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

Abstract

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.

Figure 1

Base substitution analysis of the 77-bp region of the rd29B promoter involved in ABA- and dehydration-responsive expression in transgenic tobacco. GUS activity before and after treatment was measured in 20 independent transformants for each construct and is shown as average values. MU, 4-methyl-umbelliferone. (A) Upper-strand sequences of the 77-bp fragment (wild type) and its mutated sequences (M1-M6). The monomer (x1) or tandemly repeated dimer (x2) of each 77-bp fragment containing different mutations was ligated to the −51 rd29B minimal TATA promoter-GUS construct, respectively. Dashes indicate the wild-type sequence. (B) Effect of base substitutions in the ABRE and MYB recognition sites for dehydration-responsive expression of the rd29B. Half of the leaf from T1 tobacco plant was used immediately for the assay of GUS activity (control), and the other half was dehydrated for 24 h (dry). (C) Effect of ABA treatment on the induction of the GUS reporter gene driven by the 77-bp fragment. T2 tobacco seedlings were used immediately for the assay of GUS activity (control), transferred from agar plates for hydroponic growth in water (H₂O) or 100 μM ABA (ABA) solution, or dehydrated for 24 h (dry).

Figure 2

Isolation of cDNAs encoding ABRE-binding proteins by the yeast one-hybrid system. (A) Confirmation of five isolated cDNAs encoding ABRE-binding proteins by the yeast one-hybrid system. Five plasmids containing insert DNA from AREB1, AREB2, AREB3, GBF1, and GBF3 were retransformed into yeast strains carrying the reporter genes HIS3 and lacZ under the control of the 77-bp fragment containing two ABREs. The transformants were examined for growth in the presence of 3-AT and β-galactosidase (β-gal) activity. (B) Binding specificity of proteins encoded by isolated cDNAs. Five plasmids were used for transformation into yeast carrying the reporter genes under the control of the 77-bp fragment containing mutated ABREs. (C) Activation of reporter genes in yeast by proteins encoded by isolated cDNAs. The insert DNA fragments of the five isolated cDNA clones were cloned into the yeast expression vector YepGAP and used for transformation into yeast carrying the reporter genes under the control of the 77-bp fragment containing two ABREs.

Figure 3

Comparison of deduced amino acid sequences of the AREB 1, AREB2, and AREB3 proteins and sunflower DPBF1 (21). Symbols denote identical (*) and conserved (⋅) amino acid residues in the four sequences. The boxes represent conserved regions, and dashes indicate gaps introduced to maximize alignment. Inverted characters indicates consensus sequences for various kinases (R/KXXS/T for CDPK, S/TXXD/E for CK II, S/TXK/R for PKC, K/RXXXS/T for cGMP-dependent protein kinase) appeared within the conserved regions.

Figure 4

(A) Expression of the AREB1, AREB2, and rd29B genes in response to dehydration, low temperature, high salt, or ABA. Each lane was loaded with 40 μg of total RNA from 3-week-old Arabidopsis plants that had been dehydrated (Dry), transferred to 4°C (Cold), transferred to hydroponic growth in 250 mM NaCl (NaCl), transferred to hydroponic growth in 100 μM ABA (ABA), or transferred to water (H₂O), as described in Materials and Methods. The number above each lane indicates the number of minutes or hours after the initiation of the treatment. rRNAs blotted on the membrane were visualized by staining with methylene blue.

Figure 5

Transactivation of the rd29B promoter-GUS fusion gene by AREB1 and AREB2 proteins using Arabidopsis protoplasts. (A) Expression of rd29B in response to dehydration, low temperature, high salt, or ABA in wild type (Columbia & Landsberg) or ABA-related mutants (aba2, abi1, and abi3). Plants were untreated (C), dehydrated for 10 h (D), or treated with water (H), 250 mM NaCl (N) or 100 μM ABA (A) for 5 h. (B) Schematic diagram of the effector and reporter constructs used in cotransfection experiments. The effector constructs contain the CaMV 35S promoter and TMV Ω sequence fused to AREB1, AREB2, or GBF3 cDNAs. The reporter constructs contain the 77-bp fragments of the rd29B promoter connected tandemly five times (×5) or single (×1). The promoters were fused to the −51 rd29B minimal TATA promoter-GUS construct. (C) Transactivation of the rd29B promoter-GUS fusion gene by AREB1, AREB2, and GBF3 proteins. The reporter gene driven by the 77-bp fragments tandemly repeated five times was transfected with each effector plasmid or the vector as control treatments. Transactivation experiments using protoplasts prepared from wild type or ABA-mutant (abi1, aba2, and era1) Arabidopsis leaves. To normalize for transfection efficiency, the CaMV 35S promoter-luciferase (LUC) plasmid was cotransfected in each experiment. Bars indicate the standard error of three replicates. Ratios indicate the multiples of expression compared with the value obtained with the pBI35SΩ vector, and ratio in parentheses indicates the multiples of expression compared with value obtained with the wild type transformed with pBI35SΩ vector. (D) Transactivation using the reporter construct containing the single 77-bp fragment.

Figure 6

ABA-dependent phosphorylation of recombinant AREB proteins by in-gel kinase activity assay. (A) T87 cell extracts treated with or without 100 μM ABA treatment were resolved on 10% polyacrylamide gel containing recombinant AREB proteins (AREB1a: 73G-131Q; AREB2a: 84S-133D; AREB2 bZIP domain: 335Y-401V). The protein kinase activities were analyzed as described in Materials and Methods. (B) Accumulation of the rd29B, AREB1, and AREB2 mRNAs in T87 culture cells. The rd29B mRNA was detected by reverse transcription–PCR, and the AREB1 and AREB2 mRNAs were detected by RNA gel blot analysis.