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The phytohormone abscisic acid (ABA) plays an essential role in adaptive stress responses. The hormone regulates, among others, the expression of numerous stress-responsive genes. From various promoter analyses, ABA-responsive elements (ABREs) have been determined and a number of ABRE binding factors have been isolated, although their in vivo roles are not known. Here we report that the ABRE binding factors ABF3 and ABF4 function in ABA signaling. The constitutive overexpression of ABF3 or ABF4 in Arabidopsis resulted in ABA hypersensitivity and other ABA-associated phenotypes. In addition, the transgenic plants exhibited reduced transpiration and enhanced drought tolerance. At the molecular level, altered expression of ABA/stress-regulated genes was observed. Furthermore, the temporal and spatial expression patterns of ABF3 and ABF4 were consistent with their suggested roles. Thus, our results provide strong in vivo evidence that ABF3 and ABF4 mediate stress-responsive ABA signaling. INTRODUCTIONBeing sessile, plants have the capability to adapt to adverse environmental conditions such as drought, cold, and high salt. Under these stress conditions, the plant hormone abscisic acid (ABA) level increases in vegetative tissues, triggering adaptive responses that are essential for their survival and productivity (Zeevaart and Creelman, 1988;Leung and Giraudat, 1998). Under water deficit conditions, for example, ABA induces stomatal closure, minimizing water loss through transpiration. The ABA-controlled process is vital for plant survival, and ABA-deficient and ABA-responsive mutants are susceptible to water stress. On the other hand, high levels of ABA inhibit overall plant growth (Himmelbach et al., 1998).Underlying the ABA-mediated stress responses is the transcriptional regulation of stress-responsive gene expression Busk and Pages, 1998). Numerous genes have been reported that are upregulated under stress conditions in vegetative tissues (Ingram and Bartels, 1996;Shinozaki and Yamaguchi-Shinozaki, 1997). These include a class of genes known as LEA (for LATE EMBRYOGENESIS ABUN-DANT ) genes, which are expressed abundantly in developing seed under normal conditions, osmolyte biosynthetic genes, and genes of general cellular metabolism. In general, the gene products are considered to have protective or adaptive roles under stress conditions. In addition, the expression of many regulatory genes, including various kinase/phosphatase and transcription factor genes, also is induced by abiotic stresses. Not all stress-inducible genes are regulated by ABA. However, a large number of them also are responsive to exogenous ABA, and in many cases, their induction is impaired in ABA-deficient mutants. Meanwhile, the expression of some genes, such as rbcS and CAB genes, is suppressed by ABA and stress (Bartholomew et al., 1991;Wang et al., 1996; Weatherwax et al., 1996).ABA-responsive elements (ABREs) that control ABA-and/ or stress-responsive gene expression have been determined by numerous studies , and their putative cogna...

The phytohormone abscisic acid (ABA) regulates stress-responsive gene expression during vegetative growth. The ABA regulation of many genes is mediated by a subfamily of basic leucine zipper class transcription factors referred to as ABFs (i.e. ABF1-ABF4), whose transcriptional activity is induced by ABA. Here we show that a calcium-dependent protein kinase is involved in the ABA-dependent activation process. We carried out yeast two-hybrid screens to identify regulatory components of ABF4 function and isolated AtCPK32 as an ABF4-interacting protein. AtCPK32 has autophosphorylation activity and can phosphorylate ABF4 in vitro. Mutational analysis indicated that serine-110 of ABF4, which is highly conserved among ABF family members, may be phosphorylated by AtCPK32. The serine-110 residue is essential for ABF4-AtCPK32 interaction, and transient expression assay revealed that it is also required for the normal transcriptional function of ABF4. The expression patterns and subcellular localization of AtCPK32 are similar to those of ABF4. Furthermore, its overexpression affects both ABA sensitivity and the expression of a number of ABF4-regulated genes. Together, our data demonstrate that AtCPK32 is an ABA signaling component that regulates the ABA-responsive gene expression via ABF4.

ABF2 is a basic leucine zipper protein that regulates abscisic acid (ABA)-dependent stress-responsive gene expression. We carried out yeast two-hybrid screens to isolate genes encoding ABF2-interacting proteins in Arabidopsis (Arabidopsis thaliana). Analysis of the resulting positive clones revealed that two of them encode an AP2 domain protein, which is the same as AtERF48/DREB2C. This protein, which will be referred to as DREB2C, could bind C-repeat/dehydration response element in vitro and possesses transcriptional activity. To determine its function, we generated DREB2C overexpression lines and investigated their phenotypes. The transgenic plants were ABA hypersensitive during germination and seedling establishment stages, whereas primary root elongation of seedlings was ABA insensitive, suggesting developmental stage dependence of DREB2C function. The DREB2C overexpression lines also displayed altered stress response; whereas the plants were dehydration sensitive, they were freezing and heat tolerant. We further show that other AP2 domain proteins, DREB1A and DREB2A, interact with ABF2 and that other ABF family members, ABF3 and ABF4, interact with DREB2C. Previously, others demonstrated that ABF and DREB family members cooperate to activate the transcription of an ABA-responsive gene. Our result implies that the cooperation of the two classes of transcription factors may involve physical interaction.

Arabidopsis (Arabidopsis thaliana) genome contains more than 90 armadillo (arm) repeat proteins. However, their functions are largely unknown. Here, we report that an Arabidopsis arm repeat protein is involved in abscisic acid (ABA) response. We carried out two-hybrid screens to identify signaling components that modulate ABA-responsive gene expression. Employing a transcription factor, ABF2, which controls the ABA-dependent gene expression via the G-box type ABA-responsive elements, we isolated an arm repeat protein. The ABF2-interacting protein, designated as ARIA (arm repeat protein interacting with ABF2), has another conserved sequence motif, BTB/POZ (broad complex, tramtrak, and bric-a-brac/poxvirus and zinc finger) domain, in the C-terminal region. The physiological relevance of ABF2-ARIA interaction was supported by their similar expression patterns and similar subcellular localization. Plants overexpressing ARIA are hypersensitive to ABA and high osmolarity during germination and insensitive to salt during subsequent seedling growth. By contrast, an ARIA knockout mutant exhibits ABA and glucose insensitivities. Changes in the expression levels of several ABF2-regulated genes were also observed in ARIA overexpression lines, indicating that ARIA modulates the transcriptional activity of ABF2. Together, our data indicate that ARIA is a positive regulator of ABA response.Armadillo (arm) repeat is a 42-amino acid proteinprotein interaction motif (Peifer et al., 1994;Hatzfeld, 1999;Andrade et al., 2001). The repeat was first identified in the Drosophila segment polarity gene armadillo (Riggleman et al., 1989) and since then in many eukaryotic proteins involved in cell signaling or cellular architecture. Armadillo and its vertebrate homolog b-catenin are components of the Wingless and the Wnt signaling pathways, which determine the patterning of Drosophila embryo body segments and vertebrate cell fates, respectively (Polakis, 2000). When triggered by the Wingless or Wnt growth factor signal, otherwise unstable Armadillo/b-catenin becomes stabilized, translocates into the nucleus, and together with the TCF/LEF subfamily of transcription factors, activates the Wingless/Wnt target genes. b-Catenin also plays a structural role in cell-cell adhesion by linking the transmembrane adhesion molecules cadherins to actin cytoskeleton.Pfam (http://www.sanger.ac.uk/Software/Pfam/) and SMART (http://smart.embl-heidelberg.de/) protein databases enlist more than 90 Arabidopsis (Arabidopsis thaliana) arm repeat proteins. Based on their sequence homology, these proteins can be grouped into several different subfamilies such as impotin-a, kinesin, and U-box protein families (Coates, 2003). However, the functions of the Arabidopsis and other plant arm repeat proteins have not been characterized in detail except those of ARC1 and PHOR1. ARC1 interacts with an S-locus receptor kinase of Brassica (Gu et al., 1998) and has been demonstrated to be a positive regulator of the self-incompatibility response (Stone et al., 1999). A recent ...