Overexpression of ABI5 Binding Proteins Suppresses Inhibition of Germination Due to Overaccumulation of DELLA Proteins.

Abscisic acid (ABA) and gibberellic acid (GA) antagonistically regulate many aspects of plant growth, including seed dormancy and germination. The effects of these hormones are mediated by a complex network of positive and negative regulators of transcription. The DELLA family of proteins repress GA response, and can promote an ABA response via interactions with numerous regulators, including the ABA-insensitive (ABI) transcription factors. The AFP family of ABI5 binding proteins are repressors of the ABA response. This study tested the hypothesis that the AFPs also interact antagonistically with DELLA proteins. Members of these protein families interacted weakly in yeast two-hybrid and bimolecular fluorescence complementation studies. Overexpression of AFPs in sleepy1, a mutant that over-accumulates DELLA proteins, suppressed DELLA-induced overaccumulation of storage proteins, hyperdormancy and hypersensitivity to ABA, but did not alter the dwarf phenotype of the mutant. The interaction appeared to reflect additive effects of the AFPs and DELLAs, consistent with action in convergent pathways.

ABI5-binding proteins (AFPs) alter transcription of ABA-induced genes via a variety of interactions with chromatin modifiers.

KEY MESSAGE: Overexpression of ABI5/ABF binding proteins (AFPs) results in extreme ABA resistance of seeds via multiple mechanisms repressing ABA response, including interactions with histone deacetylases and the co-repressor TOPLESS. Several ABI5/ABF binding proteins (AFPs) inhibit ABA response, resulting in extreme ABA resistance in transgenic Arabidopsis overexpression lines, but their mechanism of action has remained obscure. By analogy to the related Novel Interactor of JAZ (NINJA) protein, it was suggested that the AFPs interact with the co-repressor TOPLESS to inhibit ABA-regulated gene expression. This study shows that the AFPs that inhibit ABA response have intrinsic repressor activity in a heterologous system, which does not depend on the domain involved in the interaction with TOPLESS. This domain is also not essential for repressing ABA response in transgenic plants, but does contribute to stronger ABA resistance. Additional interactions between some AFPs and histone deacetylase subunits were observed in yeast two-hybrid and bimolecular fluorescence assays, consistent with a more direct mechanism of AFP-mediated repression of gene expression. Chemical inhibition of histone deacetylase activity by trichostatin A suppressed AFP effects on a small fraction of the ABI5-regulated genes tested. Collectively, these results suggest that the AFPs participate in multiple mechanisms modulating ABA response, including both TOPLESS-dependent and -independent chromatin modification.

Direct targets of the transcription factors ABA-Insensitive(ABI)4 and ABI5 reveal synergistic action by ABI4 and several bZIP ABA response factors.

The plant hormone abscisic acid (ABA) is a key regulator of seed development. In addition to promoting seed maturation, ABA inhibits seed germination and seedling growth. Many components involved in ABA response have been identified, including the transcription factors ABA insensitive (ABI)4 and ABI5. The genes encoding these factors are expressed predominantly in developing and mature seeds, and are positive regulators of ABA mediated inhibition of seed germination and growth. The direct effects of ABI4 and ABI5 in ABA response remain largely undefined. To address this question, plants over-expressing ABI4 or ABI5 were used to allow identification of direct transcriptional targets. Ectopically expressed ABI4 and ABI5 conferred ABA-dependent induction of slightly over 100 genes in 11 day old plants. In addition to effector genes involved in seed maturation and reserve storage, several signaling proteins and transcription factors were identified as targets of ABI4 and/or ABI5. Although only 12% of the ABA- and ABI-dependent transcriptional targets were induced by both ABI factors in 11 day old plants, 40% of those normally expressed in seeds had reduced transcript levels in both abi4 and abi5 mutants. Surprisingly, many of the ABI4 transcriptional targets do not contain the previously characterized ABI4 binding motifs, the CE1 or S box, in their promoters, but some of these interact with ABI4 in electrophoretic mobility shift assays, suggesting that sequence recognition by ABI4 may be more flexible than known canonical sequences. Yeast one-hybrid assays demonstrated synergistic action of ABI4 with ABI5 or related bZIP factors in regulating these promoters, and mutant analyses showed that ABI4 and these bZIPs share some functions in plants.

Redundant and distinct functions of the ABA response loci ABA-INSENSITIVE(ABI)5 and ABRE-BINDING FACTOR (ABF)3.

Abscisic acid-responsive gene expression is regulated by numerous transcription factors, including a subgroup of basic leucine zipper factors that bind to the conserved cis-acting sequences known as ABA-responsive elements. Although one of these factors, ABA-insensitive 5 (ABI5), was identified genetically, the paucity of genetic data for the other family members has left it unclear whether they perform unique functions or act redundantly to ABI5 or each other. To test for potential redundancy with ABI5, we identified the family members with most similar effects and interactions in transient expression systems (ABF3 and ABF1), then characterized loss-of-function lines for those loci. The abf1 and abf3 monogenic mutant lines had at most minimal effects on germination or seed-specific gene expression, but the enhanced ABA- and stress-resistance of abf3 abi5 double mutants revealed redundant action of these genes in multiple stress responses of seeds and seedlings. Although ABI5, ABF3, and ABF1 have some overlapping effects, they appear to antagonistically regulate each other's expression at specific stages. Consequently, loss of any one factor may be partially compensated by increased expression of other family members.

The Arabidopsis thaliana ABSCISIC ACID-INSENSITIVE8 encodes a novel protein mediating abscisic acid and sugar responses essential for growth.

Abscisic acid (ABA) regulates many aspects of plant growth and development, yet many ABA response mutants present only subtle phenotypic defects, especially in the absence of stress. By contrast, the ABA-insensitive8 (abi8) mutant, isolated on the basis of ABA-resistant germination, also displays severely stunted growth, defective stomatal regulation, altered ABA-responsive gene expression, delayed flowering, and male sterility. The stunted growth of the mutant is not rescued by gibberellin, brassinosteroid, or indoleacetic acid application and is not attributable to excessive ethylene response, but supplementing the medium with Glc improves viability and root growth. In addition to exhibiting Glc-dependent growth, reflecting decreased expression of sugar-mobilizing enzymes, abi8 mutants are resistant to Glc levels that induce developmental arrest of wild-type seedlings. Studies of genetic interactions demonstrate that ABA hypersensitivity conferred by the ABA-hypersensitive1 mutation or overexpression of ABI3 or ABI5 does not suppress the dwarfing and Glc dependence caused by abi8 but partially suppresses ABA-resistant germination. By contrast, the ABA-resistant germination of abi8 is epistatic to the hypersensitivity caused by ethylene-insensitive2 (ein2) and ein3 mutations, yet ABI8 appears to act in a distinct Glc response pathway from these EIN loci. ABI8 encodes a protein with no domains of known function but belongs to a small plant-specific protein family. Database searches indicate that it is allelic to two dwarf mutants, elongation defective1 and kobito1, previously shown to disrupt cell elongation, cellulose synthesis, vascular differentiation, and root meristem maintenance. The cell wall defects appear to be a secondary effect of the mutations because Glc treatment restores root growth and vascular differentiation but not cell elongation. Although the ABI8 transcript accumulates in all tested plant organs in both wild-type and ABA response mutants, an ABI8-beta-glucuronidase fusion protein is localized primarily to the elongation zone of roots, suggesting substantial post-transcriptional regulation of ABI8 accumulation. This localization pattern is sufficient to complement the mutation, indicating that ABI8 acts either at very low concentrations or over long distances within the plant body.

Regulatory networks in seeds integrating developmental, abscisic acid, sugar, and light signaling.

Progression through embryogenesis and the transition to germination is subject to regulation by many transcription factors, including those encoded by the Arabidopsis LEC1 (LEAFY COTYLEDON1), FUS3 (FUSCA3), and abscisic acid-insensitive (ABI) ABI3, ABI4, and ABI5 loci. To determine whether the ABI4, ABI5, LEC1, and FUS3 loci interact or act independently, we analyzed abi fus3 and abi lec1 double mutants. Our results show that both ABI4 and ABI5 interact genetically with both LEC1 and FUS3 in controlling pigment accumulation, suppression of vivipary, germination sensitivity to abscisic acid, gene expression during mid- and late embryogenesis, sugar metabolism, sensitivity to sugar, and etiolated growth. However, the relative strengths of the observed interactions vary among responses and may even be antagonistic. Furthermore, the interactions reveal cryptic effects of individual loci that are not detectable by analyses of single mutants. Despite these strong genetic interactions, but consistent with the disparities in peak expression of these loci, none of the ABI transcription factors appear to interact directly with either FUS3 or LEC1 in a yeast (Saccharomyces cerevisiae) two-hybrid assay system.

Regulation and role of the Arabidopsis abscisic acid-insensitive 5 gene in abscisic acid, sugar, and stress response.

Abscisic acid (ABA) and stress response from late embryonic growth through early seedling development is regulated by a signaling network that includes the Arabidopsis ABA-insensitive (ABI)5 gene, which encodes a basic leucine zipper transcription factor. We have characterized genetic, developmental, and environmental regulation of ABI5 expression. Although expressed most strongly in seeds, the ABI5 promoter is also active in vegetative and floral tissue. Vegetative expression is strongly induced by ABA, and weakly by stress treatments during a limited developmental window up to approximately 2 d post-stratification, but ABA and some stresses can induce expression in specific tissues at later stages. ABI5 expression is autoregulated in transgenic plants and yeast (Saccharomyces cerevisiae), and stress response appears to involve ABI5-dependent and -independent mechanisms. To determine whether ABI5 is necessary and/or sufficient for ABA or stress response, we assayed the effects of increased ABI5 expression on growth and gene expression. Although overexpression of ABI5 confers hypersensitivity to ABA and sugar, as previously described for ABI4 and ABI3 overexpression lines, it has relatively limited effects on enhancing ABA-responsive gene expression. Comparison of expression of eight ABI5-homologous genes shows overlapping regulation by ABI3, ABI4, and ABI5, suggestive of a combinatorial network involving positive and negative regulatory interactions.