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

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.     

The Bax lnhibitor-1 needs a functional electron transport chain for cell death suppression

Bax inhibitor-1 (BI-1) is an evolutionarily conserved cell death suppresser in animals, yeast, and plants. In this study, yeast strains carrying single-gene deletions were screened for factors related to cell death suppression by Arabidopsis BI-1 (AtBI-1). Our screen identified mutants that failed to survive Bax-induced lethality even with AtBI-1 coexpression (Bax suppressor). The Deltacox16 strain was isolated as a BI-1-inactive mutant; it was disrupted in a component of the mitochondrial cytochrome c oxidase. Other mutants defective in mitochondrial electron transport showed a similar phenotype. ATP levels were markedly decreased in all these mutants, suggesting that BI-1 requires normal electron transport activity to suppress cell death in yeast.     

Cell death suppressor Arabidopsis bax inhibitor-1 is associated with calmodulin binding and ion homeostasis

Cell death suppressor Bax inhibitor-1 (BI-1), an endoplasmic reticulum membrane protein, exists in a wide range of organisms. The split-ubiquitin system, overlay assay, and bimolecular fluorescence complementation analysis demonstrated that Arabidopsis (Arabidopsis thaliana) BI-1 (AtBI-1) interacted with calmodulin in yeast (Saccharomyces cerevisiae) and in plant cells. Furthermore, AtBI-1 failed to rescue yeast mutants lacking Ca2+ ATPase (Pmr1 or Spf1) from Bax-induced cell death. Pmr1 and Spf1, p-type ATPases localized at the inner membrane, are believed to be involved in transmembrane movement of calcium ions in yeast. Thus, the presence of intact Ca2+ ATPases was essential for AtBI-1-mediated cell death suppression in yeast. To investigate the effect of AtBI-1 on calcium homeostasis, we evaluated sensitivity against cyclopiazonic acid (CPA), an inhibitor of sarcoplasmic/endoplasmic reticulum Ca2+ ATPase in AtBI-1-overexpressing or knock-down transgenic Arabidopsis plants. These plants demonstrated altered CPA or ion stress sensitivity. Furthermore, AtBI-1-overexpressing cells demonstrated an attenuated rise in cytosolic calcium following CPA or H2O2 treatment, suggesting that AtBI-1 affects ion homeostasis in plant cell death regulation.     

Identification of novel mitochondrial membrane protein (Cdf 3) from Arabidopsis thaliana and its functional analysis in a yeast system

We screened the Arabidopsis cDNA library to identify functional suppressors of AtBI-1, a gene that suppresses cell death induced by Bax gene expression in yeast. Cdf 3 encodes a 118-amino-acid protein with a molecular mass of 25 kDa. This protein has two uncharacterized domains at amino acids residues 5-64 and 74-117. In the present study, CDF3 was found to induce growth defects in yeast and arrested yeast growth, although the cell-growth defect was somewhat less than that of Bax. Its localization in the inner mitochondria was essential for suppression of yeast-cell proliferation. The morphological abnormality of the intracellular network, which is a hallmark of AtBI-1, was attenuated by Cdf 3 expression.     

Functional association of cell death suppressor, Arabidopsis Bax inhibitor-1, with fatty acid 2-hydroxylation through cytochrome b5

Bax inhibitor-1 (BI-1) is a widely conserved cytoprotective protein localized in the endoplasmic reticulum (ER) membrane. We identified Arabidopsis cytochrome  b ₅ (AtCb5) as an interactor of Arabidopsis BI-1 (AtBI-1) by screening the Arabidopsis cDNA library with the split-ubiquitin yeast two-hybrid (suY2H) system. Cb5 is an electron transfer protein localized mainly in the ER membrane. In addition, a bimolecular fluorescence complementation (BiFC) assay and fluorescence resonance energy transfer (FRET) analysis confirmed that AtBI-1 interacted with AtCb5 in plants. On the other hand, we found that the AtBI-1-mediated suppression of cell death in yeast requires Saccharomyces cerevisiae fatty acid hydroxylase 1 (ScFAH1), which had a Cb5-like domain at the N terminus and interacted with AtBI-1. ScFAH1 is a sphingolipid fatty acid 2-hydroxylase localized in the ER membrane. In contrast, AtFAH1 and AtFAH2, which are functional ScFAH1 homologues in Arabidopsis, had no Cb5-like domain, and instead interacted with AtCb5 in plants. These results suggest that AtBI-1 interacts with AtFAHs via AtCb5 in plant cells. Furthermore, the overexpression of AtBI-1 increased the level of 2-hydroxy fatty acids in Arabidopsis, indicating that AtBI-1 is involved in fatty acid 2-hydroxylation.     

AtBI-1, a plant homologue of Bax inhibitor-1, suppresses Bax-induced cell death in yeast and is rapidly upregulated during wounding and pathogen challenge

Extensive searches have so far failed to identify functional plant homologues of the mammalian apoptotic machinery. Here we report the isolation and characterisation of an Arabidopsis thaliana homologue of human Bax Inhibitor-1, AtBI-1, isolated during a differential screen of plants challenged with the phytopathogen Pseudomonas syringae. AtBI is a member of a small gene family in Arabidopsis, members of which display extensive amino acid identity to human BI-1. AtBI-1 is also functionally similar to BI-1 in its ability to suppress the lethal phenotype in yeast conferred by expression of the mammalian proapoptotic protein, Bax. Expression of AtBI-1 is rapidly upregulated in plants during wounding or pathogen challenge, suggesting a role in responses to biotic and abiotic stress. AtBI-1 upregulation appears R gene independent and is not markedly affected by mutations required for specific classes of R genes. However, the accumulation of AtBI-1 message is significantly reduced in coi1, in which defence responses to insects, pathogens and wounding are compromised.     

Loss of Calmodulin Binding to Bax Inhibitor-1 Affects Pseudomonas-mediated Hypersensitive Response-associated Cell Death in Arabidopsis thaliana

Bax inhibitor-1 (BI-1) is a cell death suppressor protein conserved across a variety of organisms. The Arabidopsis atbi1-1 plant is a mutant in which the C-terminal 6 amino acids of the expressed BI-1 protein have been replaced by T-DNA insertion. This mutant BI-1 protein (AtBI-CM) produced in Escherichia coli can no longer bind to calmodulin. A promoter-reporter assay demonstrated compartmentalized expression of BI-1 during hypersensitive response, introduced by the inoculation of Pseudomonas syringae possessing the avrRTP2 gene, Pst(avrRPT2). In addition, both BI-1 knockdown plants and atbi1-1 showed increased sensitivity to Pst(avrRPT2)-induced cell death. The results indicated that the loss of calmodulin binding reduces the cell death suppressor activity of BI-1 in planta.Bax inhibitor-1 (At5g47120, BI-1)² is a highly conserved cell death suppressor protein that resides in the endoplasmic reticulum (ER) membranes of a range of organisms. BI-1 is important in the response of organisms to abiotic and biotic stresses. Down-regulation of BI-1 in tobacco suspension cells (BY2) induced sensitivity against starvation (1), whereas overexpression in barley induced the breakdown of mlo-mediated penetration resistance to the fungal pathogen, powdery mildew (Blumeria graminis) (2). Cultured rice cells overexpressing Arabidopsis BI-1 (AtBI-1) showed increased resistance to Magnaporthe grisea-induced hypersensitive response (HR)-like cell death, potentially confirming the role of BI-1 in HR regulation (3). Recent studies on animal and plant BI-1 indicated a close relationship with ER stress response (4–6). BI-1-deficient mice are hypersensitive to apoptosis induced by ER stress agents such as thapsigargin, tunicamycin, and brefeldin A (4). Such events correlate with decreased calcium release from the ER, and our previous study demonstrated an association of BI-1 with calcium signaling in stress-treated plant cells (7). However, the molecular mechanism by which BI-1 suppresses cell death is still unclear.Recently, Watanabe et al. (5, 8) demonstrated that an Arabidopsis T-DNA-tagged mutant, atbi1-1, was more susceptible to fungal toxin-, heat-shock-, and tunicamycin-induced cell death. The atbi1-1 plant has T-DNA inserted into the AtBI-1 protein C-terminal region, which contains potential coiled-coil structures and is essential for inhibiting both Bax-induced lethality in yeast and oxidative stress-induced cell death in plant cells as we had demonstrated earlier (9). We also found that the C-terminal 14 amino acids of AtBI-1 were capable of binding to the calmodulin molecule, a mediator of calcium signaling (7). Here, the present study directly proved the functional interaction between the highly conserved calmodulin molecule and BI-1 using a genomic mutation of the AtBI-1 gene. Such a genomic mutant showed accelerated sensitivity against Pseudomonas-induced HR cell death. The results indicated that the C-terminal-less BI-1 protein, which lost the CaM binding, was associated with reduced cell death suppression activity in vivo.     

Cucumber BAX inhibitor-1, a conserved cell death suppressor and a negative programmed cell death regulator under cold stress

Programmed cell death (PCD) is a genetically controlled and conserved process in eukaryotes during development as well as in response to pathogens and other stresses. BAX inhibitor-1 (BI-1) has been implicated as an anti-PCD factor which is highly conserved in plants. Sequence of putative cucumber BI-1 protein exhibited 77.7 % identity and 91.2 % positive value with the homologue Blast BI-1 protein of Arabidopsis thaliana (AtBI-1). This highly homologous protein to the AtBI-1 protein was named CsBI-1. This protein contains an open reading frame (ORF) of 250 amino acids with a BAX inhibitor domain and five transmembrane regions conserved among members of the BI-1 family. Primers designed by the cDNA of CsBI-1gene were used for further sequencing. Cell death in cold-stored cucumber developed concomitantly with increased expression of the CsBI-1 gene and reached maximum at day 6. However, cell death accelerated significantly after 9 d when sharp decrease of the CsBI-1 expression occurred. After warming to 20 °C, expression of the CsBI-1 gene was the highest at day 3, decreased afterwards, and the lowest expression was detected at day 9 when PCD obviously appeared. The overall results indicate that CsBI-1 is cucumber homologue of Arabidopsis thaliana AtBI-1 gene. CsBI-1 is a conserved cell death suppressor induced by cold stress and a negative regulator of PCD.     

Induction of mammalian cell death by a plant Bax inhibitor

Arabidopsis thaliana AtBI-1 is an orthologue of mammalian Bax inhibitor-1 capable of suppressing Bax-induced cell death in yeast as well as mammalian cells. Here we investigated whether or not AtBI-1 suppresses Bax-induced cell death using human fibrosarcoma HT1080 cells. Surprisingly, AtBI-1 did not block Bax-induced cell death, but it triggered apoptotic cell death in mammalian cells. The proapoptotic effect of AtBI-1 was blocked by the X-linked caspase inhibitor XIAP, suggesting that the cell death caused by AtBI-1 is similar to that caused by Bax.     

Overexpression of Bax inhibitor suppresses the fungal elicitor-induced cell death in rice (Oryza sativa L) cells

Treatment of suspension-cultured cells of rice (Oryza sativa L.) with cell wall extract of rice blast fungus (Magnaporthe grisea) elicits a rapid generation of H2O2, alkalinization of culture medium, and eventual cell death. To elucidate genes involved in these processes, we exploited SAGE (Serial Analysis of Gene Expression) technique for the molecular analysis of cell death in suspension-cultured cells treated with the elicitor. Among the downregulated genes in the elicitor-treated cells, a BI-1 gene coding for Bax inhibitor was identified. Transgenic rice cells overexpressing Arabidopsis BI-1 gene showed sustainable cell survival when challenged with M. grisea elicitor. Thus, the plant Bax inhibitor plays a functional role in regulating cell death in the rice cell culture system.     

Molecular characterization of two plant BI-1 homologues which suppress Bax-induced apoptosis in human 293 cells

To date, few homologues of animal programmed cell death (PCD) regulators have been identified in plants. Among these is the plant Bax Inhibitor-1 (BI-1) protein, which possesses, like its human counterpart, the ability to suppress Bax-induced lethality in yeast cells. As the role of BI-1 in the regulation of plant PCD remains to be elucidated, we cloned BnBI-1 and NtBI-1 from cDNA libraries of oilseed rape ( Brassica napus L.) and tobacco ( Nicotiana tabacum L.). The analysis of the deduced amino acid sequences of BnBI-1 and NtBI-1 indicated that these proteins share a relatively high level of identity with other plant BI-1 proteins (73-95%) as well as with animal BI-1 proteins (26-42%). Comparative analysis with other available plant BI-1 proteins allowed the establishment of a structural model presenting seven transmembrane domains. Moreover, transient co-transfection of Bax with BnBI-1 or NtBI-1 in human embryonic kidney 293 cells revealed that both proteins can substantially inhibit apoptosis induced by Bax overexpression. Localization studies were also conducted using stable transformation of tobacco BY-2 cells and Saccharomyces cerevisiae, or transient expression in tobacco leaves, with the fusion protein BnBI-1GFP under control of the cauliflower mosaic virus 35S promoter. All transformants showed a fluorescence pattern of distribution typical of an endoplasmic reticulum (ER) protein. Results from differential permeabilization experiments in BY-2 cells expressing BnBI-1GFP also showed that the C-terminus is located on the cytosolic side of the ER. Taken altogether, our results suggest that BI-1 is evolutionarily conserved and could act as a key regulator of a death pathway common to plants and animals.     

BI-1 regulates an apoptosis pathway linked to endoplasmic reticulum stress

Bax inhibitor-1 (BI-1) is an evolutionarily conserved endoplasmic reticulum (ER) protein that suppresses cell death in both animal and plant cells. We characterized mice in which the bi-1 gene was ablated. Cells from BI-1-deficient mice, including fibroblasts, hepatocytes, and neurons, display selective hypersensitivity to apoptosis induced by ER stress agents (thapsigargin, tunicamycin, brefeldin A), but not to stimulators of mitochondrial or TNF/Fas-death receptor apoptosis pathways. Conversely, BI-1 overexpression protects against apoptosis induced by ER stress. BI-1-mediated protection from apoptosis induced by ER stress correlated with inhibition of Bax activation and translocation to mitochondria, preservation of mitochondrial membrane potential, and suppression of caspase activation. BI-1 overexpression also reduces releasable Ca(2+) from the ER. In vivo, bi-1(-/-) mice exhibit increased sensitivity to tissue damage induced by stimuli that trigger ER stress, including stroke and tunicamycin injection. Thus, BI-1 regulates a cell death pathway important for cytopreservation during ER stress.     

Antisense down regulation of NtBI-1 in tobacco BY-2 cells induces accelerated cell death upon carbon starvation

Bax inhibitor-1 (BI-1) protein is proposed to be a conserved programmed cell death suppressor. In this report, we investigate the anti-apoptotic function of plant BI-1 by antisense (AS) down regulation of NtBI-1 in Nicotiana tabacum cv. BY-2 cells. We observed that AS cell lines were more susceptible to autophagy, internucleosomal DNA fragmentation and death than control cells when subjected to sucrose starvation and hypo-osmotic shock, in agreement with a role of BI-1 as a death inhibitor.     

Plastidic protein Cdf1 is essential in Arabidopsis embryogenesis

Arabidopsis cell growth defect factor-1 (Cdf1 in yeast, At5g23040) was originally isolated as a cell growth suppressor of yeast from genetic screening. To investigate the in vivo role of Cdf1 in plants, a T-DNA insertion line was analyzed. A homozygous T-DNA insertion mutant (cdf1/cdf1) was embryo lethal and showed arrested embryogenesis at the globular stage. The Cdf1 protein, when fused with green fluorescent protein, was localized to the plastid in stomatal guard cells and mesophyll cells. A promoter-β-glucuronidase assay found expression of Cdf1 in the early heart stage of embryogenesis, suggesting that Cdf1 was essential for Arabidopsis embryogenesis during the transition of the embryo from the globular to heart stage.     

Arabidopsis Bax inhibitor-1 functions as an attenuator of biotic and abiotic types of cell death

Programmed cell death (PCD) is a common process in eukaryotes during development and in response to pathogens and stress signals. Bax inihibitor-1 (BI-1) is proposed to be a cell death suppressor that is conserved in both animals and plants, but the physiological importance of BI-1 and the impact of its loss of function in plants are still unclear. In this study, we identified and characterized two independent Arabidopsis mutants with a T-DNA insertion in the AtBI1 gene. The phenotype of atbi1-1 and atbi1-2, with a C-terminal missense mutation and a gene knockout, respectively, was indistinguishable from wild-type plants under normal growth conditions. However, these two mutants exhibit accelerated progression of cell death upon infiltration of leaf tissues with a PCD-inducing fungal toxin fumonisin B1 (FB1) and increased sensitivity to heat shock-induced cell death. Under these conditions, expression of AtBI1 mRNA was up-regulated in wild-type leaves prior to the activation of cell death, suggesting that increase of AtBI1 expression is important for basal suppression of cell death progression. Over-expression of AtBI1 transgene in the two homozygous mutant backgrounds rescued the accelerated cell death phenotypes. Together, our results provide direct genetic evidence for a role of BI-1 as an attenuator for cell death progression triggered by both biotic and abiotic types of cell death signals in Arabidopsis.     

Molecular analysis of programmed cell death during senescence in Arabidopsis thaliana and Brassica oleracea: cloning broccoli LSD1, Bax inhibitor and serine palmitoyltransferase homologues

This study was undertaken to characterize the programmed cell death (PCD) processes that occur during detached and natural on-plant senescence and correlate them with the expression of putative regulatory genes that may be involved in the process. DNA fragmentation and TUNEL analysis of broccoli florets showed that DNA was processed into fragments of approximately 180 bp after 48 h of harvest-induced tissue senescence. Characteristic laddering patterns were also visible in Arabidopsis leaves undergoing natural on-plant senescence and during detached senescence. Several recently isolated plant proteins have been assigned a PCD role, for example, the zinc finger containing protein, LSD1 (lesion simulating disease); Bax inhibitor (BI); and serine palmitoyltransferase (SPT), an enzyme in the sphingolipid signalling pathway. Two cDNAs encoding each of these proteins were isolated from broccoli (BoBI-1, BoBI-2, BoLSD1, BoLSD2, BoSPT1, BoSPT2), and the mRNAs increased during harvest-induced senescence in floret tissue. Expression of the Arabidopsis homologues (AtBI-1, AtLSD1, AtSPT1) were also characterized during detached leaf senescence in Arabidopsis leaves. AtBI-1 expression was constitutively expressed during detached senescence, AtLSD1 expression remained constitutively low, and AtSPT1 expression increased during detached senescence.     

Plant Bax Inhibitor-1 interacts with ATG6 to regulate autophagy and programmed cell death

Autophagy is an evolutionarily conserved catabolic process and is involved in the regulation of programmed cell death during the plant immune response. However, mechanisms regulating autophagy and cell death are incompletely understood. Here, we demonstrate that plant Bax inhibitor-1 (BI-1), a highly conserved cell death regulator, interacts with ATG6, a core autophagy-related protein. Silencing of BI-1 reduced the autophagic activity induced by both N gene-mediated resistance to Tobacco mosaic virus (TMV) and methyl viologen (MV), and enhanced N gene-mediated cell death. In contrast, overexpression of plant BI-1 increased autophagic activity and surprisingly caused autophagy-dependent cell death. These results suggest that plant BI-1 has both prosurvival and prodeath effects in different physiological contexts and both depend on autophagic activity.     

The Arabidopsis BAP1 and BAP2 genes are general inhibitors of programmed cell death

Here we identify the BAP1 and BAP2 genes of Arabidopsis (Arabidopsis thaliana) as general inhibitors of programmed cell death (PCD) across the kingdoms. These two homologous genes encode small proteins containing a calcium-dependent phospholipid-binding C2 domain. BAP1 and its functional partner BON1 have been shown to negatively regulate defense responses and a disease resistance gene SNC1. Genetic studies here reveal an overlapping function of the BAP1 and BAP2 genes in cell death control. The loss of BAP2 function induces accelerated hypersensitive responses but does not compromise plant growth or confer enhanced resistance to virulent bacterial or oomycete pathogens. The loss of both BAP1 and BAP2 confers seedling lethality mediated by PAD4 and EDS1, two regulators of cell death and defense responses. Overexpression of BAP1 or BAP2 with their partner BON1 inhibits PCD induced by pathogens, the proapoptotic gene BAX, and superoxide-generating paraquat in Arabidopsis or Nicotiana benthamiana. Moreover, expressing BAP1 or BAP2 in yeast (Saccharomyces cerevisiae) alleviates cell death induced by hydrogen peroxide. Thus, the BAP genes function as general negative regulators of PCD induced by biotic and abiotic stimuli including reactive oxygen species. The dual roles of BAP and BON genes in repressing defense responses mediated by disease resistance genes and in inhibiting general PCD has implications in understanding the evolution of plant innate immunity.