate in

osmoregulation under abiotic

stress, especially when produced

in certain organs and/or organelles

ROS scavenging Suspected Trehalose could control damage

due to ROS overproduction

Antiapoptotic Demonstrated Trehalose delays PCD in plants

Suspected Trehalose could support cell surviv

under stress conditions

Signal Demonstrated Trehalose elicits plant defense

Demonstrated T6P acts as a hexokinase inhibitor

in plants

Suspected

Abbreviations: HR, hypersensitive response; PCD, programmed cell death; ROS, reactiv

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In the case of pathogen attacks, such antiapoptoticproperties can appear contradictory. In numerous plantdiseases, a PCD-like phenomenon called hypersensitiveresponse (HR) is triggered when the host recognizes apathogen effector destined to suppress immunity [78].HR involves inducing cell death at the infection point, thuslimiting the progression of the pathogen. Accumulation oftrehalose at the infection point could interfere with HR.This is in agreement with our finding using GenevestigatorV3 [77] that AtTRE is also upregulated when A. thalianaleaves are inoculated with P. syringae (AvrRpm1) com-pared with P. syringae DC3000. P. syringae (AvrRpm1)triggers HR in an incompatible reaction. Finally, it istempting to hypothesize that some pathogens producetrehalose to help overcome PCD, but data to support orrefute this assumption are not available.

Trehalose: a signal molecule during stress response?

It is known that sugars, such as sucrose, act as signalingmolecules [79,80].However, the role of trehalose in signalingremains controversial, with potentially opposite roles fortrehaloseandT6P.There is onlyweakevidence for thedirectrole of trehalose as a signalingmolecule. Trehalose interactswith lipids and proteins [81], which could represent part of asignal transduction cascade. In A. thaliana, trehalase is amembraneprotein,with theactivehydrolasedomain locatedon the apoplastic side of the plasmamembrane [28], where itmight participate in sugar sensing. There is stronger evi-dence for the idea that signaling functions are carried out byT6P. T6P inhibits hexokinase in yeast [7,82]. In plants, T6Phas been first suggested to act as an essential signal mol-ecule for growth and development [46,64]. Recent findingshave shown that such properties could be related to aninhibiting effect carried out by T6P on SnRK1 (Sucrosenon-fermenting-related Kinases 1), a key transcriptionalregulator that responds to carbon and energy supply[83,84]. Trehalose biosynthetic enzymes might have regu-

se) of trehalose in plants

Refs Adverse effect Refs

[10,41,43,44] Trehalose gene overexpression

provokes growth aberrations

[41,43,44]

[39,40,55] Trehalose is a key compound of

virulence in certain pathogens

[60,61]

[49,50,51]

[11,23] Trehalose overaccumulation could

result in developmental aberrations

[22,46,64]

[69,70] Trehalose could block ROS-induced

signaling

[76]

al Trehalose could reduce HR or PCD

under abiotic and biotic stresses

[39,40,55]

[82,83,84] T6P acts as a hexokinase inhibitor

in plants

[82–84]

Trehalose could destabilize starch

metabolism, possibly via induction

of starch synthesis and degradation

gene

[45,64]

e oxygen species; T6P, trehalose-6-phosphate.

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latory functions because they can be modified by proteinkinases and interact with 14-3-3 protein, both known fortheir signaling functions [85]. Regarding the role of kinaseproteins in plant defense activation [86], we suggest thatT6Pmight be a signalingmolecule for plant stress responsesthrough kinase activation.

Finally, the fact that both trehalose and T6P could besignaling molecules raises the question of whether stressrelated defense responses result fromadirect signaling effectof these molecules. To date, no related and relevant exper-imental evidence on the mechanism has been produced.

Concluding remarksIt seems probable that trehalose and/or T6P are involved inprotecting plants from some forms of stress, as attested bysome striking examples, such as drought-resistant pheno-types of resurrection plants, genetically engineered plantsfor trehalose production or Rhizobacteria mutants withimproved capacity for symbiosis. However, the exact mech-anisms of protection, in which trehalose is involved,remain unclear. The positive and negative effects of tre-halose in plants that have been demonstrated or aresuspected are summarized in Table 2. There are two mainareas that need further investigation. First, although thereis a strong correlation between the presence of trehaloseand better tolerance to some abiotic stresses, high concen-trations of trehalose in plants (mutant or genetically modi-fied) often lead to developmental aberrations, indicatingthat trehalose is a putative double-faced molecule. Second,the role of trehalose and/or T6P in resistance to somepathogens requires further research. Currently, one canonly assume that in some cases trehalose synthesis isstimulated by some plant–pathogen interactions. The anti-apoptotic effect of trehalose appears to work against plantdefense strategies. Furthermore, the presence of trehaloseas a constitutive sugar in most pathogens makes investi-gating this interaction difficult. It is possible to inducedefense mechanisms artificially using trehalose, but it isnot clear whether trehalose is involved in plant defenseagainst pathogens in natural situations.

We are only just beginning to comprehend the effect oftrehalose on plant physiology and further studies in higherplants are still required to better understand the role oftrehalose in plant protection. Broader molecularapproaches (transcriptomics, proteomics and metabolo-mics) would be useful for elucidating a complete view oftrehalose action in plants. Optimization of trehalose andT6P assays will help in correlating levels of these metab-olites with resulting phenotypes. In particular, the charac-terization of A. thaliana mutants affected in trehalosebiosynthesis or degradation could be a help in definingthe exact role of trehalose in defense mechanisms againstboth biotic and abiotic stresses.

AcknowledgementsWe thank Fabienne Baillieul for helpful discussions about the manuscriptand Arnaud Haudrechy for drawing trehalose molecules.

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