First published online June 12, 2003; 10.1104/pp.103.020347
Plant Physiology 132:1424-1438 (2003)
© 2003 American Society of Plant Biologists
CELL BIOLOGY AND SIGNAL TRANSDUCTION
Mechanisms of Glucose Signaling during Germination of Arabidopsis1
John Price,
Tsai-Chi Li,
Shin Gene Kang,
Jong Kuk Na and
Jyan-Chyun Jang*
Department of Horticulture and Crop Science, The Ohio State University,
Columbus, Ohio 43210
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ABSTRACT
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Glucose (Glc) signaling, along with abscisic acid (ABA) signaling, has been
implicated in regulating early plant development in Arabidopsis. It is
generally believed that high levels of exogenous Glc cause ABA accumulation,
which results in a delay of germination and an inhibition of seedling
development—a typical stress response. To test this hypothesis and
decipher the complex interactions that occur in the signaling pathways, we
determined the effects of sugar and ABA on one developmental event,
germination. We show that levels of exogenous Glc lower than previously cited
could delay the rate of seed germination in wild-ecotype seeds. Remarkably,
this effect could not be mimicked by an osmotic effect, and ABA was still
involved. With higher concentrations of Glc, previously known Glc-insensitive
mutants gin2 and abi4 exhibited germination kinetics similar
to wild type, indicating that Glc-insensitive phenotypes are not the same for
all developmental stages of growth and that the signaling properties of Glc
vary with concentration. Higher concentrations of Glc were more potent in
delaying seed germination. However, Glc-delayed seed germination was not
caused by increased cellular ABA concentration, rather Glc appeared to slow
down the decline of endogenous ABA. Except for the ABA-insensitive mutants,
all tested genotypes appeared to have similar ABA perception during
germination, where germination was correlated with the timing of ABA drop to a
threshold level. In addition, Glc was found to modulate the transcription of
genes involved in ABA biosynthesis and perception only after germination,
suggesting a critical role of the developmental program in sugar sensing. On
the basis of an extensive phenotypic, biochemical, and molecular analysis, we
suggest that exogenous Glc application creates specific signals that vary with
concentration and the developmental stage of the plant and that Glc-induced
fluctuations in endogenous ABA level generate a different set of signals than
those generated by external ABA application.
Because metabolic and structural functions require the proper amount of
carbon source, different organisms have developed the ability to sense
internal levels of sugar and accordingly adjust their cellular and metabolic
activities. These regulatory mechanisms are particularly important for plants,
because sugar production, consumption, and storage occur in the same organism.
On one hand, plants have developed sophisticated programs in managing sugar
production in source tissue and sugar storage in sink tissue; on the other
hand, a complex regulatory circuit controlling gene expression has evolved to
accommodate constant changes of sugar-dependent cellular activities
(Smeekens, 2000 ;
Coruzzi and Bush, 2001;
Coruzzi and Zhou, 2001).
Because sugars affect the expression of a diverse array of genes involved
in different cellular processes, it is proposed that distinct signaling
pathways are employed for the control of these genes. At least three types of
Glc signal transduction mechanisms have been found in plants: hexokinase
(HXK)-dependent pathways (Jang et al.,
1997; Xiao et al.,
2000), HXK-independent pathways
(Martin et al., 1997;
Mita et al., 1997;
Roitsch, 1999;
Xiao et al., 2000;
Ciereszko et al., 2001), and
glycolysis-dependent pathways that depend on the catalytic activity of HXK
(Xiao et al., 2000). Besides
gene regulation, sugars also negatively affect seed germination and early
seedling development. This developmental arrest has been used for the genetic
selection of mutants with altered response to sugars. For example,
Glc-insensitive (gin; Zhou et
al., 1998; Arenas-Huertero et
al., 2000) and Suc-insensitive (sis;
Laby et al., 2000) mutants
were isolated using 333 mM Glc and 300 mM Suc,
respectively. In contrast, Suc-uncoupled (sun) mutants were
identified in a screen on the basis of the expression of a sugar-responsive
fusion gene PC: LUC (Huijser et al.,
2000). More recently, the impaired Suc induction (isi)
mutants were isolated using a reporter gene ApL3:GUS
(Rook et al., 2001).
Surprisingly, most of these mutants turned out to be abscisic acid
(ABA)-related. For instance,
gin6/isi3/sis5/san5/sun6 is
allelic to ABA-insensitive mutant abi4-1,
gin1/isi4/san3/sis4/sre1 is allelic
to ABA deficient mutant aba2, and
gin5/isi2/sis3 is allelic to aba3
(Rolland et al., 2002). It has
been suggested that Glc-induced ABA accumulation is essential for HXK-mediated
Glc responses (Arenas-Huertero et al.,
2000). However, the role of ABA in sugar signaling, how sugar
signaling and ABA signaling crosstalk, and whether ABA-independent
sugar-signaling pathways exist are still uncertain
(Finkelstein and Gibson,
2001).
The relationship between sugar and ABA in regulating seed germination is
unclear. It is presumed that high levels of exogenous Glc cause ABA
accumulation, which results in a delay of germination and an inhibition of
early seedling development. Arenas-Huertero et al.
(2000) showed that ABA
accumulated 3- to 6-fold in seedlings when treated with high concentrations of
Glc. In contrast, endogenous ABA concentration was not changed in seedlings
when treated with low levels (e.g. 27.8 mM) of Glc
(Garciarrubio et al., 1997).
Because these studies were conducted using seedlings but not imbibed or
germinating seeds, it is not known whether exogenous sugar can induce ABA
accumulation and consequently inhibit seed germination. Intriguingly, a low to
intermediate level (<167 mM) of exogenous Glc was able to
relieve the inhibition of seed germination induced by exogenous ABA. It was
proposed that ABA inhibits seed germination through the restriction of energy
and metabolites, because germination could be restored by the addition of
metabolizable sugars and amino acids in the culture medium
(Garciarrubio et al., 1997).
These results were supported by a more recent study using 15 to 90
mM Glc, Suc, or Fru
(Finkelstein and Lynch, 2000).
However, multiple lines of evidence from this study indicated that the effect
of sugar was likely caused by the change of signaling in addition to metabolic
events. A subsequent report demonstrated that the addition of sugar inhibited
mobilization of stored lipids during germination
(To et al., 2002).
In the past, the sensitivity of a plant to sugar signals was primarily
determined by looking at overall plant growth, a process involving
germination, cotyledon greening and expansion, hypocotyl elongation, true leaf
development, and root growth. Although many mutants have been identified in
such way, their responses to sugar in each of these developmental steps are
not clear. Here, we use a simple but effective approach to systematically
determine sugar sensitivity in each mutant. We have found that some mutants
previously known to be sugar insensitive are actually sensitive to the
sugar-induced delay of seed germination. We have also provided additional
evidence for an intimate relationship between sugar and ABA in controlling
seed germination.
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RESULTS
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To understand the linkages between the sugar- and ABA-signaling pathways,
we observed how Glc and ABA affected seed germination. Germination kinetics
was chosen for study because germination can be simply and objectively scored
for all seeds in a sample and because other developmental events (like
hypocotyl elongation or cotyledon greening) appear to involve additional or
different signaling mechanisms than those for germination. Seeds, including
wild ecotypes (WT) and various mutants insensitive to Glc or ABA, were plated
on Murashige and Skoog plates with added Glc and/or ABA. To ensure uniformity
of results, all seeds were harvested and stored identically, seeds used in
tests were selected for similar size and color, the Murashige and Skoog plates
used contained the same amount of medium and were handled identically, and the
seeds were evenly spaced in grids on the plates to avoid spatial variations.
Germination experiments were repeated both as simultaneous replicate plates
and as separate experiments. The kinetics of germination was determined by
measuring the proportion of seeds at different time points in a sample where
the radicle had begun to emerge from the seed coat. To interpret the results,
we compared the rates of germination between different strains and different
treatments throughout the time course. The slope of the curves and the timing
of when the germination rates started to change revealed the differences that
occurred among the various samples and treatments.
Arabidopsis Seed Germination Is Sensitive to Low Levels of Glc
To assess the amount of Glc required to transmit a signal during
germination, seeds were grown in the presence of low Glc levels, and
germination kinetics was determined (Fig.
1). WTs Columbia (Col-0), Landsberg erecta
(Ler), and Bensheim (BE) all showed a delay in germination in the
presence of exogenous Glc. Delay of germination was evident even at 27.8
mM Glc in Col-0, which is considered well below the level (333
mM) repressive for cotyledon greening and early seedling
development (Jang et al.,
1997) or ABA accumulation
(Garciarrubio et al., 1997).
Compared with other tested ecotypes, Ler exhibited an overall shift
toward later germination, whereas BE was less sensitive to low exogenous Glc
(Fig. 1B). In all ecotypes, the
sensing of the low Glc levels occurred very early in the germination process,
where germination delay was evident as early as 12 h after transfer to
24°C in the light.
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Figure 1. Arabidopsis can sense a low level of exogenous Glc during seed germination.
Arabidopsis seeds were surface-sterilized and water-imbibed in the dark for 3
d at 4°C. Seeds were transferred to 1x Murashige and Skoog plates
containing B5 vitamins, 0.05% (w/v) MES (pH 5.7), and 0.7% (w/v) phytagar
(Invitrogen) without sugar (Murashige and Skoog) or with Glc (concentration
indicated in the figure). The plant material was incubated in the dark at
4°C for 3 d to break dormancy and was then transferred to light at
24°C. Germination kinetics was determined by measuring the time of radicle
emergence after transfer to constant light and 24°C. A, ABA biosynthetic
mutants aba2-1 and aba3-1 and ABA
perception mutant abi4 are less sensitive to Glc than their
background ecotype Col-0. B, Compared with the background ecotype, there were
no apparent changes in Glc sensitivity in either HXK (AtHXK1) loss-of-function
mutant gin2 (in Ler background) or AtHXK1 overexpresser
35SAtHXK1 (in BE background).
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The initial delay of germination was largely relieved in the ABA
biosynthetic mutant aba2-1, indicating that somehow, ABA is involved
in transmitting the signal triggered by low-level Glc. The ABA-insensitive
mutant abi4-1, which has been reported to be a transcriptional
regulator affecting ABA signaling, also reduced this delay but to a lesser
extent than did aba2-1. Another ABA biosynthetic mutant
aba3-1 also reduced this delay but was not as effective as
aba2-1 (Fig. 1A).
Unlike aba2-1, aba3-1, and abi4-1, the dominant
ABA-insensitive mutant abi1-1 appeared to be sensitive to Glc. It had
germination kinetics similar to Ler, thus ABI1 is not likely to be an
important component in transmitting a sugar signal (data not shown). The
mutant strain gin2, which is defective in AtHXK1
(Rolland et al., 2002), had
slightly quicker germination kinetics in the presence of Glc when compared
with its parental ecotype Ler
(Fig. 1B), more notable at 55.5
mM Glc. In contrast, a Glc-hypersensitive transgenic line
overexpressing AtHXK1, CaMV35S:AtHXK1, exhibited a somewhat
enhanced Glc-induced delay of germination when compared with parental ecotype
BE. Although both results were reproducible, the subtle difference suggests
that AtHXK1 may play only an indirect role in sensing low levels of Glc during
germination.
Arabidopsis Seed Germination Is Not Affected by Low Levels of
Mannitol
To test whether Glc-induced delay of seed germination was an osmotic
effect, germination kinetics was also determined for seeds plated on medium
containing mannitol, a non-metabolizable sugar. As shown in
Figure 2, a delay in
germination still occurred, but only when mannitol was at a high concentration
(333 mM), and the delay was much less effective when compared with
that caused by Glc (Fig. 2A).
This suggests that the delay in kinetics caused by low Glc (shown in
Fig. 1) is not a pure osmotic
response. The delay caused by 333 mM mannitol may be due to osmotic
stress, because aba2-1, aba3, and abi4-1 were less sensitive
to this treatment (Fig.
2B).
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Figure 2. Osmotic effect does not mimic the inhibitory effect of Glc on seed
germination. Plant material was prepared as described in
Figure 1, except that no sugar
(Murashige and Skoog) or various concentrations of either mannitol or Glc
(indicated in the legend) were added to the Murashige and Skoog plates.
Germination kinetics was determined by measuring the time of radicle emergence
after transfer cultures to constant light, 24°C. A, Compared with
mannitol, Glc is more effective in delaying seed germination. B, ABA
biosynthetic mutants aba2-1 and aba3-1 and ABA perception
mutant abi4 are less sensitive to 333 mM mannitol than the
background ecotype Col-0. C, HXK does not appear to be involved in osmotic
sensing. Compared with the background ecotype, there is no apparent difference
in germination kinetics in response to mannitol in either HXK (AtHXK1)
loss-of-function mutant gin2 (in Ler background) or AtHXK1
overexpresser 35SAtHXK1 (in BE background).
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Similar to Glc response, different ecotypes exhibited varied sensitivity to
mannitol. Ler and BE ecotypes turned out to be hypersensitive to 333
mM mannitol when compared with the Col-0. In contrast to ABA
mutants, HXK did not seem to be involved in sensing low concentrations of
mannitol because gin2 and CaMV35S:AtHXK1 displayed
a similar pattern to Ler and BE, respectively
(Fig. 2C).
Do Glc and ABA Inhibit Seed Germination through the Same
Mechanism?
It is generally accepted that high levels of Glc induce ABA accumulation,
which causes delays in development
(Arenas-Huertero et al., 2000).
However, there has not been a systematic investigation during germination
using various WT ecotypes and mutants to confirm this notion. To do this, we
compare germination kinetics between intermediate (167 mM) and high
(333 mM) levels of Glc and 1 µM exogenous ABA
(Fig. 3A). Surprisingly,
neither aba2-1/aba3-1 nor abi4-1 showed significant
difference in germination kinetics caused by 167 mM Glc when
compared with the WT. However, whereas aba2-1 and aba3-1
showed resistance to 333 mM Glc, abi4-1 exhibited a
response similar to the WT. By contrast, the abi4-1 mutant displayed
a robust resistance to 1 µM ABA that was not observed in
aba2-1, aba3-1, or WT. We also determined the role of HXK in sensing
high levels of Glc and ABA (Fig.
3B). Similar to ABA mutants, neither gin2 nor
35S:AtHXK1 displayed altered sensitivity to 167
mM Glc. Surprisingly, neither of them was significantly different
from the WT in the response to 333 mM Glc. Together, these results
raise the possibility that neither ABI4 nor AtHXK1 is involved in germination
controlled by high levels of Glc. The results also indicate that a
"Glc-insensitive" phenotype may not be universal to all
developmental stages of plant growth, because although these mutants appear to
be sensitive to high Glc levels during germination, abi4
(Arenas-Huertero et al., 2000;
Finkelstein and Lynch, 2000;
Huijser et al., 2000;
Laby et al., 2000;
Rook et al., 2001) and plants
with reduced HXK levels (Jang et al.,
1997) are less sensitive to high levels of Glc during early
seedling development.
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Figure 3. Sugar sensing is affected by developmental program, because previously
known sugar-insensitive mutants abi4 and gin2 are sensitive
to a high level of Glc during germination. Plant material was prepared as
described in Figure 1, except
that no sugar (Murashige and Skoog), or Glc (concentration indicated in the
figure) or 1 µMABA was added to the Murashige and Skoog plates.
Germination kinetics was determined by measuring the time of radicle emergence
after transfer cultures to constant light and 24°C. A, Compared with the
background ecotype Col-0, whereas ABA biosynthetic mutants aba2-1 and
aba3-1 are less sensitive to Glc, abi4 is less sensitive to
ABA. B, HXK AtHXK1 is not involved in sensing either Glc or ABA during seed
germination. Compared with the background ecotype, there is no apparent
difference in germination kinetics in either HXK (AtHXK1) loss-of-function
mutant gin2 (in Ler background) or AtHXK1 overexpresser
35SAtHXK1 (in BE background).
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Glc Modulates ABA-Induced Delay of Seed Germination
Germination kinetics provided information on another developmental effect
that we observed, partial relief of ABA-induced delay of germination in the
presence of exogenous Glc. This effect was first noted in WT seeds
(Garciarrubio et al., 1997);
we further investigated the kinetics of this relief in various Glc- and
ABA-insensitive strains. In the presence of exogenous ABA, seed germination
was usually repressed (Fig.
3A). This repression was diminished when 167 mM Glc, a
level repressive for germination but not for subsequent overall seedling
development, was also added to the medium
(Fig. 4A). Lower levels of
exogenous Glc, e.g. 16.7 mM, were also effective in diminishing the
delay in germination caused by ABA (data not shown). Interestingly, the rate
of germination was not statistically different in aba2-1, aba3-1,
abi4-1, and their parental ecotype Col-0, suggesting that the presence of
both Glc and ABA overrides the normal ABA-responsive pathways. However,
35S: AtHXK1 did show quicker relief of repression than the
parental ecotype BE in the rescue experiment
(Fig. 4A), suggesting that HXK
is likely a positive factor for Glc effect antagonistic to ABA. In a previous
report, sugar was shown in WT to suppress ABA inhibition of radicle emergence
but not seedling growth (Finkelstein and
Lynch, 2000), although the genetic components involved were not
identified. We were surprised to find that except for the ABA-insensitive
mutants, seedlings that germinated with Glc and ABA did not develop further
after germination (Fig. 4B). We
noted that plants did not develop uniformly in the absence of exogenous sugar.
Although abi4 could germinate without delay and turned green in the
presence of 1 µM ABA, less than 50% of the seedlings developed
further to form true leaves. This is in contrast to abi1, in which
more than 50% of the seedlings could form true leaves under the same
condition. The phenotypic variation between abi4 and abi1
might be due to the different background ecotype, or it is possible that ABI4
and ABI1 differ in their temporal expression or their developmental roles.
This response of abi4 may also be due to the specific characteristics
of the abi4-1 allele, because differences in phenotypic expression
were apparent in abi4-3 and abi4-4 plants treated with 3
µM ABA after 3 d of growth
(Huijser et al., 2000).
Together, these results suggest that the signaling events occurring during
germination are distinct from those affecting post-germination events and that
ABA can have important regulatory effects in post-germination growth.
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Figure 4. Glc partially relieves ABA-induced inhibition of seed germination but not
subsequent seedling development. A, Compared with the background ecotype
Col-0, Glc rescue of ABA-induced inhibition of seed germination is not altered
in either ABA biosynthetic mutants aba2-1 and aba3-1 or ABA
perception mutant abi4. However, HXK seems to be a positive regulator
for this rescue, because faster germination kinetics was observed in AtHXK1
overexpresser 35SAtHXK1 (in BE background). Plant material was
prepared as described in Figure
1, except that 167 mM Glc and 1 µM ABA
were added to the Murashige and Skoog plates. Germination kinetics was
determined by measuring the time of radicle emergence after transfer cultures
to constant light and 24°C. B, Although Glc can rescue the inhibition of
seed germination induced by ABA, subsequent seedling development only occurred
in ABA-insensitive mutants (abi1 and abi4). Seedlings of
aba2-1 (Col-0 background), abi4 (Col-0 background), and
abi1 (Ler background) were grown for 21 d in constant white
light (90 µE m–2
s–1) at 25°C. Plant material was prepared as
described in Figure 1, except
that either 333 mM Glc, 1 µM ABA, or a combination of
167 mM Glc and 1 µM ABA was added to the Murashige
and Skoog plates.
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The Role of Ethylene in Glc Signaling
On the basis of seedling development, sugar signaling has been shown to
interact with ethylene-signaling pathway
(Zhou et al., 1998;
Gazzarrini and McCourt, 2001).
Although ethylene acts as a negative regulator of ABA action during
germination and CTR1, EIN2, and ETR1 are involved in this interaction
(Beaudoin et al., 2000;
Ghassemian et al., 2000;
Gazzarrini and McCourt, 2001),
it is not clear whether these components are involved in sugar response during
germination. We determined the effect of exogenous Glc on the germination
kinetics of ethylene-signaling mutants. Similar to the abi4-1 mutant,
ctr1-1 was as sensitive to high concentrations of Glc (167 or 333
mM) as its parental ecotype
(Fig. 5), indicating either
that high Glc may have a repressive effect downstream of CTR1 in the
ethylene-signaling pathway or that high Glc represses seed germination
independent of CTR1. It is noted that the germination kinetics of Col-0 with
333 mM Glc was somewhat different in Figures
3A and
5; this is likely due to the
use of two different WT seed lots for the two sets of experiments.
Ethylene-insensitive mutants etr1-1 and ein2-1 appeared to
have a delay of germination kinetics even greater than that of ctr1-1
or WT in the presence of high concentrations of Glc
(Fig. 5). Consistent with the
idea that CTR1 is a negative regulator and EIN2 is a positive regulator of
ethylene response, we have found that whereas ctr1-1 is less
sensitive, ein2-1 is more sensitive to exogenous ABA (1
µM) during germination (data not shown). Collectively, our
results suggest that ethylene signaling antagonizes the inhibitory effect of
Glc on seed germination. ABA is likely involved in transmitting the sugar
signal because our results resemble a model showing that ethylene plays a role
opposing ABA during seed germination
(Beaudoin et al., 2000).
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Figure 5. Ethylene-signaling mutants have altered Glc response during germination.
Germination kinetics was determined in Col-0 or ethylene-signaling mutants
ctr1-1, etr1-1, or ein2. Plant material was
prepared as described in Figure
1, except that no sugar (Murashige and Skoog) or high
concentration of Glc (indicated in the figure) was added to the Murashige and
Skoog plate. Germination kinetics was determined by measuring the time of
radicle emergence after transfer to constant light and 24°C.
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Exogenous Glc Delays Decline in Endogenous ABA Concentration during
Seed Germination
To better understand how endogenous ABA affects germination, we observed
how endogenous ABA levels fluctuate over time with and without the presence of
exogenous Glc or ABA. ABA levels were measured in plant material of various
time points and treatments using the Phytodetek ABA kit (Agdia Inc., Elkhart,
IN). To minimize variation in the results, different lots of kit reagent were
tested for consistency before obtaining the final results, the most consistent
reagent lot (no. 38, mfg. date 2/99) was used for all experiments, duplicate
samples were prepared in each experiment, and each experiment was repeated.
Consistent with previous reports
(Leon-Kloosterziel et al.,
1996), the endogenous ABA concentration was significantly lower in
ABA biosynthetic mutants aba2-1 and aba3-1 than that in the
WT (Table I). This reduction
was also evident in gin1, a mutant allelic to aba2-1
(Cheng et al., 2002), compared
with its WT WS (data not shown). In contrast, the transcriptional regulator
mutant abi4-1 had endogenous ABA levels in imbibed seeds similar to
WT, indicating that ABI4 does not affect internal ABA levels in the seeds
(Table I). Cellular ABA
concentration went down in WT seeds after a 3-d incubation in 4°C water.
Similar changes in ABA concentration were observed in aba2-1 and
aba3-1. Surprisingly, the concentration of ABA in abi4-1 did
not decrease after 3 d in 4°C water, indicating that unlike
aba2-1 and aba3-1, this mutant may not have responded
normally to the cold treatment. The endogenous ABA concentration continued to
drop after the seeds were transferred to Murashige and Skoog medium containing
Glc during a 3-d incubation at 4°C in the dark. It appeared that the onset
of maximal germination occurred when endogenous ABA levels decreased to 2 ng
ABA g–1 fresh weight. Some minor variations to
this trend were noted in our data; we suspect that this is due to the limited
number of replicate experiments that we could conduct because the availability
of lot 38 reagent was limited. In Col-0, internal ABA levels dropped from 5.4
(Table I) to 1.9, 2.9, and 4.2
ng ABA g–1 fresh weight with a treatment of 0,
167, and 333 mM Glc, respectively (d 0 data in
Table II). A
concentration-dependent Glc inhibition of ABA level decline was also observed
in abi4-1 (compare data in Table
I with d 0 data in Table
III), aba2-1, and aba3-1 (data not shown).
Another trend that became apparent in both WTs and mutant strains was that in
the absence or presence of 167 mM Glc, ABA levels continued to
decline both before and after the onset of germination in the light at
25°C. Endogenous ABA concentrations in aba2-1 and aba3-1
had already dropped below the threshold level on d 0, and they continued to
decline during germination (data not shown). In contrast, internal ABA levels
began to rise after reaching a threshold low level in the WT and
abi4-1 treated with 333 mM Glc (Tables
II and
III). Together, these results
indicate that ABA levels decline as part of the normal germination process,
and this can be perturbed by high concentrations of exogenous Glc. The rebound
of ABA concentration in samples treated with 333 mM Glc on d 6 is
likely due to ABA accumulation in a portion of germinated seeds (64.4% in WT;
51% in abi4). ABA levels declined more rapidly and stayed low in the
presence of low-level Glc, whereas high exogenous Glc reduced the rate of the
ABA decline. Additionally, the timing of germination appeared to be correlated
with the decline of ABA concentration. Germination appeared to reach maximal
levels as the ABA concentration declined past a threshold value of
approximately 2 ng ABA g–1 fresh weight.
To further understand the mechanisms controlling germination, we compared
the endogenous levels of ABA present in germinating seeds treated with
exogenous Glc or ABA. In previous studies, aba2-1 was shown to be
insensitive to Glc, so we anticipated that Glc-repressed germination in WT
seeds would occur through increased biosynthesis of endogenous ABA.
Germination kinetics with 333 mM Glc and 1 µM ABA
(Figs. 3A and
5) were quite similar in Col-0,
making it seem reasonable that the two treatments repressed through a similar
mechanism. When endogenous ABA levels were measured in similarly treated Col-0
seeds, we were surprised to find that whereas ABA levels in 333 mM
Glc-treated seeds declined during germination, they accumulated to a very high
level in seeds treated with 1 µM exogenous ABA
(Table II). These results
suggest that the onset of germination may be determined by factors other than
overall ABA concentration. We cannot rule out the possibility that Glc
repression causes a localized increase of ABA in target tissues or that the
levels of ABA present in Glc-treated seeds are sufficient to cause repression.
However, because the germination kinetics of Col-0 was similar in both 333
mM Glc and 1 µM ABA, endogenous ABA concentration
alone is not sufficient to explain why these treatments had resulted in
similar germination kinetics.
Glc Affects the Expression of ABI1, ABA2, and
ABI4
Our results suggest that ABI4 is involved in plant response to a low
concentration of Glc (Fig. 1A)
and exogenous ABA (Fig. 3A). By
contrast, ABA2 is involved in plant response to both low and high
concentrations of Glc (Figs. 1A
and 3A) but not to exogenous
ABA (Fig. 3A). Because the
effects of high Glc and exogenous ABA were not identical, we hypothesized that
genes involved in ABA biosynthesis and signaling might be regulated by Glc
during germination and early seedling development. To compare gene expression
profiles with the kinetics of germination rate and cellular ABA contents, we
followed the same protocol in preparing plant material. Ecotype Col-0 dry
seeds were surface-sterilized and incubated in 4°C water for 3 d.
Cold-imbibed seeds were then transferred to a flask containing Murashige and
Skoog liquid and incubated for additional 3 d at 4°C to ensure a complete
removal of seed dormancy in the control (Murashige and Skoog without Glc or
ABA). Cultures with various treatments were then allowed to germinate and grow
in the white light at 24°C on a shaker (140 rpm). Consistent with the role
of ABA in seed dormancy, ABA2 was present in dry seeds, although the
expression level was very low and could only be detected by reverse
transcription (RT)-PCR but not by standard RNA gel-blot analysis. The low
level of ABA2 expression disappeared after seeds were imbibed in
4°C water for 3 d (Fig.
6A). In contrast, ABI4 was not detectable in WT dry seeds
but was induced after imbibition in 4°C water for 3 d. The expression of
ABI4 decreased significantly when seeds were transferred from water
to Glc-free Murashige and Skoog medium, suggesting that certain components in
Murashige and Skoog medium or developmental factors negatively regulated the
expression of ABI4, even though metabolic activities of the seeds
were relatively low when kept at 4°C
(Fig. 6A).
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Figure 6. Developmental program-dependent induction of ABA2 and
ABI4 by Glc. A, Imbibition exerts an opposing effect on the
expression of ABA2 and ABI4. Col-0 seeds were surface
disinfected and immediately collected (Dry seed), imbibed for 3 d in water at
4°C and collected, or imbibed at 4°C water for 3 d and incubated in
4°C Glc-free 1x Murashige and Skoog medium for another 3 d and then
collected. ABA2 expression was determined by RT-PCR following the
manufacturer's protocol (Panvera, Madison, WI), and ABI4 was
determined by RNA gel-blot analysis. Five micrograms of RNA was loaded in each
lane. RT-PCR of ubiquitin expression and ethidium bromide staining of rRNA
bands are shown for quality and loading controls, respectively. B, RNA
gel-blot analysis of ABA2 expression during germination. Col-0 seeds
were surface disinfected, imbibed 3 d in water at 4°C and another 3 d at
4°C in Glc-free 1x Murashige and Skoog liquid medium containing B5
vitamins and MES (pH 5.7) without sugar (control) or with Glc and/or ABA
(concentration indicated in the figure). After transferring cultures to
constant light at 24°C, samples were collected 2, 4, or 6 d after
incubation (DAI), and total RNA was isolated. Five micrograms of RNA was
loaded in each lane. Ethidium bromide staining of rRNA bands for 2-DAI samples
is shown for loading control. RNA quality and loading are similar for 4- and
6-DAI samples. C, RNA gel-blot analysis of ABI4 expression during
germination. Plant material was prepared identically to the procedure in
Figure 6B, except that samples were collected 4 h after incubation (HAI) in
addition to 2, 4, and 6 DAI. Five micrograms of RNA was loaded in each lane.
Ethidium bromide staining of rRNA bands for 4-HAI and 2-DAI samples is shown
for loading control. RNA quality and loading are similar for 4- and 6-DAI
samples. D, RNA gel-blot analysis of ABI1 expression during
germination. Plant material was prepared identically to the procedure in
Figure 6B, except that samples were collected 4 and 6 DAI. Five micrograms of
RNA was loaded in each lane. Ethidium bromide staining of rRNA bands for 4-DAI
samples is shown for loading control. RNA quality and loading are similar for
the 6-DAI samples.
|
|
Later in the germination and early seedling development, the expression of
ABA2, ABI4, and ABI1 was enhanced by Glc but was relatively
unaffected by ABA (Fig. 6,
B–D). It is possible that the effect of Glc or ABA was
limited by the developmental stage of the plants. For instance, the
ABA2 induction appeared to be correlated with the onset of
post-germination events such as hypocotyl and cotyledon development, i.e. when
WT seed germination approached 100% with each Glc treatment
(Table II), ABA2
expression reached a maximal level at the same time. Sugar concentration also
had an effect independent of germination status, because although the seeds
treated with 16.7 and 167 mM Glc were both germinated by d 4,
induction of ABA2 was clearly higher with 167 mM Glc. We
were not able to detect the presence of ABA2 transcript by either RNA
gel-blot analysis or RT-PCR in any sample 4 HAI in the light at 25°C,
where no seeds had been germinated. Compared with 167 mM Glc, 333
mM Glc was relatively ineffective in ABA2 induction,
presumably because seed germination has yet to occur
(Table II). The expression
pattern of ABI1 or ABI4 was similar to that of ABA2
except that ABI1 was not expressed in the early stage of germination
(4 HAI and 2 DAI; Fig. 6, C and
D). This contrasts with seedlings grown 2 or more weeks, where
transcription of ABI4 is induced by 333 or 389 mM Glc but
not 111 mM Glc (Arenas-Huertero
et al., 2000; Cheng et al.,
2002). These results indicate that the developmental programs of
the seeds tightly control the expression of these genes but that these
programs can be modified at specific developmental stages by the presence of
sugar. Sugar acts to stimulate ABA biosynthesis in germinated seedlings by
up-regulating genes involved in ABA biosynthesis, such as ABA2. On
the other hand, sugar also activates the expression of both ABI1 and
ABI4, both of which are implicated in ABA signaling. Nevertheless, it
is still not clear why 333 mM Glc has such a potent negative effect
on germination and early seedling development, because expression of ABA2,
ABI1, and ABI4 is only slightly affected by high Glc levels. One
possible explanation is that 333 mM Glc may affect other genes
critical for ABA metabolism, because high Glc concentration slowed down the
decline of endogenous ABA during germination (Tables
I,II,III).
This could include genes specific to the developmental stage of the plant,
because only approximately 50% of the seeds are germinated with high-level Glc
compared with nearly 100% germination with 167 mM Glc. Another
possibility is that the response to 333 mM Glc is actually a
combined response to both a sugar-specific signal and an osmotic signal,
because an equivalent concentration of mannitol clearly affected germination
in WT (Fig. 2A).
|
DISCUSSION
|
|---|
A primary role of sugar in regulating seed germination is to modulate both
cellular ABA concentration and ABA response. Germination kinetics has assisted
in discerning which genes may be involved in the transmission of the sugar or
ABA signal. Previous studies have shown that gin6, isi3, sis5, and
sun6 are allelic to abi4-1, which confer resistance to
growth-repressive levels of sugars. On the basis of these reports, we
anticipated that abi4-1 would also confer resistance to Glc during
germination. However, abi4-1 showed sensitivity to 333 mM
Glc similar to its parental ecotype Col-0. The lack of resistance to sugar in
abi4-1 indicates that ABI4 either is not essential or is redundant in
the transmission of sugar signal during germination, even though it is
involved in osmotic sensing, because abi4-1 showed reduced
sensitivity to 333 mM mannitol. Nevertheless, ABI4 is clearly
involved in Glc response after seed germination, because there is no
developmental arrest observed in abi4-1 mutant
(Fig. 4B).
This finding is consistent with studies of ABI5, in which ABI5 was shown to
be a regulator required for maintaining a germinated embryo in a quiescent
state in a narrow developmental window but not directly involved in the
control of germination (Lopez-Molina et
al., 2001). A number of other ABA-responsive elements binding bZIP
factors such as ABF3 and ABF4 (Kang et
al., 2002) also control ABA response in vegetative stage.
Overexpression of either ABI5 or ABF3/ABF4 leads to an ABA-hypersensitive
phenotype, i.e. an arrest of early seedling development under the level of ABA
non-inhibitory to the WT plants. Intriguingly, these plants also showed longer
delay of seed germination than the WT when treated with exogenous ABA. This
suggests that an unknown negative regulator of seed germination can be
trans-activated by excessive amount of either ABI5 or ABF3/ABF4, although
their default function is to control vegetative development but not seed
germination. It will be interesting to find out whether overexpression of the
ABI4 type of AP2/EREBP transcription factors will result in similar
phenotypes.
Because abi4-1 (Fig.
3A), ctr1-1 (Beaudoin
et al., 2000), and abi1-1
(Gosti et al., 1999) are
insensitive to exogenous ABA but sensitive to 333 mM Glc (Figs.
3A and
5) during seed germination, we
propose that there are different ABA-sensing mechanisms in controlling seed
germination and seedling development (Fig.
7). Sensing may involve changes in expression levels driven by the
concentration of signal molecule (Fig.
6) as well as temporal effects of different genes on the
developmental program. The latter is evident when comparing ABI4
expression during germination and seedling development, where ABI4 is
not induced by 333 mM Glc before germination
(Fig. 6C) but is induced by the
same treatment during later growth
(Arenas-Huertero et al., 2000;
Cheng et al., 2002). Unlike
aba2-1 and aba3-1, the dominant ABA-insensitive mutant
abi1-1 is sensitive to Glc
(Arenas-Huertero et al., 2000;
Huijser et al., 2000;
Laby et al., 2000). We have
found that abi1-1 has germination kinetics similar to WT (data not
shown), thus ABI1 is not likely an important component in transmitting sugar
signals during seed germination. Similar to abi4, the
ethylene-signaling mutant ctr1 was sensitive to Glc during seed
germination (Fig. 5) but not
during subsequent seedling development
(Zhou et al., 1998). Although
it has been proposed that sugar- and ethylene-signaling pathways may interact
and that ctr1 is important for both pathways
(Zhou et al., 1998;
Gibson et al., 2001), the
involvement of CTR1 is apparently restricted to certain but not all
developmental processes. Ethylene-signaling mutants etr1 and
ein2 were more sensitive to Glc during germination than the WT
(Fig. 5), suggesting that the
ethylene signal partially reduces the germination repression caused by Glc.
Exogenous ABA appears to have an effect on germination different from the
effect caused by high Glc treatment, perhaps because one treatment creates
additional signaling events that are not replicated in the other treatment.
Evidence for this possibility is apparent when one considers the response of
abi4 to 1 µM ABA and 333 mM Glc. If a
decline in ABA concentration were the sole signal for germination,
abi4 would be expected to behave similarly whether a high
concentration of Glc or ABA is present. However, abi4 germinated
robustly on ABA but behaved like WT with high-level Glc, suggesting that
something other than just ABA concentration controls the germination rates. In
addition, application of exogenous ABA causes the rise of cellular ABA
concentration to a constant high level
(Table II) that is different
from an ABA-declining curve (Table
II) created by Glc treatment. In contrast to abi4, ctr1,
and abi1, the germination kinetics of ABA biosynthetic mutants
aba2-1 and aba3-1 showed greater resistance to Glc but were
sensitive to exogenous ABA.
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Figure 7. Proposed models for Glc-, ABA-, and Glc/ABA-signaling pathways in
controlling seed germination and seedling development. ABA2 and ABA3 affect
the plant response to Glc during both germination and seedling development due
to their effects on cellular ABA concentration. Both ABI4 and CTR1 are
involved in plant response to high levels of exogenous Glc during seedling
development but not during seed germination. In contrast, ABI1, ABI4, and CTR1
are involved in plant response to exogenous ABA during seed germination but
not during seedling development (except ABI1). HXK appears to be a positive
regulator for Glc-induced rescue of seed germination inhibited by ABA.
However, plants do not develop further unless either ABI1 or ABI4 is
defective.
|
|
Our finding that decreased ABA levels coincide with the onset of
germination is consistent with a report showing precocious germination of
transgenic tobacco (Nicotiana tabacum) expressing an ABA-modulating
antibody (Phillips et al.,
1997). In this study, the ABA-modulating antibody gene was placed
under control of a seed-specific promoter, which allowed the accumulation of
antibody before seed development and reduced the concentration of free ABA. In
maturing seeds with high antibody concentration, the embryo within the seed
had green cotyledons. When the seed coat was removed, only antibody-expressing
seeds could germinate without cold treatment. These results suggest that a
reduction of free ABA in seeds triggers the developmental programs normally
associated with germination. This is also consistent with some vp
mutants in maize (Zea mays), whose seeds germinate precociously due
to a deficiency in ABA (Robertson,
1955; Neill and Parry, 1986). In our study, we show that a
reduction of ABA occurs during germination in WT Arabidopsis seeds and that
Glc can modulate the rate at which the ABA decline occurs.
Exogenously applied Glc appeared either to increase the rate of ABA
synthesis or to reduce the rate of ABA decay during germination. Much of the
findings suggests the latter alternative. Because aba2-1 and
aba3-1 are insensitive to Glc during germination and ABA2 and ABA3
are biosynthetic enzymes in the final stages of ABA biosynthesis, it seemed
reasonable to expect Glc would delay germination through induction of ABA
biosynthesis. However, from the results in Tables
I,
II,
III, it is clear that ABA
concentrations decline in germinating WT seeds even in the presence of Glc. In
addition, ABA2 transcription was not induced quickly by Glc during
early germination (Fig. 6B).
The earliest ABA2 induction was detected 2 DAI, and the induction
rate correlated with the onset of germination but not with Glc concentration.
This indicates that Glc does not increase ABA biosynthesis during germination
by transcriptional up-regulation. Also, early steps of ABA synthesis have been
shown occur in the plastids (Seo and
Koshiba, 2002). If Glc increased ABA levels in seeds, it would
suggest that proplastids are active in synthesizing ABA, which is currently
unknown. It still may be possible that Glc stimulates ABA production locally
and transiently in the seed, however, the rate of loss/decay of ABA would have
to be faster than the rate of Glc-induced synthesis.
Then why are aba2-1 and aba3-1 insensitive to Glc? The
most likely explanation is that the endogenous ABA level in aba2-1
and aba3-1 had already dropped near the threshold level (2 ng
g–1 fresh weight) after cold treatment at 4°C
(Table I). Germination cannot
be suppressed even in the presence of high levels of exogenous Glc because ABA
biosynthesis is blocked in these mutants. However, similar to the WT, the
endogenous ABA level continued to decline when these cold-treated
aba2-1 or aba3-1 seeds were transferred and incubated at
4°C in the Murashige and Skoog medium containing Glc. For example, ABA
levels in aba3-1 dropped from 2.2 to 1.1, 1.5, and 1.8 ng
g–1 fresh weight in the medium containing 0, 167,
and 333 mM Glc, respectively. These results clearly indicate that
although both aba2-1 and aba3-1 are less sensitive to
Glc-induced inhibition of seed germination, their Glc-sensing mechanism is
still operational, as evidenced by the differential decline of the endogenous
ABA concentration. Their "Glc insensitivity" is most likely due to
the block of ABA accumulation beyond the threshold low level that is required
for the inhibition of seed germination. However, this does not necessarily
mean that aba2-1 and aba3-1 affect germination identically.
Both aba2-1 and aba3-1 had similar declines in endogenous
ABA concentration, however, aba2-1 appeared to be more insensitive to
low levels of Glc during germination than aba3-1. Both
aba2-1 and aba3-1 are mutations resulting in a single amino
acid substitution (aba2-1, S264N; aba3-1, G469E;
Xiong et al., 2001;
González-Guzmán et al.,
2002). It is possible that mutation of ABA2 causes
greater reduction in ABA biosynthesis than mutation of ABA3 because
an alternate ABA biosynthetic pathway can substitute for the loss of
ABA3 but not ABA2 (Seo
and Koshiba, 2002). Because both aba2-1 and
aba3-1 are not null mutations, another possibility is that the
aba2-1 allele causes a more severe disruption in ABA biosynthesis
than the aba3-1 allele. We cannot rule out the existence of factors
other than ABA concentration that are important for determining the onset of
germination. Our results also suggest that there is not a linear relationship
between Glc concentration and the expression of genes involved in ABA
biosynthesis, because ABA2 transcription was activated to different
extents by low (16.7 mM) and medium (167 mM) levels of
Glc independent of germination status. On the basis of these observations, we
propose that sugar modulates cellular ABA concentration, but the effect of
sugar on seed germination is mediated through a combinatory effect of both
sugar and ABA. Although the modulation of ABA level by Glc appears to be
modest, this modulation might be sufficient to cause significant delay of seed
germination, which resembles the effect of exogenous ABA.
The components that transmit the Glc signal are still unknown, because we
have not found any mutants except aba2-1 and aba3-1 that are
less sensitive to both low and high concentration of Glc during seed
germination. The physiological relevance of different responses to low and
high sugar concentrations is also uncertain, however, there are some
indications that high sugar levels are associated with biotic stresses. Sugars
have been found to accumulate in Arabidopsis at the site of nematode attack
(Bockenhoff et al., 1996), and
sugar transporters are up-regulated when exposed to pathogens such as
bacteria, fungi, or nematodes (Truernit et
al., 1996; Juergensen et al.,
2003). The AtHXK1 mutant gin2, considered to be sugar
insensitive for overall growth, appeared to be slightly more sensitive to 333
mM Glc during germination than its parental ecotype, Ler,
again indicating that it may not be critical for transmitting a sugar signal
during germination. Because mutation of HXK does confer resistance to high
levels of sugar in subsequent growth
(Arenas-Huertero et al., 2000;
Rolland et al., 2002), this
suggests that the signaling functions of HXK may be active only during certain
stages of development. However, the subtle delay of seed germination does
correlate with the level of HXK when low levels of Glc were used
(Fig. 1B). This indicates that
the signaling mechanisms of HXK are also affected by sugar concentration and
that in the presence of low Glc levels, HXK might play an indirect role in
controlling seed germination. The former idea is further supported by
abi4 being sensitive to 333 mM but not <55
mM Glc.
In summary, our simple but effective analyses have provided insights into
the complicated effects of sugar in plant development. Plants do not normally
encounter either high ABA or high sugar condition except under stress.
However, varied levels of exogenous Glc can modulate internal ABA
concentration by increasing synthesis or inhibiting degradation, allowing us
to see how small fluctuations of internal ABA concentration affect the seed
germination process. This modulating effect of Glc cannot be recreated using
exogenous ABA. The process of identifying additional sugar-signaling
components remains a challenge, no matter whether they will again turn out to
be related to ABA-signaling or other hormone-signaling cascades. Global gene
expression analysis may help to distinguish the effects of sugar, ABA, and
osmotic stress, and new specific marker genes may prove to be more effective
in the genetic dissection of sugar-responsive pathways.
|
MATERIALS AND METHODS
|
|---|
Growth of Plant Material
Arabidopsis seeds were surface-sterilized and water-imbibed in the dark for
3 d at 4°C. Seeds were transferred to 1x Murashige and Skoog basal
salt mixture with B5 vitamins and 0.05% (w/v) MES (pH 5.7); Glc, ABA, and/or
0.7% (w/v) phytagar (Invitrogen, Carlsbad, CA) were also added where
indicated. For Murashige and Skoog with phytagar, plates were air-dried to
remove excess surface moisture, and seeds were individually spotted using a
1-µL sterile loop. The plant material was incubated in the dark at 4°C
for 3 d to break dormancy and then was transferred to light at 24°C.
Cultures without phytagar were shaken at 140 rpm using an orbital platform
shaker (New Brunswick, Edison, NJ) under continuous light; all other cultures
were grown with a photoperiod of 16 h of light and 8 h of dark. Germination
kinetics was determined by measuring the time of radicle emergence from
repeated experiments with duplicate plates of approximately 25 seeds each.
Quantitation of ABA Contents
ABA content was determined in seeds or germinated plants immediately after
surface sterilization, imbibition, and at various time points after light
treatment. ABA extraction and determination were performed as previously
described (Arenas-Huertero et al.,
2000). In brief, 30 to 1,000 mg of plant material was washed at
least seven times with sterile distilled water before being homogenized in 1
to 2 mL of ABA extraction buffer (10 mM HCl and 1% [w/v]
polyvinylpolypyrrolidone in methanol). The extract was mixed overnight at
4°C; the supernatant was collected, measured, and neutralized with 15
µL of 1 M NaOH mL–1 extract. ABA
content was quantified using the Phytodetek-ABA kit (AGDIA Inc.) following the
manufacturer's protocol. Raw values for ABA content were standardized to
compensate for the variations in plant mass and extraction volume.
Gene Expression Analysis
Seedlings were grown in liquid cultures containing various amounts of Glc
and/or ABA as indicated and were collected for RNA extraction at various time
points after light treatment. Total RNA was isolated from plant material using
either a standard protocol (Ausubel et al.,
1987) for non-germinating seeds or the plant RNeasy kit (Qiagen
USA, Valencia, CA) for germinated material. RNA gel-blot analysis was
performed with 5 µg of total RNA per lane using standard protocols
(Xiao et al., 2000). The
primer pairs used for the synthesis of the probes, the size of the probes, and
the accession numbers are as follows: ABI1,
5'-TTTCACCGGGATCAGATT-3' and 5'-TAGTTCGCTACCTGAGAA-3'
(497 bp; accession no. U12856); ABA2,
5'-AAAGTGGCATTGATCACT-3' and 5'-TCCTAGTCAAGCCTAGA-3'
(495 bp; accession no. AC037424.10; Jen Sheen, personal communication); and
ABI4, 5'-CACCGACTCATCAACTT-3' and
5'-CATCTGGACCATCTGAT-3' (508 bp; accession no. AF040959).
|
ACKNOWLEDGMENTS
|
|---|
We thank Arabidopsis Biological Resource Center for providing seed stocks,
Dong-mei Li for assisting in germination assays, and Dietz Bauer and Jim
Metzger for critical reading of the manuscript.
Received January 13, 2003;
returned for revision March 19, 2003;
accepted April 18, 2003.
|
FOOTNOTES
|
|---|
Article, publication date, and citation information can be found at
www.plantphysiol.org/cgi/doi/10.1104/pp.103.020347.
1 This work was supported by the Ohio Agricultural Research and Development
Center and Plant Molecular Biology and Biotechnology Program at the Ohio State
University (to J.C.J.). Salaries and research support were provided by the
state and federal funds appropriated to the Ohio Agricultural Research and
Development Center, the Ohio State University. This is manuscript number
HCS02-35. 
*
Corresponding author; e-mail
jang.40{at}osu.edu;
fax 614–292–8496.
|
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