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4 L’accumulation du régulateur négatif de la mort cellulaire programmée Bax Inhibitor-1 est favorisée par les cytokinines dans des cellules de Nicotiana tabacum

Table des matières

Ce chapitre, qui a été soumis pour publication, a été écrit suite à mes travaux et j’ai ainsi préparé toutes les figures à l’exception de la Figure 4-5 et de la Figure 4-6A. Ces dernières proviennent de travaux effectués par le deuxième auteur alors qu’il était sous la supervision du troisième auteur et ce sont eux qui m’ont fourni ces deux figures.J’ai structuré et écrit entièrement cette première version de l’article.

La mort cellulaire programmée (PCD) est un phénomène vital pour les organismes multicellulaires y compris les végétaux. À ce jour, les processus biochimiques impliqués dans le contrôle de laPCD végétale demeurent obscurs. La protéine Bax Inhibitor-1 (BI-1) est conservée entre les animaux et les végétaux et agit au sein des deux règnes en tant que régulateur négatif de la PCD par un mécanisme encore inconnu. Dans le but d’élucider la façon dont cette protéine est régulée et agit, nous avons utilisé des suspensions cellulaires de Nicotiana tabacum pour y étudier les effets d’apports exogènes de cytokinines (CK) sur l’accumulation de NtBI-1 via immunobuvardage. Nous avons découvert que NtBI-1 est induit suite à une exposition envers les CK, et ce à des concentrations permettant l’établissement d’une réponse de stress (évaluée par une réduction sévère de la croissance et l’induction de PR1a ), mais non à des concentrations induisant la PCD. L’accumulation de la protéine augmente sans modulation de l’accumulation de l’ARNm, indiquant l’implication de mécanismes de régulation post-transcriptionnels. La surexpression de NtBI-1 est modifiée quand la phosphorylation intracellulaire du 6-benzylaminopurine (Bap), une étape critique dans l’induction de la PCD par la phytohormone, est inhibée par l’utilisation d’un inhibiteur de l’adénosine kinase. De plus, la surexpression de NtBI-1 induite par le Bap nécessite une exposition suffisamment longue pour induire la réponse de stress. L’ajout de Bap est aussi accompagné d’un influx rapide de Ca2+de l’apoplaste vers le cytosol, et ce dans l’intervalle de concentration menant au stress et à la PCD et l’inhibition de l’influx par le La3+rétablit partiellement la viabilité. Cependant, l’accumulation de NtBI-1 n’est pas influencée par cet influx de Ca2+ni par aucun agent connu pour altérer l’homéostasie intracellulaire du Ca2+, suggérant que la régulation du niveau de la protéine n’est pas sensible à la signalisation par le Ca2+. Nos données indiquent un rôle pour NtBI-1 dans la réponse de stress envers les CK et suggèrent l’implication de cette protéine dans l’activité anti-sénescence de cette phytohormone.

The negative programmed cell death regulator Bax Inhibitor-1 is up-regulated by cytokinins in Nicotiana tabacum cells

Nathalie Bolduc a , Gregory N. Lamb b , Stephen G. Cessna b and Louise F. Brisson a,1

aDépartement de Biochimie et de Microbiologie, Université Laval, Québec, Qc, Canada, G1K 7P4

bDepartment of Chemistry and Biochemistry, Eastern Mennonite University, 1200 Park Rd, Harrisonburg, VA 22802, USA

1To whom correspondence should be addressed.E-mail: louise.brisson@rsvs.ulaval.ca; fax 1-418-6567176

Programmed cell death (PCD) is a vital phenomenon for multicellular organisms including plants. To date,biochemical processes involved in the control of plant PCD are only poorly understood. The protein Bax Inhibitor-1 (BI-1) is conserved between animals and plants and acts in both kingdoms as a negative regulator of PCD by an unknown mechanism. 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 the protein NtBI-1 via western analysis. We found that NtBI-1 is up-regulated following treatments with CKs at concentrations inducing a stress response (as determined by severe growth reduction and PR1a induction), but not at PCD-inducing concentrations. Interestingly, the level of the protein increased without modulation of its mRNA, indicative of a post-transcriptional mechanism. NtBI-1 protein up-regulation was altered when the intracellular phosphorylation of the CK 6-benzylaminopurine (Bap), a critical step for the Bap -induced cell death, was inhibited by the use of an adenosine kinase inhibitor. Moreover, Bap-induced NtBI-1 up-regulation required an exposure time sufficient to induce a stress response. Application of Bap was accompanied by a rapid cytosolic Ca2+pulse, and inhibition of this pulse with La3+partially restored viability. However, CK-induced NtBI-1 accumulation was not altered by prior La3+treatment, or by treatment with several other modulators of intracellular Ca2+homeostasis and signaling, suggesting that CK-dependent regulation of the NtBI-1 protein accumulation is not mediated by Ca2+signaling. Our data point toward a role for NtBI-1 in the stress response to CKs and open the possibility of its involvement in their senescence-delay activity.

Programmed cell death (PCD) is a sophisticated physiological process evolved by eukaryotes to remove unwanted, damaged or infected cells in the course of normal development or under pathological situations, with the aim of maintaining the integrity or fitness of the remaining organism or cell population (Gallaher et al. , 2001). In plants, the proper occurrence of a PCD program is essential for the establishment of the hypersensitive response (HR) following pathogen attack, and the formation of xylem vessels, root aerenchyma, pollen grains, endosperm, etc. Senescence is also a form of PCD involving whole organs such as leaves, petals and fruits, and even the whole plant body (Hoeberichts and Woltering, 2003).

Plant PCD is less documented than animal PCD. However, actual knowledge indicates that some biochemical pathways could be conserved between kingdoms, such as the involvement of reactive oxygen species (ROS), the release of cytochrome c (Cyt c ) from mitochondria and signal transduction by Ca2+fluxes (Hoeberichts and Woltering, 2003). Although some morphological similarities with animal apoptosis have been observed in plant PCD, such as cytoplasmic shrinkage and internucleosomal DNA fragmentation, plants mostly rely on their vacuole to undergo PCD (Butler and Simon, 1971; Jones, 2001), reminiscent of lysosomal/autophagic cell death in animal development (Lockshin and Zakeri, 2004).

On the other hand, while animal apoptosis is regulated by well characterized pro- and anti-apoptotic regulatory members of the Bcl-2 family, most plant PCD regulators are to be discovered, and Bcl-2-related genes have no sequence homologues in plants (Aravind et al. , 2001; Lam et al. , 2001). In animals, Bcl-2 regulates PCD by interacting with structurally similar proteins of the Bcl-2 family, such as Bax, an important inducer of PCD. These proteins act mainly by interference with proteases (caspases) activation through their effect on mitochondrial membrane integrity or endoplasmic reticulum (ER) Ca2+homeostasis (Orrenius et al. , 2003). Bax Inhibitor-1 (BI-1), a negative regulator of apoptosis in mammals (Xu and Reed, 1998), has recently emerged as an evolutionary conserved negative regulator of plant PCD (reviewed by Hückelhoven, 2004). Similar to Bcl-2, BI-1 is targeted to membranes (mainly the ER) and its sequence analysis suggests the presence of seven putative transmembrane domains (Xu and Reed, 1998; Bolduc et al. , 2003). Human BI-1 has been originally shown to suppress Bax-induced cell death in yeast and human cells (Xu and Reed, 1998). Numerous plant homologues were subsequently identified, sharing not only high sequence identity but also functionality. Effectively, plant BI-1 can counteract Bax in yeast (Kawai et al. , 1999; Sanchez et al. , 2000), in human HEK cells (Bolduc et al. , 2003) and in Arabidopsis thaliana (Kawai-Yamada et al. , 2001). Although BI-1 is a negative regulator of plant PCD (Bolduc and Brisson, 2002; Hückelhoven et al. , 2003; Matsumura et al. , 2003; Kawai-Yamada et al. , 2004), its mode of action remains unknown. However, a recent investigation by Chae and co-workers (2004) indicates that in mouse, BI-1 could act by lowering the amount of releasable Ca2+from the ER, as similarly demonstrated for Bcl-2 (Orrenius et al. , 2003).

As many aspects of plant development involve PCD, it is not surprising that hormones affect PCD regulation, specifically those generally associated with stresses, namely salicylic acid (SA), jasmonic acid (JA), abscisic acid (ABA) and ethylene (Hoeberichts and Woltering, 2003; Overmyer et al. , 2003). Cytokinins (CKs), a structurally diverse group of N6-substituted purine derivatives, are also traditionally recognized as anti-PCD hormones, principally because they promote growth and differentiation, and delay senescence. Effectively, their level declines during senescence, and exogenous application of this hormone or overexpression of the bacterial CK biosynthetic enzyme isopentenyl transferase ( ipt ) favors delayed leaf senescence (Mok, 1994). However, such a view has been recently challenged by the occurrence of CK-induced PCD in suspension cells of A. thaliana , Daucus carota and Nicotiana tabacum (Mlejnek and Procházka, 2002; Carimi et al. , 2003; Mlejnek et al. , 2003; Carimi et al. , 2004). This PCD was characterized by the occurrence of internucleosomal DNA fragmentation and potential involvement of mitochondria.

Although the molecular mechanisms underlying this phenomenon remain obscure, toxicity associated with high levels of CKs in plant tissues (due to exogenous application or increased accumulation of the hormone in transgenic plants expressing ipt ) has also been reported episodically (Ainley et al. , 1993; Martineau et al. , 1994; McCabe et al. , 2001; Rakwal et al. , 2003; Lee et al. , 2004). It is not clear whether this CK’s effect is chemical rather than hormonal, since adenine and adenosine derivatives with CK activities are efficient apoptosis inducers in animal cells (Mlejnek and Kuglik, 2000; Ishii et al. , 2002; Vermeulen et al. , 2002). However, it is clear that CK-mediated cell death necessitates the entry of the hormone into the cell and its subsequent phosphorylation, both in plant and animal cells (Mlejnek and Procházka, 2002; Mlejnek et al. , 2003). Furthermore, a correlation between high level of CKs and accumulation of stress genes, including pathogenesis-related (PR) genes, has been reported both in whole plant tissues (Memelink et al. , 1990; Martineau et al. , 1994; Rakwal et al. , 2003) or in vitro tissue cultures (Memelink et al. , 1987; Poupet et al. , 1990; Lee et al. , 2004), suggesting the induction of a stress response by CKs.

In a previous study, we showed that tobacco cell lines underexpressing NtBI-1 die prematurely when subjected to sucrose starvation (Bolduc and Brisson, 2002), a situation that favors the activation of senescence, and thus suggest a role for BI-1 in the control of this process. Interestingly, while investigating for PCD-related hormones that could modulate BI-1, tobacco cells provided with exogenous 6-benzylaminopurine (Bap), a synthetic active CK, revealed up-regulation of NtBI-1 at the protein level. Here we show that CKs induce a stress response concomitant with NtBI-1 over-accumulation in cultured tobacco cells, which depends on post-transcriptional events but not on Ca2+signaling.

To better characterize CK-induced NtBI-1 up-regulation, cultures were supplemented with increasing concentrations of four different CKs (Bap, Kin, iPA and Zea), and samples were collected after 24 h for protein analysis. Concomitantly, cell death and fresh weight increases were determined after a 96 h exposure (Tables 4-2 and 4-3). Table 4-2 summarizes the effects on cell viability of physiological to toxic CKs concentrations on N. tabacum cvs. Xanthi and Wisconsin-38. The later cells, tested only with Bap application, were slightly more susceptible to this CK than Xanthi cells, as indicated by a lower viability at 10mM. The precise concentration inducing a significant cell viability reduction in Xanthi cells varied from one CK to another, although all fell in the same concentration range. Bap and Kin induced cell death at 10mM, while iPA and Zea where only effective at 25mM (Table 4-2). Evaluation of genomic DNA integrity in Bap-treated cells revealed the occurrence of DNA laddering only for 25 and 50mM (data not shown), a characteristic of PCD also observed in others CKs-induced PCD studies. Of note, cells were less likely killed by Zea than by other CKs. Indeed, cell viability was 57% after being exposed for 96 h to 25mM Zea, while it was usually 20-40% with other CKs. Furthermore, around 30% of the cells were still viable at 100mM, while other CKs left no more than 10-20% of viable cells when exposed to 50mM.

Of interest, observations of CK-treated cultures revealed unexpected negative effects on growth at non-lethal CKs concentrations, which were quantified by fresh weight measurements (Table 4-3). Similar to their effect on cell viability, growth reduction in Xanthi cells depended on the nature of the CK. Indeed, as little as 0.1mM Bap was sufficient to reduce growth, while 5mM of iPA, and 0.5mM of Kin and Zea were required to achieve the same effect. Moreover, Bap-induced growth reduction in Xanthi cells required a lower concentration (0.1mM) than in Wisconsin-38 cells (0.5mM). Of note, cells treated with up to 10mM Bap eventually recovered and grew to a cell density comparable to those obtained with control cells, but with an increased period of growth reaching 15 days (data not shown).

We then evaluated the influence of the different CKs on NtBI-1 accumulation by immunoblots. Using Bap, up-regulation of the protein was consistently observed for 0.1-10mM, sometimes at 25mM and never at 50mM (Figure 4-1A). Moreover, the increased accumulation was sustained in the time frame analyzed (up to 96 h) for 1-10mM, and to a lower extent at 0.1mM, while at the same time the protein was almost undetectable in control cells. Interestingly, the Bap threshold for NtBI-1 up-regulation was identical to the one leading to growth reduction (Table 4-3). Thus, these data strongly suggest a tight correlation between stress-inducing Bap concentrations (as reflected by limited or absence of growth) and NtBI-1 induction. Significantly, the same correlation was observed in other cell lines, i.e. N. tabacum cv. Wisconsin-38 expressing a cytosolic aequorin (Tables 4-2 and 4-3 and Figure 4-1B) and in N. tabacum cv. SR-1 (data not shown). However, increased NtBI-1 accumulation was not as strong in Wisconsin-38 as it was in Xanthi cells, although steady-state levels were similar (compare Figures 4-1A and B). Intriguingly, the protein was barely detectable in 96 h control cells (Figure 4-1A), which is equivalent to a 7 days-post-transfer (DPT) stage. At 7 DPT, cells were mostly reaching the stationary phase and still exhibited good viability, but by 11 DPT, viability has already dropped below 70% and NtBI-1 was no detectable anymore, even with over saturated conditions of detection (Figure 4-1C and data not shown).

NtBI-1 up-regulation was not specific to Bap but was also observed with all other tested CKs, although the effective concentrations were variables. For iPA, increased accumulations were only obvious from 1-5 to 10mM (variable at 1mM), while 0.5 to 25mM Kin and 0.5 to 50mM Zea produced the same results (Figure 4-1D). Interestingly, as previously observed for Bap, increased NtBI-1 accumulation following these CKs treatments tightly correlated with concentrations that significantly induced growth impairment, which started at 5mM for iPA, and 0.5mM for Kin and Zea (Table 4-3).

A-B) Immunoblots show typical Bap dose-response profiles in cvs. Xanthi (A) and Wisconsin-38 (B) cells after treatments of 24 or 96 h.

C) BI-1 protein expression profile and cell viability in naturally senescingXanthi cultures. 5 DPT cells were used as control for viability determination.

D) Dose-response analysis of NtBI-1 accumulation in Xanthi cells after a 24 h exposure to Kin, iPA or Zea.

A time-course analysis on 0.1-50mM Bap-treated cells was performed to study the effect of CKs on NtBI-1 expression at the protein and mRNA level (Figure 4-2A, D), in relation to cell viability for toxic Bap concentrations (10-50mM; Figure 4-2C). Immunoblots revealed that BI-1 over-accumulated 12 h after Bap addition (Figure 4-2A). At 25mM, the increased accumulation was not always observed and was weaker than in 0.1-10mM Bap-treated cells (Figures 4-1A, 4-2A and data not shown). At 50mM, we observed a persistent basal level up to 12 h followed by a slight decrease at 24 h and complete disappearance thereafter (Figure 4-2A). This disappearance correlated with general proteolysis, as determined by the loss of integrity of high molecular weight proteins and Rubisco at 48 h (data not shown). Because most of these cells were already dead by 24 h (Figure 4-2C), we asked how long NtBI-1 could sustain in dying cells. Inhibition of protein synthesis by the use of Chx showed an important decrease in NtBI-1 detection from 12 h (Figure 4-2B), while most of the cells were already dead after 6 h of treatment (Figure 4-2C) and massive proteolysis detected only after 48 h of treatment (data not shown). This indicates a relatively long half-life of the protein (6-12 h, and even more), probably due to its membranous localization. This also indicates that detection of the protein up to 24 h in a situation where cells are obviously dying (i.e. 50mM Bap) could reflect some up-regulation. Indeed, in 50mM-treated cells, we observed a relatively strong increase in NtBI-1 mRNA accumulation that started between 3 and 6 h after Bap addition, followed by an almost complete disappearance after 48 h, concomitantly with massive RNA degradation (Figure 4-2D). A similar but delayed pattern was observed in 25mM-treated cells (Figure 4-2D). Surprisingly, accumulations were relatively constant over time not only for control, but also for 0.1 (not shown) and 1mM Bap (Figure 4-2D). Moreover, in 10mM-treated cells, a slight increased accumulation was only observed after 48 and 96 h (Figure 4-2D). Together with immunoblot analysis, these data clearly indicate that NtBI-1 mRNA accumulation does not necessarily reflect protein accumulation, which can be regulated at the post-transcriptional level (at least in the case described here).

A-B) Time-course analysis of the NtBI-1 protein in Xanthi cells exposed to 0-50μM Bap (A) or 50μg/ml Chx (B) as determined by immunoblots.

C) Time-course analysis of viability in Xanthi cells treated with adenine (○), Bap (10μM●; 25μM□; 50μM■ or Chx (Δ).

D) Accumulation of NtBI-1 and PR1a mRNAs in Xanthi cells exposed to 1-50μM Bap as shown by northern blots. Ethidium bromide-stained gels (lower panels) show homogenous RNA loading. Wells with reduced rRNA had degraded RNA visible in the lower part of the gels, reminiscent of massive cell death.

To address whether Bap-induced NtBI-1 up-regulation is time-dependent, cellswere incubated for 1 h with 0 to 50mM Bap, washed and then further incubated under standard conditions for 23 h. Under this short exposure, only high Bap concentrations (i.e. 10-50mM) were sufficient to stimulate NtBI-1 up-regulation but also to fully impair growth (Figure 4-4A and D). Lower concentrations had no (0.1mM Bap) or limited (1mM Bap) effect on NtBI-1 accumulation, while their influence on growth was very limited or moderate, respectively (Figure 4-4A and D). Moreover, while longer exposure to 50mM Bap was lethal, the washing procedure reducing the exposure time to 1 h restored viability (Figure 4-4C). This probably explains why NtBI-1 up-regulation can be observed in these conditions. Interestingly, the tight correlation between growth impairment and NtBI-1 induction is again observed. Of note, an exposure as short as 5 minutes to 10 and 50mM Bap was enough to induce NtBI-1 up-regulation (Figure 4-4B and data not shown).

Cells were pre-treated with different concentrations of AdkI and then exposed to Bap for 24, 48 or 96 h. Accumulation of the NtBI-1 protein was analyzed by immunoblots.

A-B) Analysis of NtBI-1 accumulation at 10 and 50 µM Bap.

C-D) Effects on cell viability and growth were evaluated 96 h after treatment.

A-B) Cells were treated for 60 minutes with different concentrations of Bap (A) or for 5-60 minutes with 50μM Bap (B) and collected 24 h later for immunoblot analysis of the NtBI-1 protein accumulation.

C-D) Effects of short treatments on cell viability and growth were evaluated 96 h after treatment.

Because of its localization to ER membranes, a Ca2+reservoir, BI-1 is suspected to exert its function through some influence on Ca2+homeostasis. CK-induced Ca2+uptake in cells of the bryophyte mosses Funaria hygrometrica and Physcomitrella patens has already been reported (Saunders and Hepler, 1983; Hahm and Saunders, 1991; Schumaker and Gizinski, 1993), and we asked if Bap-induced NtBI-1 up-regulation could be the consequence of cytosolic Ca2+signaling. For this purpose, we first measured CK-induced Ca2+ cytpulses in aequorin-transformed suspension cells (Figure 4-5). Both Bap and Kin (Figure 4-5A), but not Zea (data not shown), induced rapid and substantial Ca2+ cytpulses, each reaching a climax of greater than 1 µM Ca2+within the first minute of cytokinin application, followed by a slow sustained decrease to ½ maximal levels after three minutes. Intriguingly, extensive variability in pulse amplitude was observed from day to day (note the size of the error bars in Figure 4-5A, and compare average amplitudes in Figure 4-5A to the representative example in 4-5B). However, in several different experiments, we could determine that maximal pulse amplitude was reached with ~ 1 µM Bap and the minimal concentration required to initiate a Ca2+ cytpulse was ~ 0.1 µM (Figure 4-5B). Interestingly, this range coincided with the minimal concentration range necessary to induce NtBI-1 up-regulation as well as significant growth reduction (0.5 µM; Figure 4-1B and Table 4-3).

Aequorin-transformed and reconstituted N. tabacum cv. Wisconsin-38 cells were treated with CKs or with vehicle control solution at the time indicated by the bold arrow.

A) Cells were treated with 25μM Bap or Kin. Each trace is the average of 8 or more independent identical experiments. The region of the graph under the black bar is that during which the [Ca2+]cytvalues for both Bap- and Kin-treated cells differ statistically (p < 0.01) from the control trace. There is no significant difference between the responses generated by Bap and Kin (p > 0.05).

B) Dose-response analysis using 0.1-10μM Bap. Data shown are representative of ten independent experiments.

We investigated the role of this Ca2+ cytpulse in Bap-mediated PCD as well as in NtBI-1 up-regulation (Figure 4-6). We first noticed the potent inhibition of the CK-induced Ca2+ cytpulse by prior addition of the cation channel blocker LaCl3, or the cell-impermeant Ca2+chelator EGTA (Figure 4-6A), revealing an apoplastic origin of the Ca2+ cytpulse. However, such an inhibition using up to 1.5 mM LaCl3or the cell-impermeant Ca2+chelator Bapta did not affect Bap-induced NtBI-1 up-regulation (Figure 4-6B, C). Similar observations were made using up to 1.5 mM GdCl3or 5 mM EGTA (data not shown). On the other hand, direct modulation of cytosolic Ca2+concentration by the use of the Ca2+ionophore A23187 did not influence NtBI-1 expression pattern in control or Bap-treated cells (Figure 4-6D). Similarly, neither CaCl2nor A23187 had an influence on NtBI-1 accumulation pattern when added independently (data not shown). Interestingly, 0.5 mM LaCl3partially but significantly restored cell viability (Figure 4-6E). LaCl3had also a positive effect on growth, which was significant although limited, probably due to its toxicity (Figure 4-6F). Moreover, we also induced cell death with chemicals known to cause depletion of plant intracellular Ca2+stores, such as caffeine (Cessna et al. , 1998), mastoparan (Franklin-Tong et al. , 1996; Tucker and Boss, 1996; Takahashi et al. , 1998) or cyclopiazonic acid (Zuppini et al. , 2004). When these compounds were used at sub-lethal concentrations, the NtBI-1 protein accumulation remained unchanged, while toxic concentrations led to its down-regulation concomitant with massive cell death (data not shown), similar to the pattern observed for 50 µM Bap. These data indicate that an influx of externally-derived Ca2+into the cytosol is an early event in CK-induced PCD. Moreover, it reveals that NtBI-1 accumulation is not sensitive to alterations of the intracellular Ca2+homeostasis, and its up-regulation by CKs is not mediated by a Ca2+-sensitive pathway.

A) Wisconsin-38 cells were treated with 1.5 mM LaCl3, 5 mM EGTA or untreated (control), then immediately placed in the luminometer chamber and stimulated with 12.5 µM Bap (bold arrow). Each trace is an average of 4 or more independent experiments. The region of the graph under the black bar is that over which the [Ca2+]cytvalues for both EGTA- and La3+-treated cells differ statistically (p < 0.05) from control cells.

B-C) Xanthi cells were treated with the indicated amount of LaCl3or Bapta, and then exposed to 50 (B) or 2.5 (C) µM Bap. Cells were collected 24 h later for immunoblots analysis of NtBI-1. In (B) , Bap exposure was of 5 min as described in Figure 4.

D) Effect of [Ca2+]cytmodulation on Bap-induced up-regulation of the NtBI-1 protein. Xanthi cells were pretreated with 5 mM CaCl2in the presence or absence of 5 µM A23187, and then stimulated with 2.5 µM Bap. NtBI-1 was analyzed by immunoblots.

E-F) Cell viability and growth evaluated 96 h after co-treatment of Xanthi cells with LaCl3and Bap (as in B ). Significant differences are represented by different letters (p < 0.05; assessed from 5 independent experiments). In (E) ,Δcell viability was estimated by subtracting from the value of LaCl3-treated cells the value of their respective control (0 mM LaCl3).

In this study, we bring evidences that tobacco suspension cells present symptoms of a stress responseinvolving increased NtBI-1 accumulation when provided with exogenous CKs at physiological levels. The minimal CKs concentrations required to significantly impair growth (from 0.1 to 5mM) coincide with those promoting the NtBI-1 protein up-regulation, which are also concomitant with the PR1a mRNA induction under Bap treatments. The later mRNA encodes the most abundant PR protein in tobacco, typically associated with biotic stresses, further supporting the view of a stress response. In this context, increasing the NtBI-1 protein accumulation might help cells to survive to the insult and so be part of this stress response. This is also demonstrated by the use of any means that lowers Bap-induced stress and toxicity, such as AdkI and short treatments. In all these experiments, up-regulation occurs only for those situations promoting a stress response, revealed by a significant growth reduction. The relative effects of increasing amounts of Bap on different parameters assessed in this study are resumed in Figure 4-7. It clearly illustrates the tight relation between the NtBI-1 protein up-regulation, PR1a mRNA induction, growth impairment and cell survival. Of note, accumulation of PR1a mRNA and NtBI-1 protein were severely reduced when Bap concentration reached a toxic level, suggesting the activation at that point of a PCD pathway rather than a stress/defense pathway.

Intriguingly, we noted many discrepancies between NtBI-1 protein and mRNA levels in the course of this study. First, NtBI-1disappears from protein extracts of naturally (Figure 4-1C) or sucrose starvation-induced (cvs. Xanthi and BY-2; N. Bolduc and L. Brisson, unpublished data) senescing cultures, or when cells are treated with the ethylene precursor ACC (Table 4-1). On the other hand, NtBI-1 mRNA accumulation remains relatively stable from 3 to 7 DPT, and its counterpart in cultured Arabidopsis cells exhibits a relative constant level in aging cells (Swidzinski et al. , 2002). In all cases, the phenomenon can not be explained by general proteolysis that accompanies cell death since NtBI-1 disappearance preceded death (Figure 4-1C and unpublished data). As an anti-PCD protein, disappearance of NtBI-1 could mark the transition between a surviving mode to the execution of a PCD program. These results prompt us to speculate that cells systematically maintain a basal NtBI-1 mRNA expression level, but adjust the production of the corresponding protein as needed.

Data integrated from different periods of treatment are presented (24 h : PR1a and NtBI-1 mRNA, NtBI-1 protein, cell survival; 96 h: growth).

This hypothesis is further supported by the fact that low-moderate Bap concentrations lead to up-regulation of the NtBI-1 protein, while the accumulation of the mRNA remains relatively constant. The increased accumulation of the protein is thus regulated at the post-transcriptional level, consistent with some known effects of CKs including stimulation of protein synthesis (Fosket and Tepfer, 1978; Tepfer and Fosket, 1978). Of note, the CK-stimulated increased rate of synthesis of three ATP synthase subunits without changes of their corresponding mRNAs levels (in Lupinus luteus ) is due to specific polyribosome loading (Sherameti et al. , 2004).

Finally, we could hardly detect an up-regulation of the NtBI-1 protein under toxic Bap concentrations, while the mRNA steady-state level increased substantially (Figures 4-2 and 4-7). One could speculate that under mild stresses, protein synthesis relies on available NtBI-1 mRNA, which becomes insufficient for substantial protein production under strong stresses. Furthermore, turnover of the protein in dying cells may compensate for overaccumulation in still living cells, resulting in no apparent increased protein accumulation. Of interest, we observed similar accumulation profiles in planta in tobacco leaves undergoing massive HR following a permissive tobacco mosaic virus (TMV) infection (N. Bolduc and L. Brisson, unpublished data). Although HR was accompanied by increased NtBI-1 mRNA accumulation, extensive necrosis (and thus proteolysis) occurred within a few hours, so that accumulation of the protein remained constant and finally shut down along with massive necrosis. Similarly, Kawai-Yamada and others (2004) reported BI-1 mRNA induction following exposure to toxic levels of H2O2and SA in plant cell cultures, but we could hardly obtain correlative data at the protein level (Table 4-1). Even at sub-lethal levels, these compounds were not effective to stimulate up-regulation of the protein (Table 4-1 - 125mM), while they were effective to induce PR1a mRNA accumulation (in the case of SA; Gilbert, 1999; Norman et al. , 2004; our unpublished results). Therefore, our data clearly indicate that BI-1 mRNA accumulation does not always reflect the actual protein accumulation, and inversely protein accumulation does not always reflect mRNA accumulation. The relatively long half-life of the protein should also be taken into account while analyzing protein expression data.

Molecular mechanisms underlying CK-mediated stress response and PCDremain obscure. Intracellular CKs phosphorylation is required for their cytotoxic activity (Mlejnek and Kuglik, 2000; Ishii et al. , 2002; Mlejnek and Procházka, 2002; Mlejnek et al. , 2003; this study). Intracellular CK interconversion between the free base and the corresponding nucleoside and nucleotide forms are common (Jameson, 1994; Mok and Mok, 2001) and CK nucleotides are apparently the prominent metabolites formed immediately following cellular uptake (reviewed by Jameson, 1994). Accordingly, the nucleotide benzyladenosine monophosphate (Bad-MP) was the main metabolite (91%) detected 12 h after treatment with 30mM Bad in tobacco cells (Mlejnek et al. , 2003). Since cells are not permeable to CK-nucleotides (Kamínek et al. , 1997), the phosphorylation step could be a means to trap the molecule, but at the same time transforms it into a toxic byproduct. Indeed, Bad-MP accumulated up to 48 h into tobacco cells without apparent inactivation by conversion to another metabolite (Mlejnek et al. , 2003), which could be explained by the apparent inability of CK oxidase/deshydrogenase (CKX) to inactivate CK nucleotides (Laloue and Fox, 1989).

Intriguingly, the potent toxicity of the different CKs is unrelated to their known metabolic activity as growth promoter, Zea being usually the most potent (Skoog, 1973). On the contrary, Bap is the most effective stress and cell death inducer, followed by Kin, Zea, and finally iPA (although Zea ranks as the worst when considering only cell death induction). This is probably mostly due to their differential ability to serve as substrates for CKXs (before their eventual conversion to a nucleotide form), whose activities are dependent on the structure of the N6-side chain, with an apparent preference for isoprenoid moieties such as those exhibited by iPA and Zea (reviewed by Armstrong, 1994). Bap and Kin, with their phenolic side chain, are hence poor substrates for CKXs. Moreover, CKX more readily cleaves the side chain of iP than the hydroxylated side chain of Zea (reviewed by Armstrong, 1994), which could explain why iPA was a less potent inducer of the stress response. However, this does not provide a satisfactory explanation for the inefficiency of Zea as a cell death inducer. Similarly, Kin and Zea are not efficient cell death inducers in carrot cells (Carimi et al. , 2004). Intriguingly, Zea was inefficient to induce a Ca2+ cytpulse compared to Kin and Bap. Since our data indicate the partial involvement of the CK-mediated Ca2+ cytpulse in PCD induction, the inability of Zea to promote such an effect could be connected to its lower cell death-inducing activity.

Ca2+is an almost universal intracellular messenger and it has also been recognized as a ubiquitous signal in plant PCD and stress related situations (reviewed by Hoeberichts and Woltering, 2003; White and Broadley, 2003). CK action on Ca2+penetration into plant cells was clearly shown in the bryophyte mosses Funaria hygrometrica and Physcomitrella patens (Saunders and Hepler, 1983; Hahm and Saunders, 1991; Schumaker and Gizinski, 1993). However, evidence for this action remains poor and controversial in the case of higher plant (Mérillion et al. , 1991; Gouvêa et al. , 1997; Silverman et al. , 1998; Romanov et al. , 2002). Data presented in this study clearly show the occurrence of a rapid influx of an external Ca2+pulse into cells following Bap application and its partial involvement in the establishment of PCD. This Ca2+ cytpulse could thus represent a “Ca2+switch” (Scrase-Field and Knight, 2003) that participates to the stressed status of the cell. In animal cells, elevated Ca2+ cytis sensed and buffered by mitochondrial uptake (Reddy, 2001), but exceeding the buffering capacity leads to PTP opening and release of apoptogenic proteins such as Cyt c , which activates the apoptosome for the induction of the caspase cascade (Carafoli, 2004; Szabadkai and Rizzuto, 2004). Evidence is lacking for the presence of such a signaling pathway in plants, although Cyt c escape and occurrence of a mitochondrial permeability transition have been reported in plant PCD (Balk and Leaver, 2001; Tiwari et al. , 2002; Curtis and Wolpert, 2004). CK-induced PCD is accompanied by a rapid decrease of mitochondrial membrane potential in animal cells (Ishii et al. , 2002; Vermeulen et al. , 2002) as well as ATP depletion and Cyt c release in plant cells (Mlejnek et al. , 2003; Carimi et al. , 2003), indicative of mitochondrial dysfunctions in both cell types. Consequently, it is possible that the Ca2+switch is sensed by plant mitochondria, and/or by an array of Ca2+-sensors proteins.

It was surprising to observe substantial variations in pulses amplitudes and cell responsiveness from day to day experiments. We can speculate that the sensitivity of CK-induced Ca2+ cytpulse is potentially due to the nature of the channel involved. Shumacker and Gizinski (1993) demonstrated the involvement of voltage-dependent calcium channels (VDCCs) in CK action in moss. Opening of voltage-dependent ions channels, which is only transitory, depends of membrane depolarization, and after closing they remain refractory to further opening until repolarization (Jones, 2003). Mild stresses imposed on cultured cells (such as alteration in the shaking speed or direction) might activate the opening of such channels, making them refractory to further stimulation by CKs. Of interest, ABA-induced Ca2+ cytpulse in guard cells involves VDCCs (Yang et al. , 2004).

ROS signaling has been suggested to be a signal for BI-1 induction (Kawai-Yamada et al. , 2004). In this report we investigated whether BI-1 modulation could be mediated by Ca2+signaling. It is apparently not the case, although Bap-mediated up-regulation of the NtBI-1 protein coincided with a stress response and an influx of external Ca2+. Indeed, any modulations of the Ca2+homeostasis (including by Bap treatments) failed to change the accumulation profile of the protein. This indicates that if the cytosolic Ca2+level has any influence on NtBI-1 function, this is not via the modulation of its accumulation, although modulation of its activity can not be excluded at the moment. Conversely, a number of data point toward the involvement of plant BI-1 in Ca2+homeostasis. In metazoans, ER stresses induce the opening of ER Ca2+channels and subsequent Ca2+uptake by mitochondria, potentially leading to the activation of a death program following mitochondrial dysfunctions (Orrenius et al. , 2003). Pro-apoptotic proteins such as Bax and Bak help maintaining a higher [Ca2+]ERsteady-state (Scorrano et al. , 2003; Oakes et al. , 2005), while the anti-apoptotic Bcl-2 rather favors a lower level (reviewed by Distelhorst and Shore, 2004). This represents more (Bax) or less (Bcl-2) Ca2+release from the ER when cells are under adverse conditions integrated at the ER (e.g. accumulation of misfolded proteins), and thus more or less opportunity to reach Ca2+overload by mitochondria and subsequent PCD induction. BI-1 can counteract Bax toxicity without direct protein-protein interaction and in different cellular systems (see introduction), thus opening the possibility of a lowering effect of BI-1 on [Ca2+]ERsteady-state. Indeed, Chae and others (2004) recognized the hypersensitivity of bi-1 -/-mice cells to apoptosis induced by ER stress agents, and found that BI-1 overexpression reduces releasable Ca2+from the ER similar to Bcl-2. This definitively links the BI-1 protein to the control of Ca2+homeostasis, although how it exerts this action remains unknown. It could involve the modulation of the expression or activity of Ca2+-regulating proteins, as observed for Bcl-2 (reviewed by Distelhorst and Shore, 2004; Oakes et al. , 2005).

Taken all together,our data indicate that increasing the accumulation of the NtBI-1 protein in the course of the stress response to CKs might contribute to cell survival. BI-1 is part of a pathway where its expression level influence cellular ability to resist to carbon starvation- or senescence-induced stresses (Bolduc and Brisson, 2002), potentially via modulation of intracellular Ca2+homeostasis (Chae et al. , 2004). Modulation of BI-1 levels by CKs might be indirectly due to the activation of the stress response, although we can not exclude at the moment a direct effect of CKs via the two-component phosphorelay system (Hutchison and Kieber, 2002). Unraveling the signalization leading to this CK-mediated BI-1 up-regulation will need further studies, which could also reveal the involvement of this negative PCD regulator in the senescence delay-activities of CKs.

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