Modulation of Bax Inhibitor-1 and cytosolic Ca2+ by cytokinins in Nicotiana tabacum cells.

The protein Bax Inhibitor-1 (BI-1) has recently emerged as a negative regulator of plant programmed cell death (PCD), but how it functions at the biochemical level remains unknown. To elucidate its regulation and mode of action, we used suspension cells of Nicotiana tabacum to study the effects of cytokinins (CKs) on the expression level of NtBI-1 via western analysis. We found that the NtBI-1 protein is up-regulated following treatments with CKs at concentrations inducing a stress response (determined by growth reduction and PR1a accumulation), but not at PCD-inducing concentrations. These data point toward a role for NtBI-1 in the stress response to CKs. Application of CKs was also accompanied by a rapid cytosolic Ca(2+) pulse, and inhibition of this pulse with La(3+) or EGTA partially restored viability, indicating a signaling role for Ca(2+) in CK-induced cell death. However, CK-induced NtBI-1 accumulation was not altered by pretreatment with La(3+), nor by treatment with several modulators of intracellular Ca(2+) homeostasis and signaling, suggesting that CK-dependent regulation of NtBI-1 accumulation is not directly mediated by Ca(2+).

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.

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.