From the Department of Biology, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China
Received for publication, October 6, 2000, and in revised form, December 19, 2000
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ABSTRACT |
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cis,trans-Abscisic acid
(ABA) plays an important role in plant growth and development,
regulation of seed maturation, germination, and adaptation to
environmental stresses. Knowledge of ABA mechanisms of action and the
interactions of components required for ABA signal transduction is far
from complete. Using transient gene expression in rice protoplasts, we
observed additive and inhibitory effects between maize VP1
(Viviparous-1, a transcriptional
activator) and a dominant-negative mutant protein phosphatase, ABI1-1
(ABA-insensitive-1-1), from
Arabidopsis. Lanthanide ions were shown to be specific
agonists of ABA-inducible gene expression and to interact
synergistically with ABA and overexpressed VP1. Both VP1 and lanthanum
activities could be antagonized by coexpression of ABI1-1, which
demonstrates the specific ABA dependence of these effectors on
ABA-regulated gene expression. We obtained pharmacological evidence
that phospholipase D (PLD) functions in ABA-inducible gene expression
in rice. Antagonism of ABA, VP1, and lanthanum synergy by 1-butanol, a
specific inhibitor of PLD, was similar to the inhibition by
coexpression of ABI1-1. These results demonstrate that ABA, VP1,
lanthanum, PLD, and ABI1 are all involved in ABA-regulated gene
expression and are consistent with an integrated model whereby
La3+ acts upstream of PLD.
cis,trans-Abscisic acid
(ABA)1 modulates seed
development, dormancy, cell division, stomatal movements, and cellular
responses to environmental stresses such as drought, cold, salt,
pathogen attack, and UV light (1-3). Despite its importance in plant
growth and development, the mechanisms of ABA action are largely
unknown, and there may be more than one ABA signal transduction pathway leading to both fast (guard cell ion fluxes) and slow (gene regulation) cellular responses (4-6). Transient gene expression studies with LEA (late embryogenesis
abundant) and drought-inducible gene promoters have defined
the cis-acting elements necessary and sufficient to confer
ABA-inducible transcription (7-10). Separate ABA-responsive elements
and coupling elements function cooperatively and redundantly as an ABA
response complex (9, 11, 12).
Recent insight into the mechanisms of ABA-inducible transcription have
come from cloning of TRAB1 (transcription factor
responsible for ABA regulation), a basic
leucine zipper transcription factor that binds ABA response complex
promoter elements and VP1
(Viviparous-1), a transcriptional
activator required for ABA-regulated gene expression during seed
maturation (13-15). Other proteins interact with ABA response
complexes, VP1 orthologs, and basic leucine zipper factors, but there
is no direct evidence that these factors function in ABA signaling
(16-27). Regulation by ABA of TRAB1 and VP1 transactivation of ABA
response complexes is not at the level of DNA binding, based on
transient gene expression and in vivo footprinting assays (13, 28). Consistent with this model is the finding that the bean
ortholog of VP1, PvALF, functions to remodel chromatin independent of
exogenous ABA (29). VP1 and orthologs have distinct functions that are
not ABA-dependent, and the precise mechanism(s) of VP1 action in ABA signaling are not known (14, 15, 27, 30-35).
Genetic analysis (36, 37) of seed maturation and germination processes
in Arabidopsis has resulted in map-based cloning of the
ABI1-ABI5
(ABA-insensitive)
genes. ABI3 is the genetic equivalent of the maize
Vp1 gene and is conserved both functionally and at the
nucleotide level in monocots and dicots (3, 4, 38). The ABI4
gene shows homology to the APETELA2 family of
transcriptional regulators (39). The ABI5 gene encodes a
member of the basic leucine zipper family of transcription factors (40)
and is highly homologous to rice TRAB1, sunflower DPBF1, and
Arabidopsis ABF1-ABF4 and AREB1-AREB3 (10, 13, 24,
25).
The semidominant abi1 and abi2 mutations are the
most pleiotropic ABA-insensitive mutants in terms of physiological and
tissue-specific ABA processes (4, 41). The ABI1 and
ABI2 genes encode homologous type 2C protein Ser/Thr
phosphatases with partially redundant but distinct tissue-specific
functions in the regulation of ABA-, cold-, or drought-inducible genes
and ion channels (42, 43). Remarkably, the sole mutant alleles,
abi1-1 and abi2-1, are both missense mutations of
a conserved Gly-to-Asp mutation (G180D in abi1-1 and G168D
in abi2-1) that results in a dominant phenotype in
vivo and reduced phosphatase activity in vitro
(44-46). Plants homozygous for intragenic null suppressor alleles of
abi1-1 exhibit higher seed dormancy and enhanced ABA
sensitivity to germination inhibition and stomatal movements,
suggesting that ABI1 and probably ABI2 act as
negative regulators of ABA signaling (47). Overexpression of
abi1-1 antagonizes both up- and down-regulation of
ABA-responsive promoters (46). ABI1 acts downstream of the
ABA agonist lanthanum (48). The molecular mechanisms and targets of
ABI1 and lanthanum action are not known.
Phospholipase C (PLC) and phospholipase D (PLD) are phosphodiesterases
that hydrolyze phospholipids, producing inositol 1,4,5-trisphosphate and diacylglycerol or phosphatidic acid and the head group,
respectively. Phospholipases have been proposed to play a major role in
mediating a wide range of cellular processes in plants such as membrane trafficking, cell proliferation, cytoskeletal organization, defense responses, differentiation, reproduction, and hormone action (49, 50).
PLC and PLD have been implicated in ABA regulation of stomatal movements (51, 52). Richie and Gilroy (53) have shown that application
of phosphatidic acid to barley aleuron inhibits gibberellin-inducible We are interested in the cell biology of ABA signal transduction, from
events at the cell surface such as ligand binding (60) through
secondary messengers such as calcium, inositol 1,4,5-trisphosphate, and
cyclic ADP-ribose (61) to changes in gene expression. The activities of
overexpressed ABA signaling effectors are easily and rapidly assayed in
rice protoplasts. This capacity allows facile analysis of
pharmacological agents for interaction with ABA signaling components as
well as characterization of the molecular mechanisms of such
interactions. In this study, we have obtained pharmacological evidence
that PLD functions in ABA-inducible reporter gene expression in rice
and acts downstream of the ABA effector lanthanum. VP1 transactivation
of ABA-inducible promoters is inhibited by 1-butanol, and the extent of
1-butanol inhibition is comparable to that effected by coexpression of
the dominant-negative ABI1-1 protein. These results are consistent with
genetic studies showing an ABA requirement for VP1 action and suggest
that PLD and ABI1-1 act upstream of VP1 on a single ABA signaling pathway.
Plant Materials--
Embryonic rice suspension cultures
(Oryza sativa L. cv. IR-54) were kindly provided by Dr.
W. M. Marcotte, Jr. (Clemson University, Clemson, SC) and
propagated in MS basal medium (62). Three days after subculturing,
protoplasts were prepared and transformed with various mixtures of DNA
constructs using polyethylene glycol precipitation as previously
described (48, 60). Aliquots of transformed protoplast samples were
treated with or without pharmacological agents and ABA for 18 h in
the dark in a final volume of 0.8 ml of Krens solution (7).
Plasmid Constructs--
Plasmid pBM207 contains the wheat
(Triticum aestivum) Em (early
methionine-labeled) promoter driving expression of
Chemicals--
1-Butanol was obtained from Acros Organics
(Geel,Belgium). 2-Butanol was from Nacalai Tesque, Inc. (Kyoto,
Japan). Synthetic abscisic acid ((±)-cis,trans-abscisic
acid) and lanthanum chloride were obtained from Sigma (St. Louis,
Missouri). ABA was dissolved and stored in absolute ethanol at
Functional Assays--
Flow cytometry of live protoplasts
expressing GFP was performed on a Becton-Dickinson FACS Vantage
dual-beam instrument equipped with a 200-µm nozzle, Lysis II
acquisition and analysis software, and an Enterprise argon-ion laser
(1.3 watts) tuned to 488 nm. The sheath fluid used was Krens (7). GFP
emission detection was carried out with a fluorescein isothiocyanate
530/30-nm band-pass filter. For each sample, 10,000 protoplasts were
gated, and the weighted GFP fluorescence per 10,000 cells was
calculated as the product of the average fluorescence intensity of the
population and the number of individual cells (48) expressing above a
background threshold. After 16 h of incubation, cell
viability was determined by flow cytometry of an aliquot of protoplasts
treated for 5 min with 0.01% (w/v) fluorescein diacetate (Molecular
Probes, Inc., Eugene, OR).
For reporter enzyme assays, protoplasts were lysed in 250 µl of lysis
buffer and spun at maximum speed for 3 min in a microcentrifuge, and
the supernatant was retained. Luciferase substrate (Promega, Madison,
WI) was prepared according to the manufacturer's instructions. 10 µl
of sample extract was mixed with 50 µl of substrate, and the
luciferase activity was measured on a Zylux FB15 luminometer (Fisher Scientific, Pittsburgh, PA). GUS activities were
determined by fluorometry with 4-methylumbelliferyl glucuronide as
substrate according to Desikan et al. (60). The relative
reporter gene activity was represented as the ratio of GUS to
luciferase activities, expressed in units of 4-methylumbelliferyl
fluorescence/40 µl of extract/h and photons/10 µl of extract/min, respectively.
To characterize the maximum and specific effects of
pharmacological agents on ABA-inducible expression, it was considered important to perform interaction experiments with concentrations of
exogenous ABA that are physiologically relevant. Therefore, an ABA dose
response of Em-GUS expression was performed. The results are
shown in Fig. 1A. There was a
log-linear relationship of ABA concentration to Em promoter
expression up to 100 µM, with a correlation coefficient
of r = 0.92. There was a significant increase
(p < 0.0005) in Em-GUS transcription in
response to 1 µM ABA treatment. The maximum induction of
Em-GUS (28-fold) was observed with 100 µM ABA
treatment. The slight decrease in relative Em-GUS induction observed with a saturating ABA concentration (200 µM) may
have been caused by a negative effect of high ABA concentrations on protoplast viability (60).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-amylase production and triggers synthesis of an ABA-inducible amylase inhibitor. They have also recently shown in vitro
that ABA stimulates PLD activity in plasma membrane extracts (54). PLC
and PLD genes are up-regulated by ABA (55, 56). 1-Butanol, a specific
inhibitor of PLD (49, 57-59), inhibits ABA-induced accumulation of the
RAB (response to ABA) protein (53). However, the specificity of PLD action in secondary messenger formation and
ABA-regulated gene expression is far from understood.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-glucuronidase (GUS; encoded by uidA from
Escherichia coli) (7). Plasmids pQS264, pLSP, and pDHN7
contain the barley (Hordeum vulgare) Hva1, Hva22, and dehydrin (Dhn7) promoters driving GUS
expression, respectively (8, 9). Plasmids pBM314 and pAHC27 contain the
cauliflower mosaic virus 35 S promoter (referred to below as
35S) and the maize (Zea mays) Ubi
(ubiquitin) promoter driving GUS expression, respectively
(7, 63). Plasmid pCR559 contains the Em promoter driving a
modified S65T green fluorescent protein cDNA from Aequoria victoria (60, 64). A construct (pDH559) containing the
maize Ubi promoter driving S65T GFP was created by digesting
pCR559 with XhoI and filling in the linearized product with
reverse transcriptase before digesting with SphI to release
the Em promoter. The resulting 3.7-kilobase pair fragment
(pCR559 minus the Em promoter) was then gel-purified and
ligated with the 2-kilobase pair SphI/SmaI fragment of pAHC27, encoding the maize Ubi promoter (63).
Plasmid pCR349.13S contains the 35 S promoter driving VP1 sense
cDNA (18). Plasmid pG2 encodes the cauliflower mosaic virus 35 S
promoter-maize C4 pyruvate-orthophosphate dikinase (Ppdk35S)
promoter chimera driving the coding region of the Arabidopsis
thaliana abi1-1 dominant-negative G180D mutant allele (46).
Plasmid pDirect2.6 contains the Ubi promoter in a reversed
orientation and was used as control construct to balance the total
amount of input plasmid DNA between various treatments. Plasmid pAHC18
contains the Ubi promoter driving firefly (Photinus
pyralis) luciferase (63) and was included in
transformations to provide an internal reference for non-ABA-inducible
transient transcription in reporter enzyme assays.
20 °C as a 0.1 M stock solution. Prior to use,
required dilutions of ABA were made in Krens solution, and control
samples received the same volumes of solvents as in ABA treatments.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
1-Butanol-specific inhibition of
ABA-inducible gene expression in transiently transformed rice
protoplasts. A, ABA induces Em-GUS
expression in a dose-dependent manner. Numbers
in parentheses indicate the -fold induction of ABA-inducible
Em-GUS expression relative to the zero ABA treatment. The
correlation coefficient (r) for linear regression of the
-fold induction between 0 and 100 µM ABA is shown. The
asterisk indicates significantly different from no ABA
treatment (p < 0.0005, two-sided Student's
t test, equal variance assumed). B, 1-butanol
specifically antagonizes ABA-inducible Em-GUS expression in
a dose-dependent manner, while having no effect on 35 S
promoter activity. The asterisk indicates significantly
different from control (p < 0.01, two-sided Student's
t test, equal variance assumed). The single
dagger indicates no significant difference from control
(p 0.34, two-sided Student's t test,
equal variance assumed). The correlation coefficient (r) is
for linear regression of percent inhibition of Em-GUS
expression and 1-butanol concentration. C, increasing
concentrations of ABA decrease the inhibition of Em-GUS
expression by 1-butanol. Numbers in parentheses
are the percent relative inhibition by 0.1% 1-butanol at the given ABA
concentration. The asterisks indicate significantly
different from control (p < 0.04, two-sided Student's
t test, equal variance assumed). There is a negative
correlation (r =
0.92) between ABA concentration and
percent inhibition of Em-GUS expression. Data are the
average of two replicate experiments. D, 1-butanol
antagonizes expression of the ABA-inducible Hva1 and
Hva22 promoters. -Fold induction was calculated relative to
untreated, paired controls (set to unity) from four samples. The
asterisks indicate significantly different from ABA-treated,
no 1-butanol control (p < 0.04, two-sided paired
Student's t test, equal variance assumed). Error
bars in A, B, and D are the
means ± S.E. of three to six replicates/sample. LUC,
luciferase.
PLD is competitively inhibited by 1-butanol due to its ability to transfer the phosphatidyl moiety of the substrate to 1-butanol instead of water, producing phosphatidylbutanol that can be used as a quantitative assay of PLD activity (57). The trans-phosphatidylation reaction is unique to PLD, making 1-butanol a suitable pharmacological agent to study PLD-regulated processes (57-59). To test the role of PLD in ABA signaling in rice embryonic tissue, we measured the effect of various concentrations of 1-butanol on ABA-inducible gene expression in protoplasts at a subsaturating concentration of ABA (1 µM). The results are shown in Fig. 1B. 1-Butanol antagonized ABA-inducible Em-GUS expression in a dose-dependent manner. Significant inhibition of ABA-induced Em-GUS expression was seen with 0.01% (v/v) 1-butanol (p < 0.01). The maximum inhibition of Em-GUS expression (85%) was observed with 0.2% 1-butanol treatment. There was a good linear correlation (r = 0.88) between inhibition of Em-GUS expression and 1-butanol concentration. The 1-butanol effect was specific for ABA-inducible Em promoter activity because the non-ABA-inducible promoter constructs Ubi-GUS (data not shown) and 35S-GUS (Fig. 1B; p > 0.34) were not significantly affected.
Since pharmacological agents used at high concentrations have negative effects on cell viability, it was important to rule out the trivial possibility that 1-butanol affected transcription by a general decrease in cell activity. The results of an experiment to correlate protoplast viability with 1-butanol doses are shown in Table I. Treatment of protoplasts with 0.5% 1-butanol decreased cell viability 26% in untreated control cells, whereas 0.01% 1-butanol, a concentration that antagonized Em-GUS expression significantly (Fig. 1B), decreased cell viability only 6% (p > 0.2). These results indicate that 1-butanol inhibition of ABA-inducible gene expression is not due to its effect on cell viability.
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To provide insight into the interaction of ABA and PLD signaling, the
effect of 1-butanol on the dose dependence of ABA-inducible gene
expression was tested. The results are shown in Fig. 1C. In
response to increasing concentrations of ABA, the inhibition of
Em-GUS caused by 0.1% 1-butanol steadily decreased from
above 50% at subsaturating ABA concentrations to 29% at a saturating concentration of ABA (100 µM) (Fig. 1C). The
linear correlation coefficient for 1-butanol inhibition of
Em-GUS expression and ABA concentration was
r = 0.92.
We extended our experiments to include the ABA-inducible promoters Hva1 and Hva22 (9). Fig. 1D shows the results of 1-butanol (0.1%) inhibition experiments on promoters induced by ABA. 1-Butanol significantly antagonized expression of the ABA-inducible Em-GUS, Hva1-GUS, and Hva22-GUS reporter constructs (p < 0.04), albeit to different extents (Fig. 1D).
We sought further evidence for the specificity of 1-butanol effects on PLD activity by testing a non-active isomer of butanol, 2-butanol (52, 53, 57, 59), for antagonistic activity against ABA-inducible gene expression. The results are shown in Table II. Increasing concentrations of 1-butanol (p < 0.0002), but not 2-butanol (p > 0.25), significantly inhibited ABA-inducible Em-GUS expression (Table II).
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To place the site of action of PLD in an ABA signaling cascade, we performed interaction studies between 1-butanol and two agonists of ABA-inducible gene expression, the transcription factor VP1 and the trivalent ion lanthanum (18, 48, 65, 66). The results are shown in Table III. Overexpression of VP1 by cotransformation of the 35S-VP1 cDNA construct transactivated Em-GUS expression by 20-fold and acted in synergy with a subsaturating concentration of ABA to give 40-fold transactivation. 1-Butanol treatment of cotransformed protoplasts resulted in significant antagonism of 35S-VP1 transactivation and ABA plus 35S-VP1 synergy to a similar extent as 1-butanol inhibition of ABA induction alone (Table III). Lanthanum ion treatment (1 mM) activated Em-GUS expression by 4-fold, and a 30-fold synergistic induction was observed in response to 10 µM ABA plus lanthanum treatment. 1-Butanol also significantly inhibited Em-GUS expression induced by lanthanum or lanthanum plus ABA in synergy (Table III).
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Previous studies have placed the action of ABI1 downstream of lanthanum
(48). To further integrate the action of PLD into a single cell model
of ABA signaling, interaction and specificity studies were performed by
treating the protoplasts with 35S-VP1, lanthanum, and
Ppdk35S-ABI1-1 to compare the inhibitory effects of ABI1-1
with those of 1-butanol. In the experiments shown in Fig.
2, the specificity and activity of
effectors for ABA-inducible gene expression were quantified by flow
cytometry and traditional enzyme assays (48, 60). The non-ABA-inducible
Ubi-GFP and the ABA-inducible Dhn-GUS reporter
constructs were cotransformed with or without cotransformation of
35S-VP1 and/or Ppdk35S-ABI1-1. Aliquots of
various cotransformations were then treated with 100 µM
ABA and 1 mM lanthanum alone or in combination, and GFP and GUS expression was quantified by flow cytometry or reporter gene assays. In Fig. 2 (C and D), the cotransformed
reporters were the ABA-inducible constructs Em-GFP and
Hva22-GUS. The Ubi promoter was not activated by
ABA, lanthanum, or cotransformed 35S-VP1 and was not
inhibited by cotransformed Ppdk35S-ABI1-1 (p > 0.2, two-sided Student's t test, equal variance assumed)
(Fig. 2A). In the same samples, Dhn-GUS was
significantly induced 5.6-fold by 100 µM ABA
(p < 0.002), 2.6-fold by lanthanum (p < 0.07), and 12.6-fold by the synergistic effect of lanthanum plus ABA
treatment (p < 0.006, this induction is greater than
ABA or lanthanum alone, one-sided Student's t test, equal
variance assumed). In parallel experiments with the cotransformed
ABA-inducible reporters Em-GFP and Hva22-GUS
(Fig. 2, C and D), significant induction of the Em and Hva22 promoters by 100 µM
ABA (58.9- and 16-fold, respectively; p < 0.007) and 1 mM lanthanum (5- and 3.4-fold, respectively; p < 0.03) was observed. There was also significant
synergism upon induction of the Hva22 promoter by treatment
with ABA plus lanthanum (28.3-fold; p < 0.005, one-sided Student's t test, equal variance assumed).
Overexpression of the cotransformed 35S-VP1 construct significantly (p < 0.02) transactivated the
Dhn, Em, and Hva22 promoters by 2.2-, 23.8-, and 3.4-fold, respectively, in the absence of exogenous ABA or
lanthanum (Fig. 2, B-D). The inhibition of ABA-inducible
promoter expression by the overexpressed Ppdk35S-ABI1-1 cDNA effector construct was similar when compared separately or together for control untreated, 100 µM ABA-, lanthanum-,
or ABA/lanthanum-treated samples (Fig. 2, B-D). Taken
together, the average inhibition of the Em, Dhn,
and Hva22 promoters by abi1-1 overexpression was 54% in control, 64% in 100 µM ABA-, 69% in 1 mM lanthanum-, and 69% in ABA/lanthanum-treated
protoplasts. The average abi1-1 inhibition of the promoters
transactivated by overexpressed VP1 in the absence or presence of ABA
and lanthanum, alone or together, was 28% (control), 44% (ABA), 35%
(lanthanum), and 45% (ABA plus lanthanum).
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In the presence of overexpressed 35S-VP1 and a saturating
ABA concentration (100 µM), an increase in ABA-inducible
reporter gene expression over 100 µM ABA alone was
observed (Fig. 2, B-D), as previously reported for the
Em promoter (18, 48, 66). Furthermore, there was a
significant (p < 0.07) interaction between lanthanum
and VP1 in ABA-inducible gene expression that could be seen in the
presence or absence of 100 µM ABA (Fig. 2,
B-D). The observed magnitudes of the interactions between
ABA and 35S-VP1 and between 35S-VP1 plus
lanthanum plus ABA were additive rather than synergistic (Fig. 2,
B-D).
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DISCUSSION |
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The rice protoplast transient reporter assay system has a large
dynamic range of ABA sensitivity (Fig. 1A). This attribute, together with the ease of handling large numbers of samples, makes it
an ideal system to screen and characterize effector molecules that
interact with ABA signaling pathways. Previously, Northern blots of
rice suspension culture RNAs showed interactions between salt stress
(67) or lanthanum treatment (65) and ABA-inducible Em gene
expression. We have demonstrated here that 1-butanol, a specific
inhibitor of PLD (49, 52, 53, 57-59), but not the biologically
inactive analog 2-butanol, specifically antagonized ABA-inducible gene
expression in rice protoplasts (Fig. 1 and Table II). Taken together
with the similar results of Richie and Gilroy (53) and Jacob et
al. (52) in barley aleurone and guard cell protoplasts,
respectively, these data suggest that PLD is a conserved element in ABA
signaling cascades in plants. 1-Butanol inhibition of ABA-inducible
gene expression was strongly dose-dependent (Fig.
1B), and increasing concentrations of ABA could partially overcome the inhibition by 0.1% 1-butanol (Fig. 1C). These
results are consistent with the competitive inhibition of PLD by
1-butanol and suggest that PLD is a major element in ABA signaling
leading to gene expression. Because the inhibition by 1-butanol was not complete, other ABA signaling mechanisms may be operating in parallel to PLD. Fan et al. (68) have shown that antisense
suppression of PLD expression retards ABA-inducible leaf senescence.
Expression of PLD mRNA is induced by ABA and stresses (55),
consistent with the hypothesis that PLD activity is rate-limiting for
ABA signal amplitude. We are currently testing if overexpression of PLD
isoforms can increase ABA perception in protoplasts. Staxén et al. (51) have provided biochemical and pharmacological
evidence for the role of PLC in ABA-mediated stomatal closure. It
remains to be determined if PLC is involved in ABA-regulated gene
expression and whether there is cross-talk between PLC and PLD
pathways. PLC produces diacylglycerol and inositol 1,4,5-trisphosphate, which trigger some cellular responses to ABA such as calcium transients and stomatal closure (51). Wu et al. (61) have shown that inositol 1,4,5-trisphosphate is able to induce ABA-inducible gene expression in tomato; therefore, a PLC pathway may also operate in
ABA-inducible gene expression. Diacylglycerol can be interconverted to
phosphatidic acid by diacylglycerol kinase (50). Interestingly, major
isoforms of PLD require micromolar quantities of calcium for their
optimal activity (50, 69), suggesting that PLC action may precede
PLD-dependent ABA signaling.
We have demonstrated that 1-butanol antagonized the VP1 transactivation of Em-GUS expression to the same extent as that of ABA induction. Since both VP1 and ABA are required for Em expression in planta (66), we interpret 1-butanol inhibition of VP1 transactivation as primarily an effect on ABA-dependent processes required for VP1 activity. However, other interpretations are plausible. For example, VP1/ABI3 also has ABA-independent genetic interactions with developmental factors and some ABA-regulated promoters (30, 70, 71), and PLD may be involved downstream of these VP1-related pathways.
Lanthanide ions have been extensively used as plasma membrane calcium channel blockers (72). Hagenbeek et al. (48) have demonstrated that trivalent ions such as lanthanum and terbium specifically activate ABA-inducible promoters through an ABI1-dependent pathway in rice protoplasts. We have shown that 1-butanol inhibits lanthanum-activated and lanthanum/ABA synergistic Em-GUS expression, suggesting that PLD plays a significant downstream role in a lanthanum-mediated ABA signaling pathway. The mechanism of action of lanthanum on ABA-inducible gene expression is not known. Lanthanum interacts with membranes and is hypothesized to promote calcium release (73, 74), processes that are associated with PLC and PLD activities. The effects of ABA plus 35S-VP1 on ABA-inducible gene expression are inhibited by overexpression of ABI1-1 cDNA (Fig. 2, B-D), but to a lesser extent (39% average for Em, Hva22, and Dhn promoters) compared with 1-butanol inhibition (Table III). These results suggest that PLD and ABI1 may affect the same or similar ABA signaling pathway(s). Lanthanum and ABA act in synergy on ABA-inducible gene expression with or without coexpressed VP1, suggesting that ABA, lanthanum, and VP1 act on the same pathway. Overexpression of ABI1-1 inhibits ABA and lanthanum activation, either alone or in synergy, of the Dhn, Em, and Hva22 promoters to a similar extent (68% on average). However, inhibition by ABI1-1 of the VP1 interactions with lanthanum and ABA is, on average, lower (42%) (Fig. 2, B-D) than inhibition of ABA and lanthanum effects. This result is consistent with the existence of an ABA-independent mechanism of VP1 transactivation (30, 71). The molecular mechanisms of VP1 and ABI1 activity are not known. ABI1 may negatively regulate ABA signaling by interacting with a transcription complex that includes VP1.
Transient gene expression studies permit integration of diverse
(e.g. interspecies) trans-acting effectors into a
single system, thereby facilitating characterization and testing of
molecular mechanisms. Our results provide insight into the action of
ABA, lanthanum, PLD, ABI1, and VP1 in regulating ABA-inducible promoter activity. The data are consistent with, but do not derive, a
model of ABA action: receptor, La3+ ABI1, PLD
VP1
gene expression. However, it is also possible that these effectors
act in parallel rather than sequentially.
With the exception of an unconfirmed report in 1984 (75), no ABA
receptors have been described. Using surface plasmon resonance in
conjunction with flow cytometry, Desikan et al. (60)
provided indirect evidence for a putative ABA-receptor complex that
interacts with a cell-surface glycoprotein. A monoclonal antibody
(JIM19) generated against pea guard cell protoplasts specifically binds to plasma membrane glycoproteins and antagonizes ABA-inducible gene
expression in rice and barley (60, 76). It is noteworthy in this
context that PLC is involved in shedding of arabinogalactan proteins by
plasma membrane vesicles (77). The precise role of arabinogalactan
proteins in ABA signaling is yet to be established (78). We are
currently pursuing multiparameter correlated flow cytometric
analysis2 of cell-surface
glycoprotein markers and Em-GFP expression in response to
ABA and effectors to critically analyze the relationship of
cell-surface glycoproteins and ABA perception and signaling from the
receptor through lanthanum, PLD, ABI1, and VP1, leading to gene expression.
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ACKNOWLEDGEMENTS |
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We thank Regina Chak, Patrick Ng, and Frances Chan for technical assistance and T.-H. Ho and R. Quatrano (Washington University, St. Louis, MO), W. M. Marcotte, Jr., P. Quail (United States Department of Agriculture Plant Gene Expression Center, Albany, CA), R. Wu (Cornell University, Ithaca, NY), J. Sheen (Massachusetts General Hospital, Boston, MA), D. McCarty (University of Florida, Gainesville, FL), and M. Robertson (Commonwealth Scientific and Industrial Research Organization, Canberra, Australia) for providing constructs.
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FOOTNOTES |
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* This work was supported by Competitive Earmarked Research Grant HKUST6173/97M from the Hong Kong Research Grants Council and Grant AoE99/00.SC08 from the Hong Kong Government University Grants Council Area of Excellence Funding for Plant and Fungal Biotechnology.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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To whom correspondence should be addressed. Tel.: 852-2358-8634;
Fax: 852-2358-1559; E-mail: borock@ust.hk.
Published, JBC Papers in Press, January 3, 2001, DOI 10.1074/jbc.M009168200
2 D. Hagenbeek and C. D. Rock, manuscript in preparation.
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ABBREVIATIONS |
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The abbreviations used are:
ABA, cis,trans-abscisic acid;
PLC, phospholipase C;
PLD, phospholipase D;
GFP, green fluorescent protein;
GUS, -glucuronidase.
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