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From the
Received for publication, October 16, 2001, and in revised form, November 4, 2001
Abscisic acid (ABA) regulates seed maturation,
germination, and adaptation of vegetative tissues to environmental
stresses. The mechanisms of ABA action and the specificity conferred by signaling components in overlapping pathways are not completely understood. The ABI5 gene (ABA
insensitive 5) of Arabidopsis
encodes a basic leucine zipper factor required for ABA response in the seed and vegetative tissues. Using transient gene expression in rice
protoplasts, we provide evidence for the functional interactions of
ABI5 with ABA signaling effectors VP1
(viviparous 1) and ABI1 (ABA insensitive 1).
Co-transformation experiments with ABI5 cDNA constructs resulted in specific transactivation of the
ABA-inducible wheat Em, Arabidopsis
AtEm6, bean Abscisic acid (ABA)1 is
one of the major plant hormones and functions in regulation of seed
maturation, germination, and adaptation of vegetative tissues to
environmental stresses (1, 2). ABA acts to effect changes on multiple
physiological processes such as inducing the rapid closure of stomatal
pores to limit transpiration and by triggering slower changes in gene
expression (see Refs. 3-5 for reviews). Although these disparate
processes share genetic elements (some ABA mutants affect both
processes) and signaling intermediates such as phospholipases,
cADP-ribose, inositol 1,4,5-trisphosphate, and calcium ions
(6-9), these secondary messengers are not specific to ABA pathways.
Our knowledge of separate yet overlapping ABA and stress signal
transduction pathways is fragmentary.
Genetic analyses (10, 11) of germination processes in
Arabidopsis have resulted in map-based cloning of the
ABA-insensitive genes, ABI1-5 (12-19). The ABI1
and ABI2 genes encode homologous type 2C protein Ser/Thr
phosphatases (PP2Cs) with partially redundant but distinct
tissue-specific negative regulator functions in the regulation of ABA-,
cold-, or drought-inducible genes and ion channels (20-24). The
original 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. The substrates for ABA-regulatory protein phosphatases 2C
are not known (15, 16, 25).
The ABI3, ABI4, and ABI5 genes encode
proteins belonging to three distinct classes of transcription factors:
the basic B3 domain, APETALA2 domain, and the basic leucine zipper
(bZIP) domain families, respectively. Physiological, genetic, and
transgenic analyses of abi3, abi4, and
abi5 mutants show cross-regulation of expression, suggesting
that these genes function in a combinatorial network rather than a
regulatory hierarchy controlling seed development and ABA responses
(26).
Despite numerous biochemical studies showing binding of bZIP factors to
ABA-responsive promoter elements (27-31), until recently there was no
functional evidence for the role of bZIP factors in ABA signaling.
Cloning of ABI5 and its homologs, the Dc3-Promoter binding factors, ABA response element-binding factors (ABFs), ABA-responsive element-binding proteins (AREBs), and TRAB1
(transcription factor responsible for
ABA regulation 1), has demonstrated a
correlation between these bZIPs and ABA signaling. Members of this
family of bZIPs can bind ABA-responsive elements, heterodimerize, and have limited transactivating activities (18, 32-36). ABI5 transcript and protein accumulation, phosphorylation state, stability, and activity are highly regulated by ABA during germination and early seedling growth (18, 37). Similarly, expression of some of the
ABA-responsive element-binding protein genes is induced by ABA, and
their ability to transactivate an ABA-responsive promoter is inhibited
by the abi1-1 mutation (35).
The VP1 (viviparous 1)
gene of maize (38) is orthologous to ABI3 of
Arabidopsis (12) and encodes a transcription factor required
for ABA-regulated seed development. Structure-function studies with VP1
in transient gene expression assays have demonstrated that the
N-terminal acidic domain functions as both a transcriptional activator
and repressor (39). The conserved B2 domain is required for
transactivation of the ABA-inducible Em promoter and for
enhancing the in vitro binding of various bZIP proteins to
their cognate targets (40). The B3 domain binds specifically to
promoter sequences required for transactivation but not to
ABA-responsive cis-elements (41). The exact molecular
mechanisms of VP1/ABI3 action are not known, but it interacts
genetically with ABI4 and ABI5, possibly forming a regulatory complex
mediating seed-specific and/or ABA-inducible gene expression (26).
Recently, TRAB1 was shown to bind both ABA-responsive promoter elements
and VP1, thereby providing a mechanism for bZIP and VP1 transactivation
of ABA signaling (33). Similarly, two-hybrid assays in yeast have shown
that ABI5 forms homodimers and binds to ABI3; the B1 domain of ABI3 was
essential for these interactions (42). Regulation by ABA of TRAB1 and
VP1 transactivation was not at the level of DNA binding (33),
suggesting the existence of additional regulatory mechanisms. PvALF, a
bean ortholog of VP1 that transactivates the
We are interested in elucidating the molecular mechanisms of ABA
signaling. In this study, we utilized transient gene expression in
protoplasts from embryonic rice callus cultures to functionally analyze
the interactions of genetically defined ABA regulatory genes
(ABI5, ABI1-1, and VP1) and
pharmacological effectors (La3+, 1-butanol, and an
inhibitor of phospholipase D) (44, 45) in ABA-inducible gene
expression. We have obtained evidence that ABI5 specifically interacts
with all tested ABA signaling effectors and promoters from both
monocots and dicots, demonstrating the conservation of ABA signaling in
plants and the utility of rice protoplasts for molecular and cell
biological dissection of ABA regulatory mechanisms.
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 Murashige and Skoog medium (46). Three days after
subculturing, protoplasts were isolated and transformed with various
mixtures of DNA constructs using polyethylene glycol precipitation as
previously described (47, 48). Aliquots of transformed protoplast
samples were treated with or without ABA and pharmacological agents for
16 h in the dark in a final volume of 0.8 ml of Krens solution.
Chemicals--
1-Butanol was obtained from Acros Organics (Geel,
Belgium). Synthetic ABA and lanthanum chloride were obtained from
Sigma. Fluorescein diacetate was obtained from Molecular Probes Inc. (Eugene, OR) and was stored as 1% stock solution in acetone at Plasmid Constructs--
Plasmid pBM207 contains the wheat
(Triticum aestivum) early methionine-labeled (Em)
promoter driving the expression of Previous results have demonstrated a specific log linear dose
response to exogenous ABA of various promoters in synergy with transgene effectors in transiently transformed rice protoplasts (44,
45). To test the role of ABI5 in ABA signaling in rice protoplasts and
further examine the conservation of ABA signaling machinery among
species, we measured the effect of overexpressed ABI5 cDNA, driven
by the Ubi promoter, on various ABA-inducible promoters.
Table I shows the results of numerous
promoter activation experiments that tested the specificity and extent
of functional interactions of ABA and co-expressed ABI5. In these
experiments a construct containing the Ubi promoter alone
was transformed in the negative control samples to account for possible
DNA effects or titration of endogenous transcription factors.
Therefore, the effects observed by Ubi::ABI5
co-transformation are due to ABI5 overexpression. There was a
significant 12-fold induction of wheat Em::GUS
expression observed with 10 µM ABA treatment.
Co-transformation of Ubi::ABI5 cDNA
specifically and significantly transactivated Em::GUS expression more than 2-fold over control,
in the presence or absence of ABA (Table I). Co-expression of ABI5 also
specifically and significantly transactivated the ABA-inducible
Hva1 and Hva22 promoters of barley, the
Rice protoplasts are a facile model system for cell biological analyses
of signaling mechanisms (4, 53). We extended our analyses of the ABA
agonist lanthanum and ABA antagonist 1-butanol, a competitive and
specific inhibitor of PLD and ABA-regulated gene expression (44, 54,
55), to the
ABI5 Interacts with Abscisic Acid Signaling Effectors in
Rice Protoplasts*
,
Department of Biology, Hong Kong University
of Science and Technology, Clear Water Bay, Kowloon, Hong Kong,
China, the § Department of Molecular, Cellular, and
Developmental Biology, University of California, Santa Barbara,
Santa Barbara, California 93106, and the ¶ Department of Biology,
The Chinese University of Hong Kong, Shatin, New Territories,
Hong Kong, China
ABSTRACT
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-Phaseolin, and barley
HVA1 and HVA22 promoters. Furthermore, ABI5
interacted synergistically with ABA and co-expressed VP1, indicating
that ABI5 is involved in ABA-regulated transcription mediated by VP1.
ABI5-mediated transactivation was inhibited by overexpression of
abi1-1, the dominant-negative allele of the
protein phosphatase ABI1, and by 1-butanol, a competitive inhibitor of
phospholipase D involved in ABA signaling. Lanthanum, a trivalent ion
that acts as an agonist of ABA signaling, potentiated ABI5
transactivation. These results demonstrate that ABI5 is a key target of
a conserved ABA signaling pathway in plants.
INTRODUCTION
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DISCUSSION
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-Phas promoter, has been proposed to function
by remodeling chromatin independent of exogenous ABA (43).
EXPERIMENTAL PROCEDURES
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20 °C. ABA was dissolved and stored in absolute ethanol at
20 °C as a 0.1 M stock solution. Prior to use,
required dilutions of ABA, lanthanum chloride, and 1-butanol were made
in Krens solution, and control samples received the same volumes of
solvents as in ABA and pharmacological treatments.
-glucuronidase (GUS; encoded by
uidA from Escherichia coli) (40). The
AtEm6::GUS fusion is a translational fusion
including nine codons of the AtEm6 gene, created by ligating
a 1.2-kb XbaI-PvuII fragment of the
AtEm6 gene cloned into the XbaI and
SmaI sites of pBI101.3. Plasmid pTZ207 containing the
Vicia faba -Phaseolin promoter (49) was created by
digesting pTZ/Phas with AccI to release the 1.5-kb
-Phaseolin cDNA. The vector was end-filled with
Klenow fragment, dephosphorylated with calf intestinal phosphatase, and ligated to the 2-kb NcoI/EcoRI end-filled
fragment of pBM207 encoding GUS. Plasmids pQS264 and pLSP contain the
barley (Hordeum vulgare) Hva1 and
Hva22 promoters driving GUS expression, respectively (50).
Plasmid pBM314 (51) contains cauliflower mosaic virus 35S
(35S) promoter driving GUS expression. A construct (pDH359) containing the maize Ubi promoter (52) driving 1.4-kb
ABI5 cDNA was created by digesting pDH349
(Ubi::VP1-Myc) with EcoRI and filling
in the linearized product with Klenow fragment before digesting with
BamHI to release the VP1-Myc fragment. The resulting 4.8-kb
vector was then ligated with the 1.4-kb
BamHI/HindIII end-filled fragment of pBKA5 (18)
encoding the Arabidopsis thaliana (L.) Heynh ABI5
cDNA. Plasmid pCR349.13S contains the 35S promoter driving the VP1 sense cDNA (40). Plasmid pG2
encodes the 35S-maize C4 pyruvate-orthophosphate dikinase
(Ppdk-35S) promoter chimera driving the coding region of
Arabidopsis abi1-1 dominant-negative G180D mutant allele (25). Plasmids pG1 and pDirect2.6 were used as
controls to demonstrate the protein-specific nature of the ABI5 and
ABI1-1 effects. Plasmid pG1 is identical to pG2 except that it is wild
type at amino acid 180 (Gly) and that the phosphatase active site has
been mutated (G174D) to express a null mutant (25). Plasmid pDirect2.6
contains the Ubi promoter in a reverse orientation and was
used as a control construct to balance the total amount of input
plasmid DNA between various treatments and as a potential target for
binding of endogenous transcription factors. Plasmid pAHC18 contains
the Ubi promoter driving firefly (Photinus
pyralis) luciferase (52) and was included in transformations to
provide an internal reference for non-ABA-inducible transient transcription in reporter enzyme assays. Typically 60 µg of DNA for
reporter constructs and 40 µg of DNA for effector constructs were
used for transformations. Cell viability was measured by staining the
protoplasts with 0.01% fluorescein diacetate, and batches of
protoplasts with viability higher than 90% were used for transformations.
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-Phas promoter of bean, but not the non-ABA-inducible
35S promoter of cauliflower mosaic virus (Table I). The ABI5
transactivation functioned in synergy with exogenous ABA, based on the
observed factorial rather than additive responses of promoters to ABA
plus ABI5 treatments compared with either treatment alone (Table I).
This result demonstrates that ABI5 transactivation acted via an
ABA-specific pathway.
ABI5 transactivates the ABA-inducible barley promoters Hva1, Hva22, and
bean -Phas promoter in rice
-Phas experiments, it was 100 µM. The
values are the averages ± S.E. of four replicate transformations.
-Phas promoter of bean (49). Zheng et
al. (56) have shown that -Phaseolin accumulates up to 4%
of the total endosperm protein in transgenic rice. The -Phas promoter exhibited a relatively weak response to a
saturating dose (100 µM) of ABA in rice protoplasts
(Tables I and II), ranging from 4- to
20-fold induction because of experimental variation. 1-Butanol
treatment specifically inhibited ABA-inducible -Phas promoter activity in a dose-dependent manner (Table II).
The biologically inactive isomer 2-butanol had no effect on
ABA-inducible promoter expression (44). Lanthanum ions had a small but
significant agonist effect on the -Phas promoter and
acted in synergy with ABA (Table II), as previously observed for other
ABA-inducible promoters (44, 45, 57).
Lanthanum synergizes with and a PLD inhibitor (1-But) antagonizes ABA
induction of Phas::GUS expression
To determine whether ABI5 is regulated by the lanthanum effect, we tested the interaction of lanthanum with ABI5 transactivation of Em::GUS expression, and the results are shown in Table III. Lanthanum ion treatment (1 mM) significantly activated Em::GUS expression by 1.7-fold, and a synergistic induction was observed in response to 10 µM ABA plus lanthanum treatment (35-fold versus 17-fold induction in response to ABA alone). Co-transformed ABI5 potentiated both ABA and lanthanum induction of Em::GUS alone and in combination, because co-transformation of ABI5 resulted in a factorial increase in Em::GUS expression of 1.5-fold, 2-fold, and 1.3-fold over the 10 µM ABA treatment, the 1 mM lanthanum treatment, or both treatments, respectively, similar to the 1.7-fold transactivation over control (no ABA; Table III).
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Phospholipase C and PLD have been implicated in ABA signaling (8, 9, 44, 54, 55). To test the dependence of ABI5 transactivation of Em::GUS on PLD activity, protoplasts were co-transformed with ABI5 cDNA and were treated with or without a competitive inhibitor of PLD, 1-butanol (54). 1-Butanol significantly antagonized ABA induction and ABI5 transactivation of Em::GUS in a dose-dependent manner (Table IV), and the inhibitions by 1-butanol of ABA induction versus ABI5 transactivation were not significantly different from each other (Table IV).
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Previous studies have shown that ABA induction and VP1 transactivation
of ABA-inducible promoters are antagonized by overexpression of the
dominant-negative allele of ABI1 (25, 44, 45),
with greater than 90% inhibition possible with increasing
concentrations of effector abi1-1 construct (53).
We co-expressed ABI5 and abi1-1 (or ABI1null as
a negative control) in rice protoplasts and observed that abi1-1 significantly inhibited dose-dependent
ABA induction (by 68%) and ABI5 transactivation/ABA synergy of
Em::GUS expression by 68 and 62%, respectively
(Fig. 1A). Overexpression of
abi1-1 also inhibited ABA-inducible
Phas::GUS expression and ABA synergy with ABI5
(Fig. 1B).
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Protein interaction assays in yeast have identified the domains
required for the physical interaction of ABI5 with ABI3 (42); however,
the functional significance of the interactions is unknown. We tested
for functional interactions of ABI5 with the maize ortholog of ABI3,
VP1, on heterologous ABA-inducible promoters. Fig.
2 shows the results from ABI5 and VP1
cDNA effector construct co-transformation experiments on
transactivation and ABA synergy of the wheat Em (Fig.
2A), Arabidopsis AtEm6 (Fig.
2B), and bean -Phas (Fig. 2C)
promoters. Overexpression of ABI5 and VP1 alone transactivated all
three promoters, and both effectors synergized with ABA (Fig. 2).
Interestingly, VP1 and ABI5 had different modes of synergy with ABA on
Em::GUS expression than with AtEm6 or
-Phas. Overexpression of VP1 had a relatively stronger
transactivating effect with low (especially zero) dose treatments of
ABA (Fig. 2A). Conversely, the synergy between VP1 and ABA
was more apparent for AtEm6 and -Phas at high
ABA concentrations (Fig. 2, B and C). When both VP1 and ABI5 were co-expressed with the AtEm6::GUS
or -Phas::GUS reporters, strong synergies
between both the effectors and ABA were observed (Fig. 2, B
and C). Most strikingly, strong and significant synergistic
interactions of ABA, ABI5, and VP1 were observed with all promoters
over the range of ABA concentrations tested (Fig. 2).
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, and $) indicate
significantly different from the activation by any of the effectors
alone, p < 0.0012, 0.01, and 0.02, respectively
(paired Student's t test, equal variance assumed). The
error bars are ± S.E., three or four
replicates/sample. LUC, luciferase.
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DISCUSSION |
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We have demonstrated synergistic interactions of ABA with ABI5 and
VP1, alone and in combination, in transient gene expression of both
monocot and dicot ABA-inducible promoters in rice protoplasts. The data
presented here consistently point toward the conservation of ABA
signaling pathways between plant species. All tested ABA-inducible promoters from monocots (Hva1, Hva22, and
Em) and dicots (AtEm6 and -Phas)
were regulated by ABA in rice protoplasts, including the barley
Dehydrin promoter studied previously (45). The ABA pathway-specific pharmacological agents La3+ and 1-butanol
acted predictably on the ABA-regulated promoters, as did the maize
VP1 and Arabidopsis abi1-1 gene
products that have previously been shown to interact with each other
and the above pharmacological agents (44, 45). The strong
transactivation by VP1 of the Em promoter in the absence of
ABA (Fig. 2A) is likely explained by the observation that
the Em promoter elements sufficient for activation by ABA
and VP1 are partially separable (58).
It was shown previously that a GAL4AD-ABI5 fusion activates an AtEm6-LacZ reporter in yeast by 2-3-fold in absence of ABA (42). Presumably, this reflects an ABA-independent DNA binding event targeted to the ABI5-binding site, with transactivation accomplished by the GAL4 activation domain. Our results showing synergy of ABI5 with ABA suggest that ABA is required for ABI5 transactivation of ABA-inducible promoters. More significantly, the Arabidopsis ABI5 gene product interacted with all the tested ABA effectors, firmly supporting the conclusion that the ABA signaling mechanisms operating in rice embryonic protoplasts are conserved with those in other plants and tissues and that ABI5 activation may be the consequence, directly or indirectly, of the effectors. A similar conclusion was drawn for ABA activation of TRAB1 by Hobo et al. (33) based on observed ABA-dependent transactivation but ABA-independent DNA binding by TRAB1. The ABI5-related ABA-responsive element-binding proteins 1 and 2 did not transactivate the RD29 promoter in the absence of (AREB1,2) ABA in Arabidopsis leaf protoplasts (35), whereas another Arabidopsis ABI5 family member, ABA-response element-binding factor 3, transactivated the Em promoter in rice protoplasts in the (ABF3) absence of exogenous ABA (36), similar to ABI5 shown here.
The molecular mechanisms of the effectors studied here are not known, but there is evidence that La3+ and PLD act at the plasma membrane, suggesting that they function near to a postulated membrane-bound ABA receptor that may interact with G-protein subunits coupled to calcium and ion channels (59-62). Some early ABA signal transduction components exist in animals, suggesting that ABA signaling mechanisms may be even more broadly conserved than previously thought (63). We are currently testing whether La3+ can modulate ABA activation of PLD in plasma membrane fractions (60).
Because the abi1-1 allele acts as a dominant-negative protein phosphatase possibly acting on targets other than those of wild type PP2Cs (23, 25), it is difficult to interpret its antagonistic action on ABI5 activity (or any other ABA activity). For example, if abi1-1 "poisons" or traps some necessary ABA sensitivity components, then theoretically ABI1 could function either upstream or downstream of ABI5 activation without a discernible end result of lower ABA-inducible gene expression. Allen et al. (21) observed reduced ABA-inducible [Ca2+]cyt concentrations and S anion channel currents in the abi1-1 and abi2-1 mutants that were restored by external Ca2+, suggesting that ABI1 and ABI2 act upstream of [Ca2+]cyt to regulate anion channels. However, Grabov et al. (64) showed that abi1-1 dominant-negative protein had no detectable effect on the ABA-activation of the S-anion channel in transgenic tobacco while decreasing ABA sensitivity of K+ channels, suggesting that ABI1 function may be more flexible. Consistent with this hypothesis, Shen et al. (65) have shown that abi1-1 antagonizes only the ABA-inducible pathway of gene expression but not the ABA suppression pathway of gibberellin-inducible gene expression. Taken together, we interpret these results to support the hypothesis that ABI1 could act at or near ABI5 during transcriptional activation of ABA-inducible genes in rice. ABI5 and homologs are phosphorylated in planta (35) and are plausible targets for ABI1 activity in vitro (37), because the conserved regions contain consensus residues for protein kinases (18, 35). ABI1 did not physically interact with ABI5 in yeast two-hybrid assays (42), but this result could be due to the absence of a phosphorylated ABI5 substrate in yeast.
The activities of overexpressed ABI5 and VP1 on seed-specific reporter gene expression demonstrated here suggest that spatial, temporal, and quantitative expression of transcription factors may constitute a combinatorial mechanism conferring specificity and amplitude of ABA-inducible gene expression in plants (66). Consistent with this model is the observation that the promoters studied here are also expressed to a lesser degree in vegetative tissues in response to ABA and/or stress2 or when VP1 orthologs are ectopically expressed (26, 43). The physiological significance of a 2-4-fold increase in ABA-inducible gene expression by ABI5 in rice protoplasts is corroborated by overexpression studies with 35S::ABI5 transgenic Arabidopsis. Lopez-Molina et al. (37) have shown that there is a limited developmental time window immediately after germination when ABA-inducible ABI5 accumulation correlates with ABA-mediated growth quiescence. Three days after germination, ABI5 expression was no longer ABA-inducible, but in 35S::ABI5 transgenic plants expressing ABI5 to varying degrees there was a good correlation between ABA sensitivity to root and embryo growth inhibition and ABI5 protein levels, and 35S::ABI5 plants retained water more efficiently than wild type (37). Because the rice callus cultures used in our studies are derived from embryonic tissue, it is possible that endogenous ABA regulatory factors (such as OsVP1, OsABI5, and OsABI4) interact with overexpressed ABI5 and contribute to the observed transactivations. Two-week-old transgenic 35S::ABI5 Arabidopsis plants do not exhibit significantly elevated AtEm1 or AtEm6 expression, perhaps because of the absence of embryonic factors in vegetative tissue that interact with ABI5.3 However, bZIP protein binding to ABA-responsive elements is independent of VP1/PvAlf and dependent on ABA in vivo (33, 43, 67).
Although the exact mechanisms of VP1 action are not known, it is postulated based on several protein-protein interaction studies in yeast that VP1 could potentiate ABA-inducible gene expression by forming DNA-binding complexes with 14-3-3, histone, bZIP, zinc finger, RNA polymerase II subunit RPB5 or other proteins (43, 68-71). Mutations in VP1 and ABI3 loci have a range of pleiotropic effects on a number of developmental markers for seed maturation and germination that have different degrees of ABA-responsiveness, and ABI3 genetically interacts with developmental mutants that are not ABA-insensitive (39, 72, 73). These results suggest that VP1 and ABI3 do not have entirely conserved functions and may serve to integrate ABA signaling into a network regulating development.
Because of the ease of manipulation and high throughput of transient
gene expression assays, rice protoplasts are a good model system to
address the molecular mechanisms of ABA responses. ABI5 and VP1 are the
prototypical bZIP and ABA transcription factors, based on mutant
phenotypes (18, 19, 38). The B1 domain of ABI3 binds to the N-terminal
charged domains of ABI5 (42). There are eight closely related bZIP
members in the ABI5 family in Arabidopsis; for
many of these there exists circumstantial or functional evidence for
their involvement in ABA signaling (34-36). Likewise, there are 14 members of the VP1/ABI3 B3 domain family in
Arabidopsis (74), including two known regulators of
embryonic development: FUS3 and LEC2. Therefore,
it is likely that genetic redundancy may mask subtle, tissue-specific
ABA mutant phenotypes in planta. Structure/function
analysis, domain swapping, and co-transformation experiments with ABI5,
VP1, and family members in rice protoplasts will facilitate unraveling
the complexity of ABA-inducible transcription. For example, generating
an allelic series of ABI5 mutant cDNAs could address whether ABA,
La3+, PLD, VP1, and ABI1 modulation of ABI5 activity is
mediated through the same activation domains. Likewise, there are over
30 PP2C homologs in Arabidopsis that have conserved amino
acid residues found in ABI1 and ABI2 critical for ABA signaling (4,
25), and it is feasible to test the efficacy of these family members as
effectors of ABA-inducible gene expression in transiently transformed protoplasts. The outcomes of these studies should provide ample resources and strategies for practical applications to genetic engineering of crops with value-added seed qualities and improved productivity under environmental stress conditions.
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ACKNOWLEDGEMENTS |
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We thank Regina Chak, Patrick Ng, Francis Chan, and Noelle Roszkowski for technical assistance and M. Delseny (CNRS, France), T.-H. Ho and R. Quatrano (Washington University, St. Louis, MO), P. Quail (United States Department of Agriculture Plant Gene Expression Center, Albany, CA), J. Sheen (Massachusetts General Hospital, Boston, MA), and D. McCarty (University of Florida, Gainesville, FL) for providing constructs. We also thank W. M. Marcotte, Jr. (Clemson University, Clemson, SC) for providing rice suspension cells and constructs and Maurice S. B. Ku for critical reading of the manuscript.
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FOOTNOTES |
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* This work was supported by Grant AoE/B-07/99 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.
To whom correspondence should be addressed. Tel.:
852-2358-8634; Fax: 852-2358-1559; E-mail: borock@ust.hk.
Published, JBC Papers in Press, November 9, 2001, DOI 10.1074/jbc.M109980200
2 S. Grillo and R. S. Quatrano, personal communication.
3 R. Finkelstein, manuscript in preparation.
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ABBREVIATIONS |
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The abbreviations used are:
ABA, cis,trans-abscisic acid;
PLD, phospholipase D;
GUS, -glucuronidase encoded by uidA;
bZIP, basic leucine
zipper transcription factor;
Em, early methionine-labeled;
Ubi, Zea mays ubiquitin promoter;
35S, cauliflower mosaic virus 35S promoter;
-Phas, -Phaseolin.
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