Role of reactive oxygen species and proline cycle in anthraquinone accumulation in Rubia tinctorum cell suspension cultures subjected to methyl jasmonate elicitation

Plant Physiology and Biochemistry 49 (2011) 758e763

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Research article

Role of reactive oxygen species and proline cycle in anthraquinone accumulation in Rubia tinctorum cell suspension cultures subjected to methyl jasmonate elicitation María Perassolo, Carla Verónica Quevedo, Víctor Daniel Busto, Ana María Giulietti, Julián Rodríguez Talou* Cátedra de Microbiología Industrial y Biotecnología, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Junín 956, 1113 Ciudad Autónoma de Buenos Aires, Argentina

a r t i c l e i n f o

a b s t r a c t

Article history: Received 19 January 2011 Accepted 30 March 2011 Available online 9 April 2011

Elicitors are compounds or factors capable of triggering a defense response in plants. This kind of response involves signal transduction pathways, second messengers and events such as Reactive Oxygen Species (ROS) generation, proline accumulation and secondary metabolite production. Anthraquinone (AQs) biosynthesis in Rubia tinctorum L. involves different metabolic routes, including shikimate and 2-Cmethyl-D-erythritol-4-phosphate (MEP) pathways. It has been proposed that the proline cycle could be coupled with the pentose phosphate pathway (PPP), since the NADPþ generated by this cycle could act as a cofactor of the first enzymes of the PPP. The end-product of this pathway is erithrose-4-phosphate, which becomes the substrate of the shikimate pathway. The aim of this work was to study the effect of methyl jasmonate (MeJ), a well-known endogenous elicitor, on the PPP, the proline cycle and AQs production in R. tinctorum cell suspension cultures, and to elucidate the role of ROS in MeJ elicitation. Treatment with MeJ resulted in AQs as well as proline accumulation, which was mimicked by the treatment with a H2O2-generating system. Both MeJ-induced effects were abolished in the presence of diphenyliodonium (DPI), a NADPH oxidase inhibitor (main source of ROS). Treatment with the elicitor failed to induce PPP; therefore, this route did not turn out to be limiting the carbon flux to the shikimate pathway. Ó 2011 Elsevier Masson SAS. All rights reserved.

Keywords: Anthraquinone Diphenyliodonium Elicitation Methyl jasmonate Proline cycle Reactive oxygen species Rubia tinctorum

1. Introduction Elicitation is a well-known strategy that has been widely used to enhance secondary metabolite production in plant systems. The treatment of plant cells with an elicitor results in physiological changes and an onset of a defense response, which involves the expression of genes associated with secondary metabolite accumulation [1e3]. It has been observed that some substances normally produced by plant cells in a defense response as signal molecules can also trigger the same kind of response when applied

Abbreviations: AQs, anthraquinone(s); DPI, diphenyliodonium; G6PDH, glucose6-phosphate dehydrogenase; GDH, glutamate dehydrogenase; GI, growth index; GOD, glucose oxidase; H2O2, peroxide; ICDH, isocitrate (NADP) dehydrogenase; ICS, isochorismate synthase; JA, jasmonic acid; MeJ, methyl jasmonate; MEP, 2-Cmethyl-D-erythritol-4-phosphate; MES, 2-N-morpholino ethanesulphonic acid; O2$, superoxide; PPP, pentose phosphate pathway; ROS, reactive oxygen species; SA, salicylic acid. * Corresponding author. Tel.: þ54 11 4964 8270/34; fax: þ54 11 4964 8200/8377. E-mail address: [email protected] (J.R. Talou). 0981-9428/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.plaphy.2011.03.015

exogenously. Some examples of these compounds, known as endogenous elicitors, are jasmonic acid (JA) and its methyl ester (methyl jasmonate, MeJ), as well as salicylic acid (SA) [3]. The defense response begins with the interaction between the elicitor and a receptor localized in the plasmatic membrane or in the cytosol of the plant cell [1,2]. This process leads to the activation of effectors and to the amplification of the signal by a cascade of second messengers, which finally results in the establishment of a defense response that includes secondary metabolite production. Production of ROS is a common event in defense responses [4], being superoxide (O2$) and peroxide (H2O2) the most abundant species. They are mainly produced by membrane-bound NADPH oxidase and, to a lesser extent, by apoplastic peroxidase [1]. These compounds exhibit a wide variety of actions, including hypersensitivity reactions, cell death, cell wall reinforcement, gene activation and induction of defense compounds [1]. O2$ and H2O2 have been described as signals involved in phytoalexin induction in different plant species [5]. It has been reported that, in tomato, H2O2 is involved in the defense response to different elicitors, including MeJ [4].

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Accumulation of proline in response to different kinds of stress (drought, high salinity, UV irradiation, heavy metals, and oxidative and biotic stress) is well documented, being described to exhibit antioxidant activity, not only by reacting with ROS but also because it can stabilize and protect detoxifying enzymes [6]. This amino acid also contributes to cell recovery after stress by providing electrons to the respiratory chain and by acting as a nitrogen and carbon source [6,7]. In addition to this, proline and its degradation products have been described to induce the expression of genes related to pathogen defense [8]. It has also been reported that, under stress conditions, proline accumulation is a consequence of an increase in its biosynthesis from glutamate [7]. The defense response can also induce the pentose phosphate pathway (PPP), in order to feed secondary metabolite routes with carbon skeletons (Fig. 1). It could also provide antioxidant protection against ROS by producing NADPH. Glucose-6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49), the first enzyme of this pathway, has a protective role against oxidative stress in cell types that have no other NADPH-producing pathways [9]. It has been proposed that the PPP could be coupled to the proline cycle that occurs in the mitochondria and the cytosol [10]. The model suggests that the first enzymes in the PPP could use the NADPþ generated by the conversion of proline in the cytosol, thus driving the carbon flux towards the shikimate and other secondary metabolite pathways. Anthraquinones (AQs) are secondary metabolites with interesting therapeutic activities [11]. In Rubia tinctorum L., their biosynthesis involves several metabolic pathways (see Fig. 1): on one hand, the ring C is originated from an IPP unit derived from the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway. On the other hand, a-ketoglutarate and isochorismate are condensed to produce o-succynilbenzoate, which is the precursor of A and B rings. Isochorismate is synthesized from chorismate, the end product of

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shikimate pathway, by the isochorismate synthase (ICS) [12]. Many authors have reported an induction in AQs production in R. tinctorum cell suspension cultures in response to the treatment with different elicitors: chitosan [13], fungal polysaccharides, shear stress [14], JA and SA [15], and MeJ [12]. It was observed that the defense response against chitosan was regulated by the final activation of mitogen activated protein kinase (MAPK). This response involved a rise in intracellular Ca2þ concentration and the activation of phospholipase C (PLC), phosphoinositide 30 eOHekinase (PI3K) and proteinkinase C (PKC), whereas the adenilate ciclase (AC)/cAMP/proteinkinase A (PKA) cascade was not involved [16]. In this work, we studied the effect of MeJ elicitation on AQs production, the PPP and proline cycle, and also the role of ROS in the defense response. For the latter purpose, an inhibitor of the NADPH oxidase (diphenyliodonium, DPI) was used. 2. Results and discussion 2.1. Assays with MeJ and DPI In order to evaluate the effect of MeJ elicitation on R. tinctorum suspension cultures, and to elucidate the role of ROS in response to this elicitor, different experiments were performed. Besides control and MeJ treatment, DPI was added (to both non-treated and MeJtreated cells). This compound acts as an inhibitor of the NADPH oxidase (one of the main sources of ROS). Finally, a treatment with a H2O2-generating system was accomplished by the addition of both glucose and glucose oxidase (GOD, EC 1.1.3.4). This enzyme converts glucose into gluconic acid and produces H2O2. 2.1.1. Effect on pH, growth index (GI) and cell viability When media pH values were compared (Table 1), it was found that both MeJ treatments (MeJ alone and MeJeDPI), showed an

Fig. 1. Schematic illustration of the metabolic pathways mentioned in this work. 6PGDH: 6-phosphogluconate dehydrogenase; DHNA: 1,4-dihydroxy-2-naphthoic acid; DMAPP: 3,3-dimethylallylpyrophosphate; DXS: 1-deoxy-D-xylulose 5-phosphate synthase; G6PDH: glucose-6-phosphate dehydrogenase; GAP: glyceraldehyde-3-phosphate; GDH: glutamate dehydrogenase; ICDH: isocitrate (NADP) dehydrogenase; ICS: isochorismate synthase; IPP: isopentenyl-5-pyrophosphate; OSB: o-succinylbenzoic acid; -P: phosphate; P5C: pyrroline-5-carboxylate; P5CDH: P5C dehydrogenase; P5CR: P5C reductase; P5CS: P5C synthetase; PDH: proline dehydrogenase PEP: phosphoenolpyruvate; PPP: pentose phosphate pathway; TCA: Tricarboxylic acids.

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Table 1 pH values measured in the media culture of the cell suspensions under the different treatments: control, MeJ, DPI, MeJeDPI and glucoseeGOD. Results are expressed as mean values  standard deviation. Treatment

Time (hours) 4

Control MeJ DPI MeJ þ DPI Glucose/GOD

8

12

24

5.67  0.20 5.84  0.09 5.75  0.04 5.82  0.06 5.90  0.16 6.20  0.09 6.11  0.08 6.41  0.05 e e e 6.13  0.07 e e e 6.42  0.14 e e e 3.79  0.14

48 5.83 6.32 5.96 5.28 3.96

    

0.03 0.04 0.05 0.10 0.11

increase in pH values when compared to control, consistent with the cytoplasmatic acidification that is reported to occur in response to elicitation [1]. Similar results were observed in cells treated only with DPI. The addition of glucose and GOD resulted in a drastic acidification of extracellular pH, which may be a consequence of gluconic acid formation. Growth index (GI) of the cultures subjected to different treatments is depicted in Fig. 2A. GI of those cells treated with MeJ was similar to control cells, whereas the other treatments (DPI and MeJeDPI) resulted in significantly lower GIs when compared with control treatment (p < 0.05). Cell viability was evaluated by the ability of the cells to exclude Evans blue dye. This vital dye is capable of penetrating non-viable cells and staining them permanently. Therefore, cell viability decreases with increasing dye retention. As shown in Fig. 2B, addition of MeJ or DPI alone did not affect cell viability, whereas when both were added, more dye was retained. 2.1.2. Anthraquinone biosynthesis, accumulation of proline and G6PDH activity MeJ addition resulted in an increase in AQs production (Fig. 3A), and this effect was significantly notorious (p < 0.05) after 8 (20%),

Fig. 3. (A) AQs content, (B) Proline accumulation, and (C) G6PDH activity evaluated in protein extracts, obtained from the different cell cultures. Different letters represent significant differences between mean values (p < 0.05). Error bars indicate standard deviation.

Fig. 2. (A) Growth Index (GI) and (B) Cell Viability (evaluated as Evans blue uptake) of cell cultures under control, MeJ (100 mM), DPI (10 mM) and MeJeDPI (100 and 10 mM, respectively) treatments. Different letters mean significant differences between medias (p < 0.05). Error bars indicate standard deviation.

12 (28%), 24 (11%) and 48 (53%) hours of elicitation. In the presence of DPI, cells treated with MeJ failed to increase AQs production, which was found to be similar to control cells at both 24 and 48 h. These results indicate that H2O2 is involved in the signal transduction pathway that leads to AQs synthesis. Regarding proline accumulation (Fig. 3B), the addition of MeJ resulted in increased proline content, which was significantly higher than that in control treatment (p < 0.05) at 8 (23%), 12 (27%) and 48 h (22%) after elicitation. In both treatments with DPI, proline content was decreased after 24 and 48 h of elicitation (between 19 and 27% less, p < 0,05). Moreover, in the presence of DPI, proline biosynthesis after MeJ elicitation was similar to control values at both 24 and 48 h of elicitation. In order to evaluate PPP, the activity of the key enzyme of this pathway (G6PDH) was evaluated. As can be seen in Fig. 3C, at early stages of elicitation (4 h), G6PDH activity decreased after MeJ elicitation (p < 0.05), but then the activity of this enzyme recovered, since no significant differences were found between elicitated and control cultures.

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2.1.3. Effect of MeJ on ICDH and GDH activities Finally, we evaluated the effect of MeJ elicitation on isocitrate (NADP) dehydrogenase (ICDH, EC 1.1.1.42) and glutamate dehydrogenase (GDH, EC 1.4.1.2), two enzymes involved in a-ketoglutarate (precursor of glutamate) and glutamate (precursor of proline) biosynthesis, respectively. It has been described that one of the proposed roles of ICDH is to produce a-ketoglutarate for amino acid synthesis [17,18]. Taking this into consideration, along with the fact that proline accumulation was increased after MeJ addition, we supposed that the activity of this enzyme could be affected by this elicitor. However, in our experiments, no significant differences were found for ICDH activity when control and MeJ treatments were compared (Fig. 4A). GDH activity was drastically affected by this elicitor (Fig. 4B). GDH activity in cultures treated with MeJ showed a significant increase (p < 0.01) after 12 (100%), 24 (171%) and 48 (100%) hours of elictor addition. These findings, together with the higher proline levels that were observed, are analogous with those reported by Wang et al. [7]. These authors observed that Triticum aestivum plants subjected to saline conditions showed higher levels of accumulation of both proline and glutamate, accompanied by a concomitant increase in NADHeGDH activity. 2.2. Assays with the H2O2-generating system: Effect on growth index, cell viability, AQs content and proline accumulation in the presence of buffer MES When analyzing the effect of the H2O2-generating system, we found that glucoseeGOD treatment adversely and significantly affected G6PDH activity, cell growth and viability, as well as significantly increased AQs and proline content (data not shown). Due to the generation of gluconic acid and the low pH observed (Table 1), it

Fig. 4. (A) Specific activity of ICDH analyzed in protein extracts from the different cell cultures. Significant differences (p < 0.05) among means are expressed by different letters. (B) GDH activity observed in cell cultures subjected to the different treatments. Different letters mean significant differences between medias (p < 0.01). Error bars indicate standard deviation.

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was impossible to define if these effects were a consequence of the acidic culture conditions or because of H2O2 production. Therefore, an experiment was performed with the addition of 2-N-morpholino ethanesulphonic acid (MES) buffer (50 mM), in order to avoid a drastic decrease in media pH. Three different treatments were carried out: control (MES buffer only), gluconic acid (1.08 g/l) and glucoseeGOD (1 g/l and 5 U/ml, respectively). GI values were similar between gluconic acid and control treatments at both 24 and 48 h, whereas they were decreased after glucoseeGOD treatment (data not shown). Results for cell viability are depicted in Fig. 5A. As can be seen, there were no siginficant differences in Evans Blue uptake between gluconic acid and control treatments, while the addition of glucose and GOD resulted in a drastic rise in Evans blue uptake at both 24 and 48 h. These findings are in accordance with the earlier reports of several authors about the role of H2O2 in triggering apoptosislike cell death [19]. AQs content was significantly higher in cells treated with glucose and GOD than in control ones, showing an increase of 75% at 24 and 124% at 48 h of treatment (Fig. 5B). These findings, along with the results obtained when MeJ and DPI were used, indicate that H2O2 is a signal needed for AQs biosynthesis and is involved in the defense cascade triggered by MeJ. As for proline accumulation, the same behavior was observed (Fig. 6A), since it was increased in cells subjected to the H2O2generating system (46% at 24 and 38% at 48 h, p < 0.05). As before, proline synthesis turned out to be an event related to H2O2 production. These findings correlated well with the proposed antioxidant properties of proline (ROS scavenger and activator of alternative ROS-detoxifying pathways), described by Szabados and Savoure [6]. Regarding G6PDH activity (Fig. 6B), it was found that glucoseeGOD treatment adversely affected the behavior of this enzyme. This effect can not be attributed to the low pH values since this parameter was controlled by MES buffer.

Fig. 5. (A) Cell viability, and (B) AQs accumulation after the treatments employing MES buffer. Different letters at each time point mean significant differences (p < 0.05) between treatments. Error bars indicate standard deviation.

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4. Methods 4.1. Cell suspension cultures R. tinctorum L. suspension culture was a kind gift of Dr Rob Verpoorte (Leiden University, The Netherlands). Cells were cultured in Gamborg’s B5 medium, supplemented with 20 gL1 sucrose, 2 mg L1 2,4-dichlorophenoxyacetic acid, 0.5 mg L1 1-naphthaleneacetic acid, 0.5 mg L1 indoleacetic acid, and 0.2 mg L1 kinetin, pH: 5.75e5.80. Suspension cultures were grown in 250 mL Erlenmeyer flasks at 25  2  C on a gyratory shaker at 100 rpm with a 16-h photoperiod using cool white fluorescent lamps at a light intensity of 1.8 W/m2. Sub culturing was carried out every 7e10 days, using a three-fold dilution of cells. 4.2. Treatments

Fig. 6. (A) Proline content, and (B) G6PDH activity evaluated in control, gluconic acid and glucoseeGOD treatments. For each time point, different letters mean significant differences (p < 0.05) between treatments. Error bars indicate standard deviation.

These experiments confirm that H2O2 is a necessary signal that mediates AQs and proline accumulation in the defense response triggered by MeJ. Since no induction of G6PDH was observed, it can be concluded that PPP is not a limiting factor as a carbon donor to secondary metabolic pathways. Similar results were observed in a previous work, where the effects of glutamate and two proline analogs, azetidine-2-carboxylic acid (A2C) and thiazolidine-4carboxylic acid (T4C) were analyzed [20]. Although AQs and proline accumulation was enhanced in the presence of glutamate and T4C, no induction of PPP was observed in any of the performed treatments.

3. Conclusions In this work, we evaluated the production of AQs and the role of proline cycle in R. tinctorum cell suspension cultures after the elicitation of MeJ, and the involvement of ROS in this response. We found that 100 mM of MeJ resulted in AQs production (up to 53%), an effect that was observed at early elicitation stages (8 h) and throughout the whole experiment. Elicitor treatment also induced proline accumulation (up to 27%), along with GDH but not ICDH activity. These findings strongly suggest that proline accumulation is a consequence of an increased synthesis from glutamate, which could be originated by GDH from a-ketoglutarate. Accumulation of proline and AQs was similar to control values in the presence of DPI, an inhibitor of the main source of ROS, the NADPH oxidase. These results together with the increased production of both proline and AQs observed after the glucoseeGOD treatment (a H2O2-generating system) confirm that this ROS is involved in the induction of AQs and proline biosynthesis. G6PDH activity was not induced by MeJ elicitation; therefore, the PPP is not a limiting factor as a carbon donor to the shikimate pathway.

All the experiments were carried out with a cell suspension cultured as mentioned above. For the experiments with MeJ, DPI and glucose GOD, aproximmately 2 g of fresh weighed (FW) cells were inoculated into 25 mL of B5 fresh medium contained in 100 mL Erlenmeyer flasks. Suspension cultures were incubated for 5 days. After this time period, 4 different treatments were performed (besides control). Cells were either incubated in the presence of 100 mM MeJ, 10 mM DPI, both DPI and MeJ (10 and 100 mM, respectively) or both Glucose oxidase (GOD, EC 1.1.3.4) and glucose (5 U mL1 and 1 gL1, respectively). The addition of DPI was carried out 3 h before the addition of MeJ. For all treatments, samples were taken at 24 and 48 h after culture. In case of control and MeJ, additional samples were taken at 4, 8, and 12 h after elicitation. Two separate experiments were performed, and each one of them was carried out in triplicate. The experiments were incubated in the conditions mentioned above. In order to avoid the low pH values observed due to the production of gluconic acid (see Section 2.1.1), other experiments were performed in the presence of 2-N-morpholino ethanesulphonic acid (MES). Cell suspensions were inoculated and cultured as mentioned above. After 5 days of incubation, 50 mM MES buffer was added to all the suspension cultures. Cells were either incubated in the presence of both Glucose oxidase (GOD) and glucose (5 U mL1 and 1 gL1, respectively) or gluconic acid (1.08 gL1). Samples were taken at 24 and 48 h after culture. Two separate experiments were performed, and each one of them was carried out in triplicate. The experiments were incubated in the conditions mentioned above. 4.3. Analytical techniques The FW biomass quantification was performed according to Vasconsuelo et al. [13]. Growth index (GI) was calculated according to the following formula: GI¼(XfXi)/Xi where: Xf ¼ FW biomass after harvest; Xi ¼ inoculated FW biomass. Cell viability was evaluated by the Evans Blue exclusion test, as mentioned by Smith et al. [21]. Briefly, cell samples were incubated with the dye for 15 min, and after 3 washes with distilled water, the dye was extracted 3 times at 50  C with 1% sodium dodecyl sulfate (SDS) in methanol/water (1/1, v/v). The fractions were collected together and the absorbance was measured at 600 nm. Results were expressed as relative units (RU) with respect to control values (1 RU).

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AQs content was determined spectophotometrically, according to Schulte et al. [22], and proline concentration was evaluated as in Bates et al. [23]. Protein extracts were prepared as Moreno et al. [24] and protein concentration was determined as described by Bradford et al. [25], using bovine serum albumin (BSA) as standard. Glucose-6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49) activity was assayed according to Armero et al. [26]. Isocitrate (NADP) dehydrogenase (ICDH, EC 1.1.1.42) was analyzed as mentioned by Chen et al. [17] with some modifications. The reaction was performed in 100 mM TriseHCl buffer (pH 7.5), in the presence of 10 mM MgCl2, 2 mM isocitrate, and 1 mM NADPþ, using 15 mL of protein extract (final volume: 0.5 mL). In both cases, enzymatic activity was expressed as Specific Activity (mU/mg protein). One unit (1 U) is defined as the amount of enzyme that catalyzes the production 1 mmol of NADPH per minute at 30  C. Glutamate dehydrogenase (GDH, EC 1.4.1.2) was evaluated by the consumption of NADH, according to Turano et al. [27]. Enzymatic activity was expressed as Specific Activity (mU/mg protein). One unit (1 U) is defined as the amount of enzyme that catalyzes the consumption of 1 mmol of NADH per minute at 30  C. 4.4. Statistical analysis Significance of treatment effects was determined by using variance analysis (ANOVA). Variations between treatments means were analyzed using Tukey’s test (p ¼ 0.05). The software used for these analyses was InfoStat 2010 Version [28]. 4.5. Chemicals All the chemicals used in this work were of analytical grade and purchased from Sigma Aldrich Chemical Co (St. Louis, MO, USA). Acknowledgments This work was supported by grant PICT 14-15112 and 14-33166, ANPCyT (Agencia Nacional de Promoción Científica y Tecnológica), Argentina, and by grant UBACyT B111, Universidad de Buenos Aires. R.T.J. and G.A.M. are researchers from CONICET (Consejo Nacional de Investigaciones Científicas y Técnicas), Argentina. P.M., Q.C.V. and B.V.D. are fellows from CONICET. References [1] J. Zhao, L.C. Davis, R. Verpoorte, Elicitor signal transduction leading to production of plant secondary metabolites, Biotechnol. Adv. 23 (2005) 283e333. [2] A. Vasconsuelo, R. Boland, Molecular aspects of the early stages of elicitation of secondary metabolites in plants, Plant Sci. 172 (2007) 861e875. [3] M.E. Kolewe, V. Gaurav, S.C. Roberts, Pharmaceutically active natural product synthesis and supply via plant cell culture technology, Mol. Pharm. 5 (2008) 243e256. [4] M.L. Orozco-Cardenas, J. Narvaez-Vasquez, C.A. Ryan, Hydrogen peroxide acts as a second messenger for the induction of defense genes in tomato plants in response to wounding, systemin, and methyl jasmonate, Plant Cell 13 (2001) 179e191. [5] S.T. Perrone, K.L. McDonald, M.W. Sutherland, D.I. Guest, Superoxide release is necessary for phytoalexin accumulation in Nicotiana tabacum cells during the expression of cultivar-race and non-host resistance towards Phytophthora spp, Physiol. Mol. Plant Pathol. 62 (2003) 127e135.

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