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

Biochimie 89 (2007) 961e971 www.elsevier.com/locate/biochi

Modulation of Bax Inhibitor-1 and cytosolic Ca2þ by cytokinins in Nicotiana tabacum cells Nathalie Bolduc a,*, Gregory N. Lamb b,c, Stephen G. Cessna b,c, Louise F. Brisson a b

a De´partement de Biochimie et de Microbiologie, Universite´ Laval, Que´bec, Qc, G1K 7P4, Canada Department of Chemistry, Eastern Mennonite University, 1200 Park Rd, Harrisonburg, VA 22802, USA c Department of Biology, Eastern Mennonite University, 1200 Park Rd, Harrisonburg, VA 22802, USA

Received 12 October 2006; accepted 9 February 2007 Available online 20 February 2007

Abstract 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 Ca2þ pulse, and inhibition of this pulse with La3þ or EGTA partially restored viability, indicating a signaling role for Ca2þ in CK-induced cell death. However, CK-induced NtBI-1 accumulation was not altered by pretreatment with La3þ, nor by treatment with several modulators of intracellular Ca2þ homeostasis and signaling, suggesting that CK-dependent regulation of NtBI-1 accumulation is not directly mediated by Ca2þ. Ó 2007 Elsevier Masson SAS. All rights reserved. Keywords: Bax Inhibitor-1; Calcium; Cytokinins; Hormones; Programmed cell death

1. Introduction Programmed cell death (PCD) is a 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. In plants, the proper occurrence of a PCD program is essential throughout life, e.g. for the formation of xylem vessels, root aerenchyma

Abbreviations: Ade, adenine; Bap, 6-benzylaminopurine; CK, cytokinin; Chx, cycloheximide; DPT, days post transfer; ER, endoplasmic reticulum; iPA, N6-(2-isopentenyl)adenosine; Kin, kinetin; NtBI-1, Nicotiana tabacum orthologue of Bax inhibitor-1; PCD, programmed cell death; Zea, zeatin. * Corresponding author at present address: USDA-ARS, Plant Gene Expression Center, 800 Buchanan Street, Albany, CA 94710, USA. Tel.: þ1 510 559 5922; fax: þ1 510 559 5678. E-mail address: [email protected] (N. Bolduc). 0300-9084/$ - see front matter Ó 2007 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.biochi.2007.02.004

and pollen grains, and for the establishment of the hypersensitive response (HR) following pathogen attack [1]. Plant PCD is less documented than animal PCD. However, current 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 Ca2þ fluxes [1]. Some similarities with apoptosis, a major animal PCD form, have been observed in plant PCD, such as cytoplasmic shrinkage and internucleosomal DNA fragmentation [2,3]. However, plants mostly rely on their vacuole to undergo PCD [4,5], reminiscent of the lysosomal/autophagic PCD type in animal development [6]. While animal apoptosis is regulated by well characterized pro- and anti-apoptotic regulatory members of the Bcl-2 family, most plant PCD regulators are still to be discovered, and Bcl-2-related genes have no sequence homologues in plants [7]. In animals, the anti-apoptotic Bcl-2 regulates PCD by

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interacting with structurally similar proteins of the Bcl-2 family such as Bax, an important inducer of PCD. These proteins act competitively mainly through their effect on mitochondrial membrane integrity or endoplasmic reticulum (ER) Ca2þ homeostasis [8]. The multi-spanning membranous ER protein Bax Inhibitor-1 (BI-1), a negative regulator of apoptosis in mammals [9], has recently emerged as an evolutionarily conserved negative regulator of plant PCD. Human BI-1 has been originally shown to suppress Bax-induced cell death in yeast and human cells [9]. Numerous plant homologues were subsequently identified, sharing not only high sequence identity but also functionality. Indeed, plant BI-1 can counteract Bax in yeast [10,11], in human embryonic kidney cells [12] and in Arabidopsis thaliana [13]. Although BI-1 is a negative regulator of intrinsic plant PCD [14e17], its mode of action remains unknown. However, a recent investigation by Chae and co-workers [18] indicates that in mouse, BI-1 could act by lowering the amount of releasable Ca2þ from the ER, as similarly demonstrated for Bcl-2 [8]. Cytokinins (CKs) are a structurally diverse group of N6-substituted purine derivatives traditionally recognized as anti-PCD hormones, principally because they promote growth and differentiation and delay senescence. Specifically, their level declines during senescence, and exogenous application of this hormone or overexpression of the bacterial CK biosynthetic enzyme isopentenyl transferase (ipt) delays leaf senescence [19]. 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 [20e24]. Adenine (Ade) and adenosine derivatives with CK activities are also efficient apoptosis inducers in animal cells [25e27]. At the whole plant level, toxicity associated with high levels of CKs in plant tissues (due to exogenous application or increased accumulation in transgenic plants expressing ipt) has been reported episodically [23,28e32]. In order to better characterize the regulation of BI-1 in plant cells, we screened for compounds that could modulate BI-1 accumulation at the protein level. We observed that cultured tobacco cells exogenously provided with the active CK 6-benzylaminopurine (Bap) showed up-regulation of NtBI-1. In this paper, we describe the modulation of the NtBI-1 protein under low (stress-inducing) and high (PCD-inducing) concentrations of CKs and we show a tight correlation between the accumulation of the protein and the occurrence of a stress response. Moreover, we show that CK-induced cell death appears to be partially Ca2þ dependent, while NtBI-1 accumulation is not. 2. Materials and methods 2.1. Plant material, culture conditions and treatments Unless otherwise specified, chemicals were obtained from Sigma. CK-habituated N. tabacum cv. Xanthi cells used in this study have been in culture for at least ten years. Cells were subcultured weekly in Murashige and Skoog medium supplemented with 3% (w/v) sucrose and 4.5 mM

2,4-dichlorophenoxyacetic acid (2,4-D). The day of the subculture is referred to as day 0, while subsequent days are referred to as days post transfer (DPT). For treatments, 4-day-old (4 DPT) cells were diluted 1:1 with fresh medium (to get a larger volume of cells) and used 2 days later, thus obtaining a homogenous exponentially growing cell population equivalent to 3e4 DPT cells. Thus, cells treated for 96 h were equivalent to 7e8 DPT, when cells reach the stationary phase. When specified, CKs were added to the medium from a 5 mM stock solution, prepared by first dissolving the powder in a drop of 0.1 M HCl (final 2 mM). Control cells were treated with the CK structural analog Ade at 10 or 50 mM. All pre-treatments with LaCl3, EGTA, CaCl2, A23187 or 1,2-bis(2-aminophenoxy) ethane-N,N,N0 ,N0 -tetraacetic acid (Bapta) were done for a period of 5 min before addition of 6-benzylaminopurine (Bap). In the case of LaCl3 and EGTA, toxicity associated with these chemicals was partially circumvented by the use of a short treatment in the presence of a high Bap concentration. Five minutes after addition of 50 mM Bap, chemicals were removed from the medium by washing cells three times in MS salts, and cells were then transferred into ‘‘medium without Bap’’ (conditioned medium without Bap from same-age cultured cells). 2.2. Immunoblot and northern blot analysis Preparation of total protein extracts and immunoblot analysis using anti-BI-1 serum were carried out as described [14]. Briefly, the material was ground in liquid nitrogen, weighted in 1.5 ml centrifuge tubes, and lysis buffer was added at a ratio of 150 ml per 100 mg of powder. In preliminary experiments, quantification of protein content revealed that this method ensures a homogenous protein concentration in all samples (2 mg/ml). Gel electrophoresis was performed using 30 ml of these extracts and following protein transfer to a PVDF membrane, homogenous loading was confirmed by Ponceau Red staining (those shown present an unidentified protein of about 20 kDa). All procedures for northern blot were done according to Bolduc et al. [12]. 2.3. Fresh weight increase and cell viability determination After 96 h of treatment, cells were collected on filter papers under vacuum, weighted immediately and values were adjusted to 1 ml of culture. Value from day 0 was subtracted in order to get the gain of fresh weight per ml of culture. For determination of viability, cells were collected in microcentrifuge tubes (0.5 ml packed cell volume), and incubated for 5 min in the dark with 50 mg/ml fluorescein diacetate (FDA), which specifically stains living cells. Cells were then washed once in MS medium and resuspended in MS to a final volume of 1 ml. Intensity of cellular fluorescence was measured in a 96-well plate with a Fluoroskan Ascent FL (ThermoLabsystems, Finland) using an excitation wavelength of 488 nm and an emission bandpass filter of 527 nm. Viability in control cells remained over 95% under our experimental

N. Bolduc et al. / Biochimie 89 (2007) 961e971

conditions, and values from treated cells were expressed as percentage of control cells value. Initial results obtained were confirmed by microscopic counts using FDA, giving similar results. 2.4. Luminometry and Ca2þ quantification Luminometry of aequorin-transformed suspension cultures (N. tabacum cv. Wisconsin-38, referred to as W38) was performed as previously described [33] with minor changes. Briefly, 48 h after subculture into fresh MS medium, 1 mM coelenterazine (Nanolight Technology, Pinetop, AZ) was added to cells for 14e20 h. An aliquot (0.1 ml) was then transferred to a luminometer cuvette and placed in a Turner Designs TD 20/20 luminometer chamber. Luminescence was integrated 5 times per second. CKs or Ade were added from 100 mM stock solutions corrected to the osmolarity of the MS media (measured at 180  4 mOsM with a vapor pressure osmometer) by the addition of 180 mM sucrose. No measurable change in osmolarity or pH occurred in cell culture media after addition of either a 180 mM sucrose solution, Ade or CK in sucrose. LaCl3 and EGTA were added to the cell cultures from 50 mM stock solutions. At the end of each experiment, aequorin was discharged with a detergent solution and [Ca2þ]cyt was calculated from the resulting luminescence data as previously described [33]. 2.5. Data analysis All data presenting cell death and fresh weight increases are the mean values derived from at least three independent experiments performed on different weeks, while immunoblots are typical examples. All standard deviations and p-values were calculated using the Student’s t-test. In the case of Ca2þ quantification, these values were determined for every point on the graph; the error bars for standard deviations are shown only at select time points. 3. Results 3.1. CKs induce NtBI-1 upregulation at concentrations inducing a stress response Preliminary works suggested the modulation of NtBI-1 at the protein level following an exposure to Bap. Considering that CKs have been previously associated with PCD in cell cultures (see Section 1), we wanted to better characterize this CK-induced NtBI-1 up-regulation. Tobacco cell cultures were supplemented with increasing concentrations of four different CKs (N6-substituted purine derivatives: Bap, kinetin (Kin), N6-(2-isopentenyl)adenosine (iPA) and trans-zeatin (Zea)) and were compared to cells treated with the structural CK analog Ade (10 mM). The effects of physiological to toxic CK concentrations on cell viability, monitored after 96 h of treatment, are summarized in Table 1. The precise concentration reducing significantly the viability of Xanthi cells varied from one CK to another, although all fell in the same

963

Table 1 Cell viability in tobacco cells after treatment with various CKs mM

Viability (%)a Xanthi

0 0.01 0.1 1 10 25 50 100

Wisconsin-38

Bap

iPA

Kin

Zea

Bap

100 113 87 85 67** 21** 10** nd

100 nd 115 106 100 33* 18* nd

100 nd 99 102 74** 42** 20** 11**

100 nd 89 86 95 57* 30** 29**

100 92 97 102 37* 16** 12** nd

a Viability was determined 96 h after the beginning of the treatment and statistical analysis were done by comparison to the 10 mM Ade controls (*p < 0.05; **p < 0.001; nd, not determined).

concentration range of 10e50 mM. Bap and Kin induced cell death at 10 mM, while this required concentrations of 25 mM in the case of iPA and Zea. DNA laddering, a hallmark of PCD, was detected only for 25 and 50 mM Bap (not tested for other CKs; data not shown). Cells were less sensitive to Zea than to other CKs, with a viability of 57% after having been exposed for 96 h to 25 mM Zea, while it was 20e40% with other CKs. Furthermore, around 30% of the cells were still viable at 100 mM Zea, while other CKs left no more than 10e20% of viable cells when exposed to 50 mM. Fresh weight measurements of CK-treated cultures revealed growth impairment at non-lethal CKs concentrations (Table 2). As little as 0.1 mM Bap was sufficient to reduce growth, while 5 mM of iPA, and 0.5 mM of Kin and Zea were required to achieve the same effect. No effect on viability or growth was observed after an exposure to Ade up to 50 mM (data not shown). The influence of different CKs on NtBI-1 accumulation is presented in Fig. 1. Up-regulation of the protein was consistently observed for 0.1e10 mM Bap, weakly at 25 mM and never at 50 mM (Fig. 1A), while up to 50 mM Ade had no effect on the protein accumulation (data not shown). Moreover, the increased accumulation was sustained in the time frame analyzed (up to 96 h) for 1e10 mM, and to a lower Table 2 Growth reduction of tobacco cells after treatment with various CKs mM

Fresh weight increase (mg/ml)a Xanthi

0 0.01 0.1 0.5 1 5 10 25 50 a

Wisconsin-38

Bap

iPA

Kin

Zea

Bap

253 267 85** nd 7** nd 6** 0** 27**

233 nd 244 nd 186 51* 19** 9** 4**

263 nd 229 49* 15** nd 4** 2** 32**

287 nd 283 117* 93** nd 9** 7** 3**

176 170 111 67* 48* 36* 13** 23** 33**

Fresh weight increase was determined 96 h after the beginning of the treatment and statistical analysis were done by comparison to the 10 mM Ade controls (*p < 0.05; **p < 0.001; nd, not determined).

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Fig. 1. NtBI-1 protein accumulation is increased by CKs but decreased by aging. (A) Immunoblots show typical Bap doseeresponse profiles after treatments of 24 or 96 h. (B) Cell viability (left) and NtBI-1 protein expression profile (right) in naturally senescing Xanthi cultures. Five days post transfer (DPT) cells were used as control for viability determination. The immunoblot presented was overexposed in order to favor the detection of very low amount of protein. (C) Doseeresponse analysis of NtBI-1 accumulation in Xanthi cells after a 24 h exposure to Kin, iPA or Zea.

extent at 0.1 mM. The protein was barely detectable in 96 h control cells (Fig. 1A), which is roughly equivalent to a 7 DPT stage in our experimental conditions. At 7 DPT, cells were reaching stationary phase and still exhibited good viability, but by 11 DPT, viability has already dropped below 70% and NtBI-1 was no longer detectable, even with over saturated conditions of detection (Fig. 1B). Interestingly, the Bap threshold for NtBI-1 up-regulation is identical to the one leading to growth reduction (Table 2), revealing a tight correlation between stress-inducing Bap concentrations (as reflected by limited or absence of growth) and NtBI-1 increased accumulation. NtBI-1 up-regulation was not specific to Bap but was also observed with all other tested CKs, although the effective concentrations were variables (Fig. 1C). Increased accumulations were obvious for Kin from 0.5 to 25 mM, for iPA from 1e5 to 10 mM (variable at 1 mM), and 0.5e50 mM Zea produced the same results. As previously observed for Bap, increased NtBI-1 accumulation following these treatments tightly correlated with concentrations that significantly induced growth

impairment, which started at 5 mM for iPA, and 0.5 mM for Kin and Zea (Table 2). A time-course analysis on 0.1e50 mM Bap-treated cells revealed an increased accumulation of NtBI-1 starting around 12 h after addition of Bap at concentrations ranging from 0.1 to 10 mM (Fig. 2A). At 25 mM, a concentration that significantly impaired cell viability (Fig. 2C), the increased accumulation was not always observed and was weaker than in 0.1e10 mM Bap-treated cells (Fig. 2A). At 50 mM, we observed a sustained basal NtBI-1 level up to 12 h followed by a slight decrease at 24 h and complete disappearance thereafter (Fig. 2A). This disappearance is likely due to general proteolysis, considering the high cell death rate (Fig. 2C) and the loss of integrity of high molecular weight proteins and Rubisco starting at 48 h (Fig. 2D). Because most of these cells were already dead by 24 h, we asked how long NtBI-1 could be detected in dying cells. Inhibition of protein synthesis by the use of cycloheximide (Chx) showed an important reduction in NtBI-1 detection from 12 h (Fig. 2B), while most of the cells were already dead after 6 h of treatment (Fig. 2C), indicating a quite long half-life of the protein (6e12 h). In order to better evaluate the stress status of the Baptreated cells, accumulation of the defense/stress marker PR1a was evaluated by northern blots. The mRNA accumulated in the presence of 0.1e10 mM Bap (Fig. 2E and data not shown), while it was virtually undetectable under standard culture conditions until cells reached the stationary phase (96 h; Fig. 2E). Detectable accumulation started between 6 and 12 h and reached a peak after 48 h of treatment. Interestingly, while 0.1 and 1 mM Bap led to strong and similar PR1a mRNA accumulations, 10 mM Bap was a less potent inducer (Fig. 2E), which could be attributable to some cell death (Fig. 2C). Accordingly, when cells were treated with a toxic dose of Bap such as 25 and 50 mM, the accumulation was weak and transitory or even undetectable (Fig. 2E). The accumulation of NtBI-1 was also evaluated at the RNA level and showed a pattern quite different from the one observed at the protein level. NtBI-1 transcript accumulations were relatively constant over time for control cells and those treated with 0.1 (data not shown) and 1 mM Bap (Fig. 2E). However, in cells exposed to toxic concentrations (25e 50 mM), we observed a transient accumulation of transcript followed by a complete disappearance concomitant with a massive RNA degradation. 3.2. CKs induce cytosolic Ca2þ pulses Because of its localization to ER membranes, a Ca2þ reservoir, BI-1 is suspected to exert its function through some influence on Ca2þ homeostasis [18]. CK-induced Ca2þ uptake in cells of the bryophyte mosses Funaria hygrometrica and Physcomitrella patens has already been reported [34e36], and we asked if Bap-induced NtBI-1 up-regulation could be the consequence of cytosolic Ca2þ signaling. For this purpose, CK-induced Ca2þ cyt pulses were measured in Wisconsin-38 aequorin-transformed (W38) tobacco suspension cells. Both Bap and Kin but not Zea (Fig. 3A), induced rapid and

N. Bolduc et al. / Biochimie 89 (2007) 961e971

A

0

6

12

24

48

96

h

E

0

6

12

24

48

96

h BI-1

0 0.1

3

965

0

PR1a

1 BI-1

10 25

1

PR1a

50

B

BI-1 0

3

6

12

24

48

h

10

PR1a

Chx

cell viability ( )

C

BI-1 120 25

100

PR1a

80 60 BI-1

40 20

50

PR1a

0 0

D

0

24

6

12

48

24

72

48

96 h

h

Fig. 2. Time-course analysis of NtBI-1 and PR1a in Xanthi cells. (A,B) Cultures were exposed to 0e50 mM Bap (A) or 50 mg/ml Chx (B) and NtBI-1 was detected by immunoblot. (C) Viability after an exposure to Ade (10 mM B), Bap (10 mM ; 25 mM ,; 50 mM -) or Chx (6). (D) Coomassie blue staining of protein extracts from cells treated with 50 mM Bap. The most intense band visible from 0 to 24 h is the Rubisco. (E) Accumulation of NtBI-1 and PR1a mRNAs detected by northern blots. Ethidium bromide-stained gels (lower panels) show RNA loading. Wells with reduced rRNA had degraded RNA visible in the lower part of the gels, reminiscent of massive cell death.

substantial Ca2þ cyt pulses, each reaching a peak greater than 1 mM Ca2þ within the first minute of CK application, followed cyt by a slow sustained decrease to about the half of maximal levels after 3 min. Although variability in pulse amplitude was observed from day to day, in several different experiments we could determine that maximal pulse amplitude was reached with w1 mM Bap, and the minimal concentration required to initiate a Ca2þ cyt pulse was w0.1 mM (Fig. 3B). This concentration range is similar to that necessary to induce a stress response (including NtBI-1 up-regulation) in tobacco Xanthi cells. Since the Ca2þ cyt quantification has been performed in different cells, we verified if W38 cells responded similarly to Bap in terms of growth, cell death and NtBI-1 accumulation. NtBI-1 up-regulation was clearly observed from 0.5 to 10 mM Bap, although there was sometime a slight increased accumulation starting at 0.05 mM (Fig. 3C). Viability was not impaired until a concentration of 10 mM was applied (Table 1), while growth became significantly reduced starting at 0.05 mM (Table 2). Thus, as observed for Xanthi cells, W38 cells are highly sensitive to exogenous Bap supply, and the up-regulation of NtBI-1 coincides with stress-inducing (growth-reducing) concentrations (0.5 mM and more).

3.3. Ca2þ movement from the apoplast to the cytosol partially mediates CK effects We investigated the role of this Ca2þ cyt pulse in Bap-mediated PCD. We first noticed the potent inhibition of the CK-induced Ca2þ cyt pulse by prior addition of the cation channel blocker LaCl3, or the cell-impermeant Ca2þ chelator EGTA (Fig. 4A), revealing an apoplastic origin of the Ca2þ cyt pulse. We noted an important toxicity of these chemicals in our cellular system (massive death observed after an exposure of 24 h; N. Bolduc, unpublished data), making it difficult to investigate the effects of these compounds. To circumvent this problem, we used a short pulse of 50 mM Bap in absence or presence of La3þ or EGTA followed by washes (see Section 2). This greatly reduced the toxicity of La3þ and EGTA while still allowing the increased accumulation of NtBI-1 (and permitted about 80% of the cells to survive the Bap treatment; data not shown). LaCl3 at 0.5 mM partially but significantly restored cell viability (Fig. 4B). LaCl3 at 0.5 mM had also a positive effect on growth (Fig. 4C), allowing Bap-treated cultures to accumulate a modest but significantly higher fresh weight than the cultures not pre-treated with the chemical.

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A

1.6 1.4

[Ca2+]cyt (µm)

1.2 1 0.8 0.6 0.4 0.2 0 0

0.5

1

1.5

2

2.5

3

Time (minutes)

B cell viability ( )

15

a 5 0

Fig. 3. Bap and Kin induce a rapid and transient cytosolic Ca2þ flux. W38 cells were treated with CKs at the time indicated by the bold arrow. (A) Cells were treated with 25 mM Bap, Kin, Zea or Ade. Each trace is the average of 8 or more independent experiments. The region of the graph under the black bar is that during which the [Ca2þ]cyt values for both Bap- and Kin-treated cells differ statistically ( p < 0.01) from the Ade trace. There is no significant difference between the responses generated by Bap and Kin, or between Zea and the control Ade ( p > 0.05). (B) Doseeresponse analysis using 0.1e50 mM Bap. Data shown are representative of ten independent experiments. (C) Doseeresponse analysis of NtBI-1 accumulation in W38 cells after a 24 h exposure to 0.01e50 mM Bap. Control (C) in (B) and (C) is 10 mM Ade.

On the other hand, the washing strategy was not as effective to reduce the long term toxic effects of EGTA (reduction of viability by 20% at 5 mM, data not shown), ruling out studies on cell viability and growth. Taken together, these data indicate that an influx of externally-derived Ca2þ into the cytosol is an early event partially involved in CK-induced toxicity. 3.4. NtBI-1 protein accumulation is not sensitive to modulations of Ca2þ homeostasis Direct modulation of [Ca2þ]cyt by the use of the Ca2þ ionophore A23187 in the presence of CaCl2 did not influence

Fresh weight increase (g/ml)

a

a

50 0

50 0

0 50

0 50

µM Ade µM Bap

0.5

1.5

0.5

1.5

mM LaCl3

-5 -10

C

b

10

0.25 a 0.20 a

0.15

b

0.10 0.05

d

c

c

0.00 0 50

0.5 50

1.5 50

0 0

0.5 0

1.5 0

mM LaCl3 µM Ade

0

0

0

50

50

50

µM Bap

2þ

Fig. 4. Modulation of CK effects by Ca -signaling inhibitors. (A) W38 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 mM Bap (bold arrow). Each trace is an average of four or more independent experiments. The region of the graph under the black bar is that over which the [Ca2þ]cyt values for both EGTA- and La3þ-treated cells differ statistically ( p < 0.05) from control cells. (B,C) Xanthi cells were first treated with 0e 1.5 mM LaCl3, exposed for 5 min to the indicated amount of Ade or Bap, and washed as described in Section 2. Cell viability and growth were evaluated 96 h after the co-treatment. Significant differences are represented by different letters ( p < 0.05; assessed from five independent experiments). In (B), D cell viability was estimated by subtracting from the value of LaCl3-treated cells the value of their respective control (0 mM LaCl3).

NtBI-1 expression in control or Bap-treated cells (Fig. 5A). Similarly, neither supplemental CaCl2 nor A23187 had an influence on NtBI-1 accumulation pattern when added independently (data not shown). Inhibition of Bap-mediated Ca2þ cyt pulse using up to 1.5 mM LaCl3 or the specific cell-impermeant Ca2þ chelator Bapta were also unsuccessful to alter the Bap-induced NtBI-1 up-regulation (Fig. 5B). Similar results

N. Bolduc et al. / Biochimie 89 (2007) 961e971

967 Viability

Relative intensity

++++

+++

[Ca2+]cyt PR1a BI-1

BI-1 protein

mRNA

++

+ Growth 0

0

0.1

1

10

25

50

M

Fig. 6. Schematic representation of the kinetic relationship between increasing amounts of Bap and Ca2þ cyt , growth, viability, accumulation of NtBI-1 as well as accumulation of PR1a mRNA in tobacco cells. Data integrated from different periods of Bap treatments are presented (early effects, Ca2þ cyt ; 24 h, PR1a, NtBI-1, viability; 96 h, growth).

4.1. CKs as cell death inducers Fig. 5. Evaluation of Ca2þ-signaling modulators on NtBI-1 accumulation in Xanthi cells. (A) Effect of [Ca2þ]cyt modulation on Bap-induced up-regulation of the NtBI-1 protein. Cells were pretreated with 5 mM CaCl2 in the presence or absence of 5 mM A23187, and then stimulated with 2.5 mM Bap. The application of water or CaCl2 alone gave a pattern equivalent to A23187 alone (not shown). (B) Cells were treated with the indicated amount of LaCl3, EGTA or Bapta, and then exposed to Bap or Ade for 5 min. Cells were then washed (see Section 2) and collected 24 h later for immunoblots analysis of NtBI-1.

were obtained using up to 1.5 mM GdCl3 (data not shown) or 5 mM EGTA (Fig. 5B). Moreover, we induced cell death with chemicals known to cause depletion of plant intracellular Ca2þ stores, such as caffeine [37], mastoparan [38] or cyclopiazonic acid [39]. 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). This is very similar to the pattern observed for 50 mM Bap (Fig. 2). These data reveal that NtBI-1 protein accumulation is not sensitive to alterations of the intracellular Ca2þ homeostasis, and its up-regulation by CKs is apparently not mediated by a Ca2þ-signaling pathway. 4. Discussion In this study, we provide evidence that tobacco suspension cells respond to exogenous CKs by an increased accumulation of the NtBI-1 protein, presumably as part of a stress response. In order to characterize this response to CKs, we investigated different parameters that are summarized in Fig. 6. It illustrates the tight relation between increasing amounts of Bap versus growth, viability, NtBI-1 and PR1a accumulation, as well as the occurrence of a Ca2þ cyt pulse.

While CKs are phytohormones known to promote growth and delay senescence, the recent reports of PCD induction by Ade and adenosine derivatives (see Section 1 for references) is surprising. Considering that CKs can induce cell death in both plant and animal cells [26,27], the latter lacking the molecular machinery identified in plant cells to perceive and transduce CK signal, one could speculate that biochemical events associated with CK-induced death are different than those involved with their growth promoter activities. Accordingly, we found that the toxicity associated with different CKs is unrelated to their known metabolic behavior. While Zea is usually the most potent growth promoter [40], we found that Bap is the most effective cell death inducer, followed by Kin, iPA, and finally Zea. CK cytotoxicity could be due to their differential ability to be inactivated by CK oxidase/deshydrogenase (CKX), 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 in [41]). Bap and Kin, with their phenolic side chain, are poor substrates for CKX. However, this does not provide a satisfactory explanation for the relative inefficiency of Zea as a cell death inducer, which is also inefficient in carrot cells [23]. Hormonal effects of CKs are mediated through a phosphorelay signaling pathway in which histidine kinases play a pivotal role as CK sensors (for a review see [42]). These CK receptors, localized at the plasma membrane, directly bind a variety of natural and synthetic CKs in a highly specific manner. Since only active CKs compete for the binding [43], we can speculate that the most active CKs such as Zea are more likely trapped by receptors at the plasma membrane and less likely get into cells where they could be involved in activities related to cell death.

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4.2. CKs as stress inducers A correlation between high level of CKs and accumulation of stress genes, including pathogenesis-related (PR) genes, has been reported in both whole plant tissues [29,31,44] and in vitro tissue cultures [32,45,46], suggesting the induction of a stress response by CKs. Data presented in this study also support such a view. All tested CKs favored a stress response as measured by a reduction of growth. Furthermore, all of them promoted an up-regulation of the protein NtBI-1. The minimal CKs concentrations required to significantly impair growth (from 0.1 to 5 mM) coincide with those necessary to induce the NtBI-1 protein, as well as the PR1a mRNA (in the case of 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 the insult and so be part of this stress response. Of note, accumulation of PR1a mRNA and NtBI-1 protein were severely reduced when Bap reached a toxic level (25e50 mM), suggesting the activation at that point of a PCD pathway rather than a stress/defense pathway. Interestingly, our data also lead to the speculation that CKs could delay leaf senescence by positively regulating BI-1. 4.3. BI-1 regulation Interestingly, we noted some discrepancies between NtBI-1 protein and mRNA levels. Indeed, we observed that low-moderate Bap concentrations (0.1e10 mM) lead to a relatively stable mRNA accumulation, while the protein is up-regulated. On the other hand, we could almost not detect an up-regulation of the NtBI-1 protein under toxic Bap concentrations (or any other treatment with a relatively high cytotoxicity (N. Bolduc, unpublished data)), while the mRNA steady-state level increased substantially (Fig. 2). These data suggest a complex regulation of this protein in plants. Increased BI-1 mRNA accumulation in situations associated with enhanced cell death has already been reported in cell suspension cultures exposed to toxic concentrations of H2O2 or SA [17], in tissues undergoing senescence [12,47] and in stressed plants [11,13,15,48,49], although Matsumura and co-workers [16] reported the down-regulation of OsBI-1 in rice cells upon challenge with a fungal elicitor. However, our study is the first report of BI-1 accumulation directly at the protein level. Immunoblot analysis of cell cultures treatments with physiological to toxic concentrations of H2O2 or SA did not lead to an increased NtBI-1 accumulation (N. Bolduc, unpublished data). Similarly, induction of HR in tobacco leaves by the tobacco mosaic virus revealed a strong increased accumulation of NtBI-1 mRNA, but failed to produce any detectable NtBI-1 protein upregulation (N. Bolduc, unpublished data). All these information prompt us to speculate that under some mild stresses, the protein stability is increased, or increased protein synthesis relies on available mRNA, which becomes insufficient for substantial protein production under stronger stresses. Furthermore, the turnover of the protein in dying cells may compensate for over-accumulation in still

living cells, resulting in no apparent increased protein accumulation. Of note, increased NtBI-1 protein accumulation concomitant with a stable mRNA buildup is consistent with the direct stimulation of protein synthesis by CKs [50]. Accordingly, the CK-stimulated increased synthesis rate of three ATP synthase subunits without changes of their corresponding mRNAs levels in Lupinus luteus is due to specific polyribosome loading [51]. Clearly, more work will be necessary to understand the regulation of BI-1 in plants. 4.4. CKs, Ca2þand cell death Ca2þ is an almost universal intracellular messenger that has been recognized as an ubiquitous signal in plant PCD and stress related situations (reviewed by [1,52]). CK action on Ca2þ penetration into plant cells was clearly shown in the bryophyte mosses F. hygrometrica and P. patens [34e36]. However, evidence for this action still awaits experimental data in the case of higher plant [53e55]. Data presented in this study show the occurrence of a rapid influx of an external Ca2þ pulse into cells following Bap application and its partial involvement in the establishment of PCD. In animal cells, elevated [Ca2þ]cyt is sensed and buffered by mitochondrial uptake, but exceeding the buffering capacity leads to mitochondrial permeability transition pore opening and release of apoptogenic proteins such as Cyt c, which activates the apoptosome for the induction of the fatal proteolytic caspase cascade [56]. 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 some cases of plant PCD [2,57]. CK-induced PCD is accompanied by a rapid decrease of mitochondrial membrane potential in animal cells [26,27] as well as ATP depletion and Cyt c release in plant cells [21,22,24], indicative of mitochondrial dysfunctions in both cell types. If the CKinduced Ca2þ cyt pulse is effectively sensed by plant mitochondria and/or Ca2þ-sensors proteins, the inefficiency of Zea to promote a Ca2þ cyt pulse could explain its lower potency as a cell death inducer. This is further supported by the tight relation between Ca2þ cyt pulse amplitude generated by a given CK concentration and CK ability to induce a stress response. Indeed, we found that the lowest Bap concentration able to reduce growth (0.1 mM) is also the minimal concentration allowing a modulation of the Ca2þ cyt . On the other hand, inhibition of the Ca2þ pulse from the apoplast to the cytosol following Bap application only partially suppressed Bap toxicity (Fig. 4), and was barely beneficial to restore growth. The inhibitors themselves (La3þ and EGTA) showed some toxicity, even when used only for a short treatment, but this alone can not explain the limited improvements observed. This potentially indicates that other molecules are also involved in the signal transduction leading to cell death. Indeed, Carimi and collaborators [58] recently demonstrated in an Arabidopsis cell culture system the involvement of nitric oxide (NO) signaling in Bap-induced PCD. Their data suggest that NO synthase-derived NO would interfere with mitochondrial respiration, which is consistent with the report of ATP depletion

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and Cyt c release in the course of CK-induced PCD [21,22,24]. It is thus likely that Ca2þ and NO signaling converge in this type of PCD. Of note, NO is known to have a positive effect on Ca2þ signaling [59e62], making this gas a likely player upstream of Ca2þ in CKs-induced PCD.

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la Recherche sur la Nature et les Technologies (FQRNT), and Jeffress Research Grant #J636 to SC from the Thomas F. and Kate Miller Jeffress Memorial Trust.

References 4.5. Ca2þ and NtBI-1 We observed that Bap-mediated up-regulation of the NtBI-1 protein coincided with a stress response and a cytosolic influx of external Ca2þ. However, modulation of the Ca2þ homeostasis failed to change the accumulation profile of the protein (Fig. 5 and data not shown). This indicates that if the Ca2þ cyt 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. Considering the signaling of Bap-induced PCD by NO [58], this gas could also be involved in the regulation of NtBI-1. 5. Conclusion Our evidence of an increased accumulation of the NtBI-1 protein in the course of the stress response to CKs suggests that it may well contribute to cell survival. BI-1 is part of a pathway where its expression level positively influences cellular ability to resist to a variety of biotic and abiotic stresses including carbon starvation [14], fungal toxin and heat shock [63]. This occurs potentially via the modulation of intracellular Ca2þ homeostasis or interference with the redox status [18,64,65], although BI-1 does not interfere directly with the accumulation of ROS but rather might function downstream of it [17,63]. As an anti-PCD protein, disappearance of NtBI-1 in highly stressed cells undergoing cell death (such as after an exposure to CKs or in the course of natural senescence) could mark the transition between a surviving mode to the execution of a PCD program. Furthermore, the appearance of stress markers such as the PR1a mRNA (this study) or ROS [21] after addition of growth-reducing Bap concentrations is in the time-frame corresponding with first detection of the NtBI-1 protein up-regulation. Thus, up-regulation of the BI-1 protein level by CKs coincides with the general activation of the stress response and is likely involved in survival. Although Ca2þ appears to be partially involved in that case of PCD but not in NtBI-1 modulation, a number of data point toward the putative involvement of plant BI-1 in Ca2þ homeostasis. Indeed, BI-1 overexpression in metazoans reduces releasable Ca2þ from the ER and confers some protection against ER stresses [18,66]. Acknowledgements We are indebted to Marc Germain, Dominick Pallotta and Marc Rideau for helpful commentaries on the manuscript, and Fre´de´ric Pitre for helping with preliminary works and statistical analysis. This work was supported by grants to LB from the National Sciences and Engineering Research Council of Canada, PhD fellowship to NB from the Fond que´be´cois de

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