Download Organelle Division and Cytoplasmic Inheritance

Organelle Division and
Cytoplasmic Inheritance
The origin and basis of the transmission of organelle genomes
Tsuneyoshi Kuroiwa and Hidenobu Uchida
O
ur current understanding of
cytoplasmic inheritance is
that most instances result
from the transmission of mitochondrial and plastid genomes from
parent{s) to progeny. The prevalent
view regarding the origin of the mirochondria and plastids is that they
were modified from prokaryotic endosymbionts during eukaryotic evolution (Gray 1992, Margulis 1970).
According to this hyporhesis, mitochondria and plastids originated as
a purpIe bacteria and cyanobacteria,
respecrively (Figure 1-1). This hypothesis suggests that originaHy,
during asexual cell division in unicellular eukaryotes, organelles reproduced in the parent cell and then
separated into daughter cells {Figure
1-11). This process is called cytoplasmie inheritance in asexual reproduction.
During the evolution from haploid to diploid organisms, sexual
reproduction, which aIJows variation within a population, must have
arisen in unieellular eukaryotes thar
display unicellular isogamy [i.e., the
gametes are similar in size or activity; Figure i-lII), mu.lticellular
anisogamy (i.e., ehe gametes are
slightly different in size or activity;
Tsuneyoshi Kuroiwa is a professor in
the Department of Biological Sciences,
Graduare School of Science, University
of Tokyo, Hongo, Tokyo 113, Japan.
Hidenobu Uchida is a research associate in the Institute of Biological Seiences, University of Tsukuba, Tsukuba
305, Japan. © 1996 American Institute
of Biological Sciences.
December 1996
Cytoplasmic inheritance
occurs in asexually and
sexually reproducing
orgamsms
Figure i-IV), and oogamy (i.e., the
gametes are very different in size
and activity-they are eggs and
sperm cells, or spermsj Figure I-V).
The process by which organelles are
partitioned into gametes and zygotes
or fertilized eggs is ca Iled cytoplasrnic inheritance related to sexual reproduction. Cyroplasmic inheritance
in asexual reproduction occurs still
in vegetative growth in sexually reproducing organisms (Figure 1-111).
The transmission of organelle
DNAs of the male and female parents to their progeny represents the
next stagc in the evolution of cytoplasmie inheritance. In primitive cytoplasmic inheritance in sexual reproduction, male- and fema lederived organelles are mixed in a
zygote, and in some cases fused,
permitting recombination of their
plastid DNA (ptDNA; Sager 1972).
The recomhination of mitoehondrial
DNA (mtDNA), which depends on
mitochondrial plasmids and thus is
analogous to bacrerial sex (conjugation}, is seen in the slime mold
Physarum polycephalum (Kawano
et a1. 1995).
In more advanced forms of cytoplasmic inheritance, uni parental
transmission.of plastid genes is gen-
erally seen. Uniparental transmission of plastid genes oecurs in the
isogamous green alga Chlamydomonas reinhardtii (Sager 1954) and is
due to rapid destruction of the plastid nuclei (plastid nudeoids; complex of ptDNA and proteins) from
one parent during isogamous sexual
reproduetion (Kuroiwa et al. 1982).
Uniparental inheritance and preferential destruction of organelle nuclei containing mtDNA and ptDNA
from one parent are also apparent in
anisogamous and oogamous eukaryotes (Kuroiwa 1991).
Usually, cytoplasmie inheritance
of organelle genomes has been used
to denote events of sexual reproduetion. However, cytoplasmic inheritanee in asexual reproduetion should
also be recognized as a basic type of
cytoplasmic inheritance. In this article, we describe recenr findings
regarding the meehanisms of organelle genome transmission in
asexually reproducing red algae,
isogamous green algae, and isogamous and oogamous plants, in order
of I-V in Figure 1.
Plastid-dividing ring and
mitochondria-dividing ring
During the process of cytoplasmic
inheritance in asexual reproduction,
mitoehondria and plastids reproduce
in the parent cell and then separate
into daughter cells (Figure 1-11).
Mitochondrial DNA (mtDNA) and
plastid DNA (ptDNA) can be routinely observed as organelle nuclei
in various organisms using high-resolution epifluorescence microscopy
827
Sexual
reproduction
t'::\E99
Oogamy
.0
Isogamy
Sperm cell
~1:r or
M-.-
'"" .. e m y
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~ JV t
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Asexual
PLANTAE
FUNGI
reproduction
ANIMALIA
c
aO
C°,,- )
0:1 f(161 eh) "'0
tiJ~;~ b
9
(Kuroiwa 1991). Previous reports
using these techniques showed that
mitochondrial and plastid divisions
can be clearly separated into two
main events: division of the organelle
nuc1ei, and division of the rest of the
organelles, or organellokinesis
(mitochondriokinesis and plasridkinesis), which is analogous to cytokinesis {Kuroiwa 1982). We have
recently reviewed molecular and cellular mechanisms of mitochondrial
nuclear division (Kuroiwa et al.
1994). Hefe we describe the cytological mechan isms involved in
mitochondriokinesis and plastidklnesis in unicellular red algae, such
828
Pigure 1. Aseries of evolutionary events
in eukaryoric cells showing origin of
eukaryotes (I), asexual reproduction (II),
an~ sexual reproduction in isogamy (lII),
amsogamy (IV), and oogamy (V). When
mitochondria and plastids originated
from bacteria (Ia--c) and cyanobactena
(Id), respectively, their cytoplasmic inheritance (CIAr) occurred in llnicellu~
lar organisms with asexual reproducrion (11) and was mainrained during cell
division in isogamous, anisogamous,
and oogamous organisrns with sexual
reproducrion (III-V). When eukaryores
developed sexual reproduction, cytoplasmic inheritance (CISr) occurred in
isogamous organisms (III). The preferential digestion of organelle nuclei
(nucleoids) from the one parent occurs
soon after mating in organisms of
isogamy (III-*) and before ferrilization
in those of anisogamy (IV-*) and 00gamy (V-*). Squares e cells; large circles
with a dot in squares = cell nuclei; small
solid circles in squares = plastids, open
circles in squares e mitochondria; M =
meiosis; a =origin of life; b = ancestral
prokaryote; c = c purpIe bactenum, d :::
cyanobacterium; e = ancestral eukaryote; f ::: ancestral eukaryore with mitochondrion; and g = ancestral eukaryore
with mitochondrion and plastid.
I
d
as Cyanidioschyzon merolae and
Cyanidium caldarium, which may
be among the most primitive eukaryotes (Seckbach 1991).
There is no evidence of any significant structural elements that are
obviously related to mirochondrial
division in eukaryores, despite the
factthat mitochondrial division commonly occurs in eukaryotes and is
inhibited in P. polycephalum by the
drug cytochalasin B, which disrupts
actin fi lamen ts (Kuroiwa and
Kuroiwa 1980). By contrast, a plastid-dividing ring (PD-ring) is found
on the cytoplasmic face of the outer
membrane of the envelope at the
constricted isthmus of dividing chloroplasts in many algae and plants
(Figure 2a, b; Duckett and Ligrone
1993, Kuroiwa 1991, Mita et a1
1986, Suzuki et al. 1994). Because
these observations have followed a
general trend in which the PD-rings
in primitive unicellular eukaryotes,
such as C. caldarium, are larger rhan
those of multiplastidic cells in land
plants, we hypothesized that unknown mirochondrion-dividing rings
(MD-rings) might follow a similar
trend. Thus we set out to observe a
primitive ce1l.
The cells of C. merolae, which
has a smaller nuclear genome than
C. caldarium (Suzuki et al. 1994).
are only 2 um in diameter. They
conrain a nucleus, one mirochondrion, and one chloroplast (arranged
in rhar order), and they prcliferate
by binary division (Figure 3a; De
Luca et a1. 1978). After an extensive
search for MD-rings in various organisms, we identified an MD-ring
and a PD-ring, respecrively, in the
equatorial regions of the dividing
mitochondrion and chloroplastof
C. merolae (Figure 3aII-alV, b-f;
Knroiwa et al. 1993, 1995).
BioScience Val. 46 No. 11
The initial sign of MD-ring formation is the movement of a microbody to the region between the mitochondrion and the ceIl nucleus
(Figure 3al, aIl). An MD-ring that is
40-nm wide and 40-nm thick forms
in the cytoplasm at the point ar which
the microbody has atrached to the
mitoehondrion. Furtherrnore, the
MD- and PD-rings form at approximately the same time. The spherical
rnicrobody begins to elongate along
the MD-ring, and the end of the
microbody is associated with the
PD-ring (Figure 3aIII). When chloroplast division is in progress, the
dense material of the PD-ring appears clearly at the consrricted isthmus of the dumbbell-shaped chloroplast (Kuroiwa er al. 1995). Electron
microscopic images of six serial thin
secrions from the cell in the stage of
Figure 3alV show that the MD-ring
in the cytoplasmic side is cireular
arid becomes noticeably wider and
thicker after the completion of chloroplast division (Figure 3aIV, b-e).
In cornputer-reconstructed images of
the ceIl in the stage of Figure 3aIII,
the large but narrow MD-ring and
the small but thick PD-ring are
clearly seen at rhe equatorial regions
of the dividing mitochondrion and
chloroplast, respeetively (Figure 3g).
These results suggest that the
arnount of the substance that makes
up the MD-ring remains constant
throughout the mitochondrial division. A sharp furrow forms in the
mitochondrion as a result of MD ring coritraction, and the MD-ring
probably generates the consrricrive
force that separates the daughter
mitochondria. The MD-ring disappears, as if rnarerials of the MD~ring
are eonverted into the dividing elongated microbody. Following nuclear
division, the mierobody divides completely to form two daughter rnicrobodies, and cytokinesis occurs (Figure 3aV, aVI).
An association between ehe PDring and microbodies has also been
observed in the algae (Kuroiwa
1991). In C. caldarium, just before
the formation of the PD-ring, many
Golgi vesicles are found distributed
between the microbody and the
bundle of filaments that make up the
PD-ring. However, in C. merolae,
rhese small vesicles are not observed
at the equatorial regions of organelles
December 1996
'",
.~
. ",
'.
I:
...
' .' b
Figure 2. Electron micrographs of the PD-rings at the constricted isthmus of the
dividing chloroplasts in cells during rhe first (a) and rhe second (b) endospore
divisions in Cyanidium caldarium RK-l. The PD-rings (arrows) can be seen clearly
on the cytoplasmic side of the ourer envelope. Bar = 0.1 um,
[usr before the formation of the MDand PD-rings. Accordingly, microbodies may control the organelle
division by direct association in some
organisms, such as C. merolae, and
indirectly in C. caldarium,
Microbodies are common in eukaryotes and eonsist of at least two
types: glyoxysomes and peroxisornes,
both of which contain caralase, an
enzyme that breaks down hydrogen
peroxide into water and oxygen
(Giese 1979). To the known functions of microbodies, we can thus
add the control of mitochondrial
and plastid division, at least in certa in organisms,
The cornponents of the PD- and
MD-rings have not yet been identified. The ultrastructural behaviors
of the PD- and MD -rings observed in
c. merolae are similar to those of the
PD-ring in C. caldarium (Figure 2a,
b, Kuroiwa 1991). Several results
support rhe hypothesis that the PDring in C. caldarium is a bundle of
actinlike filaments [Kuroiwa 1991).
For one, the PD-ring of C. caldarium
is labeled by rhodamine-conjugated
phalloidin (phalloidin binds to actin
filaments, and the fluorescenee of
the attached rhodamine ean be detected by fluoreseence microscopy).
In addition, fine filaments 6-nm in
diameter (consistent with actin) were
seen in transmission electron rnicrographs of aPO-ring. Finally, cy-
tochalasin B inhibits the formation
of the PD-ring as weIl as the actin
contracrile ring that plays a role in
cytokinesis (Kuroiwa 1991). These
results suggest that the PD-ring of
C. caldarium is a bundle of actin
filaments, However, the PD-ringcannot be detected by anri-acrin antibodies raised against chieken gizzard acrins. We also examined
whether actin is conrained in the
MD- and PD-rings and the mierobody in C. merolae. However, although C. merolae does contain an
actin gene, we were unable to detect
actin filaments in the PD- and MDrings by rhodarnine-phalloidin staining and labeling with the anti-actin
antibodies (Ta kah ashi et al. 1995).
One possible explanation is that C.
caldarium and C. merolae contain a
more primitive actin or actinlike
proteins that cannot be detected by
antibodies against chicken gizzard
actins.
Why do we never see the MD-ring
around the mitochondria in organisms other than C. merolae? We
believe that there must be a structure analogous to the MD-ring in
other organisms, Perhaps the MDring is simpler in more highly evolved
organisms. The mitochondria in
higher organisms might divide with
use of a much finer ring, for example, which we cannot yet visualize,
829
a
c
Figure 3. Diagrammatic summary of organelle division (a), elecrron rnicrographs
of five serial sections of.the MD-ring from the cel! in the stage of Figure 3aIV (bf), and a videoframe photograph of the films of transmission electron micrograph
images of three consecutive serial sections of the cell in rhe stage of Figure 3aIII
(g) in Cyanidioscbyzon merolae. The major evenrs in C. merolae (a] are the
movement of the microbody to the region between rhe mitochondrion and cell
nucleus (1, Il); the forrnation of rhe MD-ring and PD-ring after association with
the microbody (II); the elongation of the microbody along the MD -ring and the
differential contraction of MD- and PD-ring (IIl); the conversion of materials
from MD- and PD-ring to the microbody (IV); the cornpl etion of mitochondrial
and chloroplast divisions (V); and the completion of a nuclear division before
cytokinesis (VI). Serial thin sections show rhat the MD-ring is a circular ring
located on the cytoplasmic side of the outer membrane (b-f). The films from three
sections were overlaid using the color image processor (g). A video-frame
photograph of the films shows that the large but fine MD-ring and the small but
thick PD-ring are visible ar the equatorial region of the dividing mitochondrion
and the dividing chloroplasr, respecrively. Asterisks = microbodies; small arrows
on mitochondria = MD-rings; large arrows on chloroplasts = PD rings; N = cell
nudei; M = mitochondria; C = chloroplasts; shaded regions = DNA-containing
regions, Bar = 0.5 um,
Immuno-electron microscopy using antihodies to the bacterial FtsZ
(filamentation temperature sensitive)
protein shows that in Eschericbia
coli and Staphylococcus aureus, the
FtsZ prorein forrns a ring at the
leading edge of the cell division site
(Bi and Lutkenhaus 1991, Lutkenhaus 1993). A similar protein may
830
be present in chloroplasts of the
higher plant Arabidopsis and thus
involved in the divi sion of eukaryotic organelles (Osteryoung and
Vierling 1995). However, a ring of
FtsZ protein has never been observed
directly in bacteria by transmission
e1ectron microscopy. Accordingly,
if we obtain antibodies against corn-
ponents of the PD- and MD-rings,
we may be able to identify these
structures in the equatorial region of
dividing organelles, even in higher
organisms.
If organelles are descended from
bacterial endosymbionts, there may
ha ve been a dividing ring such as the
FtsZ ring inside chloroplasts and
mitochondria at an early stage of
evolution (Figure I-H, Ig). However,
the nucleus eventually came to control the organelle division from the
outside, using PD- and MD-rings
made from actinlike proteins that
are encoded in the nuclear genorne
(Figure l-II).
The organelle division described
above is a step of cyroplasrnic inheritance in asexually reproducing organisms with a simple life cycle such
as C. caladrium and C. merolae.
The separation of organelles following their divisions to daughter cells
is also important for eytoplasmic
inheritance in asexual reproduction.
In rhe yeast Saccharomyces cereuisiae, uni parental (maternal or paternal) inheritance of mitochondrial
genetic markers is thought to be due
to vegetative segregation, such as
the random partitioning of mirochondria by cell division (Birky
1983). Russe! (1984) and Sodmergen
et al. (1995) have shown that in the
higher plant Plumbago zeylanica,
the internal polarity of plastids in
the generative cell generates two different sperm cells after the second
pollen mitosis. Such preferential
movement oE plastids may furnish
the key to understanding the mechanism of separation of organelles.
Relocation of the huge genes
from bacterial endosymbionts
Ir is unknown whether or not the
relocation of the huge genes from
bacterial endosymbionts to the
nuclear genome occurred before the
nucleus came to contro! organelle
di vision using PD- and MD-rings.
Because several genes in plastids have
relocated from the plasrid to the
nuclear genome as algae and land
plants evolved (Gillham 1994, Ohta
er al. 1994), we expected that the
organelles of more primitive eukaryotes might have larger genomes than
more advanced ones. The genome
sizes of the mitochondrion and plas-
BioScience Val. 46 No. 11
tid in the primitive alga C. merolae
are approximately 32 kilobase pairs
(kb) and 150 kb, respectively (Ohta
et al. 1994), and thus both genomes
are smaller than or approximately
the same size as those of the organelles of algae and land planes
(Gillham 1994, Palmer 1992, Reith
and Munholland 1993).
Beeause the genornes of free-living bacteria are larger than 1
mega base pair (Mbp; Lima-de-Faria
1983), the surprising similarity of
the organelle genome sizes from
simple red algae to land plants suggests that thenucleus must have
commandeered more than 80% of
the genome of free-living prokaryotes early in the transition from symbiotic bacteria to organelles (Figure
1-1). There must be acrive mechanisms or genes in the host nucleus to
relocate huge genes from free-living
prokaryotes ro the nucleus.
Cytoplasmic inheritance in
isogamous algae
During the evolution from haploid
to diploid organisms, sexual reproduetion evolved in unicellular eukaryotes with differentiated isogamous gametes, and a new kind of
plastid and mitoehondrial inheritanee related to sexual reproduction
may have evolved (Figure 1-III). In
this primitive cytoplasrnic inheritanee in sexual reproduction, the organelles from male and female gametes would possibly be mixed in a
zygote, and in some cases fuse to
bring about recombination of their
organelleDNA (Kawano etal. 1995}.
In rnore evolved organisms, organelle
DNA from male or female gametes
follows uniparental transrnission to
progeny. A rnechanism of uniparental inheritance that is seen in the
isogamous alga C. reinhardtii (Figure 4) is the preferential digestion in
zygotes of one parent's plastid genome by nucleases.
The transmission patterns of chloroplast and mitoehondrial genes have
been studied in many plants and
animals (Gillham 1994, Gillham et
al. 1991, Kuroiwa 1991, Sears 1980,
Smith 1988). We foeus he re on the
rnechanisrns of cytoplasmic inheritance of plastids in C. reinhardtii
from a cytological perspective, with
an emphasis on the concept of or-
December 1996
T i m e ( h) a ft e r m a t i n q
Figure 4. Summary
2
1
o
of the representative events of cell
nuclei and chloroplast n uc l e i in
young C. reinbardtii zygotes du ring
the first rwo hours
(a ), and epifluoresc ence photom icrographs of a zygote 10 minutes (b)
and 40 minutes (c)
after mixing of mt:
and mt" garnetes.
Preferenrial digestion of ptDNA from
mt: parent occurs
wirhin the first 40
minutes after mating (right half in
c). The uniparental inheritance of
ptDNA in young C,
reinhardtii zygotes
can be explained in
terms of the active
digestion hypothesis (Kuroiwa 1985,
/
mt :
rnt'
/ S ig na l
c e ll nucl eus
1991; see text). mt"
cel l n uc leus
gametes can protect
N
N
I"
their ptDNA against
calcium -de pendent
nucJease (nuclease
Cl during garnetogenesis by changing the chloro plast
matrix or ptDNA
binding proreins
surrounding mt"
ptDNA. Irnmediately after mating,
a signal mu st activate several genes
in rhe cell nucleus
of mt" origin, and
specific mRNAs
from these genes
are synthesized in
the newly formed
zygote. Proteins
encoded by these mRNAs are synthesized de novo in the cytoplasm and may be
involved in such processes as the preferential digestion of ptDNA or rhe formation
of a cell wall. Some proteins that act as inducers directly or indirectly activate
nuclease C. Nuc1ease C preferentially digests ptDNA in the chloroplast of mt:
origin . Because ptDNA of mt" origin is not affected and is transmitted to the
progeny, uniparental inheritance OCCUts. mt: = mating type minus; mt" = mating
type plus; N ;;; cell nuclei; p ;;; plastid nuclei; C ;;; chloroplast, F ;;; flagella. Bar ;;;
1 um ,
.
a
~
d
ganelle n uclei (Figure 4).
Sager (1954) identified non-Mendelian inheritance of organelJar genomes in crosses of streptomycinresistant mutanrs of C. reinhardtii.
Sager and Lane (1972) showed that
the major ptDNA from the plus
mating type (mt+), but not the minus
mating type (mr), parent was missing in 24-hour-old zygotes that had
been formed in crosses in which the
DNA of only one patent was labeled
with 15N . To explain the mechanism
of the preferential digestion of mt:
831
Mate r nal i n h e ri t a n c e
Bi p a r e nt a l i nh er itanc e
1<C:~
.
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.
@)~o
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co=~ @)
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::J
T r ice l lular p o ll e n
.
grai n
.. ..·. (f·
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p o ll e n g r a in
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o
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. CJ3
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. v.' - _....
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2 n d p o ll e n g rai n
.,., .,0
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- ~..":
... . . eH
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.. ..
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':()~
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B i c e ll u l a r p o lle n
g ra i n
M igra ti o n of
g e ne r a t i ve c e
E m b r yo g en e s i s
1st po l le n grain
I
I
I
mit o s is
\
\{5lO\
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M i r a tl il i s t y p e
ptDNA, Sager and her colleagues
proposed a model analogous to bacterial restriction-modification systerns (Sager and Ramanis 1973). AIthough extensive methylation oi mt:
832
Figure 5. Fluoreseenee photomicrographs of a vegetative nucleus, generative nuclei, plastid nuclei (small arrow), and mitoehondrial nuclei (large
arrow) in pollen grains of two angiosperms, Lycoris radiata (a, b) and
Rhododendron indicum (c) visualized
by staining with DAPI, and a schematic
representation (d) of the cell mechanisms of maternal inheritance aud biparental inheritance of organelle DNA in
angiosperms, based on whether or nor
organelle DNAs are present. Maternal
inheritanee of plastids and mitochondria of the Mirabilis and Lycoris type
may oceur in four steps: a reducrion in
the number of organelles just after the
first pollen mitosis; the preferential digestion of organelle nuclei during the
formation of sperm cells (three horizontal arrows and a single vertieal arrow in [cl)); the digestion of organelles
that do not contain DNA during fertilization (a, b}; and the amplification of
organelle DNAs du ring oogenesis, Biparental inheritance of organelles of
the Pelargonium and Rhododendron
type may be the result of the protection
of organelle DNA from nucleases during formation of sperm cells, and consequently, of organelle DNA remaining in
the cytoplasrn in generative (c) and
sperm cells. Patterns of normal green,
stripcd, and white leaves in biparental
plants may be reflected by the division
and separation of organelles from rhe
two parents during embryogenesis, G =
generative nucleus; V = vegetative
nucleus; srnall arrows = plastid nuclei;
large arrows -= mitochondrial nuclei;
EN -= egg nucleus; EPN = egg plastid
nucleus; EMN = egg rnitochondrial
nucleus, Bar (a) = 10 um; bars (h, c) =
0.1 um.
Po llen t e tr ad
d
ptDNA occurred by seven hours after mating (Sager et a1. 1984), other
groups questioned the significance
of this methylation because the
ptDNAs of both mating types were
heavily methylated in a mutant
(me-l) that exhibited typical uniparental transmission of mt" ptDNA
(Bolen et al, 1982). Moreover, addition of rnethylation inhibitors, which
prevent methylation of the ptDNA
of the mt: parenr, had 00 effect on
the normal pattern of transmission
of ptDNA by the mt" parent (Feng
and Chiang 1984).
By 1982, we established a method
for detecting small DNA molecules
(approximately 1 kb) in organelles
in vive using high-resolution fluorescence microscopy. We used this
method to analyze organelle genome
inheritance in C. reinhardtii (Kuroiwaetal.1982}andfoundthatwithin
40 rninutes after mating, the plastid
nuclei (nucleoid; complex of pro-
BioScience Val. 46 No. 11
teins and ptDNAs) from rhe mt:
parenr was completely digesced,
whereas plastid nuclei from the rnt"
parcnt remained intact (Figure 4ac). Similar observations were repeated by other groups (Coleman
and Maguire 1983, Munaut et al.
1990). Preferential digestion of plastid nuclei due to the digestion of
ptDNAs is completely synchronized
among the zygotes, although its timing depends on the culture age, strain,
and medium. Ir evidendy occurs before the fusion of both cell nuclei
and chloroplasts. We concluded that
preferential digestion of ptDNAs in
plastid nuclei from the mt: parent is
the cellular mechanism responsible
for uni parental inheritance in isogamous algae, such as C. reinbardtii,
Dictyosphaeria cavernosa, and Acetabularia caJycuJus (Kuroiwa 1991,
Kuroiwa er a1. 1982).
i What physiological events precede rhe prefererrtiai digesricn of
ptDNA after mating? Based on experiments using inhibitors of RNA
or protein synthesis, DNase inhibitors, and UV irradiation, Kuroiwa
(1985) proposed an active digestion
model as a mechanism for the uniparental inheritance of ptDNA in C.
reinhardtii (Figure 4d). According
(0 this model, specific mRNAs are
synchesized in the esr" cell nudeus in
young zygotes in response to a mating signal. Proteins encoded by these
mRNA(s) then activate calcium-dependent nuclease C, which preferenrially digests ptDNA of mr origin
(Kuroiwa 1985, 1991), leading to
uniparental inheritance. Ulrraviolet
(UV) irradiation experiments suggest that the RNAs essential for preferential digestion of ptDNA from
the mt: parent are synthesized de
novo within ten minutes after maring, and that the corresponding gene
might be linked to the mt+ genome.
Possible straregies to analyze the
preferential digestion of ptDNA include isolating and characterizing
mutants that affecr the process, and
screening for proteins or mRNAs
whose accumulation correlates with
preferential digestion of ptDNA.
Gillham et a1. (1991) isolated the
mat-3 mutant, which shows gready
increased transmission ofchloroplast
genes from the mt- parent. However, mutants have not been obtained
in which preferendal digestion of
December 1996
ptDNA from the zer parent is inhibited completely.
Two-dimensional electrophoresis
analysis has shown that in zygotes
incubated with (35S}-methonine after ma ti ng , approximately 200
polypeptides are synthesized de novo.
At least 7 of them-94 kD («), 94 kD
(ß), 94 kD (y), 52 kD, 50 kD, 38 kD,
and 20 kD-are correlated with
uniparental transmission of the
mtr chloroplast genome (Figure 4d;
Nakamura et al. 1988. Uchida et al.
1993). When mt" ceIls were cultured in the presence of an inhibitor
of DNA synthesis, 5-fluorodeoxyuridine (5-FdUrd), to reduce the presence of the ptDNA and mared with
unrreated mr cells , rhese seven
polypeptides were detected in the
zygotes, most of which showed preferential digesrion of ptDNAs (Nakamura and Kuroiwa 1989). Wurtz et
a1. (1977) reported that in mt" gametes derived from 5-FdUrd treated
cells, biparental transmissten of chloroplast genes increased up to 20%30%. If 5-FdUrd affeets the eell
nuclear genome, gene expression(s)
essential for preferential digestion
of ptDNAs from the mt" parent
would be inhibited and exceptional
zygotes would increase.
We performed differential screening between mRNAs from gametes
and cells ten minutes after mixing
rhe two types of gametes and obtained cDNA clones corresponding
to a zygote-specific gene family. The
zygote-specific genes may be releted
to the preferential desrruction of
ptDNA, the fusion of cell nuclei,
and formation of a cell wall (Figure
4d). Two clones, pZS102-1 and
pZS102-69, conrain highly similar
open reading frames (ORFs) and
were judged to be cDNAs for two
members (zyslA and zys1B,respectively) of a novel gene family (Uchida
et a1. 1993). The predieted polypeptides encoded by these genes are
small (20 kD) and contain cysreineand glutamine-rieh regions (Pigure
4d) and are unlikely to be identical
to any of the seven polypepides related to preferential digestion of
ptDNA INakamura et a1. 1988).
Recent analysis by another group
(Armbrust et al. 1993) suggests that
a protein encoded by a zygote-specific dass III gene, ezy-l, which is
linked to a mating locus-forming
gene cluster (Ferris and Goodenough
1987), may be identical to polypeptide 4 (52 kD) found by Nakamura
et a1. (1988). Based on the time
course of protein detection after
mating and the effecr of UV on the
accumulation of this prorein, both
the zyslA gene and the ezy-l gene
seem to be correlated with the preferential destruction of ptDNA of
mt: origin. Because the two corresponding peptides, ZYS-1A and ZYS1B, did not show nuclease activity,
they may playa role as inducers or
promoters of calcium-dependent
nuelease C (Figure 4d). PtDNA in
permeabilized mtr cells exhibited
increasing resistance to the nuclease
C fraction as cultures progressed,
whereas those in mr: cells remained
sensitive to nuclease C.
This finding suggests that an unknown mechanism that proteers
mt' -derived ptDNA from nuclease
C exists in the chloroplast membrane, in the chloroplast matrix, OI
in ptDNA bindingproteins surroundingptDNA (Kuroiwa 1985). Beeause
nuclease C was detected in zygotes
and in vegetative and gametie cells
of the (WO mating types (Kuroiwa
1985,1991), the enzyme must exist
in an inactive form in the cytoplasm
or lysosome of such cells until it is
activated by the mating-induced
polypeptides. VanWinkele-Swift and
Salinger (1988) reported the isolation of a mutant (mtJ-I) that seems ro
fail (0 protect its own ptDNA frorn
destruction. This mutant may define
a gene (hat is necessary to keep the
enzyme inactive until it is needed.
Cytoplasmic inheritance in
algae and land plants
Gillham (1994) has extensively reviewed the transmission of mtDNA
in slime molds, ciliates, filamenrous
fungi, and animals, and the transmission of mtDNA and ptDNA in
green algae, ferns, and lugher plants.
In this article, we focus on cytological studies of maternal inheritance
of organelle DNA in green algae and
land plants, which are anisogamous
or oogamous, in which the male
contributes litde to the zygote cytoplasm. Maternal inherirance in these
organisms has been postulated to
occur by simple physical exclusion
of mitochondria and plastids of male
833
origin by cytokinesis or fertilization
(Mogensen 1988, Sears 1980). However, we found that anisogamous
algae, such as Bryopsis maxima and
Bryopsis pulumosa, and oogarnous
organisms, such as Nitella axiliformis and Pteris uittata, show a
phenornenon similar to the preferential digestion of mtDNA and ptDNA
frorn one parent (Kuroiwa 1991). In
contrast to the isogamous organisms, the preferential digestion of
mtDNA and ptDNA occurred before fertilization in the anisogamous
algae and oogamous organisms (Figures 1-IV, V, and 5).
Correns (1909) and Baur (1909)
discovered non-Mendelian inheritance of plastid phenotype in the
higher plants Mirabillis ialapa and
Pelargonium zonale, respectively.
Interestingly, their findings corresponded to two typical patterns of
plastid genes; rhat is, maternal and
biparental transmission without
Mendelian ratios. We have carried
out eytogenetie studies of the pattern of transmission of ptDNAs in
sperm cells and compared these results with those of previous genetic
studies. We found two basic types of
organelle DNA behavior in generative arid sperm cells of pollen grains
(Miyamura er al. 1987). The first
type of behavior, exemplified by M.
jalapa, Lycoris radiata (Figure 5a,
b), and Nicotiana tabacum, is characterized by the digestion of mtDNA
and ptDNA in the sperm cells of
pollen grains (Figure 5a, h, d). The
second type of behavior, typified by
P. zonale, Oenothera biennis, and
Rhododendron indicum (Figure Sc),
is characterized by the presence of
mtDNA and ptDNA in sperm cells
in pollen tubes (Figure Se, d). Chloroplast genes in M. jalapa and P.
zonale show typical maternal transmission and biparental transmission,
respectively, in genetic crosses (Sager
1972). The cytological observations
thus agree with ehe classieal genetic
data. A similar trend was confirmed
in more than 100 angiosperm genera
(Corriveau and Coleman 1988).
These cytological results indicate that
the preferential digestion of organelle
DNA during sperm cell formation
must be responsible for maternal
inheritance in higher plants as weil
as in anisogamous and oogamous
algae and ferns (Figure I-IV, V;
834
Kuroiwa 1991).
How does the preferential digestion of organelle DNA in the generative cells oecur in higher plants? We
have observed the behavior of organelle DNA during the first pollen
mitosis in many speeies. In general,
the ratio of the volume of cytoplasm
to that of the eell nuclei appears to
be related to the preferential digesrion of organelle DNA. In plants
such as the Geraniaceae, wh ich have
a relatively large cytoplasm compared to the nucleus, the preferential destruction of organelle DNA
does not occur, and organelle DNA
remains in generative eells (Figure
5d). By contrast, the preferential
destruction of organelle DNA oceurs in planrs sueh as the Compositae,
whieh h ave a r e lat ive l y small
cytoplasmmuc'aces-r-io. The preferential destruction of organelle
DNA is also found in some plants
with a large volume of cytoplasm in
the generative cell, such as the
Liliaceae. Accordingly, a decrease
in the volume of the generative cell
by unequal cell division, or pollen
mitosis, is not directly responsible
for the destruction of mtDNA and
prDNA (Figure 5d). Nucleases are
not activated in plants such as P.
zonale and Schlumbergera russellianum that show biparental transmission of ptDNA. The low frequency
of biparental plastid inheritance that
has been documented in both Nieotiana and Petunia may be due ro a
malfunction in the system for inducing ptDNA digestion during pollen
by a calcium-dependent nuclease C;
degradation of organelles, possibly
by lysosomes, after digestion of organelle DNAs is complete; and a
huge amplification of organelle DNA
in egg cells during oogenesis.
Uniparental inheritance also inhibits the recombination of organelle
genes in ehe zygote. Thus, although
fertilization allows the recombination of nuclear genomes, events before or during fertilization prevent
the recombination of organelle DNA
from the two parents.
Why are organelle genomes transmitted uniparentally? Cosmides and
Tooby (1981) suggested that uniparental transmission is the result of
"intragenomic conflict" between
organelle genomes of maternal and
patemal origin. We suggest an alternative explanation. We believe that
the nuclear genorne of the host eeIl
must have undergone three steps to
control the independence of protoorganelles following endosymbiosis:
transfer of more than 70% of the
symbiont genome into the host's ceIl
nucleus, regulation of proroorganelle division by ring-forming
cytoskeletal protein(s), such as actin, that are eneoded by the nuclear
genome; and uni parental transmission of organelle genomes to prog~
eny to avoid the evolution of organelles through reeombination of
the organelle genes of borh parents.
Thus, the evolution of protoorganelles was effeetively terminated, and they were eonverted into
mitochondria and plastids.
formation.
Note that a huge amplification of
organelle DNA occurs during 00genesis in P. zonale, which accompanies an increase in the egg cell
volume (Kuroiwa and Kuroiwa
1992). This phenomenon complements processes such as unequal division of first pollen mitosis and
preferential digestion of organelle
DNA in generative cells to yield a
complete uniparental inheritance of
organelle genomes. We hypothesize
that elimination of organelle DNA
from sperm or sperm cells before
and during fertilization in anisogamous algae, ferns, and angiosperms
involves four steps: unequal cell division in the generative cell, which
decreases organelle number; digestion of organelle DNA, presumably
Acknowledgments
This research was supported by a
grant-aid for special promoted research (No. 06101002) from the
]apanese Ministry ofEducation, Seience and Culture.
References cited
Armbrust EV, Ferris PJ. Goodenough UW. 1993.
A mating type-linked gene cluster expressed
in Chlamydomonas zygotes parricipares in
the uniparental inherirance of the chloroplasr genome. Ce1l74: 801-811.
BaueE. 1909. Das Wesen und die Erblichkeitsverhaltnisse der "Varierares albomarginatae
hort" von Pelargonium zonale. Zeitschrift
fuer Versuchstierkunde 1: 330-351.
Bi E, Lutkenhaus J. 1991. FtsZ ring srructure
associared with division in Escherichia coli.
Nature 354: 161-164.
Birky CW jr. 1983. Relaxed cellular conrrols
BioScience Val. 46 No. 11
and organelle heredity. Science 222: 468475.
Bolen PL, Grant OM, Swincon D, Boynton JE,
Gillham NW. 1982. Extensive methylation
ofchlocoplastDNA by a nuclear gene mutation dces not affect chloroplast gene transmission in Chlamydomonas. Ce1l28: 335343.
Coleman AW, Maguire MJ. 1983. Cytological
derecrion of the basis of uniparental inhentance of plastid DNA in Chlamydomonas
moewusii. Currenr Genetics 7: 211-218.
Cerrens C. 1909. Vererbungsversuche mit blass
(gelb) grunen und bluntblattrigen Sippen bei
Mirabilis ialapa, Urtica pilulifera, und Lunariaannua .Zeitschrift Euer Vererbungslehre
1:291-329.
Corriveau jS, Coleman AW. 1988. Rapid screening rnethod ro detecr potential biparental
inheritance of plasrid DNA and results for
over 200angiospermspecies. AmericanJournal of Botany 75: 1443-1458.
Cosmides LM, Tooby J. 1981. Cyroplasmic
inheritance and inrragenomic conflicr.journal of Theoretical Biology 89: 89-129.
De Luca P, Taddei R, Verano 1. 1978.
"Cyanidioschyzon merolae"': a new alga of
thermal acidic environmenrs. Webbia 33:
37--44.
Ducken JG, Ligrone R. 1993. Plastid-dividing
rings in ferns. Annals of Botany (London)
72:619-627.
Feng TW, Chiang KS. 1984. The persistence of
mate mal inheritance in Ch/amydomonas
despite hypcmethylatlon of chloroplast DNA
induced by inhibirors. Proceedings of rhe
National Academy of Seiences of ehe United
States of America 81: 3438-3442.
Ferris PJ, Googenough UW. 1987. Transcription of novelgenes, inducing a gene linked to
the mating-rype locus, induced by Chlamydomonas fertilization. Molecular and Cellular Biology 7: 2360-2366.
Giese AC. 1979. Cell physiology. Philadelphia
(PA): W. B. Saunders.
Gillham NW. 1994. Organelle genes and genomes. OxEord (UK): Oxford University
Press.
Gillham NW, Boymon JE, Harris EH. 1991.
Transmission of plastid genes. Pages 55-92
in Bogorad L, Vasil IV, eds. The molecular
biology of plastids. San Diego (CA): Academic Press.
Gray MW. 1992. Theendosymbionthypothesis
revisited. Imernational Review of Cytology
141:233-357.
Kawano 5, Takano H, Kuroiwa T. 1995. Sexuality of mitochondria: fusion, recombination and plasmids. International Review of
Cytology 161: 49-110.
Kuroiwa H, Kuroiwa T. 1992. Giant mitochondria in the marure egg cell of Pelargonium
zonale. Protoplasma 168: 184-188,
Kuroiwa H, Ohta T, f<uroiwa T. 1996. Srudies
on the development and three-dimensional
reconstruetion of giant mitochondria and
their nuelei in egg cells ofp elargonium zonale
Ait. Protoplasma 196: 235-244.
Kuroiwa T. 1982. Mitochondrial nudei. International Review of Cytology 75: 1-59.
___. 1985. Mechanisms oE maternal inheritance of chloroplast DNA: an active digestion hypothesis. Microbiologkal Sciences 2:
267-272.
___.1991. The replication, differentiation,
December 1996
and inheritance of plastid with emphasis on
the concept of organelle nuclei. International Review of Cytology 128: 1--62.
Kuroiwa T, Kuroiwa H. 1980. Inhibition of
Physarum mitochondrial divisionbycytcchalasin B. Experientia 36: 193-194.
Kuroiwa T, Kawano S, Nishibayashi S, Saro C.
1982. Epifluorescent microscopic evidence
for matemal inheritance of chloroplasr DNA.
Nature 298: 481-483.
Kuroiwa T, Suzuki K, Kuroiwa H. 1993. Mitochondrial division by dividing apparatus (an
electron dense ring) in Cyanidioshyzon
merolae. Protoplasma 175: 173-177.
Kuroiwa T, Ohta T, Kuroiwa H, Kawano S.
1994. Molecular andcellular rnechanisms of
mitochondrial nuclear division and mitochondriokinesis. Microscopy Research and
Techniqce 27: 220-232.
Kuroiwa T, Suzuki K, and Kuroiwa H. 1995.
Mirochondria dividing ring: ultrastrucrural
basis for rhe mechanisms of mirochondrial
division in Cyanidioschyzon merolae.
Protoplasma 156: 12-23.
Lima-de-Faria A. 1983. Molecular evolution
and crganizarion of the chromosome.
Amsterdam (rhe NetherIands): Elsevier.
Lutkenhaus J. 1993. FtsZ ring in bacterial cytokinesis. Molecular Microbiology 9: 403409.
Margulis 1. 1970. Origin of eukaryotic cells.
New Haven (CT): Yale Universiry Press.
Mita T, Kanbe T, Tanaka K, Kuroiwa T. 1986.
Aringstructure around the dividing plane of
t h e Cyanidium caldarium chloroplast. Protoplasma 130:211-213.
Miyamura S, Kuroiwa T, Nagata T. 1987. Disappearance of plastid and mitochondrial
nucleoids during rhe formation of generative cells of higher plants revealed by fluorescence microscopy. Protoplasma 141: 149159.
Mogensen HL. 1988. Exclusion of male mitochondria and plastids during syngamy in
barley as a basis for maternal inheritance.
Proceedings of rhe National Academy of
Sciences oE the United States of Arnerica 85:
2594-2597.
MunautC, DombrowigzD,Matagne RF. 1990.
Detection of chloroplast D NA by using fluorescent monodonal anti-bromodeoxyuridine
antibody and analysis oE its fate during zygote formation in Chlamydomonas reinhardtii. Current Genetics 18: 259-263.
Nakamura S, Kuroiwa T. 1989, Selective elimination of chloroplast DNA by5-fluorodeoxyuridine causing no effect on preferential digestion of male chloroplast nucleoids
in Chlamydomonas. European Journal of
Cell Biology 48: 165-173.
Nakamura 5, Sato C, Kuroiwa T. 1988. Polypeptides related to preferential digestion oE male
chloroplast nucleoids in Chlamydomonas.
PlantScience56: 129-136.
Ohta N, Kawano S, Kuroiwa T. 1994. Physical
map of the plastid genome of unicellular red
alga Cyanidium caldarium strain RK-1.
Current Genetics 26: 136-138.
Osteryoung KW, Vierling E. 1995. Conserved
cell and organelle division. Nature 376: 473474.
Palmer JD. 1992. Chloroplasr and mitochondrial genome evolution in land plants. Pages
99-133 in Herrmann RG, ed. Plant gene
research: cell organelles. Vienna (Austria):
Springer-Verlag.
Reich M, Munholland J. 1993. A high-resolunon map of rhe chloroplast genome of ehe
red alga Porphyra purpurea. Plant Cell 5:
465-475.
Russel SO. 1984. Ulrrasrructure of the sperm of
Plumbago zeylanica, 2. Quantitative cyrology and three-dimensional reconstruction.
Planra 162: 385-391.
Sager R. 1954. Mendelian and non-Mendelian
inheritance of srreptomycin resistance in
Chlamydomonas reinhardtü. Proceedings of
the National Academy of Seiences of the
United Stares of America 40: 356-363.
___.1972. Cytoplasmic genes and organelles.
New York: Academic Press.
Sager R, Lane D. 1972. Molecular basis of
maremal inheritance. Proceedings of rhe
National Academy of Science of the United
Srates of America 69: 2410-2413.
Seger R, Ramanis Z. 1973. The mechanism of
maternal inheritance in Chlamydomonas:
biochemical andgenetic studies. Theoretical
and Applied Generies 43: 101-108.
Sager R, Sano H, Grabowy CT. 1984. Control
of maremal inheritance by DNA metlrylation in Chlamydomonas. Current Topics in
Microbiology and Immunology 108: 157172.
Sears BB. 1980. Elimination of plastids during
spermatogenesie and fertilization in the plant
kingdem. Plasmid4: 233-255.
Seckbach J. 1991. Systemarie problems wirh
Cyanidium caladrium and Galdieria
sulphuraria and rheir implications for molecular biology stndies. Journal of Phycology 27: 794-796.
Smith SE. 1988. Biparental inheritance of organelles and its implications in crap improvemem. Plant Breeding Reviews 6: 361393.
Sodmergen GH, ehen ZM, Hu ZM, Guo FL,
Guan XL. 1995. Male gametophyte developmeßt inPlumbago zeylanica:cytoplasm localization and cell detennination in the early
generative celL Protoplasma 186: 79-86.
Suzuki K, Ehara T, Osafune H, Kawano S,
Kuroiwa T. 1994. Behavior of mitochondria, chloroplast and their nuclei during the
mitotic eyde in the ultramicro alga
Cyanidioschyzon mero/ae. European Jour·
nal of Cell Biology 63: 280-288.
Takahashi H, Takano H, Yokoyama A, Hara
Y, Kawano S, Toh-e A, Kuroiwa T. 1995.
Isolation, characterization and chromosomal
mapping of an actin gene from the primitive
red aiga Cyanidioschyzon merolae. Current
Genetks 28: 484-490.
Uchida H, Kawano S, Sato H, Kuroiwa T. 1993.
Isolation and characterization of novel genes
which are expressed during the very early
stage of zygote formation in Chlamydomonas
reinhardtü. Current Genetics 24: 290-300.
VanWinkle-Swift KP, Salinger AP. 1988. Loss
of mt' derived zygotic chloroplast DNA is
associated with alethal allele in Chlamydomonas monoica. Current Genetks 13:
331-337.
Wurtz EA, Boynton JE, Gillham NW. 1977.
Perturbation of chloroplast DNA amounts
and chloroplasr gene transmission in
ChJamydomonas by 5-fluorodeoxyuridine.
Proceedings oE the National Academy of
Sciences of the United Srates of America 74:
4552-4556.
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