- Email: [email protected]
Phytoalexin production and cell death in elicited carrot cell suspension cultures
Plant Science, 77 ( 1991) 261--266
26I
Elsevier Scientific Publishers Ireland Ltd.
Phytoalexin production and cell death in elicited carrot cell suspension cultures F l a v i a M a r i n e l l i a, S i m o n a D i G r e g o r i o b a n d V i t t o r i a N u t i R o n c h i b "Scuola Normale Superiore, P.--a Cavalieri 7. 56100 Pisa and Dipartimento di Chimica e Chhnica Imlustrialc. Universita di Pisa, Via Risorgimento 35. 56100 Pisa and hlstituto di Mutagencsi e D(fferen-ianwnto. CNR, Via Sve:ia I0. 56124 Pisa (Ital)')
(Received July 30th, 1990; revision received March 6th, 1991; accepted April 12th, 1991) The production of the carrot phytoalexin, 6-methoxymellein,and its immediate biosynthetic precursor, 6-hydroxymellein,was induced in suspension cultures by biotic elicitors (Aspergillus niger pectinase and purified endo-polygalacturonasefrom Fusarium moniliforme) and by the abiotic elicitor HgCI2. Aspergillus niger pectinase and HgCI2 elicited intracellular accumulation of 6-hydroxymellein, whereas Fusarium moniliforme endo-polygalacturonaseinduced extracellular 6-methoxymelleinproduction in the culture medium. Culture conditions such as mediumcompositionand 2,4-dichlorophenoxyacetic acid (2,4-D) concentration influenced the amounts of both dihydroisocoumarinssynthetized. Moreover, elicitors were phytotoxicand their addition to carrot cell suspension cultures caused an increase of extracellular pH and medium conductivity. Key words: Daucus carota; suspension cultures; elicitor; phytoalexin; cell death; hypersensitive response
Introduction One important plant inducible defence response is the biosynthesis of phytoalexins, which are low molecular weight secondary metabolites with antimicrobial activity [1,2]. Another inducible defence reaction, often associated with phytoalexin production, is the hypersensitive response (HR) which consists of a rapid and localized death of plant cells to prevent colonization by the pathogens [3,4]. An interesting model for the investigation of plant defence reactions is represented by the cell suspension cultures of D a u c u s carota. The carrot phytoalexin, a dihydroisocoumarin known as 6-methoxymellein (6-MM), is produced in carrot cell suspension cultures following infection by C h a e t o m i u m g l o b o s u m or treatment with elicitors such as pronase, pectinase and oligosaccharides from carrot cell walls [5--8]. Recently, we have reported that two different preparations of pecCorrespomlence to: Flavia Marinelli. Laboratorio di Fisiologia Vegetale. Dipartimento di Biologia vegetale, Universita "'La Sapienza". P.zzale Aldo Moro, 5, 00185 Rome. Italy.
tinolytic enzymes, a pectinase from Aspergillus niger and a purified endo-polygalacturonase from Fusarium moniliforme, act as elicitors of phytoalexin biosynthesis in carrot cell suspension cultures [9]. In this paper we investigate the elicitor-activity of these pectinolytic enzymes and of the abiotic elicitor HgCI2. The influence of culture conditions on phytoalexin biosynthesis, the phytotoxicity of the elicitors and their effects on the appearance of HR symptoms such as cell death, electrolyte leakage and extracellular pH alkalinization have also been studied.
Methods Plant m a t e r i a l and culture m e t h o d
Carrot cell suspension cultures were initiated from hypocotyl of D a u c u s carota cv. St. Valery and maintained in 50 ml of Gamborg's B5 medium [10] supplemented with 20 g/l sucrose, 2.26 /xM 2,4-D and 1.1 #M 6-benzylaminopurine (6-BAP) in 300-ml Erlenmeyer flasks. The flasks were incubated in the dark at 25°C on a gyratory shaker at 80 strokes/min. Carrot cells were subcultured
0168-9452/91/$03.50 © 1991 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
262
every 10--12 days by inoculating 2 ml of packed cell volume (PCV) into 50 ml of fresh medium. For the experiments in Murashige and Skoog medium [11] or with different concentrations of 2,4-D, cells were collected by filtration, washed twice by centrifugation with fresh medium and inoculated at the same concentration (2 ml PCV).
Viability of cultured carrot cells Viability of cultured carrot cells was determined by counting the fluorescent and the nonfluorescent cells after incubation with fluorescein diacetate (Sigma), as previously described [12]. Fluorescein diacetate (2/A) from a stock solution (4 mg/ml acetone) was added to 1 ml aliquots of carrot suspension cells. Cells were incubated for 10 min, concentrated by centrifugation and analysed at 460 nm. The results are presented as a mean from at least three replicates, and at least 100 cells were observed in each determination.
Extraction and determination 6-hydroxynwllein (6-HM )
q[' 6-MM
and
6-MM and 6-HM were obtained from cultured carrot cells and media by extracting with ethylacetate at 40°C for I h. Combined extracts were filtered through dry sodium sulphate, evaporated to dryness and resuspended in I ml ethyl-acetate. Quantitative determination of 6-MM and 6-HM in cell and media extracts was performed by reverse phase H P L C on a Cl8-silica gel column (12.5 cm x 4.6 mm) under the following conditions: sample volume, 6/~1; flow, 1 ml/min; starting pressure, 170 arm: solvent programme, equilibration for 10 rain at 40'/, methanol in water, linear gradient from 40 to 60% methanol over 10 min, linear gradient from 60 to 80% methanol over 15 min and washing by 90% methanol for 15 rain; UV detection, at 267 rim.
6-MM previously isolated from infected roots and 6-HM obtained by its chemical demethylation with BBr 3 in CH2CI_~, were used as standards [9]. Results
Assay of compounds with elicitor activity After 8--10 days subculture, 500 /A of the following solutions were added to 50 ml of cell suspension: (a) Solution A: A. niger pectinase (Sigma P5146) (0.3--300 units/ml) in 200 mM sodium acetate buffer (pH 5.2). (b) Solution B: purified F. moniliforme endo-polygalacturonase (EC 3.2.1.15) (0.3--60 units/ml), kindly provided by Prof. F. Cervone (Dept. of Plant Biology, University 'La Sapienza' Rome, Italy), in 200 mM sodium acetate buffer (pH 5.2). (c) Solution C: 0.001 100 mM HgCI2 in distilled water. All elicitor solutions were filtered through 0.2 #m Millipore filters before use. As a control, suspension cultures were treated with equal volumes of 200 mM sodium acetate buffer (pH 5.2), or with heat-inactivated enzymes (100°C, I h). At different times after elicitation, cell viability, extracellular pH and conductivity were monitored; cells were harvested by filtration, frozen in liquid nitrogen and stored at -80°C; media were collected and conserved at -20°C. All experiments were repeated at least three times and the presented data are the mean values.
Elicitation q/phytoalexin production hy pectinolytic enzymes We tested two different preparations of fungal pectinolytic enzymes as elicitors of phytoalexin production in carrot cell suspension cultures: a commercial preparation of pectinase from A. niger and a highly purified endo-polygalacturonase from F. monil!lbrme. Incubation of carrot cells with A. niger pectinase induced the production of a dihydroisocoumarin, identified as 6-HM which is considered to be the immediate biosynthetic precursor of the carrot phytoalexin 6-MM [9]. After a lag of 10 h, increasing amounts of 6-HM accumulated inside the carrot cells, reaching the maximum (110 ~g/100 ml culture) after 50 h of treatment. Significant quantities of 6-HM or 6-MM were not detected in the medium (Fig. IA). Cells treated with F. monil(/brme endopolygalacturonase did not accumulate significant amounts of 6-HM but, after a lag of 10 h, produced considerable quantities of 6-MM in the medium, reaching the maximum (220 /~g/100 ml culture) at 50 h (Fig. 1B).
263 i. Effect of medium composition and 2.4-D concentration on dihydroisoecoumarin production by cultured carrot cells treated for 48 h with 300 mu/ml culture of pectinase from (A) A. niger or of endo-polygalacturonase from (B) F.
120
AI
Table
100 80
o
moniliJbrme Medium
60
~, 40
"6 o_
20
2'o
4'0
6'0
&
Time of elieRotion (hr)
250
IZ'
200
-0
~ " ~ - -20 ~
,
= ~
-
_,_
40 60 80 Time of elicltatlon (hr)
C
--.-I_
1O0
6-MM a (p_g/culture)
6-HM a (p,g/culture)
( A ) A. niger Murashige and Skoog + 2.26 #M 2,4-D Gamborg's B5 + 2.26/~M 2,4-D Gamborg's B5 + 4.52 p,M 2,4-D Gamborg's B5 + 9.04 #M 2.4-D
9 ± 4
50 + 10
15 ± 3
II0 ± 10
10 ± 4
57 ± 7
9 + 5
60 ± 5
(B) F. monili/brme Murashige and Skoog + 2.26#M 2,4-D Gamborg's B5 + 2.26#M 2.4-D Gamborg's B5 + 4.52 #M 2,4-D Gamborg's B5 + 9.04 ~M 2,4-D
100 ± 10
24 ± 4
220 ± 18
20 ± 6
100 ± 18
22 ± 7
95 ± 5
24 ± 7
-v
120
I. Time-course of 6-MM and 6-HM production in cultured carrot cells elicited by 300 #g/ml culture of Aspergillus niger pectinase (A) and k2 moniliJbrmeendo-polygalacturonase ( B ) 6-MM inside (O) and outside (@) the cells; 6-HM inside (r'l) and outside (11) the cells.
aAverage of three determinations ± S.D.
Fig.
H e a t - d e n a t u r e d A. niger pectinase a n d F. monili[brme e n d o - p o l y g a l a c t u r o n a s e did not induce any 6 - H M or 6 - M M p r o d u c t i o n (data not shown).
lnjluenee of culture medium and 2,4-D concentrations on 6 - M M and 6-HM production The~effect of the culture m e d i u m on the p r o d u c tion of 6 - H M a n d 6 - M M , elicited respectively by pectinase from A. niger or e n d o - p o l y g a l a c t u r o n a s e from F. monil(forme, is shown in T a b l e I. T h e elicitor-induced biosynthesis either of 6 - H M or 6 - M M was more active in the G a m b o r g ' s B5 m e d i u m [10] t h a n in the M u r a s h i g e a n d Skoog m e d i u m [ 11 ]. T a b l e I also shows the i n h i b i t o r y effect o f 2,4-D
o n the p r o d u c t i o n of 6 - H M a n d 6 - M M , elicited respectively by A. niger pectinase or F. moniliforme e n d o - p o l y g a l a c t u r o n a s e . T h e observed effect of 2,4-D on the d i h y d r o i s o c o u m a r i n p r o d u c t i o n did n o t depend on the differential rate of culture growth, since equal quantitites of cells (5 g fresh wt.) had been elicited a n d then analysed for phytoalexin p r o d u c t i o n .
Cell death, extracellu&r p H anti conductiviO' variations after elieitation by pectinolytie enzymes Time-course of cell viability reduction, extracellular pH a n d m e d i u m c o n d u c t i v i t y variations were followed after elicitation by pectinase from A. niger a n d e n d o - p o l y g a l a c t u r o n a s e from F. moniliforme. Figures 2 and 3 show only the data on the elicitation by F. monili['orme e n d o polygalacturonase, the results with A. niger pectinase being nearly identical. A b o u t 40% of the cells were killed after 48 h t r e a t m e n t with e n d o - p o l y g a l a c t u r o n a s e , whereas
264
more than 95% of the control cells remained alive; a partial increase of culture viability was observed after 60 h of elicitation (Fig. 2). Toxicity of endo-polygalacturonase and pectinase was almost completely lost if the enzymes had been heat-denatured (data not shown). Other effects caused by the addition of endopolygalacturonase, as well as of pectinase, are shown in Fig. 3: an alkalinization of the culture medium (0.5--1 pH unit) during the first 10 h of treatment and a net conductivity increase after 20--30 h of elicitation. Moreover, cell browning, commonly attributed to phenylpropanoid metabolism activation, and a decrease of growth rate, determined by measuring fresh weight after 48 h of treatment, were also observed (data not shown).
400
A .....__A
A
7,0
i
380
'
y'Y" .
.
.... • . . . - - ~ o ~ O
360
.
.
o
"'""A
~
//.~ ....... . . . . . . . .
340~. ""
6,0 ~: e . "--.. "Q,
5,5 "'O ................. •
320l
3000
6,5
¢o
2'0
3'0
4'0
Time of elicitation (hr)
5'0
e'0
~'05'°
Fig. 3. Variations ofextracellular pH and medium conductivity in carrot cell suspension cultures elicited by 300 #g/ml culture of F. moniliforme endo-polygalacturonase. Extracellular pH of elicited ( A ) and control ( • ) cells: medium conductivity of elicited ( o ) and control ( • ) cells.
Toxicity and eficitor-activity of mercuric chloride Cell viability and phytoalexin production were also followed after treatment with different concentrations of the abiotic elicitor HgCI2, previously reported to induce 6-MM accumulation in carrot root slices but not in carrot cell suspension cultures [5,13]. 6-HM, but not 6-MM, was produced following treatment of cultured carrot cells with HgCI2 (Fig. 4). Cultured carrot cells treated with HgC12 solutions from 0.01 #M to 0.01 mM produced increasing amounts of 6-HM (up to 80 #g/100 ml) and their cell viability was progressively reduced, being 100
90 b._~..~........... • .~
O0 70 60
"~
so
• = u=
40 30 20 10
.-
o~
................. • .................. • ................. • ..............
Discussion The comparison of the results presented in this paper with the data previously reported by other
60
IO0
70
~. '~"~.. \"~
80
y'"
-'" ....
50
..
"'""
~_- 50 = 40 O o 311
""" "',,A-" ""
4o
"",
6b
6o
60
~ 7o
....... A
~'"
2b
4
less than 10% the cells still viable after exposure to 0.01 mM. At concentrations over 0.1 mM, 6-HM was present only in the trace levels normally detected in control cultures, suggesting that almost all the cells had been killed before the induction of phytoalexin production.
16o
"\
12o
Time of elicitotion (hr)
Fig. 2. Time-course of cell viability in carrot cell suspension cultures elicited by 300 #g/ml culture of F. moniliforme endopolygalacturonase. Cell viability of elicited (A) and control (&) cells.
°i 30
2O 10 O0
.F,
20
0,0001
0,001 0,01 HgCI2(mM)
0,1
I ,a
10
Fig, 4. 6-HM accumulation (t'l) and cell viability (A) in carrot cell suspension cultures treated for 24 h with different concentrations of HgCI 2.
265 authors [6--8] on the elicitor-induction of 6-MM biosynthesis in cultured carrot cells, points out some interesting differences: the production of higher amounts of 6-MM in the culture medium instead of inside the cells, and the induction of the intracellular accumulation of 6-HM, which has never been detected in carrot cell suspension cultures. As we have previously discussed [9], these differences are partially inherent to the experimental system, which includes the selected cell line, the nature and the amount of the elicitors and the optimization of the culture conditions. In this regard, the lower yield of 6-MM obtained by other authors [6---8] is probably due to the use of Murashige and Skoog medium and of high concentration of 2,4-D, which exert an inhibitory effect on dihydroisocoumarin biosynthesis. The production of 6-HM instead of 6-MM appears to depend on the nature of the elicitor preparation tested: we have used the abiotic elicitor HgCI2, an highly purified preparation of endo-polygalacturonase from F. moniliforme and a crude preparation of pectinase from A. niger in which endo-polygalacturonase is only a minor component. In these last two cases the elicitor activity seems to be associated with the enzymatic activity, but we cannot exclude the possibility that in the crude pectinase preparation a factor different from endo-polygalacturonase is responsible for eliciting 6-HM. However, the physiological and metabolic correlation between the two dihydroisocoumarins in the carrot response to elicitation is not clear yet: 6-HM has been reported to be the immediate biosynthetic precursor of 6-MM [14], but it has also been suggested that 6-HM is a product of 6-MM detoxification metabolism by carrot cells themselves [15]. Our finding that 6-HM accumulates inside the cells while 6-MM is produced extracellularly, suggests a metabolic compartmentalization of these two dihydroisocoumarins and offers a useful system for further investigation on their correlation as well as on their role in defence response of carrots against microorganisms. Our aim is now to understand the different action of elicitors on 6-MM and 6-HM biosynthesis; at present we can only suggest that the conversion of 6-HM into 6-MM is inhibited when cultured
carrot cells are treated by pectinase preparation and HgCI2 solutions. In this regard, we are currently trying to identify in elicited carrot cells the inducible 6-HM O-methyltransferase activity, detected for the first time by Kurosaki and Nishi [14] in elicitor-treated carrot roots. Our attention is also focused on a wider description of the carrot cell reactions to the treatment with different biotic and abiotic elicitors. Preliminary data on the elicitor-induced hypersensitive response show that A. niger pectinase, F. moniliforme endo-polygalacturonase and HgCI 2 solutions cause the death of cultured carrot cells. In this regard, it is worth considering the hypothesis [2,16,17] that plant cell death, causing the release of endogenous elicitors, induces phytoalexin production in neighbouring alive cells or tissues. This hypothesis may explain the elicitor activity of different biotic and abiotic factors, all of which are toxic to plant cells. Nevertheless, the existence of a causal correlation between cell damage and phytoalexin induction is still unclear; for example, Hoffman et al. [13,18] showed that cell death is not an absolute prerequisite for 6-MM accumulation, which is, in fact, induced by nontoxic elicitors such as ethylene. Electrolyte leakage and variation of the proton concentration inside/outside the cells are also phenomena often associated with tissue necrosis and hypersensitive response [3,4,19]. An increase of medium conductivity and extracellular pH similar to the one described in this paper, has previously been observed in elicited cell suspension cultures [3,20]; an ionic exchange response consisting in a concomitant K+efflux/H + influx, has been reported by Aktinson et al. [41 in suspension cultures of tobacco cells challenged with a hypersensitive strain of Pseudomonas syringae.
Acknowledgements We thank Prof. F. Cervone and G. Salvi for supplying purified endo-polygalacturonase from F. moniliforme and for helpful discussions; Ugo Zanelli for measuring cell viability; T. Van Boxel for drawing pictures and G. Cecchi for preparing the manuscript.
266
References I
2
3
4
5
6
7
8
9
10
I1
J. Ebel and H. Grisebach, Defense strategies of soybean against the fungus Phytophtora me~asl~erma f.sp. ,~t~v('hwa: a molecular analysis. Trends Biochem. Sci., 13 11988) 23 27. R.A. Dixon, P.M. Dey and C . L Lamb, Phytoalexins: enzymology and molecular biology. Adv. Enzyrnol. Relat. Areas Mol. Biol., 55 (1983) 1 135. S.W. Hutcheson, A. Collmer and C.J. Baker, Elicitation of hypersensitive response by P,~'etldo/~lontt,~ .~l'rinL,ae. Physiol. Plant., 76(1989) 155 163. M.M. Atkinson, J.S. Huang and J.A. Knopp, The hypersensitive reaction o1" tobacco to P,~'etl~h~nlon~l.~' ,~|'ro ingae pv. pisi. Plant Physiol., 79 (1985) 843 847. F. Kurosaki and A. Nishi, Isolation and antimicrobial activity of the phytoalexin 6-methoxymellein from cultured carrot cells. Phytochemistry, 22 (1983) 669 672. F. Kurosaki and A. Nishi, Elicitation ofphytoalexin production in cultured carrot cells. Physiol. Plant Pathol., 24 (1984} 169 176. F. Kurosaki, Y. Tsurusawa and A. Nishi, Phytoalexin production in cultured carrot cells treated with pcctinolytic enzymes. Phytochemistry, 24 (1985) 1479 1480. F. Kurosaki, Y. Tsurusawa and A. Nishi, Partial purification and characterization of clicitors for 6-methoxymellein production in cultured carrot cells. Physiol. Plant Pathol., 27 (1985} 209 217. F. Marinelli, V. Nuti Ronchi, D. Pini and P. Salvadori, Induction of 6-methoxymellein and 6-hydroxymcllein production in carrot cells. Phytochemistry, 29 ([990) 849 851. O.L. Gamborg, R.A. Miller and K. Ojima, Nutrient requirements of suspension cultures of soybean root cells. Exp. Cell Res., 50 (1968) 151 158. T. Murashige and F. Skoog, A revised medium l\~r rapid growth and bioassay with tobacco tissue culture. Physiol. Plant., 15 (1962) 4 7 3 ~ 9 7 .
12
13
14
15
16
17
18
19
20
J.M. Widholm, The use of fluorescein diacetate and phenosafranine for determination of viability of cultured plant cells. Stain Technol., 47 (1972) 189--194. R. Hoffman and J.B. Heale, Cell death, 6-methoxymellein accumulation, and induced resistance to Botrytis einerea in carrot root slices. Physiol. Mol. Plant Pathol., 30 (1987) 67--75. F. Kurosaki and A. Nishi, A methyltransferase for synthesis of the phytoalexin 6-methoxymellein in carrot cells. FEBS Lett., 227 (1988) 183 186. F. Kurosaki, Y. Tsurusawa and A. Nishi, Production and metabolism of 6-methoxymellein in cultured carrot cells. Physiol. Plant Pathol., 25 (1984) 313 322. M.G. Hahn, P. Bucheli, F. Cervone, S.H. Doares, R.A. O'Neill, A. Darvill and P. Albersheim, The roles of cell wall costituents in plant pathogen interactions, in: E. Nester and T. Kosuge (Eds.), Plant-Microbe Interactions, Vol. 3, McGraw Hill, 1989, pp. 131 181. S.H. Doares, P. Bucheli, P. Albersbeim and A.G. Darvill, Host-pathogen interactions. XXXIV. A heat-labile activity secreted by a fungal phytophatogen releases fragments of plant cell walls that kill plant cells. Mol. Plant-Microbe Interact., 2 (1989) 346 353. R. Hoffman, E. Roebroeck and J.B. Heale, Effects of ethylene biosynthesis in carrot root slices on 6-methoxymellein accumulation and resistance to Botrytis cinerea. Physiol. Plant, 73 (1988) 71 76. J.M. Dow and J.A. Callow, Leakage of electrolytes from isolated leaf mesophyll cells of tomato induced by glycopeptides from culture filtrates of Fuh,ia juh,a (Cooke) Ciferri (syn Clado~sporiumJuh'um). Physiol. Plant. Patho[., 15 (1979) 27 34. A.R. Ayers, J. Ebel, B. Valent and P. Albersheim, Hostpathogen interaction X. Fractionation and biological activity of an elicitor isolated from the mycelial walls of Phytophthora megasperma var. sojae. Plant Physiol., 57 (1976) 7 6 ~ 7 6 5 .
26I
Elsevier Scientific Publishers Ireland Ltd.
Phytoalexin production and cell death in elicited carrot cell suspension cultures F l a v i a M a r i n e l l i a, S i m o n a D i G r e g o r i o b a n d V i t t o r i a N u t i R o n c h i b "Scuola Normale Superiore, P.--a Cavalieri 7. 56100 Pisa and Dipartimento di Chimica e Chhnica Imlustrialc. Universita di Pisa, Via Risorgimento 35. 56100 Pisa and hlstituto di Mutagencsi e D(fferen-ianwnto. CNR, Via Sve:ia I0. 56124 Pisa (Ital)')
(Received July 30th, 1990; revision received March 6th, 1991; accepted April 12th, 1991) The production of the carrot phytoalexin, 6-methoxymellein,and its immediate biosynthetic precursor, 6-hydroxymellein,was induced in suspension cultures by biotic elicitors (Aspergillus niger pectinase and purified endo-polygalacturonasefrom Fusarium moniliforme) and by the abiotic elicitor HgCI2. Aspergillus niger pectinase and HgCI2 elicited intracellular accumulation of 6-hydroxymellein, whereas Fusarium moniliforme endo-polygalacturonaseinduced extracellular 6-methoxymelleinproduction in the culture medium. Culture conditions such as mediumcompositionand 2,4-dichlorophenoxyacetic acid (2,4-D) concentration influenced the amounts of both dihydroisocoumarinssynthetized. Moreover, elicitors were phytotoxicand their addition to carrot cell suspension cultures caused an increase of extracellular pH and medium conductivity. Key words: Daucus carota; suspension cultures; elicitor; phytoalexin; cell death; hypersensitive response
Introduction One important plant inducible defence response is the biosynthesis of phytoalexins, which are low molecular weight secondary metabolites with antimicrobial activity [1,2]. Another inducible defence reaction, often associated with phytoalexin production, is the hypersensitive response (HR) which consists of a rapid and localized death of plant cells to prevent colonization by the pathogens [3,4]. An interesting model for the investigation of plant defence reactions is represented by the cell suspension cultures of D a u c u s carota. The carrot phytoalexin, a dihydroisocoumarin known as 6-methoxymellein (6-MM), is produced in carrot cell suspension cultures following infection by C h a e t o m i u m g l o b o s u m or treatment with elicitors such as pronase, pectinase and oligosaccharides from carrot cell walls [5--8]. Recently, we have reported that two different preparations of pecCorrespomlence to: Flavia Marinelli. Laboratorio di Fisiologia Vegetale. Dipartimento di Biologia vegetale, Universita "'La Sapienza". P.zzale Aldo Moro, 5, 00185 Rome. Italy.
tinolytic enzymes, a pectinase from Aspergillus niger and a purified endo-polygalacturonase from Fusarium moniliforme, act as elicitors of phytoalexin biosynthesis in carrot cell suspension cultures [9]. In this paper we investigate the elicitor-activity of these pectinolytic enzymes and of the abiotic elicitor HgCI2. The influence of culture conditions on phytoalexin biosynthesis, the phytotoxicity of the elicitors and their effects on the appearance of HR symptoms such as cell death, electrolyte leakage and extracellular pH alkalinization have also been studied.
Methods Plant m a t e r i a l and culture m e t h o d
Carrot cell suspension cultures were initiated from hypocotyl of D a u c u s carota cv. St. Valery and maintained in 50 ml of Gamborg's B5 medium [10] supplemented with 20 g/l sucrose, 2.26 /xM 2,4-D and 1.1 #M 6-benzylaminopurine (6-BAP) in 300-ml Erlenmeyer flasks. The flasks were incubated in the dark at 25°C on a gyratory shaker at 80 strokes/min. Carrot cells were subcultured
0168-9452/91/$03.50 © 1991 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
262
every 10--12 days by inoculating 2 ml of packed cell volume (PCV) into 50 ml of fresh medium. For the experiments in Murashige and Skoog medium [11] or with different concentrations of 2,4-D, cells were collected by filtration, washed twice by centrifugation with fresh medium and inoculated at the same concentration (2 ml PCV).
Viability of cultured carrot cells Viability of cultured carrot cells was determined by counting the fluorescent and the nonfluorescent cells after incubation with fluorescein diacetate (Sigma), as previously described [12]. Fluorescein diacetate (2/A) from a stock solution (4 mg/ml acetone) was added to 1 ml aliquots of carrot suspension cells. Cells were incubated for 10 min, concentrated by centrifugation and analysed at 460 nm. The results are presented as a mean from at least three replicates, and at least 100 cells were observed in each determination.
Extraction and determination 6-hydroxynwllein (6-HM )
q[' 6-MM
and
6-MM and 6-HM were obtained from cultured carrot cells and media by extracting with ethylacetate at 40°C for I h. Combined extracts were filtered through dry sodium sulphate, evaporated to dryness and resuspended in I ml ethyl-acetate. Quantitative determination of 6-MM and 6-HM in cell and media extracts was performed by reverse phase H P L C on a Cl8-silica gel column (12.5 cm x 4.6 mm) under the following conditions: sample volume, 6/~1; flow, 1 ml/min; starting pressure, 170 arm: solvent programme, equilibration for 10 rain at 40'/, methanol in water, linear gradient from 40 to 60% methanol over 10 min, linear gradient from 60 to 80% methanol over 15 min and washing by 90% methanol for 15 rain; UV detection, at 267 rim.
6-MM previously isolated from infected roots and 6-HM obtained by its chemical demethylation with BBr 3 in CH2CI_~, were used as standards [9]. Results
Assay of compounds with elicitor activity After 8--10 days subculture, 500 /A of the following solutions were added to 50 ml of cell suspension: (a) Solution A: A. niger pectinase (Sigma P5146) (0.3--300 units/ml) in 200 mM sodium acetate buffer (pH 5.2). (b) Solution B: purified F. moniliforme endo-polygalacturonase (EC 3.2.1.15) (0.3--60 units/ml), kindly provided by Prof. F. Cervone (Dept. of Plant Biology, University 'La Sapienza' Rome, Italy), in 200 mM sodium acetate buffer (pH 5.2). (c) Solution C: 0.001 100 mM HgCI2 in distilled water. All elicitor solutions were filtered through 0.2 #m Millipore filters before use. As a control, suspension cultures were treated with equal volumes of 200 mM sodium acetate buffer (pH 5.2), or with heat-inactivated enzymes (100°C, I h). At different times after elicitation, cell viability, extracellular pH and conductivity were monitored; cells were harvested by filtration, frozen in liquid nitrogen and stored at -80°C; media were collected and conserved at -20°C. All experiments were repeated at least three times and the presented data are the mean values.
Elicitation q/phytoalexin production hy pectinolytic enzymes We tested two different preparations of fungal pectinolytic enzymes as elicitors of phytoalexin production in carrot cell suspension cultures: a commercial preparation of pectinase from A. niger and a highly purified endo-polygalacturonase from F. monil!lbrme. Incubation of carrot cells with A. niger pectinase induced the production of a dihydroisocoumarin, identified as 6-HM which is considered to be the immediate biosynthetic precursor of the carrot phytoalexin 6-MM [9]. After a lag of 10 h, increasing amounts of 6-HM accumulated inside the carrot cells, reaching the maximum (110 ~g/100 ml culture) after 50 h of treatment. Significant quantities of 6-HM or 6-MM were not detected in the medium (Fig. IA). Cells treated with F. monil(/brme endopolygalacturonase did not accumulate significant amounts of 6-HM but, after a lag of 10 h, produced considerable quantities of 6-MM in the medium, reaching the maximum (220 /~g/100 ml culture) at 50 h (Fig. 1B).
263 i. Effect of medium composition and 2.4-D concentration on dihydroisoecoumarin production by cultured carrot cells treated for 48 h with 300 mu/ml culture of pectinase from (A) A. niger or of endo-polygalacturonase from (B) F.
120
AI
Table
100 80
o
moniliJbrme Medium
60
~, 40
"6 o_
20
2'o
4'0
6'0
&
Time of elieRotion (hr)
250
IZ'
200
-0
~ " ~ - -20 ~
,
= ~
-
_,_
40 60 80 Time of elicltatlon (hr)
C
--.-I_
1O0
6-MM a (p_g/culture)
6-HM a (p,g/culture)
( A ) A. niger Murashige and Skoog + 2.26 #M 2,4-D Gamborg's B5 + 2.26/~M 2,4-D Gamborg's B5 + 4.52 p,M 2,4-D Gamborg's B5 + 9.04 #M 2.4-D
9 ± 4
50 + 10
15 ± 3
II0 ± 10
10 ± 4
57 ± 7
9 + 5
60 ± 5
(B) F. monili/brme Murashige and Skoog + 2.26#M 2,4-D Gamborg's B5 + 2.26#M 2.4-D Gamborg's B5 + 4.52 #M 2,4-D Gamborg's B5 + 9.04 ~M 2,4-D
100 ± 10
24 ± 4
220 ± 18
20 ± 6
100 ± 18
22 ± 7
95 ± 5
24 ± 7
-v
120
I. Time-course of 6-MM and 6-HM production in cultured carrot cells elicited by 300 #g/ml culture of Aspergillus niger pectinase (A) and k2 moniliJbrmeendo-polygalacturonase ( B ) 6-MM inside (O) and outside (@) the cells; 6-HM inside (r'l) and outside (11) the cells.
aAverage of three determinations ± S.D.
Fig.
H e a t - d e n a t u r e d A. niger pectinase a n d F. monili[brme e n d o - p o l y g a l a c t u r o n a s e did not induce any 6 - H M or 6 - M M p r o d u c t i o n (data not shown).
lnjluenee of culture medium and 2,4-D concentrations on 6 - M M and 6-HM production The~effect of the culture m e d i u m on the p r o d u c tion of 6 - H M a n d 6 - M M , elicited respectively by pectinase from A. niger or e n d o - p o l y g a l a c t u r o n a s e from F. monil(forme, is shown in T a b l e I. T h e elicitor-induced biosynthesis either of 6 - H M or 6 - M M was more active in the G a m b o r g ' s B5 m e d i u m [10] t h a n in the M u r a s h i g e a n d Skoog m e d i u m [ 11 ]. T a b l e I also shows the i n h i b i t o r y effect o f 2,4-D
o n the p r o d u c t i o n of 6 - H M a n d 6 - M M , elicited respectively by A. niger pectinase or F. moniliforme e n d o - p o l y g a l a c t u r o n a s e . T h e observed effect of 2,4-D on the d i h y d r o i s o c o u m a r i n p r o d u c t i o n did n o t depend on the differential rate of culture growth, since equal quantitites of cells (5 g fresh wt.) had been elicited a n d then analysed for phytoalexin p r o d u c t i o n .
Cell death, extracellu&r p H anti conductiviO' variations after elieitation by pectinolytie enzymes Time-course of cell viability reduction, extracellular pH a n d m e d i u m c o n d u c t i v i t y variations were followed after elicitation by pectinase from A. niger a n d e n d o - p o l y g a l a c t u r o n a s e from F. moniliforme. Figures 2 and 3 show only the data on the elicitation by F. monili['orme e n d o polygalacturonase, the results with A. niger pectinase being nearly identical. A b o u t 40% of the cells were killed after 48 h t r e a t m e n t with e n d o - p o l y g a l a c t u r o n a s e , whereas
264
more than 95% of the control cells remained alive; a partial increase of culture viability was observed after 60 h of elicitation (Fig. 2). Toxicity of endo-polygalacturonase and pectinase was almost completely lost if the enzymes had been heat-denatured (data not shown). Other effects caused by the addition of endopolygalacturonase, as well as of pectinase, are shown in Fig. 3: an alkalinization of the culture medium (0.5--1 pH unit) during the first 10 h of treatment and a net conductivity increase after 20--30 h of elicitation. Moreover, cell browning, commonly attributed to phenylpropanoid metabolism activation, and a decrease of growth rate, determined by measuring fresh weight after 48 h of treatment, were also observed (data not shown).
400
A .....__A
A
7,0
i
380
'
y'Y" .
.
.... • . . . - - ~ o ~ O
360
.
.
o
"'""A
~
//.~ ....... . . . . . . . .
340~. ""
6,0 ~: e . "--.. "Q,
5,5 "'O ................. •
320l
3000
6,5
¢o
2'0
3'0
4'0
Time of elicitation (hr)
5'0
e'0
~'05'°
Fig. 3. Variations ofextracellular pH and medium conductivity in carrot cell suspension cultures elicited by 300 #g/ml culture of F. moniliforme endo-polygalacturonase. Extracellular pH of elicited ( A ) and control ( • ) cells: medium conductivity of elicited ( o ) and control ( • ) cells.
Toxicity and eficitor-activity of mercuric chloride Cell viability and phytoalexin production were also followed after treatment with different concentrations of the abiotic elicitor HgCI2, previously reported to induce 6-MM accumulation in carrot root slices but not in carrot cell suspension cultures [5,13]. 6-HM, but not 6-MM, was produced following treatment of cultured carrot cells with HgCI2 (Fig. 4). Cultured carrot cells treated with HgC12 solutions from 0.01 #M to 0.01 mM produced increasing amounts of 6-HM (up to 80 #g/100 ml) and their cell viability was progressively reduced, being 100
90 b._~..~........... • .~
O0 70 60
"~
so
• = u=
40 30 20 10
.-
o~
................. • .................. • ................. • ..............
Discussion The comparison of the results presented in this paper with the data previously reported by other
60
IO0
70
~. '~"~.. \"~
80
y'"
-'" ....
50
..
"'""
~_- 50 = 40 O o 311
""" "',,A-" ""
4o
"",
6b
6o
60
~ 7o
....... A
~'"
2b
4
less than 10% the cells still viable after exposure to 0.01 mM. At concentrations over 0.1 mM, 6-HM was present only in the trace levels normally detected in control cultures, suggesting that almost all the cells had been killed before the induction of phytoalexin production.
16o
"\
12o
Time of elicitotion (hr)
Fig. 2. Time-course of cell viability in carrot cell suspension cultures elicited by 300 #g/ml culture of F. moniliforme endopolygalacturonase. Cell viability of elicited (A) and control (&) cells.
°i 30
2O 10 O0
.F,
20
0,0001
0,001 0,01 HgCI2(mM)
0,1
I ,a
10
Fig, 4. 6-HM accumulation (t'l) and cell viability (A) in carrot cell suspension cultures treated for 24 h with different concentrations of HgCI 2.
265 authors [6--8] on the elicitor-induction of 6-MM biosynthesis in cultured carrot cells, points out some interesting differences: the production of higher amounts of 6-MM in the culture medium instead of inside the cells, and the induction of the intracellular accumulation of 6-HM, which has never been detected in carrot cell suspension cultures. As we have previously discussed [9], these differences are partially inherent to the experimental system, which includes the selected cell line, the nature and the amount of the elicitors and the optimization of the culture conditions. In this regard, the lower yield of 6-MM obtained by other authors [6---8] is probably due to the use of Murashige and Skoog medium and of high concentration of 2,4-D, which exert an inhibitory effect on dihydroisocoumarin biosynthesis. The production of 6-HM instead of 6-MM appears to depend on the nature of the elicitor preparation tested: we have used the abiotic elicitor HgCI2, an highly purified preparation of endo-polygalacturonase from F. moniliforme and a crude preparation of pectinase from A. niger in which endo-polygalacturonase is only a minor component. In these last two cases the elicitor activity seems to be associated with the enzymatic activity, but we cannot exclude the possibility that in the crude pectinase preparation a factor different from endo-polygalacturonase is responsible for eliciting 6-HM. However, the physiological and metabolic correlation between the two dihydroisocoumarins in the carrot response to elicitation is not clear yet: 6-HM has been reported to be the immediate biosynthetic precursor of 6-MM [14], but it has also been suggested that 6-HM is a product of 6-MM detoxification metabolism by carrot cells themselves [15]. Our finding that 6-HM accumulates inside the cells while 6-MM is produced extracellularly, suggests a metabolic compartmentalization of these two dihydroisocoumarins and offers a useful system for further investigation on their correlation as well as on their role in defence response of carrots against microorganisms. Our aim is now to understand the different action of elicitors on 6-MM and 6-HM biosynthesis; at present we can only suggest that the conversion of 6-HM into 6-MM is inhibited when cultured
carrot cells are treated by pectinase preparation and HgCI2 solutions. In this regard, we are currently trying to identify in elicited carrot cells the inducible 6-HM O-methyltransferase activity, detected for the first time by Kurosaki and Nishi [14] in elicitor-treated carrot roots. Our attention is also focused on a wider description of the carrot cell reactions to the treatment with different biotic and abiotic elicitors. Preliminary data on the elicitor-induced hypersensitive response show that A. niger pectinase, F. moniliforme endo-polygalacturonase and HgCI 2 solutions cause the death of cultured carrot cells. In this regard, it is worth considering the hypothesis [2,16,17] that plant cell death, causing the release of endogenous elicitors, induces phytoalexin production in neighbouring alive cells or tissues. This hypothesis may explain the elicitor activity of different biotic and abiotic factors, all of which are toxic to plant cells. Nevertheless, the existence of a causal correlation between cell damage and phytoalexin induction is still unclear; for example, Hoffman et al. [13,18] showed that cell death is not an absolute prerequisite for 6-MM accumulation, which is, in fact, induced by nontoxic elicitors such as ethylene. Electrolyte leakage and variation of the proton concentration inside/outside the cells are also phenomena often associated with tissue necrosis and hypersensitive response [3,4,19]. An increase of medium conductivity and extracellular pH similar to the one described in this paper, has previously been observed in elicited cell suspension cultures [3,20]; an ionic exchange response consisting in a concomitant K+efflux/H + influx, has been reported by Aktinson et al. [41 in suspension cultures of tobacco cells challenged with a hypersensitive strain of Pseudomonas syringae.
Acknowledgements We thank Prof. F. Cervone and G. Salvi for supplying purified endo-polygalacturonase from F. moniliforme and for helpful discussions; Ugo Zanelli for measuring cell viability; T. Van Boxel for drawing pictures and G. Cecchi for preparing the manuscript.
266
References I
2
3
4
5
6
7
8
9
10
I1
J. Ebel and H. Grisebach, Defense strategies of soybean against the fungus Phytophtora me~asl~erma f.sp. ,~t~v('hwa: a molecular analysis. Trends Biochem. Sci., 13 11988) 23 27. R.A. Dixon, P.M. Dey and C . L Lamb, Phytoalexins: enzymology and molecular biology. Adv. Enzyrnol. Relat. Areas Mol. Biol., 55 (1983) 1 135. S.W. Hutcheson, A. Collmer and C.J. Baker, Elicitation of hypersensitive response by P,~'etldo/~lontt,~ .~l'rinL,ae. Physiol. Plant., 76(1989) 155 163. M.M. Atkinson, J.S. Huang and J.A. Knopp, The hypersensitive reaction o1" tobacco to P,~'etl~h~nlon~l.~' ,~|'ro ingae pv. pisi. Plant Physiol., 79 (1985) 843 847. F. Kurosaki and A. Nishi, Isolation and antimicrobial activity of the phytoalexin 6-methoxymellein from cultured carrot cells. Phytochemistry, 22 (1983) 669 672. F. Kurosaki and A. Nishi, Elicitation ofphytoalexin production in cultured carrot cells. Physiol. Plant Pathol., 24 (1984} 169 176. F. Kurosaki, Y. Tsurusawa and A. Nishi, Phytoalexin production in cultured carrot cells treated with pcctinolytic enzymes. Phytochemistry, 24 (1985) 1479 1480. F. Kurosaki, Y. Tsurusawa and A. Nishi, Partial purification and characterization of clicitors for 6-methoxymellein production in cultured carrot cells. Physiol. Plant Pathol., 27 (1985} 209 217. F. Marinelli, V. Nuti Ronchi, D. Pini and P. Salvadori, Induction of 6-methoxymellein and 6-hydroxymcllein production in carrot cells. Phytochemistry, 29 ([990) 849 851. O.L. Gamborg, R.A. Miller and K. Ojima, Nutrient requirements of suspension cultures of soybean root cells. Exp. Cell Res., 50 (1968) 151 158. T. Murashige and F. Skoog, A revised medium l\~r rapid growth and bioassay with tobacco tissue culture. Physiol. Plant., 15 (1962) 4 7 3 ~ 9 7 .
12
13
14
15
16
17
18
19
20
J.M. Widholm, The use of fluorescein diacetate and phenosafranine for determination of viability of cultured plant cells. Stain Technol., 47 (1972) 189--194. R. Hoffman and J.B. Heale, Cell death, 6-methoxymellein accumulation, and induced resistance to Botrytis einerea in carrot root slices. Physiol. Mol. Plant Pathol., 30 (1987) 67--75. F. Kurosaki and A. Nishi, A methyltransferase for synthesis of the phytoalexin 6-methoxymellein in carrot cells. FEBS Lett., 227 (1988) 183 186. F. Kurosaki, Y. Tsurusawa and A. Nishi, Production and metabolism of 6-methoxymellein in cultured carrot cells. Physiol. Plant Pathol., 25 (1984) 313 322. M.G. Hahn, P. Bucheli, F. Cervone, S.H. Doares, R.A. O'Neill, A. Darvill and P. Albersheim, The roles of cell wall costituents in plant pathogen interactions, in: E. Nester and T. Kosuge (Eds.), Plant-Microbe Interactions, Vol. 3, McGraw Hill, 1989, pp. 131 181. S.H. Doares, P. Bucheli, P. Albersbeim and A.G. Darvill, Host-pathogen interactions. XXXIV. A heat-labile activity secreted by a fungal phytophatogen releases fragments of plant cell walls that kill plant cells. Mol. Plant-Microbe Interact., 2 (1989) 346 353. R. Hoffman, E. Roebroeck and J.B. Heale, Effects of ethylene biosynthesis in carrot root slices on 6-methoxymellein accumulation and resistance to Botrytis cinerea. Physiol. Plant, 73 (1988) 71 76. J.M. Dow and J.A. Callow, Leakage of electrolytes from isolated leaf mesophyll cells of tomato induced by glycopeptides from culture filtrates of Fuh,ia juh,a (Cooke) Ciferri (syn Clado~sporiumJuh'um). Physiol. Plant. Patho[., 15 (1979) 27 34. A.R. Ayers, J. Ebel, B. Valent and P. Albersheim, Hostpathogen interaction X. Fractionation and biological activity of an elicitor isolated from the mycelial walls of Phytophthora megasperma var. sojae. Plant Physiol., 57 (1976) 7 6 ~ 7 6 5 .