Abscisic Acid Signal Transduction Pathway Research Articles

Arabidopsis plants deficient in plastidial glyceraldehyde-3-phosphate dehydrogenase show alterations in abscisic acid (ABA) signal transduction: interaction between ABA and primary metabolism.

Abscisic acid (ABA) controls plant development and regulates plant responses to environmental stresses. A role for ABA in sugar regulation of plant development has also been well documented although the molecular mechanisms connecting the hormone with sugar signal transduction pathways are not well understood. In this work it is shown that Arabidopsis thaliana mutants deficient in plastidial glycolytic glyceraldehyde-3-phosphate dehydrogenase (gapcp1gapcp2) are ABA insensitive in growth, stomatal closure, and germination assays. The ABA levels of gapcp1gapcp2 were normal, suggesting that the ABA signal transduction pathway is impaired in the mutants. ABA modified gapcp1gapcp2 gene expression, but the mutant response to the hormone differed from that observed in wild-type plants. The gene expression of the transcription factor ABI4, involved in both sugar and ABA signalling, was altered in gapcp1gapcp2, suggesting that their ABA insensitivity is mediated, at least partially, through this transcriptional regulator. Serine supplementation was able partly to restore the ABA sensitivity of gapcp1gapcp2, indicating that amino acid homeostasis and/or serine metabolism may also be important determinants in the connections of ABA with primary metabolism. Overall, these studies provide new insights into the links between plant primary metabolism and ABA signalling, and demonstrate the importance of plastidial glycolytic glyceraldehyde-3-phosphate dehydrogenase in these interactions.

Identification and characterization of bZIP‐type transcription factors involved in carrot (Daucus carota L.) somatic embryogenesis

Seed dormancy is an important adaptive trait that enables seeds of many species to remain quiescent until conditions become favorable for germination. Abscisic acid (ABA) plays an important role in these developmental processes. Like dormancy and germination, the elongation of carrot somatic embryo radicles is retarded by sucrose concentrations at or above 6%, and normal growth resumes at sucrose concentrations below 3%. Using a yeast one-hybrid screening system, we isolated two bZIP-type transcription factors, CAREB1 and CAREB2, from a cDNA library prepared from carrot somatic embryos cultured in a high-sucrose medium. Both CAREB1 and CAREB2 were localized to the nucleus, and specifically bound to the ABA response element (ABRE) in the Dc3 promoter. Expression of CAREB2 was induced in seedlings by drought and exogenous ABA application; whereas expression of CAREB1 increased during late embryogenesis, and reduced dramatically when somatic embryos were treated with fluridone, an inhibitor of ABA synthesis. Overexpression of CAREB1 caused somatic embryos to develop slowly when cultured in low-sucrose medium, and retarded the elongation of the radicles. These results indicate that CAREB1 and CAREB2 have similar DNA-binding activities, but play different roles during carrot development. Our results indicate that CAREB1 functions as an important trans-acting factor in the ABA signal transduction pathway during carrot somatic embryogenesis.

Intracellular Ca2+ stores could participate to abscisic acid-induced depolarization and stomatal closure in Arabidopsis thaliana

In Arabidopsis thaliana cell suspension, abscisic acid (ABA) induces changes in cytosolic calcium concentration ([Ca2+]cyt) which are the trigger for ABA-induced plasma membrane anion current activation, H+-ATPase inhibition, and subsequent plasma membrane depolarization. In the present study, we took advantage of this model to analyze the implication of intracellular Ca2+ stores in ABA signal transduction through electrophysiological current measurements, cytosolic Ca2+ activity measurements with the apoaequorin Ca2+ reporter protein and external pH measurement. Intracellular Ca2+ stores involvement was determined by using specific inhibitors of CICR channels: the cADP-ribose/ryanodine receptor (Br-cADPR and dantrolene) and of the inositol trisphosphate receptor (U73122). In addition experiments were performed on epidermal strips of A. thaliana leaves to monitor stomatal closure in response to ABA in presence of the same pharmacology. Our data provide evidence that ryanodine receptor and inositol trisphosphate receptor could be involved in ABA-induced (i) Ca2+ release in the cytosol, (ii) anion channel activation and H+-ATPase inhibition leading to plasma membrane depolarization and (iii) stomatal closure. Intracellular Ca2+ release could thus contribute to the control of early events in the ABA signal transduction pathway in A. thaliana.

Involvement of NADPH oxidase NtrbohD in the rapid production of H 2O 2 induced by ABA in cultured tobacco cell line BY-2

The mechanisms for the production of hydrogen peroxide (H 2O 2) induced by abscisic acid (ABA) were investigated in suspension culture cells of tobacco BY-2 cells. The results showed that the immediate generation of H 2O 2, which was mainly derived from superoxide dismutase-catalyzed dismutation of superoxide radical, was significantly induced by ABA. Furthermore, treatment of the cultured tobacco cells with ABA resulted in a time-dependent quick increase in plasma membrane (PM) NADPH oxidase activity, which coincided on time and magnitude with the elevation in ABA-induced accumulation of H 2O 2. Moreover, these enhanced effects were pronouncedly inhibited by two NADPH oxidase inhibitors, diphenylene iodonium and imidazole, suggesting that PM NADPH oxidase is involved in the rapid accumulation of H 2O 2 in cultured tobacco cells. In addition, analysis of the expression level of NtrbohD, a PM NADPH oxidase gene in tobacco, by RT-PCR and protein gel blot revealed that the gene at both mRNA and protein levels was upregulated by ABA, indicating that NtrbohD participates in the ABA-stimulated rapid production of H 2O 2 in tobacco culture cells. Taken together, these findings suggest that ABA induces the rapid accumulation of reactive oxygen species via NADPH oxidase in suspension culture cells of tobacco, and that NADPH oxidase and H 2O 2 appear to be important components in ABA signal transduction pathway in plants.

The Induction of Abscisic-Acid-Mediated Drought Tolerance is Independent of Ethylene Signaling in Arabidopsis Plants Responding to a Harpin Protein

Harpin proteins from plant pathogenic bacteria can activate distinct signaling pathways and cause multiple effects in plants. When Arabidopsis thaliana (Arabidopsis) plants are treated with the HrpNEa harpin produced by Erwinia amylovora, a bacterial pathogen that causes fire blight in rosaceous plants, abscisic acid (ABA) is stimulated to mediate drought tolerance, and ethylene signaling is activated to regulate plant growth enhancement and insect resistance. It is unclear if ABA and ethylene signaling interacts in response to harpin proteins. Here we report that ethylene signaling is dispensable to the induction of ABA-mediated drought tolerance in Arabidopsis. In wild-type (WT) plants growing under drought stress conditions, ABA, but not ethylene, was required for HrpNEa to promote cellular adaptive responses and decrease drought severity of plants. During the induction of drought tolerance in HrpNEa-treated WT plants, expression of the ABA signaling gene ABI2 was induced coincidently with decreases in transcripts of ETR1, which encodes an ethylene receptor, and several other genes that are also involved in ABA and ethylene signal transduction pathways. In response to HrpNEa, the Arabidopsis etr1-1 mutant developed drought tolerance similarly as did WT, but the abi2-1 mutant did not, suggesting that sensing of ABA is essential, but sensing of ethylene is not. Consistently, the induction of drought tolerance was abolished by inhibiting WT to synthesize ABA, instead of ethylene. Our results suggest that HrpNEa treatment enables plants to prioritize the ABA signal transduction pathway over ethylene signaling in accordance with the real-time requirement to survive under drought stress conditions.

A molecular timetable for apical bud formation and dormancy induction in poplar.

The growth of perennial plants in the temperate zone alternates with periods of dormancy that are typically initiated during bud development in autumn. In a systems biology approach to unravel the underlying molecular program of apical bud development in poplar (Populus tremula x Populus alba), combined transcript and metabolite profiling were applied to a high-resolution time course from short-day induction to complete dormancy. Metabolite and gene expression dynamics were used to reconstruct the temporal sequence of events during bud development. Importantly, bud development could be dissected into bud formation, acclimation to dehydration and cold, and dormancy. To each of these processes, specific sets of regulatory and marker genes and metabolites are associated and provide a reference frame for future functional studies. Light, ethylene, and abscisic acid signal transduction pathways consecutively control bud development by setting, modifying, or terminating these processes. Ethylene signal transduction is positioned temporally between light and abscisic acid signals and is putatively activated by transiently low hexose pools. The timing and place of cell proliferation arrest (related to dormancy) and of the accumulation of storage compounds (related to acclimation processes) were established within the bud by electron microscopy. Finally, the identification of a large set of genes commonly expressed during the growth-to-dormancy transitions in poplar apical buds, cambium, or Arabidopsis thaliana seeds suggests parallels in the underlying molecular mechanisms in different plant organs.

Reduced embryo sensitivity to abscisic acid in a sprouting-susceptible sorghum (Sorghum bicolor) variety is associated with altered ABA signalling

AbstractIn the work reported in this paper, we attempted to elucidate the nature of the different abscisic acid (ABA) sensitivities presented by developing embryos from sorghum [Sorghum bicolor (L.) Moench] lines with contrasting pre-harvest sprouting (PHS) behaviour (Redland B2, susceptible; IS 9530, resistant). We explored two different hypotheses for a possible mechanism: (1) a different functionality of the ABA signalling pathway, and (2) a different rate of ABA degradation/conjugation in the apoplast of embryos from these genotypes. To assess the first possibility, we used an ABA-responsive gene (Rab17) as a reporter of changes in endogenous ABA content, which were artificially induced in embryos from both genotypes by means of fluridone application immediately after anthesis, to reduce ABA content, and embryo incubation in the presence of ABA. A defect in ABA signalling should be seen as a level of Rab17 expression that is independent of endogenous ABA content. For testing the second possibility at two stages of development, embryos from both lines were isolated and incubated in water for different periods. ABA concentrations in embryos and the incubation media were quantified through radioimmunoassay. In contrast to our findings for the resistant IS 9530 line, Rab17 expression did not respond to changes in ABA levels in sensitive Redland B2 embryos. The ABA degradation/conjugation rates in embryos and incubation media did not show clear differences between sorghum lines for any of the developmental stages analysed. These results suggest that a disruption in the ABA signal transduction pathway in Redland B2 underlies the low ABA sensitivity shown by embryos from this line.

Transcriptional Responses to Gibberellin and Abscisic Acid in Barley Aleurone

Cereal aleurone has been established as a model system to investigate giberrellin (GA) and abscisic acid (ABA) responses. Using Barley 1 GeneChip, we examined the mRNA accumulation of over 22 000 genes in de-embryonated barley aleurone treated with GA and ABA. We observed that 1 328 genes had more than a threefold change in response to GA treatment, whereas 206 genes had a more than threefold change in response to ABA treatment. Interestingly, approximately 2.5-fold more genes were up-regulated than down-regulated by ABA. Eighty-three genes were differentially regulated by both GA and ABA. Most of the genes were subject to antagonistic regulation by ABA and GA, particularly for genes related to seed maturation and germination, such as genes encoding late embryogenesis abundant proteins and storage mobilization enzymes. This supports the antagonistic roles of GA and ABA in seed maturation and seed germination. Interestingly, we observed that a significant percentage of the genes were coordinately regulated by both GA and ABA. Some GA-responsive genes encoded proteins involved in ethylene, jasmonate, brassinosteroid and auxin metabolic and signaling transduction pathways, suggesting their potential interaction with the GA response. We also identified a group of transcription factor genes, such as MYB and Homeobox genes, that were differentially regulated by GA. In addition, a number of GA- and/or ABA-responsive genes encoded components potentially involved in GA and ABA signal transduction pathway. Overall, the present study provides a comprehensive and global view of transcript expression accompanying the GA and ABA response in barley aleurone and identifies a group of genes with potential regulatory functions in GA- and ABA-signaling pathways for future functional validation. (Managing editor: Li-Hui Zhao)

Role of Abscisic Acid in Seed Dormancy

Seed dormancy is an adaptive trait that improves survival of the next generation by optimizing the distribution of germination over time. The agricultural and forest industries rely on seeds that exhibit high rates of germination and vigorous, synchronous growth after germination; hence dormancy is sometimes considered an undesirable trait. The forest industry encounters problems with the pronounced dormancy of some conifer seeds, a feature that can lead to non-uniform germination and poor seedling vigor. In cereal crops, an optimum balance is most sought after; some dormancy at harvest is favored because it prevents germination of the physiologically mature grain in the head prior to harvest (that is, preharvest sprouting), a phenomenon that leads to considerable damage to grain quality and is especially prominent in cool moist environments. The sesquiterpene abscisic acid (ABA) regulates key events during seed formation, such as the deposition of storage reserves, prevention of precocious germination, acquisition of desiccation tolerance, and induction of primary dormancy. Its regulatory role is achieved in part by cross-talk with other hormones and their associated signaling networks, via mechanisms that are largely unknown. Quantitative genetics and functional genomics approaches will contribute to the elucidation of genes and proteins that control seed dormancy and germination, including components of the ABA signal transduction pathway. Dynamic changes in ABA biosynthesis and catabolism elicit hormone-signaling changes that affect downstream gene expression and thereby regulate critical checkpoints at the transitions from dormancy to germination and from germination to growth. Some of the recent developments in these areas are discussed.

Cross-talk between calcium and reactive oxygen species originated from NADPH oxidase in abscisic acid-induced antioxidant defence in leaves of maize seedlings.

The signal interactions between calcium (Ca2+) and reactive oxygen species (ROS) originated from plasma membrane NADPH oxidase in abscisic acid (ABA)-induced antioxidant defence were investigated in leaves of maize (Zea mays L.) seedlings. Treatment with ABA led to significant increases in the activity of plasma membrane NADPH oxidase, the production of leaf O2-, and the activities of several antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX) and glutathione reductase (GR). However, such increases were blocked by the pretreatment with Ca2+ chelator EGTA or Ca2+ channel blockers La3+ and verapamil, and NADPH oxidase inhibitors such as diphenylene iodonium (DPI), imidazole and pyridine. Treatment with Ca2+ also significantly induced the increases in NADPH oxidase activity, O2- production and the activities of antioxidant enzymes, and the increases were arrested by pretreatment with the NADPH oxidase inhibitors. Treatment with oxidative stress induced by paraquat, which generates O2-, led to the induction of antioxidant defence enzymes, and the up-regulation was suppressed by the pretreatment of Ca2+ chelator and Ca2+ channel blockers. Our data suggest that a cross-talk between Ca2+ and ROS originated from plasma membrane-bound NADPH oxidase is involved in the ABA signal transduction pathway leading to the induction of antioxidant enzyme activity, and Ca2+ functions upstream as well as downstream of ROS production in the signal transduction event in plants.

The abi1-1 mutation blocks ABA signaling downstream of cADPR action.

Arabidopsis thaliana abscisic acid insensitive 1-1 (abi1-1) is a dominant mutant that is insensitive to the inhibition of germination and growth by the plant hormone, abscisic acid (ABA). The mutation severely decreases the catalytic activity of the ABI1 type 2C protein phosphatase (PP2C). However, the site of action of the abi1-1/ABI1 in the ABA signal transduction pathway has not yet been determined. Using single cell assays, we showed that microinjecting mutant abi1-1 protein inhibited the activation of RD29A-GUS and KIN2-GUS in response to ABA, cyclic ADP-ribose (cADPR), and Ca2+. The inhibitory effect of the mutant protein, however, was reversed by co-microinjection of an excess amount of the ABI1 protein. In transgenic Arabidopsis plants, overexpression of abi1-1 rendered the plants insensitive to ABA during germination, whereas overexpression of ABI1 did not have any apparent effect. Moreover, transgenic plants overexpressing abi1-1 were blocked in the induction of ABA-responsive genes; however, overexpression of ABI1 did not affect gene expression. Taken together, our results demonstrate that abi1-1 is likely to be a dominant negative mutation and ABI1 likely acts downstream of cADPR in the ABA-signaling pathway. Our results on ABI1 overexpression in Arabidopsis are not compatible with a negative regulatory role of this phosphatase in ABA responses.

Abscisic acid-induced transcription is mediated by phosphorylation of an abscisic acid response element binding factor, TRAB1.

The rice basic domain/Leu zipper factor TRAB1 binds to abscisic acid (ABA) response elements and mediates ABA signals to activate transcription. We show that TRAB1 is phosphorylated rapidly in an in vivo labeling experiment and by phosphatase-sensitive mobility shifts on SDS-polyacrylamide gels. We had shown previously that a chimeric promoter containing GAL4 binding sites became ABA inducible when a GAL4 binding domain-TRAB1 fusion protein was present. This expression system allowed us to assay the ABA response function of TRAB1. Using this system, we show that Ser-102 of TRAB1 is critical for this function. Because no ABA-induced mobility shift was observed when Ser-102 was replaced by Ala, we suggest that this Ser residue is phosphorylated in response to ABA. Cell fractionation experiments, as well as fluorescence microscopy observations of transiently expressed green fluorescent protein-TRAB1 fusion protein, indicated that TRAB1 was localized in the nucleus independently of ABA. Our results suggest that the terminal or nearly terminal event of the primary ABA signal transduction pathway is the phosphorylation in the nucleus of preexisting TRAB1.

De Novo Activation of the β-Phaseolin Promoter by Phosphatase or Protein Synthesis Inhibitors

The promoter for the phaseolin (phas) bean seed protein gene adopts an inactive chromatin structure in leaves of transgenic tobacco. This repressive architecture, which confers stringent spatial regulation, is disrupted upon transcriptional activation during embryogenesis in a process that requires the presence of both a transcription factor (PvALF) and abscisic acid (ABA). Toward determining the need for de novo synthesis of proteins other than PvALF in transcriptional activation we explored the effect of several eukaryotic protein synthesis inhibitors. Surprisingly, cycloheximide (CHX), emetine, and verrucarin A were able to induce transcription from the phas promoter in tobacco and bean leaf tissue in the absence of either PvALF or ABA. This induction was decreased by the replication inhibitors hydroxyurea and aphidicolin but not by genistein or mimosine. Since protein phosphatases and kinases are essential components of the ABA signal transduction pathway, it is conceivable that CHX is also capable of inducing phosphorylation of proteins usually involved in ABA-mediated activation. Interestingly, okadaic acid, an inhibitor of serine/threonine phosphatase, also strongly activated transcription from the phas promoter. In contrast, the protein synthesis inhibitors anisomycin and puromycin did not activate transcription from the phas promoter, nor did the tyrosine phosphatase inhibitors phenylarsine oxide and sodium orthovanadate. These discrete but different results on transcriptional activation may reflect specific modes of action of the inhibitors, or they may reflect differential interactions of the inhibitors or of downstream events resulting from inhibitor activity with presently unknown components of the transcriptional activation system.

Dissection of abscisic acid signal transduction pathways in barley aleurone layers.

Abscisic acid (ABA) induces genes that are highly expressed during late embryogenesis, but suppresses gibberellin (GA)-responsive genes essential for seed germination and seedling growth. Promoter elements necessary and sufficient for ABA up- and down-regulation of gene expression have been previously defined in barley aleurone layers. We have studied the effect of a protein phosphatase 2C, ABI1, an ABA-inducible protein kinase, PKABA1, and a transcription factor, VP1, on ABA action in a barley aleurone transient expression system. The observations have allowed us to dissect ABA signal transduction pathways leading to either induction or suppression of gene expression. The ABA induction of embryogenesis genes is highly inhibited in the presence of a mutated protein phosphatase 2C, encoded by the abi1-1 dominant mutant gene that is known to block ABA responses in Arabidopsis. However, the abi1-1 gene product has no effect on the ABA suppression of a GA-responsive alpha-amylase gene. On the other hand, PKABA1 suppresses the expression of alpha-amylase genes, but has little effect on ABA up-regulated genes. Therefore, it appears that ABA induction and suppression follow two separate signal transduction pathways with the former inhibited by ABI1 and the latter modulated by PKABA1. The presence of VP1 enhances the ABA induction of late embryogenesis genes, but also suppresses germination specific genes. A schematic model based on these observations is presented to explain the effect of these regulatory proteins on ABA-mediated gene expression.

Distinct abscisic acid signaling pathways for modulation of guard cell versus mesophyll cell potassium channels revealed by expression studies in Xenopus laevis oocytes.

Regulation of guard cell ion transport by abscisic acid (ABA) and in particular ABA inhibition of a guard cell inward K(+) current (I(Kin)) is well documented. However, little is known concerning ABA effects on ion transport in other plant cell types. Here we applied patch clamp techniques to mesophyll cell protoplasts of fava bean (Vicia faba cv Long Pod) plants and demonstrated ABA inhibition of an outward K(+) current (I(Kout)). When mesophyll cell protoplast mRNA (mesophyll mRNA) was expressed in Xenopus laevis oocytes, I(Kout) was generated that displayed similar properties to I(Kout) observed from direct analysis of mesophyll cell protoplasts. I(Kout) expressed by mesophyll mRNA-injected oocytes was inhibited by ABA, indicating that the ABA signal transduction pathway observed in mesophyll cells was preserved in the frog oocytes. Co-injection of oocytes with guard cell protoplast mRNA and cRNA for KAT1, an inward K(+) channel expressed in guard cells, resulted in I(Kin) that was similarly inhibited by ABA. However, oocytes co-injected with mesophyll mRNA and KAT1 cRNA produced I(Kin) that was not inhibited by ABA. These results demonstrate that the mesophyll-encoded signaling mechanism could not substitute for the guard cell pathway. These findings indicate that mesophyll cells and guard cells use distinct and different receptor types and/or signal transduction pathways in ABA regulation of K(+) channels.

Cis-elements and trans-factors that regulate expression of the maize Cat1 antioxidant gene in response to ABA and osmotic stress: H2O2 is the likely intermediary signaling molecule for the response.

The mechanisms by which the maize antioxidant Cat1 gene responds to abscisic acid (ABA) and osmotic stress have been investigated. Results show that during late embryogenesis, Cat1 expression in vivo is independent of endogenous ABA levels. However, exogenously applied ABA significantly enhances Cat1 expression. Transient assays using particle bombardment show that the proximal ABRE2 element on the Cat1 promoter is responsible for the induction of Cat1 expression by ABA. We further show that ABA induces the expression of Cat1 via the interaction between ABRE2 and one of its binding proteins, CBF1 (Cat1 binding factor 1). Using ABA-deficient mutant embryos, we show that osmotic stress induces Cat1 expression through two alternate signal transduction pathways: an ABA signaling pathway leading to the interaction between the ABRE2 motif and CBF1, and a pathway via the interaction of ABRE2 and CBF2 (Cat1 binding factor 2) that is independent of ABA. The data presented clearly suggest that hydrogen peroxide (H2O2) plays an important intermediary role in the ABA signal transduction pathway leading to the induction of the Cat1 gene.

ATP binding cassette modulators control abscisic acid-regulated slow anion channels in guard cells

In animal cells, ATP binding cassette (ABC) proteins are a large family of transporters that includes the sulfonylurea receptor and the cystic fibrosis transmembrane conductance regulator (CFTR). These two ABC proteins possess an ion channel activity and bind specific sulfonylureas, such as glibenclamide, but homologs have not been identified in plant cells. We recently have shown that there is an ABC protein in guard cells that is involved in the control of stomatal movements and guard cell outward K+ current. Because the CFTR, a chloride channel, is sensitive to glibenclamide and able to interact with K+ channels, we investigated its presence in guard cells. Potent CFTR inhibitors, such as glibenclamide and diphenylamine-2-carboxylic acid, triggered stomatal opening in darkness. The guard cell protoplast slow anion current that was recorded using the whole-cell patch-clamp technique was inhibited rapidly by glibenclamide in a dose-dependent manner; the concentration producing half-maximum inhibition was at 3 &mgr;M. Potassium channel openers, which bind to and act through the sulfonylurea receptor in animal cells, completely suppressed the stomatal opening induced by glibenclamide and recovered the glibenclamide-inhibited slow anion current. Abscisic acid is known to regulate slow anion channels and in our study was able to relieve glibenclamide inhibition of slow anion current. Moreover, in epidermal strip bioassays, the stomatal closure triggered by Ca2+ or abscisic acid was reversed by glibenclamide. These results suggest that the slow anion channel is an ABC protein or is tightly controlled by such a protein that interacts with the abscisic acid signal transduction pathway in guard cells.

Abscisic acid-induced stomatal closure mediated by cyclic ADP-ribose.

Abscisic acid (ABA) is a plant hormone involved in the response of plants to reduced water availability. Reduction of guard cell turgor by ABA diminishes the aperture of the stomatal pore and thereby contributes to the ability of the plant to conserve water during periods of drought. Previous work has demonstrated that cytosolic Ca2+ is involved in the signal transduction pathway that mediates the reduction in guard cell turgor elicited by ABA. Here we report that ABA uses a Ca2+-mobilization pathway that involves cyclic adenosine 5'-diphosphoribose (cADPR). Microinjection of cADPR into guard cells caused reductions in turgor that were preceded by increases in the concentration of free Ca2+ in the cytosol. Patch clamp measurements of isolated guard cell vacuoles revealed the presence of a cADPR-elicited Ca2+-selective current that was inhibited at cytosolic Ca2+ >/= 600 nM. Furthermore, microinjection of the cADPR antagonist 8-NH2-cADPR caused a reduction in the rate of turgor loss in response to ABA in 54% of cells tested, and nicotinamide, an antagonist of cADPR production, elicited a dose-dependent block of ABA-induced stomatal closure. Our data provide definitive evidence for a physiological role for cADPR and illustrate one mechanism of stimulus-specific Ca2+ mobilization in higher plants. Taken together with other recent data [Wu, Y., Kuzma, J., Marechal, E., Graeff, R., Lee, H. C., Foster, R. & Chua, N.-H. (1997) Science 278, 2126-2130], these results establish cADPR as a key player in ABA signal transduction pathways in plants.

Promoter switches specific for abscisic acid (ABA)‐induced gene expression in cereals

Abscisic acid (ABA) has been shown to regulate many physiological and developmental processes which are often mediated by the induction and suppression of gene expression. Herein, we review the progress made in the understanding of gene expression regulated by ABA, with the emphasis on cis‐ and trans‐acting elements controlling gene expression. Promoter sequences containing an ACGT‐core have been shown by several groups to be necessary for the ABA‐induced gene expression. However, similar ACGT‐core‐containing sequences are also necessary for responses to a variety of environmental and physiological cues. To address the question what determines the response specificity, we have studied two barley ABA inducible genes and defined the modular nature of ABA response complex (ABRC), the promoter unit necessary and sufficient for ABA induction of gene expression. ABRCs of these genes consist of a 10‐bp element with an ACGT‐core (ACGT‐box) and a coupling element (CE1 or CE3). These ABRCs function in both seed and vegetative tissues. Genetic analysis has led to the cloning of genes, such as maize Viviparous 1 (VP1), involved in the regulation of sensitivity of plants to ABA. In seeds, ABA induction of the ABRC containing CE3, but not the ABRC with CE1, is enhanced in the presence of the transcription regulator, encoded by the VP1 gene, indicating these two ABRCs are mediated by different ABA signal transduction pathways. Other potential signal transduction components mediating ABA signal transduction pathways are also discussed.

Rapid Germination of a Barley Mutant Is Correlated with a Rapid Turnover of Abscisic Acid Outside the Embryo.

In our study of the role of abscisic acid (ABA) in controlling the germination of barley grains, we tested a barley mutant line with a gigantum appearance (Hordeum distichum cv Quantum) for an ABA-insensitive phenotype by assaying germination in the presence of 10-4 M ABA. Dissected embryos of the mutant germinated at least 10 h earlier than did those of the wild type. The half-maximal concentrations of ABA inhibitory for germination were determined to be 5 x 10-4 M for the mutant and 10-6 M for the wild type. Expression of an ABA-induced Rab gene was studied to determine ABA responsiveness. The ABA concentration required for a half-maximal induction of Rab gene expression was 4 x 10-6 M in isolated embryos of both the mutant and wild type. This result suggests that ABA signal transduction pathways were not affected in the mutant. When isolated embryos were allowed to imbibe in water, ABA was released from the mutant and wild-type embryos at the same rate. However, the free ABA level in the incubation medium of the mutant showed a much faster decrease than that of the wild type, as demonstrated by two independent ABA assay methods (high-performance liquid chromatography and enzyme-linked immunosorbent assay). Our results suggest that turnover of ABA outside the embryo is a determining factor in the germination of barley seeds.