AREB1 is a transcription activator of novel ABRE-dependent ABA signaling that enhances drought stress tolerance in Arabidopsis

Abstract

ABSCISIC ACID-RESPONSIVE ELEMENT BINDING PROTEIN1 (AREB1) (i.e., ABF2) is a basic domain/leucine zipper transcription factor that binds to the abscisic acid (ABA)-responsive element (ABRE) motif in the promoter region of ABA-inducible genes. Here, we show that expression of the intact AREB1 gene on its own is insufficient to lead to expression of downstream genes under normal growth conditions. To overcome the masked transactivation activity of AREB1, we created an activated form of AREB1 (AREB1DeltaQT). AREB1DeltaQT-overexpressing plants showed ABA hypersensitivity and enhanced drought tolerance, and eight genes with two or more ABRE motifs in the promoter regions in two groups were greatly upregulated: late embryogenesis abundant class genes and ABA- and drought stress-inducible regulatory genes. By contrast, an areb1 null mutant and a dominant loss-of-function mutant of AREB1 (AREB1:RD) with a repression domain exhibited ABA insensitivity. Furthermore, AREB1:RD plants displayed reduced survival under dehydration, and three of the eight greatly upregulated genes were downregulated, including genes for linker histone H1 and AAA ATPase, which govern gene expression and multiple cellular activities through protein folding, respectively. Thus, these data suggest that AREB1 regulates novel ABRE-dependent ABA signaling that enhances drought tolerance in vegetative tissues.

Figure 1.

Expression of the AREB1 Gene and Subcellular Localization of the AREB1 Protein. (A) Structure of AREB1 family proteins. NLS, nuclear localization signal. C1 to C4 indicate conserved domains within the family. (B) Expression profiles of AREB family genes in response to dehydration, high salt, or ABA. Each lane was loaded with 20 μg of total RNA from 3-week-old Arabidopsis plants that had been dehydrated (DRY), transferred to hydroponic growth in 250 mM NaCl (NaCl), transferred to hydroponic growth in 100 μM ABA (ABA), or transferred to water (H₂O). rRNAs are shown as equal loading controls. A band located in the center of each column indicates a transcript that corresponds to each gene. (C) Phylogenetic tree of AREB family proteins. Proteins were aligned using ClustalX software, and the tree was constructed using MEGA software. (D) Nuclear localization of AREB1 protein in onion epidermal cells: fluorescent images of GFP, fluorescent images stained with propidium iodide (PI), and merged images (GFP/PI). (E) Patterns of AREB1 promoter-driven GUS expression in seedlings at different ages or in different tissues: (a) 2-d-old seedling, (b) 5-d-old seedling, (c) cotyledon, (d) primary leaf, (e) 2-week-old seedling, (f) 2-week-old seedling treated with 50 μM ABA, (g) flower, (h) immature silique, (i) seeds from (h). Bars = 0.5 mm in (a) to (d) and (g) to (i) and 5.0 mm in (e) and (f). (F) Expression of AREB1 and RD29B in wild-type and 35S-AREB1 plants (line 6) induced by 50 μM ABA treatment. Representative data are shown. Each lane was loaded with 15 μg of total RNA from 2-week-old Arabidopsis plants. rRNAs are shown as equal loading controls.

Figure 2.

The N-Terminal Conserved Region of AREB1 Functions as a Transcriptional Activation Domain in Protoplasts Derived from Arabidopsis T87 Cultured Cells. All transactivation experiments were performed 3 to 10 times, and results from one representative experiment are shown. Bars indicate standard deviation; n = 3 to 5. (A) Scheme of the effector and reporter constructs used in the transactivation analysis with AREB1 bZIP DNA binding domain. The effector constructs contain the CaMV 35S promoter and TMV Ω sequence fused to AREB1 cDNA fragments encoding different portions of AREB1. The reporter construct, RD29B-GUS, contains 77-bp fragments of the RD29B promoter connected tandemly five times. The promoters were fused to the −51 RD29B minimal TATA promoter–GUS construct. Nos-T, nopaline synthase terminator. (B) Transactivation domain analysis of AREB1 using N-terminal deletion constructs. Protoplasts were cotransfected with the RD29B-GUS reporter and the effector construct (shown on the left) carrying an N-terminal truncated form of AREB1 cDNA or pBI-35SΩ (vector). To normalize for transfection efficiency, the pBI35SΩ-LUC reporter was cotransfected as a control in each experiment. Bars indicate standard deviation of three replicates. “Relative activity” indicates the multiples of expression compared with the value obtained with the pBI221-35SΩ vector control. Top numbers indicate amino acid numbers of AREB1. P, Q, R, S, T, and U indicate the partial region of the AREB1 cDNA. The region P, Q, R, or U includes a conserved domain (solid rectangle), C1, C2, C3, or C4, respectively, in Figure 1A. (C) Schematic diagram of the effector and reporter constructs used in the transactivation analysis with the GAL4 DNA binding domain. The effector plasmids encoding the GAL4 DNA binding domain are fused to AREB1 cDNA fragments encoding different portions of AREB1. The GUS reporter construct, GAL4-GUS, containing GAL4 binding sites, is fused to the minimal promoter of CaMV 35S. (D) N-terminal conserved P region of AREB1 contains sufficient domain for transcriptional activation. Protoplasts were cotransfected with the GAL4-GUS reporter and the effector construct expressing a portion of AREB1 or the vector DNA. (E) AREB1ΔQT is a constitutive active form of AREB1. Protoplasts were cotransfected with the RD29B-GUS reporter and the effector construct expressing intact AREB1, AREB1ΔQT, or AREB1ΔP/RT, or vector DNA.

Figure 3.

AREB1ΔQT Is a Constitutive Active Form of AREB1 in Planta. (A) RNA gel blot analysis of AREB1 and RD29B expression in wild-type, vector control (vector), 35S-AREB1, and 35S-AREB1ΔQT plants. Two-week-old seedlings were either not treated (−) or treated (+) with ABA for 7 h. Each lane contained 10 μg of total RNA. Two lines of the 35S-AREB1 plants (3 and 6) and three lines of the 35S-AREB1ΔQT plants (5, 12, and 26) are shown. rRNAs on ethidium bromide–stained gel are shown as equal loading controls. (B) Growth phenotype of 35S-AREB1ΔQT (line 5) and 35S-AREB1 (line 6) plants that were grown for 3 weeks on GM agar plates containing 1% sucrose. (C) Maximum rosette radius (i.e., length of the longest rosette leaf) of each plant on a GM agar plate containing 3% sucrose was measured 3 weeks after stratification. Three independent lines of wild-type plants and nine independent lines of 35S-AREB1ΔQT plants were used. Bars indicate standard deviation; n = 7. (D) Expression profile of downstream genes identified by microarray analysis (Table 1) in 35S-AREB1ΔQT plants (line 5). Two-week-old seedlings were either not treated (−) or treated (+) with ABA for 7 h. Each lane contained 7 μg of total RNA. Three to eight independent lines were used, and results from one representative experiment are shown. rRNAs on ethidium bromide–stained gel are shown as equal loading controls.

Figure 4.

35S-AREB1ΔQT Plants Are Hypersensitive to ABA. (A) Growth of 35S-AREB1ΔQT (line 5) and 35S-AREB1 (line 6) plants on GM agar plates containing 0, 0.5, or 1.0 μM ABA. Seeds were germinated and grown on GM agar plates for 6 d; representative plants are shown. (B) ABA dose response of root growth. Seeds were germinated and grown on GM agar plates containing various concentrations of ABA and 1% sucrose. Root elongation was measured 6 d after stratification, and relative growth compared with that on ABA-free medium is indicated. Bars indicate standard deviation; n = 26 to 38. The experiments were performed three or more times, sometimes using different transgenic lines, and the results were consistent.

Figure 5.

Enhanced Drought Tolerance in 35S-AREB1ΔQT Plants. (A) Enhanced tolerance to drought in the 35S-AREB1ΔQT plants (lines 12 and 26). Watering was withheld from 3-week-old plants for 12 d, then rewatering for 10 d, before the photograph was taken. Number codes indicate number of surviving plants out of total number. (B) Enhanced ability of 35S-AREB1ΔQT plants (line 12) to survive the dehydration condition. Three-week-old transgenic and wild-type plants were grown on GM agar plates, transferred to Petri dishes, left unwatered for 6 h, and then rewatered. (C) Increased survival rates of the 35S-AREB1ΔQT plants (lines 12 and 26) under dehydration. Water was withheld for 5 to 6 h from 3-week-old plants and then survival rates were counted. Surviving plants were scored on the second day. Survival rates and standard deviations (bars) were calculated from results of three independent experiments. (D) Water loss rates of 35S-AREB1ΔQT (lines 12 and 26) plants. Each data point represents the mean of duplicate measurements (n = 7 each). Error bars represent standard deviation. (E) Standardized water content of 35S-AREB1ΔQT (lines 12 and 26) plants. Details as in (D). Some error bars are smaller than the symbols. (F) Stomatal aperture of 35S-AREB1ΔQT plants (line 12). Stomatal guard cells were observed in the middle of the watering cycle.

Figure 6.

Analysis of AREB1 Loss-of-Function Mutants. (A) Scheme of the Arabidopsis AREB1 gene. Exons (open boxes) and introns (lines) are indicated. The position of the T-DNA insertion is shown (not to scale). (B) Schematic representation of the 35S-AREB1:RD construct used for expression of the chimeric repressor with a modified version of the EAR-motif RD (SRDX), consisting of 12 peptides. (C) Expression levels of AREB1 in the areb1 knockout mutant were determined by RT-PCR using total RNAs isolated from 2-week-old plants with or without 6-h treatment of 100 μM ABA or dehydration and grown on GM agar plates. Primers for detection of a truncated form of AREB1 mRNA, generated by T-DNA insertion into the first intron of AREB1, have T-DNA– and AREB1-specific binding sequences. The arrow and asterisks indicate expression of AREB1 and a larger band, respectively. TUB1, β-1 tubulin transcript as a control. (D) RNA gel blot analysis of AREB1 mRNA in wild-type and 35S-AREB1:RD plants (lines 4 and 8) in the absence or presence of 50 μM ABA for 7 h. Eight micrograms of total RNA from 3-week-old seedlings was probed with AREB1 cDNA. (E) Growth of the mutant areb1 and 35S-AREB1:RD plants (line 4) on GM agar plates containing 0, 1.0, or 3.0 μM ABA, supplemented with 1% sucrose. Seeds were germinated and grown on the medium for 2 weeks. Bars = 25 mm. (F) ABA dose response of primary root growth. Seeds were geminated and grown on GM plates containing 0.25% Gelrite, 1% sucrose, and various concentrations of ABA. Primary root elongation was measured 19 d after stratification, and relative growth compared with that on ABA-free medium is indicated. Bars indicate standard deviation; n = 15 to 31. (G) Growth phenotypes of areb1 and 35S-AREB1:RD (line 4) plants that were grown for 3 weeks on GM agar plates supplemented with 1% sucrose. (H) Maximum rosette radius (i.e., length of the longest rosette leaf) of each plant on a GM agar plate containing 3% sucrose was measured 3 weeks after stratification. Three independent lines of wild-type plants, one line of the areb1 T-DNA insertion mutant, and seven independent lines of 35S-AREB1:RD plants were used. Bars indicate standard deviation; n = 7. (I) The fusion of the RD to AREB1 creates a repressor. Arabidopsis protoplasts were cotransfected with the RD29B-GUS reporter and the effector construct expressing AREB1 or AREB1:RD, or vector DNA. The RD29B-GUS reporter plasmid and the transient assay system are described in the legend of Figure 2. (J) Expression profile of downstream genes identified by microarray analysis (Table 1) in 35S-AREB1:RD plants (line 4). Two-week-old seedlings were either not treated (−) or treated (+) with ABA for 7 h. Each lane contained 7 μg of total RNA. Three to eight independent lines were used; results from one representative experiment are shown. (K) Difference in recovery after rehydration among 35S-AREB1ΔQT (line 12), 35S-AREB1:RD (line 4), and wild-type plants. Transgenic and wild-type plants were grown on GM agar plates for 2 weeks, transferred to Petri dishes, left unwatered for 4 h, and then rewatered. The photograph was taken 2 d after rewatering. (L) Quantification of the survival rates of the wild-type and 35S-AREB1:RD plants (lines 4 and 8) after rehydration. Water was withheld for 5 h from 3-week-old plants and then survival rates were counted. Surviving plants were scored on the second day. Survival rates and standard deviations were calculated from the results of four independent experiments (n = 10 each). Bars indicate standard deviations.

Figure 7.

A Model of the Regulation of ABA Signaling by AREB1. AREB1 is postulated to mainly regulate the expression of stress-responsive genes via ABRE sequences in vegetative tissues.