Plant Cell Reports

Plant Cell Reports (1996) 15:945-948

9 Springer-Verlag1996

The role of Ca 2 + in elicitation of phytoalexin synthesis in cell culture of onion (Allium eepa L.) Alexander Dmitriev, Julia Djatsok, and Dmitry Grodzinsky Laboratory of Molecular Plant Pathology, Institute of Cell Biology and Genetic Engineering, 148 Zabolotnogo Str., Kiev 252143, Ukraine Received 26 January 1996/Revised version received 10 February 1996 - Communicated by W. Barz

Summary. Treatment of Allium cepa L. cellsuspension cultures with a biotic elicitor derived from the fungus Botrytis cinerea, resulted in phytoalexin synthesis. Two phytoalexins, 5-octylcyclopenta-l,3-dione and 5-hexyl-cyclopenta-l,3dione, were accumulated in cultured onion cells. Removal of extracelhilar Ca 2+ by the calcium chelator ethylene glycol bis(b-aminoethyl ether) N,N'-tetraacetic acid abolished the elicitor-mediated phytoalexin synthesis. The calcium channel blockers, verapamil and 8-N,N-(dimethylamino)octyl-3,4,5trimethoxybenzoate caused similar effects, whereas the addition of the Ca 2+ ionophore A23187 enhanced the accumulation of phytoalexins in the absence of the elicitor. Increase in the cytoplasmic Ca 2+ concentration in elicitor-treated onion cells was observed as monitored by the fluorescent calcium indicator indo-1. These observations suggest that Ca 2+ acts as a second messenger in the regulation of phytoalexin synthesis in cultured onion cells. Key words: Allium cepa - Ca 2+ - Elicitor Phytoalexin synthesis - Second messenger -Signal transduction Abbreviations: A23187: 4-bromo-ealcium ionophore; cAMP: adenosine 3',5'-cyclic monophosphate; [Ca2+]cyt: cytoplasmic Ca2+ concentration; EGTA: ethylene glycol bis(b-aminoethyl ether) N,N'-tetraacetic acid; EtOH: ethanol; Et20: diethyl ether; fr.wt: fresh weight; HR: hypersensitive response; PIPES: piperazine N,N'bis(2-ethanesulfonic a c i d ) ; TMB-8:[8-N,N(dimethylamino)] octyl-3,4,5-trimethoxy-benzoate;Tsh tsibulin Correspondence to: A. Dmitriev

Introduction Plant disease resistance to pathogens often depends on ,whether the plant is able to recognise the pathogen early in the infection process. Recognition of pathogens triggers a large range of inducible defence responses. The induced mechanisms associated with the HR include strengthening of the plant cell wall by alterations in the synthesis of cellwall structural proteins (Lamb et al. 1989), production of hydrolytic enzymes that attack fungi and bacteria (Boller 1985), and synthesis of low molecular weight antimicrobial phytoalexins. The latter compounds are accumulated at sites of attempted infections and are considered to play an important role in restriction of pathogen growth (Mansfield 1982). In our research on the induced defence responses in plants, two onion phytoalexins have been discovered and identified as 5-octyl-cyclopenta-l,3dione (Tsibulin ld) and 5-hexyl-cyclopenta-l,3dione (Tsibulin 2d) (Dmitriev et al. 1990). Phytoalexin synthesis in plant cells is triggered by so called elicitors, substances of low molecular weight released by the microbe or the plant or both (Darvill and Albersheim 1984). As shown for soybean (Schmidt and Ebel 1987) and wheat (Kogel et al. 1991), the elicitor binds to a receptor localised in the plasma membrane of the plant. However, intracellular signalling events following the binding o f the elicitor to its receptor are poorly understood (Scheel and Parker 1990). The aim of this study was to investigate the effect of various reagents which are known to modulate the intracellular level of Ca 2+ on phytoalexin production in cultured onion cells.

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Materials and m e t h o d s Chemicals. Calcium channel blockers A23187 and indo-1 (pentapotassium salt) were obtained from Sigma. TMB-8 was added to cell suspensions from concentrated stock solutions in EtOH. Final EtOH concentration did not exceed 0.5% (v/v). Equivalent Volumes of EtOH, citrate buffer or water were added to controls. Cell cultures. Cell-suspension cultures of onion (Allium cepa L.) were maintained in a modified Murashige-Skoog medium (Murashige and Skoog 1962), containing 2% (w/v) sucrose,. and 1-naphthaleneacetic acid (0.5 mg 1-1 medium), pH 5.7 (Djatsok et al. 1994). Culture flasks (100 ml) containing 20 ml of sterilised medium were inoculated with 1.0 g cells (fr. wt) and grown at 26~ in the dark on a reciprocal shaker at 110 rpm. Cultures were transferred to fresh medium every 10 d. Growth was monitored by measuring cell fresh weight and conductivity of the medium. Elicitor treatment. A biotic elicitor from Botrytis cinerea was prepared according to Perkovskaya et al. (1991). Glucose equivalents were determined as described by Ayres et al. (1976). 1 mg of fungal elicitor preparation after autoclaving was suspended in 200 I11 of 0.1 M sodium acetate buffer (pH 5.2), and added to a 5-ml sample of a 10-day-old culture of onion cells. The control culture received only acetate buffer. The cultures were incubated at 26~ harvested after 24 h incubation by filtration and the contents of phytoalexins were determined. Extraction and determination of phytoalexins. Harvested cells were homogenised in redistilled 96% (v/v) EtOH (at least 10 ml EtOH g-1 fr. wt cells). The extract was evaporated in vacuo at 40~ The aqueous phase was reextracted with benzol. The C6H6 fraction was evaporated up to complete removal of H20 and resuspended in hexane-Et20 (3:1). The residue was removed by centrifugation (3000 g) and the filtered supernatant was further purified by gel filtration. The contents of Tsl ld and 2d were determined by HPLC as described previously in detail (Dmitriev et al. 1990). Induction and inhibition of Tsl l d and 2d synthesis in cultured onion cells. 5 ml cultures of early stationary phase (10-dayold) were transferred into test tubes and 50-~tl aliquots of filter-sterilised solutions of EGTA (500 mM, Na-citrate buffer, pH 5.0), verapamil (5 mM, dissolved in water) and TMB-8 (30 ~M, dissolved in EtOH) were added to affect Tsl Id and 2d production. Ca2+ concentration in the onion culture medium was 5 mM, except in test tubes with EGTA where Ca2+ was absent in the medium. After incubation for 24 h, the cells were harvested by filtration. Phytoalexins accumulated in the cells were determined by HPLC. Viability of onion ceils was determined by Evan's blue exclusion (Gaff and Okong'OGola 1971).

Estimation of [Ca2+]cyt with a fluorescent dye. Change in [Ca2+]cyt of cultured onion cells was monitored by indo-1loaded cells (Timmers et al. 1991). Dye loading was accomplished after washing the cells twice in an isotonic wash solution (WS) containing 10 mM dimethyl glutaric acid (pH 4.5). Washed cells were resuspended in WS to which was added indo-1 (final concentration 20 ~Vl). Cells were incubated in this solution for 1 h at room temperature. Loading of the calcium indicator was terminated by washing the cells with 10 mM Pipes-NaOH (pH 6.0) . After addition of the elicitor, the cells were removed to measure the fluorescence at an excitation wavelength of 355 nm and an emission wavelength of 405 nm on a spectrofluorometer MFTX-2 (Zima et al. 1994). Ca2+ levels were estimated with the use of 4bromo-calcium ionophore A23187 (10 p,M) to determine fluorescence limits (Kao et al. 1989). Results

The addition o f the elicitor prepared f r o m B. cinerea to a suspension culture o f onion cells caused a marked increase in phytoalexin synthesis in the culture. W h e n the elicitor (at a final concentration o f 0.2 mg m1-1) w a s added to a 10-day-old culture o f onion cells, a fifty- to sixty-fold increase in Tsl ld and 2d production was generally observed 24 h after the elicitor treatment (Fig. 1). The effects o f various reagents which are known to change the level o f [Ca2+]eyt in other systems were examined in the presence or absence o f the elicitor. Tsl l d and 2d synthesis was appreciably 60 .~ 50 "~ 40 r

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Fig. 1. Tsl ld and 2d accumulation in cultured onion cells. A 10-day-old onion cell culture (5 ml) was pretreated with the reagents for 30 rain, then successively treated with the elicitor for 24 h. 1 Control; 2 Elicitor from B. cinerea (0.2 mg ml-1); 3 EGTA (5 mM) + elicitor; 4 Verapamil (50 ~NI) + elicitor; 5 TMB-8 (0.3 IxM) + elicitor. Phytoalexin synthesis was determined after a 24 h incubation, and the results were expressed as means of four independent experiments. S.E. did not exceed 6%.

947 suppressed2+ when EGTA, a specific chelating agent for Ca , was added to the elicitor-treated culture. Verapamil and TMB-8 (Kojima et al. 1986) are well known antagonists of Ca 2+ channels and appreciably inhibited the elicitor-induced phytoalexin synthesis in onion cells. After these treatments viability and cell number were examined and no appreciable difference was observed between control and treated cells. The addition of calcium ionophore A23187 to the cultured onion cells caused a marked enhancement of phytoalexin accumulation even in the absence of fungal elicitor (Fig. 2). Maximal accumulation of phytoalexins was observed in the presence of both elicitor and A23187, their effects being synergistic.

level of 0.32 • 0.06 ~M. A marked increase in the [Ca2+]cyt was observed aider 2-3 min of the addition of the elicitor, after which it decreased gradually. 0.9

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Fig. 3. Change in [Ca2+]cyt in onion cell culture treated with elicitor from B. cinerea (0.2 nag ml-1). Indo-1 was loaded in cultured onion cells and elicitor was added to the cell suspension at time zero (indicated by arrow). Change in fluorescence was measured at regular intervals, and [Ca2+]cyt was calculated according to (Timmers et al. 1991). Mean values of six independent experiments are shown. S.E. did not exceed 3%.

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Fig. 2. Effects of elicitor and ionophore A23187 on phytoalexin synthesis. Cultured onion cells were treated with elicitor from B. cinerea (0.2 mg ml'l), or with ionophore A23187 (10 laM) or both. The control culture received acetate buffer in place of elicitor. Data represent the relative amounts of Tsl ld and 2d 24 h after elicitation. Mean values of four independent experiments are shown, each sample was tested in duplicate. S.E. did not exceed 6%. When the ionophore was added without the elicitor the maximal amounts ofTsl ld and 2d were ca. 20% of that induced by the elicitor. Therefore extra- and intracellular Ca 2+ may play a significant role in the mechanism by which the biotic elicitor induces accumulation of phytoalexins in onion cell cultures. To confirm whether the intracellular calcium concentration is actually raised by the addition of the elicitor, [Ca2+]c~ change was monitored by the fluorescent calcium indicator indo-1. After cultured onion cells had been loaded with indo-1 for 1 h, the effect of the elicitor was tested on fluorescent intensity of the cell suspension (Fig. 3). The Ca 2+ level of onion cytoplasm was estimated at a basal

When indo-I-loaded onion ceils were kept without addition of elicitor, fluorescence response was essentially unchanged, indicating that the leakage of the Ca2+-sensitive form of the dye from the cells was, if present, negligible. In control indo-1unloaded cells, increase in the photometrical response against elicitor was not recorded. This observation suggested that the fluorescence change reflects the change in [Ca2+]oyt but did not result from other metabolic changes of onion cells caused by the elicitor. Discussion Expression of active defence mechanisms in plantpathogen interactions represents a stimulus response coupling system in which signal transduction is likely to be a feature. Elicitation of phytoalexin synthesis therefore represents a useful model for studying the signal perception and transduction mechanisms (Smith 1991). Stab and Ebel (1987) have demonstrated that antagonists of Ca 2+ flux into cells inhibited the induction of glyceollin synthesis normally observed in soybean cells treated with a glucan elicitor from Phytophthora megasperma.

948 However Kendra and Hadwiger (1987) have shown that pisatin synthesis induced in pea tissue in response to treatment with either a chitosan elicitor or Fusarium solani is apparently independent of Ca 2+. The results presented here show that addition of EGTA obviously inhibited phytoalexin accumulation in onion cells. The treatment of cultured onion cells with the calcium ionophore A23187, which causes the elevation of intracellular Ca 2+, induced the production of both Tsl ld and 2d. In addition, several reagents, which are known to suppress the increase of cytoplasmic Ca 2+ concentration, were shown to inhibit the elicitor-promoted phytoalexin production in cultured onion cells. Treatment with theophylline which causes the increase in the concentration of intracellular cAMP, enhanced the biosynthesis of both phytoalexins (data not shown). Although Tsl ld and 2d accumulation was detected after 24 h, their synthesis started after ca. 6 h, and then the content increased with time. So the initial step of the phytoalexin induction seemed to occur immediately after the addition of elicitor. This was confirmed by the marked increase in [Ca2+]oyt which was observed 2-3 min after the addition of elicitor and then decreased gradually. The timing of the peak and the profile of the transient increase in [Ca2+]cyt of elicitor-treated cells were similar to those of carrot cells treated with cAMP (Kawasaki and Nishi 1993) and of other higher plant cells treated with various stimuli such as light and growth regulators (Bush and Jones 1990). Fluorescent dyes were used to observe increases in [Ca2+]cyt during stomatal closure and pollen tube growth (Gilroy et al. 1990). However, when closure was initiated by abscisic acid, the change in [Ca2+]cyt was highly variable (Schroeder and Hagiwara 1990). These results are just indications of the complex responses we can expect from a Ca 2+based signal transduction system in plant cells. The results obtained suggest that Ca 2+ acts as a second messenger in the regulation of phytoalexin synthesis in cultured onion cells. To elucidate the overall intracellular signal transduction system operating in the induced synthesis of phytoalexins in onion cells, further characterisation of other related message systems, such as turnover of phospholipids in the cellular membrane should be examined in detail.

Acknowledgements. This workwas supported in part by grant (No. U5F000) from the International Science Foundation (New York). References

Ayres A, Ebel J, Rinelli F et al. (1976) Plant Physiol. 57: 751-759 Boiler T (1985) In: Key J, Kosuge T, (eds) Cellular and molecular biology of plant stress, AR Liss,New York, pp 247-262 Bush D, Jones R (1990) Plant Physiol. 93:841-845 Darvill A, Albersheim P (1984) Annu. Rev. Plant Physiol. 35:243-275 Djatsok Y, Gushcha N, Dmitriev A (1994)Physiol. Biochem. Cultivat. Plants 26:399-404 Dmitriev A, Tverskoy L, Koslovsky A, Kovtun A. (1990) Mol. Plant Pathol. 37:235-244 Gaff D, Okong'O-Gola O (1971) J. Exp. Bot. 22: 756758 Gilroy S, Read N, Trewavas A (1990) Nature 346: 769771 Kao J, Harootunian A, Tsien R (1989) Biol. Chem. 264:8179-8184 Kawasaki F, Nishi A (1993) Arch. Biochem. Biophys. 302:144-151 Kendra D, Hadwiger L (1987) Physiol. Mol.' Plant Pathol. 31:337-348 Kogel G, Beissmann B, Reisener H, Kogel K. (1991) Planta 183:164-169 Kojima I, Shibata H, Ogata E (1986) Bioch. Biophys. Acta 888:25-32 Lamb C, Lawton M, Dron M, Dixon R (1989) Cell 56: 215-224 Mansfield J (1982) In: Bailey J, Mansfield J, (eds) Phytoalexins, Blackie, Glasgow, pp 253-288 Murashige T, Skoog F (1962) Physiol. Plant. 15: 473497 Perkovskaya GY, Bader AI, Dmitriev AP (1991) Biopolim. Cell 7:91-94 ScheelD, Parker J (1990) Z. Naturforsch. 45c: 569575 Schmidt W, Ebel J (1987) Proc. Natl. Acad. Sci. USA 84, 4117-4121 Schroeder J, Hagiwara W (1990) Proc. Nail. Acad. Sci. USA 87, 9305-9309 Smith C (1991) In: Smith C, (ed) Biochemistry and molecular biology of plant-pathogen interactions, Clarendon Press, Oxford, pp 255-270 Stab M, Ebel J (1987) Arch. Biochem. Biophys. 257: 416-423 Timmers A, Reiss H, Schell J (1991) Cell Calcium 12, 515-521 Zima V, Djatsok O, Gantsyrin V (1994) Fiziologitsnii Zyrnal, 40:112-116

The role of Ca(2+) in elicitation of phytoalexin synthesis in cell culture of onion (Allium cepa L.).

Treatment of Allium cepa L. cellsuspension cultures with a biotic elicitor derived from the fungus Botrytis cinerea, resulted in phytoalexin synthesis...
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