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Ca2+‐dependent and Ca2+‐independent excretion modes of salicylic acid in tobacco cell suspension culture

Ca2+‐dependent and Ca2+‐independent excretion modes of salicylic acid in tobacco cell suspension... Abstract 14C‐salicylic acid (SA) was used to monitor SA metabolism and its regulation in tobacco cell suspension culture. Two SA concentrations (20 μM and 200 μM) were used for comparison. SA was quickly taken up in both treatments, and the 200 μM‐treated cells absorbed approximately 15 times that of 20 μM‐treated cells within 5 min. More than 85% and 50% of the absorbed SA were excreted in free form to the culture medium within 5 h from cells treated with 200 μM and 20 μM SA, respectively. SA excretion was significantly inhibited by EGTA and the inhibition could be reversed by the addition of exogenous Ca2+to the culture medium in the 200 μM SA treatment. However, EGTA had little or no effect on SA excretion in the 20 μM SA treatment. The data suggest that tobacco suspension‐cultured cells may contain both Ca2+‐dependent and Ca2+‐independent pathways for SA excretion. Reduced glutathione (an active oxygen species scavenger), staurosporine (a protein kinase inhibitor), and cycloheximide (an inhibitor of de novo protein synthesis) also blocked intracellular SA excretion to the culture medium in the 200 μM but not in the 20 μM SA treatment. These data support the existence of alternative SA excretion pathways in tobacco suspension‐cultured cells. Tobacco cells may use both Ca2+‐dependent and Ca2+‐independent excretion pathways to cope with different intracellular SA status, and the pathway influenced by EGTA, reduced glutathione, staurosporine, and cycloheximide is activated by SA at 200 μM, but not at 20 μM. Salicylic acid, excretion, Ca2+, glutathione, staurosporine. Introduction Salicylic acid (SA, 2‐hydroxybenzoic acid) is a natural cellular component of many plants including tobacco, cucumber, rice, wheat, cotton, tomato, and Arabidopsis (Raskin et al., 1990). SA has been considered as a natural growth regulator with numerous functions in plants (Raskin, 1992) including promotion of bud formation and growth of tobacco callus in culture (Lee and Skoog, 1965), induction of flowering in Lemna gibba G3 and Lemna paucicostata 151 (Cleland and Ajami, 1974), regulation of heat production in the inflorescence of an Arum lily (Raskin et al., 1987, 1989) and induction of resistance to TMV in tobacco (White, 1979; Malamy et al., 1990; Gaffney et al., 1993). The concentration of endogenous SA varied significantly from tissue to tissue, and from species to species. In thermogenic plants such as Dioon hildebrandtii, the SA amount could be as high as 100 μg g−1 fresh weight in male cones. However, it could be as low as less than 0.01 μg g−1 fresh weight in leaves of Nicotiana tabacum and Zea mays (Raskin et al., 1990). In plant–microbe interactions, the endogenous SA concentration could increase 10–20‐fold in TMV‐infected leave (Malamy et al., 1990). SA existed as both free and conjugate forms during plant development (Cooper‐Driver et al., 1972) and plant–microbe interactions (Malamy et al., 1992). The roles of SA and active oxygen species in plant disease resistance and cell growth/death are subject areas of intensive study (Lam et al., 1999). Many mutants with altered defence mechanisms, disease resistance and growth characters have been isolated from Arabidopsis (Glazebrook, 1999; Martin, 1999). A dominant gain‐of‐function Arabidopsis mutant, accelerated cell death 6 (acd6), exhibited elevated intracellular SA level, patches of dead and enlarged cells, reduced stature, increased defence and resistance to Pseudomonas syringae. When the elevated intracellular SA level was lowered with bacterial over‐expressed nahG protein in transgenic plants, all these altered phenotypic characters were eliminated (Rate et al., 1999). In tobacco BY2 cells, SA could arrest the cell cycle progression at G0/G1 or G2 phase (Perennes et al., 1999). In Arabidopsis, ozone‐induced cell death required a higher SA level to potentiate the activation of an oxidative burst and a cell death pathway, which resulted in apparent ozone sensitivity (Rao and Davis, 1999). These reports therefore suggest that intracellular SA concentration and SA signalling pathway(s) are associated with the functions controlling cell growth, cell death, and defence. It was reported that SA could induce active oxygen species generation in tobacco (Chen et al., 1993). In parsley suspension cultures, salicylic acid enhanced H2O2 production (Kauss and Jeblick, 1994). It was demonstrated that 500 μM SA caused superoxide generation followed by an increase in cytosolic calcium in tobacco cell suspension culture (Kawano et al., 1998). Thus, involvement of Ca2+ influx and active oxygen species generation as components of SA signalling is suggested. SA has been previously applied to tobacco cell suspension culture to study its metabolism, signalling, effects on gene expression, and cellular physiology. It was reported that 20 μM SA could induce cyanide‐resistance respiration without affecting normal cell function in tobacco suspension culture (Kapulnik et al., 1992). However, significant reduction of O2 consumption and heat production was observed in 200 μM SA‐treated cells (Kapulnik et al., 1992). A SA‐inducible gene was isolated which encoded a 48 kDa putative mitogen‐activated protein (MAP) kinase from tobacco (Zhang and Klessig, 1997). The synthesis of MAP kinase mRNA was induced by SA in a dose‐dependent manner with concentrations more than 50 μM and maximal induction by 500 μM in tobacco cell suspension. Therefore, use of SA up to 500 μM is appropriate and can be used to study its effects on particular gene induction and cellular physiology in suspension culture. In order to perform functions as described above, SA must enter the cells before inducing particular gene expression. In animals, SA can be transported across the plasma membrane via non‐ionic diffusion and carrier‐mediated mechanisms (Chatton and Roch‐Ramel, 1992; Takanaga et al., 1994). It was reported that Lemna gibba G3 plants took up nearly 90% of the SA applied at 10 μM SA within 30 min (Ben‐Tal and Cleland, 1982). In tobacco cell suspension cultures rapid SA uptake was also observed in both 20 μM (Kapulnik et al., 1992) and 200 μM (Chen and Kuc’, 1999) treatments. SA uptake was pH‐dependent (Chen and Kuc’, 1999) which indicated the possible usage of a non‐ionic diffusion mechanism similar to that of the animal system, namely SA transport across plasma membrane (Gutknecht, 1990). Most of the absorbed SA was excreted to the culture medium via a Ca2+‐dependent pathway in 200 μM SA treatment (Chen and Kuc’, 1999). Here it is reported that tobacco cells may have both Ca2+‐dependent and Ca2+‐independent pathways for SA excretion in suspension culture. The one affected by EGTA, reduced glutathione, staurosporine, and cycloheximide is activated by 200 μM SA. Depending on SA concentrations, tobacco cells may use both Ca2+‐dependent and Ca2+‐independent excretion pathways to cope with different intracellular SA status. Materials and methods Plant materials and chemicals Nicotiana tabacum cv. KY 14 cell suspension culture was maintained in the dark at room temperature on an orbital shaker (150 rpm). Cells were grown in 3% (w/v) sucrose Murashige and Skoog (MS) medium containing 1 μg ml−1 nicotinic acid, 10 μg ml−1 thiamine‐HCl, 1 μg ml−1 pyridoxine‐HCl, 100 μg ml−1 myo‐inositol, and 1 μg ml−1 2,4‐dichlorophenoxyacetic acid (2,4‐D). Cell suspensions were maintained every 7 d using a 1:2 (v/v) dilution of fresh medium or used for experiments in a 1:1 (v/v) dilution of fresh medium for 3 d. Chemicals were purchased from the following companies: [7‐14C]SA from New England Nuclear (NEN); cycloheximide (CHX), reduced glutathione, staurosporine, EGTA, and 2,4‐D, from Sigma; MS salt mixture from GIBCO BRL; thin layer chromatography (TLC) silica gel G plate from Analtech Inc. SA was applied in free acid form. Measurement of fresh weight Twenty ml of tobacco cell suspension was mixed with 30 ml of fresh culture medium containing sufficient SA to make final concentrations of 20 μM and 200 μM, respectively. Cells were harvested daily for fresh weight determination. Measurement of [14C]SA radioactivity To determine the radioactivity from [14C]SA, 2 ml of cell suspension was mixed with 3 ml of fresh culture medium. A stock containing both unlabelled and 14C‐labelled SA was used to bring the final concentrations to 200 μM and 20 μM in 50 ml Falcon disposable centrifuge tubes. The 200 μM and 20 μM treatments contained 0.76 μM (0.2 μCi) and 0.076 μM (0.02 μCi) [14C]SA, respectively, for all experiments except the one for time‐course of SA changes. In that experiment the [14C]SA amount was increased three times to 0.228 μM (0.06 μCi) for the 20 μM treatment, but remained the same for the 200 μM treatment. Cells were collected at intervals after SA addition by vacuum filtration, and washed with phosphate‐buffered saline (0.14 M NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, 1.5 mM KH2PO4, pH 7.5). The collected cells were weighed and transferred to 7 ml liquid scintillation vials containing 400 μl 60% HClO4 and 100 μl 30% H2O2, then held at 80 °C in a water bath for 2 h before the addition of 15 ml Fisher ScintiVerse II liquid scintillation fluid. To determine [14C]SA in the culture medium, 1 ml of culture filtrate was mixed directly with 15 ml Fisher ScintiVerse II liquid scintillation fluid, then radioactivity was measured using a Packard 2200CA Liquid Scintillation Analyser. TLC Tobacco cells and culture filtrate from 200 μM and 20 μM SA treatments were collected separately at intervals as described above. The forms of SA‐derived compounds were determined using TLC. Cells were first ground using a mortar and pestle in 1.8 ml 90% methanol before being transferred to a 2 ml screw‐capped microcentrifuge tube and shaken overnight at 4 °C on an orbital shaker (150 rpm). The samples were centrifuged for 5 min to pellet down the cell debris and the supernatant was transferred to a 2 ml vial for freeze‐drying. The freeze‐dried material was resuspended in 600 μl H2O (pH 2.0) and extracted twice with an equal volume of ethyl ether. Both the ether and aqueous fractions from c. 100 and 50 mg fresh weights of 200 and 20 μM SA‐treated cells, respectively, were applied to TLC silica gel G plates and developed in 1‐butanol:methanol:acetic acid (80:15:15, by vol.). To determine the form of SA‐derived compounds in culture filtrate, samples were collected separately at intervals after SA addition. Twenty μl of culture filtrate of 200 μM SA treatment was applied directly to TLC silica gel G plates and developed in chloroform:ethyl acetate:acetic acid (60:40:5, by vol.). For 20 μM SA treatment, 200 μl of culture filtrate was freeze‐dried and redissolved in a small amount of H2O (pH 2.0) before being applied to TLC silica gel G plates and developed in the same solvent mixture. Chemical treatment Tobacco cell suspensions were pretreated with EGTA (5 mM), reduced glutathione (0.75 mM), or staurosporine (2 μM or 5 μM) for 15 min before SA (200 μM or 20 μM) addition. Cycloheximide (10 or 20 μg ml−1) was added together with SA to the cell suspension. Cells were harvested at intervals for EGTA treatment or 5 h after SA addition for reduced glutathione, staurosporine, CHX, and reversion by exogenous Ca2+ of EGTA inhibition experiments. Radioactivity from cells was measured as described above. Results Growth rates of tobacco cells in suspension at various SA concentrations were measured. The fresh weight of cells in 20 μM SA 7 d after subculture was similar to that of the controls, however, those in 200 μM SA was reduced by 15% (Fig. 1). Therefore, the two SA concentrations that caused slight different effects on cell growth were used in the following experiments. The radioactive [14C]SA was used to monitor the metabolism of exogenous SA after being added to cell suspensions. Tobacco cells took up c. 35% (145760 cpm) and 23% (46248 cpm) of the total applied SA (412750 cpm and 202112 cpm) within 5 min in the 200 μM and 20 μM treatments, respectively. The total radioactivity then decreased in cells but increased in culture medium by 5 h after the 200 μM SA treatment. In the 20 μM SA, total cellular radioactivity continued to increase until 1 h after treatment (52713 cpm), then, decreased gradually. More than 85% and 50% of the absorbed SA was secreted to the culture medium, and approximately 4% (14958/412750 cpm) and 10% (20400/202112 cpm) of the applied SA were found in cells at 5 h in the 200 μM and 20 μM treatments, respectively (Fig. 2). The average recovery of [14C]SA in cultures (cells plus medium) was about 89%, and the specific activity of [14C]SA was kept relatively constant during the treatment. The results showed that most of the SA taken up by cells was excreted to the culture medium during the following incubation period, and SA excretion was the major mechanism responsible for the decrease of cellular SA as monitored by radioactivity. Decarboxylation which removed the [7‐14C]carboxyl group from radioactive labelled salicylic acid contributed to the reduction of cellular radioactivity. It was reported that tobacco leaves showed a transient total net SA increase from 2 h to 4 h after exogenous SA application (Seo et al., 1995). From 4–6 h after treatment, the total net SA decreased again to almost the basal line of 0 h. Similar results were also observed earlier (Hennig et al., 1993). It was speculated that the total net SA increase after SA treatment may be due to an increase of de novo SA production or to an inhibition of SA conversion to other derivative (Seo et al., 1995). In tobacco cell suspension culture, the total SA remained relatively constant within the first 2 h after treatment and decreased in the following incubation from 3–6 h. No net SA increase after SA treatment was observed (Kapulnik et al., 1992). The conflict (net SA increase after SA treatment) observed between SA‐treated tobacco leaves and cell suspension culture may be due to the different systems used. It has been previously demonstrated that the decrease of the SA amount was mostly due to the excretion of intracellular SA to the culture medium (Chen and Kuc’, 1999). These data agree with the report of Kapulnik et al. (Kapulnik et al., 1992) and conclude that SA treatment does not cause net SA increase in tobacco cell suspension culture unlike the phenomenon observed in treated tobacco leaves. Thin layer chromatography showed that the radioactivity of a band corresponding to the free form of SA was lowest at 5 min when compared with others including zero in 200 μM SA treatment (Fig. 3A). In 20 μM SA treatment, a band of the same RF value as free SA was detected. The radioactivity of this band was lowest at 1 h (Fig. 3B). The results demonstrated that tobacco cells excreted free SA to the culture medium during both the 200 μM and 20 μM SA treatments. Cells contained more than one form of SA during both the 200 μM and 20 μM SA treatments (Fig. 4). Two radioactive bands with different RF values were detected using TLC in 200 μM SA treatment (Fig. 4A). The upper band had the same RF value as free SA and was present in the ether fraction. The other band with a lower RF value than free SA was present in the aqueous fraction. This less intense band is probably a glucoside conjugate of SA. In the 20 μM SA treatment, three bands were found in the aqueous fraction (Fig. 4B). The upper band had the same RF value as free SA and was also present in the ether fraction. The other two bands had lower RF values than free SA and were absent in the ether fraction. Both the 2‐O‐glucoside and glucose ester had been reported as conjugates of salicylic acid in tobacco (Enyedi et al., 1992; Edwards, 1994). In Cucumis sativus, three major compounds derived from SA were gentisic acid‐5‐O‐glucoside, SA‐O‐glucoside, and the glucose ester of SA (Cooper‐Driver et al., 1972). Thus, SA could be metabolized to more than one conjugated form. The additional forms detected in 20 μM SA as compared to 200 μM SA suggests that the amount of SA influences the way that SA conjugates were formed and that several mechanisms for SA conjugation exist in tobacco cells. The effects of inhibitors on SA excretion were investigated. A greater SA amount was found in EGTA‐pretreated cells than untreated controls at all time intervals analysed in the 200 μM SA treatment. However, EGTA had little or no effect in the 20 μM SA treatment and a similar intracellular SA level was detected in both the EGTA‐pretreated sample and the untreated control (Fig. 5A). Enhanced SA influx or reduced SA efflux by EGTA pretreatment is a possible explanations for the results. If enhanced SA influx is the cause of increased SA retention in cells, the intracellular SA level in EGTA‐pretreated cells should be higher than the untreated control in both the 20 μM and 200 μM SA treatments. However, the EGTA‐pretreated cells and the untreated control contained similar intracellular SA levels in 20 μM SA treatment (Fig. 5A). The data, therefore, favour a reduced SA efflux by EGTA as the explanation for the enhanced retention of intracellular SA in the 200 μM SA treatment. SA loss from treated cells to the culture medium was markedly inhibited by 5 mM EGTA in the 200 μM SA treatment (Fig. 5A), and the inhibition by EGTA could be reversed in a dose‐dependent manner by adding exogenous Ca2+to the cell suspensions. Addition of 20 mM Ca2+ to the 5 mM EGTA‐pretreated cells resulted in a similar intracellular SA level to that of SA‐alone control (Fig. 5B). These data, therefore, suggest that tobacco suspension‐cultured cells may have both Ca2+‐dependent and Ca2+‐independent pathways for SA excretion. The Ca2+‐dependent excretion pathway is likely to be activated by SA at 200 μM but not at 20 μM. Reduced glutathione (0.75 mM), a known oxidative stress scavenger, and staurosporine (2 μM or 5 μM), a potent protein kinase inhibitor, also caused a significant elevation of intracellular SA in the 200 μM SA treatment (3–5‐fold that of the untreated control), whereas, it had little or no effect in the 20 μM SA treatment (Fig. 6A, B). Cycloheximide (10 or 20 μg ml−1), an inhibitor of de novo protein synthesis, also resulted in a striking increase in the intracellular SA level in the 200 μM SA treatment (about 4–5‐fold that of the untreated control) but not in 20 μM (Fig. 6C). These results also support the existence of alternative pathways for SA excretion in tobacco cells in suspension culture. Depending on SA concentrations, tobacco cells may utilize either pathway for SA excretion, and the one influenced by EGTA, reduced glutathione, staurosporine, and cycloheximide is specifically activated by SA at 200 μM but not at 20 μM. Fig. 1. View largeDownload slide Effects of salicylic acid (0 μM, 20 μM or 200 μM) on tobacco cell growth in suspension culture. Data are means of three independent experiments with one replicate per experiment. Bars indicate the standard error. Fig. 1. View largeDownload slide Effects of salicylic acid (0 μM, 20 μM or 200 μM) on tobacco cell growth in suspension culture. Data are means of three independent experiments with one replicate per experiment. Bars indicate the standard error. Fig. 2. View largeDownload slide Time‐course of the radioactivity and total salicylic acid (SA) changes in cells and culture filtrate at intervals after the addition of 200 μM or 20 μM SA containing [14C]SA to cell suspension cultures. Data are means of three independent experiments with one replicate per experiment. Bars indicate standard error. Fig. 2. View largeDownload slide Time‐course of the radioactivity and total salicylic acid (SA) changes in cells and culture filtrate at intervals after the addition of 200 μM or 20 μM SA containing [14C]SA to cell suspension cultures. Data are means of three independent experiments with one replicate per experiment. Bars indicate standard error. Fig. 3. View largeDownload slide Time‐course and the forms of salicylic acid (SA) detected by TLC in the culture filtrate at intervals after the addition of 200 μM or 20 μM SA containing [14C]SA to cell suspension cultures. (A) 200 μM SA; (B) 20 μM SA. In (A), 20 μl of culture filtrate was applied directly to a TLC plate. In (B), 200 μl of culture filtrate was concentrated, before being applied to a TLC plate. The bands came from exposure of X‐ray film to the plates. The experiment was done twice and representative data of one experiment are shown. Fig. 3. View largeDownload slide Time‐course and the forms of salicylic acid (SA) detected by TLC in the culture filtrate at intervals after the addition of 200 μM or 20 μM SA containing [14C]SA to cell suspension cultures. (A) 200 μM SA; (B) 20 μM SA. In (A), 20 μl of culture filtrate was applied directly to a TLC plate. In (B), 200 μl of culture filtrate was concentrated, before being applied to a TLC plate. The bands came from exposure of X‐ray film to the plates. The experiment was done twice and representative data of one experiment are shown. Fig. 4. View largeDownload slide Time‐course and forms of salicylic acid (SA) detected by autoradiography of TLC of extracts of tobacco cells at intervals after the addition of 200 μM (A) or 20 μM SA (B) containing [14C]SA to cell suspension cultures. For sample preparation and application to TLC plates, refer to Materials and methods. The experiment was repeated twice and representative data of one experiment are shown. Fig. 4. View largeDownload slide Time‐course and forms of salicylic acid (SA) detected by autoradiography of TLC of extracts of tobacco cells at intervals after the addition of 200 μM (A) or 20 μM SA (B) containing [14C]SA to cell suspension cultures. For sample preparation and application to TLC plates, refer to Materials and methods. The experiment was repeated twice and representative data of one experiment are shown. Fig. 5. View largeDownload slide Effects of EGTA and Ca2+ on intracellular [14C]SA accumulation in tobacco cell suspensions. (A) Changes of [14C]SA radioactivity in 200 μM or 20 μM SA‐treated cells at intervals after SA addition. (B) Reversion of 5 mM EGTA‐inhibited SA excretion by the addition of exogenous Ca2+. The experiment was repeated twice and data from one is shown here. Fig. 5. View largeDownload slide Effects of EGTA and Ca2+ on intracellular [14C]SA accumulation in tobacco cell suspensions. (A) Changes of [14C]SA radioactivity in 200 μM or 20 μM SA‐treated cells at intervals after SA addition. (B) Reversion of 5 mM EGTA‐inhibited SA excretion by the addition of exogenous Ca2+. The experiment was repeated twice and data from one is shown here. Fig. 6. View largeDownload slide Effects of (A) reduced glutathione (0.75 mM), (B) staurosporine (2 μM or 5 μM) or (C) cycloheximide (CHX, 10 or 20 μg ml−1) on cellular [14C]SA radioactivity in 200 μM and 20 μM SA‐treated cells. Data are means of three replicates with standard error bars for one experiment. Fig. 6. View largeDownload slide Effects of (A) reduced glutathione (0.75 mM), (B) staurosporine (2 μM or 5 μM) or (C) cycloheximide (CHX, 10 or 20 μg ml−1) on cellular [14C]SA radioactivity in 200 μM and 20 μM SA‐treated cells. Data are means of three replicates with standard error bars for one experiment. Discussion SA is an endogenous molecule with hormone activity in plants. In animal systems, a putative carrier‐mediated transporter for SA had been reported in the human colon adenocarcinoma cell line Caco‐2 (Takanaga et al., 1994). However, it is not clear whether plants contain similar, putative SA transporters. In this study, tobacco cells in suspension culture excreted most of the exogenous SA in the form of free SA in both the 200 μM and 20 μM treatments (Figs 2, 3). It was reported that SA glucoside (SAG) injected into the intercellular space of tobacco leaves was probably converted into free SA before entering the cells (Hennig et al., 1993). The results of this study agree with the report and demonstrate that the free but not conjugated SA is the form transported across plasma membranes. The function of SA excretion is unclear. However, accumulation of SA in cells may affect physiological functions and cause cytotoxicity (Kapulnik et al., 1992). Thus, a suggested role for SA excretion is probably associated with detoxification. Tobacco cells contained free SA and possibly water‐soluble SA conjugates in both the 200 μM and 20 μM SA treatments (Fig. 4) as reported previously (Kapulnik et al., 1992) in 20 μM SA‐treated cells in suspension culture. The functions of free and conjugated forms of SA in cells were unclear. It was reported that PR‐1a gene expression was induced in tobacco leaves injected with hydrolysable SA glucoside (SAG), but not with an unhydrolysable SAG analogue thio‐SAG (TSAG) (Hennig et al., 1993). These data suggest the free SA as the active form responsible for SA function in cells. In roots of Vicia faba and Fagopyrum esculentum, exogenous SA was conjugated into O‐glucoside as a detoxification mechanism (Schulz et al., 1993). Therefore, a possible role of conjugated SA as a detoxification mechanism is suggested in tobacco suspension‐cultured cells. SA is likely to be stored in the vacuole (Ben‐Tal and Cleland, 1982). The absorbed SA was excreted into the culture medium in both the 200 μM and 20 μM SA treatments (Fig. 2). SA excretion was blocked by inhibitors in the 200 μM, but not in the 20 μM SA treatment (Figs 5, 6). It was reported that 500 μM SA rapidly increased cytosolic Ca2+concentration within 10 s after application in tobacco cell suspension culture (Kawano et al., 1998). In lactating rat mammary tissue, SA stimulated external Ca2+ influx and calcium‐dependent K+ efflux (Shennan, 1992). The results from EGTA experiments (Fig. 5) agree with these reports and suggest that 200 μM SA also caused an increase of cytosolic Ca2+ concentration. It in turn may act as a signalling component for the activation of an SA excretion pathway. SA has been demonstrated to elevate active oxygen species in tobacco plants (Chen et al., 1993), and in parsley and tobacco cell suspension cultures (Kauss and Jeblick, 1994; Kawano et al., 1998). This study's results showed that SA excretion was blocked by reduced glutathione, an active oxygen species scavenger, in the 200 μM SA treatment (Fig. 6A). The data are consistent with those reports and suggest that 200 μM SA could generate active oxygen species, which are likely to function as a signalling component for the activation of a SA excretion pathway in suspension‐cultured cells. SA excretion by cells treated with 200 μM but not 20 μM SA was inhibited greatly by staurosporine (Fig. 6A). It was reported that SA could induce a 48 kDa mitogen‐activated protein (MAP) kinase in a dose‐dependent manner with concentrations more than 50 μM in tobacco cell suspension culture (Zhang and Klessig, 1997). Whether a dose‐dependent induction of putative protein kinase activities is required for SA excretion, similar to that report (Zhang and Klessig, 1997) requires further investigation. Cycloheximide blocked SA excretion in the 200 μM, but not in the 20 μM SA treatment (Fig. 6C). The data suggest that tobacco cells may contain both constitutive and inducible SA excretion pathways, and the inducible one is activated by 200 μM SA. The requirement for de novo synthesized proteins for SA excretion in 200 μM SA treatment is possibly due to (1) an inducible carrier‐mediated transporter for SA excretion, (2) the inducible signals/signal transduction pathways that link to activate the Ca2+‐dependent SA excretion mechanism or (3) both. Tobacco cells may have both Ca2+‐dependent and Ca2+‐independent pathways for SA excretion. It is not clear how these excretion pathways are activated and regulated under different SA doses. However, approximately 35% (70 μM) and 23% (4.6 μM) of the total applied SA was taken up by cells treated with 200 μM and 20 μM SA, respectively, 5 min after addition. The deduced intracellular SA concentration in 200 μM‐treated cells (c. 1.0 mM) is much higher than that in 20 μM‐treated cells (c. 65 μM). The great difference (c. 15 times) of intracellular SA levels may activate alternative signal components and/or signal transduction pathways, as observed from the results of Figs 5 and 6. Whether a putative controlling mechanism exists which utilizes the intracellular SA level as a regulatory threshold to activate particular signalling components and/or signal transduction pathways for SA excretion needs more investigations. It is concluded that tobacco cells may contain both Ca2+‐dependent and Ca2+‐independent pathways for SA excretion in suspension culture. The one influenced by EGTA, reduced glutathione, staurosporine, and cycloheximide is activated by SA at 200 μM but not at 20 μM. Tobacco cell suspension cultures may be used as a suitable system to study SA transport, SA signalling, and detoxification of xenobiotics. 4 To whom correspondence should be addressed. Fax: +886 2 27827954. 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Ca2+‐dependent and Ca2+‐independent excretion modes of salicylic acid in tobacco cell suspension culture

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Oxford University Press
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0022-0957
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1460-2431
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10.1093/jexbot/52.359.1219
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Abstract

Abstract 14C‐salicylic acid (SA) was used to monitor SA metabolism and its regulation in tobacco cell suspension culture. Two SA concentrations (20 μM and 200 μM) were used for comparison. SA was quickly taken up in both treatments, and the 200 μM‐treated cells absorbed approximately 15 times that of 20 μM‐treated cells within 5 min. More than 85% and 50% of the absorbed SA were excreted in free form to the culture medium within 5 h from cells treated with 200 μM and 20 μM SA, respectively. SA excretion was significantly inhibited by EGTA and the inhibition could be reversed by the addition of exogenous Ca2+to the culture medium in the 200 μM SA treatment. However, EGTA had little or no effect on SA excretion in the 20 μM SA treatment. The data suggest that tobacco suspension‐cultured cells may contain both Ca2+‐dependent and Ca2+‐independent pathways for SA excretion. Reduced glutathione (an active oxygen species scavenger), staurosporine (a protein kinase inhibitor), and cycloheximide (an inhibitor of de novo protein synthesis) also blocked intracellular SA excretion to the culture medium in the 200 μM but not in the 20 μM SA treatment. These data support the existence of alternative SA excretion pathways in tobacco suspension‐cultured cells. Tobacco cells may use both Ca2+‐dependent and Ca2+‐independent excretion pathways to cope with different intracellular SA status, and the pathway influenced by EGTA, reduced glutathione, staurosporine, and cycloheximide is activated by SA at 200 μM, but not at 20 μM. Salicylic acid, excretion, Ca2+, glutathione, staurosporine. Introduction Salicylic acid (SA, 2‐hydroxybenzoic acid) is a natural cellular component of many plants including tobacco, cucumber, rice, wheat, cotton, tomato, and Arabidopsis (Raskin et al., 1990). SA has been considered as a natural growth regulator with numerous functions in plants (Raskin, 1992) including promotion of bud formation and growth of tobacco callus in culture (Lee and Skoog, 1965), induction of flowering in Lemna gibba G3 and Lemna paucicostata 151 (Cleland and Ajami, 1974), regulation of heat production in the inflorescence of an Arum lily (Raskin et al., 1987, 1989) and induction of resistance to TMV in tobacco (White, 1979; Malamy et al., 1990; Gaffney et al., 1993). The concentration of endogenous SA varied significantly from tissue to tissue, and from species to species. In thermogenic plants such as Dioon hildebrandtii, the SA amount could be as high as 100 μg g−1 fresh weight in male cones. However, it could be as low as less than 0.01 μg g−1 fresh weight in leaves of Nicotiana tabacum and Zea mays (Raskin et al., 1990). In plant–microbe interactions, the endogenous SA concentration could increase 10–20‐fold in TMV‐infected leave (Malamy et al., 1990). SA existed as both free and conjugate forms during plant development (Cooper‐Driver et al., 1972) and plant–microbe interactions (Malamy et al., 1992). The roles of SA and active oxygen species in plant disease resistance and cell growth/death are subject areas of intensive study (Lam et al., 1999). Many mutants with altered defence mechanisms, disease resistance and growth characters have been isolated from Arabidopsis (Glazebrook, 1999; Martin, 1999). A dominant gain‐of‐function Arabidopsis mutant, accelerated cell death 6 (acd6), exhibited elevated intracellular SA level, patches of dead and enlarged cells, reduced stature, increased defence and resistance to Pseudomonas syringae. When the elevated intracellular SA level was lowered with bacterial over‐expressed nahG protein in transgenic plants, all these altered phenotypic characters were eliminated (Rate et al., 1999). In tobacco BY2 cells, SA could arrest the cell cycle progression at G0/G1 or G2 phase (Perennes et al., 1999). In Arabidopsis, ozone‐induced cell death required a higher SA level to potentiate the activation of an oxidative burst and a cell death pathway, which resulted in apparent ozone sensitivity (Rao and Davis, 1999). These reports therefore suggest that intracellular SA concentration and SA signalling pathway(s) are associated with the functions controlling cell growth, cell death, and defence. It was reported that SA could induce active oxygen species generation in tobacco (Chen et al., 1993). In parsley suspension cultures, salicylic acid enhanced H2O2 production (Kauss and Jeblick, 1994). It was demonstrated that 500 μM SA caused superoxide generation followed by an increase in cytosolic calcium in tobacco cell suspension culture (Kawano et al., 1998). Thus, involvement of Ca2+ influx and active oxygen species generation as components of SA signalling is suggested. SA has been previously applied to tobacco cell suspension culture to study its metabolism, signalling, effects on gene expression, and cellular physiology. It was reported that 20 μM SA could induce cyanide‐resistance respiration without affecting normal cell function in tobacco suspension culture (Kapulnik et al., 1992). However, significant reduction of O2 consumption and heat production was observed in 200 μM SA‐treated cells (Kapulnik et al., 1992). A SA‐inducible gene was isolated which encoded a 48 kDa putative mitogen‐activated protein (MAP) kinase from tobacco (Zhang and Klessig, 1997). The synthesis of MAP kinase mRNA was induced by SA in a dose‐dependent manner with concentrations more than 50 μM and maximal induction by 500 μM in tobacco cell suspension. Therefore, use of SA up to 500 μM is appropriate and can be used to study its effects on particular gene induction and cellular physiology in suspension culture. In order to perform functions as described above, SA must enter the cells before inducing particular gene expression. In animals, SA can be transported across the plasma membrane via non‐ionic diffusion and carrier‐mediated mechanisms (Chatton and Roch‐Ramel, 1992; Takanaga et al., 1994). It was reported that Lemna gibba G3 plants took up nearly 90% of the SA applied at 10 μM SA within 30 min (Ben‐Tal and Cleland, 1982). In tobacco cell suspension cultures rapid SA uptake was also observed in both 20 μM (Kapulnik et al., 1992) and 200 μM (Chen and Kuc’, 1999) treatments. SA uptake was pH‐dependent (Chen and Kuc’, 1999) which indicated the possible usage of a non‐ionic diffusion mechanism similar to that of the animal system, namely SA transport across plasma membrane (Gutknecht, 1990). Most of the absorbed SA was excreted to the culture medium via a Ca2+‐dependent pathway in 200 μM SA treatment (Chen and Kuc’, 1999). Here it is reported that tobacco cells may have both Ca2+‐dependent and Ca2+‐independent pathways for SA excretion in suspension culture. The one affected by EGTA, reduced glutathione, staurosporine, and cycloheximide is activated by 200 μM SA. Depending on SA concentrations, tobacco cells may use both Ca2+‐dependent and Ca2+‐independent excretion pathways to cope with different intracellular SA status. Materials and methods Plant materials and chemicals Nicotiana tabacum cv. KY 14 cell suspension culture was maintained in the dark at room temperature on an orbital shaker (150 rpm). Cells were grown in 3% (w/v) sucrose Murashige and Skoog (MS) medium containing 1 μg ml−1 nicotinic acid, 10 μg ml−1 thiamine‐HCl, 1 μg ml−1 pyridoxine‐HCl, 100 μg ml−1 myo‐inositol, and 1 μg ml−1 2,4‐dichlorophenoxyacetic acid (2,4‐D). Cell suspensions were maintained every 7 d using a 1:2 (v/v) dilution of fresh medium or used for experiments in a 1:1 (v/v) dilution of fresh medium for 3 d. Chemicals were purchased from the following companies: [7‐14C]SA from New England Nuclear (NEN); cycloheximide (CHX), reduced glutathione, staurosporine, EGTA, and 2,4‐D, from Sigma; MS salt mixture from GIBCO BRL; thin layer chromatography (TLC) silica gel G plate from Analtech Inc. SA was applied in free acid form. Measurement of fresh weight Twenty ml of tobacco cell suspension was mixed with 30 ml of fresh culture medium containing sufficient SA to make final concentrations of 20 μM and 200 μM, respectively. Cells were harvested daily for fresh weight determination. Measurement of [14C]SA radioactivity To determine the radioactivity from [14C]SA, 2 ml of cell suspension was mixed with 3 ml of fresh culture medium. A stock containing both unlabelled and 14C‐labelled SA was used to bring the final concentrations to 200 μM and 20 μM in 50 ml Falcon disposable centrifuge tubes. The 200 μM and 20 μM treatments contained 0.76 μM (0.2 μCi) and 0.076 μM (0.02 μCi) [14C]SA, respectively, for all experiments except the one for time‐course of SA changes. In that experiment the [14C]SA amount was increased three times to 0.228 μM (0.06 μCi) for the 20 μM treatment, but remained the same for the 200 μM treatment. Cells were collected at intervals after SA addition by vacuum filtration, and washed with phosphate‐buffered saline (0.14 M NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, 1.5 mM KH2PO4, pH 7.5). The collected cells were weighed and transferred to 7 ml liquid scintillation vials containing 400 μl 60% HClO4 and 100 μl 30% H2O2, then held at 80 °C in a water bath for 2 h before the addition of 15 ml Fisher ScintiVerse II liquid scintillation fluid. To determine [14C]SA in the culture medium, 1 ml of culture filtrate was mixed directly with 15 ml Fisher ScintiVerse II liquid scintillation fluid, then radioactivity was measured using a Packard 2200CA Liquid Scintillation Analyser. TLC Tobacco cells and culture filtrate from 200 μM and 20 μM SA treatments were collected separately at intervals as described above. The forms of SA‐derived compounds were determined using TLC. Cells were first ground using a mortar and pestle in 1.8 ml 90% methanol before being transferred to a 2 ml screw‐capped microcentrifuge tube and shaken overnight at 4 °C on an orbital shaker (150 rpm). The samples were centrifuged for 5 min to pellet down the cell debris and the supernatant was transferred to a 2 ml vial for freeze‐drying. The freeze‐dried material was resuspended in 600 μl H2O (pH 2.0) and extracted twice with an equal volume of ethyl ether. Both the ether and aqueous fractions from c. 100 and 50 mg fresh weights of 200 and 20 μM SA‐treated cells, respectively, were applied to TLC silica gel G plates and developed in 1‐butanol:methanol:acetic acid (80:15:15, by vol.). To determine the form of SA‐derived compounds in culture filtrate, samples were collected separately at intervals after SA addition. Twenty μl of culture filtrate of 200 μM SA treatment was applied directly to TLC silica gel G plates and developed in chloroform:ethyl acetate:acetic acid (60:40:5, by vol.). For 20 μM SA treatment, 200 μl of culture filtrate was freeze‐dried and redissolved in a small amount of H2O (pH 2.0) before being applied to TLC silica gel G plates and developed in the same solvent mixture. Chemical treatment Tobacco cell suspensions were pretreated with EGTA (5 mM), reduced glutathione (0.75 mM), or staurosporine (2 μM or 5 μM) for 15 min before SA (200 μM or 20 μM) addition. Cycloheximide (10 or 20 μg ml−1) was added together with SA to the cell suspension. Cells were harvested at intervals for EGTA treatment or 5 h after SA addition for reduced glutathione, staurosporine, CHX, and reversion by exogenous Ca2+ of EGTA inhibition experiments. Radioactivity from cells was measured as described above. Results Growth rates of tobacco cells in suspension at various SA concentrations were measured. The fresh weight of cells in 20 μM SA 7 d after subculture was similar to that of the controls, however, those in 200 μM SA was reduced by 15% (Fig. 1). Therefore, the two SA concentrations that caused slight different effects on cell growth were used in the following experiments. The radioactive [14C]SA was used to monitor the metabolism of exogenous SA after being added to cell suspensions. Tobacco cells took up c. 35% (145760 cpm) and 23% (46248 cpm) of the total applied SA (412750 cpm and 202112 cpm) within 5 min in the 200 μM and 20 μM treatments, respectively. The total radioactivity then decreased in cells but increased in culture medium by 5 h after the 200 μM SA treatment. In the 20 μM SA, total cellular radioactivity continued to increase until 1 h after treatment (52713 cpm), then, decreased gradually. More than 85% and 50% of the absorbed SA was secreted to the culture medium, and approximately 4% (14958/412750 cpm) and 10% (20400/202112 cpm) of the applied SA were found in cells at 5 h in the 200 μM and 20 μM treatments, respectively (Fig. 2). The average recovery of [14C]SA in cultures (cells plus medium) was about 89%, and the specific activity of [14C]SA was kept relatively constant during the treatment. The results showed that most of the SA taken up by cells was excreted to the culture medium during the following incubation period, and SA excretion was the major mechanism responsible for the decrease of cellular SA as monitored by radioactivity. Decarboxylation which removed the [7‐14C]carboxyl group from radioactive labelled salicylic acid contributed to the reduction of cellular radioactivity. It was reported that tobacco leaves showed a transient total net SA increase from 2 h to 4 h after exogenous SA application (Seo et al., 1995). From 4–6 h after treatment, the total net SA decreased again to almost the basal line of 0 h. Similar results were also observed earlier (Hennig et al., 1993). It was speculated that the total net SA increase after SA treatment may be due to an increase of de novo SA production or to an inhibition of SA conversion to other derivative (Seo et al., 1995). In tobacco cell suspension culture, the total SA remained relatively constant within the first 2 h after treatment and decreased in the following incubation from 3–6 h. No net SA increase after SA treatment was observed (Kapulnik et al., 1992). The conflict (net SA increase after SA treatment) observed between SA‐treated tobacco leaves and cell suspension culture may be due to the different systems used. It has been previously demonstrated that the decrease of the SA amount was mostly due to the excretion of intracellular SA to the culture medium (Chen and Kuc’, 1999). These data agree with the report of Kapulnik et al. (Kapulnik et al., 1992) and conclude that SA treatment does not cause net SA increase in tobacco cell suspension culture unlike the phenomenon observed in treated tobacco leaves. Thin layer chromatography showed that the radioactivity of a band corresponding to the free form of SA was lowest at 5 min when compared with others including zero in 200 μM SA treatment (Fig. 3A). In 20 μM SA treatment, a band of the same RF value as free SA was detected. The radioactivity of this band was lowest at 1 h (Fig. 3B). The results demonstrated that tobacco cells excreted free SA to the culture medium during both the 200 μM and 20 μM SA treatments. Cells contained more than one form of SA during both the 200 μM and 20 μM SA treatments (Fig. 4). Two radioactive bands with different RF values were detected using TLC in 200 μM SA treatment (Fig. 4A). The upper band had the same RF value as free SA and was present in the ether fraction. The other band with a lower RF value than free SA was present in the aqueous fraction. This less intense band is probably a glucoside conjugate of SA. In the 20 μM SA treatment, three bands were found in the aqueous fraction (Fig. 4B). The upper band had the same RF value as free SA and was also present in the ether fraction. The other two bands had lower RF values than free SA and were absent in the ether fraction. Both the 2‐O‐glucoside and glucose ester had been reported as conjugates of salicylic acid in tobacco (Enyedi et al., 1992; Edwards, 1994). In Cucumis sativus, three major compounds derived from SA were gentisic acid‐5‐O‐glucoside, SA‐O‐glucoside, and the glucose ester of SA (Cooper‐Driver et al., 1972). Thus, SA could be metabolized to more than one conjugated form. The additional forms detected in 20 μM SA as compared to 200 μM SA suggests that the amount of SA influences the way that SA conjugates were formed and that several mechanisms for SA conjugation exist in tobacco cells. The effects of inhibitors on SA excretion were investigated. A greater SA amount was found in EGTA‐pretreated cells than untreated controls at all time intervals analysed in the 200 μM SA treatment. However, EGTA had little or no effect in the 20 μM SA treatment and a similar intracellular SA level was detected in both the EGTA‐pretreated sample and the untreated control (Fig. 5A). Enhanced SA influx or reduced SA efflux by EGTA pretreatment is a possible explanations for the results. If enhanced SA influx is the cause of increased SA retention in cells, the intracellular SA level in EGTA‐pretreated cells should be higher than the untreated control in both the 20 μM and 200 μM SA treatments. However, the EGTA‐pretreated cells and the untreated control contained similar intracellular SA levels in 20 μM SA treatment (Fig. 5A). The data, therefore, favour a reduced SA efflux by EGTA as the explanation for the enhanced retention of intracellular SA in the 200 μM SA treatment. SA loss from treated cells to the culture medium was markedly inhibited by 5 mM EGTA in the 200 μM SA treatment (Fig. 5A), and the inhibition by EGTA could be reversed in a dose‐dependent manner by adding exogenous Ca2+to the cell suspensions. Addition of 20 mM Ca2+ to the 5 mM EGTA‐pretreated cells resulted in a similar intracellular SA level to that of SA‐alone control (Fig. 5B). These data, therefore, suggest that tobacco suspension‐cultured cells may have both Ca2+‐dependent and Ca2+‐independent pathways for SA excretion. The Ca2+‐dependent excretion pathway is likely to be activated by SA at 200 μM but not at 20 μM. Reduced glutathione (0.75 mM), a known oxidative stress scavenger, and staurosporine (2 μM or 5 μM), a potent protein kinase inhibitor, also caused a significant elevation of intracellular SA in the 200 μM SA treatment (3–5‐fold that of the untreated control), whereas, it had little or no effect in the 20 μM SA treatment (Fig. 6A, B). Cycloheximide (10 or 20 μg ml−1), an inhibitor of de novo protein synthesis, also resulted in a striking increase in the intracellular SA level in the 200 μM SA treatment (about 4–5‐fold that of the untreated control) but not in 20 μM (Fig. 6C). These results also support the existence of alternative pathways for SA excretion in tobacco cells in suspension culture. Depending on SA concentrations, tobacco cells may utilize either pathway for SA excretion, and the one influenced by EGTA, reduced glutathione, staurosporine, and cycloheximide is specifically activated by SA at 200 μM but not at 20 μM. Fig. 1. View largeDownload slide Effects of salicylic acid (0 μM, 20 μM or 200 μM) on tobacco cell growth in suspension culture. Data are means of three independent experiments with one replicate per experiment. Bars indicate the standard error. Fig. 1. View largeDownload slide Effects of salicylic acid (0 μM, 20 μM or 200 μM) on tobacco cell growth in suspension culture. Data are means of three independent experiments with one replicate per experiment. Bars indicate the standard error. Fig. 2. View largeDownload slide Time‐course of the radioactivity and total salicylic acid (SA) changes in cells and culture filtrate at intervals after the addition of 200 μM or 20 μM SA containing [14C]SA to cell suspension cultures. Data are means of three independent experiments with one replicate per experiment. Bars indicate standard error. Fig. 2. View largeDownload slide Time‐course of the radioactivity and total salicylic acid (SA) changes in cells and culture filtrate at intervals after the addition of 200 μM or 20 μM SA containing [14C]SA to cell suspension cultures. Data are means of three independent experiments with one replicate per experiment. Bars indicate standard error. Fig. 3. View largeDownload slide Time‐course and the forms of salicylic acid (SA) detected by TLC in the culture filtrate at intervals after the addition of 200 μM or 20 μM SA containing [14C]SA to cell suspension cultures. (A) 200 μM SA; (B) 20 μM SA. In (A), 20 μl of culture filtrate was applied directly to a TLC plate. In (B), 200 μl of culture filtrate was concentrated, before being applied to a TLC plate. The bands came from exposure of X‐ray film to the plates. The experiment was done twice and representative data of one experiment are shown. Fig. 3. View largeDownload slide Time‐course and the forms of salicylic acid (SA) detected by TLC in the culture filtrate at intervals after the addition of 200 μM or 20 μM SA containing [14C]SA to cell suspension cultures. (A) 200 μM SA; (B) 20 μM SA. In (A), 20 μl of culture filtrate was applied directly to a TLC plate. In (B), 200 μl of culture filtrate was concentrated, before being applied to a TLC plate. The bands came from exposure of X‐ray film to the plates. The experiment was done twice and representative data of one experiment are shown. Fig. 4. View largeDownload slide Time‐course and forms of salicylic acid (SA) detected by autoradiography of TLC of extracts of tobacco cells at intervals after the addition of 200 μM (A) or 20 μM SA (B) containing [14C]SA to cell suspension cultures. For sample preparation and application to TLC plates, refer to Materials and methods. The experiment was repeated twice and representative data of one experiment are shown. Fig. 4. View largeDownload slide Time‐course and forms of salicylic acid (SA) detected by autoradiography of TLC of extracts of tobacco cells at intervals after the addition of 200 μM (A) or 20 μM SA (B) containing [14C]SA to cell suspension cultures. For sample preparation and application to TLC plates, refer to Materials and methods. The experiment was repeated twice and representative data of one experiment are shown. Fig. 5. View largeDownload slide Effects of EGTA and Ca2+ on intracellular [14C]SA accumulation in tobacco cell suspensions. (A) Changes of [14C]SA radioactivity in 200 μM or 20 μM SA‐treated cells at intervals after SA addition. (B) Reversion of 5 mM EGTA‐inhibited SA excretion by the addition of exogenous Ca2+. The experiment was repeated twice and data from one is shown here. Fig. 5. View largeDownload slide Effects of EGTA and Ca2+ on intracellular [14C]SA accumulation in tobacco cell suspensions. (A) Changes of [14C]SA radioactivity in 200 μM or 20 μM SA‐treated cells at intervals after SA addition. (B) Reversion of 5 mM EGTA‐inhibited SA excretion by the addition of exogenous Ca2+. The experiment was repeated twice and data from one is shown here. Fig. 6. View largeDownload slide Effects of (A) reduced glutathione (0.75 mM), (B) staurosporine (2 μM or 5 μM) or (C) cycloheximide (CHX, 10 or 20 μg ml−1) on cellular [14C]SA radioactivity in 200 μM and 20 μM SA‐treated cells. Data are means of three replicates with standard error bars for one experiment. Fig. 6. View largeDownload slide Effects of (A) reduced glutathione (0.75 mM), (B) staurosporine (2 μM or 5 μM) or (C) cycloheximide (CHX, 10 or 20 μg ml−1) on cellular [14C]SA radioactivity in 200 μM and 20 μM SA‐treated cells. Data are means of three replicates with standard error bars for one experiment. Discussion SA is an endogenous molecule with hormone activity in plants. In animal systems, a putative carrier‐mediated transporter for SA had been reported in the human colon adenocarcinoma cell line Caco‐2 (Takanaga et al., 1994). However, it is not clear whether plants contain similar, putative SA transporters. In this study, tobacco cells in suspension culture excreted most of the exogenous SA in the form of free SA in both the 200 μM and 20 μM treatments (Figs 2, 3). It was reported that SA glucoside (SAG) injected into the intercellular space of tobacco leaves was probably converted into free SA before entering the cells (Hennig et al., 1993). The results of this study agree with the report and demonstrate that the free but not conjugated SA is the form transported across plasma membranes. The function of SA excretion is unclear. However, accumulation of SA in cells may affect physiological functions and cause cytotoxicity (Kapulnik et al., 1992). Thus, a suggested role for SA excretion is probably associated with detoxification. Tobacco cells contained free SA and possibly water‐soluble SA conjugates in both the 200 μM and 20 μM SA treatments (Fig. 4) as reported previously (Kapulnik et al., 1992) in 20 μM SA‐treated cells in suspension culture. The functions of free and conjugated forms of SA in cells were unclear. It was reported that PR‐1a gene expression was induced in tobacco leaves injected with hydrolysable SA glucoside (SAG), but not with an unhydrolysable SAG analogue thio‐SAG (TSAG) (Hennig et al., 1993). These data suggest the free SA as the active form responsible for SA function in cells. In roots of Vicia faba and Fagopyrum esculentum, exogenous SA was conjugated into O‐glucoside as a detoxification mechanism (Schulz et al., 1993). Therefore, a possible role of conjugated SA as a detoxification mechanism is suggested in tobacco suspension‐cultured cells. SA is likely to be stored in the vacuole (Ben‐Tal and Cleland, 1982). The absorbed SA was excreted into the culture medium in both the 200 μM and 20 μM SA treatments (Fig. 2). SA excretion was blocked by inhibitors in the 200 μM, but not in the 20 μM SA treatment (Figs 5, 6). It was reported that 500 μM SA rapidly increased cytosolic Ca2+concentration within 10 s after application in tobacco cell suspension culture (Kawano et al., 1998). In lactating rat mammary tissue, SA stimulated external Ca2+ influx and calcium‐dependent K+ efflux (Shennan, 1992). The results from EGTA experiments (Fig. 5) agree with these reports and suggest that 200 μM SA also caused an increase of cytosolic Ca2+ concentration. It in turn may act as a signalling component for the activation of an SA excretion pathway. SA has been demonstrated to elevate active oxygen species in tobacco plants (Chen et al., 1993), and in parsley and tobacco cell suspension cultures (Kauss and Jeblick, 1994; Kawano et al., 1998). This study's results showed that SA excretion was blocked by reduced glutathione, an active oxygen species scavenger, in the 200 μM SA treatment (Fig. 6A). The data are consistent with those reports and suggest that 200 μM SA could generate active oxygen species, which are likely to function as a signalling component for the activation of a SA excretion pathway in suspension‐cultured cells. SA excretion by cells treated with 200 μM but not 20 μM SA was inhibited greatly by staurosporine (Fig. 6A). It was reported that SA could induce a 48 kDa mitogen‐activated protein (MAP) kinase in a dose‐dependent manner with concentrations more than 50 μM in tobacco cell suspension culture (Zhang and Klessig, 1997). Whether a dose‐dependent induction of putative protein kinase activities is required for SA excretion, similar to that report (Zhang and Klessig, 1997) requires further investigation. Cycloheximide blocked SA excretion in the 200 μM, but not in the 20 μM SA treatment (Fig. 6C). The data suggest that tobacco cells may contain both constitutive and inducible SA excretion pathways, and the inducible one is activated by 200 μM SA. The requirement for de novo synthesized proteins for SA excretion in 200 μM SA treatment is possibly due to (1) an inducible carrier‐mediated transporter for SA excretion, (2) the inducible signals/signal transduction pathways that link to activate the Ca2+‐dependent SA excretion mechanism or (3) both. Tobacco cells may have both Ca2+‐dependent and Ca2+‐independent pathways for SA excretion. It is not clear how these excretion pathways are activated and regulated under different SA doses. However, approximately 35% (70 μM) and 23% (4.6 μM) of the total applied SA was taken up by cells treated with 200 μM and 20 μM SA, respectively, 5 min after addition. The deduced intracellular SA concentration in 200 μM‐treated cells (c. 1.0 mM) is much higher than that in 20 μM‐treated cells (c. 65 μM). The great difference (c. 15 times) of intracellular SA levels may activate alternative signal components and/or signal transduction pathways, as observed from the results of Figs 5 and 6. Whether a putative controlling mechanism exists which utilizes the intracellular SA level as a regulatory threshold to activate particular signalling components and/or signal transduction pathways for SA excretion needs more investigations. It is concluded that tobacco cells may contain both Ca2+‐dependent and Ca2+‐independent pathways for SA excretion in suspension culture. The one influenced by EGTA, reduced glutathione, staurosporine, and cycloheximide is activated by SA at 200 μM but not at 20 μM. Tobacco cell suspension cultures may be used as a suitable system to study SA transport, SA signalling, and detoxification of xenobiotics. 4 To whom correspondence should be addressed. Fax: +886 2 27827954. 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Journal

Journal of Experimental Botany – Oxford University Press

Published: Jun 1, 2001

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