Dissection of Arabidopsis Bax inhibitor-1 suppressing Bax-, hydrogen peroxide-, and salicylic acid-induced cell death

Abstract

Overexpression of plant Bax Inhibitor-1 (BI-1) was able to suppress Bax-mediated cell death in yeast and Arabidopsis. Here, we demonstrate that reactive oxygen species production induced by the ectopic expression of Bax was insensitive to the coexpression of AtBI-1. Similarly, H2O2- or salicylic acid-mediated cell death also was suppressed in tobacco BY-2 cells overexpressing AtBI-1. To define the functional domain of AtBI-1 as a cell death suppressor, a truncated series of the AtBI-1 protein was analyzed in yeast possessing a galactose-inducible mammalian Bax. The results showed that DeltaC-AtBI-1 (with the C-terminal 14 amino acids deleted) lost the ability to sustain cell growth. Furthermore, a mutant protein in which the C-terminal seven amino acid residues of AtBI-1 were replaced with others lacking a coiled-coil structure failed to inhibit cell death, suggesting that the C-terminal region is essential for the inhibition of cell death. We also noted that the C-terminal hydrophilic region was interchangeable between animal and plant Bax inhibitors.

Figure 1.

Cytological Observation of ROS Production in Plants Expressing Bax. To detect superoxide radical (O₂⁻), 3-week-old transgenic Arabidopsis plants treated with DEX were stained with NBT. Each plant was treated with 5 μM DEX for 7 h to induce Bax. Detached leaves of non-DEX-treated ([A], [D], and [G]) or DEX-treated transgenic plants possessing Bax ([B], [E], and [H]) or DEX-treated Bax+AtBI-GFP plants ([C], [F], and [I]) were infiltrated with NBT solution. After the stained leaves were boiled in acetic acid:glycerol:ethanol to remove the chlorophyll, evidence of ROS production was observed with a light microscope. (D) to (I) show magnified images of (A) to (C). Formazan precipitates are indicated with arrowheads in (H) and (I). The presence of the formazan precipitate indicates the location of O₂⁻. The experiment was repeated at least three times, and representative photographs are presented.

Figure 2.

Suppression of Ion Leakage from Detached Leaves of Transgenic Arabidopsis Expressing Both Bax and AtBI-1. (A) Three-week-old Bax and Bax+AtBI-GFP1 transgenic plants were used in the experiments. Detached leaves were incubated with (+) or without (−) 5 μM DEX in distilled water at 23°C for 4 days, and photographs were taken. (B) Detached leaves from transgenic plants containing pTA7002 (vector control; pTA), pTA-Bax (Bax), Bin-AtBI-GFP (AtBI-GFP), or both pTA-Bax and Bin-AtBI-GFP (Bax+AtBI-GFP1 to Bax+AtBI-GFP3) were incubated with 5 μM DEX in distilled water at 23°C. Bax+AtBI-GFP1 to Bax+AtBI-GFP3 are independent lines. The electrical conductivity of the solution was measured with a conductivity meter and is indicated as a relative value. n = 5.

Figure 3.

Inhibition of H₂O₂-Induced Cell Death by AtBI-1. (A) Induction of cell death by exogenously supplied H₂O₂ in tobacco BY-2 suspension cells. Evans blue treatment was performed at 0, 2, 6, 10, and 20 h after the addition of 3 mM H₂O₂, and dead cells showing shrunken blue cytoplasm were scored with a light microscope. Data shown are means ± SE of three experiments. (B) Comparison of AtBI-1 (AtBI-GFP2) and control (GFP1) cells stained with Evans blue at 18 h after treatment with 3 mM H₂O₂. (C) Effects of AtBI-1 or ΔC-AtBI-1 on H₂O₂-induced cell death. Tobacco BY-2 cells expressing GFP, AtBI-GFP, or ΔC-AtBI-GFP were incubated with H₂O₂ (0 to 4 mM) for 18 h. Dead cells were scored, and the results are expressed as means ± sd of at least three experiments. WT, wild type. (D) Turnover of H₂O₂ after its addition to transgenic cell lines expressing GFP, AtBI-GFP, or ΔC-AtBI-GFP. One millimolar H₂O₂ was added to each cell line, and the H₂O₂ concentration of the medium was measured after 5 min. Data shown are means ± SE of three experiments. gfw, grams fresh weight.

Figure 4.

Inhibition of SA-Induced Cell Death by AtBI-1. Tobacco BY-2 cells expressing GFP, AtBI-GFP, or ΔC-AtBI-GFP were treated with 500 μM (A) or 750 μM (B) SA and then cultured for 18 h before examination with Evans blue staining. Dead cells stained with Evans blue were scored, and values are expressed as percentages. Data shown are means ± SE of more than three replications. (C) shows microscopic images of cells at 18 h after 750 μM SA treatment. WT, wild type.

Figure 5.

Accumulation of AtBI-1 mRNA in Arabidopsis Cells under Oxidative Stress or in NahG Transgenic Plants. (A) Arabidopsis suspension cells (3 days old) were treated with H₂O₂ (1 and 3 mM) or SA (500 and 750 μM) for 24 h under continuous shaking. Equal amounts of total RNAs (10 μg) were electrophoresed on a 1.2% formaldehyde agarose gel and transferred to a membrane. An α-³²P-dCTP–labeled 3′ untranslated region of AtBI-1 cDNA was used as a probe. (B) Analysis of AtBI-1 mRNA accumulation in NahG transgenic plants. Total RNAs (20 μg) from 2-week-old wild-type (WT) and NahG transgenic plants were electrophoresed and hybridized. The middle panels in (A) and (B) show gels stained with ethidium bromide for rRNA. The bottom panels show relative levels of AtBI-1 expression in Arabidopsis suspension cells treated with H₂O₂ and SA (A) and NahG transgenic plants (B). Quantitative measurements of RNA gel blots were performed using the BAS1500 imaging plate scanner.

Figure 6.

Evaluation of Cell Death Inhibition Activity in a Truncated Series of AtBI-1 Proteins to Bax. (A) Schemes of truncated AtBI-1 proteins. ΔN-, ΔC-, and ΔNC-AtBI-1 were constructed using PCR as described in Methods. The red bar shown in the C-terminal region of AtBI-1 indicates the conserved domain exhibited in (C). The results of cell death suppression from (B) are shown at right. ++, suppressed; + weakly suppressed; −, not suppressed. TM1 to TM7, transmembrane domains 1 to 7. (B) The full-length AtBI-1 (AtBI), constructed AtBI-1 mutants (ΔN, ΔC, and ΔNC), and the empty vector pYX112 (pYX) were transformed to a yeast strain possessing galactose-inducible mammalian Bax and streaked on glucose (Glu)- or galactose (Gal)-containing medium. The photograph was taken after 3 days of incubation at 30°C. (C) Amino acid comparisons of the C-terminal region of BI-1 proteins from various organisms. AtBI-1, Arabidopsis thaliana (Kawai et al., 1999); OsBI-1, Oryza sativa (Kawai et al., 1999); BnBI-1, Brassica napus; NtBI-1, Nicotiana tabacum; HvBI-1, Hordeum vulgare; HsBI-1, Homo sapiens; RnBI-1, Rattus norvegicus; MmBI-1, Mus musculus; PoBI-1, Paralichthys olivaceus. Red letters (K and R) indicate positively charged amino acids. Blue letters (D and E) indicate negatively charged amino acids. (D) The probability of forming the coiled-coil structure calculated using the COILs program (available at http://www.ch.embnet.org/software/COILS_form.html). A putative coiled-coil region is predicted in the C-terminal region of the AtBI-1 protein. aa, amino acids.

Figure 7.

Localization of a C-Terminal Truncated AtBI-GFP (ΔC-AtBI-GFP) Protein in Yeast and Plant Cells. Distribution of GFP ([A], [D], [G], and [J]), AtBI-GFP ([B], [E], [H], and [K]), and ΔC-AtBI-GFP ([C], [F], [I], and [L]) in yeast ([A] to [F]) or tobacco BY-2 ([G] to [L]) was observed with a fluorescence microscope (DMRD; Leica) ([A] to [F]) or a confocal laser scanning microscope (BX50; Olympus) ([G] to [L]). Fluorescence ([A] to [C] and [G] to [I]) and bright-field ([D] to [F] and [J] to [L]) images are displayed.

Figure 8.

Comparison of the Cell Death–Suppression Activity of C-Terminal Mutants of AtBI-1. (A) Various mutations of the C terminus of AtBI-1 were constructed, and their ability to suppress Bax-induced cell death was analyzed. Mutated series of AtBI-1 (AtBI28 to AtBI33) were constructed using PCR as described in Methods. The amino acids replaced are underlined. The estimated charges (+, −, or none) of the replaced regions and calculated scores for the coiled-coil structure also are indicated. In AtBI33, the C-terminal 14 amino acids were replaced with the 9 amino acids of human BI-1. (B) Spot assay of cell death–suppression activity in yeast. Yeast transformed with plasmids containing mutagenized AtBI-1 genes were cultured in SD medium containing glucose. After 1 day of shaking, the OD₆₀₀ of each culture was adjusted to 0.2 and diluted to 0.0002. Each dilution was spotted on SD medium containing glucose (Glu) or galactose (Gal). The results show the growth of each line after 2 days (Glu) and 4 days (Gal) of incubation at 30°C. The results of cell death suppression are shown at right: +, suppressed; −, weak suppression or not suppressed.

Figure 9.

Scheme of the Role of the BI-1 Protein in the ROS-Induced Cell Death Pathway. ROS cause mitochondrial permeability transition, ATP depletion, release of cytochrome c from the mitochondrial membranes, and lipid peroxidation, which cause ion leakage and cell death (Rao and Davis, 1999; Houot et al., 2001; Maxwell et al., 2002; Tiwari et al., 2002). BI-1 proteins, as components of the ER membrane, are involved in counteracting “oxidative stress.”