Journal of Experimental Botany, Vol. 54, No. 382, pp. 533-537,
January 1, 2003
© 2003 Oxford University Press
Is trehalose-6-phosphate a regulator of sugar metabolism in plants?
Received 8 april 2002; Accepted 29 August 2002
Centre for Novel Agricultural Products, Department of Biology, University of York, Heslington, York YO10 5YW, UK
1 To whom correspondence should be addressed. Fax: +44 (0)1904 432860. E-mail: iag1{at}york.ac.uk
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Abstract |
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 Top Abstract IntroductionThe occurrence of trehalose...Trehalose-6-phosphate is...References |
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It has recently emerged that many higher plants can synthesize trace amounts of trehalose. In arabidopsis disruption of the first step of trehalose synthesis, catalysed by trehalose-6-phosphate synthase (TPS), has lethal consequences, demonstrating an important physiological role. It is not yet clear what the precise function of trehalose synthesis is, but there is mounting evidence that trehalose-6-phosphate is implicated in the regulation of sugar metabolism. Further work is necessary to confirm this hypothesis and determine the underlying mechanism.
Key words: Arabidopsis, trehalose, trehalose-6-phosphate, trehalose-6-phosphate synthase.
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Introduction |
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TopAbstractIntroductionThe occurrence of trehalose...Trehalose-6-phosphate is...References |
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Trehalose (
-D-glucopyranosyl-[1,1]--D-glucopyranoside) is a non-reducing disaccharide sugar composed of two glucose units joined by an , -1, 1 linkage. It is widely distributed in nature (Elbein, 1974) and functions as a stress protection metabolite and storage carbohydrate (Goddijn and van Dun, 1999). The biosynthesis of trehalose has been best studied in Escherichia coli and Saccharomyces cerevisiae and involves a two-step process catalysed by trehalose-6-phosphate synthase (TPS) and trehalose-6-phosphate phosphatase (TPP). Trehalose-6-phosphate is formed from glucose-6-phosphate and uridine-5-diphosphoglucose by TPS and is then dephosphorylated to trehalose by TPP (Fig. 1).
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The occurrence of trehalose may be ubiquitous in plants |
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TopAbstractIntroductionThe occurrence of trehalose...Trehalose-6-phosphate is...References |
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Although the presence of trehalose is well documented in a few highly desiccation-tolerant plants such as Selaginella lepidophylla and Myrothamnus flabellifolius (Müller et al., 1995), a general role for this sugar in angiosperms was dismissed until quite recently. The inability to detect trehalose led to the suggestion that the majority of higher plants had lost the ability to produce it (Crowe et al., 1992). However, the activity of trehalase, an enzyme responsible for the breakdown of trehalose to glucose, is present in numerous plants (Müller et al., 1995). By applying an inhibitor of trehalase called Validamycin A, Goddijn et al. (1997) were able to establish that tobacco (Nicotiana tabacum) and potato (Solanum tuberosum) plants could accumulate detectable levels of trehalose.
Subsequently, genes encoding TPS were cloned from Arabidopsis thaliana and S. lepidophylla and their function proven by complementation of a S. cerevisiae tps1
mutant (Blazquez et al., 1998; Zentella et al., 1999). These proteins contain a TPS domain at the N-terminus and a putative TPP domain at the C-terminus. However, the TPP domain lacks two consensus sequences that are conserved in phosphatases (LDYD|GD|T|LM| and GDDRSD; Thaller et al., 1998) and does not appear to be functional (Zentella et al., 1999). AtTPS1 is expressed in all arabidopsis tissues (Eastmond et al., 2002). Analysis of the arabidopsis genome reveals that there are 
11 TPS homologues that can be ascribed to one of two classes depending on the presence of phosphatase boxes in their TPP domain (Table 1). This same classification is obtained by homology comparison with the S. cerevisiae TPS1 and TPS2 genes that encode the TPS and TPP, respectively (Leyman et al., 2001). Class I consists of four genes (including AtTPS1) that lack phosphatase boxes and class II consists of seven genes that contain phosphatase boxes. In agreement with this classification, all of the class I genes contain 16 introns with conserved exon intron boundaries whereas the class II genes contain two or three introns. This suggests an early evolutionary divergence of the two classes. Vogel et al. (2001) have reported that two class II TPS homologues (AtTPS7 and AtTPS8), although expressed, appear to lack both TPS and TPP activity.
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In S. cerevisiae trehalose synthesis is carried out by a holoenzyme complex, which consists of TPS1, TPS2 and regulatory subunits TSL1 and TPS3 (Bell et al., 1998). One possible explanation for the existence of plant proteins containing TPS and TPP domains that lack catalytic activity is that they play a role in the formation of a complex. Interestingly, AtTPS1 contains a unique N-terminal extension, which is not found in other arabidopsis TPS homologues and shares sequence homology with parts of TSL1 (Leyman et al., 2001). This domain might substitute for the regulatory role of TSL1 (Leyman et al., 2001). TPS has also been identified as a 14-3-3 binding protein, along with key regulatory enzymes of plant primary metabolism such as nitrate reductase and sucrose phosphate synthase (Moorhead et al., 1999). It remains to be determined whether 14-3-3 binding regulates trehalose synthesis.
Arabidopsis also contains TPP homologues (Class III), which lack a TPS domain (Table 1). Two class III proteins were isolated by multi-copy suppression of the heat-sensitive phenotype of the S. cerevisiae tps2 mutant (Vogel et al., 1998). However, although the proteins contain phosphatase boxes the level of homology to known TPP proteins is relatively low and it has been suggested that their true physiological function might be to phosphorylate substrates other than T-6-P (Leyman et al., 2001). Trehalase genes have been identified from soybean (Glycine max) and arabidopsis (Aeschbacher et al., 1999; Müller et al., 2001). In contrast to TPS, arabidopsis appears to house a single trehalase gene (Table 1).
A survey of gene sequence databases shows that cDNA sequences have been described for the majority of the arabidopsis genes putatively associated with trehalose metabolism (Table 1). Only two members of class I and one member of class III are not represented (Table 1). This constitutes evidence that the majority, if not all, of these genes are expressed. Work now needs to focus on temporal and spatial regulation of the different genes throughout plant development. TPS homologues have also been described in a taxonomically diverse set of plant species including wheat (Triticum aestivum), soybean, potato, tomato (Lycopersicon esculentum), and the common ice plant (Mesembryanthemum crystallinum). It is emerging that trehalose may, in fact, be ubiquitous among higher plants, but that the levels are generally extremely low. These data strongly suggest that there is a generic role for trehalose synthesis.
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Trehalose-6-phosphate is implicated in the regulation of sugar metabolism |
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TopAbstractIntroductionThe occurrence of trehalose...Trehalose-6-phosphate is...References |
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Researchers have genetically engineered plants to synthesize trehalose in an attempt to increase their desiccation tolerance. Surprisingly, over-expression of heterologous TPS genes from S. cerevisiae or E. coli in plants resulted in significant morphological growth defects and altered metabolism (Goddijn et al., 1997; Romero et al., 1997). These phenotypes could best be interpreted in the context of changes in carbon allocation between source and sink tissues and led to speculation that some aspect of trehalose metabolism might be implicated in sugar signalling (Goddijn and Smeekens, 1998; Goddijn and van Dun, 1999).
Although the over-expression data were suggestive of an important role for trehalose metabolism in plants the phenotypes described could be due to ‘knock-on’ effects caused by perturbation of metabolism. More recently, a genetic approach has been used to establish directly that AtTPS1 is essential in arabidopsis (Eastmond et al., 2002). Disruption of AtTPS1 leads to an embryo lethal phenotype. Embryo development is arrested at the onset of seed maturation when storage reserve deposition is initiated. TPS1 is transiently up-regulated at this same developmental stage and is required for the full expression of seed maturation marker genes (2S2 and OLEOSN2). Sucrose levels in the seed also increase at this stage (Eastmond et al., 2002) and may be involved in triggering maturation via sugar signalling (Wobus and Weber, 1999). In vitro culture of tps1 mutant embryos at low sucrose concentrations partially overcomes the block in development suggesting that the AtTPS1 function is linked in some way to sugar metabolism (Eastmond et al., 2002). A preliminary report on the complementation of tps1 with the E. coli TPS gene fused to the AtTPS1 promoter suggests that it is the catalytic activity of the enzyme rather than some regulatory property of the protein that is essential for arabidopsis embryo development (Schluepmann et al., 2001).
Plants could therefore require T-6-P or trehalose. Paul et al. (2001) have reported that tobacco plants expressing E. coli TPS accumulate more T-6-P and display increased rates of photosynthesis per unit leaf area under saturating light, whereas those expressing TPP display reduced photosynthetic rates. These data suggest a correlation with T-6-P levels rather than trehalose and imply that T-6-P either directly or indirectly controls carbon assimilation. Exogenous trehalose has also been shown to affect plant metabolism and gene expression (Müller et al., 1998; Wingler et al., 2000). However, it remains to be proven whether these effects are physiologically relevant.
In some yeasts, including S. cerevisiae, TPS1 plays a critical role in the regulation of glycolysis (Thevelein and Hohmann, 1995). Teusink et al. (1998) have proposed that glycolysis operates via an autocatalytic (or ‘turbo’) principle in which ATP is consumed to drive the catabolism of glucose prior to it being replenished by subsequent metabolism (Fig. 1). As a consequence, when the supply of glucose increases abruptly, glycolysis is predisposed to use ATP faster than it can be generated, causing metabolism to stall (Teusink et al., 1998). In S. cerevisiae the tps1 mutant is unable to grow on glucose (Thevelein and Hohmann, 1995) and it appears that TPS1 is required to restrict the influx of glucose into glycolysis thereby preventing a stall (Teusink et al., 1998). The phenotype of transgenic tobacco over-expressing E. coli TPS (Paul et al., 2001) and the phenotype of the arabidopsis tps1 mutant (Eastmond et al., 2002) could also be interpreted as the consequence of glycolytic deregulation.
The mechanism by which TPS1 controls glycolysis in yeasts is not fully understood, but the predominant site of action is believed to be the initial enzymatic step, catalysed by hexokinase (HXK) (Thevelein and Hohmann, 1995) (Fig. 1). TPS1 is also necessary for carbon catabolite repression of gene expression in S. cerevisiae (Thevelein and Hohmann, 1995), which is believed to operate through HXKII-mediated sugar signalling (Entian and Frohlich, 1984). In plants, sugars also act as global regulators of gene expression (Smeekens, 2000) and a similar sensing role has been proposed for HXK (Jang et al., 1997). T-6-P is a potent inhibitor of S. cerevisiae HXKII in vitro (Blazquez et al., 1993) providing a potential means of biochemical regulation. However, restoration of wild-type T-6-P levels in tps1 only partially rescues glycolytic function suggesting that the TPS1 gene product is also required for the correct control of glycolysis (Noubhani et al., 2000; Bonini et al., 2000). Furthermore, it has been reported that expression of a T-6-P-insensitive HXK from Schizosaccharomyces pombe in either wild type or HXK-deficient S. cerevisiae does not cause deregulation of glycolysis, suggesting that hexokinase might not be the sole target of T-6-P/TPS1 regulation (Leyman et al., 2001).
In arabidopsis T-6-P is not an inhibitor of AtHXK1 or AtHXK2 activity in vitro (Eastmond et al., 2002). Similarly, T-6-P has no effect on HXK activity from spinach (Spinacia oleracea) leaf extracts (Wiese et al., 1999). Furthermore, although mutations in HXKII can rescue growth of tps1 on glucose in S. cerevisiae (Thevelein and Hohmann 1995), attempts to rescue arabidopsis tps1 embryo growth in vivo using HXK antisense suppression and in vitro using the inhibitor glucosamine have failed (Eastmond et al., 2002). These data do not support a role for HXK in T-6-P-mediated regulation in plants and point towards another cellular target. However, it cannot be dismissed that arabidopsis contains a T-6-P-sensitive HXK since the gene family has not been exhaustively studied.
Although TPS is clearly important in higher plants much remains to be ascertained about the physiological role of trehalose synthesis. It is probable that this role is generic and involves the regulation of sugar metabolism by T-6-P. However, definitive proof of this hypothesis remains to be provided and will depend on identifying cellular target(s) of T-6-P action.
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