ABA-responsive Element Binding Research Articles

TaABF1, ABA response element binding factor 1, is related to seed dormancy and ABA sensitivity in wheat ( Triticum aestivum L.) seeds

A seed-specific bZIP type transcription factor involved in abscisic acid (ABA) signal transduction of developing wheat ( Triticum aestivum L.) seeds is TaABF1, ABA response element binding factor 1. The expression of TaABF1 was examined in wheat cultivars possessing different levels of seed dormancy and ABA sensitivity at 40–50 days after pollination. Zenkoujikomugi, Norin61, and OW104 were dormant cultivars showing high sensitivity to ABA. Chihokukomugi, RL4137, and Chinese Spring, all non-dormant cultivars, show low sensitivity to ABA. Tamaizumi, AUS1408, and Kitakei-1354 represent intermediate levels of seed dormancy and sensitivity to ABA. The relation between seed dormancy and ABA sensitivity is high ( r = 0.89) in these cultivars. Relative amounts of transcripts of TaABF1 in embryos revealed high coefficients of correlation to seed dormancy ( r = 0.78) and ABA sensitivity ( r = 0.86). These results suggest that TaABF1 plays a role in the regulation of seed dormancy and ABA sensitivity in wheat.

Two Closely Related Subclass II SnRK2 Protein Kinases Cooperatively Regulate Drought-Inducible Gene Expression

The subclass III group of SNF1-related protein kinase 2 (SnRK2) members is known to play an important role in ABA and osmotic stress signaling in Arabidopsis; however, the roles of other subclasses remain elusive. Here, we established a double mutant of SRK2C/SnR2.8 and SRK2F/SnRK2.7 to investigate the functions of subclass II SnRK2s. Microarray analysis suggested that subclass II SnRK2s regulate some drought-responsive genes involving ABA-responsive element binding transcription factors (AREB/ABF) and their targets, and quantitative reverse transcription- PCR confirmed that those genes were down-regulated significantly in srk2cf. This study indicates that subclass II SnRK2s also play important roles in drought stress signaling in Arabidopsis.

A tomato bZIP transcription factor, SlAREB, is involved in water deficit and salt stress response

Abiotic stresses such as cold, water deficit, and salt stresses severely reduce crop productivity. Tomato (Solanum lycopersicum) is an important economic crop; however, not much is known about its stress responses. To gain insight into stress-responsive gene regulation in tomato plants, we identified transcription factors from a tomato cDNA microarray. An ABA-responsive element binding protein (AREB) was identified and named SlAREB. In tomato protoplasts, SlAREB transiently transactivated luciferase reporter gene expression driven by AtRD29A (responsive to dehydration) and SlLAP (leucine aminopeptidase) promoters with exogenous ABA application, which was suppressed by the kinase inhibitor staurosporine, indicating that an ABA-dependent post-translational modification is required for the transactivation ability of SlAREB protein. Electrophoretic mobility shift assays showed that the recombinant DNA-binding domain of SlAREB protein is able to bind AtRD29A and SlLAP promoter regions. Constitutively expressed SlAREB increased tolerance to water deficit and high salinity stresses in both Arabidopsis and tomato plants, which maintained PSII and membrane integrities as well as water content in plant bodies. Overproduction of SlAREB in Arabidopsis thaliana and tomato plants regulated stress-related genes AtRD29A, AtCOR47, and SlCI7-like dehydrin under ABA and abiotic stress treatments. Taken together, these results show that SlAREB functions to regulate some stress-responsive genes and that its overproduction improves plant tolerance to water deficit and salt stress.

Comparative analysis of phytohormone-responsive phosphoproteins in Arabidopsis thaliana using TiO2-phosphopeptide enrichment and mass accuracy precursor alignment

Protein phosphorylation/dephosphorylation is a central post-translational modification in plant hormone signaling, but little is known about its extent and function. Although pertinent protein kinases and phosphatases have been predicted and identified for a variety of hormone responses, classical biochemical approaches have so far revealed only a few candidate proteins and even fewer phosphorylation sites. Here we performed a global quantitative analysis of the Arabidopsis phosphoproteome in response to a time course of treatments with various plant hormones using phosphopeptide enrichment and subsequent mass accuracy precursor alignment (MAPA). The use of three time points, 1, 3 and 6 h, in combination with five phytohormone treatments, abscisic acid (ABA), indole-3-acetic acid (IAA), gibberellic acid (GA), jasmonic acid (JA) and kinetin, resulted in 324,000 precursor ions from 54 LC-Orbitrap-MS analyses quantified and aligned in a data matrix with the dimension of 6000 x 54 using the ProtMax algorithm. To dissect the phytohormone responses, multivariate principal/independent components analysis was performed. In total, 152 phosphopeptides were identified as differentially regulated; these phosphopeptides are involved in a wide variety of signaling pathways. New phosphorylation sites were identified for ABA response element binding factors that showed a specific increase in response to ABA. New phosphorylation sites were also found for RLKs and auxin transporters. We found that different hormones regulate distinct amino acid residues of members of the same protein families. In contrast, tyrosine phosphorylation of the G alpha subunit appeared to be a common response for multiple hormones, demonstrating global cross-talk among hormone signaling pathways.

The bZIP transcription factor OsABF1 is an ABA responsive element binding factor that enhances abiotic stress signaling in rice

A number of basic leucine zipper (bZIP) transcription factors are known to function in stress signaling in plants but few have thus far been functionally characterized in rice. In our current study in rice, we have newly isolated and characterized the OsABF1 (Oryza sativa ABA responsive element binding factor 1) gene that encodes a bZIP transcription factor. Its expression in seedling shoots and roots was found to be induced by various abiotic stress treatments such as anoxia, salinity, drought, oxidative stress, cold and abscisic acid (ABA). Subcellular localization analysis in maize protoplasts using GFP fusion vectors indicated that OsABF1 is a nuclear protein. In a yeast experiment, OsABF1 was shown to bind to ABA responsive elements (ABREs) and its N-terminal region was necessary to transactivate the downstream reporter gene. The homozygous T-DNA insertional mutants Osabf1-1 and Osabf1-2 were more sensitive in response to drought and salinity treatments than wild type plants. Furthermore, the upregulated expression of some ABA/stress-regulated genes in response to ABA treatment was suppressed in these Osabf1 mutants. Our current results thus suggest that OsABF1 is involved in abiotic stress responses and ABA signaling in rice.

The interface between metabolic and stress signalling.

It is becoming increasingly clear that stress and metabolic signalling networks interact and that this interaction is important in plant responses to herbivory, pathogen attack, drought, cold, heat and osmotic stresses including salinity. At the interface between these two major signalling systems are the hormone abscisic acid (ABA) and signalling factors including protein kinases and transcription factors. This briefing reviews links between ABA, stress and sugar signalling, focusing on the roles of sucrose non-fermenting-1-related protein kinases (SnRKs), SnRK1-activating protein kinases (SnAKs), calcium-dependent protein kinases (CDPKs) and ABA response element binding proteins (AREBPs, which are transcription factors). Links between stress and nitrogen / amino acid signalling are also described, including the roles of a protein kinase called general control non-derepressible (GCN)-2 in regulating protein synthesis through phosphorylation of the alpha-subunit of translation initiation factor-2 (eIF2alpha) in response not only to decreases in amino acid levels but also to a range of stresses. Evidence of a link between sugar and amino acid signalling is explored, with nitrate reductase being a target for regulation by both SnRK1 and GCN2 through different mechanisms; possible links between SnRK1 and GCN2 via a pathway including the protein kinase target of rapamycin (TOR)-1 are described. The significance of these interactions to the concept of signalling networks as opposed to simple cascades and pathways, and the importance of the subject in the context of the predicted increase in severity and range of stresses that plants will have to withstand as a result of global climate change are discussed.

Arabidopsis mutant deficient in 3 abscisic acid-activated protein kinases reveals critical roles in growth, reproduction, and stress

Abscisic acid (ABA) is an important phytohormone regulating seed dormancy, germination, seedling growth, and plant transpiration. We report here an Arabidopsis triple mutant that is disrupted in 3 SNF1-related protein kinase subfamily 2 (SnRK2s) and nearly completely insensitive to ABA. These SnRK2s, SnRK2.2, SnRK2.3, and SnRK2.6 (also known as OST1), are activated by ABA and can phosphorylate the ABA-responsive element binding factor family of b-ZIP transcription factors, which are important for the activation of ABA-responsive genes. Although stomatal regulation of snrk2.6 and seed germination and seedling growth of the snrk2.2/2.3 double mutant are insensitive to ABA, ABA responses are still present in these mutants, and the growth and reproduction of these mutants are not very different from those of the WT. In contrast, the snrk2.2/2.3/2.6 triple mutant grows poorly and produces few seeds. The triple mutant plants lose water extremely fast when ambient humidity is not high. Even on 50 muM ABA, the triple mutant can germinate and grow, whereas the most insensitive known mutants cannot develop on 10 muM ABA. In-gel kinase assays showed that all ABA-activated protein kinase activities are eliminated in the triple mutant. Also, the expression of ABA-induced genes examined is completely blocked in the triple mutant. These results demonstrate that the protein kinases SnRK2.2, SnRK2.3, and SnRK2.6 have redundant functions, and suggest that ABA signaling is critical for plant growth and reproduction.

Dual role for 14‐3‐3 proteins and ABF transcription factors in gibberellic acid and abscisic acid signalling in barley (Hordeum vulgare) aleurone cells

The balance of gibberellins [gibberellic acid (GA)] and abscisic acid (ABA) is a determining factor during transition of embryogenesis and seed germination. Recently, we showed that 14-3-3 proteins are important in ABA signalling in barley aleurone cells. Using 14-3-3 RNAi constructs in the barley aleurone transient expression system, we demonstrate here that silencing of each 14-3-3 isoform suppresses GA induction of the alpha-amylase gene. 14-3-3 Proteins interact with ABA-responsive element (ABRE) binding factors HvABF1, 2 and 3, and here we show that these transcription factors also interact with the ABA-responsive kinase PKABA1, a kinase that mediates cross-talk between the GA and ABA pathway. ABF1 and ABF2 have a function in both signalling pathways as: (1) ectopic expression of wild-type ABF1 and mutant ABF2, lacking the 14-3-3 interaction domain, transactivates the ABA inducible HVA1 gene; and (2) GA induction of the alpha-amylase gene is repressed by ectopic expression of wild-type ABF1 and 2. Mutant ABF1 and 2 were still effective repressors of GA signalling. In summary, our data provide evidence that 14-3-3 proteins and members of the ABF transcription factor family have a regulatory function in the GA pathway and suggest that PKABA1 and ABF transcription factors are cross-talk intermediates in ABA and GA signalling.

Characterization of promoter elements required for expression and induction by sucrose of the Arabidopsis COX5b-1 nuclear gene, encoding the zinc-binding subunit of cytochrome c oxidase

Arabidopsis COX5b-1 encodes an isoform of the zinc binding subunit 5b of mitochondrial cytochrome c oxidase. A promoter region required for expression and induction by sucrose of this gene was analyzed using plants stably transformed with mutagenized promoter fragments fused to the gus reporter gene. Promoter dependent expression is absolutely dependent on a G-box present at -228 from the translation start site. This element interacts in vitro and in vivo with transcription factors from the bZip family, preferentially with the abscisic acid-responsive element binding factor AREB2/ABF4. A region located upstream of the G-box (-333/-259) contains elements with the core sequence ATCATT and distalB-like sequences (CCACTTG) that are required for expression in vegetative tissues. These sequences bind different sets of proteins present in plant nuclear extracts and participate in induction by sucrose (ATCATT) and abscisic acid (distalB) of the COX5b-1 promoter. We propose that the COX5b-1 promoter has acquired novel regulatory mechanisms during evolution after gene duplication. These novel mechanisms have allowed the diversification of expression patterns, but also the conservation of some responses that, as induction by sucrose, are shared by COX5b-1 and other genes encoding components of the mitochondrial respiratory chain. Conservation of these responses may be a pre-requisite for the successful incorporation of new regulatory elements in this class of genes.

Arabidopsis sucrose non‐fermenting‐1‐related protein kinase‐1 and calcium‐dependent protein kinase phosphorylate conserved target sites in ABA response element binding proteins

AbstractEvidence is provided that plant transcription factors of the ABA response element binding protein (AREBP) class are phosphorylated by sucrose non‐fermenting‐1‐related protein kinase‐1 (SnRK1) and a calcium‐dependent protein kinase at highly conserved target sites. Two target sites for SnRK1 were identified in AREBPs using a specially developed motif search pipeline. Peptides containing the AREBP sites were phosphorylated by purified SnRK1 in vitro and by calcium‐dependent and calcium‐independent activities present in soluble protein extracted from Arabidopsis seedlings grown in liquid culture. Most (69–77%) of the calcium‐independent phosphorylation of these peptides was removed by immunoprecipitation using anti‐SnRK1 antisera, linking this activity unambiguously to SnRK1. It is possible that the protein kinase responsible for the calcium‐dependent activity was SnRK3, a protein kinase that is related to SnRK1 and which has similar requirements for target site recognition. However, the involvement of other calcium‐dependent protein kinases cannot be ruled out.

Positive role of a wheat HvABI5 ortholog in abiotic stress response of seedlings

ABA-responsive element binding protein (AREB) and ABA-responsive element binding factor (ABF), members of the basic region/leucine zipper (bZIP)-type protein family, act as major transcription factors in ABA-responsive gene expression under abiotic stress conditions in Arabidopsis. Barley HvABI5 and rice transcription factor responsible for ABA regulation 1 (TRAB1) are homologues of AREB/ABF and are expressed in drought- and ABA-treated seedlings. However, no direct evidence has shown an association of an AREB/ABF-type transcription factor with stress tolerance in cereals. To understand the molecular basis of abiotic stress tolerance through a cereal AREB/ABF-type transcription factor, a wheat HvABI5 ortholog, Wabi5, was isolated and characterized. Wabi5 expression was activated by low temperature, drought and exogenous ABA treatment, and its expression pattern differed between two wheat accessions with distinct levels of stress tolerance and ABA sensitivity. Wabi5-expressing transgenic tobacco plants showed a significant increase in tolerance to abiotic stresses such as freezing, osmotic and salt stresses and a hypersensitivity to exogenous ABA in the seedling stage compared with wild-type plants. Expression of a GUS reporter gene under the control of promoters of three wheat cold-responsive/late embryogenesis abundant (Cor/Lea) genes, Wdhn13, Wrab18 and Wrab19, was enhanced by ectopic Wabi5 expression in wheat callus and tobacco plants. These results clearly indicated that WABI5 functions as a transcriptional regulator of the Cor/Lea genes in multiple abiotic stress responses in common wheat.

The wheat PKABA1-interacting factor TaABF1 mediates both abscisic acid-suppressed and abscisic acid-induced gene expression in bombarded aleurone cells

To investigate the crosstalk of abscisic acid (ABA) and gibberellin (GA) signaling in wheat (Triticum aestivum), we have focused on the transcription factor TaABF1. TaABF1 (a member of the ABA response element binding factor family) physically interacts with PKABA1, a signaling component in the ABA-suppression of GA-induced gene expression in cereal grains. Constitutive expression of TaABF1 in aleurone cells of imbibing grains completely eliminated GA-induced expression from the Amy32b promoter. In addition to its effect on Amy32b, TaABF1 strongly stimulated expression from the ABA-inducible HVA1 and HVA22 promoters. Overexpression of TaABF1 fully substituted for exogenous ABA in the induction of these two promoters. The introduction of a construct directing RNA inhibition (RNAi) of TaABF1 did not prevent either ABA-mediated or PKABA1-mediated suppression of Amy32b expression. Similarly, the RNAi construct did not prevent ABA-induction of HVA1. These results suggest that another protein may act redundantly with TaABF1 during cereal imbibition. Although TaABF1 mRNA was downregulated during imbibition of afterripened grains, transcript levels were not significantly altered by exogenous GA or ABA, suggesting that upregulation of TaABF1 at the mRNA level is not required for its role in ABA signaling. We propose a model in which TaABF1 is involved in two separate branches of ABA signaling. In this model, TaABF1 acts downstream of PKABA1 in ABA-suppression of GA-induced gene expression, and participates (independently of PKABA1) in the stimulation of ABA-induced genes.

A rice dehydration-inducible SNF1-related protein kinase 2 phosphorylates an abscisic acid responsive element-binding factor and associates with ABA signaling

By a differential cDNA screening technique, we have isolated a dehydration-inducible gene (designated OSRK1) that encodes a 41.8 kD protein kinase of SnRK2 family from Oryza sativa. The OSRK1 transcript level was undetectable in vegetative tissues, but significantly increased by hyperosmotic stress and Abscisic acid (ABA). To determine its biochemical properties, we expressed and isolated OSRK1 and its mutants as glutathione S-transferase fusion proteins in Escherichia coli. In vitro kinase assay showed that OSRK1 can phosphorylate itself and generic substrates as well. Interestingly, OSRK1 showed strong substrate preference for rice bZIP transcription factors and uncommon cofactor requirement for Mn(2+) over Mg(2+). By deletion of C-terminus 73 amino acids or mutations of Ser-158 and Thr-159 to aspartic acids (Asp) in the activation loop, the activity of OSRK1 was dramatically decreased. OSRK1 can transphosphorylate the inactive deletion protein. A rice family of abscisic acid-responsive element (ABRE) binding factor, OREB1 was phosphorylated in vitro by OSRK1 at multiple sites of different functional domains. MALDI-TOF analysis identified a phosphorylation site at Ser44 of OREB1 and mutation of the residue greatly decreased the substrate specificity for OSRK1. The recognition motif for OSRK1, RQSS is highly similar to the consensus substrate sequence of AMPK/SNF1 kinase family. We further showed that OSRK1 interacts with OREB1 in a yeast two-hybrid system and co-localized to nuclei by transient expression analysis of GFP-fused protein in onion epidermis. Finally, ectopic expression of OSRK1 in transgenic tobacco resulted in a reduced sensitivity to ABA in seed germination and root elongation. These findings suggest that OSRK1 is associated with ABA signaling, possibly through the phosphorylation of ABF family in vivo. The interaction between SnRK2 family kinases and ABF transcription factors may constitute an important part of cross-talk mechanism in the stress signaling networks in plants.

Gene expression patterns reveal tissue‐specific signaling networks controlling programmed cell death and ABA‐ regulated maturation in developing barley seeds

Gene expression patterns covering over 10,000 seed-expressed sequences were analyzed by macroarray technology in maternal tissue (mainly pericarp) and filial endosperm and embryo during barley seed development from anthesis until late maturation. Defined sets of genes showing distinct expression patterns characterized both tissue type and major developmental phases. The analysis focused on regulatory networks involved in programmed cell death (PCD) and abscisic acid (ABA)-mediated maturation. These processes were similar in the different tissues, but typically involved the expression of alternative members of a common gene family. The analysis of co-expressed gene sets and the identification of cis regulatory elements in orthologous rice gene 'promoter' regions suggest that PCD in the pericarp is mediated by distinct classes of proteases and is under the hormonal control of both jasmonic acid (JA) and ethylene via ethylene-responsive element binding protein (EREBP) transcription factors (TFs). On the other hand, PCD in endosperm apparently involves only the ethylene pathway, but employs distinct gene family members from those active in the pericarp, and a different set of proteases and TFs. JA biosynthetic genes are hardly activated. Accordingly, JA levels are high in the pericarp but low in the endosperm during middle and late developmental stages. Similarly, genes acting in the deduced ABA biosynthetic pathway and signaling network differ between endosperm and embryo. ABA in the endosperm appears to exert an influence over storage product synthesis via SNF1 kinase. In the embryo, ABA seems to influence the acquisition of desiccation tolerance via ABA response element binding factors, but the data also suggest the existence of an ABA-independent but interactive pathway acting via the dehydration-responsive element binding (DREB) 2A TF.

Characterization of three homologous basic leucine zipper transcription factors (bZIP) of the ABI5 family during Arabidopsis thaliana embryo maturation

The Arabidopsis thaliana genome contains approximately 80 genes encoding basic leucine zipper transcription factors, divided into 11 groups. Abscisic Acid-Insensitive 5 (ABI5) is one of the 13 members of group A and is involved in ABA signalling during seed maturation, and germination. Seven other members of this group are expressed during seed maturation, but only one of them (Enhanced Em Level, EEL) has been functionally characterized during this developmental phase. Since EEL and two other group A genes, AtbZIP67 and AREB3 (ABA-Responsive Element Binding protein 3), display similar mRNA temporal expression in whole siliques, it is suspected that they might share some overlapping functions. To address this question, the proteins' tissular and subcellular localization in transgenic Arabidopsis were precisely characterized, using translational fusions with a green fluorescent protein (GFP) expressed under the corresponding bZIP promoter. It was found that the three fusion proteins were expressed with a largely overlapping pattern and constitutively localized in the nuclei. An RNA interference approach (RNAi) was then used to knock out the expression of all three genes simultaneously. Endogenous EEL, AREB3, and AtbZIP67 transcripts could be specifically reduced, but no visible defects could be observed during seed maturation.

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.

Arabidopsis basic leucine zipper transcription factors involved in an abscisic acid-dependent signal transduction pathway under drought and high-salinity conditions.

The induction of the dehydration-responsive Arabidopsis gene, rd29B, is mediated mainly by abscisic acid (ABA). Promoter analysis of rd29B indicated that two ABA-responsive elements (ABREs) are required for the dehydration-responsive expression of rd29B as cis-acting elements. Three cDNAs encoding basic leucine zipper (bZIP)-type ABRE-binding proteins were isolated by using the yeast one-hybrid system and were designated AREB1, AREB2, and AREB3 (ABA-responsive element binding protein). Transcription of the AREB1 and AREB2 genes is up-regulated by drought, NaCl, and ABA treatment in vegetative tissues. In a transient transactivation experiment using Arabidopsis leaf protoplasts, both the AREB1 and AREB2 proteins activated transcription of a reporter gene driven by ABRE. AREB1 and AREB2 required ABA for their activation, because their transactivation activities were repressed in aba2 and abi1 mutants and enhanced in an era1 mutant. Activation of AREBs by ABA was suppressed by protein kinase inhibitors. These results suggest that both AREB1 and AREB2 function as transcriptional activators in the ABA-inducible expression of rd29B, and further that ABA-dependent posttranscriptional activation of AREB1 and AREB2, probably by phosphorylation, is necessary for their maximum activation by ABA. Using cultured Arabidopsis cells, we demonstrated that a specific ABA-activated protein kinase of 42-kDa phosphorylated conserved N-terminal regions in the AREB proteins.

ABFs, a Family of ABA-responsive Element Binding Factors

Abscisic acid (ABA) plays an important role in environmental stress responses of higher plants during vegetative growth. One of the ABA-mediated responses is the induced expression of a large number of genes, which is mediated by cis-regulatory elements known as abscisic acid-responsive elements (ABREs). Although a number of ABRE binding transcription factors have been known, they are not specifically from vegetative tissues under induced conditions. Considering the tissue specificity of ABA signaling pathways, factors mediating ABA-dependent stress responses during vegetative growth phase may thus have been unidentified so far. Here, we report a family of ABRE binding factors isolated from young Arabidopsis plants under stress conditions. The factors, isolated by a yeast one-hybrid system using a prototypical ABRE and named as ABFs (ABRE binding factors) belong to a distinct subfamily of bZIP proteins. Binding site selection assay performed with one ABF showed that its preferred binding site is the strong ABRE, CACGTGGC. ABFs can transactivate an ABRE-containing reporter gene in yeast. Expression of ABFs is induced by ABA and various stress treatments, whereas their induction patterns are different from one another. Thus, a new family of ABRE binding factors indeed exists that have the potential to activate a large number of ABA/stress-responsive genes in Arabidopsis.