Download The role of trehalose-6-phosphate synthase in Arabidopsis embryo

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Biochemical Society Transactions (2005) Volume 33, part 1
The role of trehalose-6-phosphate synthase
in Arabidopsis embryo development
L.D. Gómez, S. Baud and I.A. Graham1
CNAP, Department of Biology, University of York, Heslington, York YO10 5YW, U.K.
Abstract
We previously showed that trehalose-6-phosphate synthase 1 (TPS1), which catalyses the first step in
trehalose synthesis, is essential for embryo maturation in Arabidopsis [Eastmond, van Dijken, Spielman,
Kerr, Tissier, Dickinson, Jones, Smeekens and Graham (2002) Plant J. 29, 225–235]. The tps1 mutant embryos
develop more slowly than wild type. Patterning in the tps1 embryos appears normal but they do not
progress past the torpedo stage to cotyledon stage, which is when storage reserves start to accumulate in
the expanding cotyledons. Our initial data led to the hypothesis that trehalose metabolism plays a key role
in regulating storage reserve accumulation by allowing the embryo to respond to the dramatic increase in
sucrose levels that occurs at the torpedo stage of embryo development. More recent data demonstrate that
while the tps1 mutant is blocked in the developmental progression of embryos from torpedo to cotyledon
stage the expression of genes involved in the accumulation of storage reserves proceeds in a similar
fashion to wild type. Thus it appears that induction of metabolic processes required for accumulation of
storage reserves in tps1 occurs independently of the developmental stage and instead follows a temporal
programme similar to wild-type seeds in the same silique.
Introduction
Seed development in Arabidopsis is generally divided into
three phases. In the first, known as embryo morphogenesis,
a series of programmed cell divisions results in the basic cell
patterning of the mature plant being established. The torpedo
stage embryo represents the end of this phase at which stage
the root and shoot meristems are formed and small unexpanded cotyledons are evident [1]. The second phase is
dominated by the expansion of the cotyledons and hypocotyl and the associated accumulation of storage reserve compounds. A significant amount of cell division as well as
cell expansion occurs during this phase with the number of
cotyledon cells increasing substantially from torpedo through
to mid-late cotyledon stage [2]. During the final phase,
the mature seed is subjected to desiccation after which the
embryo reaches a stage of quiescence [3]. This developmental
programme is underpinned by coordinate regulation of gene
expression including for example those involved in storage
reserve accumulation during the second phase of development
[4]. A large number of embryo-lethal Arabidopsis mutants
that fail to produce viable seed due to disruption in essential embryo development (EMB) genes have been identified
[5]. These mutants range from those disrupted in basic housekeeping functions to specific signalling processes essential for
embryo development. Until recently trehalose metabolism
was not thought to play any significant role in development
in higher plants but a number of lines of evidence have led
Key words: Arabidopsis, embryo development, reserve accumulation, trehalose-6-phosphate,
TPS1.
Abbreviations used: TPP, trehalose phosphate phosphatase; TPS, trehalose-6-P synthase; T-6-P,
trehalose-6-P.
1
To whom correspondence should be addressed (email [email protected]).
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Biochemical Society
to a revision of this view [6,7]. Key among these was the
discovery that trehalose-6-P synthase 1 (TPS1) is required for
progression past the torpedo stage of embryo development
[7] leading to it being classified as an EMB gene [5].
The non-reducing disaccharide trehalose is widely distributed in nature, playing a role in stress protection and
carbohydrate storage. Since the demonstration that manipulation of the trehalose biosynthetic pathway results in profound effects on plant growth and development in mature plants [6], the function of the trehalose pathway in higher
plants has been a matter of intense research. Although in a
few desiccation-tolerant plants trehalose is present in concentrations compatible with a role in stress protection, in
most plants the amounts (0.15 mg g−1 dry weight) are too
low to support such a role [8]. The biosynthesis of trehalose
occurs via a phosphorylated intermediate, trehalose-6-P
(T-6-P), with two steps catalysed by the enzymes trehalose6-P synthase (TPS) and trehalose phosphate phosphatase
(TPP). In Saccharomyces cereviseae the TPS1 protein and
T-6-P play a key role in regulating the flux of carbon into
glycolysis through the regulation of hexokinase [9,10]. The
demonstration that the E. coli otsA gene, which encodes a
protein with the same catalytic activity as TPS1 but which is
significantly divergent at the amino acid level, indicates that
the Arabidopsis tps1 mutant phenotype is due to the lack of
T-6-P rather than an inherent property of the TPS1 protein.
A reverse genetics approach was used for the isolation
of transposon insertions in the first and second exons of
the AtTPS1 gene (tps1-2 and tps1-1 mutants, respectively)
[7]. A third allele with a T-DNA insertion in the last exon
of the gene has also recently been identified in the SAIL
collection [11]. All of these tps1 alleles are recessive and show
Nutrient Sensing through the Plasma Membrane of Eukaryotic Cells
Figure 1 Comparison of embryo development in tps1 and
wild type
The graph shows the length of wild-type and tps1 embryos over 18 days
after flowering (DAF). The images show progressive development of
wild-type embryos from torpedo stage at 6 DAF through early, mid
and late-cotyledon stages. Tps1 embryo development is delayed, reaching late globular 6 DAF and torpedo stage 12–15 DAF. Images are not to
scale.
of the TPS and TPP genes occurs in a wide range of tissues
and is controlled by stimuli such as nutritional status, abiotic
stress, circadian cycle, and in stomatal guard cells following
ABA (abscisic acid) treatment [12,13]. Different members of
the TPS family are upregulated under specific conditions,
with for example TPS5 being induced by sucrose feeding [14]
and TPS1 being induced during embryo development [7,11].
The lack of complementation of the tps1 mutant by any of the
other TPS genes and the absence of any reports to date of
similar phenotypes caused by mutation of any of the other
TPS genes is consistent with TPS1 playing a unique role in
plant development.
Since the metabolic targets of T-6-P are unknown, a
transcriptomic analysis of the changes produced by alterations in trehalose metabolism represents a valuable approach
to identify the processes controlled by this metabolite. Recent
analysis of the transcriptional changes induced by altered
levels of T-6-P in plants overexpressing TPS, TPP and
trehalase or treatments involving feeding different carbon
sources were studied using the Affymetrix 8200 gene chip
[14]. Clusters of genes that correlated with alterations in
levels of T-6-P were identified and the majority of these were
associated with stress signalling, with only two genes from the
clusters being associated with primary metabolism, namely
aldose 1-epimerase and a SNF-related kinase, AKIN11 [14].
The effect of altered T-6-P levels thus appears to extend well
beyond primary metabolism.
Storage reserve accumulation
in tps1 mutants
a wrinkled seed phenotype. The development of the tps1
embryo follows a normal growth and patterning process, but
the rate of growth is decreased. Consequently, at the time
when the wild-type embryos reach the end of the maturation
phase, the tps1 embryos have only reached torpedo or early
cotyledon stage in the same siliques. This delay in progression
of tps1 relative to wild type is observed from early stages of
seed development, becoming more marked as development
proceeds (Figure 1).
AtTPS1 is also expressed in a variety of mature plant
tissues [7]. Recently, a dexamethasone-inducible transcription
system has been used to drive expression of a TPS1 transgene
in the tps1 mutant during seed development, allowing mature
tps1 plants to be recovered [11]. Tps1 plants obtained in
this way remain in the vegetative phase and do not produce
inflorescences unless the TPS1 transgene is again induced.
Interestingly, the growth region in roots of rescued tps1
plants appears to be reduced in the absence of TPS1 transgene
expression [11].
The Arabidopsis genome contains approximately 11 TPS
and 10 TPP homologues, while there is one gene encoding a
trehalase enzyme. Microarray databases show that expression
In wild-type embryos storage reserve gene expression and
reserve accumulation are massively induced at the transition
from torpedo to early cotyledon phase. Interestingly, storage
reserve gene induction and reserve accumulation have been
reported to still occur in a number of embryo lethal mutants
that are arrested as early as the globular stage of embryo
development [15,16]. Thus it appears that induction of
metabolic processes required for accumulation of storage
reserves occurs independently of the developmental stage and
instead follows a temporal programme similar to wild-type
seeds in the same silique. Such a temporal programme could
be regulated for example by phytohormones such as ABA and
metabolites such as soluble sugars [17]. On the basis of RTPCR experiments we previously reported that two storage
reserve-related gene transcripts, namely oleosin and napin,
were not induced in tps1 torpedo stage embryos. We have
subsequently performed a more detailed analysis using several
storage reserve promoter–reporter gene lines crossed into the
tps1 mutant (Table 1). A line carrying the At2S3 promotor
fused with the coding sequence of GFP [18] was crossed
into the tps1 mutant in order to follow the temporal and
spatial pattern of expression. In the wild-type background,
At2S3::GFP fluorescence is first observed at late torpedo stage
in the embryo axis and then spreads throughout the whole
embryo as development proceeds to the cotyledon stage.
At2S3-GFP is also expressed in torpedo stage tps1, such that
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Biochemical Society Transactions (2005) Volume 33, part 1
Table 1 Reserve proteins promoter–reporter activity in
tps1-arrested embryos
Promoter–reporter activity
At2S3::GFP
β-conglycinin::GUS
DC8::GUS
WT torpedo
++
−
−
WT cotyledon
tps1
+++
++
+++
+++
+++
+++
at the end of silique development it gives a spatial pattern of
expression similar to the late torpedo stage wild type. We have
also introduced the GUS reporter driven by the promoter of
the β subunit of β-conglycinin and the DC8 promoter [19,20]
into tps1. In contrast to At2S3::GFP, the expression of DC8
and β-conglycinin promoters in tps1 shows levels similar
to the wild type (Table 1). These data contrast with our
initial observation on the expression of oleosin and napin
transcripts by RT-PCR, but are consistent with other reports
showing that cell differentiation proceeds separately from
morphogenesis. These results using promoter reporter lines
to monitor induction of storage reserve related genes in tps1
are consistent with transcriptomic, metabolite and electron
microscopic based studies in our laboratory, which all show
that the cellular differentiation in torpedo stage tps1 embryos
is more typical of cotyledon stage wild-type embryos that are
accumulating storage reserves than torpedo stage wild-type
embryos (data not shown). Separate programmes therefore
appear to operate during embryo development governing cell
differentiation and morphogenesis in the tps1 mutant.
Is T-6-P involved in the regulation
of cell growth and division?
The principal phenotypic defect of the tps1 mutant is the
progressive delay in morphogenesis during phase 1 of embryo
development rather than a strict block imposed at the torpedo
stage (Figure 1). The fact that cellular differentiation in the
morphogenically delayed tps1 mutant proceeds in a similar
fashion to that in wild-type embryos suggests that cell growth
and division are affected in this mutant. How then does T-6-P
mediate this effect on embryo development? On the basis
of the S. cerevisiae model of T-6-P regulation of carbon flux
into glycolysis and the observation that sucrose levels increase
significantly during the torpedo stage of embryo development
one possible explanation is that T-6-P plays a similar role in
plants to that in yeast. However, the biochemical analysis of a
number of plant hexokinases shows that they are not inhibited
by T-6-P unlike the S. cereviseae enzyme [7,21]. It is possible
that other enzymes or proteins associated with primary
carbon metabolism could be direct targets for regulation by
T-6-P but to date none have been identified. Transcriptomic
data from mature plant tissues discussed above [14] along
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with the fact that the tps1 mutant is able to undergo cellular
differentiation including incorporation of soluble sugars and
amino acids into storage reserve compounds suggest that
the primary defect may not be associated directly with the
regulation of metabolism.
Little is known about the linkage between nutritional
status and growth and division of plant cells. Work in
Arabidopsis cell cultures has shown that two of the D-type
cyclin genes are regulated by sugars suggesting that at least
some of the control occurs at the transcriptional level [22]. It
is possible that disruption of trehalose metabolism may either
directly or indirectly affect the crosstalk that must occur to
coordinate nutritional status with cell growth and division.
This would account for the delayed embryo development in
the tps1 mutant. Further genetic and biochemical studies are
needed to test the validity of this hypothesis.
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Received 18 October 2004