Research Focus

Plant development: introducing trehalose metabolism

Matthew Ramon1,2 and Filip Rolland1,2

1 Department of Molecular Microbiology, VIB, Leuven, Belgium2 Laboratory of Molecular Cell Biology, Institute of Botany and Microbiology, K.U.Leuven, Kasteelpark Arenberg 31,

B-3001 Leuven-Heverlee, Flanders, Belgium

Update TRENDS in Plant Science Vol.12 No.5

Trehalose metabolism, a short side-branch of primarycarbon metabolism that is controlled by a surprisinglylarge gene family, is emerging as an important newregulatory pathway in plants. Two recent studies byNamiko Satoh-Nagasawa et al. and Leonardo Gomezet al. have highlighted its novel and possibly pivotal rolein coordinating carbon supply with plant growth anddevelopment.

Developmental mutants affected in trehalosemetabolismThe classical ramosa mutants define an inflorescencebranching pathway in maize and were described severaldecades ago. In the past year, three RAMOSA genes havebeen cloned and characterized in detail [1–3]. Remarkably,Namiko Satoh-Nagasawa et al. [3] have recently shownthat one of them, RA3 (RAMOSA3), encodes a functionaltrehalose-6-phosphate phosphatase (TPP), a metabolicenzyme involved in the synthesis of the sugar trehalose.Surprising? Perhaps not. Another recent paper, by Leo-nardo Gomez et al., describes how a functional trehalose-6-phosphate synthase gene, TPS1, is essential for embryodevelopment in Arabidopsis thaliana [4]. Furthermore,TPS1 function also appears to be required for normalvegetative development and floral transition in Arabidop-sis, suggesting that trehalose metabolism plays a crucialrole in several key aspects of plant development [5].

Whereas alternative pathways exist in prokaryotes, inyeast and plants, biosynthesis of trehalose, a non-reducingdisaccharide composed of two glucose units, typically occursin two steps. The intermediate trehalose-6-phosphate (T6P)is first synthesized from UDP-glucose and glucose-6-P bytrehalose-6-phosphate synthase (TPS) and then convertedto trehalose by trehalose-6-phosphate phosphatase (TPP)[6] (Figure 1). Although in many organisms, ranging frombacteria and fungi to invertebrates, trehalose is an import-ant reserve carbohydrate and stress-protectant, most vas-cular plants, with the exception of some desert resurrectionspecies, only seem to accumulate minute amounts of thissugar [7]. Nevertheless, based on similarity with the singleyeast TPS (Class I) and TPP (Class II and III) genes, 21putative TPS and TPP genes have been found in Arabidop-sis, suggesting that there are more novel and regulatoryfunctions for trehalose metabolism in plant growth anddevelopment to be discovered [8–11] (Figure 2).

Corresponding author: Rolland, F. (filip.rolland@bio.kuleuven.be).Available online 6 April 2007.

www.sciencedirect.com 1360-1385/$ – see front matter � 2007 Elsevier Ltd. All rights reserve

Trehalose metabolism controls specificdevelopmental transitionsDetailed and comprehensive analysis of the embryo-lethalphenotype of Arabidopsis tps1 mutants has not onlyrevealed severely delayed embryo morphogenesis andgrowth but also that TPS1 is required for developmentto progress past the so-called torpedo stage at the end ofearly embryo morphogenesis [4,12]. This stage is charac-terized by a marked transition from mainly cell divisionactivity for basic cell patterning to cell expansion anddifferentiation, and the accumulation of storage com-pounds [13]. Interestingly, this transition is associatedwith changes in the nature of carbon supply. Whereasearly cell division is mainly driven by high hexose levelsprovided by maternal cell wall invertases, hydrolysis ofsucrose by increased sucrose synthase activity in theembryo during the maturation phase, is associated withredirection of development to cell expansion and storagereserve accumulation [4,13]. Consistent with a specific roleduring this transition, TPS1 expression is transientlyupregulated at this stage [5]. Remarkably, although tps1embryos have significantly reduced cell division rates,their transcriptome and metabolic profile and degree ofdifferentiation are more similar to those of wild-type (WT)cotyledon-stage embryos of the same age. Thus, TPS1disruption uncouples embryo growth and morphogenesisfrom cellular differentiation and associated transcriptionalchanges, which appear to follow a separate, temporalprogram [4].

Embryo-lethality complicates analysis of TPS functionin adult plants. However, when wrinkled tps1 seeds werecold-stratified and incubated on agar medium for anextended period of time, some of them eventually didgerminate but only to develop tiny plantlets, suggestingthat TPS also has a role in vegetative development [4]. Inan earlier study, transient expression of TPS1 behind adexamethasone (DEX) inducible promoter was used tobypass embryo lethality [5]. Vegetative growth of theseplants was indeed significantly retarded and they failedto flower. Continuous application of DEX induced flower-ing, albeit severely delayed, confirming that TPS1 alsohas a function in Arabidopsis floral transition. Moreover,DEX-induced ectopic expression resulted in reduced api-cal dominance, with increased inflorescence branching[5].

This brings us back to RA3. The ra3 mutants, deficientin a Class III-type TPP enzyme, are similarly character-ized by an uncontrolled branching of the tassels and ears,the male and female maize inflorescences, respectively.

d. doi:10.1016/j.tplants.2007.03.007

Figure 1. Main pathway of trehalose metabolism in plants and yeast. Synthesis of

trehalose-6-phosphate from UDP-glucose and glucose-6-phosphate is catalyzed by

trehalose-6-phosphate synthase (TPS) activity. Trehalose-6-phosphate is then

converted to trehalose by trehalose-6-phosphate phosphatase (TPP) activity.

Finally, trehalose can be hydrolyzed by trehalase generating two glucose

molecules [6].

186 Update TRENDS in Plant Science Vol.12 No.5

Before production of the axillary inflorescence meristems,no phenotypic difference can be observed between WTand ra3 plants. However, once inflorescence axillary mer-istem initiation sets in, a general loss of determinacybecomes apparent, suggesting that TPP activity isrequired for establishing the accurate identity and deter-minacy of the axillary meristems [3]. Consistent with arestriction of the phenotype to the inflorescences, RA3expression is confined to cells surrounding the axillarymeristems [3]. Interestingly, like embryo development,the transition to flowering is associated with markedalterations in metabolic flux; induction of flowering coin-cides with starch mobilization and a transient increase inleaf carbohydrate export to the shoot apical meristem,possibly directly affecting floral meristem identity geneexpression [14].

Figure 2. The 22 genes encoding putative trehalose metabolism enzymes in Arabidops

terminal extension only found in plant TPS1. TPS5–TPS11 (Class II) are more similar t

putative TPS enzymes. Arabidopsis, like other plant species, also contains an additiona

part of yeast TPP, but containing conserved phosphatase boxes. Two of these, TPPA

Arabidopsis also encodes a single trehalase enzyme.

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Trehalose-6-phosphate: link between carbohydratemetabolism and development?The developmental transitions affected in mutants intrehalose metabolism genes are associated with alterationsin carbon fluxes to and sugar demand in meristematictissue. Consistently, root length of tps1 seedlings is alsoseverely reduced with a short meristematic region,suggesting functions for TPS1 in root meristem establish-ment or activity as well [5]. The mutant phenotypesobserved with both tps and tpp mutants, and the partialrescue of the Arabidopsis tps1 mutant phenotypes byexpression of the distantly related E. coli TPS1 orthologOtsA, also suggest that the metabolic intermediate T6Prather than the TPS1 protein itself is responsible for thedevelopmental effects [3,4,15]. A recent study on the devel-opment of a sensitive and specific assay for T6P quantifi-cation clearly showed that changes in sugar levels lead tolarge and rapid changes in T6P content, indicating that thisintermediate can act as a signal for the sugar status inplants [16]. Consistently, embryo development of excisedtorpedo staged tps1 embryos can be partially rescued onlower thanphysiological, inplanta concentrationsof sucrose[12].

Possible mechanisms of trehalose-6-phosphateaction in growth and developmentThe effects of altered T6P levels have been studied intransgenic plants overexpressing microbial TPS andTPP orthologs. These studies revealed that T6P is indis-pensable for efficient carbohydrate use and that T6P

is thaliana. TPS1–TPS4 (Class I) are most similar to the yeast TPS, except for an N-

o the yeast TPP. Catalytic activity has not been demonstrated yet for any of these

l family of smaller proteins (Class III), with only limited similarity to the C-terminal

and TPPB, have already been shown to be active TPP enzymes [8–11]. Finally,

Update TRENDS in Plant Science Vol.12 No.5 187

depletion (by high heterologous TPP activity) causes anaccumulation of sugar-phosphates [15]. This is an inter-esting observation because yeast tps1 mutants are unableto grow on glucose because of an unrestricted, ATP-deplet-ing influx of sugar into glycolysis, a phenotype that issuppressed by inactivation of HXK2 [17]. However,although T6P was shown to inhibit yeast HXK2 activityin vitro, this is not the case for the plant hexokinase.Decreased HXK activity also does not rescue tps1 embryolethality [12]. Possibly, T6P acts further downstream inglycolysis. Alternatively, sugar phosphate accumulationcould be due to impaired starch synthesis becausesucrose-induced redox activation of ADP-glucose pyropho-sphorylase (AGPase) is severely attenuated when T6Plevels are depleted [18].

In any case, a regulatory role for T6P in directing carbonfluxes is also apparent from the starch and cell wall pectinaccumulation in tps1 embryos [4]. Being synthesized fromG6P and UDP-glucose at the crossroads of primary carbonmetabolism, T6P would indeed be excellently positioned toact as a link between metabolism and growth and devel-opment. The expression of several TPS and TPP genes isalso strongly regulated by changes in sugar levels (e.g. Ref.[19]).

Although ra3 mutations have clear effects on axillarymeristem identity, RA3 expression is localized in cellssurrounding the meristems and not within, suggestingthat T6P (or the RA3 protein itself) can act non-cell-autonomously as a mobile, short-range signal [3]. How-ever, the mechanisms involved could be diverse andalthough adding T6P to isolated chloroplasts was alsofound to trigger post-translational redox-activation ofAGPase (suggesting a role for T6P in cytosol to chloroplastsignaling) [18], no direct metabolic or signaling targetshave been identified yet. Micro-array analyses of Arabi-dopsis seedlings have also shown that the expression ofseveral, mostly stress-related, genes is correlated withT6P levels, suggesting that T6P modulates transcriptionrelatively directly [11]. Moreover, double mutant andexpression analyses in maize have indicated that RA3acts upstreamand controls expression of theRA1 putativetranscription factor in regulating inflorescence meristemidentity [3]. Possibly, trehalose metabolism also controlsdevelopment more indirectly by interfering with sugarsignaling [20].

Challenges and opportunitiesIt is clear from the phenotypes described above that T6Plevels need to be tightly controlled, possibly involvingfunctional redundancy of several proteins. However, inArabidopsis, only one TPS protein (TPS1) appears to beactive, suggesting that control could be exerted mainly atthe level of T6P breakdown by TPP activity. Determiningthe dynamics of flux through the trehalose pathway inresponse to altered sugar availability and developmentalcueswould contribute to our understanding of its regulatoryaction. A major challenge is the accurate measurement ofthe minute amounts of this metabolite in plant tissues [16].Moreover, elucidation of its function in specific cell typeswillrequire an even higher, in situ resolution. Differential tis-sue- and cell-type-specific expression of several TPP and

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class II TPS genes (see http://www.genevestigator.ethz.ch)possibly contributes to the specific effects on development.In addition, even though no catalytic activity has beenreported yet for TPS2–TPS4 (putative TPS) and TPS5–TPS11 (putative TPP) proteins, regulatory functions forthese proteins (or proteins with demonstrated activity likeTPS1 and RA3), independent of catalytic activity, cannot beexcluded.

In conclusion, these recent publications establish planttrehalose research as a new and exciting field, affectingnearly all aspects of plant development. Interactionsbetween carbon metabolism and development are numer-ous and crucial but also complex, requiring the develop-ment of new tools and multidisciplinary approaches.Trehalose metabolism genes and proteins have turnedup in various ‘omics’ analyses and can be expected to beisolated in many more seemingly unrelated mutant andfunctional screens. In light of the latest findings, theyshould not be overlooked or discarded as irrelevantfalse positives, but rather be seen as an opportunity fordiscovery.

AcknowledgementsWe gratefully acknowledge financial support from the FlandersInteruniversity Institute for Biotechnology (VIB; M.R) and the ResearchFoundation-Flanders (FWO; F.R.).

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