Journal of General Microbiology (1989), 135, 1559-1566.
Printed in Great Britain
1559
Transmission Pattern of Mitochondria1 DNA during Plasmodium
Formation in Physarum polycephalum
By S H I G E Y U K I K A W A N O * A N D T S U N E Y O S H I K U R O I W A
Department of Biology, Faculty of Science, University of Tokyo, Hongo, Tokyo 113, Japan
(Received 8 November 1988; revised 1 February 1989; accepted 2 March 1989)
The transmission pattern of mitochondrial DNA (mtDNA) was studied during plasmodium
formation in Physarum polycephalum. Plasmodia were generated by matings between pairs of
amoebal strains carrying mtDNA molecules that were distinguishable by restriction
endonuclease digestion. The transmission of mtDNA was uniparental in every case; the
plasmodia always carried mtDNA with the restriction pattern of only one of the two parental
types. In each mating pair, one strain consistently acted as mtDNA donor, but this strain did not
always act as mtDNA donor when combined in other mating pairs. The identity of the mtDNA
donor in each pair was not determined by the different types of mtDNA molecules present or by
different alleles of matB or matC, two mating-type loci which regulate amoebal fusion. The
results suggested that alleles of a third mating-type locus, matA, which controls zygote
development, might form a hierarchy such that the mtDNA donor in any cross would be the
strain of higher status. The deduced hierarchy was matA2 > m a t A I l > matAl2 > matA1.
INTRODUCTION
Much has been learned about the rules and mechanisms of inheritance of mitochondrial and
chloroplast genomes (reviewed by Gillham, 1978; Birky, 1978; Dujon, 1981). In many
organisms, these organelle genomes are transmitted to progeny predominantly or entirely by
only one parent. This uniparental inheritance has usually been attributed in oogamous species to
failure of organelles from the male gamete to enter the egg, or to the presence in the male gamete
of comparatively few organelles. Thus the determination of organelle transmission in such
species is dependent upon sexual differentiation. In isogamous species, however, there is no
significant sexual differentiation and sexual incompatibility, where it exists, is defined in terms
of mating types. It is not clear whether there is any general relationship between mating types
and the uniparental inheritance of organelle genomes in such species.
The acellular slime mould Physarum polycephalum is an isogamous species in which the
mating-type system appears particularly complex, and it is therefore of some interest to
determine the way in which mitochondrial inheritance is regulated in this organism. In sexual
development (crossing), haploid amoebae of P. polycephalum act as isogametes, fusing in pairs to
form diploid zygotes which develop into macroscopic, diploid plasmodia by successive mitotic
cycles in the absence of cell division (reviewed by Dee, 1982, 1987). In this process,
mitochondrial DNA (mtDNA) is transmitted uniparentally ; the plasmodia formed by crossing
carry mtDNA from only one of the parent amoebae (Kawano et al., 1987a). Crossing is under
the control of a mating-type system which comprises three unlinked loci : matA, matB and matC
(Dee, 1966, 1978; Youngman et al., 1979; Kirouac-Brunet et al., 1981; Shinnick et al., 1978;
Kawano et al., 1987b). To cross efficiently, amoebae must carry different alleles of at least matA
and matB; for each of these loci, full compatibility results when any two alleles are combined
from among a set of at least thirteen. Heteroallelism is additionally necessary for matC, which
Abbreviation: mtDNA, mitochondrial DNA.
0001-5243 0 1989 SGM
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S. KAWANO A N D T. KUROIWA
has at least three alleles, when crosses are carried out under conditions of elevated pH or reduced
ionic strength.
In an earlier study (Kawano et al., 1987a), we demonstrated uniparental inheritance of
mtDNA in crosses involving one particular pair of strains, and obtained suggestive evidence
that there might be a consistent bias in favour of the mtDNA from one of these strains. The aim
of the present work was to determine whether consistent bias in mtDNA transmission was a
feature of other crosses in P . polycephalum and, if so, to investigate whether such bias could be
correlated with particular mtDNA genomes or amoebal mating types.
METHODS
Strains. Principal amoebal strains are listed in Table 1. Amoebae were cultured at 29.5 "C with Escherichia coli
on SM-1 plates, as described by Kawano et al. (19876). Stocks of amoebal strains were maintained as desiccated
cysts on silica gel granules (Anderson et al., 1983). Plasmodia were cultured at 26 "C on MEA (malt extract agar;
Kawano et al., 19876) or as microplasmodia in SDM (semidefined liquid medium; Daniel & Baldwin, 1964).
Sporulation was induced on MEA plates, essentially as detailed by Wheals (1970). Germination of spores and
cloning of amoebal progeny were done according to the methods of Anderson (1979) but used SM-1 instead of
liver infusion agar. Amoebal strains were checked by phase-contrast and DAPI-fluorescence microscopy
(Nishibayashi et al., 1987) to ensure that all strains showed similar cell sizes and contents of mitochondria and
mtDNA.
Generationofplasmodia and mating-type analysis. To obtain plasmodia by mating, lo5 amoebae of each of two
strains carrying unlike matA, matB and sometimes matC alleles were mixed at 26 "C in 'concentrated' live E. coli
suspension and plated as described by Youngman et a/. (1981). Following plasmodium formation, small agar
blocks carrying plasmodia were cut from the mating plates and transferred to MEA for further growth. Fully
grown macroplasmodia on MEA were transferred to SDM for culture as microplasmodia (Daniel & Baldwin,
1964). Since it has been suggested that crossing may sometimes result in the formation of unstable plasmodial
heterokaryons containing both fused and unfused nuclei of the two parental amoebal strains (Dee & Anderson,
1984), tests of sample plasmodia from all crosses were carried out to determine whether their progeny, obtained by
sporulation, showed appropriate segregations for alleles of the unlinked matA and matB loci. These analyses were
carried out essentially as described by Youngman et al. (1979). Table 2 shows that the parental :recombinant ratios
for the amoebal progeny of all these plasmodia did not differ significantly from the expected 1 :1 ratio (P> 0.05;
chi-square tests).
Restriction endonuclease analysis of mtDNA. Mitochondria were isolated from microplasmodia or surface
plasmodia of each strain as described by Kawano et al. (1983). Mitochondria1 DNA was prepared from isolated
mitochondria by centrifugation in Hoechst 33258/CsCl density gradients according to Hudspeth et al. (1980).
Restriction endonucleases were obtained from New England Biolabs and the Takara Shuzo Co. Digests were done
in buffers recommended by the manufacturers. Electrophoresis and photography of 1-2 % (w/v) agarose slab gels
were as described by Kawano et al. (1983).
Table 1 . Amoebal strains
Designation
mtDNA
type
Genotype
RA669
RA670
RA555
matAl matB4 matC3
matA2 matBl matC3
matAl matBl matCl
RA689
matA2 matB4 matCl
HI28
matA12 matBS
-
NG9
matAll matB6
-
02 1
022
023
024
matAl
matAll
matA1
matAl2
-
matB4
matB6
matB4
matBS
;
N
Reference
Progeny of a x i; Kawano et al. (19876)
Progeny of a x i; Kawano et al. (19876)
Progeny of a x i; S. Kawano & R. W. Anderson,
unpublished
Progeny of a x i; S. Kawano & R. W. Anderson,
unpublished
Progeny of plasmodial strain Hi; Kawano et al.
(1987a)
Progeny of plasmodial strain Ng; Kawano et al.
(1987~)
Progeny of RA669 x NG9
Progeny of RA670 x NG9
Progeny of RA669 x HI28
Progeny of RA670 x HI28
-, Indicates that this allele is unknown, but is not matCl, 2 or 3 at least.
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Transmission pattern of Physarum mtDNA
k
b
1
2
3
4
5
6
7
8
1
2
3
4
5
6
7
8
Fig. 1. Uniparental transmission pattern of mtDNA during plasmodium formation in two mating
pairs involving three parental amoebal strains: RA670 (lane al), RA669 (lane bl), and NG9 (lanes a2
and b2). Mitochondria1 DNAs were digested with BstNI. Digestion patterns for six plasmodia formed
in matings of NG9 with RA670 are shown in lanes a3-a8 ; digestion patterns for plasmodia formed in
matings of NG9 with RA669 are shown in lanes b3-48.
Table 2. Recombination of matA and matB alleles among progeny obtained by sporulation of
sample crosses
Number of amoebal progeny
Cross
RA670
RA669
RA670
NG9
RA669
RA689
RA555
RA689
RA555
oz1
023
(matA2
(matAl
(matA2
(matA22
(matAI
(matA2
(matAZ
(matA2
(matAZ
(matAI
(matAI
matBI)
matB4)
matBI)
matB6)
matB4)
matB4)
matBI)
matB4)
matBI)
matB4)
matB4)
x
x
x
x
x
x
x
x
x
x
x
NG9
NG9
HI28
HI28
HI28
NG9
NG9
HI28
HI28
022
024
(matAl2
(matAII
(matAI2
(matAI2
(matAI2
(matAII
(matAlZ
(matAI2
(matAI2
(matAII
(matA12
matB6)
matB6)
matB5)
matB5)
matB5)
matB6)
matB6)
matB5)
matB5)
matB6)
matB5)
Parental
Recombinant
21
16
16
17
19
18
20
16
15
11
16
16
15
13
14
12
16
17
12
14
20
18
RESULTS
Uniparental transmission of mtDNA during plasmodium formation in two mating pairs involving
three amoeba1 strains
We showed in our earlier study (Kawano et al., 1987a) that plasmodia carrying only one type
of mtDNA (N-type) were formed when an amoebal strain carrying N-type mtDNA was crossed
with a strain carrying mtDNA of a second type (M-type). To determine whether this bias in
favour of N-type mtDNA would also occur in other crosses, two additional M-type strains
(RA669 and RA670) were crossed with the original N-type strain (NG9). Plasmodia were
obtained from six independent mixtures of each crossing pair and mtDNA from each
plasmodium was isolated and analysed by restriction endonuclease digestion. The two mtDNA
types of the parents were readily distinguished on the basis of differences in their restriction
patterns (lanes 1 and 2 in Fig. 1). Analysis of mtDNA from the crossed plasmodia showed
uniparental inheritance of mtDNA in every case; within each plasmodium, mtDNA of only a
single, parental type was present (lanes 3-8 in Fig. 1). Moreover, the mtDNA type transmitted
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S. KAWANO AND T. KUROIWA
1
2
3
1
2
3
1
2
3
Fig. 2. Uniparenral transmission pattern of mtDNA during plasmodium formation in three mating
pairs involving four amoebal strains: RA670 (lane al), NG9 (lane bl), RA669 (lane cl) and HI28 (lanes
a2, b2 and c2). Mitochondria1 DNAs were digested with HindIII. Digestion patterns for plasmodia
formed in the matings are shown as follows: RA670 x HI28 (lane a3), NG9 x HI28 (lane b3) and
RA669 x HI28 (lane c3).
to crossed plasmodia was reproducible for each pair of amoebal strains. However, the two pairs
of strains gave different results; N-type mtDNA was displaced by M-type mtDNA in
NG9 x RA670, but M-type was displaced by N-type in NG9 x RA669. Thus it appeared that
the ability of a strain to act as mtDNA donor in a particular cross must depend upon some
property other than mtDNA type.
Hierarchy of mtDNA transmission among amoebal strains
The results of the initial crosses, NG9 x RA669 and NG9 x RA670, suggested the possibility
of a hierarchical relationship between amoebal strains, such that the strain of superior status in a
cross would always act as donor of mtDNA to the plasmodia that were formed. For the strains
used in these crosses, Table 3 (a) shows that the hierarchy would be RA670 > NG9 > RA669. To
test this idea fully for the three strains, it was desirable to follow the inheritance of mtDNA in
one further cross, RA669 x RA670, but this was not possible because both strains carried
mtDNA of the same type (M-type). An indirect approach was therefore employed: the
inheritance of mtDNA was followed in crosses of each of the three original strains with a fourth
strain, HI28. This strain carried mtDNA of H-type, which showed a restriction pattern that
could be readily distinguished from the M-type and N-type patterns of the original strains. The
results of these crosses are shown in Fig. 2 and Table 3(b). Like the previous crosses, these
additional crosses all showed uniparental transmission of mtDNA. The H-type mtDNA of HI28
was not transmitted to the plasmodia formed in mixtures with RA670 or NG9 (Fig. 2a, b), but
was transmitted to all plasmodia obtained from the cross RA669 x HI28 (Fig. 2c). This pattern
of transmission was consistent with the hierarchy proposed for the original three strains, placing
HI28 lower in the hierarchy than RA670 or NG9, but higher than RA669 (Table 3b).
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Transmission pattern of Physarum mtDNA
Table 3. Relationship between mtDNA transmission hierarchy and mating types
mtDNA type
of plasmodia
Cross*
(a) RA670 ( M ) x NG9 (N)
RA669 (M) x NG9 (N)
M
N
Hierarchy
RA670 > NG9
RA669 < NG9
RA670 > NG9 > RA669
matA2
matB1
matC3
(b) RA670 (M) x HI28 (H)
NG9 (N) x HI28 (H)
RA669 (M) x HI28 (H)
M
N
H
(M)
(M)
(M)
(M)
x
x
x
x
NG9
NG9
HI28
HI28
(N)
(N)
(H)
(H)
M
N
M
H
matA1
matB4
matC3
-
RA670 > HI28
NG9
> HI28
RA669 < HI28
RA670 > NG9 > HI28
matA2
matBl
matC3
(c) RA689
RA555
RA689
RA555
matAfl
matB6
matAll
matB6
-
RA689
RA555
RA689
RA555
RA689 > NG9
matA2
matB4
matel
matAl2
matB.5
-
>
<
>
<
>
matAll
matB6
-
NG9
NG9
HI28
HI28
HI28
matAl2
matB.5
-
> RA669
matA1
matB4
matC3
> RA555
matAl
matBl
matel
* Letters in parentheses indicate mtDNA type.
Relationship between mtDNA transmission hierarchy and mating type
Numerous changes that occur during crossing of P . polycephalum amoebae are either directly
or indirectly controlled by the mating-type system (see Anderson et al., 1986). It was therefore
plausible that different alleles of one of the three multiallelic mating-type loci might be
responsible for the different positions of amoeba1 strains within the hierarchy of mtDNA
transmission. It is clear from Table 3(a, b) that matC did not determine position in the hierarchy, since the strains of highest and lowest status both carried the same allele, matC3. The
results were consistent with a hierarchy based upon matA in which matA2 > m a t A l l >
matAl2 > matAI, or upon matB in which matBl > matB6 > matB5 > matB4. Further crosses
with two additional strains were therefore carried out to determine whether either hypothetical
hierarchy would correctly predict the transmission patterns of the mtDNAs. These additional
strains, RA555 and RA689, were both derived from the same parent as RA669 and RA670, and
both carried mtDNA of M-type.
RA555 carried the same matB allele as RA670, while RA689 carried the same matB allele as
RA669. If the hierarchy of mtDNA transmission were determined by matB alleles, it would be
expected that RA555 would be the mtDNA donor in crosses with NG9 and HI28, and that NG9
and HI28 would both be mtDNA donors in crosses with RA689. Table 3(c) shows that this
prediction was not fulfilled. Thus mtDNA transmission was not determined by matB alleles.
RA555 carried the same matA allele as RA669, while RA689 carried the same matA allele as
RA670. If hierarchy of mtDNA transmission were determined by matA alleles, it would be
expected that RA689 would act as mtDNA donor in crosses with NG9 and HI28, and that NG9
and HI28 would act as mtDNA donors in crosses with RA555. This prediction agreed precisely
with the observations (Table 3c). Thus the results were consistent with the hypothesis that
mtDNA transmission was determined by matA alleles.
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S . KAWANO AND T . KUROIWA
Table 4. Transmissionpattern of mtDNA in reciprocal crosses
mtDNA type
of plasmodia
Cross
(a) *RA669
021
(b) *RA669
023
(rnatAl matB4; M-type)
(matAI matB4; N-type)
(matAl matB4; M-type)
(matAI matB4; H-type)
x
x
x
x
NG9
022
HI28
024
(matAZZ matB6; N-type)
(matAZl matB6; M-type)
(matA12 matB5; H-type)
(matAZ2 matB5; M-type)
N
M
H
M
* These crosses are also in Table 3.
Analysis of mtDNA transmission in reciprocal crosses
The uniparental transmission of mtDNA was further investigated by comparing pairs of
reciprocal crosses in which mtDNA types and mating types were exchanged (Table 4). The
results of these reciprocal crosses confirmed the earlier conclusion that mtDNA transmission
did not depend upon mtDNA type, and were consistent with the hierarchy of matA alleles
proposed above. In the cross RA669 x NG9, for example, M-type mtDNA carried by the
matA1 matB4 strain was displaced by N-type mtDNA carried by the matA11 matB6 strain
(Table 4a). To carry out the reciprocal cross, a matAI rnatB4 strain carrying N-type mtDNA
and a matAl I matB6 strain carrying M-type mtDNA were derived as progeny of other crosses
(see Table 1). The two new strains, OZl and 0 2 2 , were then mixed together and the
transmission of mtDNA was determined by restriction endonuclease digestion. Despite the
exchange of mtDNA types, it was again the matA11 matB6 strain that donated the mtDNA to
crossed plasmodia (Table 4a). Similar results were also obtained from a second pair of reciprocal
crosses (Table 4b).
DISCUSSION
The results presented in this paper provide further support for our earlier conclusion that
transmission of mtDNA to crossed plasmodia was uniparental; every plasmodium carried
mtDNA showing the restriction pattern of only one parent. As was suspected on the basis of our
earlier data, the mtDNA type that was lost from each combination of strains was not random,
but was consistent among all replicates of each experiment. The simplest types of explanation
for such results would attribute the biased transmission of mtDNA types to some property of the
mtDNA itself, such that competition or interaction between mtDNA molecules would occur
during the 30-40 mitotic cycles separating zygote formation and the earliest time at which
plasmodia could be analysed. For example, a small difference in the frequencies of replication of
molecules of two mtDNA types could be compounded over this period, leading to the effective
elimination of one type. That such explanations are untenable is shown by our observations that
the relationships between N-type and M-type mtDNA or between M-type and H-type mtDNA
were inconsistent from cross to cross. Some character of amoeba1 strains other than mtDNA
type itself must be responsible for biased uniparental transmission of mtDNA.
It is well documented in another isogamous species, Saccharomyces cereuisiae, that
consistently biased uniparental inheritance can be correlated with a bias in the mtDNA
contents of the mating strains (Birky et al., 1978). In such biased crosses, one parent contains
more mtDNA molecules than the other, and the greater the disproportion in mtDNA content,
the greater the frequency of zygotes that are uniparental for mitochondrial alleles from the
majority parent. Although diploid, polyploid or aneuploid amoebae sometimes arise as progeny
of crosses in P.polycephalum (Mohberg, 1977), we can exclude any bias in mtDNA content in
this work because the strains were all shown by phase-contrast and DAPI-fluorescence
microscopy to be approximately equal in size and in content of mitochondria and mtDNA.
In yet another isogamous organism, Chlamydomonas, Boynton et al. (1987) have demonstrated
that mtDNA transmission is correlated with mating type; meiotic progeny of sexual zygotes
normally receive mitochondrial genomes from the mt- parent and a chloroplast genome from the
mt+ parent. The mating-type system of Physarum is more complex than that of Chlamydomonas,
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Transmission pattern of Physarum mtDNA
1565
being both multilocus and multiallelic. The matB and matC loci both regulate zygote formation,
apparently by influencing the probability that amoebae will fuse (Youngman et al., 1981;
Shinnick et al., 1978); the results in Table 3 show that neither locus can be a major determinant
of mtDNA transmission. The third mating-type locus, matA, regulates the development of
zygotes into plasmodia (Youngman et al., 1981). The idea that this regulation of zygote
development by matA might somehow include the control of mtDNA transmission is an
attractive one, and our results suggest that the simple relationship of two mt alleles in
Chlamydomonas may have a counterpart in Physarum in a hierarchical relationship between the
multiple alleles of rnatA. A more conclusive test of the relationship between matA alleles and
mtDNA transmission would have been to determine the matA genotypes and mtDNA
transmission behaviours of a large number of progeny of a cross. Unfortunately, however, easily
scored mitochondrial mutants are not available in this organism, and it has not been feasible to
analyse mtDNA from a large number of plasmodia using the relatively difficult and laborious
method of restriction endonuclease digestion presently employed.
Even if conclusive evidence can be obtained to link matA and mtDNA transmission, the
mechanism by which uniparental inheritance occurs in Physarum is likely to remain obscure
until methods are available to study the precise behaviour of mitochondria and mtDNA
molecules during the earliest stages of plasmodial development.
We are grateful to Dr R. W. Anderson for many stimulating and critical discussions and for the gift of some
amoeba1 strains. We thank Miss T. Nanba and Mrs K. Mori for their skilful technical assistance. This work was
supported in part by Grant no. 61740407 (S. K.) and 6274041 1 (S. K.) from the Ministry of Education, Science and
Culture of Japan.
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