Functional dissection of an abscisic acid (ABA)-inducible gene reveals two independent ABA-responsive complexes each containing a G-box and a novel cis-acting element.

To elucidate the mechanism by which abscisic acid (ABA) regulates gene expression, the promoter of the barley ABA-responsive HVA22 gene has been analyzed by both loss- and gain-of-function studies. Previous reports indicate that G-box sequences, which are present in genes responding to a variety of environmental and physiological cues, are involved in ABA response. However, our data suggest that G-box sequences are necessary but not sufficient for ABA response. Instead, an ABA response complex consisting of a G-box, namely, ABRE3 (GCCACGTACA), and a novel coupling element, CE1 (TGCCACCGG), is sufficient for high-level ABA induction, and replacement of either of these sequences abolishes ABA responsiveness. We suggest that the interaction between G-box sequences, such as ABRE3 in the HVA22 gene, and CE-type sequences determines the specificity in ABA-regulated gene expression. Our results also demonstrate that the ABA response complex is the minimal promoter unit governing high-level ABA induction; four copies of this 49-bp-long complex linked to a minimal promoter can confer more than 100-fold ABA-induced gene expression. In addition to ABA response complex 1, composed of ABRE3 and CE1, the HVA22 promoter contains another ABA response complex. The ABA responsiveness of this ABA response complex 2 relies on the interaction of G-box (ABRE2; CGCACGTGTC) with another yet unidentified coupling element. These two complexes contribute incrementally to the expression level of HVA22 in response to ABA.     

Dehydration-Stress-Regulated Transgene Expression in Stably Transformed Rice Plants

To confer abscisic acid (ABA) and/or stress-inducible gene expression, an ABA-response complex (ABRC1) from the barley (Hordeum vulgare L.) HVA22 gene was fused to four different lengths of the 5′ region from the rice (Oryza sativa L.) Act1 gene. Transient assay of β-glucuronidase (GUS) activity in barley aleurone cells shows that, coupled with ABRC1, the shortest minimal promoter (Act1–100P) gives both the greatest induction and the highest level of absolute activity following ABA treatment. Two plasmids with one or four copies of ABRC1 combined with the same Act1–100P and HVA22(I) of barley HVA22 were constructed and used for stable expression of uidA in transgenic rice plants. Three Southern blot-positive lines with the correct hybridization pattern for each construct were obtained. Northern analysis indicated that uidA expression is induced by ABA, water-deficit, and NaCl treatments. GUS activity assays in the transgenic plants confirmed that the induction of GUS activity varies from 3- to 8-fold with different treatments or in different rice tissues, and that transgenic rice plants harboring four copies of ABRC1 show 50% to 200% higher absolute GUS activity both before and after treatments than those with one copy of ABRC1.     

Isolation of carrot basic leucine zipper transcription factor using yeast one-hybrid screening

Abscisic acid (ABA) is involved in various physiological and developmental processes, including stress responses and seed maturation. Many ABA-regulated genes associated with these processes have been identified and analyzed. Previously, we identified 2 important elements in the promoter of the carrotDcECP31 gene: motif X (CACACGTGGG), which is like an ABA-responsive element (ABRE), and motif Y (CACACGTATC). Together, these are sufficient for embryo-specific ABA-inducible promoter activity. We also showed that motif X functions is an enhancerlike element and that motif Y participates in ABA responsiveness. In this study, we isolated the nuclear protein that interacts with motif Y of theDcECP31 promoter. We performed yeast one-hybrid screening using integrated motif Y as bait and isolated clones. Sequence analysis revealed that clone 22 included the carboxyl-terminal half of bZIP, which contains the basic and leucine zipper domains and binds to G-boxes containing the sequence ACGT. This result supports the hypothesis that carrot C-ABI3, a homologue of theArabidopsis ABI3 protein, functions as a coactivator that interacts with the G-box via protein-protein contacts and suggests that the complex controls the expression of theDcECP31 gene.     

The promoter-elements of some abiotic stress-inducible genes from cereals interact with a nuclear protein from tobacco

In this communication, we report the binding of abscisic acid responsive elements (ABREs) of rice Osem, namely motif A and motif B, with a cognate trans-acting factor present in the nuclear extract of tobacco leaf. The binding is specific as both the complexes were disrupted with an excess of homologous non-radioactive DNA like motif A or motif B themselves or with cis-elements of rice Rab16A, motif I (ABRE) and motif IIa (non-ACGT ABRE-like sequences). Four tandem repeats of ABRE from wheat Em (4X ABRE) or two tandem repeats of Em ABRE, plus two copies of coupling element (CE1) from barley HVA22 (2X ABRC), also showed specific complexes, that were competed out by an excess of homologous competitors like motif I, motif IIa, motif A, motif B, 4X ABRE and 2X ABRC, but not by the unrelated 4X DRE sequence. Elution of the protein from all the complexes showed a single 26 kDa polypeptide band. Introgression of two of the above synthetic promoters 4X ABRE and 2X ABRC, each fused with minimal promoter of cauliflower mosaic virus 35S (CaMV 35S), could induce the expression of the reporter gene β-glucuronidase (gus) in transgenic tobacco in response to high NaCl concentration, dehydration or abscisic acid, but not at the constitutive level, proving that they can be used as efficient stress-inducible promoters. Our work shows both in vivo and in vitro activity of the promoters from monocot genes in the model dicot plant tobacco.     

BPH1, a novel substrate receptor of CRL3, plays a repressive role in ABA signal transduction

Key message

BPH1 acts as a substrate receptor of CRL3 complex and negatively regulates ABA-mediated cellular responses. The study on its function provides information that helps further understand the relationship between ABA signaling and UPS.

Abstract

Abscisic acid (ABA) plays a crucial role in a variety of cellular processes, including seed dormancy, inhibition of seedling growth, and drought resistance in plants. Cullin3-RING E3 ligase (CRL3) complex is a type of multi-subunit E3 ligase, and BTB/POZ protein, a component of CRL3 complex, functions as a receptor to determine a specific substrate. To elucidate the CRL3 complex that participates in ABA-mediated cellular processes, we first investigated ABA-inducible BTB/POZ genes based on data from the AtGenExpress Visualization Tool (AVT). We then isolated an ABA-inducible gene encoding a potential CRL3 substrate receptor in Arabidopsis, BPH1 (BTB/POZ protein hypersensitive to ABA 1). The isolate gene has a BTB/POZ domain and a NPH3 domain within its N-terminal and C-terminal region, respectively. Yeast two-hybrid and co-immunoprecipitation assays showed that BPH1 physically interacted with cullin3a, a scaffold protein of CRL3, suggesting that it functions as an Arabidopsis CRL3 substrate receptor. The functional mutation of BPH1 caused delayed seed germination in response to ABA and enhanced sensitivity by NaCl and mannitol treatments as ABA-related stresses. Moreover, bph1 mutants exhibited enhanced stomatal closure under ABA application and reduced water loss when compared with wild-type, implying their enhanced tolerance to drought stress. Based on the information from microarray/AVT data and expression analysis of various ABA-inducible genes between wild-type and bph1 plants following ABA treatments, we concluded loss of BPH1 resulted in hyper-induction of a large portion of ABA-inducible genes in response to ABA. Taken together, these results show that BPH1 is negatively involved in ABA-mediated cellular events.

     

ACGT-containing abscisic acid response element (ABRE) and coupling element 3 (CE3) are functionally equivalent

ACGT-containing ABA response elements (ABREs) have been functionally identified in the promoters of various genes. In addition, single copies of ABRE have been found to require a cis-acting, coupling element to achieve ABA induction. A coupling element 3 (CE3) sequence, originally identified as such in the barley HVA1 promoter, is found approximately 30 bp downstream of motif A (ACGT-containing ABRE) in the promoter of the Osem gene. The relationship between these two elements was further defined by linker-scan analyses of a 55 bp fragment of the Osem promoter, which is sufficient for ABA-responsiveness and VP1 activation. The analyses revealed that both motif A and CE3 sequence were required not only for ABA-responsiveness but also for VP1 activation. Since the sequences of motif A and CE3 were found to be similar, motif-exchange experiments were carried out. The experiments demonstrated that motif A and CE3 were interchangeable by each other with respect to both ABA and VP1 regulation. In addition, both sequences were shown to be recognized by a VP1-interacting, ABA-responsive bZIP factor TRAB1. These results indicate that ACGT-containing ABREs and CE3 are functionally equivalent cis-acting elements. Furthermore, TRAB1 was shown to bind two other non-ACGT ABREs. Based on these results, all these ABREs including CE3 are proposed to be categorized into a single class of cis-acting elements.     

The stress- and abscisic acid-induced barley gene HVA22: developmental regulation and homologues in diverse organisms

Abscisic acid (ABA) induces the expression of a battery of genes in mediating plant responses to environmental stresses. Here we report one of the early ABA-inducible genes in barley (Hordeum vulgare L.), HVA22, which shares little homology with other ABA-responsive genes such as LEA (late embryogenesis-abundant) and RAB (responsive to ABA) genes. In grains, the expression of HVA22 gene appears to be correlated with the dormancy status. The level of HVA22 mRNA increases during grain development, and declines to an undetectable level within 12 h after imbibition of non-dormant grains. In contrast, the HVA22 mRNA level remains high in dormant grains even after five days of imbibition. Treatment of dormant grains with gibberellin (GA) effectively breaks dormancy with a concomitant decline of the level of HVA22 mRNA. The expression of HVA22 appears to be tissue-specific with the level of its mRNA readily detectable in aleurone layers and embryos, yet undetectable in the starchy endosperm. The expression of HVA22 in vegetative tissues can be induced by ABA and environmental stresses, such as cold and drought. Apparent homologues of this barley gene are found in phylogenetically divergent eukaryotic organisms, including cereals, Arabidopsis, Caenorhabitis elegans, man, mouse and yeast, but not in any prokaryotes. Interestingly, similar to barley HVA22, the yeast homologue is also stress-inducible. These observations suggest that the HVA22 and its homologues encode a highly conserved stress-inducible protein which may play an important role in protecting cells from damage under stress conditions in many eukaryotic organisms.     

Calcium-dependent induction of novel proteins by abscisic acid in wheat aleurone tissue of different developmental stages

Aleurone tissue of mature wheat (Triticum aestivum L. cv. Sappo) grains make novel polypeptides in response to abscisic acid (ABA), but only in the presence of Ca²⁺. Effects of ABA plus Ca²⁺ include up- and down-modulation of other polypeptides. The ABA-induced polypeptides appear not to be the 21-kilodalton (kDa) amylase inhibitor which has been reported to be ABA-inducible in barley.Aleurone tissue from developing grains of different ages failed to respond to ABA plus Ca²⁺ in any way. Endogenous ABA levels were determined by monoclonal radioimmunoassay in developing, mature, and sensitised developing tissues. The ABA level rose to a maximum at 35 days post anthesis but was not detectable in mature cells. Developing layers sensitised to gibberellic acid (GA) showed decreased levels of ABA, similar to those in mature tissue, concurrent with acquired responsiveness to GA in respect of its induction of -amylase. However, these sensitised cells still remained non-responsive to added ABA in terms of modulation of polypeptide pattern, though they did respond to ABA in the blocking of GA-induced -amylase production. The role of protein phosphorylation in signal transduction was examined. The implications of these findings are discussed with reference to the role of ABA in developing and mature aleurone cells.Abbreviations ABA Abscisic acid - dpa days post anthesis - GA₃ Gibberellic acid - kDa kilodalton - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis - TCA trichloroacetic acid     

Abscisic acid promotes novel DNA-binding activity to a desiccation-related promoter of Craterostigma plantagineum

Abscisic acid-treated callus of the resurrection plant Craterostigma plantagineum tolerates extreme desiccation. Nuclear proteins from tolerant callus bind specific sequence elements in the promoter region of the ABA and desiccation-inducible CDeT27–45 gene. One specific region of the promoter, which is protected from DNAse I treatment by DNA-binding activities, is different from previously reported ABA response elements. Four complexes of nuclear proteins and this DNA region are detected by electrophoretic mobility shift assay: two of these complexes (I and II) are readily detectable in untreated samples and are increased by ABA treatment while two other complexes (III and IV) accumulate only following ABA treatment and are prevented from accumulating by protein synthesis inhibitors. When a fragment containing the novel binding site is deleted from the wild-type promoter the ABA responsiveness of the promoter is removed; however, gain of function experiments using synthetic promoters in a protoplast transient assay suggest that besides the binding site other promoter elements are required. A second region of the promoter, containing the sequence element ACGT which is found in abscisic acid response elements, is also bound by nuclear proteins. The level of this second binding activity is similar in both untreated and ABA-treated cells and promoter/reporter gene constructs which contain only the four ACGT elements of the CDeT27–45 promoter are not ABA responsive in a C. plantagineum transient assay system