Download Is trehalose-6-phosphate a regulator of sugar metabolism in plants?

Journal of Experimental Botany, Vol. 54, No. 382,
Regulation of Carbon Metabolism Special Issue, pp. 533±537, January 2003
DOI: 10.1093/jxb/erg039
Is trehalose-6-phosphate a regulator of sugar metabolism
in plants?
Peter J. Eastmond, Yi Li and Ian A. Graham1
Centre for Novel Agricultural Products, Department of Biology, University of York, Heslington, York YO10 5YW, UK
Received 8 april 2002; Accepted 29 August 2002
Abstract
It has recently emerged that many higher plants can
synthesize trace amounts of trehalose. In arabidopsis
disruption of the ®rst 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 con®rm this hypothesis and determine
the underlying mechanism.
Key words: Arabidopsis, trehalose, trehalose-6-phosphate,
trehalose-6-phosphate synthase.
Introduction
Trehalose (a-D-glucopyranosyl-[1,1]-a-D-glucopyranoside) is a non-reducing disaccharide sugar composed of
two glucose units joined by an a, a-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-6phosphate is formed from glucose-6-phosphate and
uridine-5-diphosphoglucose by TPS and is then dephosphorylated to trehalose by TPP (Fig. 1).
The occurrence of trehalose may be ubiquitous
in plants
Although the presence of trehalose is well documented in a
few highly desiccation-tolerant plants such as Selaginella
1
lepidophylla and Myrothamnus ¯abellifolius (MuÈller 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 (MuÈller 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 tps1D
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 classi®cation 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 classi®cation, 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
To whom correspondence should be addressed. Fax: +44 (0)1904 432860. E-mail: [email protected]
Journal of Experimental Botany, Vol. 54, No. 382, ã Society for Experimental Biology 2003; all rights reserved
534 Eastmond et al.
Fig. 1. A schematic diagram of the pathway of trehalose metabolism
and its relationship to glycolysis in Saccharomyces cerevisiae. G-6-P
is glucose-6-phosphate, UDPG is uridine-5-diphosphoglucose, T-6-P is
trehalose-6-phosphate, TRE is trehalose, TPS is trehalose-6-phosphate
synthase, TPP is trehalose-6-phosphate phosphatase, and HXK is
hexokinase.
TPS homologues (AtTPS7 and AtTPS8), although expressed, appear to lack both TPS and TPP activity.
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 identi®ed
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 heatsensitive phenotype of the S. cerevisiae tps2D 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 identi®ed from soybean
(Glycine max) and arabidopsis (Aeschbacher et al., 1999;
MuÈller 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.
Trehalose-6-phosphate is implicated in the
regulation of sugar metabolism
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
signi®cant 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
Regulation of sugar metabolism 535
Table 1. A list of putative genes that are likely to be involved in trehalose metabolism in Arabidopsis thaliana
Class I, II and III sequences were retrieved from the Munich Information Centre for Protein Sequences (MIPS) arabidopsis annotation database
(http://mips.gsf.de/proj/thal/db/index.html) based on psiBLAST searches with both AtTPS1 and AtTPPB as query sequences. mRNA is described
as `Yes' when either EST or cDNA sequences corresponding to each gene are present in public databases.
Gene
MIPS code
TPS domain
TPP domain
Phosphatase boxes
mRNA
Function
Reference
Class I: putative trehalose-6-phosphate synthases with an N-terminal TPS domain, but without phosphatase boxes in their C-terminal TPP domain
AtTPS1
At1g78580
+
+
±
Yes
Yes
Blazquez et al., 1998
AtTPS2
At1g16980
+
+
±
Yes
+
+
±
No
AtTPS3
At1g17000a
AtTPS4
At4g27550
+
+
±
No
Class II: putative trehalose-6-phosphate synthases/phosphatases with both N-terminal TPS and C-terminal TPP domains
AtTPS5
At4g17770
+
+
+
Yes
AtTPS6
At1g68020
+
+
+
Yes
AtTPS7
At1g06410
+
+
+
Yes
No
Vogel et al., 2001
AtTPS8
At1g70290
+
+
+
Yes
No
Vogel et al., 2001
AtTPS9
At1g23870
+
+
+
Yes
AtTPS10
At1g60140
+
+
+
Yes
AtTPS11
At2g18700
+
+
+
Yes
Class III: putative trehalose-6-phosphate phosphatases consisting only of the TPP domain
AtTPPA
At5g51460
±
+
+
AtTPPB
At1g78090
±
+
+
At1g22210
±
+
+
At1g35910
±
+
+
At2g22190
±
+
+
At4g12430
±
+
+
At4g22590
±
+
+
a
At4g39770
±
+
+
At5g10100
±
+
+
At5g65140
±
+
+
Yes
Yes
Yes
Yes
No
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Vogel et al., 1998
Vogel et al., 1998
Trehalase
Yes
Yes
MuÈller et al., 2001
a
At4g24040
Indicates putative pseudogenes.
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 (MuÈller 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 tps1D mutant is unable to grow on glucose
(Thevelein and Hohmann, 1995) and it appears that TPS1
is required to restrict the in¯ux 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
536 Eastmond et al.
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 tps1D 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 HXKde®cient 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 tps1D 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, de®nitive 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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