- Email: [email protected]
Toxic effects of phaseollin on plant cells
Physiological Plant PatholoQ (1977) 10, 22l-227
Toxic effects of phaseollin on plant cells R.
A.
SKIPP,~
C.
SELBY
and J. A.
BAILEY
Agricultural ResearchCouncil Plant Growth Substanceand SystemicFungi&& Unit, Wye College ( University of London), Ashfoord,Kent TN25 5AH, U.K. (Acceptedfor publication Januav
1977)
Plant cells, from bean and tobacco suspension cultures and from pods and hypocotyls of bean, were rapidly killed following immersion in solutions containmg 30 pg phaseollin per ml. These concentrations inhibited the respiration of bean cells within 2 min of treatment and also reduced the growth of bean suspension cultures during a 3 week incubation period. Thereafter, as a result of a few cells surviving, the rate of growth of the suspension cultures increased and significant amounts of dry matter were produced. The total amount of phaseollin present in the suspension cultures and its concentration in the growth medium decreased during the incubation period. These findings are discussed in relation to the similarities between the effects of phaseollm on plant and on fungal cells, and to their relevance to current concepts about the formation and r6le of phaseollin in infected plant tissues.
INTRODUCTION
Recent studies on the biological activity of phaseollin, an isoflavanoid phytoalexin produced by dwarf bean (Phaseolus vulgaris), have shown that this compound causes drastic changes in a range of fungal species. Spores of the bean pathogen, Colletotrichum lindemuthianum, were killed within 2 min of exposure to a solution containing 10 pg phaseollin per ml [13] and similar concentrations exerted fungicidal effects on the germ tubes of ten different fungi [lsl]. Phaseollin has also been shown to cause lysis of mammalian erythrocytes [17]. Experiments with higher plants have been mainly directed to an examination of effects on permeability and respiration [17], rather than on the viability of treated cells. In view of the possible significance of such an effect, a more extensive study was undertaken using cells of suspension cultures, epidermis and endocarp of P. vulgaris and those of suspension cultures of Mcotiana tabacum. MATERIALS
AND
METHODS
Suspension cultures were derived from callus induced from sterile bean seedling hypocotyl segments (Phaseolus vulgaris cv. Kievitsboon Koekoek) and were maintained on the medium of Murashige & Skoog [.ZO] containing O-5 mg/l, 2,4-d& chlorophenoxy acetic acid (2,4-D) and 0.1 mg/l kinetin (Medium I). The suspension cultures were shaken at 80 rev/min at 25 “C and subcultured every 14 days. Bag,
t Present address: Plant Diseases Division Sub-Station, Palmerston North, New Zealand. (Reprint requests
c/o Grasslands to J. A. B.)
Division,
D.S.I.R.,
Private
222
R. A. Skipp,
C. Selby
and
J. A. Bailey
Cell suspension cultures from tobacco pith (Nicotiuna tabacum cv. Winsconsin 38) were maintained under the same conditions, except that 1 mg/l !&4n was used (Medium II). The suspension cultures consisted of clumps of cells, 1 to 5 mm in diameter, and large numbers of individual cells. Flasks were allowed to stand for a few minutes to permit the aggregated cells to settle, and the “supernatant”, which contained mostly single cells, was removed with a pipette for testing. The epidermal cells used were present in thin strips of tissue cut from hypocotyls of beans (cv. Prince), which had been grown at 20 “C for 10 days in an illuminated growth room. Bean endocarp cells were present in strips cut similarly from the internal linings of pods (cv. Comtesse de Chambord) obtained locally. Crystalline phaseollin, obtained from virus-infected hypocotyls of bean [3], was dissolved in either ethanol or dimethyl sulphoxide (DMSO) and added to aqueous test solutions to give a range of concentrations. Where necessary, extra solvent was added to make their final concentrations 2.0 and 0.5% (v/v) respectively. In experiments testing the effects of phaseollin on the viability of cells from suspension cultures, ethanolic solutions were added to McCartney bottles containing 10 ml of “supernatant”. The tubes were placed on a roller mixer (Luckham Ltd) and samples of about O-5 ml were removed for microscopic examination at intervals during incubation at 25 “C. One hundred cells were examined in each of three drops of treated “supernatant” and the number alive and dead were recorded. Living cells had obviously intact protoplasts and usually possessed cytoplasmic strands in which streaming was evident. Dead cells showed granulation of the cytoplasm and often shrinkage of the protoplast and nucleus. “Supernatant” was also added to an equal volume of distilled water containing 10 pg Rhodamine G per ml. After about 5 min treatment, dead cells appeared pink and living cells colourless. Tissue containing epidermal or endocarp cells was submerged in 1 ml of a solution of phaseollin and 2.0% ethanol in distilled water contained within a small watch glass. The respiration rates of bean suspension cultures were measured with an oxygen electrode (Rank Brothers, Cambridge), The electrode was calibrated assuming that air-saturated water contains 0.26 ~01 oxygen per ml at 25 “C [7] and zero oxygen concentration was obtained by the addition of a few sodium dithionite crystals, Five ml of a 7-day-old suspension culture were added to the electrode chamber and the lid was inserted, care being taken to prevent air bubbles being trapped. After a steady rate of oxygen uptake had been obtained, 150 pg phaseollin in O-1 ml of ethanol were injected with a syringe through the narrow bore in the chamber lid. The rate of 0, uptake of these cells and those treated with ethanol only were measured at regular intervals. The effect of phaseollin on the growth of suspension cultures of bean was measured using four replicate flasks containing 50 ml sterilized Medium I, to which phaseollin, dissolved in DMSO, was added. The final concentration of DMSO was 0.04 or 0.5%. The flasks were inoculated with 5 ml of a 14-day-old culture and 14 or 21 days later the resulting cells were obtained by filtration, dried at 70 “C for 48 h and weighed. Phaseollin was extracted from these cells by boiling them in ethanol and from the supernatant by partitioning with diethyl ether. The
Toxic effects of phaseollin
on plant cells
223
weight of phaseollin in these extracts was measured by ultraviolet following purification by thin-layer chromatography [3]. RESULTS E$eEt of phaseollin
on the viability
spectrophotometry
of plant cells
Cells treated with 30 pg phaseollin per ml for 1 h are illustrated in Plates 1 and 2. Cells of bean and tobacco suspension cultures [Plate 1 (a) and (c)] and cells of bean endocarp and epidermis (Plate 2) were dead and appeared granular. Cells exposed to 2% ethanol remained alive, the intact cytoplasm often showing streaming [Plate l(b) and (d)]. Further studies were made to determine the time course of the effect of phaseollin on bean suspension cells. Table 1 shows the percentage of living cells in a suspenTAISLE 1 on the viability of cells from
Effect of phmeollin Concentration of phaseollin k&4 0 10 20 30 50
Stainedb 88 88 83 70 5
% cells found 10 Unstained” 85 84 81 69 1
alive’
after
Stainedb a3 86 84 24 0
bean suspension
exposure to phaseollin 50 UnstainedG
cultures for:
(min)
Stainedb
76 78 78 18 0
a See text for criteria of viability. Results are the mean of three replicates b Sample mixed with equal volume of an aqueous solution of Rhodamine and examined after 5 min. c Cells examined immediately after removal of sample without Rhodamine
100 UnstainedC
85 88 78 14 0
76 83 73 18 0
of 100 cells. G (10 pg/ml) G treatment.
sion containing 1.7 x lo5 cells/ml after exposure to phaseollin at concentrations ranging from 0 to 50 pg/ml for 10, 50 and 100 min. Samples treated with Rhodamine G before microscopic examination gave results similar to those obtained with unstained cells. During incubation, the proportion of living cells in the controls (2% ethanol) remained constant (76 to 88%), whereas, in a solution containing 50 pg phaseollin per ml, less than 5% of cells were found alive after 10 min and all were killed after 50 min. Cells were also killed at a concentration of 30 pg/ml, less than 20% of cells being alive after treatment for 100 min. Levels below 30 pgg/ml had little effect on cell viability. In another experiment, using an undetermined but apparently less dense cell suspension, all cells were killed within 30 mm of being placed in a solution containing 30 pg phaseollin per ml. Effect of phmeollin
on the respiration
of bean cells in suspension culture
As seen in Table 2, the addition of phaseollin to give a concentration of 30 pg/ml in an actively respiring suspension of bean cells caused a rapid fall in the rate of oxygen uptake. A similar but slower reduction occurred when cells were treated with 2% ethanol.
224
R. A. Skipp, TABLE
Effect
of phaseollin
Oxygen trode.
0
Control (2% ethanol) Phaseollin (30 pg/ml)
9.7kO.6 9-92 1.1
uptake by a 7-day-old suspension The values are the means of three
of bean
susjension
J. A. Bailey
cells
of oxygen uptake (nmol/ml/min) treatment for: (mm) 2 5
Treatment
and
2
on the respiration Rate
C. Selby
7*7+_oci 4.2 + 0.6
6.220.4 3.350.7
culture was measured measurements.
after 10 4.2cO.6 1*9&O-2
using
an oxygen
elec-
Effect of phaseollin on the growth of bean cells in suspensionculture Table 3 shows the weight of cells harvested 2 and 3 weeks after flasks of growth medium containing phaseollin at concentrations ranging from 0 to 30 pg/ml (dissolved in O-04 and 0.5% DMSO respectively) had been inoculated with a suspension of bean cells. In all cases, there was a net increase in weight during incubation
Effect Phaseollm concentration b%/~) 0 2 5 10 15 30 Weight of inoculum a Mean b Yields 0 Yields
of phaseollin
TABLE 3 on the growth of bean suspension Dry
weight
Experiment
cultures
of harvested cells (md” lb Experiment 26
420 + 30 357 * 37 355 + 124 391+36 248 & 149 79+38
460+98 5295 78 353*83 292 + 20 108+24
19+3-5
43+2*1
weight and standard deviation of cehs from four replicate flasks. measured after 2 weeks. Concentration of DMSO was 0.04%. measured after 3 weeks. Concentration of DMSO was 0.5%.
but compared with that which occurred in the absence of phaseollin, this was reduced by approximately 40% by 15 pg phaseollin per ml and by approximately 85% by 30 pg/ml. At the time of sampling, microscopical examination showed that, although many cell aggregates appeared alive, most individual cells ( > 80%) in cultures treated with 30 pg phaseollin per ml were dead. The usual high proportion of living cells was found in the controls. In subsequent experiments, incubation was extended to 7 weeks to determine whether cells which survived exposure to 30 pg phaseollin per ml eventually produced significant growth. The weight of cells produced in a typical experiment was 375 + 9 mg, thus approaching the weights achieved during 3 weeks’ incubation of control flasks (Table 3).
PLATE 1. Light micrographs of cells from suspension cultures of bean and tobacco showing the effects of treatment with phaseollin (30 pg/ml) in 2% e th anol for 1 h. (a) Bean cells treated with phaseollin. (b) Bean cells treated only with 2% ethanol. (c) Tobacco cells treated with phaseollin. (d) Tobacco cells treated only with 2% ethanol. Note granulation in cells treated with phaseollin. (Magnifications x 750.)
[facing
page 224
cells
PLATE 2. Light micrographs of bean (b) 1 h after they had been treated
hypocotyl epidermal cells (a) and bean pod endocarp with phaseollin (30 pg/ml) in 2% ethanol.
Toxic effects of phaseollin
225
on plant cells
Lass of phaseollin from suspensioncultures of bean cells Table 4 shows the amount of phaseollin in cells and medium, 1 day and 3 weeks after flasks of medium containing 26.5 pg phaseollin per ml had been inoculated TABLE
Loss of ad&d phaseollin
from
suspension
cultures
4
of bean cells during prolonged
incubation
Weight of phaseollin kg per flask) after incubation for : (days) 1
21
Medium Cells
414+ 58 490+45
44k24 138+55
Total
904
182
62 73
9 77
o/o recovery y0 recovery
from
similar
but uninoculated
flasks
Phaseollin dissolved in DMSO was added to duplicate flasks containing 50 ml of Medium I to give a total of 1458 pg phaseollin per flask (26.5 pg/ml) and a final DMSO concentration of 0.5% (v/v). The flasks were inoculated with 5.0 ml of a 14day-old suspension of bean cells and incubated at 25 “Cl.
with a suspension of bean cells. One day after inoculation phaseollin was distributed between the cells and medium. Three weeks later the pattern of distribution had changed little. However, during this time, the total amount of phaseollin extracted from the flasks had fallen to only 15% of that recovered after incubation for 1 day. DISCUSSION
The results presented show that plant cells, like those of fungi [13, 141, are rapidly affected by concentrations of phaseollin above about 20 pg/ml. Respiration of bean cells was greatly inhibited after contact with phaseollin (30 pg/ml) for only 2 min, and about 80% were dead within 60 min. VanEtten & Bateman [17] did not detect any effect of phaseollin (47 pg/ml) on the endogenous or exogenous respiration of 1 to 2 mm thick slices of bean hypocotyl tissue. However, their finding that a similar concentration caused a rapid increase in the leakage of pre-absorbed rubidium ions from this tissue would be consistent with the type ofeffect reported here. Little is known about the phytotoxicity of other phytoalexins with the exception of pisatin. This compound inhibited the growth of wheat roots [6J and reduced the growth of pea suspension cultures [I]. Recent observations on its effect on plant cells have shown that high levels of pisatin (300 pg/ml) caused shrinkage of protoplasts from the walls of pea epidermal cells and burst isolated pea protoplasts [12]. Comparing these levels with those found effective in the present study, it would seem that pisatin is less toxic to plant cells than phaseollin. Similar conclusions were reached on the relative fungicidal activities of these two compounds [la]. Further similarities between the effects of phaseollin on plant and fungal tissues become apparent when results of experiments on the growth of bean suspension
226
R. A. Skipp, C. Selby and J. A. Bailey
cultures in the presence of phaseollin are compared with those obtained in a similar study using mycelium of Colletotrichum lindemuthianum in liquid culture [ 131. Measurements of the amounts of phaseollin present in these fungal cultures have shown that the distribution of phaseollin changes during incubation, phaseollin being removed from the medium and taken up by the mycelium. Eventually, the phaseollin was metabolized by the fungus and as the amount of phaseollin in the system decreased, growth was resumed. Similarly, cells of bean suspension cultures took up more than half the added phaseollin within 24 h. After 3 weeks, by which time little growth had occurred, the amount of phaseollin in both the supernatant and cells was very low. Continued incubation produced substantial growth. Present concepts about the formation of phytoalexins and the regulation of their levels in plant tissues may require re-evaluation in view of the findings presented above. In the Phytoalexin theory of Muller & Bijrger [9], the biogenesis of these compounds is conceived as a process resulting from the death of host cells. Much evidence has been presented to support this proposition, particularly from studies on the formation of phaseollin in dwarf bean. These have shown that agents which cause cell death initiate the production of phaseollin [Z, 4, 5, 15, 161, there being none detectable in the tissues before cell death is observed. Thereafter, the concentration of phaseollin increased to a very high level which was maintained for a long period and was localized within the dead tissue [4]. However, it has also been suggested that living cells can produce phaseollin [II, see also 81. Bean pod endocarp tissue was not killed by the fungal polypeptide, Monilicolin A, yet small amounts of phaseollin diffused into drops of liquid from the treated tissue [II]. During our work (unpublished data), untreated bean suspension cultures were also found to contain low levels of phaseollin, but as a small proportion of cells were dead, it was not possible to determine its origin. The two findings might be reconciled if phaseollin is synthesized in living cells but is also metabolized by them, so that small amounts only are ever present, and it only accumulates to high levels when cell death occurs. However, in this work phaseollin has been shown to kill plant cells and so it is also possible that cellular necrosis occurs as a consequence, not a prerequisite, of the accumulation of phaseollin. Thus, resistance and necrosis might be the result of a shift from a low basal level of phaseollin to one in which higher and ultimately phytotoxic levels are reached. Any proper interpretation of the relationship between phaseollin synthesis and cellular necrosis must await the development of more sensitive techniques so that detailed time-course studies of the occurrence of these processes can be examined. We thank Professor R. L. Wain for his continued support. REFERENCES 1. BAILEY, J. A. (1970). Pisatin production by tissue cultures of P&-urn sativum L. 3oumal of General Microbiolopy 61, 409-415. 2. BAILEY, J. A. (1973). Phaseollin accumulation in Pha.seolus vulgaris following infection by fungi, bacteria and a virus. In: The Third Long Ashton Plants’ Response, Ed. by R. J. W. Byrde & C. V. London and New York.
Symposium, Cutting, pp.
Fungal Pathogenicity 337-353. Academic
and the Press,
Toxic effects of phaseollin
on plant
ceils
227
3. BAILEY, J. A. & BURDEN, R. S. (1973). Biochemical changes and phytoalexin accumulation in Phoseolus vulgaris following cellular browning caused by tobacco necrosis virus. Physiological Plant Pathology 3, 171-177. 4. BAILEY, J. A. & DEVERALL, B. J. (1971). Formation and activity of phaseollin in the interaction between bean hypocotyls (Phaseolus vulgaris) and physiological races of Colletottihum lindemuthianum. Physiological Plant Pathology 1, 435d49. 5. BAILEY, J. A. & INGHAM J. L. (1971). Phaseollin accumulation in bean (Phaseolus vulgaris) in response to infection by tobacco necrosis virus and the rust Uromyces appendiculatus. Physiological Plant Patholog 1 451-456. 6. CRUICKSHANK, I. A. M. & PERRIN, D. R. (1961). Studies on phytoalexins. III. The isolation, assay, and general properties of a phytoalexin from Pisum sativum L. Australian Journal of Biological Science 14, 336-348. 7. ESTABROOK, R. W. (1967). Mitochondrial respiratory control and the polarographic measurement of ADP : 0 ratios. In Methods in Enzymology, Ed. by R. W. Estabrook & M. E. Pullman, Vol. X, pp. 41-47. Academic Press, London and New York. 8. MANSFIELD, J. W., HARGREAVES, A. J. & BOYLE, F. C. (1974). Phytoalexin production by live cells in broad bean leaves infected with Botrytis cinerea. Jvature 252, 316-317. 9. MULLER, K. 0. & BURGER, H. (1948). Experimentelle Untersuchungen tiber die PhytobhthoraResistenz der Kartoffel. Arbeiten aus der biologischen Reichsanstalt fiir Land- u. Forstwirtschaft Berlin 23, 189-231. medium for rapid growth and bioassays with 10. MURASHIGE, T. & SKOOG, F. (1962). A revised tobacco tissue cultures. Physiologia plaatarum 15, 473-479. 11. PATTON, J., GOODCHILD, D. J. & CRUICKSHANK, I. A. M. (1974). Phaseollin production by live bean endocarp. Physiological Plant Pathology 4, 167-171. 12. SHIRAISHI, T., OKU, H., ISONO, M. & Ouom, S. (1975). The injurious effect of pisatin on the plasma membrane of pea. Plant and Cell Physiology 16, 939-942. 13. SKIPP, R. A. & BAILEY, J. A. (1976). The effect of phaseollm on the growth of Colletotrichum linakmuthianum in bioassays designed to measure fimgitoxicity. Physiological Plant Pathology 9, 253-263. 14. SIUPP, R. A. & BAILEY, J. A. (1977). The fungitoxicity of some isoflavanoid phytoalexins measured using several different~ types of bioassay. Physiological Plant Pathology 11, (in press). 15. SKIPP, R. A. & DEVERALL, B. J. (1972). Relationships between timgal growth and host changes visible by light microscopy during infection of bean hypocotyls (Phaseolus vulgaris) susceptible and resistant to physiological races of Colletotrichum lindemuthianum. Physiological Plant Pathology 2, 357-374. 16. SKIPP, R. A. & DEVERALL, B. .I. (1973). Studies on cross-protection in the anthracnose disease of bean. Physiological Plant Fa&logy 5, 299-313. 17. VANETTEN, H. D. & BATEMAN, D. F. (1971). Studies on the mode of action of the phytoalexin phaseollin. Phytopathology 61, 1363-1372.
Toxic effects of phaseollin on plant cells R.
A.
SKIPP,~
C.
SELBY
and J. A.
BAILEY
Agricultural ResearchCouncil Plant Growth Substanceand SystemicFungi&& Unit, Wye College ( University of London), Ashfoord,Kent TN25 5AH, U.K. (Acceptedfor publication Januav
1977)
Plant cells, from bean and tobacco suspension cultures and from pods and hypocotyls of bean, were rapidly killed following immersion in solutions containmg 30 pg phaseollin per ml. These concentrations inhibited the respiration of bean cells within 2 min of treatment and also reduced the growth of bean suspension cultures during a 3 week incubation period. Thereafter, as a result of a few cells surviving, the rate of growth of the suspension cultures increased and significant amounts of dry matter were produced. The total amount of phaseollin present in the suspension cultures and its concentration in the growth medium decreased during the incubation period. These findings are discussed in relation to the similarities between the effects of phaseollm on plant and on fungal cells, and to their relevance to current concepts about the formation and r6le of phaseollin in infected plant tissues.
INTRODUCTION
Recent studies on the biological activity of phaseollin, an isoflavanoid phytoalexin produced by dwarf bean (Phaseolus vulgaris), have shown that this compound causes drastic changes in a range of fungal species. Spores of the bean pathogen, Colletotrichum lindemuthianum, were killed within 2 min of exposure to a solution containing 10 pg phaseollin per ml [13] and similar concentrations exerted fungicidal effects on the germ tubes of ten different fungi [lsl]. Phaseollin has also been shown to cause lysis of mammalian erythrocytes [17]. Experiments with higher plants have been mainly directed to an examination of effects on permeability and respiration [17], rather than on the viability of treated cells. In view of the possible significance of such an effect, a more extensive study was undertaken using cells of suspension cultures, epidermis and endocarp of P. vulgaris and those of suspension cultures of Mcotiana tabacum. MATERIALS
AND
METHODS
Suspension cultures were derived from callus induced from sterile bean seedling hypocotyl segments (Phaseolus vulgaris cv. Kievitsboon Koekoek) and were maintained on the medium of Murashige & Skoog [.ZO] containing O-5 mg/l, 2,4-d& chlorophenoxy acetic acid (2,4-D) and 0.1 mg/l kinetin (Medium I). The suspension cultures were shaken at 80 rev/min at 25 “C and subcultured every 14 days. Bag,
t Present address: Plant Diseases Division Sub-Station, Palmerston North, New Zealand. (Reprint requests
c/o Grasslands to J. A. B.)
Division,
D.S.I.R.,
Private
222
R. A. Skipp,
C. Selby
and
J. A. Bailey
Cell suspension cultures from tobacco pith (Nicotiuna tabacum cv. Winsconsin 38) were maintained under the same conditions, except that 1 mg/l !&4n was used (Medium II). The suspension cultures consisted of clumps of cells, 1 to 5 mm in diameter, and large numbers of individual cells. Flasks were allowed to stand for a few minutes to permit the aggregated cells to settle, and the “supernatant”, which contained mostly single cells, was removed with a pipette for testing. The epidermal cells used were present in thin strips of tissue cut from hypocotyls of beans (cv. Prince), which had been grown at 20 “C for 10 days in an illuminated growth room. Bean endocarp cells were present in strips cut similarly from the internal linings of pods (cv. Comtesse de Chambord) obtained locally. Crystalline phaseollin, obtained from virus-infected hypocotyls of bean [3], was dissolved in either ethanol or dimethyl sulphoxide (DMSO) and added to aqueous test solutions to give a range of concentrations. Where necessary, extra solvent was added to make their final concentrations 2.0 and 0.5% (v/v) respectively. In experiments testing the effects of phaseollin on the viability of cells from suspension cultures, ethanolic solutions were added to McCartney bottles containing 10 ml of “supernatant”. The tubes were placed on a roller mixer (Luckham Ltd) and samples of about O-5 ml were removed for microscopic examination at intervals during incubation at 25 “C. One hundred cells were examined in each of three drops of treated “supernatant” and the number alive and dead were recorded. Living cells had obviously intact protoplasts and usually possessed cytoplasmic strands in which streaming was evident. Dead cells showed granulation of the cytoplasm and often shrinkage of the protoplast and nucleus. “Supernatant” was also added to an equal volume of distilled water containing 10 pg Rhodamine G per ml. After about 5 min treatment, dead cells appeared pink and living cells colourless. Tissue containing epidermal or endocarp cells was submerged in 1 ml of a solution of phaseollin and 2.0% ethanol in distilled water contained within a small watch glass. The respiration rates of bean suspension cultures were measured with an oxygen electrode (Rank Brothers, Cambridge), The electrode was calibrated assuming that air-saturated water contains 0.26 ~01 oxygen per ml at 25 “C [7] and zero oxygen concentration was obtained by the addition of a few sodium dithionite crystals, Five ml of a 7-day-old suspension culture were added to the electrode chamber and the lid was inserted, care being taken to prevent air bubbles being trapped. After a steady rate of oxygen uptake had been obtained, 150 pg phaseollin in O-1 ml of ethanol were injected with a syringe through the narrow bore in the chamber lid. The rate of 0, uptake of these cells and those treated with ethanol only were measured at regular intervals. The effect of phaseollin on the growth of suspension cultures of bean was measured using four replicate flasks containing 50 ml sterilized Medium I, to which phaseollin, dissolved in DMSO, was added. The final concentration of DMSO was 0.04 or 0.5%. The flasks were inoculated with 5 ml of a 14-day-old culture and 14 or 21 days later the resulting cells were obtained by filtration, dried at 70 “C for 48 h and weighed. Phaseollin was extracted from these cells by boiling them in ethanol and from the supernatant by partitioning with diethyl ether. The
Toxic effects of phaseollin
on plant cells
223
weight of phaseollin in these extracts was measured by ultraviolet following purification by thin-layer chromatography [3]. RESULTS E$eEt of phaseollin
on the viability
spectrophotometry
of plant cells
Cells treated with 30 pg phaseollin per ml for 1 h are illustrated in Plates 1 and 2. Cells of bean and tobacco suspension cultures [Plate 1 (a) and (c)] and cells of bean endocarp and epidermis (Plate 2) were dead and appeared granular. Cells exposed to 2% ethanol remained alive, the intact cytoplasm often showing streaming [Plate l(b) and (d)]. Further studies were made to determine the time course of the effect of phaseollin on bean suspension cells. Table 1 shows the percentage of living cells in a suspenTAISLE 1 on the viability of cells from
Effect of phmeollin Concentration of phaseollin k&4 0 10 20 30 50
Stainedb 88 88 83 70 5
% cells found 10 Unstained” 85 84 81 69 1
alive’
after
Stainedb a3 86 84 24 0
bean suspension
exposure to phaseollin 50 UnstainedG
cultures for:
(min)
Stainedb
76 78 78 18 0
a See text for criteria of viability. Results are the mean of three replicates b Sample mixed with equal volume of an aqueous solution of Rhodamine and examined after 5 min. c Cells examined immediately after removal of sample without Rhodamine
100 UnstainedC
85 88 78 14 0
76 83 73 18 0
of 100 cells. G (10 pg/ml) G treatment.
sion containing 1.7 x lo5 cells/ml after exposure to phaseollin at concentrations ranging from 0 to 50 pg/ml for 10, 50 and 100 min. Samples treated with Rhodamine G before microscopic examination gave results similar to those obtained with unstained cells. During incubation, the proportion of living cells in the controls (2% ethanol) remained constant (76 to 88%), whereas, in a solution containing 50 pg phaseollin per ml, less than 5% of cells were found alive after 10 min and all were killed after 50 min. Cells were also killed at a concentration of 30 pg/ml, less than 20% of cells being alive after treatment for 100 min. Levels below 30 pgg/ml had little effect on cell viability. In another experiment, using an undetermined but apparently less dense cell suspension, all cells were killed within 30 mm of being placed in a solution containing 30 pg phaseollin per ml. Effect of phmeollin
on the respiration
of bean cells in suspension culture
As seen in Table 2, the addition of phaseollin to give a concentration of 30 pg/ml in an actively respiring suspension of bean cells caused a rapid fall in the rate of oxygen uptake. A similar but slower reduction occurred when cells were treated with 2% ethanol.
224
R. A. Skipp, TABLE
Effect
of phaseollin
Oxygen trode.
0
Control (2% ethanol) Phaseollin (30 pg/ml)
9.7kO.6 9-92 1.1
uptake by a 7-day-old suspension The values are the means of three
of bean
susjension
J. A. Bailey
cells
of oxygen uptake (nmol/ml/min) treatment for: (mm) 2 5
Treatment
and
2
on the respiration Rate
C. Selby
7*7+_oci 4.2 + 0.6
6.220.4 3.350.7
culture was measured measurements.
after 10 4.2cO.6 1*9&O-2
using
an oxygen
elec-
Effect of phaseollin on the growth of bean cells in suspensionculture Table 3 shows the weight of cells harvested 2 and 3 weeks after flasks of growth medium containing phaseollin at concentrations ranging from 0 to 30 pg/ml (dissolved in O-04 and 0.5% DMSO respectively) had been inoculated with a suspension of bean cells. In all cases, there was a net increase in weight during incubation
Effect Phaseollm concentration b%/~) 0 2 5 10 15 30 Weight of inoculum a Mean b Yields 0 Yields
of phaseollin
TABLE 3 on the growth of bean suspension Dry
weight
Experiment
cultures
of harvested cells (md” lb Experiment 26
420 + 30 357 * 37 355 + 124 391+36 248 & 149 79+38
460+98 5295 78 353*83 292 + 20 108+24
19+3-5
43+2*1
weight and standard deviation of cehs from four replicate flasks. measured after 2 weeks. Concentration of DMSO was 0.04%. measured after 3 weeks. Concentration of DMSO was 0.5%.
but compared with that which occurred in the absence of phaseollin, this was reduced by approximately 40% by 15 pg phaseollin per ml and by approximately 85% by 30 pg/ml. At the time of sampling, microscopical examination showed that, although many cell aggregates appeared alive, most individual cells ( > 80%) in cultures treated with 30 pg phaseollin per ml were dead. The usual high proportion of living cells was found in the controls. In subsequent experiments, incubation was extended to 7 weeks to determine whether cells which survived exposure to 30 pg phaseollin per ml eventually produced significant growth. The weight of cells produced in a typical experiment was 375 + 9 mg, thus approaching the weights achieved during 3 weeks’ incubation of control flasks (Table 3).
PLATE 1. Light micrographs of cells from suspension cultures of bean and tobacco showing the effects of treatment with phaseollin (30 pg/ml) in 2% e th anol for 1 h. (a) Bean cells treated with phaseollin. (b) Bean cells treated only with 2% ethanol. (c) Tobacco cells treated with phaseollin. (d) Tobacco cells treated only with 2% ethanol. Note granulation in cells treated with phaseollin. (Magnifications x 750.)
[facing
page 224
cells
PLATE 2. Light micrographs of bean (b) 1 h after they had been treated
hypocotyl epidermal cells (a) and bean pod endocarp with phaseollin (30 pg/ml) in 2% ethanol.
Toxic effects of phaseollin
225
on plant cells
Lass of phaseollin from suspensioncultures of bean cells Table 4 shows the amount of phaseollin in cells and medium, 1 day and 3 weeks after flasks of medium containing 26.5 pg phaseollin per ml had been inoculated TABLE
Loss of ad&d phaseollin
from
suspension
cultures
4
of bean cells during prolonged
incubation
Weight of phaseollin kg per flask) after incubation for : (days) 1
21
Medium Cells
414+ 58 490+45
44k24 138+55
Total
904
182
62 73
9 77
o/o recovery y0 recovery
from
similar
but uninoculated
flasks
Phaseollin dissolved in DMSO was added to duplicate flasks containing 50 ml of Medium I to give a total of 1458 pg phaseollin per flask (26.5 pg/ml) and a final DMSO concentration of 0.5% (v/v). The flasks were inoculated with 5.0 ml of a 14day-old suspension of bean cells and incubated at 25 “Cl.
with a suspension of bean cells. One day after inoculation phaseollin was distributed between the cells and medium. Three weeks later the pattern of distribution had changed little. However, during this time, the total amount of phaseollin extracted from the flasks had fallen to only 15% of that recovered after incubation for 1 day. DISCUSSION
The results presented show that plant cells, like those of fungi [13, 141, are rapidly affected by concentrations of phaseollin above about 20 pg/ml. Respiration of bean cells was greatly inhibited after contact with phaseollin (30 pg/ml) for only 2 min, and about 80% were dead within 60 min. VanEtten & Bateman [17] did not detect any effect of phaseollin (47 pg/ml) on the endogenous or exogenous respiration of 1 to 2 mm thick slices of bean hypocotyl tissue. However, their finding that a similar concentration caused a rapid increase in the leakage of pre-absorbed rubidium ions from this tissue would be consistent with the type ofeffect reported here. Little is known about the phytotoxicity of other phytoalexins with the exception of pisatin. This compound inhibited the growth of wheat roots [6J and reduced the growth of pea suspension cultures [I]. Recent observations on its effect on plant cells have shown that high levels of pisatin (300 pg/ml) caused shrinkage of protoplasts from the walls of pea epidermal cells and burst isolated pea protoplasts [12]. Comparing these levels with those found effective in the present study, it would seem that pisatin is less toxic to plant cells than phaseollin. Similar conclusions were reached on the relative fungicidal activities of these two compounds [la]. Further similarities between the effects of phaseollin on plant and fungal tissues become apparent when results of experiments on the growth of bean suspension
226
R. A. Skipp, C. Selby and J. A. Bailey
cultures in the presence of phaseollin are compared with those obtained in a similar study using mycelium of Colletotrichum lindemuthianum in liquid culture [ 131. Measurements of the amounts of phaseollin present in these fungal cultures have shown that the distribution of phaseollin changes during incubation, phaseollin being removed from the medium and taken up by the mycelium. Eventually, the phaseollin was metabolized by the fungus and as the amount of phaseollin in the system decreased, growth was resumed. Similarly, cells of bean suspension cultures took up more than half the added phaseollin within 24 h. After 3 weeks, by which time little growth had occurred, the amount of phaseollin in both the supernatant and cells was very low. Continued incubation produced substantial growth. Present concepts about the formation of phytoalexins and the regulation of their levels in plant tissues may require re-evaluation in view of the findings presented above. In the Phytoalexin theory of Muller & Bijrger [9], the biogenesis of these compounds is conceived as a process resulting from the death of host cells. Much evidence has been presented to support this proposition, particularly from studies on the formation of phaseollin in dwarf bean. These have shown that agents which cause cell death initiate the production of phaseollin [Z, 4, 5, 15, 161, there being none detectable in the tissues before cell death is observed. Thereafter, the concentration of phaseollin increased to a very high level which was maintained for a long period and was localized within the dead tissue [4]. However, it has also been suggested that living cells can produce phaseollin [II, see also 81. Bean pod endocarp tissue was not killed by the fungal polypeptide, Monilicolin A, yet small amounts of phaseollin diffused into drops of liquid from the treated tissue [II]. During our work (unpublished data), untreated bean suspension cultures were also found to contain low levels of phaseollin, but as a small proportion of cells were dead, it was not possible to determine its origin. The two findings might be reconciled if phaseollin is synthesized in living cells but is also metabolized by them, so that small amounts only are ever present, and it only accumulates to high levels when cell death occurs. However, in this work phaseollin has been shown to kill plant cells and so it is also possible that cellular necrosis occurs as a consequence, not a prerequisite, of the accumulation of phaseollin. Thus, resistance and necrosis might be the result of a shift from a low basal level of phaseollin to one in which higher and ultimately phytotoxic levels are reached. Any proper interpretation of the relationship between phaseollin synthesis and cellular necrosis must await the development of more sensitive techniques so that detailed time-course studies of the occurrence of these processes can be examined. We thank Professor R. L. Wain for his continued support. REFERENCES 1. BAILEY, J. A. (1970). Pisatin production by tissue cultures of P&-urn sativum L. 3oumal of General Microbiolopy 61, 409-415. 2. BAILEY, J. A. (1973). Phaseollin accumulation in Pha.seolus vulgaris following infection by fungi, bacteria and a virus. In: The Third Long Ashton Plants’ Response, Ed. by R. J. W. Byrde & C. V. London and New York.
Symposium, Cutting, pp.
Fungal Pathogenicity 337-353. Academic
and the Press,
Toxic effects of phaseollin
on plant
ceils
227
3. BAILEY, J. A. & BURDEN, R. S. (1973). Biochemical changes and phytoalexin accumulation in Phoseolus vulgaris following cellular browning caused by tobacco necrosis virus. Physiological Plant Pathology 3, 171-177. 4. BAILEY, J. A. & DEVERALL, B. J. (1971). Formation and activity of phaseollin in the interaction between bean hypocotyls (Phaseolus vulgaris) and physiological races of Colletottihum lindemuthianum. Physiological Plant Pathology 1, 435d49. 5. BAILEY, J. A. & INGHAM J. L. (1971). Phaseollin accumulation in bean (Phaseolus vulgaris) in response to infection by tobacco necrosis virus and the rust Uromyces appendiculatus. Physiological Plant Patholog 1 451-456. 6. CRUICKSHANK, I. A. M. & PERRIN, D. R. (1961). Studies on phytoalexins. III. The isolation, assay, and general properties of a phytoalexin from Pisum sativum L. Australian Journal of Biological Science 14, 336-348. 7. ESTABROOK, R. W. (1967). Mitochondrial respiratory control and the polarographic measurement of ADP : 0 ratios. In Methods in Enzymology, Ed. by R. W. Estabrook & M. E. Pullman, Vol. X, pp. 41-47. Academic Press, London and New York. 8. MANSFIELD, J. W., HARGREAVES, A. J. & BOYLE, F. C. (1974). Phytoalexin production by live cells in broad bean leaves infected with Botrytis cinerea. Jvature 252, 316-317. 9. MULLER, K. 0. & BURGER, H. (1948). Experimentelle Untersuchungen tiber die PhytobhthoraResistenz der Kartoffel. Arbeiten aus der biologischen Reichsanstalt fiir Land- u. Forstwirtschaft Berlin 23, 189-231. medium for rapid growth and bioassays with 10. MURASHIGE, T. & SKOOG, F. (1962). A revised tobacco tissue cultures. Physiologia plaatarum 15, 473-479. 11. PATTON, J., GOODCHILD, D. J. & CRUICKSHANK, I. A. M. (1974). Phaseollin production by live bean endocarp. Physiological Plant Pathology 4, 167-171. 12. SHIRAISHI, T., OKU, H., ISONO, M. & Ouom, S. (1975). The injurious effect of pisatin on the plasma membrane of pea. Plant and Cell Physiology 16, 939-942. 13. SKIPP, R. A. & BAILEY, J. A. (1976). The effect of phaseollm on the growth of Colletotrichum linakmuthianum in bioassays designed to measure fimgitoxicity. Physiological Plant Pathology 9, 253-263. 14. SIUPP, R. A. & BAILEY, J. A. (1977). The fungitoxicity of some isoflavanoid phytoalexins measured using several different~ types of bioassay. Physiological Plant Pathology 11, (in press). 15. SKIPP, R. A. & DEVERALL, B. J. (1972). Relationships between timgal growth and host changes visible by light microscopy during infection of bean hypocotyls (Phaseolus vulgaris) susceptible and resistant to physiological races of Colletotrichum lindemuthianum. Physiological Plant Pathology 2, 357-374. 16. SKIPP, R. A. & DEVERALL, B. .I. (1973). Studies on cross-protection in the anthracnose disease of bean. Physiological Plant Fa&logy 5, 299-313. 17. VANETTEN, H. D. & BATEMAN, D. F. (1971). Studies on the mode of action of the phytoalexin phaseollin. Phytopathology 61, 1363-1372.