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Plant Physiol. (1999) 119: 1017-1024
Mannose Inhibits Arabidopsis Germination via a
Hexokinase-Mediated Step1
Jónatas V. Pego*,
Peter J. Weisbeek, and
Sjef C.M. Smeekens
Department of Botanical Ecology and Evolutionary Biology
(J.V.P., S.C.M.S.), and Department of Molecular Cell Biology
(J.V.P., P.J.W., S.C.M.S.), University of Utrecht,
Padualaan 8, 3584 CH Utrecht, The Netherlands
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ABSTRACT |
Low concentrations of the
glucose (Glc) analog mannose (Man) inhibit germination of Arabidopsis
seeds. Man is phosphorylated by hexokinase (HXK), but the absence of
germination was not due to ATP or phosphate depletion. The addition of
metabolizable sugars reversed the Man-mediated inhibition of
germination. Carbohydrate-mediated regulation of gene expression
involving a HXK-mediated pathway is known to be activated by Glc,
Man, and other monosaccharides. Therefore, we investigated whether Man
blocks germination through this system. By testing other Glc analogs,
we found that 2-deoxyglucose, which, like Man, is phosphorylated by
HXK, also blocked germination; no inhibition was observed with
6-deoxyglucose or 3-O-methylglucose, which are not
substrates for HXK. Since these latter two sugars are taken up at a
rate similar to that of Man, uptake is unlikely to be involved in the
inhibition of germination. Furthermore, we show that mannoheptulose, a
specific HXK inhibitor, restores germination of seeds grown in the
presence of Man. We conclude that HXK is involved in the Man-mediated
repression of germination of Arabidopsis seeds, possibly via energy
depletion.
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INTRODUCTION |
Among the many regulatory systems and signals in plants,
carbon-metabolite-mediated gene regulation has been receiving growing attention in the past few years (for reviews, see Farrar, 1991 ; Sheen,
1994; Koch, 1996; Jang and Sheen, 1997; Smeekens and Rook, 1997). Many
plant genes are controlled by sugars and are involved in such diverse
processes as photosynthesis, storage protein accumulation, and starch,
lipid, and nitrogen metabolism (Hattori et al., 1990, 1991; Nakamura et
al., 1991; Karrer and Rodriguez, 1992; Krapp et al., 1993; McLaughlin
and Smith, 1994; Chevalier et al., 1996). In general, when sugar
concentrations in the plant increase, there is repression of the genes
involved in photosynthesis and in the mobilization of starch, lipid,
and protein storage reserves. At the same time, genes required for
storing carbon metabolites for future use are induced. There are
numerous examples of genes whose expression is regulated by sugars, and
carbon-metabolite-mediated regulation of gene expression appears to be
a central and fundamental mechanism common to all higher plants (Koch,
1996).
Whereas the understanding of sugar regulation of gene expression in
plants is still in its early stages, a considerable amount of
information is available from research in bacteria (Magasanik, 1961;
Ullmann, 1985; Saier, 1989) and yeast (Gancedo, 1992; Johnston and
Carlson, 1992; Trumbly, 1992; Thevelein, 1994; Ronne, 1995). In yeast
the repression of Glc-repressible genes is triggered by HXK, the enzyme
that acts first in glycolysis by phosphorylating Glc to Glc-6-P,
originating a signal that is perceived by the GLC7 complex (Tu and
Carlson, 1995). This activates the downstream SSN6/TUP1 complex, which,
by binding to the transcription factor MIG1, modulates the chromatin
structure, thus repressing gene expression. However, the connection
between these components is unknown. In the absence of Glc a pathway
involving several protein complexes, including the SNF2-containing
complex, reverses the SSN6/TUP1-mediated block of gene expression so
that genes can be transcribed. An important question that remains is
how HXK triggers the signaling cascade. Neither Glc-6-P nor other
downstream glycolytic intermediates are capable of triggering this
repression. The HXK-dependent pathway is clearly of great importance
and seems to be at least partially conserved in eukaryotes. HXK has
been implicated in sugar-mediated gene regulation in yeast (Thevelein, 1994), animals (Vaulont and Kahn, 1994), and plants (Jang and Sheen,
1997).
In plants HXK activity has long been known as an important regulator of
glycolysis, but recently several lines of evidence have suggested that
it also has a sensor function in the sugar signal transduction pathway
(Graham et al., 1994; Jang and Sheen, 1994; Jang et al., 1997). This
points to possible similarities with the yeast and animal systems, in
which HXK and glucokinase have an identical function (Entian and
Fröhlich, 1984; Pilkins et al., 1994; Heimberg et al., 1996). By
analogy with the yeast system, and taking into account the large number
and different functions of genes regulated by sugars, there are
probably several interacting, highly complex sugar-signaling pathways
in plants. To determine the molecular components of these pathways it
became evident that a genetic approach would be of great
interest.
We developed a screening strategy for the isolation of sugar-sensing
mutants, and isolated a number of sun (Suc
uncoupled) mutants (Dijkwel et al., 1996, 1997; Smeekens
and Rook, 1997; Van Oosten et al., 1997). During the characterization
of these mutants, germination on several different sugars was tested.
Unexpectedly, low concentrations of Man, a Glc analog that can also be
phosphorylated by HXK, blocked germination of Arabidopsis seeds. Man
has recently been shown to be capable of specifically repressing
several plant genes via the HXK pathway (Graham et al., 1994; Jang and
Sheen, 1994) with greater efficiency than Glc. Here we provide evidence that Man represses germination through this HXK pathway.
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MATERIALS AND METHODS |
Plant Material and Growth Conditions
The Columbia (glabrous) ecotype of Arabidopsis (Lehle Seeds, Round
Rock, TX) was used in all experiments except those involving sun mutants, which were isolated in a C24 ecotype
background. The corresponding ecotypes were used as controls in the
experiments described here. Seeds were surface-sterilized for 12 min in
20% commercial bleach, and rinsed four times with sterile, ultrapure water (Milli-Q, Millipore). Seeds were then sown onto sterile Murashige
and Skoog (1962) medium containing vitamins (Duchefa, Haarlem, The
Netherlands), and solidified with 0.7% plant agar (Duchefa). The
different sugars and metabolites were added to this medium as indicated
below. Sowing was carried out in a small volume of 0.1% agarose that
was allowed to dry. Plates were placed at 4°C in the dark for 2 d to promote germination, and were then transferred to 22°C and a
16-h/8-h light/dark cycle at d 0.
Germination Assays
All measurements of germination frequencies were obtained at d 8 unless stated otherwise. In the absence of a universal definition, in
this paper we define germination as the emergence of 1 mm or more of
the radicle from the seed coat.
ATP Measurements
Approximately 50 seeds or seedlings were harvested from the agar
plates and immediately frozen and ground in liquid nitrogen. The
samples were then centrifuged for 5 min at 14,000 rpm in microtubes. One-hundred microliters of the supernatant was added to 100 µL of
25-times-diluted ATP assay mix solution from a bioluminescent assay kit
(Sigma). Light emission was immediately measured 3 times for 10 s
each in a luminometer (model 1253, Bio-Orbit, Turku, Finland), and the
average value was taken. Protein quantification was performed according
to the method of Bradford (1976), using 100 µL of sample and 1 mL of
Bradford reagent, and allowing the reaction to proceed for 15 min.
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RESULTS |
Man Represses Germination of Arabidopsis Seeds
Growth of Arabidopsis seeds on several different sugars was
tested. It was found that Man, a Glc epimer at the second carbon atom,
repressed germination in a concentration-dependent manner (Fig.
1). In this and subsequent experiments,
the addition of increasing concentrations of Man to the agar medium led
to a decrease in the percentage of seeds that germinated. In the
absence of sugars in the medium the germination frequency was nearly
100%. However, even with a concentration as low as 7.5 mM,
germination was virtually abolished by Man. At lower concentrations the
seeds germinated but growth was halted at an early stage. This effect was shown not to be osmotic, since germination and growth were normal
when 15 mM mannitol or sorbitol was substituted for Man. The addition of similar concentrations of Glc to the medium also did
not affect germination frequencies. Like other metabolizable sugars,
Glc induces increased growth of Arabidopsis seedlings (Rook et al.,
1998).
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| Figure 1.
Man represses germination of wild-type Arabidopsis
seeds in a concentration-dependent manner. Seeds were plated in the
absence of sugar (control) and on 2, 5, 7.5, and 15 mM Man.
Fifteen millimolar mannitol (15 Mtl) was taken as an osmotic control.
Approximately 200 seeds were used for each data point in each
experiment. Values presented are the average of three independent
experiments. Germination was scored at d 8.
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Phosphate and ATP Levels and Repression of Germination
Upon entry into the plant cell, Man is phosphorylated by HXK,
yielding Man-6-P, a process that requires ATP. Man-6-P is then only
slowly processed by the plant, since the enzymes required for this are
either absent or exist in very low concentrations (Sheu-Hwa et al.,
1975; Walker and Sivak, 1986). Due to this property, Man has been used
in the past to provoke ATP and phosphate depletion in adult leaves of
some plant species (Siegl and Stitt, 1990; Van Quy and Champigny,
1992). The Man concentrations used in these studies (50-200
mM) were much higher than the ones we used (7.5 mM). We carried out a series of experiments to determine
whether the repression of germination by Man could be due to phosphate or ATP depletion.
Since phosphate is readily taken up from the medium into plant tissues,
even at concentrations as low as 0.01 µM (Russel and Martin, 1953; Barber and Loughman, 1967; Bieleski, 1973; Lin and Hanson, 1974; Lin, 1979), we added up to 75 mM phosphate to
medium containing 7.5 mM Man, and found that the repression
of germination remained unaltered. This was attempted with combinations
of various phosphate concentrations, different phosphate salts, and at
different pH values, with consistent results (data not shown). Possible ATP depletion was tested by directly measuring ATP levels. Seeds were
plated out both in the absence and in the presence of 0.5, 7.5, and 50 mM Man. The lowest concentration of Man (0.5 mM) still allowed germination, but 7.5 and 50 mM repressed it entirely. A concentration of 50 mM Man has previously been used to provoke ATP depletion in
detached adult wheat leaves (Van Quy and Champigny, 1992). The ATP
levels were then measured at 24-h intervals for several days (Fig.
2).
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| Figure 2.
Man does not deplete ATP in wild-type Arabidopsis
seeds. Seeds were plated out at d  2 in the absence (control,  ) or
in the presence of 0.5 ( ), 7.5 ( ), or 50 (×)
mM Man. After being submitted to a 48-h cold treatment at
4°C to promote germination, they were placed at 22°C at d 0. Measurements were taken at 24-h intervals to determine the ATP and
protein levels in the seedlings. Approximately 50 seeds or seedlings
were used for each measurement per experiment. prot., Protein.
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Upon germination in the absence of sugars, ATP levels expressed on a
protein basis decreased 100- to 1000-fold, but if germination was
blocked by Man, ATP levels did not change. When germination was
repressed, the overall protein content measured in the soluble fraction
was lower than when germination occurred. This explains why the
[ATP]/µg protein ratio shown in Figure 2 decreased in germinated
seedlings (control and 0.5 mM Man) and should not be directly compared with that of the samples that did not germinate (7.5 and 50 mM Man). We conclude that at the seed stage the
presence of Man in the external medium did not greatly affect the
internal ATP concentration, since the ATP levels in seeds treated with 7.5 or 50 mM Man were not greatly affected. This suggests
that it was not due to a lack of ATP that germination was
blocked. At d 0 and 1, before germination had occurred, the ATP levels were in the same range in all seeds, showing that there was sufficient ATP to allow germination, even in the seeds on 50 mM Man.
This was observed in several independent experiments.
To further investigate a possible effect of Man on ATP or phosphate
levels, seeds were plated out on Murashige and Skoog medium containing
7.5 mM Man and 75 mM phosphate. In the absence
of Man, germination took place on d 2. In the presence of Man,
germination still had not occurred by d 4. The nongerminated seeds were
briefly rinsed with sterile water and transferred to Man-free, 75 mM phosphate Murashige and Skoog medium. If phosphate or
ATP depletion were preventing germination we would not expect seeds to
germinate upon transfer. If, on the contrary, phosphate and ATP levels
remained sufficiently high, and if Man were repressing germination via another mechanism, germination upon transfer would be expected. Upon
transfer the seeds did germinate, although with signs of stress,
as indicated by elevated anthocyanin levels (not shown). These results
suggest that in the presence of Man there was sufficient ATP and
phosphate in the seeds to allow germination.
Further evidence for this came from the observation that the addition
of Glc to Man-containing medium (Fig. 3)
was capable of restoring germination. This effect was already visible
when 2.5 mM Glc was added together with 7.5 mM
Man, and became stronger with increasing Glc/Man concentration ratios.
Identical results were obtained when Glc was substituted for other
metabolizable sugars such as Suc and Fru (data not shown). Seeds did
not germinate on medium containing 7.5 mM Man with 60 mM mannitol, showing that this effect is specific for Glc.
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| Figure 3.
Glc relieves the repression of germination caused
by Man. All values are expressed as millimolar concentrations. In lane
1, seeds were germinated in the absence of any sugar (control), whereas
in the remaining lanes 7.5 mM Man was present. Lanes 2 to
6, Germination was gradually restored with the addition of increasing
Glc concentrations. In lane 7, 60 mM mannitol (60 Mtl) was
added together with Man as an osmotic control. Germination was scored
at d 8, and approximately 200 wild-type seeds were taken for each
data point. The results represent the average of three independent
experiments.
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Glc Analogs and Germination
After testing several Glc analogs, we found that 5 mM
2-deoxyglucose also repressed Arabidopsis germination. The addition of
Glc (30 mM) was capable of fully reversing this effect, as it had been with Man (not shown). In addition to Man and
2-deoxyglucose, two other Glc analogs were tested for their effect on
the germination of Arabidopsis seeds. Whereas 10 mM Man or
2-deoxyglucose repressed germination, the same concentration of
6-deoxyglucose, 3-O-methylglucose, Fru, or Glc did not (Fig.
4). All of these Glc analogs are taken up
by the plant (Lin et al., 1984; Komor et al., 1985), but 6-deoxyglucose and 3-O-methylglucose cannot be phosphorylated by HXK. Man
and 2-deoxyglucose are phosphorylated by this enzyme into Man-6-P and
2-deoxyglucose-6-P, respectively (Sols et al., 1958; Dixon and Webb,
1979), in the same manner in which HXK normally converts Glc to
Glc-6-P. However, whereas Glc-6-P is further processed by the cell,
yielding energy and serving as a carbon source, Man-6-P and
2-deoxyglucose-6-P do not enter glycolysis at a significant rate (Egyud
and Whelan, 1963; Bessel and Thomas, 1973; Herold and Lewis, 1977;
Harris et al., 1986). This difference in the effects on germination
between these two classes of Glc analogs suggested the possible
involvement of HXK, but not Glc transporters, in the repression of
germination by Man.
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| Figure 4.
Effect of Glc analogs on germination.
Approximately 200 wild-type seeds were plated on media containing Man,
2-deoxyglucose (2-dG), 6-deoxyglucose (6-dG),
3-O-methylglucose (3-OMG), Fru, or Glc in 10 mM concentrations. In lane 1, seeds were sown in the
absence of external sugars (control).
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Mannoheptulose Inhibits the Effect of Man on Germination
Mannoheptulose is a specific, competitive HXK inhibitor (Sols et
al., 1958; Coore and Randle, 1963; Salas et al., 1965) and is not
metabolized by the plant cell. We confirmed this by plating seeds on a
medium containing 100 mM mannoheptulose and allowing them
to grow. The external addition of metabolizable sugars is known to lead
to increased growth rate, greater root elongation, and increased starch
and anthocyanin accumulation (Rook et al., 1998). However, in the
present study, seedlings grown in the presence of 100 mM
mannoheptulose remained similar to those grown in the absence of
external sugars. Furthermore, we observed no effect of 100 mM mannoheptulose on germination frequencies of Arabidopsis seeds. If HXK was involved in carbohydrate-mediated repression of
germination then this repression should be relieved by inhibiting this
enzyme. For this purpose we plated seeds on 5 mM
Man-containing medium in the absence and in the presence of 50 and 100 mM mannoheptulose (Fig. 5),
and found that this HXK inhibitor did overcome the effect of Man on
germination in a concentration-dependent manner. A 10- to 20-fold molar
excess of mannoheptulose was required for this effect, suggesting
that mannoheptulose may have lower affinity for HXK than Man, as was
observed for yeast HXK (Sols et al., 1958). These results suggest
the involvement of HXK in signaling Man-induced repression of
germination.
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| Figure 5.
Mannoheptulose (MHL), a specific HXK inhibitor,
is capable of restoring germination to Man-repressed wild-type seeds.
Seeds were plated in the absence of sugars (control) and on 5 mM Man to which 50 and 100 mM mannoheptulose
was added. Approximately 150 seeds were used for each data point and
germination was scored at d 8.
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Germination of sun6 on Man
In the sun6 mutant 3% Suc is not capable of repressing
the gene for plastocyanin and several other genes, as it is in the wild-type plant. Although Suc is a disaccharide, it can readily be
converted to Glc and Fru, both of which are HXK substrates. Van Oosten
et al. (1997) showed that in sun6 photosynthesis is no
longer repressed by the Glc analog 2-deoxyglucose, and that sun6 seedling development is insensitive to high Glc
concentrations that arrest development in wild-type seedlings. They
proposed that the sun6 mutation affects a process involved
in the HXK-mediated signal transduction pathway. If this were the case,
and if Man inhibits germination via HXK, one would expect
sun6 to germinate on Man-containing medium. Therefore, we
plated seeds in the presence of 7.5, 10, and 15 mM Man and measured the germination frequencies at d 8. A greater insensitivity to Man was observed for sun6
in the entire range of concentrations tested but was most clear with 7.5 mM Man (Fig.
6). sun6 displayed a
percentage of germination several times higher than that of the wild
type, suggesting that Man blocks germination via a HXK-mediated
pathway, as our previous work indicated.
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| Figure 6.
Germination frequency of sun6
(black bars) and wild type (white bars) in the absence of external
sugars (control) and on 7.5, 10, and 15 mM Man-containing
medium. Approximately 80 seeds per line were used in each experiment
and results represent the average of three independent experiments.
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DISCUSSION |
Carbon metabolite levels directly and indirectly influence
virtually every metabolic process in the life of a plant. Much has been
published about how metabolites regulate a given process at the enzyme
level, but far less is known about how this regulation occurs at the
gene-expression level. Carbon-metabolite-mediated regulation of gene
expression seems to be of fundamental importance for plant functioning.
In yeast, plant, and animal systems an important role for HXK has been
proposed in sensing and signaling of the sugar status. Exactly how
HXK transmits this signal to downstream elements in the pathway is
unknown.
We have shown that the HXK substrate Man represses germination of
Arabidopsis seeds at concentrations as low as 5 mM in the growth medium. All metabolizable sugars tested were able to reverse this effect. This was also true for seeds whose germination had been
inhibited by the Glc analog 2-deoxyglucose (data not shown). Although
monosaccharides such as Glc and Fru can also be phosphorylated by HXK
and should have an effect similar to that of Man or 2-deoxyglucose on
gene expression, they can be further metabolized and used as energy and
carbon sources. With Man and 2-deoxyglucose this occurs to a much
lesser extent, so we propose that this is why germination is restored
by metabolizable sugars. 6-Deoxyglucose and
3-O-methylglucose, two other Glc analogs, were taken up
by the plant with the same efficiency as Glc, but unlike Glc, Man, or
2-deoxyglucose, they could not be phosphorylated by HXK. The
finding that these HXK-nonphosphorylatable Glc analogs did not repress
germination shows that it is not via sugar uptake that Man represses
germination. Furthermore, it suggests that repression occurs via a
HXK-mediated pathway.
In the presence of 7.5 mM Man, both ATP and phosphate were
present in sufficient amounts to allow germination. Although Man has
been reported to provoke ATP and phosphate depletion in detached leaves
of adult plants, in our system we did not observe any significant reduction in seed ATP levels at the time of germination. This may be
explained by the significantly higher Man concentrations that are used
for phosphate and ATP depletion. In addition, to our knowledge,
phosphate and ATP depletion have never been measured previously in
Arabidopsis (or in any other plant at the seed stage). By plating
seeds on 7.5 mM Man and transferring them to Man-free medium at d 4, we were able to show that they remained fully viable and
capable of germinating.
Phosphate has previously been implicated in metabolite-mediated gene
regulation (Sadka et al., 1994; Takeda et al., 1994; Berger et al.,
1995), but this was not supported by our results. Several different
phosphate salts at various concentrations and pH values were tested,
but even when 75 mM phosphate was added to the medium, the
effect of 7.5 mM Man was not overcome. Jang and Sheen
(1994) and Graham et al. (1994) previously reached this same
conclusion. By coelectroporation of phosphate with Man into a maize
protoplast culture, Jang and Sheen (1994) showed that phosphate did not
relieve the repressive effect of Man on expression of photosynthesis
genes. It is possible that the genes studied by these different groups
belong to two different branches of the sugar-signaling pathway, one
branch involving phosphate and the other not. The genes involved in our
system probably belong to the latter.
To confirm that HXK is involved in the repression of germination by
Man, we carried out experiments using mannoheptulose, a specific
competitive inhibitor of this enzyme. The observation that seeds
germinate on Man when mannoheptulose is present is strong evidence that
a metabolic signal is capable of halting germination in Arabidopsis,
and that this signal is transmitted via a HXK-mediated pathway. Since
it is by this enzyme that Man is phosphorylated and since Man-6-P is
only slowly metabolized, HXK is likely to be the first component of the
pathway. This supports previous independent studies by Graham et al.
(1994) in cucumber and by Jang and Sheen (1994, 1997) and Jang et al.
(1997) in maize and Arabidopsis, which proposed an identical function
for HXK in carbohydrate-induced gene regulation.
Our results suggest that in Arabidopsis seeds the phosphorylation of
Man by HXK triggers a signaling cascade leading to the repression of
genes needed for germination. The possibility that this signal could
also activate genes whose products block germination must not be
excluded. Further research is needed to determine the nature of the
genes involved. Free Man is not found in green plants except in trace
amounts in some species, during the breakdown of reserve mannans of
seeds, and during storage in certain vegetative organs (Herold and
Lewis, 1977). In Arabidopsis Man leads to the accumulation of Man-6-P,
which is thought to be only slowly metabolized. Seed lipid and protein
reserves are mobilized to generate Suc and amino acids for germination,
which are then transported and used for growth. The expression of
several genes involved in this process is repressed by sugars,
including Man (Graham et al., 1994). One could speculate that Man
blocks germination by interfering with this process. Man present in the
medium is phosphorylated by HXK, potentially halting the mobilization
of seed reserves. The addition of an external energy and carbon source,
such as Glc, Fru, or Suc, would restore germination. Although competing with Man for HXK, mannoheptulose contributes to germination by reducing
the HXK-mediated signal.
Carbon-metabolite-mediated gene regulation in plants is expected to be
complex in view of the dozens of components known to be involved in
yeast (Thevelein, 1994; Smeekens and Rook, 1997). A higher degree of
complexity is expected due to the greater metabolic complexity and
multicellular nature of plants, and also because plants are capable of
synthesizing their own sugars via photosynthesis. By investigating the
effect of Man on germination, we are probably only looking at a
HXK-specific sugar-signaling pathway or a HXK-mediated metabolic
effect.
The availability of the sugar-signaling sun6 mutant, which
was previously isolated in our laboratory (Dijkwel et al., 1997), allowed us to further test whether Man blocks germination via a
HXK-mediated process. sun6 was isolated for its
insensitivity to Suc, a disaccharide that can readily be converted to
Glc and Fru, both of which are HXK substrates. Photosynthesis and
expression of photosynthesis genes show a reduced sensitivity to
repression by the Glc analog 2-deoxyglucose in sun6 compared
with the wild type. Seedling development in this mutant has also been
found to be insensitive to high concentrations of Glc (Van Oosten et al., 1997), and Van Oosten et al. (1997) proposed that the
sun6 mutation affects a process involved in the HXK-mediated
signal transduction pathway. When tested for its capacity to germinate on Man, we found that sun6 displays a significantly greater
insensitivity to this Glc analog than the wild type. This further
supports the conclusion that Man blocks germination via a HXK-mediated
pathway. sun6 may be mutated in HXK itself or in any of the
pathway's downstream components. We are currently attempting to clone
the gene.
Based on the findings in the present study, we have carried out screens
on Arabidopsis ethyl methanesulfonate and T-DNA- and transposon-tagged
mutant collections, and have isolated putative mutants in the
HXK-mediated sugar-sensing and -signaling pathway. The basis of this
screen is that such mutants are expected to germinate on Man-containing
medium and a considerable number of mig
(Man insensitive germination)
mutants have been obtained.
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FOOTNOTES |
1
This work was financially supported by the
Fundação para a Ciência e Tecnologia, Lisbon,
Portugal (grant no. PRAXIS XXI/BD/3103/94 to J.V.P.).
*
Corresponding author; e-mail j.pego{at}bio.uu.nl; fax
31-30-2513655.
Received July 14, 1998;
accepted December 3, 1998.
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ABBREVIATIONS |
Abbreviation:
HXK, hexokinase.
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Abstract
Introduction