Plant Cell Physiol. 49(3): 345–361 (2008)
doi:10.1093/pcp/pcn012, available online at www.pcp.oxfordjournals.org
ß The Author 2008. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.
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The Assembly of the FtsZ Ring at the Mid-Chloroplast Division Site Depends on
a Balance Between the Activities of AtMinE1 and ARC11/AtMinD1
Makoto T. Fujiwara 1, 2, *, Haruki Hashimoto 2, Yusuke Kazama 1, Tomoko Abe 1, Shigeo Yoshida 1,
Naoki Sato 2 and Ryuuichi D. Itoh 1, 3, *
1
RIKEN, Hirosawa 2-1, Wako, Saitama, 351-0198 Japan
Department of Life Sciences, University of Tokyo, Komaba 3-8-1, Tokyo, 153-8902 Japan
3
Department of Chemistry, Biology and Marine Science, Faculty of Science, University of the Ryukyus, Senbaru 1, Nishihara, Okinawa,
903-0213 Japan
2
transcription–PCR; Ws, Wassilewskija; WT, wild type; YFP,
yellow fluorescent protein.
Chloroplast division comprises a sequence of events that
facilitate symmetric binary fission and that involve prokaryoticlike stromal division factors such as tubulin-like GTPase
FtsZ and the division site regulator MinD. In Arabidopsis, a
nuclear-encoded prokaryotic MinE homolog, AtMinE1, has
been characterized in terms of its effects on a dividing or
terminal chloroplast state in a limited series of leaf tissues.
However, the relationship between AtMinE1 expression and
chloroplast phenotype remains to be fully elucidated. Here,
we demonstrate that a T-DNA insertion mutation in
AtMinE1 results in a severe inhibition of chloroplast
division, producing motile dots and short filaments of
FtsZ. In AtMinE1 sense (overexpressor) plants, dividing
chloroplasts possess either single or multiple FtsZ rings
located at random intervals and showing constriction depth,
mainly along the chloroplast polarity axis. The AtMinE1
sense plants displayed equivalent chloroplast phenotypes to
arc11, a loss-of-function mutant of AtMinD1 which forms
replicating mini-chloroplasts. Furthermore, a certain population of FtsZ rings formed within developing chloroplasts
failed to initiate or progress the membrane constriction of
chloroplasts and consequentially to complete chloroplast
fission in both AtMinE1 sense and arc11/atminD1 plants.
Our present data thus demonstrate that the chloroplast
division site placement involves a balance between the
opposing activities of AtMinE1 and AtMinD1, which acts to
prevent FtsZ ring formation anywhere outside of the midchloroplast. In addition, the imbalance caused by an
AtMinE1 dominance causes multiple, non-synchronous
division events at the single chloroplast level, as well as
division arrest, which becomes apparent as the chloroplasts
mature, in spite of the presence of FtsZ rings.
Introduction
The faithful inheritance of chloroplasts upon cell
division is essential for the maintenance of the photosynthetic activities of plants. The preservation of a certain
chloroplast number per cell is usually attained by symmetric
binary fission of pre-existing organelles (Possingham and
Lawrence 1983). Chloroplast division is considered to be
a combination of two tasks: division site selection and
scission of chloroplast envelope membranes, which is
equivalent to cytokinesis. The mechanisms by which
chloroplasts constrict in order to divide have been the
subject of much study during the last decade. The tubulinrelated GTPase FtsZ is a central component of the
constriction machinery and is of endosymbiotic origin. In
bacteria, the FtsZ proteins assemble into a ring structure
(Z-ring) just beneath the plasma membrane at the mid-cell
(Bi and Lutkenhaus 1991, Ma et al. 1996). The Z-ring
functions as the scaffold for the assembly of other cell
division proteins (Rothfield et al. 2005). The bacterial-like
FtsZ gene, AtFtsZ1-1, was the first nuclear-encoded
chloroplast
division
component
identified
from
Arabidopsis thaliana (Osteryoung and Vierling 1995,
Osteryoung et al. 1998), and it has now been shown that
most, if not all, higher and lower plants contain nuclear
genes with high similarity to FtsZ (Stokes and Osteryoung
2003, Miyagishima et al. 2004).
Arabidopsis and other higher plants also possess two
types of FtsZ homolog, FtsZ1 and FtsZ2, which are
phylogenetically distinct and functionally non-redundant
(El-Kafafi et al. 2005, Yoder et al. 2007). Like bacterial
FtsZ, both types of chloroplast FtsZ form a ring on the
stromal surface of the inner envelope membranes at the midpoint, both before and during the organelle constriction
Keywords: Arabidopsis thaliana — Binary fission — FtsZ
ring — Leaf development — Min system.
Abbreviations: CaMV, cauliflower mosaic virus; Col,
Columbia; GFP, green fluorescent protein; LB, left border; Ler,
Landsberg erecta; PDS, potential division site; RT–PCR, reverse
*Corresponding authors: Makoto T. Fujiwara, E-mail, [email protected]; Fax, þ81-3-5454-6998;
Ryuuichi D. Itoh, E-mail, [email protected]; Fax, þ81-98-895-8576
345
346
The chloroplast Min system
(McAndrew et al. 2001, Miyagishima et al. 2001, Mori et al.
2001, Vitha et al. 2001, Kuroiwa et al. 2002). Division site
selection, which usually confines the division plane only at
the mid-chloroplast, is another aspect of chloroplast division
and, to date, two soluble stromal components of bacterial
origin have been known to play a role in the division site
selection of chloroplasts. In the rod-shaped bacterium
Escherichia coli, the process of cell division site selection is
well characterized. In E. coli, MinD is a plasma membraneassociated ATPase and cooperates with MinC, an inhibitor
of FtsZ polymerization, to prevent Z-ring formation at all
potential division sites (PDSs), except for the mid-cell
(RayChaudhuri et al. 2000). A MinD homolog, AtMinD1,
has been isolated from Arabidopsis (Colletti et al. 2000,
Kanamaru et al. 2000), and reduced or elevated expression
of AtMinD1 leads to asymmetric chloroplast division or
chloroplast division inhibition, respectively.
AtMinD1 is capable of forming homodimers, and this
is required for chloroplast division site placement, since the
arc11 mutation (A296G) of AtMinD1 that collapses
functional homodimerization abolishes the symmetrical
placement of constriction sites at the mid-chloroplast
(Marrison et al. 1999, Fujiwara et al. 2004). The second
known chloroplast division site determinant is AtMinE1, an
Arabidopsis homolog of bacterial MinE (Itoh et al. 2001,
Maple et al. 2002, Reddy et al. 2002). In E. coli, MinE is
located in a mobile cylindrical or coiled structure moving
from the mid-cell to the pole, and stimulates both ATPase
activity and the subsequent membrane release of centripetal
MinD molecules (reviewed in Rothfield et al. 2005). This
ensures Z-ring assembly only at the mid-cell. Consistent
with this E. coli mechanism, reduced or elevated AtMinE1
expression levels lead to chloroplast division inhibition
(Itoh and Yoshida 2001) or multiple constriction of dividing
chloroplasts (Maple et al. 2002), respectively. Nevertheless,
the general and consistent tendency toward a paucity of
mini-chloroplasts in AtMinE1-overexpressing plants (Itoh
et al. 2001, Reddy et al. 2002, Maple et al. 2002) cannot be
adequately explained by the E. coli Min system model.
These results from Arabidopsis also differ from those of the
spherical cyanobacterium Synechocystis sp. PCC6803,
where null mutation of the minE gene affects cell shape
and division only slightly (Mazouni et al. 2004), in spite of
the phylogenetic position of cyanobacteria as the ancestor
of the chloroplast.
For a more integrated understanding of the chloroplast
division machinery, the physical and functional interactions
among membrane fission proteins and division site selection
proteins will need to be elucidated. Maple et al. (2005)
performed exhaustive physical interaction mapping analysis
of all known stromal chloroplast division proteins of Arabidopsis, and demonstrated that AtMinE1 and AtMinD1,
but not FtsZ (AtFtsZ1-1 and AtFtsZ2-1) or GC1,
can physically interact to form both homodimers and
heterodimers. Another study by the same group has shown
that AtMinD1 is a Ca2þ-dependent ATPase whose activity is
stimulated by AtMinE1 (Aldridge and Møller 2005).
Although the roles of AtMinE1 and AtMinD1 in
chloroplast division have been proposed by analogy with
the bacterial Min system model of division site selection, it
is essential to determine experimentally the precise roles of
these molecules in chloroplasts. For this purpose, we have
analyzed the effects of both the overexpression and loss of
function of AtMinE1 or AtMinD1 upon the dynamics of
FtsZ1 (AtFtsZ1-1). We demonstrate that the spatial
distribution of the FtsZ ring is controlled by a balance
between the activities of AtMinE1 and AtMinD1, which
have opposing roles and jointly negatively regulate FtsZ
ring formation except at the mid-chloroplast.
Results
A T-DNA insertional mutation reveals the indispensability of
AtMinE1 during chloroplast division
Since the cloning of a prokaryotic-like minE in the
chloroplast genome of a green alga Chlorella vulgaris
(Wakasugi et al. 1997), minE-like sequences have been
found in chloroplast and nuclear genomes from a wide
range of photosynthetic eukaryotes, including algae, moss
and seed plants (Douglas and Penny 1999, Hortensteiner
et al. 2000, Itoh et al. 2001, Hayashida et al. 2005; see
Supplementary Fig. S1). The Arabidopsis minE homolog
(AtMinE1) is a single nuclear gene, and is the sole
eukaryotic minE that has so far been functionally characterized. We previously showed that either the overexpression or repression of AtMinE1 leads to a decreased
number of enlarged chloroplasts per leaf cell, demonstrating
that normal expression levels of AtMinE1 are essential for
correct chloroplast division (Itoh and Yoshida 2001, Itoh
et al. 2001). However, antisense transgenic plants, that
maintain only 25–33% of the wild-type (WT) AtMinE1
transcript levels (Itoh and Yoshida 2001), tend to suppress
the abnormal chloroplast phenotype during successive plant
generations. This is supported by our observations that
495% of the T2 transgenic plants in all lines appear WT by
the T3 generation (data not shown). This gene silencing
effect made it difficult to determine the conclusive roles of
AtMinE1, although the necessity of this gene for correct
chloroplast division was unequivocal.
To clarify further the role of AtMinE1, we searched the
public resource databases and found a T-DNA insertion
line, Flag_056G07, in which an Agrobacterium-derived
T-DNA is located at the AtMinE1 locus (Fig. 1A, B).
Genomic PCR using total DNA from Flag_056G07
confirmed the location of T-DNA at intron 1 of AtMinE1
(Fig. 1B), which corresponds to the N-terminal Ser/Thr-rich
The chloroplast Min system
B
A
WT (Ws)
ATG
atminE1
LB
347
T-DNA
TGA
AtMinE1
Chr
I
II
I
Intron 1
e1
e2
200 bp
AMD TSD
AtMinE1
229 aa
WT
C
(kb)
S/T-rich
Extension
C-terminal
Extension
atminE1
e2 LB e2 LB
M 1& 1& 1& 1&
e
e
e
e
2.0
1.5
1.2
1.0
0.8
0.6
D
WT
(kb)
1.0
0.8
0.6
0.5
1
M
25
30
35
atminE1
40
30
35
40
(cycles)
AtMinE1
22
2
25
22
25
UBQ10
E
Chl
WT
Merged
WT
DIC
(kb)
1.0
0.8
0.6
0.5
M
20
25
20
25
30
(cycles)
AtMinE1
22
atminE1
AtMinE1 sense
30
25
22
25
UBQ10
Fig. 1 Transgenic Arabidopsis plants in which nuclear-encoded AtMinE1 harbors a T-DNA insert. (A) Appearance of 20-day-old seedlings
grown in soil. Arabidopsis thaliana wild-type (WT) ecotype Wassilewskija (Ws) and the atminE1 mutant (Flag_056G07) were grown under
normal growth conditions. (B) Insertion of T-DNA into AtMinE1 in the Arabidopsis line Flag_056G07. The site of T-DNA insertion at intron
1, corresponding to downstream of Lys34 (arrowhead) in the N-terminal S/T-rich extension region, is indicated. The sites of two
oligonucleotide primers, e1 and e2, and a primer specific for the T-DNA left border (LB) are also indicated. (C) Isolation of a homozygous
mutant of atminE1. Genomic DNA from WT and Flag_056G07 plants was amplified by PCR using e1 and e2 and between e1 and LB to
detect non-mutated (1.6 kb product, arrowhead 1) and T-DNA-mutated AtMinE1 (0.6 kb product, arrowhead 2), respectively.
(D) Comparison of the levels of AtMinE1 transcripts among atminE1, AtMinE1 sense, and their parental WT lines. RT–PCR was performed
using e1 and e2 and total RNA purified from WT (Ws and Col in the upper and lower panels, respectively), atminE1 (upper panels)
and AtMinE1 sense (lower panels) plants. An equal amount of aliquots from the PCRs was analyzed by electrophoresis in an ethidium
bromide-containing agarose gel. RT–PCR for the UBQ10 mRNA was also performed as a normalizing control. M, size markers (C, D).
(E) Aberrant chloroplast morphology in mature leaf mesophylls of atminE1 plant. Chloroplasts in primary and secondary leaf mesophylls
from 3-week-old seedlings of WT (Ws) and atminE1 were observed by non-destructive epifluorescence microscopy. Differential
interference contrast (DIC), chlorophyll autofluorescence (Chl) and merged images are shown. Bars, 10 mm.
region of this gene that functions as a chloroplast-targeting
sequence (Itoh et al. 2001). Homozygous T-DNA mutant
plants were then isolated from heterozygous plants
(Fig. 1A, C). Similar to most other mutations that perturb
chloroplast division, such as the accumulation and replication
of chloroplasts (arc) (Pyke 1997, Marrison et al. 1999), the
homozygous atminE1 mutation results in no deleterious
effects on overall plant morphology and reproduction.
By reverse transcription–PCR (RT–PCR), we detected
a considerably low level of RNA splicing of AtMinE1 in
the atminE1 mutant, as compared with that in the WT
(Fig. 1D). The amount of RT–PCR products from atminE1
at 35 amplification cycles, where the amplification seems to
be in a logarithmic phase, was roughly similar to that from
the WT at 25 amplification cycles. When normalized by the
UBQ10 control, the level of the correctly spliced AtMinE1
transcripts in atminE1 was estimated to be approximately
2,000-fold reduced compared with that in the WT.
348
Table 1
The chloroplast Min system
Number of chloroplasts per leaf mesophyll cell in the atminE1 mutanta
Plant and
total no.
of cells
No. of chloroplasts per cell (%)
0
1
2
3
4
5–20
21–40
41–60
61–80
81–100 4100
WT (Ws) 56 0 (0.0) 0 (0.0)
0 (0.0)
0 (0.0) 0 (0.0) 0 (0.0) 9 (16.1) 24 (42.9) 15 (26.8) 5 (8.9) 3 (5.4)
atminE1 117 0 (0.0) 53 (45.3) 46 (39.3) 16 (13.7) 2 (1.7) 0 (0.0) 0 (0.0)
0 (0.0)
0 (0.0) 0 (0.0) 0 (0.0)
a
This experiment was performed using the third leaves of the 1-month-old plants. Mean chloroplast number per cell is 60.1 and 1.7 for
WT (Ws) and atminE1 plants, respectively.
The morphology of the atminE1 chloroplasts was next
microscopically characterized using expanded mature
leaves. Chloroplast autofluorescence imaging revealed
either single or a few giant chloroplasts per mesophyll
cell, whereas a homogeneous population of round to
ellipsoidal chloroplasts of 4–6 mm in length, and 40–100
per cell, are distributed in WT cells (Fig. 1E). These
chloroplast abnormalities in atminE1 persisted during leaf
development and through successive plant generations (data
not shown). For the linkage analysis, the atminE1 mutant
homozygous for the T-DNA insertion was backcrossed with
the WT to produce the hemizygous F1 plants, all of which
had a restored phenotype with respect to giant chloroplast
formation (data not shown). In 568 F2 progeny, the ratio of
the restored and the giant chloroplast phenotypes was 3.1 : 1
(431 : 137 individuals for actual data), and 90 individuals,
which were arbitrarily selected from the 137 individuals
with the giant chloroplast phenotype, were all homozygous
for the T-DNA insertion (data not shown). This analysis
confirmed that the aberrant chloroplast phenotype was
tightly associated with the presence of a homozygous
T-DNA insertion within the AtMinE1 gene. This giant
chloroplast phenotype of atminE1 is distinct from that of
arc5 or arc11 which contains enlarged and dumbbellshaped chloroplasts or heterologous chloroplasts, respectively (Pyke and Leech 1994, Marrison et al. 1999), but
closely resembles that of arc6, a mutant with an extreme
division inhibition phenotype (Pyke et al. 1994)
(Supplementary Fig. S2). Moreover, the average chloroplast
number per leaf mesophyll cell in atminE1 was found to be
1.7, and no mesophyll cells lacking chloroplasts could be
observed (Table 1). In partial support of this, several
AtMinE1 antisense plants with reduced levels (i.e. 25–33%
of WT) of AtMinE1 transcripts (Itoh and Yoshida 2001)
displayed intermediary division inhibition phenotypes
between WT and atminE1, with respect to chloroplast
number and morphology, suggesting an AtMinE1
expression level–chloroplast phenotype relationship
(Supplementary Fig. S2D). Taken together, these data
demonstrate that AtMinE1 is indispensable for chloroplast
division.
Formation of mini-chloroplasts as a terminal effect of
AtMinE1 overexpression
The effects of AtMinE1 overexpression on photosynthetic tissues have been previously investigated, and these
studies led to the identification of expanded, heteromorphic
chloroplasts with either decreased numbers in mature leaf
mesophylls (Itoh et al. 2001, Reddy et al. 2002), marked size
heterogeneity (Maple et al. 2002, Reddy et al. 2002) or
hypocotyl chloroplasts that display asymmetric or multiple
constriction sites (Maple et al. 2002). However, the use of
different plant tissues and methodologies, in addition to
the lack of quantitative analyses, has suggested that
the chloroplast phenotypes resulting from AtMinE1 overexpression may not be fully resolved (Fujiwara and Sato
2004).
In an attempt to elucidate this properly, we
re-examined chloroplast morphology in both the mature
and developing leaves of the AtMinE1 sense (overexpressor)
transgenic plants. The AtMinE1 sense transgenic line, mEs
#02, displays normal growth and high accumulation
(120-fold) of AtMinE1 transcripts relative to the WT
(Itoh et al. 2001). RT–PCR using an oligo(dT) as the
primer for first-strand cDNA synthesis confirmed the
occurrence of stable AtMinE1 overexpression (4100-fold
accumulation of sense transcripts), but not co-suppression
as seen in the case of GC1 (Maple et al. 2004), in the
mEs #02 seedlings of the T4 generation (Fig. 1D). In
living mature leaves of the T4 transgenic plants, minichloroplasts, tiny spherical chloroplasts 52 mm in diameter,
appeared frequently, and co-existed with enlarged
chloroplasts in a population of leaf mesophyll cells
(Fig. 2J, Supplementary Fig. S2E and Table 2). Simultaneously, the presence of a few or even a single giant
chloroplast and the reduction of chloroplast number per
cell, as we described earlier (Itoh et al. 2001), were also
consistently observed in our current study (Supplementary
Fig. S2E). Thus, there are two aspects of the terminal
phenotypes observed in the AtMinE1 sense chloroplasts:
striking, but occasionally negligible, size heterogeneity of
chloroplasts and the universal reduction of chloroplast
number per cell.
The chloroplast Min system
A
B
C
D
F
J
349
E
G
H
I
K
L
Fig. 2 Distinctly aberrant chloroplast morphologies resulting from both the loss of function and gain of function of AtMinE1 in
developing green tissues of Arabidopsis. Chloroplasts from the primary and secondary leaf petioles of 2-week-old seedlings were observed
by microscopy using DIC optics. (A) WT (Col). (B) WT (Ler). (C) WT (Ws). (D) AtMinE1 antisense. (E) atminE1. (F, G, J, K) AtMinE1 sense.
(H, I, L) arc11 (atminD1). The sites of the chloroplast division plane at the early and late stages of division, which were distinguished from
the contact sites of two chloroplasts, are indicated by white and black arrowheads, respectively. Mini-chloroplasts present in AtMinE1
sense and arc11 mutant cells are indicated by arrows (I, J). Images in (J, K, L) are derived from isolated cells from unfixed leaf tissues.
Bars, 10 mm.
Quantitative analysis of the effects of AtMinE1 overexpression on chloroplast division in developing leaves
Chloroplast division in higher plants takes place
concurrently with the development of chloroplasts (Pyke
1999). In order to define precisely the pre-terminal
phenotypes of chloroplasts in AtMinE1 overexpressor
plants, we performed a quantitative analysis of the
chloroplast division states at an early stage of seedling
development using leaf petioles from WT and mEs #02
(Fig. 3, Supplementary Fig. S3 and Table 3). WT
chloroplasts of 4–6 mm length in non-division states and
7–13 mm in dividing states by symmetric binary fission were
350
The chloroplast Min system
Table 2 Frequency of the formation of mini-chloroplasts in
leaf mesophyll cells of the AtMinE1 sense and arc11 plantsa
Plant
AtMinE1 sense
arc11
No. of minichloroplastsb/
total no. of
chloroplasts (%)
39/447 (8.7%)
46/582 (7.9%)
a
Primary leaves from 1-month-old AtMinE1 sense and arc11 plants
were examined.
b
A mini-chloroplast was defined as a chloroplast52 mm in diameter.
observed (Fig. 2A–C). AtMinE1 T-DNA mutant or (selected) antisense chloroplasts were found to be severely prevented from dividing, resulting in the generation of giant
chloroplasts with a reduced number per cell (Fig. 2D, E). In
contrast, AtMinE1 sense chloroplasts displayed distinct phenotypes in terms of their size, shape and division state: tiny to
expanded, or highly elongated organelles, having single or
multiple constriction sites were evident (Figs. 2F, G, 3).
Multiple arrayed chloroplasts of the AtMinE1 sense
plants that had two or more (up to seven) division
constriction sites constituted approximately 35% of the
leaf petiole (cortex) chloroplasts. These were classified into
three types on the basis of the spatial relationships inside the
chloroplasts, ‘parallel’ (84.7%), ‘unparallel’ (7.6%) and
‘fusion’ (7.6%) (Fig. 3A and Tables 3, 4). The major
‘parallel’ type harbors division planes that are placed
perpendicularly or obliquely to the long axis of the
chloroplast, and also display a random nature in their
intrachloroplast distribution (e.g. spaced at 1–20 mm intervals) and constriction progress (Fig. 3B, D, and
Supplementary Fig. S3). In this type, mini-chloroplasts
can arise from not only the poles, but also from an internal
region of the chloroplasts by asymmetric binary or multiple
fission (Fig. 2F). Interestingly, these mini-chloroplasts are
further capable of division by binary fission (Fig. 2J).
Identification of the ‘unparallel’ and ‘fusion’ chloroplast
types showed complexity in the spatial determination of the
division sites in the absence of AtMinE1 repression.
Two-lobed chloroplasts of the AtMinE1 sense plants,
which constituted approximately 25% of the chloroplasts in
the leaf petiole region (Table 3), were found to follow a
symmetric binary fission in 50% of the cases (Fig. 3C,
yellow region). This preference of the division site for the
mid-chloroplast was found to be the case irrespective of
chloroplast size, and is unexpectedly high if a simple
assumption of a non-preferential selection for a division
site along the chloroplast length is made. No other regular
patterns for division sites could be found.
It is noteworthy that the comparative characterizations revealed equivalent chloroplast phenotypes in
AtMinD1-inactivated plants (Marrison et al. 1999, Colletti
et al. 2000) and AtMinE1 sense plants. The classification of
multiple arrayed chloroplasts in AtMinE1 sense (Fig. 3)
plants was applicable to those of the arc11 (atminD1)
mutant (Fujiwara et al. 2004) (Table 4). Our quantitative
data on the chloroplast division states, as well as microscopic imagery of both developing and mature leaves of
arc11, are very similar to the findings in the AtMinE1 sense
plants (Fig. 2, Tables 3, 4, and Supplementary Fig. S2E, I).
In addition, mini-chloroplasts from the arc11 mutant were
generated from various parts of the chloroplast body and
were also found to be capable of division (Fig. 2I; data
not shown).
Combining data on loss-of-function and gain-offunction (overexpression) analyses of AtMinD1 and
AtMinE1 mutants (summarized in Fujiwara and Sato
2004, Aldridge et al. 2005, this study), we speculate that
AtMinD1 and AtMinE1 have opposing roles in the
regulation of chloroplast division: either AtMinD1 overexpression or AtMinE1 inactivation results in a complete
block of chloroplast division, whereas their opposing effects
lead to aberrant chloroplast division site placements, but
result in almost equivalent phenotypes.
Ultrastructural analysis of chloroplasts in transgenic
AtMinE1 plants
To gain further insights into the ultrastructure of
chloroplasts in AtMinE1 transgenic plants, transmission
electron microscopy was performed using mature and
developing leaves from plants grown under normal conditions. As a result, chloroplasts in the AtMinE1 sense plants
were found to develop poor grana stacks at an early stage of
their development, whereas the AtMinE1 antisense or
atminE1 plants had chloroplasts with normally organized
WT thylakoid systems, such as the layered grana and the
directional distribution of thylakoid membranes (Fig. 4;
data not shown). This unusual interior of the AtMinE1
sense chloroplasts is limited to the developmental stages,
however, and is compromised as the leaves mature. In
matured leaves, the AtMinE1 sense, antisense and atminE1
chloroplasts are indistinguishable apart from grossly
expanded envelope membranes that appear variably misshapen between individual organelles (Itoh et al. 2001)
(Fig. 4; data not shown). These observations suggest that
AtMinE1 overexpression causes a certain delay or an
alteration in chloroplast development, directly or indirectly,
during leaf development.
Aberrant FtsZ ring formation underlies symmetric,
asymmetric and multiple chloroplast division in the
AtMinE1 overexpressor and arc11 plants
To investigate the structure and behavior of FtsZ
in chloroplasts with defects in division site placement,
we generated transgenic Arabidopsis plants, which express a
The chloroplast Min system
351
Binary fission
A
d1
d1
WT
a1
a1 a2
a2
Multiple fission
Binary fission
Parallel
d1
AtMinE1
sense
Parallel and Unparallel
d2 d1
d3
d1
d2
d4
a3
a3
a1
a4
a5
a2
a2
a1
Fusion
d5
a2
a1
Number of division plane
per chloroplast
B
5
WT
AtMinE1 sense
4
3
2
1
0 1
5
10
15
20
25
30
35
38
Chloroplast length (µm)
D
WT
No. 1
AtMinE1 sense
No. 2
a1
d1
a2
d2
a3
10 µm
30
No. 3
20
No. 4
Diameter of division plane
Chloroplast length (µm)
C
No. 5
10
No. 6
No. 7
No. 8
0
10
20
30
40
Position of division plane from
pole (% of chloroplast length)
50
No. 9
0
10
30
20
Chloroplast length (µm)
Fig. 3 Number, distribution and diameter of division planes in the chloroplasts of AtMinE1-overexpressing plants. Dividing chloroplasts
in the primary and secondary leaf petioles of 2-week-old seedlings were characterized statistically (see also Supplementary Fig. S3).
(A) Patterns of chloroplast division in WT (Col) and AtMinE1 sense plants. Multiple divisions of AtMinE1 sense chloroplasts are further
classified into ‘Parallel’, ‘Parallel and Unparallel’ and ‘Fusion’ patterns on the basis of their division plane distributions. Positions of the
division planes (black lines; d1, d2,..) and long axes (red lines; a1þ a2þ) in the chloroplasts are also indicated. Bar, 10 mm. (B) Number of
division planes along the chloroplast length in WT and AtMinE1 sense plants. Chloroplasts with 1–5 constriction sites in the WT (filled
circles; sample number n ¼ 30) and AtMinE1 sense plants (open circles; n ¼ 50) are included on the plot. (C) Distribution of the division
planes in AtMinE1 sense chloroplasts undergoing binary fission. The position of the division plane from the pole (% of chloroplast length) is
shown for binary dividing chloroplasts of the WT (filled circles; n ¼ 25) and AtMinE1 sense (open circles; n ¼ 40). It is noteworthy that
60% of AtMinE1 sense chloroplasts (indicated in the yellow region which covers all WT chloroplasts) undergo central binary fission
independently of their length. (D) Distribution and diameter of division planes in AtMinE1 sense chloroplasts undergoing multiple
divisions. The positions (open squares) and diameters (vertical lines) of the division planes along the long axes of the chloroplasts
(horizontal lines) for an arbitrary selection of nine organelles are indicated. For the definition of division planes (d1, d2,..) and their intervals
(a1, a2,..), see also (A).
352
Table 3
The chloroplast Min system
Comparison of the number of division plane(s) in dividing chloroplasts of leaf petiole cellsa
Plantb
No. of division planes per chloroplasts (%)
0
WT (Col)
AtMinE1 sense
AtMinE1 antisense
atminE1
arc11
152
59
58
55
60
(76.0%)
(38.3%)
(87.9%)
(100%)
(40.8%)
1
48
41
7
0
37
(24.0%)
(26.6%)
(10.6%)
(0%)
(25.2%)
2
0
25
1
0
27
3
(0%)
(16.2%)
(1.5%)
(0%)
(18.4%)
0
22
0
0
13
(0%)
(14.3%)
(0%)
(0%)
(8.8%)
Total no. of
chloroplasts
4
0
5
0
0
4
(0%)
(3.2%)
(0%)
(0%)
(2.7%)
44
0
2
0
0
6
(0%)
(1.3%)
(0%)
(0%)
(4.1%)
200
154
66
55
147
(100%)
(100%)
(100%)
(100%)
(100%)
a
Primary leaves from 2-week-old plants were sampled. Chloroplasts with obvious constriction site(s) were examined.
Control data of WT plants for atminE1 (Ws) and arc11 (Ler) are omitted from this table.
b
Table 4 Classification of the division plane distribution
patterns in multiple arrayed chloroplasts of AtMinE1 sense
and arc11 mutant plantsa
Plant
No. of division planes (%)b
Parallel
Total no. of
division
planes (%)
Unparallel Fusion
AtMinE1 100 (84.7%) 9 (7.6%) 9 (7.6%) 118 (100%)
sense
arc11
143 (87.7%) 13 (8.0%) 7 (4.3%) 163 (100%)
a
The same tissues as analyzed in Table 3 were examined.
See also Fig. 3A.
b
full-length AtFtsZ1–green fluorescent protein (GFP) fusion
under the control of an upstream genomic sequence of
AtFtsZ1-1. Of the 16 stable lines obtained, a dozen lines
showed giant or weakly expanded chloroplasts in leaf cells
(Supplementary Fig. S4), as a known consequence of the
overexpression of an AtFtsZ1–GFP (Vitha et al. 2001). To
avoid such artifactual effects of FtsZ overexpression on
chloroplast division, we chose two transgenic lines, for
further experiments, which were found to express AtFtsZ1–
GFP moderately without affecting normal chloroplast
division but to be sufficient for visualization of the
chloroplast FtsZ ring. In these lines, the GFP-labeled
FtsZ (FtsZ1) ring appears at the mid-chloroplast as a
continuous, thin and smooth line (Fig. 5A, B) in the same
manner as previously confirmed by the refined immunofluorescence staining (Vitha et al. 2001).
By crossing with the above transformants, we introduced the AtFtsZ1–GFP gene into the nuclear genome of
AtMinE1 sense or arc11 mutant plants. As expected, the
obtained F2 (second filial) plants in their respective backgrounds grew normally, had chloroplasts with aberrant
division site(s) and expressed chloroplast stroma-targeted
AtFtsZ1–GFP in their leaf cells (data not shown).
Epifluorescence microscopy was subsequently performed
on the developing leaves of these same lines at the F2 or F3
generation, as well as on the parental AtFtsZ1–GFPexpressing line as a control.
Our results revealed the formation of up to 11 FtsZ
rings in both the AtMinE1 sense and arc11 chloroplasts
with similar patterns. In binary dividing chloroplasts, which
we define as chloroplasts with a single FtsZ ring or a sole
constriction site, the FtsZ ring was found to be located at a
central or non-central division site from mini- to enlarged
chloroplasts (see Supplementary Fig. S5). The morphology
and behavior of the ring in terms of constriction depth until
separation of the two daughter chloroplasts were comparable with those of the WT, indicating their functionality
(Fig. 5C). However, two characteristic features of FtsZ were
evident during the chloroplast division cycle of AtMinE1
sense and arc11 mutant plants. One is the frequent
appearance of extra FtsZ rings, which are separate from
the contractile FtsZ ring and are seemingly merged with the
chloroplast periphery (Fig. 5C, yellow double arrowheads).
The other is an occasional thin filamentous structure
adjacent to a true FtsZ ring, which might be a complete
or incomplete ring or a spiral, and is mostly seen at earlier
stages of chloroplast division (Fig. 5C, white double
arrowheads). It remains unknown whether these latter
structures disappear at a later stage, assemble into the main
ring or grow into a separate division ring, as the AtFtsZ1–
GFP signal in live tissues was too weak to trace by timelapse microscopy.
The multiple dividing chloroplasts represent extended
phenotypes of the binary dividing chloroplasts. All the
constriction sites, which were obvious in live chloroplasts,
corresponded to the sites of the FtsZ ring (not all the FtsZ
rings seemed to be localized at the constriction sites)
(Fig. 5D). These include our rare observations of two
FtsZ rings/filaments connecting probably at the chloroplast
periphery and explaining the basis of fused division planes
in AtMinE1 sense or arc11 chloroplasts (Fig. 5D, see arc11).
A number of FtsZ rings were also found to be located
at non-constriction sites at random intervals, mostly
The chloroplast Min system
A
C
B
D
353
E
F
G
H
Fig. 4 Ultrastructure of developing chloroplasts in AtMinE1 transgenic plants. Electron micrographs of chloroplasts in the developing leaf
mesophylls of 2-week-old seedlings. (A, B) WT (Col). (C, D) AtMinE1 antisense. (E, F) AtMinE1 sense. (G, H) atminE1. The ultrastructure of
chloroplasts in the WT (Ws) was substantially similar to that of WT (Col) chloroplasts (data not shown). Bars, 5 mm (A, C, E, G) and 1 mm
(B, D, F, H).
perpendicular or oblique to the long axis of the chloroplast
(see Supplementary Fig. S6). Both these and the extra FtsZ
ring(s) in binary dividing chloroplasts may represent
initiation of the chloroplast division cycle at their sites,
which would promote chloroplast elongation according to
their polarity and membrane constriction formations.
Taken together, these data demonstrate that AtMinE1
overexpression or AtMinD1 inactivation leads to the altered
assembly and misplacement of chloroplast FtsZ ring(s),
which are licensed to form before completion of the
preceding chloroplast division at the other sites. Arrayed
chloroplasts with varying constriction depths can thus be
produced.
Altered FtsZ dynamics in chloroplasts of the atminE1 mutant
The above data and Vitha et al. (2003) demonstrated
the formation of multiple FtsZ rings in chloroplasts of the
AtMinE1 overexpressor, arc11, and AtMinD1 antisense
plants, and the fragmentation of FtsZ filaments in
chloroplasts of the AtMinD1 overexpressor plants.
Whereas these results suggest the mutually antagonistic
roles of AtMinD1 and AtMinE1 in the assembly of
chloroplast FtsZ, demonstration of the FtsZ dynamics in
chloroplasts of the AtMinE1-dysfunctional plants is lacking. Therefore, we introduced the AtFtsZ1–GFP gene into
the nuclear genome of atminE1 plants by the same methods
as employed for the AtMinE1 sense and arc11 plants
(see the previous section), and observed the petioles of the
F2 plants by epifluorescence microscopy. The AtFtsZ1–
GFP signal was quite weak, but was observed exclusively
within enlarged chloroplasts of atminE1. The atminE1
chloroplasts contained the mixture of numerous dots and
short filaments of AtFtsZ1–GFP (Fig. 5E), in the same
fashion as the AtMinD1 sense chloroplasts did (Vitha et al.
2003). Some FtsZ filaments appeared to emanate from FtsZ
dots (Fig. 5E, triple arrowhead), which might represent the
‘seeds’ of FtsZ filaments that would be masked in closed
FtsZ rings of WT chloroplasts. Surprisingly, these FtsZ dots
and filaments in atminE1 chloroplasts were motile
(Supplementary Movie S1). It is noteworthy that this is
the first observation, to our knowledge, to provide evidence
for the mobility of cytoskeleton-like elements within
plastids. Such motility of the FtsZ dots and filaments
might be due to the instability of the FtsZ assembly, which
would be caused by the stoichiometric dominance of
AtMinE1 against AtMinD1. These observations on
atminE1 plants further validate our working model on the
chloroplast Min system, where AtMinD1 and AtMinE1
have opposing roles in the FtsZ assembly upon chloroplast
division.
FtsZ ring-mediated chloroplast division can be arrested
during leaf development in the AtMinE1 overexpressor and
arc11 plants
To elucidate the differences between the morphologies
of developing and mature chloroplasts in AtMinE1 sense
and arc11 plants (Fig. 2, and Supplementary Fig. S2), we
extensively analyzed the chloroplast morphology and the
configuration of the FtsZ ring during chloroplast development. To this end, we conducted observations of leaf
petioles from the middle to upper regions (Fig. 6A). In the
third to fourth leaf petioles, a gradient of chloroplast
development along the petiole polarity was evident from the
criteria of the chloroplast size and the intensity of
chlorophyll autofluorescence, while in the first to second
leaf petioles such a gradient was obscure, and chloroplasts
were wholly in a more mature state as compared with those
in the corresponding region of the third to fourth leaf
354
The chloroplast Min system
A
B
Division Stage
IV
Chl
Merged
C
Stage I – II
Merged
Stage IV – V
GFP
Merged
GFP
Merged
AtMinE1 sense
GFP
arc11
Multiple division
arc11
AtMinE1
sense
arc11
E
atminE1
Symmetric division
ii
AtMinE1
sense
Asymmetric division
i
TopMiddle Middle
D
Merged
WT
GFP
Top
V
Non-GFP
III
AtFtsZ–GFP
II
GFP
I
Fig. 5 FtsZ rings and disorganized FtsZ filaments in chloroplasts of AtMinE1 overexpressor, arc11 and atminE1 plants. A chimeric gene
comprising an AtFtsZ1-1 cDNA fused to the green fluorescent protein (GFP) gene was introduced into living cells of WT (Col) Arabidopsis,
and was expressed stably under the control of the genomic upstream region of AtFtsZ1-1. AtMinE1 sense, arc11 and atminE1 plants were
crossed with the AtFtsZ1–GFP-expressing transgenic Arabidopsis line. Chloroplasts in the primary to fourth leaf petioles from 2-week-old
seedlings were observed by epifluorescence microscopy. (A) Behavior of the FtsZ ring during chloroplast division. Images of GFP, Chl
and merged (GFP, green color; Chl, red color) signals are shown. Note that, in addition to the ring structure, faint diffusion signals of
AtFtsZ1–GFP existed over the chloroplasts, even in the presence of weak noise generated by chloroplast autofluorescence (see right
panels). (B) The ring structure of FtsZ1 observed at different foci by microscopy. The focal plane was shifted from the top to the middle of a
chloroplast (i) attached to the cell periphery of the leaf epidermis (indicated by arrows). An intact FtsZ ring observed in a focal plane is
shown in (ii). (C) Central and non-central positioning of the FtsZ ring in binary dividing chloroplasts of AtMinE1 sense and arc11 plants.
Images of GFP fluorescence and merged images of GFP (green color) and chlorophyll autofluorescence (red color) are shown. White and
yellow double arrowheads indicate a thin filamentous structure adjacent to a true FtsZ ring and extra FtsZ rings separated from the FtsZ ring
located at the constriction site, respectively. (D) Numerous and asymmetric distribution of FtsZ rings in multiple dividing chloroplasts of
AtMinE1 sense and arc11 plants. The single and double arrowheads indicate the positions of the FtsZ ring at the constriction and nonconstriction sites, respectively. Identification of the constriction sites was performed carefully by observation of the greatly magnified
images of autofluorescence-emitting chloroplasts (not shown). The arrow indicates FtsZ that has formed into an incomplete or harsh
filament. (E) Numerous FtsZ dots and short, disorganized FtsZ filament fragments in enlarged chloroplasts of atminE1 mutant plants. A triple
arrowhead indicates the FtsZ dot which looks like it is emanating from the short FtsZ filament. Bars, 3 mm.
The chloroplast Min system
Lo
A
we
r
Mi
dd
le
Up
pe
355
r
First Leaf
Third Leaf
This study
(Fig. S7)
Third Leaf Petiole
B
First Leaf Petiole
Upper
Lower
Middle
Upper
DIC
Chl
Middle
Fujiwara et al. (2004) This study
and this study (Fig. 6) (Fig. S7)
D
Third-to-Fourth Leaf
Petiole, Middle
Third-to-Fourth Leaf First-to-Second Leaf
Petiole, Upper
Petiole, Upper-Middle
C
Fig. 6 Alterations in chloroplast morphology and in the configuration of the FtsZ rings during chloroplast development. Chloroplasts in
the first layer of cortical cells at the adaxial side of the leaf petiole of AtMinE1 sense seedlings were observed by epifluorescence
microscopy (see Fig. 5 for plant materials). (A) The leaf petiole region used for microscopic characterizations. The first to second and the
third to fourth leaf petioles were dissected into three regions (upper, middle and lower) (see left panels), and the upper and middle regions
were selected to view elongated cortical cells in the central petiole area. The right panel (the rectangle in the upper left panel was
magnified and rotated) provides information on the distribution of cortex cells and precise areas of microscopic observations as indicated
underneath the image. Cortical cells in the central petiole area were previously used to characterize arc11 chloroplasts (Fujiwara et al.
2004). Note that marginal tissues of the upper and middle petiole regions correspond to mesophylls at the base of the leaf blade, and the
observation data are shown in Supplementary Fig. S7. (B) Gradient of chloroplast development in the leaf petioles. Signals of chlorophyll
autofluorescence (Chl) from the first and the third leaf petioles of 2-week-old WT (Col) seedlings were detected by epifluorescence
microscopy with the fixed excitation condition. Chloroplast development along the petiole polarity is evident from the criteria of the
chloroplast size and the intensity of chlorophyll autofluorescence. (C) AtFtsZ1–GFP fluorescent patterns during chloroplast development.
Chloroplasts in different leaf petiole regions from 2-week-old seedlings were observed. The chloroplast morphology and the FtsZ
ring/filament structures are markedly altered with the progress of chloroplast development. Arrowheads and double arrowheads indicate
mild or deep constriction sites, respectively. (D) No detection of the chloroplast FtsZ ring in leaf petioles of 2-month-old AtMinE1 sense
seedlings. Diffusive fluorescence signals, but not any types of distinct structures, were detected in chloroplasts. Bars, 1 mm (A, left), 100 mm
(A, right), 10 mm (B) and 3 mm (C, D).
356
The chloroplast Min system
petioles (Fig. 6B). We found that in cortical cells at the
upper to middle regions of the first to second leaf petioles
from AtMinE1 sense plants, the chloroplasts could still
retain single, double or multi-parallel FtsZ rings or
filaments in their expanded and non-constricted shapes
(Fig. 6C). Similar configurations of chloroplast FtsZ rings/
filaments were also observed in the corresponding regions
of leaf petioles from arc11 plants (data not shown). These
FtsZ structures seemed to be largely influenced by the
envelope membrane morphology, and were not necessarily
complete rings. If all of the FtsZ rings in those expanded
chloroplasts act to execute membrane fission, we would
expect that dumbbell-shaped or multiple-arrayed chloroplasts would be evident in older leaf cells. However, such
organelles were rarely detected, indicating that division of
giant chloroplasts is very unlikely. Instead, the frequency
of FtsZ ring(s) was observed to decrease (Fig. 6D), keeping
pace with the leaf age, suggesting protein depolymerization
or degradation inside the chloroplasts. In addition, our
observations at the middle petiole region of the third to
fourth leaves found that in chloroplasts having mild or deep
constriction(s) with co-localizing FtsZ ring(s) (Fig. 6C, single
or double arrowheads), and in some expanding lobes of
dividing chloroplasts, deformed FtsZ rings/filaments could
have already been detected (see Fig. 5D, arrow). Thus, at
least a certain population of FtsZ filaments/rings observed in
mature giant chloroplasts are reminiscent of FtsZ rings
generated at developing stages, but not newly formed.
We extended our observation to the marginal tissues of
the petiole (see Fig. 6A for the precise position), which
correspond to mesophylls at the base of the leaf blade, from
AtMinE1 sense and arc11 plants. In the marginal tissues,
chloroplasts appeared to be developmentally more mature
than those in the central petiole area, and contained FtsZ
filaments/rings formed at both the constriction and nonconstriction sites (Supplementary Fig. S7), in the same
manner as did chloroplasts in the central, upper to middle
area of the first to second petioles (Fig. 6C). This confirms
frequent failure of chloroplast division during leaf development in AtMinE1 sense and arc11 plants, in spite of the
formation of FtsZ rings. In addition, this might explain our
previous observation (Itoh et al. 2001) that mesophyll cells
in mature leaves from AtMinE1 sense plants contained
giant, occasionally multilobed, chloroplasts.
These observations support the contention that the
FtsZ ring-mediated division–constriction processes can be
arrested and that not all the chloroplast FtsZ rings in
AtMinE1 sense and arc11 plants will complete chloroplast
fission. Such uncoupling of FtsZ ring formation and
chloroplast division may explain the universal reduction
of chloroplast number per cell, as well as the pronounced
chloroplast heterogeneity in mature leaf cells of AtMinE1
sense and arc11 plants.
Discussion
Symmetric cell division is a universal process from
bacteria to plants, fungi and animals, and it has long been
documented that chloroplasts also undergo symmetric
binary fission (Possingham and Lawrence 1983). The
regulatory mechanisms underlying chloroplast division
have been the subject of much debate and speculation
(Hashimoto 2003, Møller 2004, Lopez-Juez and Pyke 2005),
but we show in our current study in A. thaliana that this
event is achieved by a negative mechanism, involving at
least three conserved factors, FtsZ, MinD and MinE.
We demonstrate that AtMinE1 is essential for chloroplast division. The atminE1 mutation causes chloroplast
and FtsZ fragmentation phenotypes that are almost
identical to the arc6 mutation (Pyke et al. 1994, Vitha
et al. 2003) and to AtMinD1 overexpression (Colletti et al.
2000, Kanamaru et al. 2000, Vitha et al. 2003). Also,
consistent with our former data showing that the AtMinE1
promoter is activated at the shoot apex (Itoh et al. 2001), a
critical role for AtMinE1 in proplastid replication was
implied. atminE1 leaf mesophyll cells can contain a single or
a few giant chloroplasts. This raised the question of how the
plastids are replicated to keep pace with cell division, as
presumably plastid replication occurs at least once per cell
division cycle, and an almost equal number of plastids are
properly segregated and inherited by the daughter cells.
This implies that a plastid partition mechanism during cell
proliferation is still active in the absence of AtMinE1, and
that this is independent of, or is distantly associated with,
the AtMinE1-mediated plastid division mechanism.
Dual effects of AtMinE1 overexpression upon chloroplast
division
Despite seemingly different results and conclusions
from earlier studies on AtMinE1 sense (overexpressor)
chloroplasts, using different methods, plant tissues, and in
backgrounds with differing AtMinE1 mRNA levels (Itoh
et al. 2001, Maple et al. 2002, Reddy et al. 2002), we
contend that these sets of findings are not at all contradictory, but in fact complement each other. The results of
our present comprehensive analyses, including our observation of replicable mini-chloroplasts, emphasize two aspects
of chloroplast abnormalities resulting from AtMinE1
overexpression: varied chloroplast heterogeneity in terms
of size and shape, and a decrease in the chloroplast number
per cell. On the other hand, we have revealed two significant new findings regarding the nature of the regulatory mechanisms underlying chloroplast division site
placement. The first is a negative mechanism for symmetric
binary fission. In AtMinE1 sense chloroplasts, division
constriction can occur at any region along the chloroplast
length, including the mid-chloroplast. Non-synchronous
The chloroplast Min system
division constrictions, as well as elongated but unfixed
chloroplast shapes in AtMinE1 sense plants, indicate the
random nature of the spatial and temporal coordination of
the chloroplast division site(s). The second is preferential
division site selection at the mid-chloroplast. AtMinE1
overexpression causes not only asymmetric or multiple, but
also a high frequency of symmetric chloroplast binary
fissions. It might be possible that unequal distribution of
chloroplast division factors occurs upon asymmetric chloroplast division in AtMinE1 sense plants. This should lead to
differences in amounts of each of the division factors among
chloroplasts, in a chloroplast to chloroplast or cell to cell
dependent manner. If this is the case, chloroplasts containing appropriately balanced amounts of division factors by
chance would reasonably divide symmetrically in the same
fashion as those in WT plants do. Nevertheless, we prefer a
more straightforward interpretation; a high percentage of
symmetrically dividing chloroplasts in AtMinE1 sense
plants might represent an inherent preference for midchloroplast Z-ring positioning by FtsZ, even when the Min
system is not functioning properly.
Conserved and opposing roles of AtMinE1 and AtMinD1 in
chloroplast division
We have shown that the dividing and terminal
chloroplast phenotypes of the AtMinE1 overexpressor and
arc11 mutant plants are almost equivalent, whereas no
comparative studies on this issue have been conducted so
far. Reciprocally, the chloroplast and the FtsZ assembly
phenotypes of the atminE1 mutant plants (this study) and
the AtMinD1 overexpressor plants (Vitha et al. 2003) have
also been proved to be substantially equivalent. This implies
an inhibitory role for AtMinD1 and an antagonistically
facilitative role for AtMinE1 in formation and/or maintenance of FtsZ filaments in chloroplasts (Fig. 7). The
balance of AtMinD1 and AtMinE1 protein activities
determines the symmetric binary fission of WT chloroplasts.
When AtMinD1 dominates AtMinE1 by either AtMinD1
overexpression or AtMinE1 inactivation, chloroplast division is completely blocked via the production of motile dots
and short filaments of FtsZ, resulting in the generation of
several giant chloroplasts per mesophyll cell. Conversely, in
AtMinE1 overexpression and AtMinD1 inactivation, aberrant division site placement of chloroplasts takes place via
the alterations of the assembly and distribution of the FtsZ
ring along the chloroplast polarity axis (see below). This
results in a chloroplast heterogeneity ranging from negligible to remarkable in terms of both size and shape, and in a
decreased number of chloroplasts per cell.
This highly sensitive relationship is in accord with the
E. coli cell division model, where a complex interplay of
MinD and MinE controls FtsZ ring placement
(RayChaudhuri et al. 2000, Rothfield et al. 2005). In the
357
Division Phenotype
A
Terminal Phenotype
AtMinE1
AtMinD1
FtsZ1 ring
B
C
AtMinE1
AtMinD1
or
AtMinE1
AtMinD1-ox
AtMinE1-ox
AtMinD1
or
AtMinE1
AtMinD1
FtsZ1 ring
Fig. 7 Summary of the Min expression level–chloroplast phenotype relationship in Arabidopsis. (A) In leaf cells, the normal (WT)
expression levels of AtMinD1 and AtMinE1 ensure the symmetric
binary fission of chloroplasts, which is mediated by placement of
the FtsZ ring at the mid-chloroplast both before and during
envelope membrane constriction (McAndrew et al. 2001, Vitha
et al. 2001). (B) T-DNA inactivation of AtMinE1 (this study) or
overexpression of AtMinD1 (Colletti et al. 2000, Kanamaru et al.
2000, Vitha et al. 2003) causes the generation of numerous dots
and short filaments of FtsZ, instead of the closed FtsZ ring as
observed in WT chloroplasts, and hence severe inhibition of
chloroplast division, resulting in the formation of a single or few
chloroplasts per cell. (C) The overexpression of AtMinE1 (Itoh et al.
2001, Maple et al. 2002, Reddy et al. 2002, this study) or the
underexpression or loss of function of AtMinD1 (Marrison et al.
1999, Colletti et al. 2000, Fujiwara et al. 2004) leads to a
heterologous population of chloroplasts per cell in mature leaves.
The underlying cellular mechanism involves the formation of single
or multiple FtsZ ring(s), mostly along the chloroplast polarity axis,
that induces symmetric, asymmetric or multiple divisions of
chloroplasts in developing leaves. Furthermore, occasional failures
of FtsZ rings to make chloroplast division constriction, which
become apparent as the chloroplasts mature, are likely to
contribute to the number reduction and size heterogeneity of
chloroplasts in cells (this study).
case of MinE, its overexpression permits cell division at all
PDSs, and its inactivation leads to complete division block
(de Boer et al. 1989). Despite certain differences between
these two systems, as represented by the absence of a MinC
homolog in chloroplasts and the involvement of the
ARC3 protein in the division site placement of chloroplasts
(Maple et al. 2007, reviewed in Glynn et al. 2007), the
conservation of the overall functions of MinD and MinE in
both E. coli cells and chloroplasts is surprising. The fact that
the introduction of E. coli MinC into tobacco chloroplasts
358
The chloroplast Min system
blocks their division might further emphasize this functional
conservation (Tavva et al. 2005). On the contrary, there
seems to be less conservation between Min systems of the
chloroplast and the cyanobacterium Synechocystis sp.
PCC6803, whose minD null mutant often displays the
spiraled cell shape and whose minE null mutant remains
capable of division and thus is not lethal, unlike E. coli and
the chloroplast (Mazouni et al. 2004). The greater similarity
of the chloroplast Min system to that of E. coli, rather than
to that of Synechocystis, is intriguing, in view of the
phylogenetic position of cyanobacteria as the bacteria most
related to the direct origin of the chloroplast.
AtMinE1 overexpression and the arc11 mutation affect both
the assembly and the distribution of the FtsZ ring
Although there has been much speculation, no analyses
of the FtsZ structure in chloroplasts that had been impaired
in division site placement have been reported previously,
with the one exception of immunofluorescence staining of
FtsZ in AtMinD1 antisense chloroplasts (Vitha et al. 2003).
To this end, we generated transgenic Arabidopsis plants that
express an AtFtsZ1–GFP fusion protein and observed the
established stromal FtsZ ring in chloroplasts (Miyagishima
et al. 2001, Mori et al. 2001, Vitha et al. 2001). In
overcoming some of the difficulties resulting from high
autofluorescence activity in chloroplasts and in the cell
walls of live tissues, very low signals from ring-formed
AtFtsZ1–GFP proteins were detectable in dividing chloroplasts. The stable expression of AtFtsZ2-1–GFP and
transient expression of AtFtsZ1–GFP were not sufficient
to allow us to see the FtsZ ring in our present experiments
(Supplementary Fig. S4; data not shown). This is a different
outcome from that of a previous study that used AtFtsZ21–yellow fluorescent protein (YFP) or AtFtsZ1–YFP
constructs by transient expression (Maple et al. 2005).
This difference might be due to the linker length between
the AtFtsZ2-1 and GFP inserts required to retain protein
conformation and/or to distinct transgene expression levels.
It is also possible that this is dependent upon the chloroplast
division stages under observation.
We have further characterized the underlying mechanism of symmetric, asymmetric and multiple chloroplast
division by disrupting the Arabidopsis Min system. The
spatial distribution and assembly of the FtsZ ring are
perturbed equally in AtMinE1 sense and arc11 plants, but
each FtsZ ring formed along elongated chloroplasts can
function as a division ring in these plants. Abnormalities
in FtsZ ring assembly are seen as frequent formations of a
thin filamentous structure in close proximity to the ring.
This structure might be an intermediate before assembly of
the main or a separate FtsZ ring, or a disassembled form of
the FtsZ ring. Whereas the last option may be true of late
division stage WT chloroplasts (Vitha et al. 2001), we favor
the first two possibilities. The first of these might mean a
delay or inefficiency in FtsZ ring assembly, and the second
might explain the existence of parallel FtsZ rings distributed
in proximity along the chloroplast length (Fig. 5D and
Supplementary Fig. S6; Vitha et al. 2003). To resolve this
issue, it will be essential to trace the behavior of the FtsZ
ring during the course of chloroplast division.
For quantitative characterization of the distribution of
division planes in AtMinE1 sense chloroplasts, we used true
constriction sites as well as the FtsZ ring sites in
chloroplasts within a limited region of the leaf petioles
(Fig. 3 and Supplementary Fig. S5). The fact that certain
division sites can be blocked or disappear at later stages of
chloroplast development by membrane expansion (see
Fig. 6) showed that these evaluations were appropriate
and timely. Our results might permit a hypothesis of
disequilibrium of division components among division sites
marked by FtsZ rings, including preferential assembly of
the chloroplast division machinery in binary dividing
chloroplasts at a site of an FtsZ ring close to the midchloroplast.
Evidence for prevention of FtsZ ring-mediated chloroplast
division processes
The information gained by viewing the chloroplast
FtsZ ring was more revealing than we had expected and
resulted in the identification of a novel pathway of
chloroplast division inhibition during leaf development in
AtMinE1 sense and arc11 plants. Our observations reveal
that not all the chloroplast FtsZ rings can complete
chloroplast division in these plants. Since at least a
population of the FtsZ rings in developing leaf cells actually
function in chloroplast constriction and fission, it is likely
that insufficiency or disequilibrium of the dosage of
responsible components for generating division constriction
force (Yoshida et al. 2006) determines the fate of the
division process for each site: successful membrane fission
or loss of constriction. It is also possible the AtMinE1
overexpression or arc11 mutation causes a delay in the
chloroplast constriction, even when a sufficient level of the
constriction proteins is provided. In either or both cases, a
prolonged term of chloroplast division would lead to
promotion of chloroplast elongation in the AtMinE1 sense
and arc11 plants, in a way that does not disrupt the
chloroplast polarity. Moreover, development-associated
chloroplast membrane growth may serve to contribute to
the above effects, e.g. as the chloroplasts mature, the
balance of the membrane fission and the division inhibition
should shift towards the latter, due to increased needs for
the constriction proteins to conduct fission of the chloroplast membranes enclosing an increased volume. A former
description of the competency of the chloroplast size to
enable division upon their development (Marrison et al.
The chloroplast Min system
1999), as well as the inhibitory effects of AtMinE1
overexpression on chloroplast division (Itoh et al. 2001,
Reddy et al. 2002), would make sense in this regard.
Two MscS-like ion channel proteins, MSL2 and
MSL3, have recently been found to control chloroplast
size and shape, and also to co-localize with AtMinE1 on the
inner chloroplast envelope membranes (Haswell and
Meyerowitz 2006). Considering their critical roles in the
establishment of plastid polarity in certain plant tissues,
such as the leaf epidermis, it is presumed that Min protein
activity in leaf tissues of the AtMinE1 sense and arc11
plants is premised by the biological activity of MSL2 and
MSL3 on the chloroplast membrane. It would be interesting
to investigate whether AtMinE1 is involved in the ion
channel activity of chloroplast MSLs. In addition, comparative characterizations of the plastid morphology in
plant tissues between the AtMinE1 sense, atminE1 and the
msl mutant plants should also be a matter for investigation
in future studies.
Materials and Methods
Plant materials and growth conditions
Arabidopsis thaliana (L.) Heynh. ecotypes Columbia (Col),
Landsberg erecta (Ler) and Wassilewskija (Ws) were used as WT
plants in this study. Transgenic AtMinE1 overexpressors (mEs #02;
Col background; Itoh et al. 2001) and knockdown lines (mEas
#107, Col background; Itoh and Yoshida 2001) of Arabidopsis
expressing the gene in the sense and antisense orientation under the
control of the cauliflower mosaic virus (CaMV) 35S promoter,
respectively, were also used. Three recessive arc mutants, arc5
(arc5-1, Ler background; Pyke and Leech 1994), arc6 (arc6-2, Ler
background; Pyke et al. 1994) and arc11 (arc11-1, Ler background;
Marrison et al. 1999), were obtained from the Arabidopsis
Biological Resource Center (ABRC; Ohio State University,
Columbus, OH, USA). A T-DNA insertion mutant of AtMinE1,
Flag_056G07 (DLFTV7T3, Ws background), was also obtained
from the Institut National de la Recherche Agronomique (INRA,
Versailles, France) (Samson et al. 2002). These plant seeds were
surface-sterilized with 70% (v/v) ethanol and 1% (w/v) sodium
hypochlorite, 0.1% (v/v) Tween-20, and sown on soil (Golden
Peatban, Sakata Seed, Yokohama, Japan) or on 0.7% (w/v) agarcontaining Murashige–Skoog medium (Wako Jun-yaku, Osaka,
Japan) supplemented with Gamborg’s B5 vitamins (Wako Junyaku) and 2% (w/v) sucrose. Unless otherwise specified, the seeds
were placed under darkness at 48C for 3 d, and then germinated
and grown at 238C under continuous white light illumination
(100 mE m–2 s–1).
Characterization of the atminE1 mutant and the AtMinE1 overexpressor plants
Total DNA from the primary leaves of Ws and Flag_056G07
seedlings was extracted using the DNeasy Plant Mini Kit (Qiagen,
Tokyo, Japan). A T-DNA mutation at the genomic AtMinE1 locus
in individual seedlings was confirmed by PCR (Ex Taq polymerase,
TAKARA SHUZO, Kyoto, Japan) using the extracted DNA and
two AtMinE1-specific oligonucleotide primers, e1 50 -TCGA
ATTCATGGCGATGTCTTCTGGAAC-30 (restriction site underlined) and e2 50 -CCGGATCCTCACTCTGGAACATAAA
359
AATCG-30 , and a T-DNA left border (LB)-specific primer
50 -CTACAAATTGCCTTTTCTTATCGAC-30 . For the linkage
analysis, individuals hemizygous for the Flag_056G07-derived
T-DNA insertion were generated by backcrossing the atminE1
mutant with the WT. The F2 seeds were obtained by selfpollination and germinated on Murashige–Skoog agar plates.
The chloroplast phenotype of a cotyledon from each 12-day-old
seedlings and the genotype with respect to the T-DNA insertion
were analyzed by means of epifluorescence microscopy (see the
next section) and genomic PCR using the primer set e1/e2. For
RT–PCR analyses, total RNA from whole seedlings of 2-week-old
WT (Ws and Col), Flag_056G07 (T-DNA-homozygous) and mEs
#02 (at the T4 generation) plants was extracted using the GenElute
Total RNA Miniprep Kit (Sigma-Aldrich, St. Louis, MO, USA).
Approximately 0.5 mg of total RNA was subjected to reverse
transcription using the ReverTra-Plus Kit (Toyobo, Osaka,
Japan), including the oligo(dT) primer, according to the manufacturer’s instructions. The single-stranded cDNA products were
then subjected to PCR using Ex Taq polymerase and the e1 and e2
primers. The amplification conditions were one cycle for 3 min at
948C, followed by 25–40 cycles for 20 s at 948C, 30 s at 628C, and
20 s at 728C. As a control for RT–PCR, cDNA for the Arabidopsis
UBQ10 gene was amplified using 50 -TAAAAACTTTCTCTCAA
TTCTCTCT-30 and 50 -TTGTCGATGGTGTCGGAGCTT-30
under the above conditions. Both genomic and RT–PCR
products were subjected to electrophoresis in an ethidium
bromide-containing agarose gel.
Epifluorescence microscopy
Whole plant seedlings or organs were mounted under glass
coverslips, and observed by epifluorescence microscopy (IX70,
Olympus, Tokyo, Japan, equipped with a Hamamatsu ORCA-ER,
Hamamatsu, Japan) using 20 N.A. 0.5 and 100 N.A. 1.35
objective lenses (Olympus). Digital black and white images were
imported into the RGB channels of Adobe Photoshop (Adobe
Systems, San Jose, CA, USA) to obtain the final merged images.
Electron microscopy
Leaf sections of 2 mm square were punched out from the
central region of leaves and fixed with 2.5% glutaraldehyde in
0.1 M sodium cacodylate (pH 7.2) on ice for 2 h. After the samples
were rinsed three times for 10 min each in the same buffer, they
were post-fixed with 1% OsO4 in the same buffer at 48C overnight.
The samples were then rinsed with the same buffer, dehydrated
with a graded acetone series and embedded in Spurr’s resin (Spurr
1969). Thin sections were stained with uranyl acetate followed by
lead citrate and examined in a JEOL 1200 EX electron microscope
(Tokyo, Japan).
Expression of the AtFtsZ1–GFP fusion gene
Total DNA from whole seedlings of Col was purified using
the DNeasy Plant Mini Kit (Qiagen). A 1.7 kb fragment of
genomic DNA containing a 1.6 kb AtFtsZ1-1 upstream promoter
region was amplified by PCR using PfuTurbo DNA polymerase
(Stratagene, La Jolla, CA, USA) and the oligonucleotide primers
Z1-1 50 -GGGGATCCTTATAACAACCCTAATCTTC-30 and
Z1-3 50 -TCTAGAACTTGCGCAAATGC-30 . A 1.3 kb insert
containing the entire coding sequence of AtFtsZ1-1 was also
amplified with the primers Z1-2 50 -ATGGCGATAATTCCGT
TAGC-30 and Z1-4 50 -AAGCCATGGAGAAGAAAAGTCTA
CGGGGA-30 from an Arabidopsis cDNA library (MATCH
MAKER, Clontech, Mountain View, CA, USA). The two overlapping PCR products were annealed and the full-length region
360
The chloroplast Min system
was amplified with Z1-1 and Z1-4. The resulting PCR product
(2.9 kb) was then introduced into the CaMV35S-sGFP(S65T)-NOS
vector (Niwa et al. 1999) by simultaneously removing the CaMV
35S promoter to yield pZ1-GFP. A BamHI–EcoRI fragment of
pZ1-GFP, comprising a 1.6 kb AtFtsZ1-1 upstream region, the
AtFtsZ1-1 open reading frame (cDNA), the sGFP(S65T) gene and
the NOS terminator, was introduced into pBI101 by simultaneously removing the uidA and NOS terminator genes. The
resulting plasmid, pBI-Z1-GFP, was employed for Agrobacteriummediated Arabidopsis transformation by the floral dip method
(Clough and Bent 1998). Sixteen transformed (T1) Arabidopsis
seedlings were selected on kanamycin- (50 mg l–1, Nacalai Tesque,
Kyoto, Japan) containing Murashige–Skoog agar plates. T2 and T3
seedlings were used for microscopy.
For transient expression experiments, the full-length
AtFtsZ1–GFP gene was amplified with Z1-SalI (Fujiwara and
Yoshida 2001) and Z1-4, digested with SalI and NcoI, and ligated
into CaMV35S-sGFP(S65T)-NOS under the CaMV 35S promoter
to yield p35S-Z1-GFP. This vector was then introduced into the
hypocotyl cells of 6-day-old Col seedlings by particle bombardment (Itoh et al. 2001). Cells emitting GFP signals were observed
by epifluorescence microscopy.
Supplementary material
Supplementary material mentioned in the article is available to online subscribers at the journal website www.pcp.
oxfordjournals.org.
Funding
The Ministry of Education, Culture, Sports, Science
and Technology of Japan (12740452 and 17770039 to
R.D.I.; 14760068 and 17780077 to M.T.F.); the Special
Postdoctoral Research Program of RIKEN (to M.T.F. and
R.D.I.); the 21st Century COE program of University of the
Ryukyus (to R.D.I.); the Research Program for the Study
on Genesis of Matter from the Ministry of Education,
Culture, Sports, Science and Technology of Japan (to T.A.).
Acknowledgments
The authors thank the Arabidopsis Biological Resource
Center (Ohio State University, USA) and Drs. Kevin A. Pyke
(University of Nottingham, UK), Joanne L. Marrison and Rachel
M. Leech (University of York, UK) for providing the arc mutant
seeds, the INRA Versailles Genomic Resource Center (France) for
the atminE1 (Flag_056G07) seeds, and Dr. Yasuo Niwa
(University of Shizuoka, Japan) for the sGFP(S65T) vector.
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(Received September 20, 2007; Accepted January 15, 2008)