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PKC pathway in Chitosan-induced anthraquinone production by Rubia tinctorum L. cell cultures
Plant Science 165 (2003) 429 /436 www.elsevier.com/locate/plantsci
Involvement of the PLC/PKC pathway in Chitosan-induced anthraquinone production by Rubia tinctorum L. cell cultures Andrea Vasconsuelo a, Ana Marı´a Giuletti a, Gabriela Picotto b, Julia´n RodriguezTalou a, Ricardo Boland b,* a
Ca´tedra de Microbiologı´a Industrial y Biotecnologı´a, Facultad de Farmacia y Bioquı´mica, Universidad de Buenos Aires. Junı´n 956, (1113) Buenos Aires, Argentina b Departamento de Biologı´a, Bioquı´mica y Farmacia, Universidad Nacional del Sur. San Juan 670, (8000) Bahia Blanca, Argentina Received 9 October 2002; received in revised form 12 March 2003; accepted 30 April 2003
Abstract The signal transduction pathway by which the plant elicitor Chitosan affects secondary metabolite formation in Rubia tinctorum was investigated. Chitosan significantly stimulated ( /100%) anthraquinone (Aqs) synthesis in cultured R. tinctorum. The action of the elicitor could be greatly reduced by inhibition of phospholipase C (neomycin, U-73122) and protein kinase C (PKC) (calphostin C, bisindolylmaleimide, PKC down-regulation). The phorbol ester PMA mimicked the effects of Chitosan on Aqs production. Furthermore, a marked increase in PKC activity and PKC a associated to the cell membranes was observed in response to the elicitor. Compound 2-APB, a blocker of IP3 receptor-mediated release of Ca2 from inner stores, inhibited Chitosan stimulation of Aqs formation. These results indicate that Chitosan modulation of Aqs levels in R. tinctorum involves activation of the phopholipase C/PKC pathway, with the Ca2 -dependent PKC a isoform playing a major role. # 2003 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Rubia tinctorum L.; Chitosan; Elicitation; PLC/PKC; Signal transduction
1. Introduction Madder roots are source of anthraquinone (Aqs) pigments, secondary metabolites of commercial and pharmacological interest. Madder, including Rubia tinctorum , produces various kinds of Aqs colorants, such as purpurin, xanthopurpurin and alizarin, the latter being the most important of them [1]. In general, the synthesis of different secondary metabolites by plant cell and organ cultures in vitro occurs in low yields thus limiting their commercial application [2]. Aqs production by cultured cells of R. tinctorum is feasible [3]. Elicitation is an interesting strategy employed to enhance productivity of secondary metabolites. The elicitors are defined as compounds that not only induce accumulation of phythoalexins or other compounds in plants [4], but also stimulate diverse types of defense
* Corresponding author. Tel.: /54-291-459-5101x2430; fax: /54291-459-5130. E-mail address: [email protected] (R. Boland).
response-like changes in membrane permeability, membrane depolarization and production of reactive oxygen species [5]. Several studies have provided information on the sequence of signaling events involved in plant defense pathways [5,6]. The first is the perception of elicitor by plant cells. This process is more complicated than the simple idea of the existence of specific receptors for each elicitor kind at the cellular membrane. Many plant pathogens are non-invasive and remain external to the plasma membrane, and sometimes, to the cell wall. Also, there is evidence of diffusible elicitor’s molecules capable of transversing the cell wall [7]. The second step is the intracellular transduction of the elicitor’s stimuli and finally, the synthesis and transport of defense molecules. Previous reports suggest that some of the cellular functions triggered by elicitors in plants are dependent on protein phosphorylation/dephosphorylation mechanisms that resemble those described in animal cells [8,9]. Although much less is known about these processes in plants, modifications of proteins by phosphorylation in
0168-9452/03/$ - see front matter # 2003 Elsevier Science Ireland Ltd. All rights reserved. doi:10.1016/S0168-9452(03)00208-5
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response to diverse stimuli have been observed [6]. Protein kinases depending on calcium and lipids for their activation, were partially purified from Amaranthus tricolor [10] and recently, a 70 kDa protein kinase, whose activity is stimulated by diacylglicerol and phorbol esters in the presence of calcium, was purified from maize [11]. Moreover, a functional homolog of mammalian protein kinase C (PKC) was identified in potato and directly related to the elicitor’s response [12]. However, the purification and detailed biochemical analyses of lipid- and calcium-stimulated kinases in plants are still an open field to investigate. The polysaccharide Chitosan has been widely used as elicitor. Among various responses, it induces plants to increase lignin biosynthesis and cell wall lignification leading to stronger cell walls that are less penetrable by pathogens, stimulate the accumulation of jasmonic acid and also of phenol, which is known to be harmful to fungi [4,12 /15]. Chitosan is the deacetylated form of Chitin, the major component of exoskeletons of insects and crustacea and can be found in the cell walls of some fungi. Chitosan is obtained by treatment of Chitin under strong alkaline conditions [16]. Production of Aqs in R. tinctorum cell cultures elicited with Pythium aphadermatum has been shown [17]. Similar effects of Chitosan have been observed in another species of Rubiaceae (Rubia akane Nakai) [14,18]. Thus R. tinctorum represents a convenient experimental model to investigate the intracellular mechanisms that are activated by an elicitor. Specifically, the present work was designed to study the involvement of the phopholipase C (PLC)/PKC signal transduction pathway in the production of Aqs induced by Chitosan in R. tinctorum cell cultures.
GIBCO BRL (Gaithersburg, MD). All the other reagents used were of analytical grade.
2.2. Cell cultures R. tinctorum cells were a gift from Dr Rob Verpoorte (Leiden University, The Netherlands). The cells were cultured in B5 medium [19] containing 2 g/l sucrose, 2 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D), 0.5 mg/l 1naphthaleneacetic acid and indoleacetic acid and 0.2 mg/ l kinetin. After the pH was set at 5.75 /5.80, the medium was sterilized by autoclaving (1 at, 20 min). Cultures were grown in 250-ml flasks at 25 8C on a gyratory shaker (100 rpm), applying a photoperiod of 16 h. The cells were subcultured every 7 days by threefold dilution into fresh medium. Experiments were performed using 125-ml flasks with exponentially grown cells.
2.3. Subcellular fractionation Cells (7 /10 g) were homogenized in TES buffer (50 mM Tris /HCl pH 7.4, 1 mM EDTA, 250 mM sucrose), 1 mM DTT, containing protease inhibitors (0.3 mM phenylmethylsulfonyl fluoride (PMSF); 20 mg/ml leupeptin; 20 mg/ml aprotinin), with a manual homogenizer under ice using 1 ml buffer/g cell. The homogenate was filtered through two layers of nylon mesh and then centrifuged for 10 min at 9000 /g . The supernatant was centrifuged again for 1 h at 105 000 /g to obtain cytosolic and microsomal subcellular fractions. The pelleted microsomes were resuspended in the same buffer with 0.5% Triton X-100. Protein concentration was measured by the method of Bradford [20] using bovine serum albumin as standard.
2. Materials and methods 2.4. Anthraquinone determination 2.1. Materials The ionophore A23187, neomycin, bisindolylmaleimide, calphostin C, phorbol 12-myristate 13-acetate (PMA), 4-alpha PMA, compounds U-73122 and U73343, synthetic peptide glycogen synthase (GS) (PLSRTLSVAAKK), Chitosan, Immobilon P (polyvinylidene difluoride, PVDF) membranes and all the medium components were purchased from Sigma Chemical Co (St. Louis, MO). Compound 2-APB (2aminoethoxy-diphenylborate) was obtained from Calbiochem; [g-32P]-ATP and the chemiluminescence blot detection kit (ECL) were obtained from New England Nuclear (Chicago, IL). Molecular weight colored markers were bought from BioRad Laboratories (Richmond, CA). PKC a antibody and anti-rabbit IgG horseradish peroxidase-conjugated antibody were from
The concentration of Aqs produced by R. tinctorum was determined by spectrophotometry [21]. First, cells (0.1 g) were extracted with boiling 80% aqueous ethanol usually twice until the tissue was colorless. Absorbances of the extract and the medium were then measured at 434 nm using the molar extinction coefficient of alizarin (o434 /5.5 /103). The extinction coefficients of different antraquinones do not vary significantly. For instance, the difference between the molar extintions of alizarin, ruberythic acid or rubiadin are less than 5% under the measurement conditions used in the present work (Ref. [21]). Results represent the total content of Aqs (medium and cells). The results were calculated as mmol Aqs/g cell fresh weight. When expressing Aqs levels with respect to cell dry weight the same relative changes in response to Chitosan were observed.
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2.5. Elicitation The elicitation process was carried out with Chitosan (b-1,4-linked glucosamine). A stock solution was prepared by dissolving Chitosan in 1% aqueous acetic acid by stirring overnight and then sterilized at 120 8C for 20 min. The elicitor was added at a final concentration of 200 mg/l during the exponential growth phase of cell cultures and incubated for 24/48 h. In those experiments in which specific modulators (neomycin, U73122, U-73343, calphostin C, bisindolylmaleimide, 2APB, ionophore A23187) were used to mimic or block elicitor effects, their stock solutions and dilutions were prepared and sterilized by filtration. Dose-response studies for each modulator were performed.
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with PBS-T, the membranes were incubated with antirabbit IgG horseradish peroxidase-conjugated antibodies (1:10 000 in PBS-T containing 5% non-fat dry milk). Immunoreactive proteins were developed by means of enhanced chemiluminescence (ECL). The apparent molecular weight of reactive bands was estimated by reference to a wide size range of protein markers. 2.8. Statistical analysis Statistical significance of data was evaluated using Student’s t-test [25] and probability values below 0.05 (P B/0.05) were considered significant. Quantitative data are expressed as means9/S.D. from the indicated set of experiments.
2.6. Protein kinase C assay PKC was assayed by measuring the incorporation of P from [g-32P]-ATP into the synthetic GS peptide as described previously [22]. The reaction mixture (60 ml) contained 20 mM Tris/HCl (pH 7.4), 10 mM MgCl2, 25 mM GS, 50 mM [g-32P]-ATP (0.2 mCi per assay) and the sample to be assayed in the presence of 1 mM CaCl2, 60 mg/ml phosphatidylserine and 3 mg/ml 1,2-dioleyl-rac glycerol (DG), or 1 mM EGTA. Reactions were initiated by addition of [g-32P]-ATP and incubated for 10 min at 30 8C. Assay conditions were selected so that phosphorylation had linear dependence on incubation time and enzyme concentration. The reactions were stopped onto Whatman P-81 phosphocellulose papers that were immediately soaked in 75 mM phosphoric acid and washed three times in the same solution (10 min each), dried and the radioactivity counted in scintillation mixture [23]. The activity measured in the presence of EGTA was considered unspecific and substracted from activities obtained when Ca2, phosphatidylserine and DG were present in the incubation mixture. Protein concentrations were estimated by the method of Bradford using bovine serum albumin as standard [20].
3. Results and discussion
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Protein phosphorylation participates in many elicitorinduced defense responses in a number of systems [26 / 28]. Although generally the responsible kinases are unknown, it has been shown that mammalian homologs of PKC and mitogenic activated protein kinases (MAPKs) are activated in response to different elicitors [9,12,29]. This study was carried out to ascertain whether a homolog of mammalian PKC is involved in Chitosan elicitation in R. tinctorum cells. Aqs levels in cultured R. tinctorum were markedly enhanced after 24 h of treatment with Chitosan (200 mg/ l). The increases in Aqs induced by the elicitor in most experiments were greater than 70%, generally varying between 100 and 150% above controls. The exposure of the cells to the elicitor for 48 h did not augment further the production of Aqs (Fig. 1). PLC generates the second messengers required for PKC activation. The effects of two well-known PLC antagonists, neomycin and U-73122, on the accumulation of Aqs in response to Chitosan were then examined.
2.7. Western blot analysis Protein samples were mixed with one-fourth of sample buffer (400 mM Tris/HCl pH 6.8, 10% SDS, 50% glycerol, 500 mM DTT and 2 mg/ml bromophenol blue), boiled for 5 min and resolved by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) according to the method of Laemmli [24]. Fractionated proteins were electrotransferred to polyvinylidene fluoride membranes (Immobilon-P; PVDF) and then blocked for 1 h at room temperature with 5% non-fat dry milk in PBS containing 0.1% Tween-20 (PBS-T). Blots were incubated with antiPKC a (3 mg/ml) antibody overnight at 4 8C in PBS-T containing 5% non-fat dry milk. After several washings
Fig. 1. Increased Aqs production by R. tinctorum cultures in response to Chitosan. Cells suspensions of R. tinctorum were incubated with or without (control) 200 mg/l Chitosan during 24 or 48 h. Total culture Aqs content was determined by spectrophotometry as described in Section 2. Each value represents the mean of three independent determinations9/S.D. *P B/0.05, with respect to the controls.
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Table 1 Effect of PLC inhibitors neomycin, U-73122 and its inactive analog U73343 on Aqs production by elicited cultures Without inhibitors Neomycin U-73122
U-73343
(mmol Aqs/g F wt.) Controls 18.139/1.30 Chitosan 30.439/3.50*
15.59/0.95 17.419/3.37 17.99/3.0 20.59/3.16 21.839/1.92 29.69/1.93*
The cells were preincubated for 20 /30 min with compounds neomycin (100 mM), U-73122 (10 mM) or U-73343 (10 mM) before elicitation (Chitosan, 200 mg/l, 24 h). Aqs content was determined as in Fig. 1. Each value represents the mean of three independent determinations9/S.D. * P B/0.05, with respect to the corresponding control.
Table 1 shows that 100 mM neomycin added to R. tinctorum cells 30 min before addition of Chitosan reduces the stimulatory effect of the elicitor on Aqs production by 60%. Similar treatment conditions with neomycin have been shown to inhibit PLC in other plant systems [30,31]. Neomycin exhibits an affinity for phosphatidylinositol (PI) and phosphatidylinositol bisphosphate (PIP2), and accordingly this compound is used as an inhibitor of PI-turnover [32]. In mammalian cells inositol phospholipid turnover plays an important role in signal transduction of external stimuli across the plasma membrane through second messenger systems [32,33]. The signal involves the phospholipid-specific PLC activity, which cleaves phosphatidylinositol-4,5bisphosphate to yield inositol trisphosphate (IP3) and diacylglycerol (DAG). The first acts on intracellular calcium store mobilization in plant and animal cells [34] and DAG activates PKC [35]. There is evidence showing inhibitory effects of neomycin on different events of plant life [30,36], such as the elicitor-induced defense response, deflagellation and the expression of hyperosmotic stress-inducible genes [31,37]. In addition, the rapid turnover of phosphatidylinositol metabolites has been demonstrated in a variety of plant signal responses [31,37]. As shown in Table 1, 10 mM of compound U73122, another PLC blocker that impairs coupling of Gq/11 with the enzyme [38], inhibited Chitosan-dependent Aqs synthesis by about 64%. No greater inhibition of Chitosan effects was observed at 15 mM U-73122. U73343, an inactive analogue of U-73122 [39], had negligible effects on the Aqs accumulation by Chitosan in these cells (Table 1). Taking together, these results argue in favour of the participation of PLC in the signal transduction pathway activated by Chitosan. PKC is not a single protein but instead a family of serine/threonine kinases. At least ten isoforms of this enzyme are known and classified according to their activation requirements [40]. The classical or conventional PKC isozymes a, bI, bII and g require DAG, phosphatidylserine, and calcium for activation, whereas the novel PKC isoforms d, m, h and u are calcium
independent [41]. Atypical isozymes (z, l and i) are insensitive to both DAG and calcium [35]. DAGinduced activation of PKC can be mimicked by phorbol esters [42,43]. PMA (0.5 mM) increased the levels of Aqs in cultured R. tinctorum to the same extent as Chitosan while the inactive analogue of PMA, 4-a-phorbol [46] did not stimulate secondary metabolite production (Fig. 2). Adding PMA at higher concentrations (3 mM) to the medium for 24 h prior to Chitosan treatment in order to down-regulate PKC [44,45] abolished the effects of the elicitor on Aqs synthesis (Fig. 3). Moreover, the PKC inhibitors, calphostin C (1 mM) and bisindolylmaleimide (0.05 mM), having different structures and modes of action, suppressed the increase in Aqs levels induced by Chitosan by 90 and 50%, respectively (Fig. 4). These pharmacological agents used at the same concentration range as in this work, have been shown by other authors to effectively act as specific inhibitors of a plant PKC homolog [12]. The involvement of PKC in elicitation of R. tinctorum was further investigated. The activity of the enzyme was measured using a specific PKC substrate [22] in subcellular fractions from control and Chitosan-treated (200 mg/l, 24 h) cultures. Chitosan enhanced PKC activity of the microsomal fraction tenfold over control values (Fig. 5A), while in the other subcellular fractions no significant differences were found (data not given). When PKC activity was assayed with phosphatidylserine and in the absence of Ca2 and DAG, no differences between control and Chitosan-treated cells were observed implying that specific PKC isozymes mediate the elicitor effects (data not given). In agreement with these observations, Western blot analysis revealed a marked increase in the amounts of PKC a holoenzyme (80 kDa) and its catalytic fragment (50 kDa) associated to microsomal membranes from R. tinctorum cells upon treatment with the elicitor (Fig. 5B). It is known that activation of PKC isoforms induces their translocation from the cytosol to the cell membranes [46,47]. The
Fig. 2. Effect of phorbol esters on Aqs synthesis by R. tinctorum cultures. R. tinctorum cells were treated with Chitosan (200 mg/l), PMA or its inactive analog 4a-phorbol (0.5 mM each) for 24 h, followed by determination of Aqs content as in Fig. 1. Each value represents the mean of three independent determinations9/S.D. *P B/ 0.05, with respect to the controls.
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Fig. 3. Down-regulation of PKC with high concentration of PMA obliterates Chitosan-dependent Aqs production. R. tinctorum cell cultures incubated in the absence or presence of 3 mM PMA during 24 h were treated with Chitosan (200 mg/l) for further 24 h, followed by determination of Aqs content as in Fig. 1. *P B/0.05, with respect to the controls.
described changes in PKC activity and immunoreactivity suggest that Chitosan stimulates both the synthesis and translocation to the membrane of PKC a. However, effects of the elicitor on other PKC isoforms cannot be excluded. The elevation of the intracellular Ca2 concentration is necessary for activation of the calcium-dependent PKC a isozyme by Chitosan, in addition to the DAG generated by PLC. Regulation of intracellular Ca2 levels involves modulation of either Ca2 release from inner stores by activation of the PLC/IP3 pathway, or stimulation of extracellular Ca2 entry across calcium channels in the plasma membrane, or both [48,49]. 2APB represents an appropriate tool to study the involvement of intracellular Ca2 mobilization as this compound interferes with activation of the IP3 receptor at the endoplasmic reticulum stores [50,51]. As shown in Table 2, 2-APB at 5 /50 mM significantly decreased Aqs production of R. tinctorum cells in response to Chitosan, total inhibition being achieved at a concentration of 15
mM. However, exposure of the cells to the calcium ionophore A-23187 (0.25, 0.5 and 1 mM) did not induce production of Aqs (Table 2), suggesting that in addition to Ca2, other messenger systems, e.g. DAG, required for PKC activation, also mediate the action of the elicitor. In summary, by means of specific inhibitors or stimulators of phospholipase C and PKC, the effects of Chitosan could be blocked or mimicked, respectively, thus providing evidence which indicates that the elicitor causes Aqs accumulation in R. tinctorum at least through activation of the PLC/PKC pathway. The marked elevation in PKC activity induced by Chitosan supports this contention. The requirement of an increase in intracellular Ca2 shown by the experiments with 2APB and the translocation of PKC a to membranes further suggests that this calcium-dependent isoform plays a major role in elicitation by Chitosan. However, the participation of signaling cascades other than PKC cannot be ruled out. In fact, this could explain the
Fig. 4. Effects of PKC inhibitors on Chitosan-induced Aqs formation by R. tinctorum cultures. R. tinctorum cultured cells were incubated with and without Chitosan (200 mg/l) in the absence or presence of 0.05 mM bisindolylmaleimide or 1 mM calphostin C, followed by determination of Aqs content as in Fig. 1. Each value represents the mean of three independent determinations9/S.D. *P B/0.05, with respect to the controls.
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Fig. 5. Stimulation of PKC activity and PKC a translocation by Chitosan in cultured R. tinctorum cells. Cell suspensions were treated with Chitosan (200 mg/l) for 24 h followed by homogenization and subcellular fractionation. PKC activity (A) and PKC a immunoreactivity (B) were assayed in the microsomal fraction as described in Section 2. PKC activity data are the mean of two independent determinations9/S.D. A representative immunoblot of PKC a subcellular distribution in control and Chitosan-treated cells is shown. The bands detected of 80 and 50 kDa correspond to the PKC a holoenzyme and its catalytic fragment, respectively. H, total homogenate; C, cytosol; M, microsomal fraction.
Table 2 Effects of intracellular calcium modulators on Chitosan-induced Aqs synthesis (mmol Aqs/g F wt.) Control Chitosan 5 mM 2-APB 5 mM 2-APB/Chitosan 15 mM 2-APB 15 mM 2-APB/Chitosan 50 mM 2-APB 50 mM 2-APB/Chitosan
17.229/4.2 47.39/10.17 13.959/0.35 29.459/0.77 249/1.41 18.789/5.25* 22.559/3 30.39/0.98*
A23187 0.25 mM 0.5 mM 1 mM
13.859/0.21 15.639/0.53 14.989/0.25
Cell cultures were treated with Chitosan (200 mg/l) for 24 h in the absence and presence of 2-APB, or the ionophore A23187 alone to mimic the elicitor effect, at the indicated concentrations, followed by determination of Aqs content as in Fig. 1. Each value represents the mean of three independent determinations9/S.D. * P B/0.05, with respect to the effect of chitosan in the absence of 2APB.
partial blockage of Chitosan effects by the specific inhibitors used. Besides, PKC has been shown to act as an upstream activator mediating agonist effects on MAPK in animal cells [52,53]. As MAPK stimulation in response to different elicitors have been demonstrated in plant cells [9,29], the possibility that the putative PKC acts through the MAPK pathway should be taken into consideration. Also, cytoplasmic Ca2 and protein kinases increase the synthesis of jasmonic acid [54], an intermediate signal in elicitor-induced Aqs production in R. tinctorum [55]. It is clear that the signaling pathways involved in Chitosan-induced Aqs production should be further investigated. The knowledge of the intracellular mechanisms that are modulated by any given elicitor may prove useful to understand the mode of action of different pathogens on plants and to improve the production in vitro of industrial secondary metabolites.
Acknowledgements This research was supported by a grant (PICT 9906772) from the Agencia Nacional de Promocio´n
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Cientı´fica y Tecnolo´gica (ANPCyT), Argentina. A. Vasconsuelo is recipient of a research fellowship from ANPCyT. We wish to thank Dr Rob Verpoorte (Div. of Pharmacognosy, LACDR, Leiden University, The Netherlands) for the gift of cultured R. tinctorum cells.
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Involvement of the PLC/PKC pathway in Chitosan-induced anthraquinone production by Rubia tinctorum L. cell cultures Andrea Vasconsuelo a, Ana Marı´a Giuletti a, Gabriela Picotto b, Julia´n RodriguezTalou a, Ricardo Boland b,* a
Ca´tedra de Microbiologı´a Industrial y Biotecnologı´a, Facultad de Farmacia y Bioquı´mica, Universidad de Buenos Aires. Junı´n 956, (1113) Buenos Aires, Argentina b Departamento de Biologı´a, Bioquı´mica y Farmacia, Universidad Nacional del Sur. San Juan 670, (8000) Bahia Blanca, Argentina Received 9 October 2002; received in revised form 12 March 2003; accepted 30 April 2003
Abstract The signal transduction pathway by which the plant elicitor Chitosan affects secondary metabolite formation in Rubia tinctorum was investigated. Chitosan significantly stimulated ( /100%) anthraquinone (Aqs) synthesis in cultured R. tinctorum. The action of the elicitor could be greatly reduced by inhibition of phospholipase C (neomycin, U-73122) and protein kinase C (PKC) (calphostin C, bisindolylmaleimide, PKC down-regulation). The phorbol ester PMA mimicked the effects of Chitosan on Aqs production. Furthermore, a marked increase in PKC activity and PKC a associated to the cell membranes was observed in response to the elicitor. Compound 2-APB, a blocker of IP3 receptor-mediated release of Ca2 from inner stores, inhibited Chitosan stimulation of Aqs formation. These results indicate that Chitosan modulation of Aqs levels in R. tinctorum involves activation of the phopholipase C/PKC pathway, with the Ca2 -dependent PKC a isoform playing a major role. # 2003 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Rubia tinctorum L.; Chitosan; Elicitation; PLC/PKC; Signal transduction
1. Introduction Madder roots are source of anthraquinone (Aqs) pigments, secondary metabolites of commercial and pharmacological interest. Madder, including Rubia tinctorum , produces various kinds of Aqs colorants, such as purpurin, xanthopurpurin and alizarin, the latter being the most important of them [1]. In general, the synthesis of different secondary metabolites by plant cell and organ cultures in vitro occurs in low yields thus limiting their commercial application [2]. Aqs production by cultured cells of R. tinctorum is feasible [3]. Elicitation is an interesting strategy employed to enhance productivity of secondary metabolites. The elicitors are defined as compounds that not only induce accumulation of phythoalexins or other compounds in plants [4], but also stimulate diverse types of defense
* Corresponding author. Tel.: /54-291-459-5101x2430; fax: /54291-459-5130. E-mail address: [email protected] (R. Boland).
response-like changes in membrane permeability, membrane depolarization and production of reactive oxygen species [5]. Several studies have provided information on the sequence of signaling events involved in plant defense pathways [5,6]. The first is the perception of elicitor by plant cells. This process is more complicated than the simple idea of the existence of specific receptors for each elicitor kind at the cellular membrane. Many plant pathogens are non-invasive and remain external to the plasma membrane, and sometimes, to the cell wall. Also, there is evidence of diffusible elicitor’s molecules capable of transversing the cell wall [7]. The second step is the intracellular transduction of the elicitor’s stimuli and finally, the synthesis and transport of defense molecules. Previous reports suggest that some of the cellular functions triggered by elicitors in plants are dependent on protein phosphorylation/dephosphorylation mechanisms that resemble those described in animal cells [8,9]. Although much less is known about these processes in plants, modifications of proteins by phosphorylation in
0168-9452/03/$ - see front matter # 2003 Elsevier Science Ireland Ltd. All rights reserved. doi:10.1016/S0168-9452(03)00208-5
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response to diverse stimuli have been observed [6]. Protein kinases depending on calcium and lipids for their activation, were partially purified from Amaranthus tricolor [10] and recently, a 70 kDa protein kinase, whose activity is stimulated by diacylglicerol and phorbol esters in the presence of calcium, was purified from maize [11]. Moreover, a functional homolog of mammalian protein kinase C (PKC) was identified in potato and directly related to the elicitor’s response [12]. However, the purification and detailed biochemical analyses of lipid- and calcium-stimulated kinases in plants are still an open field to investigate. The polysaccharide Chitosan has been widely used as elicitor. Among various responses, it induces plants to increase lignin biosynthesis and cell wall lignification leading to stronger cell walls that are less penetrable by pathogens, stimulate the accumulation of jasmonic acid and also of phenol, which is known to be harmful to fungi [4,12 /15]. Chitosan is the deacetylated form of Chitin, the major component of exoskeletons of insects and crustacea and can be found in the cell walls of some fungi. Chitosan is obtained by treatment of Chitin under strong alkaline conditions [16]. Production of Aqs in R. tinctorum cell cultures elicited with Pythium aphadermatum has been shown [17]. Similar effects of Chitosan have been observed in another species of Rubiaceae (Rubia akane Nakai) [14,18]. Thus R. tinctorum represents a convenient experimental model to investigate the intracellular mechanisms that are activated by an elicitor. Specifically, the present work was designed to study the involvement of the phopholipase C (PLC)/PKC signal transduction pathway in the production of Aqs induced by Chitosan in R. tinctorum cell cultures.
GIBCO BRL (Gaithersburg, MD). All the other reagents used were of analytical grade.
2.2. Cell cultures R. tinctorum cells were a gift from Dr Rob Verpoorte (Leiden University, The Netherlands). The cells were cultured in B5 medium [19] containing 2 g/l sucrose, 2 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D), 0.5 mg/l 1naphthaleneacetic acid and indoleacetic acid and 0.2 mg/ l kinetin. After the pH was set at 5.75 /5.80, the medium was sterilized by autoclaving (1 at, 20 min). Cultures were grown in 250-ml flasks at 25 8C on a gyratory shaker (100 rpm), applying a photoperiod of 16 h. The cells were subcultured every 7 days by threefold dilution into fresh medium. Experiments were performed using 125-ml flasks with exponentially grown cells.
2.3. Subcellular fractionation Cells (7 /10 g) were homogenized in TES buffer (50 mM Tris /HCl pH 7.4, 1 mM EDTA, 250 mM sucrose), 1 mM DTT, containing protease inhibitors (0.3 mM phenylmethylsulfonyl fluoride (PMSF); 20 mg/ml leupeptin; 20 mg/ml aprotinin), with a manual homogenizer under ice using 1 ml buffer/g cell. The homogenate was filtered through two layers of nylon mesh and then centrifuged for 10 min at 9000 /g . The supernatant was centrifuged again for 1 h at 105 000 /g to obtain cytosolic and microsomal subcellular fractions. The pelleted microsomes were resuspended in the same buffer with 0.5% Triton X-100. Protein concentration was measured by the method of Bradford [20] using bovine serum albumin as standard.
2. Materials and methods 2.4. Anthraquinone determination 2.1. Materials The ionophore A23187, neomycin, bisindolylmaleimide, calphostin C, phorbol 12-myristate 13-acetate (PMA), 4-alpha PMA, compounds U-73122 and U73343, synthetic peptide glycogen synthase (GS) (PLSRTLSVAAKK), Chitosan, Immobilon P (polyvinylidene difluoride, PVDF) membranes and all the medium components were purchased from Sigma Chemical Co (St. Louis, MO). Compound 2-APB (2aminoethoxy-diphenylborate) was obtained from Calbiochem; [g-32P]-ATP and the chemiluminescence blot detection kit (ECL) were obtained from New England Nuclear (Chicago, IL). Molecular weight colored markers were bought from BioRad Laboratories (Richmond, CA). PKC a antibody and anti-rabbit IgG horseradish peroxidase-conjugated antibody were from
The concentration of Aqs produced by R. tinctorum was determined by spectrophotometry [21]. First, cells (0.1 g) were extracted with boiling 80% aqueous ethanol usually twice until the tissue was colorless. Absorbances of the extract and the medium were then measured at 434 nm using the molar extinction coefficient of alizarin (o434 /5.5 /103). The extinction coefficients of different antraquinones do not vary significantly. For instance, the difference between the molar extintions of alizarin, ruberythic acid or rubiadin are less than 5% under the measurement conditions used in the present work (Ref. [21]). Results represent the total content of Aqs (medium and cells). The results were calculated as mmol Aqs/g cell fresh weight. When expressing Aqs levels with respect to cell dry weight the same relative changes in response to Chitosan were observed.
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2.5. Elicitation The elicitation process was carried out with Chitosan (b-1,4-linked glucosamine). A stock solution was prepared by dissolving Chitosan in 1% aqueous acetic acid by stirring overnight and then sterilized at 120 8C for 20 min. The elicitor was added at a final concentration of 200 mg/l during the exponential growth phase of cell cultures and incubated for 24/48 h. In those experiments in which specific modulators (neomycin, U73122, U-73343, calphostin C, bisindolylmaleimide, 2APB, ionophore A23187) were used to mimic or block elicitor effects, their stock solutions and dilutions were prepared and sterilized by filtration. Dose-response studies for each modulator were performed.
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with PBS-T, the membranes were incubated with antirabbit IgG horseradish peroxidase-conjugated antibodies (1:10 000 in PBS-T containing 5% non-fat dry milk). Immunoreactive proteins were developed by means of enhanced chemiluminescence (ECL). The apparent molecular weight of reactive bands was estimated by reference to a wide size range of protein markers. 2.8. Statistical analysis Statistical significance of data was evaluated using Student’s t-test [25] and probability values below 0.05 (P B/0.05) were considered significant. Quantitative data are expressed as means9/S.D. from the indicated set of experiments.
2.6. Protein kinase C assay PKC was assayed by measuring the incorporation of P from [g-32P]-ATP into the synthetic GS peptide as described previously [22]. The reaction mixture (60 ml) contained 20 mM Tris/HCl (pH 7.4), 10 mM MgCl2, 25 mM GS, 50 mM [g-32P]-ATP (0.2 mCi per assay) and the sample to be assayed in the presence of 1 mM CaCl2, 60 mg/ml phosphatidylserine and 3 mg/ml 1,2-dioleyl-rac glycerol (DG), or 1 mM EGTA. Reactions were initiated by addition of [g-32P]-ATP and incubated for 10 min at 30 8C. Assay conditions were selected so that phosphorylation had linear dependence on incubation time and enzyme concentration. The reactions were stopped onto Whatman P-81 phosphocellulose papers that were immediately soaked in 75 mM phosphoric acid and washed three times in the same solution (10 min each), dried and the radioactivity counted in scintillation mixture [23]. The activity measured in the presence of EGTA was considered unspecific and substracted from activities obtained when Ca2, phosphatidylserine and DG were present in the incubation mixture. Protein concentrations were estimated by the method of Bradford using bovine serum albumin as standard [20].
3. Results and discussion
32
Protein phosphorylation participates in many elicitorinduced defense responses in a number of systems [26 / 28]. Although generally the responsible kinases are unknown, it has been shown that mammalian homologs of PKC and mitogenic activated protein kinases (MAPKs) are activated in response to different elicitors [9,12,29]. This study was carried out to ascertain whether a homolog of mammalian PKC is involved in Chitosan elicitation in R. tinctorum cells. Aqs levels in cultured R. tinctorum were markedly enhanced after 24 h of treatment with Chitosan (200 mg/ l). The increases in Aqs induced by the elicitor in most experiments were greater than 70%, generally varying between 100 and 150% above controls. The exposure of the cells to the elicitor for 48 h did not augment further the production of Aqs (Fig. 1). PLC generates the second messengers required for PKC activation. The effects of two well-known PLC antagonists, neomycin and U-73122, on the accumulation of Aqs in response to Chitosan were then examined.
2.7. Western blot analysis Protein samples were mixed with one-fourth of sample buffer (400 mM Tris/HCl pH 6.8, 10% SDS, 50% glycerol, 500 mM DTT and 2 mg/ml bromophenol blue), boiled for 5 min and resolved by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) according to the method of Laemmli [24]. Fractionated proteins were electrotransferred to polyvinylidene fluoride membranes (Immobilon-P; PVDF) and then blocked for 1 h at room temperature with 5% non-fat dry milk in PBS containing 0.1% Tween-20 (PBS-T). Blots were incubated with antiPKC a (3 mg/ml) antibody overnight at 4 8C in PBS-T containing 5% non-fat dry milk. After several washings
Fig. 1. Increased Aqs production by R. tinctorum cultures in response to Chitosan. Cells suspensions of R. tinctorum were incubated with or without (control) 200 mg/l Chitosan during 24 or 48 h. Total culture Aqs content was determined by spectrophotometry as described in Section 2. Each value represents the mean of three independent determinations9/S.D. *P B/0.05, with respect to the controls.
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Table 1 Effect of PLC inhibitors neomycin, U-73122 and its inactive analog U73343 on Aqs production by elicited cultures Without inhibitors Neomycin U-73122
U-73343
(mmol Aqs/g F wt.) Controls 18.139/1.30 Chitosan 30.439/3.50*
15.59/0.95 17.419/3.37 17.99/3.0 20.59/3.16 21.839/1.92 29.69/1.93*
The cells were preincubated for 20 /30 min with compounds neomycin (100 mM), U-73122 (10 mM) or U-73343 (10 mM) before elicitation (Chitosan, 200 mg/l, 24 h). Aqs content was determined as in Fig. 1. Each value represents the mean of three independent determinations9/S.D. * P B/0.05, with respect to the corresponding control.
Table 1 shows that 100 mM neomycin added to R. tinctorum cells 30 min before addition of Chitosan reduces the stimulatory effect of the elicitor on Aqs production by 60%. Similar treatment conditions with neomycin have been shown to inhibit PLC in other plant systems [30,31]. Neomycin exhibits an affinity for phosphatidylinositol (PI) and phosphatidylinositol bisphosphate (PIP2), and accordingly this compound is used as an inhibitor of PI-turnover [32]. In mammalian cells inositol phospholipid turnover plays an important role in signal transduction of external stimuli across the plasma membrane through second messenger systems [32,33]. The signal involves the phospholipid-specific PLC activity, which cleaves phosphatidylinositol-4,5bisphosphate to yield inositol trisphosphate (IP3) and diacylglycerol (DAG). The first acts on intracellular calcium store mobilization in plant and animal cells [34] and DAG activates PKC [35]. There is evidence showing inhibitory effects of neomycin on different events of plant life [30,36], such as the elicitor-induced defense response, deflagellation and the expression of hyperosmotic stress-inducible genes [31,37]. In addition, the rapid turnover of phosphatidylinositol metabolites has been demonstrated in a variety of plant signal responses [31,37]. As shown in Table 1, 10 mM of compound U73122, another PLC blocker that impairs coupling of Gq/11 with the enzyme [38], inhibited Chitosan-dependent Aqs synthesis by about 64%. No greater inhibition of Chitosan effects was observed at 15 mM U-73122. U73343, an inactive analogue of U-73122 [39], had negligible effects on the Aqs accumulation by Chitosan in these cells (Table 1). Taking together, these results argue in favour of the participation of PLC in the signal transduction pathway activated by Chitosan. PKC is not a single protein but instead a family of serine/threonine kinases. At least ten isoforms of this enzyme are known and classified according to their activation requirements [40]. The classical or conventional PKC isozymes a, bI, bII and g require DAG, phosphatidylserine, and calcium for activation, whereas the novel PKC isoforms d, m, h and u are calcium
independent [41]. Atypical isozymes (z, l and i) are insensitive to both DAG and calcium [35]. DAGinduced activation of PKC can be mimicked by phorbol esters [42,43]. PMA (0.5 mM) increased the levels of Aqs in cultured R. tinctorum to the same extent as Chitosan while the inactive analogue of PMA, 4-a-phorbol [46] did not stimulate secondary metabolite production (Fig. 2). Adding PMA at higher concentrations (3 mM) to the medium for 24 h prior to Chitosan treatment in order to down-regulate PKC [44,45] abolished the effects of the elicitor on Aqs synthesis (Fig. 3). Moreover, the PKC inhibitors, calphostin C (1 mM) and bisindolylmaleimide (0.05 mM), having different structures and modes of action, suppressed the increase in Aqs levels induced by Chitosan by 90 and 50%, respectively (Fig. 4). These pharmacological agents used at the same concentration range as in this work, have been shown by other authors to effectively act as specific inhibitors of a plant PKC homolog [12]. The involvement of PKC in elicitation of R. tinctorum was further investigated. The activity of the enzyme was measured using a specific PKC substrate [22] in subcellular fractions from control and Chitosan-treated (200 mg/l, 24 h) cultures. Chitosan enhanced PKC activity of the microsomal fraction tenfold over control values (Fig. 5A), while in the other subcellular fractions no significant differences were found (data not given). When PKC activity was assayed with phosphatidylserine and in the absence of Ca2 and DAG, no differences between control and Chitosan-treated cells were observed implying that specific PKC isozymes mediate the elicitor effects (data not given). In agreement with these observations, Western blot analysis revealed a marked increase in the amounts of PKC a holoenzyme (80 kDa) and its catalytic fragment (50 kDa) associated to microsomal membranes from R. tinctorum cells upon treatment with the elicitor (Fig. 5B). It is known that activation of PKC isoforms induces their translocation from the cytosol to the cell membranes [46,47]. The
Fig. 2. Effect of phorbol esters on Aqs synthesis by R. tinctorum cultures. R. tinctorum cells were treated with Chitosan (200 mg/l), PMA or its inactive analog 4a-phorbol (0.5 mM each) for 24 h, followed by determination of Aqs content as in Fig. 1. Each value represents the mean of three independent determinations9/S.D. *P B/ 0.05, with respect to the controls.
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Fig. 3. Down-regulation of PKC with high concentration of PMA obliterates Chitosan-dependent Aqs production. R. tinctorum cell cultures incubated in the absence or presence of 3 mM PMA during 24 h were treated with Chitosan (200 mg/l) for further 24 h, followed by determination of Aqs content as in Fig. 1. *P B/0.05, with respect to the controls.
described changes in PKC activity and immunoreactivity suggest that Chitosan stimulates both the synthesis and translocation to the membrane of PKC a. However, effects of the elicitor on other PKC isoforms cannot be excluded. The elevation of the intracellular Ca2 concentration is necessary for activation of the calcium-dependent PKC a isozyme by Chitosan, in addition to the DAG generated by PLC. Regulation of intracellular Ca2 levels involves modulation of either Ca2 release from inner stores by activation of the PLC/IP3 pathway, or stimulation of extracellular Ca2 entry across calcium channels in the plasma membrane, or both [48,49]. 2APB represents an appropriate tool to study the involvement of intracellular Ca2 mobilization as this compound interferes with activation of the IP3 receptor at the endoplasmic reticulum stores [50,51]. As shown in Table 2, 2-APB at 5 /50 mM significantly decreased Aqs production of R. tinctorum cells in response to Chitosan, total inhibition being achieved at a concentration of 15
mM. However, exposure of the cells to the calcium ionophore A-23187 (0.25, 0.5 and 1 mM) did not induce production of Aqs (Table 2), suggesting that in addition to Ca2, other messenger systems, e.g. DAG, required for PKC activation, also mediate the action of the elicitor. In summary, by means of specific inhibitors or stimulators of phospholipase C and PKC, the effects of Chitosan could be blocked or mimicked, respectively, thus providing evidence which indicates that the elicitor causes Aqs accumulation in R. tinctorum at least through activation of the PLC/PKC pathway. The marked elevation in PKC activity induced by Chitosan supports this contention. The requirement of an increase in intracellular Ca2 shown by the experiments with 2APB and the translocation of PKC a to membranes further suggests that this calcium-dependent isoform plays a major role in elicitation by Chitosan. However, the participation of signaling cascades other than PKC cannot be ruled out. In fact, this could explain the
Fig. 4. Effects of PKC inhibitors on Chitosan-induced Aqs formation by R. tinctorum cultures. R. tinctorum cultured cells were incubated with and without Chitosan (200 mg/l) in the absence or presence of 0.05 mM bisindolylmaleimide or 1 mM calphostin C, followed by determination of Aqs content as in Fig. 1. Each value represents the mean of three independent determinations9/S.D. *P B/0.05, with respect to the controls.
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Fig. 5. Stimulation of PKC activity and PKC a translocation by Chitosan in cultured R. tinctorum cells. Cell suspensions were treated with Chitosan (200 mg/l) for 24 h followed by homogenization and subcellular fractionation. PKC activity (A) and PKC a immunoreactivity (B) were assayed in the microsomal fraction as described in Section 2. PKC activity data are the mean of two independent determinations9/S.D. A representative immunoblot of PKC a subcellular distribution in control and Chitosan-treated cells is shown. The bands detected of 80 and 50 kDa correspond to the PKC a holoenzyme and its catalytic fragment, respectively. H, total homogenate; C, cytosol; M, microsomal fraction.
Table 2 Effects of intracellular calcium modulators on Chitosan-induced Aqs synthesis (mmol Aqs/g F wt.) Control Chitosan 5 mM 2-APB 5 mM 2-APB/Chitosan 15 mM 2-APB 15 mM 2-APB/Chitosan 50 mM 2-APB 50 mM 2-APB/Chitosan
17.229/4.2 47.39/10.17 13.959/0.35 29.459/0.77 249/1.41 18.789/5.25* 22.559/3 30.39/0.98*
A23187 0.25 mM 0.5 mM 1 mM
13.859/0.21 15.639/0.53 14.989/0.25
Cell cultures were treated with Chitosan (200 mg/l) for 24 h in the absence and presence of 2-APB, or the ionophore A23187 alone to mimic the elicitor effect, at the indicated concentrations, followed by determination of Aqs content as in Fig. 1. Each value represents the mean of three independent determinations9/S.D. * P B/0.05, with respect to the effect of chitosan in the absence of 2APB.
partial blockage of Chitosan effects by the specific inhibitors used. Besides, PKC has been shown to act as an upstream activator mediating agonist effects on MAPK in animal cells [52,53]. As MAPK stimulation in response to different elicitors have been demonstrated in plant cells [9,29], the possibility that the putative PKC acts through the MAPK pathway should be taken into consideration. Also, cytoplasmic Ca2 and protein kinases increase the synthesis of jasmonic acid [54], an intermediate signal in elicitor-induced Aqs production in R. tinctorum [55]. It is clear that the signaling pathways involved in Chitosan-induced Aqs production should be further investigated. The knowledge of the intracellular mechanisms that are modulated by any given elicitor may prove useful to understand the mode of action of different pathogens on plants and to improve the production in vitro of industrial secondary metabolites.
Acknowledgements This research was supported by a grant (PICT 9906772) from the Agencia Nacional de Promocio´n
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Cientı´fica y Tecnolo´gica (ANPCyT), Argentina. A. Vasconsuelo is recipient of a research fellowship from ANPCyT. We wish to thank Dr Rob Verpoorte (Div. of Pharmacognosy, LACDR, Leiden University, The Netherlands) for the gift of cultured R. tinctorum cells.
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