SEARCH

SEARCH BY CITATION

Keywords:

  • abscisic acid;
  • AREB/ABF;
  • drought stress;
  • transcriptional regulation;
  • bZIP protein;
  • phosphorylation

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

A myriad of drought stress-inducible genes have been reported, and many of these are activated by abscisic acid (ABA). In the promoter regions of such ABA-regulated genes, conserved cis-elements, designated ABA-responsive elements (ABREs), control gene expression via bZIP-type AREB/ABF transcription factors. Although all three members of the AREB/ABF subfamily, AREB1, AREB2, and ABF3, are upregulated by ABA and water stress, it remains unclear whether these are functional homologs. Here, we report that all three AREB/ABF transcription factors require ABA for full activation, can form hetero- or homodimers to function in nuclei, and can interact with SRK2D/SnRK2.2, an SnRK2 protein kinase that was identified as a regulator of AREB1. Along with the tissue-specific expression patterns of these genes and the subcellular localization of their encoded proteins, these findings clearly indicate that AREB1, AREB2, and ABF3 have largely overlapping functions. To elucidate the role of these AREB/ABF transcription factors, we generated an areb1 areb2 abf3 triple mutant. Large-scale transcriptome analysis, which showed that stress-responsive gene expression is remarkably impaired in the triple mutant, revealed novel AREB/ABF downstream genes in response to water stress, including many LEA class and group-Ab PP2C genes and transcription factors. The areb1 areb2 abf3 triple mutant is more resistant to ABA than are the other single and double mutants with respect to primary root growth, and it displays reduced drought tolerance. Thus, these results indicate that AREB1, AREB2, and ABF3 are master transcription factors that cooperatively regulate ABRE-dependent gene expression for ABA signaling under conditions of water stress.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

The plant hormone abscisic acid (ABA) regulates many important processes in plants, including seed germination and dormancy, stomatal closure, and adaptation to water stress (Finkelstein et al., 2002; Himmelbach et al., 2003). To elucidate the role of ABA, many approaches, including forward, reverse, and chemical-genetic analyses, have been utilized, and they have facilitated our understanding of ABA biosynthesis, perception, and signal transduction (Nambara and Marion-Poll, 2005; Santner and Estelle, 2009; Yamaguchi-Shinozaki and Shinozaki, 2006). Water-deficit stresses such as drought and high salinity are some of the most adverse conditions for plants, and they can cause fatal damage to plants and seriously affect crop productivity. Under such conditions, various biochemical and physiological responses, including the expression of many stress-inducible genes that function in stress tolerance and ABA accumulation, are triggered. Numerous drought stress-responsive genes have been reported, and many are induced by ABA (Busk and Pagès, 1998; Rock, 2000; Yamaguchi-Shinozaki and Shinozaki, 2006).

By analyzing the promoters of ABA-inducible genes, a conserved cis-element, designated the ABA-responsive element (ABRE; PyACGTGG/TC), was identified (Bray, 1994; Giraudat et al., 1994; Busk and Pagès, 1998). Subsequent analyses revealed that ABA-responsive gene expression needs multiple ABREs or the combination of an ABRE with a coupling element (CE) as a functional promoter (Marcotte et al., 1989; Shen and Ho, 1995; Shen et al., 1996; Hobo et al., 1999; Narusaka et al., 2003). Furthermore, recent genome-wide analyses have demonstrated that the ABRE core motif is equally abundant in Arabidopsis and rice (Oryza sativa) (Zhang et al., 2005; Gómez-Porras et al., 2007). In addition to ABRE motifs, MYC and MYB recognition sites also have important roles in ABA signaling for some stress-inducible genes such as RD22 (Abe et al., 1997, 2003).

The ABRE-binding (AREB) proteins or ABRE-binding factors (ABFs) were isolated by using ABRE sequences as bait in yeast one-hybrid screenings (Choi et al., 2000; Uno et al., 2000). The AREB/ABFs encode basic-domain leucine zipper (bZIP) transcription factors and belong to a group-A subfamily, which comprises nine homologs in the Arabidopsis genome, and they harbor three N-terminal and one C-terminal conserved domains (Choi et al., 2000; Finkelstein and Lynch, 2000; Lopez-Molina and Chua, 2000; Uno et al., 2000; Bensmihen et al., 2002; Jakoby et al., 2002; Kim et al., 2002; Suzuki et al., 2003). Among them, AREB1/ABF2, AREB2/ABF4, and ABF3 are induced by dehydration, high salinity, or ABA treatment in vegetative tissues (Fujita et al., 2005), and their gain-of-function mutants show enhanced drought stress tolerance (Kang et al., 2002; Kim et al., 2004; Fujita et al., 2005). These independent analyses imply that the three AREB/ABF transcription factors are functionally redundant. However, comprehensive analyses of AREB1, AREB2, and ABF3 have not been reported.

Although AREB/ABF transcription factors are induced by abiotic stresses, only AREB1 is also reported to be regulated by ABA-dependent phosphorylation. AREB1 requires ABA for full activation (Fujita et al., 2005), and its activity is regulated by ABA-dependent multi-site phosphorylation of the conserved domains (Furihata et al., 2006). Further analyses revealed that SNF1-related protein kinase 2 proteins (SnRK2s) such as SRK2D/SnRK2.2, SRK2E/SnRK2.6, and SRK2I/SnRK2.3 play a key role in the ABA-dependent phosphorylation of AREB1 (Furihata et al., 2006; Fujii et al., 2007; Fujii and Zhu, 2009). Similarly, the rice AREB1 ortholog TRAB1 is phosphorylated by the SnRK2 protein kinase ortholog SAPK, and the wheat AREB1 ortholog TaABF is phosphorylated by the SnRK2 ortholog PKABA1 in vitro (Johnson et al., 2002; Kobayashi et al., 2005). Since SnRK2 protein kinases phosphorylate Ser/Thr residues at R-X-X-S/T sites in the conserved regions of AREB/ABF proteins (Kobayashi et al., 2005; Furihata et al., 2006), these observations raise the hypothesis that the ABA-dependent phosphorylation of AREB/ABF transcription factors is commonly needed for their full activation.

In this study we report that AREB1, AREB2, and ABF3 have largely overlapping functions, which are well conserved in land plants. Our results also indicate that all AREB/ABF transcription factors, both in the dicot Arabidopsis and in the monocot rice, require ABA for full activation. We also identify novel downstream genes of the AREB/ABF transcription factors, such as many LEA class and group-Ab PP2C genes and transcription factors, by large-scale transcriptome analysis of the areb1 areb2 abf3 triple mutant in Arabidopsis. The triple mutant also displayed enhanced ABA insensitivity and reduced drought stress tolerance in comparison to the single and double knockout mutants of the AREB/ABF transcription factors. Thus, we demonstrate that these AREB/ABFs are master transcription factors that regulate the ABRE-dependent expression of water-stress-responsive genes in ABA signaling in response to water stresses in a cooperative manner.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

ABA is required for full activation of AREB/ABF transcription factors in Arabidopsis and rice

Three AREB/ABF family transcription factors, AREB1, AREB2, and ABF3, are in the same clade in the phylogenetic tree of the bZIP subfamily, and they are upregulated by dehydration, high salt, and exogenous ABA treatments in vegetative tissues (Fujita et al., 2005). In addition, the tissue-specific expression patterns of these genes (Kang et al., 2002; Fujita et al., 2005) and the subcellular localization of these proteins, as demonstrated with transgenic plants expressing AREB/ABF proteins fused to the C-terminus of GFP under the control of native promoters, are similar under both control and stress conditions (Figures 1 and S1 in Supporting Information). Although AREB1, AREB2, and ABF3 are localized in nuclei in various tissues, it remains unclear whether these are functionally redundant homologs.

image

Figure 1.  Cellular localization of GFP-AREB1, GFP-AREB2, and GFP-ABF3 proteins in roots and leaf epidermis. Confocal images of GFP fluorescence in root tips (a), root elongation zone (b), and leaf epidermal tissues (c) of 35Spro:GFP, AREB1pro:GFP-AREB1, AREB2pro:GFP-AREB2, and ABF3pro:GFP-ABF3 plants. The GFP fluorescence images and images merged with Nomarski images are shown. Bars = 50 μm.

Download figure to PowerPoint

Previously, we showed that ABA-induced modification of AREB1 is required to fully activate expression of ABRE-dependent downstream target genes (Fujita et al., 2005; Furihata et al., 2006). However, it was reported that transgenic Arabidopsis plants expressing unmodified AREB2/ABF4 or ABF3 could affect downstream genes and that they exhibit several remarkable phenotypes (Kang et al., 2002). To clarify whether AREB2 and ABF3 need ABA for full activation, we performed transient expression analysis using Arabidopsis leaf mesophyll protoplasts and a reporter plasmid, which was constructed by fusing five tandem copies of a 77-bp fragment of the Arabidopsis RD29B promoter containing two ABRE motifs (Uno et al., 2000) to the β-glucuronidase (GUS) gene (Figure 2a). Expression of AREB2 and ABF3 as well as of AREB1 in protoplasts produced a drastic activation of the RD29B-GUS reporter gene in the presence of ABA compared with that in the absence of ABA, whereas expression of a constitutively active form of AREB1, AREB1ΔQT, resulted in a marked activation of the reporter gene even in the absence of ABA (Figure 2a). Similar results were obtained with expression of water-stress-responsive rice AREB/ABF orthologs, OsAREB1, OsAREB2, and OsAREB8, in rice mesophyll protoplasts (Figures 2b and S2). Thus, these data indicate that AREB/ABF transcription factors in Arabidopsis and rice need exogenous application of ABA for full activation.

image

Figure 2.  AREB/ABF transcription factors require ABA for full activation. All transactivation experiments were performed two to three times, and results from a representative experiment are shown. Bars indicate SD; = 3. ‘Relative activity’ indicates the multiples of expression compared with the value obtained with the vector control. (a) AREB1, AREB2, and ABF3 are fully activated by application of ABA. Schemes of the effector and reporter constructs used in the transactivation analysis are shown on the left. The effector constructs contain the CaMV 35S promoter and tobacco mosaic virus sequence fused to AREB1, AREB2, ABF3, or AREB1ΔQT cDNA fragments. The reporter construct RD29B-GUS contains five tandem repeats of a 77-bp fragment of the RD29B promoter. The promoters were fused to the −51 RD29B minimal TATA promoter–GUS construct. The central panel shows the structure of each effector gene. Open boxes indicate conserved domains within the family. Protoplasts were co-transfected with the RD29B-GUS reporter and the effector construct. To normalize for transfection efficiency, the pBI35SΩ-LUC reporter was co-transfected as a control in each experiment. Nos-T, nopaline synthase terminator. (b) OsAREB1, OsAREB2, and OsAREB8 are fully activated by application of ABA. The effector constructs contain the maize ubiquitin promoter fused to OsAREB1, OsAREB2, or OsAREB8 cDNA fragments. Protoplasts were co-transfected with the RD29B-GUS reporter and the effector construct. To normalize for transfection efficiency, the pUbi-LUC reporter was co-transfected as a control in each experiment.

Download figure to PowerPoint

AREB/ABF transcription factors interact with an SnRK2 protein kinase, SRK2D/SnRK2.2, in nuclei

Recently, we showed that three SnRK2 protein kinases, SRK2D/SnRK2.2, SRK2E/SnRK2.6, and SRK2I/SnRK2.3, play a central role as positive regulators of ABA signaling in seeds (Nakashima et al., 2009) and in response to water stress and that they co-localize and interact with AREB1 in plant cell nuclei (Fujita et al., 2009). Our bimolecular fluorescence complementation (BiFC) analyses also revealed that AREB2 and ABF3, in addition to AREB1, can interact with SRK2D (Figure S3), which is a key determinant of ABA sensitivity (Fujita et al., 2009) and the most abundant component of these three kinases (Fujii et al., 2007). This interaction occurred in the nucleus in transfected onion epidermal cells (Figure S3). These results favor the view that AREB2 and ABF3 as well as AREB1 function downstream of SRK2D, SRK2E, and SRK2I to activate target genes in ABA signaling.

AREB1, AREB2, and ABF3 interact with each other in plant cell nuclei

As AREB1, AREB2, and ABF3 are bZIP-type transcription factors (Fujita et al., 2005), it was assumed that these AREB/ABF transcription factors form dimers to function in vivo, based on previous findings for bZIP transcription factors (Schütze et al., 2008). To test this possibility, we carried out a BiFC analysis (Walter et al., 2004). All of the samples co-expressing the N-terminal half of yellow fluorescent protein (YFPN) fused to AREB1, AREB2, or ABF3 and those co-expressing the C-terminal half of YFP (YFPC) fused to AREB1, AREB2, or ABF3 yielded YFP fluorescence in the nucleus, whereas all of the samples expressing only YFPN-AREB1, -AREB2, or -ABF3 failed to give a YFP signal (Figure 3). These results strengthen the hypothesis that AREB1, AREB2, and ABF3 function through the formation of homo- or heterodimers with each other.

image

Figure 3.  Bimolecular fluorescence complementation (BiFC) visualization of interaction of AREB1, AREB2, and ABF3 with each other following transient expression in onion epidermal cells. Yellow and cyan fluorescent protein (YFP and CFP, respectively) fluorescence and merged images from the same field of transfected cells are shown for each transfection combination. A 35Spro:CFP (pGKX-CFP) control plasmid was always co-bombarded to identify transformed cells prior to the analysis of YFP fluorescence. In each transfected cell, YFP fluorescence was normalized against CFP fluorescence. Bars = 50 μm.

Download figure to PowerPoint

Generation of multiple mutants of areb1, areb2, and abf3 in all possible combinations

Our findings so far indicate that AREB1, AREB2, and ABF3 have largely overlapping functions. Previously, overexpression studies of AREB/ABF family transcription factors in combination with loss-of-function approaches with single knockout mutants and chimeric repressors with a repression domain revealed that AREB1, AREB2, and ABF3 are transcriptional activators of ABA signaling in response to drought stress (Kang et al., 2002; Kim et al., 2004; Fujita et al., 2005). However, because such ectopic expression approaches might have caused unnatural conditions, these overexpression phenotypes need to be interpreted with caution. Additionally, considering the potential functional redundancy of AREB/ABF family transcription factors, the loss-of-function approaches with single knockout mutants may not be appropriate. Hence, considering the possibility that AREB1, AREB2, and ABF3 are to some extent functionally redundant, we analyzed multiple mutants of areb1, areb2, and abf3 in all possible combinations to overcome these problems with respect to overexpression approaches and functional redundancy.

We obtained T-DNA insertion lines of AREB1, AREB2, and ABF3 in the Columbia accession from the Arabidopsis Biological Resource Center (ABRC) at Ohio State University (Figure 4a) and constructed a series of single, double, and triple homozygous mutant combinations. Semi-quantitative reverse transcriptase PCR (RT-PCR) analysis confirmed that expression of AREB1, AREB2, and ABF3 was abolished in these single and multiple mutants (Figure 4b). All mutants showed the same growth phenotype compared with wild-type (WT) plants on agar plates with germination medium (GM) under normal conditions (Figure 4c,d). In contrast, while none of the single T-DNA mutants had any obvious phenotype in comparison with WT plants, the inflorescence heights of the areb2 abf3 and areb1 abf3 double and areb1 areb2 abf3 triple mutants appeared to be slightly shorter than those of WT plants (Figure 4e,f).

image

Figure 4.  Growth phenotypes of areb1, areb2, and abf3 single mutants and all combinations of multiple mutants. (a) Scheme of AREB1, AREB2, and ABF3 genes. Exons are indicated as thick lines and introns as thin lines. The position of the T-DNA insertion is shown. Arrowheads indicate the positions of primers used in (b). (b) Expression levels of the AREB1, AREB2, and ABF3 genes in each knockout mutant were determined by RT-PCR with total RNA isolated from 12-day-old wild-type (WT) and mutant seedlings using AREB1, AREB2, ABF3, and TUB1 primers. (c) Photographs of knockout mutants and WT plants grown for 3 weeks on germination medium (GM) agar plates. (d) Maximum rosette radius of each plant on a GM agar plate 3 weeks after stratification. The experiment was performed twice, and a representative result is shown. Bars indicate SD; = 5. (e) Photographs of knockout mutants and WT plants grown for 3 weeks on GM agar plates and 2 weeks on soil. (f) Inflorescence heights of each plant on soil 5 weeks after stratification. The experiment was performed twice, and a representative result is shown. Bars indicate SD; = 10.

Download figure to PowerPoint

The areb1 areb2 abf3 triple mutant displays reduced drought tolerance

Slight growth inhibition was observed in the soil-grown areb2 abf3 and areb1 abf3 double and areb1 areb2 abf3 triple mutants (Figure 4e,f). However, no remarkable differences were observed in the plants grown on GM agar plates (Figure 4c,d). Taken together with previous findings that AREB1, AREB2, and ABF3 are positive regulators of ABA signaling in response to drought stress (Kang et al., 2002; Kim et al., 2004; Fujita et al., 2005), it is possible that this slight growth inhibition in the soil reflected enhanced sensitivity to drought stress. To test this possibility, we examined the difference in recovery after dehydration with plants grown on soil (Figure 5a). The survival rate of the areb1 areb2 abf3 triple mutant was markedly reduced in comparison with those of the other single and double mutants and WT plants, suggesting that AREB1, AREB2, and ABF3 all confer drought stress tolerance in a redundant manner. These findings are not inconsistent with the hypothesis about the slight growth inhibition. However, at the same time we cannot exclude the possibility that other factors may also be associated with the growth phenotype, because the difference in the survival rates between the single and double mutants is not as clear as was seen in the inflorescence height.

image

Figure 5.  Drought-tolerance phenotypes of areb1, areb2, and abf3 single mutants and all combinations of multiple mutants. (a) Survival rates of knockout mutants and wild-type (WT) plants. Photographs show plants before and after stress treatment. Watering was withheld from 3-week-old plants for 11–12 days, then plants were rewatered for 1 week before the photograph was taken. Survival rates were calculated from the results of five independent experiments ( 14 for each experiment). **< 0.01, by t-test. (b) Rates of water loss from 4-week-old plants at 25°C and 70% relative humidity (RH). The experiment was performed twice, and a representative result is shown. Bars indicate SD; = 3. (c) Standardized water content of 4-week-old plants at 25°C and 70% RH. The experiment was performed twice, and a representative result is shown. Bars indicate SD; = 3.

Download figure to PowerPoint

Next, to assess whether enhanced sensitivity to drought stress in the mutants is due to altered transpiration rates, we measured water loss rates and standardized water contents of whole plants under dehydration conditions (Figure 5b,c). A slightly higher reduction was observed for both the water loss rates and the standardized water contents of the areb1 areb2 abf3 triple mutant in comparison with WT, but this difference gradually decreased. This suggests that the function of AREB1, AREB2, and ABF3 may be partially associated with stomatal closure under conditions of water stress.

The areb1 areb2 abf3 triple mutant exhibits more resistance to ABA in primary root growth than do the other single and double mutants

Previous studies reported that single T-DNA insertion mutants of AREB1, AREB2, or ABF3 display a comparatively weak ABA-insensitive phenotype (Kim et al., 2004; Fujita et al., 2005). Here, to evaluate the effect of multiple mutations of AREB1, AREB2, and ABF3 on ABA sensitivity, we germinated seeds of these mutants on GM agar plates containing various concentrations of ABA. During the germination process, none of the single, double, or triple mutants showed any obvious difference in ABA sensitivity in comparison to WT plants (Figure 6a). To test the ABA sensitivities of these mutants during the vegetative stage, we transferred 4-day-old seedlings germinated on GM agar plates to the same medium with or without ABA (Figure 6b,c). The length of the primary root was then measured 7 days later. An ABA dose-dependent inhibition of primary root growth could be observed for each line. The primary root growth of multiple and abf3 single mutants was significantly less inhibited by 50 μm ABA when compared with that of WT plants. At 50 μm ABA, the areb1 areb2 abf3 triple mutant was more resistant to ABA than were the other mutants examined. These data suggest that AREB1, AREB2, and ABF3 have largely overlapping functions in ABA signaling during the vegetative stage.

image

Figure 6.  The ABA insensitivity of the areb1 areb2 abf3 triple mutant. (a) Dose-response of germination to ABA. Seeds were germinated on germination medium (GM) agar plates containing various concentrations of ABA and 1% sucrose, and seeds with emerged radicles were counted after 2 days. Experiments were performed in triplicate ( 30 each). Bars indicate SD. (b) Photographs of seedlings at 1 week after transfer to control agar plates (GM medium with 1% sucrose) or plates containing 50 μm ABA. Seedlings were 4 days old at the time of transfer. Bars = 1 cm. (c) The ABA dose response of primary root growth. Quantification of relative primary root length of seedlings treated as described in (b) at 11 days after stratification. Bars indicate SD; = 7. *< 0.05; **< 0.01, by t-test.

Download figure to PowerPoint

Stress-responsive gene expression is remarkably impaired in the areb1 areb2 abf3 triple mutant

The areb1 areb2 abf3 triple mutant clearly showed reduced drought stress tolerance and enhanced insensitivity to ABA in primary root growth. To examine the role of AREB1, AREB2, and ABF3 in the transcriptional network in response to water stress, we compared the expression profiles of 12-day-old areb1 areb2 abf3 triple mutants with those of WT plants under both normal and stress conditions using an Agilent Arabidopsis 3 Oligo Microarray (44K feature format). After 6 h of treatment with 50 μm ABA (Fujita et al., 2009; E-MEXP-2116), dehydration stress (E-MEXP-2294), and 250 mm NaCl (E-MEXP-2293), 147, 58, and 176 genes, respectively, showed reduced expression levels (≥4-fold) in the triple mutant in comparison with the expression levels in the WT (< 0.05). According to Genevestigator stimulus data sets (https://www.genevestigator.com/) (Zimmermann et al., 2004) and our microarray data (Fujita et al., 2009; E-MEXP-2187), most of the genes showing decreased expression levels in the triple mutant are responsive to ABA, dehydration, osmotic, and/or salt stresses, indicating that the areb1 areb2 abf3 triple mutant was remarkably impaired in water-stress- and ABA-dependent gene expression (Figure 7a, Tables 1 and S1). Further, we confirmed the expression of stress-marker genes (Fujita et al., 2005), including RD29B, AIL1, and RAB18, identified by microarray analysis using quantitative real-time RT-PCR (qRT-PCR) (Figure 7b). These findings are consistent with the water-stress-sensitive and ABA-insensitive phenotypes of the triple mutant. Significantly, 84% (32 genes) of the top 38 downregulated genes whose expression was more than eightfold reduced in the triple mutant under two or more stress treatments when compared with WT plants carry two or more ABREs or CEs in their promoter regions (Table 1). Thus, these data obtained with the triple mutant strengthen the previous findings that AREB1, AREB2, and ABF3 contribute to the regulation of ABRE-dependent gene expression in response to water stress during the vegetative stage.

image

Figure 7.  Osmotic stress-dependent gene expression is globally impaired in the areb1 areb2 abf3 triple mutant. (a) Heat maps indicate stimulus responsiveness of top 100 genes showing reduced expression levels in the areb1 areb2 abf3 triple mutant in comparison with the wild-type (WT) expression level after 6 h of treatment with 50 μm ABA (Fujita et al., 2009; E-MEXP-2116), dehydration stress (E-MEXP-2294), or 250 mm NaCl (E-MEXP-2293), as determined from Genevestigator stimulus data sets (https://www.genevestigator.com/) (Zimmermann et al., 2004). The ‘No treatment’ column displays the stimulus responsiveness of all 46 genes showing reduced expression levels in the areb1 areb2 abf3 triple mutant in comparison with the WT expression level without stress treatment (E-MEXP-2295). Data were not available for some genes. (b) Gene expression profiles of stress-marker genes analyzed using quantitative real-time RT-PCR. For each gene, the expression level in the WT plants under non-stressed conditions was defined as 1.0. Data represent means and standard deviations of three replicate reactions.

Download figure to PowerPoint

Table 1.   Representative genes regulated by AREB1, AREB2, and ABF3, identified by microarray analysis of the areb1 areb2 abf3 triple mutant
AGI codeaDescriptionbDehydration 6 hdNaCl 6 heABA 6 hfStress inducibilitygAREB1 ΔQThAREB1 pahNo. of ABREsiNo. of CEsi
P valuecFold changeP valuecFold changeP valuecFold change
  1. Listed are genes whose expression was more than eightfold reduced in the areb1 areb2 abf3 triple mutant compared with wild-type (WT) plants under at least two of three stress conditions: dehydration, NaCl, and ABA.

  2. aAGI, Arabidopsis Genome Initiative.

  3. bDescription as given by The Institute for Genomic Research database.

  4. cP values <0.05 were studied.

  5. gData on inducibility were based on our microarray analysis (Fujita et al., 2009; E-MEXP-2187). D, dehydration 6 h; S, high salinity (NaCl) 6 h; A, ABA 6 h.

  6. hThe symbol # indicates genes upregulated by overexpression of constitutively active forms of AREB1, AREB1ΔQT (Fujita et al., 2005) and AREB1pa (Furihata et al., 2006).

  7. iNumber of ABRE (ABA-responsive element) and CE (coupling element) core sequences in 1-kb promoter region from the TAIR database.

Transcriptional regulation
 At3g22830.1HsfA6B3.0 × 10−512.92.0 × 10−436.9  DSA  00
 At5g43840.1HsfA6A6.9 × 10−411.21.7 × 10−651.91.2 × 10−523.9DSA  10
 At4g21440.1AtMYB1025.1 × 10−68.44.3 × 10−622.12.2 × 10−24.6DSA  10
LEA protein
 At3g53040.1Late embryogenesis abundant protein, putative  2.4 × 10−415.65.4 × 10−48.4DSA  72
 At3g02480.1ABA-responsive protein-related9.1 × 10−44.52.9 × 10−555.28.0 × 10−1043.7DSA #63
 At4g21020.1Late embryogenesis abundant domain-containing protein  8.4 × 10−312.91.2 × 10−420.2SA  63
 At4g36600.1Late embryogenesis abundant domain-containing protein  1.1 × 10−470.35.0 × 10−446.2DSA  62
 At1g52690.1Late embryogenesis abundant protein, putative1.7 × 10−632.52.3 × 10−5144.11.0 × 10−540.7DSA #52
 At2g40170.1EM6  1.6 × 10−659.26.5 × 10−532.4DSA  52
 At5g66400.1RAB18  5.7 × 10−513.01.4 × 10−513.5DSA##51
 At5g06760.1Late embryogenesis abundant group 1 domain-containing protein5.0 × 10−716.72.0 × 10−680.32.2 × 10−76.7DSA  42
 At2g42560.1Late embryogenesis abundant domain-containing protein2.7 × 10−24.12.3 × 10−461.68.0 × 10−528.3DSA  41
 At2g35300.1Late embryogenesis abundant group 1 domain-containing protein  1.9 × 10−634.75.2 × 10−613.4DSA  41
 At3g51810.1EM11.0 × 10−27.41.7 × 10−560.31.7 × 10−312.3DSA  40
 At3g15670.1Late embryogenesis abundant protein, putative5.3 × 10−56.03.4 × 10−652.92.1 × 10−639.5DSA  40
 At3g17520.1AIL18.5 × 10−58.02.9 × 10−6116.51.7 × 10−877.2DSA##32
Signaling
 At1g05100.1MAPKKK184.4 × 10−511.93.2 × 10−59.98.9 × 10−34.5DSA  52
 At1g07430.1Protein phosphatase 2C, putative (HAI2)2.2 × 10−411.69.6 × 10−710.97.3 × 10−77.7DSA  50
 At2g29380.1Protein phosphatase 2C, putative (HAI3)  2.3 × 10−638.28.8 × 10−518.9DSA  30
Cell wall
 At5g16190.1Glycosyl transferase family 2 protein (CSLA11)  2.3 × 10−516.34.3 × 10−6102.3  11
Lipid metabolism
 At5g59310.1Lipid transfer protein 4 (LTP4)2.0 × 10−410.91.5 × 10−215.71.9 × 10−616.6DSA# 21
Stress
 At5g52300.1RD29B/LTI653.2 × 10−37.71.1 × 10−550.91.7 × 10−745.3DSA##50
 At3g58450.1Universal stress protein (USP) family protein  1.9 × 10−48.71.3 × 10−611.3DSA  31
Protein
 At1g60190.1Armadillo/beta-catenin repeat family protein1.1 × 10−411.62.3 × 10−517.2  DSA  53
 At1g64110.1AIA1  1.9 × 10−522.61.3 × 10−614.9SA# 20
Development
 At5g13170.1SAG29  2.2 × 10−711.61.7 × 10−815.8A# 40
 At3g13672.1Seven in absentia family protein  2.1 × 10−719.56.3 × 10−69.1SA  10
Transport
 At1g04560.1AWPM-19-like membrane family protein6.8 × 10−55.42.5 × 10−4117.01.3 × 10−335.5DSA #22
 At2g41190.1Amino acid transporter family protein  2.0 × 10−820.97.1 × 10−78.2DSA  10
Others
 At1g54870.1Short-chain dehydrogenase/reductase family protein  1.2 × 10−413.32.3 × 10−530.4SA  62
 At5g15500.1Ankyrin repeat family protein4.0 × 10−320.57.9 × 10−6157.81.7 × 10−730.6DSA  60
Not assigned
 At3g63060.1Circadian clock coupling factor, putative5.5 × 10−414.92.3 × 10−642.1  DSA  87
 At5g50360.1Expressed protein5.5 × 10−525.41.6 × 10−551.17.0 × 10−617.9DSA  62
 At5g45690.1Expressed protein  1.5 × 10−450.75.6 × 10−580.7DSA  41
 At5g40790.1Hypothetical protein1.3 × 10−29.71.6 × 10−324.51.9 × 10−315.9DSA  31
 At5g66780.1Expressed protein  8.9 × 10−533.99.4 × 10−623.4DSA  31
 At5g01300.1Phosphatidyl-ethanolamine-binding family protein  1.6 × 10−611.11.5 × 10−510.4DSA  12
 At2g47770.1Benzodiazepine receptor-related9.6 × 10−540.82.2 × 10−549.77.1 × 10−612.5DSA #00

Numerous regulatory and LEA class genes are positively regulated by AREB/ABF transcription factors

To estimate the cellular function of AREB/ABF downstream genes, which are defined here as downregulated genes with a ratio of ≥4.0 in the areb1 areb2 abf3 triple mutant in response to water stress when compared with that of WT plants, we conducted a comprehensive gene ontology analysis with the PageMan profiling tool (Usadel et al., 2006). The commonly downregulated genes in the triple mutant in response to dehydration, high salinity, and ABA treatments significantly included LEA class genes (including RD29B, AIL1, RAB18, EM1, and EM6), ABA-regulated genes (including RD20, LTP4, and SAG29), and MYB transcription factor genes (including AtMYB74, AtMYB79, AtMYB102, and AtMYB121), and group-A type-2C protein phosphatase (PP2C) genes (including AHG1, AHG3, HAI1, HAI2, and HAI3) (Figure S4 and Table S2), while no commonly upregulated genes could be found under these stress conditions (Table S3). These results support previous findings that AREB1, AREB2, and ABF3 are the positive regulators in ABA/water-stress signaling. In addition, the pie charts showing the results of ontology analysis under stress conditions are similar, suggesting that AREB/ABF-directed molecular responses to dehydration, high salinity, and ABA treatments are also similar (Figure S5). Thus, many regulatory genes encoding transcription factors, protein kinases, and phosphatases, and functional genes encoding LEA class proteins are positively regulated by AREB/ABF transcription factors under conditions of water stress (Table 1).

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

The data presented here provide conclusive evidence that AREB1, AREB2, and ABF3 have largely overlapping functions in ABA signaling in response to water stress. In addition to temporal and spatial gene expression profiles in response to water stress (Uno et al., 2000; Kang et al., 2002; Kim et al., 2004; Fujita et al., 2005), these AREB/ABF transcription factors behave similarly in terms of protein localization (Figures 1 and S1), dimer formation (Figure 3), and interaction with the SRK2D protein kinase in nuclei (Figure S3). Moreover, the data from the transient expression analysis in protoplasts indicated that all AREB/ABF transcription factors in Arabidopsis and rice need exogenous application of ABA for full activation. This supports the view that ABA-dependent modification is required for full activation of all AREB/ABF transcription factors in Arabidopsis and rice. Taken together with previous findings on the relationships between AREB/ABF-type transcription factors and SnRK2 protein kinases in Arabidopsis and rice (Uno et al., 2000; Kobayashi et al., 2005; Furihata et al., 2006), these observations suggest that ABA-dependent phosphorylation of AREB/ABF-type transcription factors by SnRK2 protein kinases may be involved in the activation of AREB/ABFs in both monocot and dicot plants. Furthermore, our phylogenetic analysis (Figure S2a) appears to support the idea that the AREB/ABF-SnRK2 system is ubiquitous and conserved in embryophytes. Thus, the elucidation of the AREB/ABF-SnRK2 system may contribute to the creation of drought-tolerant varieties of land plants.

On the other hand, our results also show that AREB1, AREB2, and ABF3 do not completely overlap in their functions. AREB1 appears to differ in some degree from AREB2 and ABF3 in its cellular protein localization in the root tips (Figures 1a and S1a) and its role in drought stress tolerance (Figure 5; Kim et al., 2004). This may be related to variations in the lists of target genes and in the roles of each AREB/ABF (Kim et al., 2004; Fujita et al., 2005). In fact, it was reported that AREB1 plays more important roles than AREB2 and ABF3 at the post-germination stage rather than the germination stage (Kim et al., 2004; Fujita et al., 2005), and in glucose signaling rather than ABA/stress signaling (Kim et al., 2004). On the other hand, ABF3 seems to be more effective than the other AREB/ABFs in its influence on plant height (Figure 4e,f), the ABA response in the single and multiple mutants (Figure 6b,c), and the expression of downstream genes such as RD29B, AIL1, and RAB18 (Figure 7b). This may be due to the greater transactivation activity of ABF3 than of AREB1 or AREB2, which we observed even without ABA treatment in the protoplast transient expression analysis (Figure 2a). Each AREB/ABF protein may play a specific role in addition to the shared roles with other AREB/ABFs. However, since these AREB/ABFs can form hetero- or homodimers to function in vivo (Figure 3), and since other group-A bZIP transcription factors might also be involved in the stress response, it will be difficult to clarify the specific role of each AREB/ABF in response to ABA and drought stress. Collectively, our results indicate that AREB1, AREB2, and ABF3 have largely overlapping functions, but they may also have specific roles. Overall, it is clear that these AREB/ABFs function cooperatively in ABA signaling in response to water stress.

Our data on the areb1 areb2 abf3 triple mutant clearly strengthen the previous view that the AREB/ABF transcription factors AREB1, AREB2, and ABF3, play a pivotal role in ABRE-dependent gene expression in ABA signaling under conditions of water stress in planta. Our large-scale transcriptome analysis of the areb1 areb2 abf3 triple mutant comprehensively revealed novel AREB/ABF downstream genes in response to water stress, including many LEA class genes, PP2C genes, and genes for transcription factors (Tables 1, S1 and S2). Moreover, two or more ABRE/CE motifs can be seen in the promoter region of most of these genes, suggesting that most of the highly downregulated target genes (by more than a fourfold decrease) in the areb1 areb2 abf3 triple mutant are directly regulated by the AREB/ABF transcription factors, which recognize ABRE motifs, under conditions of water stress. The transcriptome data are also consistent with previous results (Kang et al., 2002; Kim et al., 2004; Fujita et al., 2005) indicating that RAB18 and RD29B are regulated by all three AREB/ABFs (Table 1). However, we cannot precisely compare these results because the reported numbers of genes affected by the ectopic expression of AREB/ABFs are far fewer than the numbers of genes affected by the areb1 areb2 abf3 triple mutation, and because the experimental methods and conditions were different. The strategy of using multiple knockout mutants appears to overcome some problems associated with overexpression approaches and with functional redundancy, which would affect the results of loss-of-function analyses using single knockout mutants. The analysis of the areb1 areb2 abf3 triple mutant in this work allowed us to determine many more downstream genes of all three transcription factors and to extend the functional analysis of AREB/ABF transcription factors in response to water stress. Our data show that the areb1 areb2 abf3 triple mutation severely impairs the expression of many ABA- and water-stress-responsive genes (Figure 7, Tables 1, S1 and S2), resulting in enhanced drought sensitivity (Figure 5) and in reduced sensitivity to ABA in primary root growth during the vegetative stage (Figure 6). Consequently, these findings show that AREB/ABF-regulated ABRE-dependent gene expression is a crucial transcriptional response to water stress.

Significantly, many transcription factors were identified as downstream genes of AREB/ABF transcription factors (Tables 1 and S1). Although some downstream transcription factors did not harbor two or more ABRE/CE motifs in the promoter region, all of them were upregulated by water stress. These data reveal that AREB/ABF transcription factors play a central role in stress-responsive gene expression by regulating downstream transcription factors directly or indirectly. Although it remains unclear whether the AREB/ABF transcription factors are regulated only by ABA or directly by water stress, AREB/ABF transcription factors appear to control many downstream transcription factors in various stages and tissues as a result (Figure S4). Thus, these findings suggest that AREB/ABF transcription factors can mediate the temporal and spatial expression of ABA- and water-stress-responsive genes through downstream transcription factors.

The LEA class proteins are widely assumed to play crucial roles in cellular tolerance under conditions of dehydration and cold stress (Battaglia et al., 2008; Bies-Ethève et al., 2008). Newly identified downstream genes of AREB/ABF transcription factors include many LEA class proteins (Figure S5, Tables 1 and S1), which include hydrophilic LEA-like or LEA proteins that typically accumulate during the late stage of embryogenesis or in response to dehydration and cold stress (Ramanjulu and Bartels, 2002). In Arabidopsis, there are 51 genes encoding LEA class proteins (Hundertmark and Hincha, 2008), and 29 of these genes are induced by dehydration (Fujita et al., 2009). Notably, almost half (13 of 29) of the dehydration-inducible LEA class genes showed impaired expression in the triple mutant, and these carry two or more ABRE/CE motifs in the promoter region (Tables 1 and S1). These findings indicate that these three AREB/ABF transcription factors regulate the expression of many LEA class protein genes to confer cellular tolerance under water stress conditions. In fact, because the areb1 areb2 abf3 triple mutation seems to be associated with the downregulation of LEA class protein genes (Tables 1 and S1) rather than an altered transpiration rate (Figure 5b,c), the impaired drought stress tolerance ability of the triple mutant may be more preferentially attributed to a decrease in the level of LEA class proteins.

Many group-A PP2Cs such as AHG1, AHG3, HAI1, HAI2, and HAI3, were significantly downregulated in the areb1 areb2 abf3 triple mutant under conditions of water stress (Tables 1 and S1). Intriguingly, all five of these PP2Cs belong to the group-Ab PP2C subfamily (Schweighofer et al., 2004; Xue et al., 2008) and carry two or more ABRE motifs (Fujita et al., 2009), supporting the notion that all five group-Ab PP2Cs are direct targets of AREB/ABF transcription factors. Both AHG1 and AHG3 were shown to be involved in ABA signaling in seeds (Yoshida et al., 2006; Nishimura et al., 2007), whereas HAI1, HAI2, and HAI3 were found to exhibit striking upregulation in response to ABA in the vegetative phase (Fujita et al., 2009). Collectively, these findings suggest that these AREB/ABF-regulated group-Ab PP2Cs may have a specific role in ABA signaling in response to water stress, unlike the related group-Aa PP2Cs (Xue et al., 2008) such as ABI1 and ABI2. Although recent reports have revealed that group-A PP2Cs play a crucial role in ABA signaling by interacting with PYR/PYL/RCARs, which are newly identified soluble ABA receptors (Park et al., 2009; Ma et al., 2009), and SRK2D/SnK2.2 protein kinase (Park et al., 2009; Fujita et al., 2009), further studies are required for understanding the role of AREB/ABF-regulated group-Ab PP2Cs in ABA signaling under water stress conditions.

Experimental procedures

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Plant materials, growth conditions, and generation of transgenic plants

Plants [Arabidopsis thaliana ecotype Columbia (Col)] were grown, transformed, and treated as described previously (Fujita et al., 2005). The T-DNA insertion lines of areb1 (SALK_002984), areb2 (SALK_069523), and abf3 (SALK_096965) were obtained from the ABRC. A series of multiple areb1, areb2, and abf3 mutants in the Col ecotype were constructed by genetic crosses and were screened using primers recommended by the ABRC. We confirmed that the AREB1, AREB2, and ABF3 genes were not expressed in these single and multiple mutants using RT-PCR analysis as described (Fujita et al., 2005). The detailed procedure for construction of pGreenII-based plasmids for plant transformation is described in Appendix S1 and Table S4. The plant transformation vectors were transformed into Arabidopsis by the vacuum infiltration method using Agrobacterium tumefaciens strain GV3101 as described previously (Fujita et al., 2005).

Transient expression assays with Arabidopsis and rice protoplasts

Transient expression assays using protoplasts derived from Arabidopsis and rice leaf mesophyll cells were performed as described (Yoo et al., 2007; Bart et al., 2006) with minor modifications. The detailed procedures for protoplast transformation and construction of effector plasmids are described in Appendix S1 and Table S4. The RD29B-GUS reporter plasmid (Uno et al., 2000), which was kindly provided by T. Hattori (Nagoya University, Japan), was co-transfected with each effector plasmid. The pBI35S-LUC reporter plasmid (Fujita et al., 2005) or the ubiquitin–luciferase plasmid (Nakashima et al., 2007) were used as an internal control in transactivation experiments using protoplasts derived from mesophyll cells of Arabidopsis or rice, respectively.

BiFC analysis

Construction of the pUCSPYNE- and pUCSPYCE-based plasmids for BiFC analysis (Walter et al., 2004) is described in Appendix S1 and Table S4. Analysis of transient expression in onion epidermal cells was performed as described previously (Fujita et al., 2005). The YFP and cyan fluorescent protein (CFP) fluorescence was observed with a confocal laser scanning microscope (LSM5 PASCAL, Carl Zeiss, http://www.zeiss.com/).

Drought tolerance assays, analysis of plant water relations, and ABA analysis

Analyses of drought tolerance, plant water relations, and ABA sensitivity in germination were performed as described previously (Fujita et al., 2005). For ABA sensitivity assays at the seedling stage, seeds were sown on GM agar plates supplemented with 1% sucrose and then stratified at 4°C for 4 days. Four-day-old seedlings were transferred to 0.5× MS plates containing 1% sucrose with or without ABA. The plates were sealed doubly with Micropore surgical tape (3M Health Care, http://www.3m.com/) and incubated vertically at 22°C under continuous illumination.

Microarray analysis and quantitative RT-PCR

Total RNA was isolated with RNAiso reagent (Takara, http://www.takara-bio.com/) from 12-day-old seedlings of the areb1 areb2 abf3 triple mutant and WT plants grown on GM agar plates with or without stress treatments of 50 μm ABA, dehydration, or 250 mm NaCl for 6 h. Genome-wide transcriptome analysis using the Arabidopsis 3 Oligo Microarray Kit (Agilent Technologies, http://www.home.agilent.com/agilent/home.jspx) was conducted with total RNA as described previously (Fujita et al., 2005). For each biological replicate, material from five plants was pooled to make a single sample for RNA purification. Two independent RNA samples were used for each experiment. The reproducibility of the microarray analysis was verified by a dye swap in each experiment. To search for cis-acting elements in promoters 1-kb upstream sequences were retrieved from The Arabidopsis Information Resource (TAIR). Heat maps were constructed with the top 100 downregulated genes in each experiment, and the stimuli responsiveness of each gene was derived from Genevestigator stimulus data sets. The qRT-PCR analyses were carried out with total RNA using an ABI 7500 real-time PCR system as described previously (Qin et al., 2008). The primers used in this work are listed in Table S4. All microarray design and data have been deposited in the ArrayExpress database (http://www.ebi.ac.uk/microarray-as/ae/; accession numbers E-MEXP-2116, E-MEXP-2187, E-MEXP-2293, E-MEXP-2294, and E-MEXP-2295).

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

We thank E. Ohgawara, K. Amano, and E. Kishi for their excellent technical assistance; K. Yoshiwara for microarray analysis; and M. Toyoshima for skillful editorial assistance. This work was supported by the Targeted Proteins Research Program (TPRP); the Ministry of Education, Culture, Sports, Science & Technology; the Program for Promotion of Basic Research Activities for Innovative Biosciences (BRAIN); and the Ministry of Agriculture, Forestry and Fisheries (MAFF), Japan.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information
  • Abe, H., Yamaguchi-Shinozaki, K., Urao, T., Iwasaki, T., Hosokawa, D. and Shinozaki, K. (1997) Role of Arabidopsis MYC and MYB homologs in drought- and abscisic acid-regulated gene expression. Plant Cell, 9, 18591868.
  • Abe, H., Urao, T., Ito, T., Seki, M., Shinozaki, K. and Yamaguchi-Shinozaki, K. (2003) Arabidopsis AtMYC2 (bHLH) and AtMYB2 (MYB) function as transcriptional activators in abscisic acid signaling. Plant Cell, 15, 6378.
  • Bart, R., Chern, M., Park, C., Bartley, L. and Ronald, P. (2006) A novel system for gene silencing using siRNAs in rice leaf and stem-derived protoplasts. Plant Methods, 2, 13.
  • Battaglia, M., Olvera-Carrillo, Y., Garciarrubio, A., Campos, F. and Covarrubias, A. (2008) The enigmatic LEA proteins and other hydrophilins. Plant Physiol. 148, 624.
  • Bensmihen, S., Rippa, S., Lambert, G., Jublot, D., Pautot, V., Granier, F., Giraudat, J. and Parcy, F. (2002) The homologous ABI5 and EEL transcription factors function antagonistically to fine-tune gene expression during late embryogenesis. Plant Cell, 14, 13911403.
  • Bies-Ethève, N., Gaubier-Comella, P., Debures, A., Lasserre, E., Jobet, E., Raynal, M., Cooke, R. and Delseny, M. (2008) Inventory, evolution and expression profiling diversity of the LEA (late embryogenesis abundant) protein gene family in Arabidopsis thaliana. Plant Mol. Biol. 67, 107124.
  • Bray, E.A. (1994) Alterations in gene expression in response to water deficit. In Stress-Induced Gene Expression in Plants (Basra, A.S., ed.). Amsterdam: Harwood Academic, pp. 123.
  • Busk, P. and Pagès, M. (1998) Regulation of abscisic acid-induced transcription. Plant Mol. Biol. 37, 425435.
  • Choi, H., Hong, J., Ha, J., Kang, J. and Kim, S.Y. (2000) ABFs, a family of ABA-responsive element binding factors. J. Biol. Chem. 275, 17231730.
  • Finkelstein, R.R. and Lynch, T.J. (2000) The Arabidopsis abscisic acid response gene ABI5 encodes a basic leucine zipper transcription factor. Plant Cell, 12, 599609.
  • Finkelstein, R.R., Gampala, S.S. and Rock, C.D. (2002) Abscisic acid signaling in seeds and seedlings. Plant Cell, 14(Suppl), S15S45.
  • Fujii, H. and Zhu, J. (2009) Arabidopsis mutant deficient in 3 abscisic acid-activated protein kinases reveals critical roles in growth, reproduction, and stress. Proc. Natl Acad. Sci. USA, 106, 83808385.
  • Fujii, H., Verslues, P.E. and Zhu, J.K. (2007) Identification of two protein kinases required for abscisic acid regulation of seed germination, root growth, and gene expression in Arabidopsis. Plant Cell, 19, 485494.
  • Fujita, Y., Fujita, M., Satoh, R., Maruyama, K., Parvez, M.M., Seki, M., Hiratsu, K., Ohme-Takagi, M., Shinozaki, K. and Yamaguchi-Shinozaki, K. (2005) AREB1 is a transcription activator of novel ABRE-dependent ABA signaling that enhances drought stress tolerance in Arabidopsis. Plant Cell, 17, 34703488.
  • Fujita, Y., Nakashima, K., Yoshida, T. et al. (2009) Three SnRK2 protein kinases are the main positive regulators of abscisic acid signaling in response to water stress in Arabidopsis. Plant Cell Physiol. 50, in press.
  • Furihata, T., Maruyama, K., Fujita, Y., Umezawa, T., Yoshida, R., Shinozaki, K. and Yamaguchi-Shinozaki, K. (2006) Abscisic acid-dependent multisite phosphorylation regulates the activity of a transcription activator AREB1. Proc. Natl Acad. Sci. USA, 103, 19881993.
  • Giraudat, J., Parcy, F., Bertauche, N., Gosti, F., Leung, J., Morris, P., Bouvier-Durand, M. and Vartanian, N. (1994) Current advances in abscisic acid action and signalling. Plant Mol. Biol. 26, 15571577.
  • Gómez-Porras, J., Riaño-Pachón, D., Dreyer, I., Mayer, J. and Mueller-Roeber, B. (2007) Genome-wide analysis of ABA-responsive elements ABRE and CE3 reveals divergent patterns in Arabidopsis and rice. BMC Genomics, 8, 260.
  • Himmelbach, A., Yang, Y. and Grill, E. (2003) Relay and control of abscisic acid signaling. Curr. Opin. Plant Biol. 6, 470479.
  • Hobo, T., Kowyama, Y. and Hattori, T. (1999) A bZIP factor, TRAB1, interacts with VP1 and mediates abscisic acid-induced transcription. Proc. Natl Acad. Sci. USA, 96, 1534815353.
  • Hundertmark, M. and Hincha, D. (2008) LEA (late embryogenesis abundant) proteins and their encoding genes in Arabidopsis thaliana. BMC Genomics, 9, 118.
  • Jakoby, M., Weisshaar, B., Droge-Laser, W., Vicente-Carbajosa, J., Tiedemann, J., Kroj, T. and Parcy, F. (2002) bZIP transcription factors in Arabidopsis. Trends Plant Sci. 7, 106111.
  • Johnson, R.R., Wagner, R.L., Verhey, S.D. and Walker-Simmons, M.K. (2002) The abscisic acid-responsive kinase PKABA1 interacts with a seed-specific abscisic acid response element-binding factor, TaABF, and phosphorylates TaABF peptide sequences. Plant Physiol. 130, 837846.
  • Kang, J.Y., Choi, H.I., Im, M.Y. and Kim, S.Y. (2002) Arabidopsis basic leucine zipper proteins that mediate stress-responsive abscisic acid signaling. Plant Cell, 14, 343357.
  • Kim, S., Ma, J., Perret, P., Li, Z. and Thomas, T. (2002) Arabidopsis ABI5 subfamily members have distinct DNA-binding and transcriptional activities. Plant Physiol. 130, 688697.
  • Kim, S., Kang, J., Cho, D., Park, J. and Kim, S. (2004) ABF2, an ABRE-binding bZIP factor, is an essential component of glucose signaling and its overexpression affects multiple stress tolerance. Plant J. 40, 7587.
  • Kobayashi, Y., Murata, M., Minami, H., Yamamoto, S., Kagaya, Y., Hobo, T., Yamamoto, A. and Hattori, T. (2005) Abscisic acid-activated SNRK2 protein kinases function in the gene-regulation pathway of ABA signal transduction by phosphorylating ABA response element-binding factors. Plant J. 44, 939949.
  • Lopez-Molina, L. and Chua, N. (2000) A null mutation in a bZIP factor confers ABA-insensitivity in Arabidopsis thaliana. Plant Cell Physiol. 41, 541547.
  • Ma, Y., Szostkiewicz, I., Korte, A., Moes, D., Yang, Y., Christmann, A. and Grill, E. (2009) Regulators of PP2C phosphatase activity function as abscisic acid sensors. Science, 324, 10641068.
  • Marcotte, W.J., Russell, S. and Quatrano, R. (1989) Abscisic acid-responsive sequences from the em gene of wheat. Plant Cell, 1, 969976.
  • Nakashima, K., Tran, L.S., Van Nguyen, D., Fujita, M., Maruyama, K., Todaka, D., Ito, Y., Hayashi, N., Shinozaki, K. and Yamaguchi-Shinozaki, K. (2007) Functional analysis of a NAC-type transcription factor OsNAC6 involved in abiotic and biotic stress-responsive gene expression in rice. Plant J. 51, 617630.
  • Nakashima, K., Fujita, Y., Kanamori, N. et al. (2009) Three Arabidopsis SnRK2 protein kinases, SRK2D/SnRK2.2, SRK2E/SnRK2.6/OST1 and SRK2I/SnRK2.3, involved in ABA signaling are essential for the control of seed development and dormancy. Plant Cell Physiol. 50, 13451363.
  • Nambara, E. and Marion-Poll, A. (2005) Abscisic acid biosynthesis and catabolism. Annu. Rev. Plant Biol. 56, 165185.
  • Narusaka, Y., Nakashima, K., Shinwari, Z.K., Sakuma, Y., Furihata, T., Abe, H., Narusaka, M., Shinozaki, K. and Yamaguchi-Shinozaki, K. (2003) Interaction between two cis-acting elements, ABRE and DRE, in ABA-dependent expression of Arabidopsis rd29A gene in response to dehydration and high-salinity stresses. Plant J. 34, 137148.
  • Nishimura, N., Yoshida, T., Kitahata, N., Asami, T., Shinozaki, K. and Hirayama, T. (2007) ABA-Hypersensitive Germination1 encodes a protein phosphatase 2C, an essential component of abscisic acid signaling in Arabidopsis seed. Plant J. 50, 935949.
  • Park, S., Fung, P., Nishimura, N. et al. (2009) Abscisic acid inhibits type 2C protein phosphatases via the PYR/PYL family of START proteins. Science, 324, 10681071.
  • Qin, F., Sakuma, Y., Tran, L.S. et al. (2008) Arabidopsis DREB2A-interacting proteins function as RING E3 ligases and negatively regulate plant drought stress-responsive gene expression. Plant Cell, 20, 16931707.
  • Ramanjulu, S. and Bartels, D. (2002) Drought- and desiccation-induced modulation of gene expression in plants. Plant Cell Environ. 25, 141151.
  • Rock, C. (2000) Pathways to abscisic acid-regulated gene expression. New Phytol. 148, 357396.
  • Santner, A. and Estelle, M. (2009) Recent advances and emerging trends in plant hormone signalling. Nature, 459, 10711078.
  • Schütze, K., Harter, K. and Chaban, C. (2008) Post-translational regulation of plant bZIP factors. Trends Plant Sci. 13, 247255.
  • Schweighofer, A., Hirt, H. and Meskiene, I. (2004) Plant PP2C phosphatases: emerging functions in stress signaling. Trends Plant Sci. 9, 236243.
  • Shen, Q. and Ho, T. (1995) 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. Plant Cell, 7, 295307.
  • Shen, Q., Zhang, P. and Ho, T. (1996) Modular nature of abscisic acid (ABA) response complexes: composite promoter units that are necessary and sufficient for ABA induction of gene expression in barley. Plant Cell, 8, 11071119.
  • Suzuki, M., Ketterling, M., Li, Q. and McCarty, D. (2003) Viviparous1 alters global gene expression patterns through regulation of abscisic acid signaling. Plant Physiol. 132, 16641677.
  • Uno, Y., Furihata, T., Abe, H., Yoshida, R., Shinozaki, K. and Yamaguchi-Shinozaki, K. (2000) Arabidopsis basic leucine zipper transcription factors involved in an abscisic acid-dependent signal transduction pathway under drought and high-salinity conditions. Proc. Natl Acad. Sci. USA, 97, 1163211637.
  • Usadel, B., Nagel, A., Steinhauser, D. et al. (2006) PageMan: an interactive ontology tool to generate, display, and annotate overview graphs for profiling experiments. BMC Bioinformatics, 7, 535.
  • Walter, M., Chaban, C., Schutze, K. et al. (2004) Visualization of protein interactions in living plant cells using bimolecular fluorescence complementation. Plant J. 40, 428438.
  • Xue, T., Wang, D., Zhang, S., Ehlting, J., Ni, F., Jakab, S., Zheng, C. and Zhong, Y. (2008) Genome-wide and expression analysis of protein phosphatase 2C in rice and Arabidopsis. BMC Genomics, 9, 550.
  • Yamaguchi-Shinozaki, K. and Shinozaki, K. (2006) Transcriptional regulatory networks in cellular responses and tolerance to dehydration and cold stresses. Annu. Rev. Plant Biol. 57, 781803.
  • Yoo, S., Cho, Y. and Sheen, J. (2007) Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis. Nat. Protoc. 2, 15651572.
  • Yoshida, T., Nishimura, N., Kitahata, N., Kuromori, T., Ito, T., Asami, T., Shinozaki, K. and Hirayama, T. (2006) ABA-hypersensitive germination3 encodes a protein phosphatase 2C (AtPP2CA) that strongly regulates abscisic acid signaling during germination among Arabidopsis protein phosphatase 2Cs. Plant Physiol. 140, 115126.
  • Zhang, W., Ruan, J., Ho, T., You, Y., Yu, T. and Quatrano, R. (2005) Cis-regulatory element based targeted gene finding: genome-wide identification of abscisic acid- and abiotic stress-responsive genes in Arabidopsis thaliana. Bioinformatics, 21, 30743081.
  • Zimmermann, P., Hirsch-Hoffmann, M., Hennig, L. and Gruissem, W. (2004) GENEVESTIGATOR. Arabidopsis microarray database and analysis toolbox. Plant Physiol. 136, 26212632.

Supporting Information

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Figure S1. Cellular localization of GFP-AREB1, GFP-AREB2, and GFP-ABF3 proteins in roots under stress conditions. Confocal images of GFP fluorescence in root tips (a) and root elongation zone (b) of AREB1pro:GFP-AREB1, AREB2pro:GFP-AREB2, and ABF3pro:GFP-ABF3 plants with or without 100 μm ABA or 250 mm NaCl treatment for 1 h. GFP fluorescence images and images merged with Nomarski images are shown. Bars = 50 μm.

Figure S2. Gene expression profiles of the rice AREB/ABF orthologs OsAREB1, OsAREB2, and OsAREB8 analyzed by using qRT-PCR. (a) An unrooted phylogenetic tree of AREB/ABF-like proteins. Phylogeny was calculated by a neighbor-joining method based on the alignment shown in (b) using only sites with no gaps. Bootstrap values (1000 replicates) are shown at branches with >50% support. (Bar = 0.05 substitutions per site). (b) A multiple alignment of conserved regions from AREB/ABF-like proteins of Arabidopsis, rice, Selaginella moellendorffii and Physcomitrella patens. Protein sequences corresponding to five conserved regions in AREB/ABF-like proteins are shown. Black, dark gray and light gray backgrounds indicate 100%, 80% and 60% conservation of similar residues, respectively. Asterisks and arrowheads indicate conserved phosphorylation motifs (R-X-X-S/T) and highly conserved residues in bZIP transcription factors, respectively. (c) For each gene, the expression level under nonstressed conditions was defined as 1.0. Data represent means and standard deviations of three replicate reactions.

Figure S3. BiFC visualization of AREB1, AREB2, or ABF3 interaction with SRK2D transiently expressed in onion epidermal cells. YFP and CFP fluorescence and merged images from the same field of transfected cells are shown for each transfection combination. A 35Spro:CFP (pGKX-CFP) control plasmid was always co-bombarded to identify transformed cells prior to the analysis of YFP fluorescence. In each transfected cell, YFP fluorescence was normalized against CFP fluorescence. Bars = 50 μm.

Figure S4. Gene expression patterns of transcription factors that function downstream of AREB/ABF transcription factors. The expression data for transcription factors, for which expression was significantly downregulated in the areb1 areb2 abf3 triple mutant, were collected from a public database, the eFP browser (http://bbc.botany.utoronto.ca/efp/cgi-bin/efpWeb.cgi). The expression data for At1g07900 were not available on the eFP browser. This figure displays a part of the data from the ‘abiotic stress’ category. The signal threshold of a log2 ratio was manually adjusted to 1. Signal intensities are shown below each figure.

Figure S5. Functional categorization of downregulated genes in the areb1 areb2 abf3 triple mutant. Downregulated genes in the areb1 areb2 abf3 triple mutant under stress conditions, for which expression ratios were greater than four times (P < 0.05), were categorized based on the Pageman program (Usadel et  al., 2006).

Table S1. Representative genes regulated by AREB1, AREB2, and ABF3, identified by microarray analysis of the areb1 areb2 abf3 triple mutant.

Table S2. Hierarchical categories significantly found in the downregulated gene sets in the areb1 areb2 abf3 triple mutant.

Table S3. Hierarchical categories significantly found in the upregulated gene sets in the areb1 areb2 abf3 triple mutant.

Table S4. Primer pairs used in this study.

Appendix S1. Supplementary experimental procedures.

As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer-reviewed and may be re-organized for online delivery, but are not copy-edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.

FilenameFormatSizeDescription
TPJ_4092_sm_R1AppendixS1_final.doc36KSupporting info item
TPJ_4092_sm_FigS1090810.tif3244KSupporting info item
TPJ_4092_sm_FigS2090810c.tif1030KSupporting info item
TPJ_4092_sm_FigS3090929.tif986KSupporting info item
TPJ_4092_sm_FigS4090810.tif3370KSupporting info item
TPJ_4092_sm_FigS5091112.tif789KSupporting info item
TPJ_4092_sm_Sup_Fig_Legends_final.doc24KSupporting info item
TPJ_4092_sm_TableS1_091112c.xls40KSupporting info item
TPJ_4092_sm_TableS2_091112d.xls23KSupporting info item
TPJ_4092_sm_TableS3_091112d.xls23KSupporting info item
TPJ_4092_sm_TableS4_091112_ab.xls24KSupporting info item

Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.