Abstract Abscisic Acid Signaling Research Articles

Core Components of Abscisic Acid Signaling and Their Post-translational Modification.

Abscisic acid (ABA) is a major phytohormone that regulates plant growth, development, and abiotic/biotic stress responses. Under stress, ABA is synthesized in various plant organs, and it plays roles in diverse adaptive processes, including seed dormancy, growth inhibition, and leaf senescence, by modulating stomatal closure and gene expression. ABA receptor, clade A protein phosphatase 2C (PP2C), and SNF1-related protein kinase 2 (SnRK2) proteins have been identified as core components of ABA signaling, which is initiated via perception of ABA with receptor and subsequent activation or inactivation by phosphorylation/dephosphorylation. The findings of several recent studies have established that the post-translational modification of these components, including phosphorylation and ubiquitination/deubiquitination, play important roles in regulating their activity and stability. In this review, we discuss the functions of the core components of ABA signaling and the regulation of their activities via post-translational modification under normal and stress conditions.

SKIP Regulates ABA Signaling through Alternative Splicing in Arabidopsis.

Abscisic acid (ABA) plays key roles in plant development and responses to abiotic stresses. A wide number of transcriptional and posttranslational regulatory mechanisms of ABA signaling are known; however, less is known about the regulatory roles of alternative splicing. In this work, we found that SKIP, a splicing factor, positively regulates ABA signaling. SKIP binds to the pre-mRNA of ABA signaling-related genes, such as PYL7, PYL8, ABI1, HAB1 and ABI5, to regulate their splicing. The precursor mRNA alternative splicing of several PYL receptors, PP2C phosphatases and ABF transcriptional factors is disrupted by the skip-1 mutation. The abnormal alternative splicing in skip-1 represses the expression of ABA-positive regulators, including PYLs and ABFs, and activates the expression of ABA-negative regulators, such as PP2Cs, which confers ABA hyposensitive phenotype of skip-1. We also found that ABA-mediated genome-wide alternative splicing and differential gene expression are changed by the skip-1 mutation. The number of the differential splicing events is increased by skip-1; however, the number of differential expressed genes in response to ABA is reduced by skip-1. Our results reveal a principle on how a splicing factor regulates ABA signaling and ABA-mediated genome-wide alternative splicing.

Roles of abscisic acid in regulating ripening and quality of strawberry, a model non-climacteric fruit.

Abscisic acid (ABA) is a dominant regulator of ripening and quality in non-climacteric fruits. Strawberry is regarded as a model non-climacteric fruit due to its extensive genetic studies and proven suitability for transgenic approaches to understanding gene function. Strawberry research has contributed to studies on color, flavor development, and fruit softening, and in recent years ABA has been established as a core regulator of strawberry fruit ripening, whereas ethylene plays this role in climacteric fruits. Despite this major difference, several components of the interacting genetic regulatory network in strawberry, such as MADS-box and NAC transcription factors, are similar to those that operate in climacteric fruit. In this review, we summarize recent advances in understanding the role of ABA biosynthesis and signaling and the regulatory network of transcription factors and other phytohormones in strawberry fruit ripening. In addition to providing an update on its ripening, we discuss how strawberry research has helped generate a broader and more comprehensive understanding of the mechanism of non-climacteric fruit ripening and focus attention on the use of strawberry as a model platform for ripening studies.

Role of MaABI5-like in abscisic acid-induced cold tolerance of 'Fenjiao' banana fruit.

Abscisic acid (ABA) is a phytohormone essential for plants to respond to various environmental stresses, and abscisic acid-insensitive 5 (ABI5) is a basic leucine zipper transcription factor of the ABA signaling pathway. Exogenous ABA induces cold tolerance in bananas; however, the role of MaABI5-like in ABA-induced cold tolerance remains unexplored. The present study found that exogenous ABA alleviated chilling injury of 'Fenjiao' banana, induced the accumulation of endogenous ABA, unsaturated fatty acids, and flavonoid content, and reduced the saturated fatty acid content. Moreover, ABA treatment upregulated the transcription levels of MaABI5-like, fatty acid desaturation genes, and flavonoid synthesis-related genes during cold storage. More interestingly, MaABI5-like directly interacted with the promoter of genes related to fatty acid desaturation (MaFAD3-1, MaFAD3-4, MaFAD3-5, MaFAD6-2, MaFAD6-3) and flavonoid synthesis (MaPAL-like, MaPAL-like1, MaC4H-like3, Ma4CL-like1, Ma4CL-like10, MaCHS6-4-like, and MaFLS) and activated their expressions. Furthermore, the transient overexpression of MaABI5-like in 'Fenjiao' banana fruit and ectopic expression in tomato plants enhanced cold tolerance and upregulated fatty acid desaturation and flavonoid synthesis-related gene transcript levels. The reduced expression of MaABI5-like by virus-induced gene silencing in 'Fenjiao' banana increased chilling injury and downregulated the expression of fatty acid desaturation and flavonoid synthesis-related genes. Thus, the study indicates that MaABI5-like regulates ABA-induced cold tolerance by increasing unsaturated fatty acid and flavonoid content.

Tomato SlPP2C5 Is Involved in the Regulation of Fruit Development and Ripening.

Abscisic acid (ABA) regulates plant development mainly through its signaling, in which ABA binds to receptors to inhibit type 2C protein phosphatases (PP2Cs). The exact roles of PP2Cs in fruit development are still unclear. In this work, we verify that tomato SlPP2C5 works as a negative regulator in ABA signaling during fruit development. SlPP2C5 was inhibited by both monomeric and dimeric ABA receptors SlPYLs through ABA dose-dependent way, and it interacted physically with SlPYLs and SlSnRK2s. SlPP2C5 was highly expressed in fruits induced by exogenous ABA. Plants with overexpressed SlPP2C5 had lower sensitivity to ABA, which showed faster seed germination and primary root growth compared to Wild type (WT), while SlPP2C5-suppressed plants were more sensitive to ABA. SlPP2C5-over-expression (OE) delayed fruit ripening onset, while SlPP2C5-RNAi advanced fruit ripening. Alteration of SlPP2C5 expression impacts fruit quality parameters as well, including pericarp thickness, fruit shape index, seed number and weight and the soluble solid content. RNA-seq analysis revealed that there were significant expression differences of genes related to ethylene release and lycopene synthesis between WT and both SlPP2C5-OE and SlPP2C5-RNAi lines with an inversed variation. Taken together, our findings demonstrate that SlPP2C5 plays an important role in the regulation of fruit development, ripening and quality.

A Clathrin-Related Protein, SCD2/RRP1, Participates in Abscisic Acid Signaling in Arabidopsis.

Abscisic acid (ABA) plays important roles in many aspects of plant growth and development, and responses to diverse stresses. Although much progress has been made in understanding the molecular mechanisms of ABA homoeostasis and signaling, the mechanism by which plant cells integrate ABA trafficking and signaling to regulate plant developmental processes is poorly understood. In this study, we used Arabidopsis STOMATAL CYTOKINESIS DEFECTIVE 2/RIPENING-REGULATED PROTEIN 1 (SCD2/RRP1) mutants and overexpression plants, in combination with transcriptome and protein-interaction assays, to investigate SCD2/RRP1 involvement in the integration of ABA trafficking and signaling in seed germination and seedling growth. Manipulation of SCD2/RRP1 expression affected ABA sensitivity in seed germination and seedling growth, as well as transcription of several ABA transporter genes and ABA content. RNA-sequencing analysis of Arabidopsis transgenic mutants suggested that SCD2/RRP1 was associated with ABA signaling via a type 2C protein phosphatase (PP2C) protein. The N- and C-terminal regions of SCD2/RRP1 separately interacted with both PYRABACTIN RESISTANCE 1 (PYR1) and ABA INSENSITIVE 1 (ABI1) on the plasma membrane, and SCD2/RRP1 acted genetically upstream of ABI1. Interestingly, ABA inhibited the interaction of SCD2/RRP1 with ABI1, but did not affect the interaction of SCD2/RRP1 with PYR1. These results suggested that in Arabidopsis SCD2/RRP1participates in early seed development and growth potentially through clathrin-mediated endocytosis- and clathrin-coated vesicle-mediated ABA trafficking and signaling. These findings provide insight into the mechanism by which cells regulate plant developmental processes through ABA.

Functional analysis of SlNCED1 in pistil development and fruit set in tomato (Solanum lycopersicum L.)

Abscisic acid (ABA) is an important regulator of many plant developmental processes, although its regulation in the pistil during anthesis is unclear. We investigated the role of 9-cis-epoxycarotenoid dioxygenase (SlNCED1), a key ABA biosynthesis enzyme, through overexpression and transcriptome analysis in the tomato pistil. During pistil development, ABA accumulates and SlNCED1 expression increases continually, peaking one day before full bloom, when the maximum amount of ethylene is released in the pistil. ABA accumulation and SlNCED1 expression in the ovary remained high for three days before and after full bloom, but then both declined rapidly four days after full bloom following senescence and petal abscission and expansion of the young fruits. Overexpression of SlNCED1 significantly increased ABA levels and also up-regulated SlPP2C5 expression, which reduced ABA signaling activity. Overexpression of SlNCED1 caused up-regulation of pistil-specific Zinc finger transcription factor genes SlC3H29, SlC3H66, and SlC3HC4, which may have affected the expression of SlNCED1-mediated pistil development-related genes, causing major changes in ovary development. Increased ABA levels are due to SlNCED1 overexpresson which caused a hormonal imbalance resulting in the growth of parthenocarpic fruit. Our results indicate that SlNCED1 plays a crucial role in the regulation of ovary/pistil development and fruit set.

The Assay of Abscisic Acid-Induced Stomatal Movement in Leaf Senescence.

Abscisic acid (ABA) is a sesquiterpenoid (15-carbon) hormone that comprehensively regulates plant stress responses, development, and senescence. Stomata are epidermal pores on plant surface used for exchanging gases such as carbon dioxide, water vapor, and oxygen. One of the mechanisms that ABA regulates leaf senescence is to control stomatal movement and thus water loss during leaf senescence. Here we describe the procedure of measuring stomatal movement in response to ABA treatments, which will provide a useful protocol to investigate ABA signaling in leaf senescence.

Cloning and characterization of a novel Gladiolus hybridus AFP family gene (GhAFP-like) related to corm dormancy

Abscisic acid (ABA) is an important phytohormone controlling seed dormancy. AFPs (ABA INSENSITIVE FIVE BINDING PROTEINS) are reported to be negative regulators of the ABA signaling pathway. The involvement of AFPs in dormant vegetative organs remains poorly understood. Here, we isolated and characterized a novel AFP family member from Gladiolus dormant cormels, GhAFP-like, containing three conserved domains of the AFP family. Quantitative PCR analysis revealed that GhAFP-like was expressed in dormant organs and its expression was down-regulated along with corm storage. GhAFP-like was verified to be a nuclear-localized protein. Overexpressing GhAFP-like in Arabidopsis thaliana not only showed weaker seed dormancy with insensitivity to ABA, but also changed the expression of some ABA related genes. In addition, a primary root elongation assay showed GhAFP-like may involve in auxin signaling response. The results in this study indicate that GhAFP-like acts as a negative regulator in ABA signaling and is related to dormancy.

Isolation and characterization of two ABRE-binding proteins: EABF and EABF1 from the oil palm

Abscisic acid (ABA) is an important phytohormone involved in the abiotic stress resistance in plants. The ABA-responsive element (ABRE) binding factors play significant roles in the plant development and response to abiotic stresses, but none so far have been isolated and characterized from the oil palm. Two ABA-responsive cDNA clones, named EABF and EABF1, were isolated from the oil palm fruits using yeast one-hybrid system. The EABF had a conserved AP2/EREBP DNA-binding domain (DNA-BD) and a potential nuclear localization sequence (NLS). No previously known DNA-BD was identified from the EABF1 sequence. The EABF and EABF1 proteins were classified as DREB/CBF and bZIP family members based on the multiple sequence alignment and phylogenetic analysis. Both proteins showed ABRE-binding and transcriptional activation properties in yeast. Furthermore, both proteins were able to trans-activate the down-stream expression of the LacZ reporter gene in yeast. An electrophoretic mobility shift assay revealed that in addition to the ABRE sequence, both proteins could bind to the DRE sequence as well. Transcriptional analysis revealed that the expression of EABF was induced in response to the ABA in the oil palm fruits and leaves, but not in roots, while the EABF1 was constitutively induced in all tissues. The expressions of both genes were strongly induced in fruits in response to the ABA, ethylene, methyl jasmonate, drought, cold and high-salinity treatments, indicating that the EABF and EABF1 might act as connectors among different stress signal transduction pathways. Our results indicate that the EABF and EABF1 are novel stress-responsive transcription factors, which are involved in the abiotic stress response and ABA signaling in the oil palm and could be used for production of stress-tolerant transgenic crops.

Proteomics and Functional Analyses of Pepper Abscisic Acid–Responsive 1 (ABR1), Which Is Involved in Cell Death and Defense Signaling

Abscisic acid (ABA) is a key regulator of plant growth and development, as well as plant defense responses. A high-throughput in planta proteome screen identified the pepper (Capsicum annuum) GRAM (for glucosyltransferases, Rab-like GTPase activators, and myotubularins) domain-containing ABA-RESPONSIVE1 (ABR1), which is highly induced by infection with avirulent Xanthomonas campestris pv vesicatoria and also by treatment with ABA. The GRAM domain is essential for the cell death response and for the nuclear localization of ABR1. ABR1 is required for priming cell death and reactive oxygen species production, as well as ABA-salicylic acid (SA) antagonism. Silencing of ABR1 significantly compromised the hypersensitive response but enhanced bacterial pathogen growth and ABA levels in pepper. High levels of ABA in ABR1-silenced plants antagonized the SA levels induced by pathogen infection. Heterologous transgenic expression of ABR1 in Arabidopsis thaliana conferred enhanced resistance to Pseudomonas syringae pv tomato and Hyaloperonospora arabidopsidis infection. The susceptibility of the Arabidopsis ABR1 putative ortholog mutant, abr1, to these pathogens also supports the involvement of ABR1 in disease resistance. Together, these results reveal ABR1 as a novel negative regulator of ABA signaling and suggest that the nuclear ABR1 pool is essential for the cell death induction associated with ABA-SA antagonism.

ABA transport factors found in Arabidopsis ABC transporters

Abscisic acid (ABA) is a phytohormone that plays an important role in responses to environmental stresses as well as seed maturation and germination. Intracellular signaling by ABA has been rigorously investigated in relation to stomatal guard-cell regulation, seed germination, and abiotic stress responses. However, intercellular regulation of ABA, including the molecular basis of ABA transport systems, has hardly been examined in any plant species. Based on genetic and biochemical analyses, we present evidence that one of the ATP-binding cassette (ABC) transporter genes, AtABCG25, encodes a protein that functions as an ABA exporter through the plasma membrane and is involved in the intercellular ABA signaling pathway. The ABC-type transporter is conserved in model species from E. coli to humans and is reported to transport various metabolites or signaling molecules in an ATP-dependent manner. At same time, another ABC transporter in Arabidopsis, AtABCG40, was independently reported to function as an ABA importer in plant cells. These findings strongly suggest the active control of ABA transport between plant cells, and they provide a novel impetus for examining ABA intercellular regulation.

HAB1–SWI3B Interaction Reveals a Link between Abscisic Acid Signaling and Putative SWI/SNF Chromatin-Remodeling Complexes in Arabidopsis

Abscisic acid (ABA) has an important role for plant growth, development, and stress adaptation. HYPERSENSITIVE TO ABA1 (HAB1) is a protein phosphatase type 2C that plays a key role as a negative regulator of ABA signaling; however, the molecular details of HAB1 action in this process are not known. A two-hybrid screen revealed that SWI3B, an Arabidopsis thaliana homolog of the yeast SWI3 subunit of SWI/SNF chromatin-remodeling complexes, is a prevalent interacting partner of HAB1. The interaction mapped to the N-terminal half of SWI3B and required an intact protein phosphatase catalytic domain. Bimolecular fluorescence complementation and coimmunoprecipitation assays confirmed the interaction of HAB1 and SWI3B in the nucleus of plant cells. swi3b mutants showed a reduced sensitivity to ABA-mediated inhibition of seed germination and growth and reduced expression of the ABA-responsive genes RAB18 and RD29B. Chromatin immunoprecipitation experiments showed that the presence of HAB1 in the vicinity of RD29B and RAB18 promoters was abolished by ABA, which suggests a direct involvement of HAB1 in the regulation of ABA-induced transcription. Additionally, our results uncover SWI3B as a novel positive regulator of ABA signaling and suggest that HAB1 modulates ABA response through the regulation of a putative SWI/SNF chromatin-remodeling complex.

Identification of Two Protein Kinases Required for Abscisic Acid Regulation of Seed Germination, Root Growth, and Gene Expression in Arabidopsis

Abscisic acid (ABA) is an important phytohormone regulating various plant processes, including seed germination. Although phosphorylation has been suggested to be important, the protein kinases required for ABA signaling during seed germination and seedling growth remain elusive. Here, we show that two protein kinases, SNF1-RELATED PROTEIN KINASE2.2 (SnRK2.2) and SnRK2.3, control responses to ABA in seed germination, dormancy, and seedling growth in Arabidopsis thaliana. A snrk2.2 snrk2.3 double mutant, but not snrk2.2 or snrk2.3 single mutants, showed strong ABA-insensitive phenotypes in seed germination and root growth inhibition. Changes in seed dormancy and ABA-induced Pro accumulation consistent with ABA insensitivity were also observed. The snrk2.2 snrk2.3 double mutant had a greatly reduced level of a 42-kD kinase activity capable of phosphorylating peptides from ABF (for ABA Response Element Binding Factor) transcription factors. ABA-induced expression of several genes whose promoters contain an ABA response element (ABRE) was reduced in snrk2.2 snrk2.3, suggesting that the mechanism of SnRK2.2 and SnRK2.3 action in ABA signaling involves the activation of ABRE-driven gene expression through the phosphorylation of ABFs. Together, these results demonstrate that SnRK2.2 and SnRK2.3 are redundant but key protein kinases that mediate a major part of ABA signaling in Arabidopsis.

Enhancement of Abscisic Acid Sensitivity and Reduction of Water Consumption in Arabidopsis by Combined Inactivation of the Protein Phosphatases Type 2C ABI1 and HAB1

Abscisic acid (ABA) plays a key role in plant responses to abiotic stress, particularly drought stress. A wide number of ABA-hypersensitive mutants is known, however, only a few of them resist/avoid drought stress. In this work we have generated ABA-hypersensitive drought-avoidant mutants by simultaneous inactivation of two negative regulators of ABA signaling, i.e. the protein phosphatases type 2C (PP2Cs) ABA-INSENSITIVE1 (ABI1) and HYPERSENSITIVE TO ABA1 (HAB1). Two new recessive loss-of-function alleles of ABI1, abi1-2 and abi1-3, were identified in an Arabidopsis (Arabidopsis thaliana) T-DNA collection. These mutants showed enhanced responses to ABA both in seed and vegetative tissues, but only a limited effect on plant drought avoidance. In contrast, generation of double hab1-1 abi1-2 and hab1-1 abi1-3 mutants strongly increased plant responsiveness to ABA. Thus, both hab1-1 abi1-2 and hab1-1 abi1-3 were particularly sensitive to ABA-mediated inhibition of seed germination. Additionally, vegetative responses to ABA were reinforced in the double mutants, which showed a strong hypersensitivity to ABA in growth assays, stomatal closure, and induction of ABA-responsive genes. Transpirational water loss under drought conditions was noticeably reduced in the double mutants as compared to single parental mutants, which resulted in reduced water consumption of whole plants. Taken together, these results reveal cooperative negative regulation of ABA signaling by ABI1 and HAB1 and suggest that fine tuning of ABA signaling can be attained through combined action of PP2Cs. Finally, these results suggest that combined inactivation of specific PP2Cs involved in ABA signaling could provide an approach for improving crop performance under drought stress conditions.

Transcriptome Analysis Reveals Specific Modulation of Abscisic Acid Signaling by ROP10 Small GTPase in Arabidopsis

Abscisic acid (ABA) is a hormone that modulates a variety of agronomically important growth and developmental processes and various stresses responses, but its signal transduction pathways remain poorly understood. ROP10, a member of ROP small GTPases in Arabidopsis (Arabidopsis thaliana), is a plasma membrane-associated protein specifically involved in negative regulation of ABA responses. To dissect the ROP10-mediated ABA signaling, we carried out transcriptome analysis using the Arabidopsis full-genome chip. Our analysis revealed a total of 262 and 125 genes that were, respectively, up- and down-regulated (> or =2-fold cutoff) by 1 mum ABA in wild type (Wassilewskija [Ws]); 42 up-regulated and 38 down-regulated genes have not been identified in other studies. Consistent with the nonpleiotropic phenotypes of rop10-1, only three genes were altered in rop10-1 in the absence of ABA treatment. In response to 1 microm ABA, 341 and 127 genes were, respectively, activated and repressed in rop10-1. Interestingly, a particular subset of 21 genes that were not altered by 1 microm ABA in Ws but only activated in rop10-1 was identified. Reverse transcription-polymerase chain reaction analysis revealed the existence of three distinct categories of ABA dose-response patterns. One novel category is characterized by their ABA unresponsiveness in Ws and activation in rop10-1 at 1 microm but not 10 and 100 microm of ABA. This indicates that ROP10 gates the expression of genes that are specific to low concentrations of ABA. Furthermore, almost all of these 21 genes are known to be highly induced by various biotic and abiotic stresses. Consequently, we found that rop10-1 enhanced the sensitivity of seed germination inhibition to mannitol and sodium chloride. Our results suggest that ROP10 negatively regulates ABA responses by specifically and differentially modulating the ABA sensitivity of a subset of genes including protein kinases and zinc-finger family proteins.

Leucine-Rich Repeat Receptor-Like Kinase1 Is a Key Membrane-Bound Regulator of Abscisic Acid Early Signaling in Arabidopsis

Abscisic acid (ABA) is important in seed maturation, seed dormancy, stomatal closure, and stress response. Many genes that function in ABA signal transduction pathways have been identified. However, most important signaling molecules involved in the perception of the ABA signal or with ABA receptors have not been identified yet. Receptor-like kinase1 (RPK1), a Leu-rich repeat (LRR) receptor kinase in the plasma membrane, is upregulated by ABA in Arabidopsis thaliana. Here, we show the phenotypes of T-DNA insertion mutants and RPK1-antisense plants. Repression of RPK1 expression in Arabidopsis decreased sensitivity to ABA during germination, growth, and stomatal closure; microarray and RNA gel analysis showed that many ABA-inducible genes are downregulated in these plants. Furthermore, overexpression of the RPK1 LRR domain alone or fused with the Brassinosteroid-insensitive1 kinase domain in plants resulted in phenotypes indicating ABA sensitivity. RPK1 is involved in the main ABA signaling pathway and in early ABA perception in Arabidopsis.

A novel inhibitor of 9-cis-epoxycarotenoid dioxygenase in abscisic acid biosynthesis in higher plants.

Abscisic acid (ABA) is a major regulator in the adaptation of plants to environmental stresses, plant growth, and development. In higher plants, the ABA biosynthesis pathway involves the oxidative cleavage of 9-cis-epoxycarotenoids, which may be the key regulatory step in the pathway catalyzed by 9-cis-epoxycarotenoid dioxygenase (NCED). We developed a new inhibitor of ABA biosynthesis targeting NCED and named it abamine (ABA biosynthesis inhibitor with an amine moiety). Abamine is a competitive inhibitor of NCED, with a Ki of 38.8 microm. In 0.4 m mannitol solution, which mimics the effects of osmotic stress, abamine both inhibited stomatal closure in spinach (Spinacia oleracea) leaves, which was restored by coapplication of ABA, and increased luminescence intensity in transgenic Arabidopsis containing the RD29B promoter-luciferase fusion. The ABA content of plants in 0.4 m mannitol was increased approximately 16-fold as compared with that of controls, whereas 50 to 100 microm abamine inhibited about 50% of this ABA accumulation in both spinach leaves and Arabidopsis. Abamine-treated Arabidopsis was more sensitive to drought stress and showed a significant decrease in drought tolerance than untreated Arabidopsis. These results suggest that abamine is a novel ABA biosynthesis inhibitor that targets the enzyme catalyzing oxidative cleavage of 9-cis-epoxycarotenoids. To test the effect of abamine on plants other than Arabidopsis, it was applied to cress (Lepidium sativum) plants. Abamine enhanced radicle elongation in cress seeds, which could be due to a decrease in the ABA content of abamine-treated plants. Thus, it is possible to think that abamine should enable us to elucidate the functions of ABA in cells or plants and to find new mutants involved in ABA signaling.

Localization, ion channel regulation, and genetic interactions during abscisic acid signaling of the nuclear mRNA cap-binding protein, ABH1.

Abscisic acid (ABA) regulates developmental processes and abiotic stress responses in plants. We recently characterized a new Arabidopsis mutant, abh1, which shows ABA-hypersensitive regulation of seed germination, stomatal closing, and cytosolic calcium increases in guard cells (V. Hugouvieux, J.M. Kwak, J.I. Schroeder [2001] Cell 106: 477-487). ABH1 encodes the large subunit of a dimeric Arabidopsis mRNA cap-binding complex and in expression profiling experiments was shown to affect mRNA levels of a subset of genes. Here, we show that the dimeric ABH1 and AtCBP20 subunits are ubiquitously expressed. Whole-plant growth phenotypes of abh1 are described and properties of ABH1 in guard cells are further analyzed. Complemented abh1 lines expressing a green fluorescent protein-ABH1 fusion protein demonstrate that ABH1 mainly localizes in guard cell nuclei. Stomatal apertures were smaller in abh1 compared with wild type (WT) when plants were grown at 40% humidity, and similar at 95% humidity. Correlated with stomatal apertures from plants grown at 40% humidity, slow anion channel currents were enhanced and inward potassium channel currents were decreased in abh1 guard cells compared with WT. Gas exchange measurements showed similar primary humidity responses in abh1 and WT, which together with results from abh1/abi1-1 double-mutant analyses suggest that abh1 shows enhanced sensitivity to endogenous ABA. Double-mutant analyses of the ABA-hypersensitive signaling mutants, era1-2 and abh1, showed complex genetic interactions, suggesting that ABH1 and ERA1 do not modulate the same negative regulator in ABA signaling. Mutations in the RNA-binding protein sad1 showed hypersensitive ABA-induced stomatal closing, whereas hyl1 did not affect this response. These data provide evidence for the model that the mRNA-processing proteins ABH1 and SAD1 function as negative regulators in guard cell ABA signaling.

Gibberellins and seed development in maize. II. Gibberellin synthesis inhibition enhances abscisic acid signaling in cultured embryos.

Abscisic acid (ABA) is required for seed maturation in maize (Zea mays L.) and other plants. Gibberellins (GAs) are also present in developing maize embryos, and mutual antagonism of GAs and ABA appears to govern the choice between precocious germination or quiescence and maturation. Exogenous ABA can also induce quiescence and maturation in immature maize embryos in culture. To examine the role of GAs versus ABA in regulating maize embryo maturation, the effects of modulating GA levels were compared with those of ABA in embryos cultured at successive stages of development. The effects of GA synthesis inhibition or exogenous GA application differed markedly in embryos at different stages of development, indicating changes in both endogenous GA levels and in the capacity for GA synthesis as embryogenesis and maturation progress. In immature embryos, the inhibition of GA synthesis mimicked the effects of exogenous ABA, as shown by the suppression of germination, the acquisition of anthocyanin pigments, and the accumulation of a variety of maturation-phase mRNAs. We suggest that GA antagonizes ABA signaling in developing maize embryos, and that the changing hormone balance provides temporal control over the maturation phase.