Convergence of Light and ABA signaling on the ABI5 promoter

Abstract

Light is one of the most important environmental cues regulating multiple aspects of plant growth and development, and abscisic acid (ABA) is a plant hormone that plays important roles during many phases of the plant life cycle and in plants' responses to various environmental stresses. How plants integrate the external light signal with endogenous ABA pathway for better adaptation and survival remains poorly understood. Here, we show that BBX21 (also known as SALT TOLERANCE HOMOLOG 2), a B-box (BBX) protein previously shown to positively regulate seedling photomorphogenesis, is also involved in ABA signaling. Our genetic data show that BBX21 may act upstream of several ABA INSENSITIVE (ABI) genes and ELONGATED HYPOCOTYL 5 (HY5) in ABA control of seed germination. Previous studies showed that HY5 acts as a direct activator of ABI5 expression, and that BBX21 interacts with HY5. We further demonstrate that BBX21 negatively regulates ABI5 expression by interfering with HY5 binding to the ABI5 promoter. In addition, ABI5 was shown to directly activate its own expression, whereas BBX21 negatively regulates this activity by directly interacting with ABI5. Together, our study indicates that BBX21 coordinates with HY5 and ABI5 on the ABI5 promoter and that these transcriptional regulators work in concert to integrate light and ABA signaling in Arabidopsis thaliana.

Figure 1. The bbx21 mutants are hypersensitive to ABA and NaCl during germination.

(A) ABA and NaCl hypersensitivity phenotypes of bbx21-1 mutants. All seedlings were grown vertically on germination medium (GM) plates with or without 1 µM ABA or 100 mM NaCl for 7 d after stratification. (B) Root lengths of the wild type (Col) and bbx21-1 grown on GM with or without 1 µM ABA or 100 mM NaCl. Relative root lengths compared with those of Col grown on GM plates are indicated. Values are means ± SD (n = 20). ***P<0.001 (Student's t test) for the differences between bbx21-1 and the wild type. (C–G) Germination rates of the bbx21-1 (Col) and bbx21-2 (Ler) mutants and their corresponding wild type controls under mock (C) and various concentrations of ABA (D and E) or NaCl (F and G) treatments. Germination rate was determined from three replicates (>150 seeds from each genotype), and error bars represent SD.

Figure 2. The bbx21 mutants lost water more slowly than did the wild type plants.

(A–B) Water loss from the detached leaves of the wild type, bbx21-1, and bbx21-2. Results are means of three replicates, and error bars represent SD. (C) Representative images of stomata of wild type and bbx21 mutant plants treated with mock or 0.5 µM ABA. Bar = 10 µm. (D) Ratios of stomatal aperture length to width. Three independent experiments were performed with similar results. Data were from one experiment with 30 stomata cells from leaves of three different plants with triple replicates. Data are means ±SEs. **P<0.01 (Student's t test) for the differences between bbx21 and the wild type.

Figure 3. Expression pattern of BBX21.

(A) Expression pattern of BBX21 during germination. (B) The expression levels of BBX21 in the wild type and aba1 mutants. Data are means of three independent experiments, and error bars represent SD.

Figure 4. Germination rates of bbx21, abi and abi bbx21 mutants.

Germination rates of the bbx21, abi2, abi3, abi4, abi5 and abi bbx21 mutants under mock (A, C, E and G) and 5 µM of ABA (B, D, F and H) treatments. Germination rate was determined from three replicates (>150 seeds from each genotype), and error bars represent SD.

Figure 5. The hy5 bbx21 mutants are insensitive to ABA during germination.

Germination rates of Col, bbx21-1, hy5-215 and hy5-215 bbx21-1 mutants under mock (A) and 5 µM ABA (B) treatments. Error bars represent SD.

Figure 6. BBX21 regulates the expression of ABI3 and ABI5.

Expression levels of ABI3 (A) and ABI5 (B) in 2-d-old germinating seeds of Col, hy5-215, bbx21-1, and hy5-215 bbx21-1 mutant treated with mock or 0.5 µM ABA.

Figure 7. BBX21 negatively regulates ABI5 expression by interfering with HY5 binding to the ABI5 promoter.

(A) Schematic representation of constructs used in the transient transfection assays in Arabidopsis protoplasts. Arrows after the 35S promoter indicate the transcription start site, and −2 kb indicate the length of the ABI5 promoter that was fused to the firefly luciferase to create the reporter construct. (B) Activation of ABI5p:LUC by indicated combinations of proteins. Error bars represent SE (n = 3). (C) EMSAs showing that increasing amounts of His-BBX21 protein (lanes 2 to 6) decreased the binding of GST-HY5 to the ABI5 promoter.

Figure 8. ABI5 directly binds to its own promoter.

(A) Diagram of the promoter fragments of the ABI5 promoter and the sequence of a subfragment containing three G-box motifs in the C fragment. The adenine residue of the translational start codon (ATG) was assigned position +1, and the numbers flanking the sequences of the subfragment were counted based on this number. A, B, C, and D indicate the corresponding promoter fragments used in yeast one-hybrid assays shown in (B). (B) Yeast one hybrid assays showing that ABI5 binds to the C fragments of its own promoter. Empty vector expressing AD domain alone was used as negative controls. (C) Diagram of the wild type (wt) and mutant (mu) ABI5 subfragments used to drive LacZ reporter gene expression in yeast one-hybrid assays. Wild type G-box elements are shown in red, and nucleotide substitutions in the mutant fragments are underlined. (D) Yeast one-hybrid assays showing that all three G-box motifs mediate ABI5 binding to its promoter. The subfragment of the ABI5 promoter was mutated to abolish G-box1, 2, 3 alone, or both G-box2 and 3, and used to drive LacZ reporter gene expression. In these assays, the respective CACGTG was mutated to CttttG to facilitate mutagenesis reactions.

Figure 9. BBX21 physically interacts with ABI5.

(A) Schematic representation of the domain structures of BBX21 showing mutations in the B-box domain. (B) Yeast two-hybrid interactions between BBX21 and ABI5 proteins. Error bars indicate SE (n = 3). (C) In vitro pull down of ABI5 with BBX21. The GST-ABI5 proteins pulled down with 6×His-BBX21 were detected by anti-GST antibody. Input, 5% of the purified GST-tagged target proteins used in pull-down assays.

Figure 10. A working model depicting how BBX21 works in concert with HY5 and ABI5 on the ABI5 promoter to integrate light and ABA signaling.

HY5 acts as a transcriptional activator of ABI5 expression . ABI5 directly binds to its own promoter through three typical G-box motifs, and activates the expression of itself. BBX21 acts as a negative regulator of ABI5 expression, possibly by interfering with the binding of HY5 and ABI5 to the ABI5 promoter. ABI5 may play a positive role in photomorphogenesis .