296 1
Jouml of General Microbiology (1 984), 130, 2961-296% Printed in Grvur Britain
Induction by Ethylene of Macrocyst Formation in the Cellular Slime Mould
Dictyosteium mucoroides
By A I K O A M A G A I
Department of Botany, Faculty of Science, Kyoto Unicersity. Kyoto 606, Japan
(Received 30 April 1984)
A hitherto unidentified volatile substance(s) is known to induce macrocyst formation in a strain
(Dm 7 ) of Diciyosteliurn rnucoroides and in a mutant (MF 1) derived from it. The properties of
this substance suggested that it might be ethylene, and here it is shown that this is indeed the
case. The addition of ethylene to MF I cells, in conditions otherwise favouring sorocarp formation, induced the formation of macrocysts. Conversely, the addition of mercury perchlorate, an
absorbent of ethylene, inhibited macrocyst formation and induced sorocarp formation under
conditions otherwise favouring macrocyst formation. Two inhibitors of ethylene synthesis,
aminooxyacetic acid and aminoethoxyvinyl glycine, also inhibited macrocyst formation.
Production and release of ethylene by D. mucoroides cells was confirmed by gas
chromatography.
INTRODUCTION
After ceasing to grow, slime mould cells usually construct a sorocarp consisting of an apical
mass of spores and a supporting cellular stalk. Under certain environmental conditions,
however, some strains of Dictyosteliurn rnucoroides form a macrocyst as a final developmental
structure via a morphogenetic pathway dissimilar to sorocarp formation (Blaskovics & Raper,
1957). The macrocyst of the cellular slime mould has been considered to represent a true sexual
phase with zygote formation and transient diploidy (Machnes & Francis, 1974). The initial sign
of macrocyst formation by Dm 7 cells is the formation of large aggregates that are subdivided
into smaller masses (precyst),each of which is surrounded by a fibrillar sheath. At the centre of
the precyst there arises a cytophagiccell (giant cell) which proceeds to engulf all the other cells in
the precyst, The enlarged cytophagic cell finally becomes surrounded by a thick wall to form the
mature macrocyst (Filosa & Dengler, 1972).
Development toward sorocarp or macrocyst formation can be controlled by environmental
conditions. Dm 7 cells form sorocarps in the light and macrocysts in the dark (Weinkauf &
Filosa, 1965). However, in the presence of charcoal the cells form sorocarpseven in the dark, suggesting that volatile substanct(s), absorbed by charcoal, are necessary for macrocyst formation
(Weinkauf & Filosa, 1965). The volatile substance must also be lipid soluble, because the cells
form macrocysts in water (Filosa & Dengler, 1972), but sorocarps in mineral oil (M. F.Filosa,
personal communication). Furthermore, the removal of C02,an antagonist of ethylene (Abeles,
1973), seems to enhance the effect of the volatile substance (Filosa, 1979). From these
observations, ethylene was considered as a candidate for the volatile substance, and several
kinds of experiment were done to determine whether or not ethylene is a naturally occurring inducer of macrocyst formation.
A mutant, MF 1, isolated by M.F. Filosa from Drn 7, forms macrocysts in the light as well as
in the dark, and also produces the volatile substance absorbed by charcoal (Filosa, 1979). Since
~~
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Abbreviutwm: AOA, aminooxyacetic acid; AVG. aminoeihoxyvinyl glycine.
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A. AMAGAI
the developmental pathway of MF 1 can be controlled by changing the amount of the volatile
substance, even in the light (Amagai & Filosa, 1984), the mutant cells were used mainly in the
present work.
METHODS
Organisms and nrlrivafion. A spontaneouslyoccumng mutant, MF 1* isolated from the wild-type Dicryosrelium
mucoroidcJ-7 (Dm 7) was UItdin addition to Dm 7. Eschcrichia cdi B/r was used aa a source of f d . Spomof MF 1
were obtained from sorocarps formed over activated charcoal (Filosa, 1979). A 0.15 ml sample of a dense suspension of Dm 7 or MF I &porntugether with E. cdi was spread with a glasa rod over the surface of nutrient agar
(Bonner, 1967). The agar plates were incubated at 22 "C under a General Electric cool white fluorescent lamp.
After 1 d, vegetative amoebae were collected and shaken in 20 mu-phaphatc buffer, pH 6.2, with E. coli for 21 h
(Gerisch, 1960). Amoebae were harvested during the growth phase and washed three times in Bonner's saline
(Bonner, 1947). A sample of cell suspension was put on 2 ml of 2% plain agar in a glw Petri dish (3 crn diameter).
After the amoebae had settled onto the ruhtratum for 30 min, excess water was removed and the surface allowed
to dry. The plates were then incubated for 48 h at 22 "C in the light. The find developmental f o m were observed
under a dissecting m i c m p e .
€ t h y b e uppJkarh. The 3 cm diameter plates on which MF 1 cells had been deposited were transferred into
glass dishes of 9 cm diamter and covered with the same size of glass dishes (air space; 250 cm3). A 3 ml volume of
1 %ethylenegas was injected into the air space through a narrow space between the two facing dishes, using a 5 ml
@arr ryringe, and after the dishes were sealed with vinyl tape and P a r a h they were dipped in water to minimize
gas leakage, and incubated at 22 "C.
Dctrcriorr of e f h y h c by gar chmmatogruphy. A 1 ml ruspcnsion of washed cells in Bonner's saline in a 20 ml
Erlenmeyer flask, tightly scaled with a silicone stopper, was shaken gently in the dark for 4% h at 100 r.p.m.
Ethylene released from the cells was determined by a slight modification of Imaseki's method (Imascki et af.,
1968). An a i r / w mixture (2 ml) was assayed by gas chromatography (Shimazu, GC7A type) on an activated
alumina (-80 mesh, Nishio Product) column (3.1 m x 3.2 mm) operated at 75 "C with helium (50 ml rnin-') as
the carrier gar. Quantification of the ethylene peak was carried out with a printing integrator attached to the
apparatus. All ethylene concentrations were adjusted for ethylene contamination in the air.
E f l h uf hMifors.Agar (I ml) containing 20 mu-MES bdier @H 7.0) on which the MF 1 cells had been d t
pariced at 2 x 106 cells cm-2 was transferred onto I ml of agar containing various concentrations of m i n e
oxyrroetic acid (AOA; Sigma)or aminoethoxyvinylglycine (AVG) in 20 mu-MES buffer @H7.0). This was followed by incubation at 22°C. AVG was kindly supplied by Dr A. Stempel, Research Division, Hoffman
LaRoche, Nulley, NJ, USA.
C02applicutlorr. HarvestedMF 1 cells were plated on 2 ml of plain agar in the lid of a glass dirh (4 cm diameter)
at 1 x 106 cellscm-z. A similar lid with a small notch in it was placed over it and the two sealed with vinyl tape (air
space M ans).Pure CO2 (3 ml) was injected into the air space through the notch,using a 10 ml plastic syringe.The
hole cawed by the needle waa d d quickly with vinyl tape. As controls, plates were incubated without added
c02*
RESULTS
To examine whether ethylene is involved in macrocyst formation, the effects of ethylene removal and application were first determined. An agar plate with MF 1 cells deposited at 2 x 106
cells cm-* was staled over a glass plate containing 1 ml mercury perchlorate (0-25 M - H ~ O
in
2.0 M-HCIO,)as an absorbent for olefines such as ethylene (Young et OJ., 1952). Under these conditions, the cells formed sorocarps instead of macrocysts. Controls had perchlorate alone and
normally thcsc cells formed macrocysts, though they sometimes stopped their development at
the aggregation stage. This incomplete development may have been due to the absorption by the
perchlorate of basic gases necessary for macrocyst formation,
Table 1 shows that exogenouslyadded ethylene (100 p.p.m.)induces macrocyst formation. Although MF l celis which developed in a large glass dish (air space 250 cm3)formed sorocarps due
to the reduced concentration of the volatile substance (Amagai & Filosa, 1984), the addition of
ethylcnc clearly induced macrocysts provided the cell density was high enough. At low cell density, however, cells usually stopped developing at thc aggregation stage, perhaps due to subthreshhold amounts of another substance(s) that might be required for complete induction of
macrocysts. While a relatively high concentration of ethylene was applied in these experiments,
a lower concentration of ethylene (1 p.p,m.) was also effective.
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2963
Ethylene and slime mould development
Table 1. Eflects of ethyiene on MF I cells
The 3 cm diameter glass dishes on which MF I cells had been deposited weft transferrod into 9 cm diameter &us dishes whicb were faced together with the same size dasr dishes (air space 250 an3).A
3 ml vdwne of 1% ethylene gas was injected into the air space, and the dabs dishes sealed with vinyl
tape, wrapped with Parafilm,dipped in water and incubated for 48 hat 22 "C in the light. % means the
proportions of the experimental cases which showed the developmental forms indicated in the Table.
Cell density
(cells c m - 2 )
No. of
experiments
2 x 10'
Ethylene
Macrocysts
(100 p.p.m.)
3
33
(%I
66
4
25
0
86
0
0
75
0
7
2x104
Aggregates
(%)
0
14
29
Sorocarps
(%I
0
100
0
100
0
71
Table 2. Reieme uf ethyiewfiom Dm 7 u d MF I cells
A 1 ml suspensionof Dm 7 or MF 1 cells was put into a 20 ml Erlenmeyer flak. The flasks were sealed
tightly with a silicone stopper and ihaken gently in the dark for 48 b at 100 r.p.m.; 2 ml of the sir/#as
mixture waa then taken from a flask and the ethylene rekased from the cells was arrayed by 8chromatography.
Amount of ethylene released @.p.m.)
Cell density
(cells rn1-l)
t
A
Dm7
I
1.7 x lo-'
3.5 x 10-2
2.4 x lom2
3.3 x 10-2
I x
2 x I@
MF 1
Table 3. Eflects of inhibitors of ethylene synthesis on macrocyst formution
Agar (I ml)containing 20 m - M E S buffer (pH 7-0) on which the MF 1 cells had been d e p i t c d at
2 x 106 cells cm-* was transferred onto 1 ml of agar containing various concentration6of AOA or AVG
in 20 mu-MES bufTer @H 74). This was followed by incubation for 48 h at 22 "C in the light.
Final
inhibitor
mncn (M)
0
1 x 10-4
5 x 10-4
1 x 10-3
I x 10-2
Final developmental forms
r
A
AOA
Macrocysts
Macrocysts
Somcarps
No aggregation
luD
ND,
AVG
3
Macmysts
Macrocysts
Macrocysts and aggregates
Macrocysts and a-gatca
Aggregatea
Not determined.
To measure ethylene production by cells, gases released by cells into the air space were analysed by gas chromatography, with special reference to ethylene. In general, it was necessary to
use a large number of cells to produce measurable amounts of ethylene and 80 a suspension culture system with high cell densities was adopted. The results obtained indicate that ethylene was
actually produced and released by both Dm 7 and the MF 1 derivative at almost the same levels
(Table 2).
Since AOA and AVG art known to inhibit the synthesis of l-aminocyclopropane-1carbolic
acid (ACC), an intermediate in the conversion of methionine to ethylene (Boller et al., 1979;
Amrheim & Wenker, 1979), their effects on macrocyst formation were examined. As shown in
Table 3, AOA inhibited macrocyst formation and induced sorocarp formation at 5 x
M.
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2964
A. AMAGAl
Fig. I . Development of MF 1 cells with and without added C02.(u) Macrocysts formed without added
COZ after 48 h incubation; (b) soracarps formed in the presence of 10% CO1after 24 h incubation; (c)
large aggregates formed without added C02after 10 h incubation; (d)normal size aggregates formed in
the presence of 10% C02after 10 h incubation. Photographs were taken under a dissecting microscope.
Magnification. 21 x .
Above
M-AOA, cells never aggregated. Although higher concentrations of AVG had
inhibitory effects on macrocyst formation, sorcxarps never formed under these conditions.
When cells developed under an atmosphere of 10% C02,an antagonist of ethylene (Abeles,
1973), sorocarps, rather than macrocysts, were formed (Fig. 1b). When C 0 2was applied to cells
at the aggregation stage, it induced many tips on the surface of an aggregate within 2 h, resulting
in the formation of many sorocarps. During macrocyst formation, the size of the aggregate initially formed was quite large (Fig. Ic), as compared with that formed in the presence of C 0 2
(Fig. Id).
DISCUSSION
It is generally accepted that ethylene is a potent hormone which regulates many events in
plant growth and development (Abeles, 1973). Ethylene is produced naturally by higher plants
and also by fungi (Ilag & Curtis, 1968) and bacteria (Freebairn & Buddenhagen, 1964), but its
function in fungi and bacteria is not yet known. This is the first demonstration that ethylene
regulates development in the slime mould.
Macrocysts always formed when Drn 7 and MF 1 cells were starved in submerged and shaken
conditions, but in contrast, cells plated on agar were capable of either macrocyst or sorocarp forDownloaded from www.microbiologyresearch.org by
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Erhjhlene and slime mould development
2965
mation, depending upon the atmospheric conditions. Therefore, it would be interesting to know
the relationship between the developmental pathway and the amount of ethylene produced by
the cells plated on agar.
Carbon dioxide promotes sorocarp formation in the appropriate conditions and one of the
earliest signs of this is the formation of protruding tips on the aggregates. It is therefore possible
that there is an antagonistic relationship between tip and macrocyst formation. In this context,
it is noteworthy that CAMP,a chemotactic substance (Konijn, 1976), greatly inhibits macrocyst
formation (Amagai & Filosa, 1934). In addition, as the aggregates formed with C 0 2 were
smaller than those formed without C 0 2and since C 0 2and ethylene appear to be mutual antagonists, it is possible that ethylene may also be involved in regulating the sire of the aggregate. As
pointed out previously (Bonner & Hoffman, 1963). there may be substances which control the
spacing of aggregation territories and the orientation of sorocarps, and the present results hint
that these may be COz and ethylene.
Although MF 1 cells mainly were used in this work, it is reasonable to suppose that ethylene
also promotes macrocyst formation in Dm 7 cells, since COz switched Dm 7 cells incubated in
the dark from macrocyst to sorocarp formation (data not shown).
All of the results presented here strongly suggest that ethylene may function as a macrocystinducing hormone in slime mould development. Taken together with our previous findings
(Amagai & Filosa, 1984), it is of particular importance to know more precisely the relative role
of ethylene and CAMP in controlling the choice of developmental pathways.
I thank Dr M.F. F i l m of The University of Toronto, Canada, Dr Y.Maeda of Tohoku University, Dr H .
lmascki of Nagoya University and Dr I. Takeuchi of Kyoto University for their helpful suuestions, and Drs 1.
Shimitu and M. DeI of Kyoto University for the technical advice and the use of gas chromatography, I also thank
Dr A. Stempel, Research Division, Hoffman LaRoche, Nutley. NJ. USA, for the generous gift of AVG.
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