Septum Formation in Amyloplasts Produces Compound
Granules in the Rice Endosperm and is Regulated by Plastid
Division Proteins
Min-Soo Yun1,2 and Yasushi Kawagoe1,*
1Division
of Plant Sciences, National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba, 305-8602 Japan
*Corresponding author: E-mail, [email protected]; Fax, +81-29-838-7417
(Received July 13, 2010; Accepted July 28, 2010)
Storage tissues such as seed endosperm and tubers store
starch in the form of granules in the amyloplast. In the
rice (Oryza sativa) endosperm, each amyloplast produces
compound granules consisting of several dozen polyhedral,
sharp-edged and easily separable granules; whereas in other
cereals, including wheat (Triticum aestivum), barley (Hordeum
vulgare) and maize (Zea mays), each amyloplast synthesizes
one granule. Despite extensive studies on mutants of starch
synthesis in cereals, the molecular mechanisms involved in
compound granule synthesis in rice have remained elusive.
In this study, we expressed green fluorescent protein (GFP)
fused to rice Brittle1 (BT1), an inner envelope membrane
protein, to characterize dividing amyloplasts in the rice
endosperm. Confocal microscopic analyses revealed that
a septum-like structure, or cross-wall, containing BT1–GFP
divides granules in the amyloplast. Plastid division proteins
including FtsZ, Min and PDV2 play significant roles not
only in amyloplast division, but also in septum synthesis,
suggesting that amyloplast division and septum synthesis
are related processes that share common factors. We propose
that successive septum syntheses which create sections
inside the amyloplast and de novo granule synthesis in
each section are primarily responsible for the synthesis of
compound granules.
Keywords: Amyloplast division • Compound granule • FtsZ •
Min • Septum • Starch.
Abbreviations: BT1, Brittle 1; DAF, days after flowering; EM,
electron microscopy; GFP, green fluorescent protein; IEM,
inner envelope membrane; ISA1, isoamylase1; OEM, outer
envelope membrane; RNAi, RNA interference; RT–PCR, reverse
transcription–PCR
Introduction
The size and shape of starch granules vary between species
and the tissues from which they are purified (Jane et al. 1994,
Shapter et al. 2008). Most potential industrial applications of
starch are mediated by its granule size (Ji et al. 2004). One granule, or simple granule, develops in each amyloplast in the
endosperm of maize (Zea mays) (Katz et al. 1993). The Triticeae
species, including wheat (Triticum aestivum) and barley
(Hordeum vulgare), share a unique type of bimodal amyloplast,
producing either one large lenticular A-type granule or one
small spherical or ovoid B-type granule (Evers 1971). In contrast,
in the rice endosperm, each amyloplast produces up to several
dozen granules, each of which is typically 3–8 µm, polyhedral
and sharp edged (Yun and Kawagoe 2009, Matsushima et al.
2010). The compound granules of rice endosperm are easily
separable from the adjacent granules by conventional purification procedures. Despite comprehensive studies on a number
of mutants of starch synthesis in cereal endosperm (Nakamura
2002, Hannah and James 2008), the mechanisms involved in
the synthesis of different granule structures remain elusive.
The plastid division apparatus evolved primarily from the
cell division apparatus of the cyanobacterial progenitor of chloroplasts (Yang et al. 2008). The binary fission of chloroplasts
depends on proteins that form complexes on the stromal side
of the inner envelope membrane (IEM) and the cytosolic side of
the outer envelope membrane (OEM) (Maple and Møller
2007a, Yang et al. 2008). Unlike the binary fission of chloroplasts
in the leaf of rice and Arabidopsis thaliana, amyloplasts in the
rice endosperm divide simultaneously at multiple sites (Yun
and Kawagoe 2009). The apparently different modes of division
between chloroplast and amyloplast suggest that plants have
acquired multiple mechanisms of plastid division.
FtsZ1 and FtsZ2 proteins in higher plants are homologs of
the bacterial FtsZ protein (Stokes et al. 2000, El-Kafafi et al.
2005, Yoder et al. 2007, Fujiwara et al. 2009a). Bacterial FtsZ
forms a ring structure called the Z ring at division sites. This Z
ring provides a scaffold for recruiting at least 10 additional
proteins that link the Z ring to the synthesis of a new cell wall
(Margolin 2005, Adams and Errington 2009). In Escherichia coli,
synthesis of the division septum is accompanied by constriction
of the outer membrane, whereas in Bacillus subtilis, a septal
cross-wall of peptidoglycan initially divides the cell (Adams and
Regular Paper
2Present address: Food Resource Division, National Food Research Institute, 2-1-12 Kannondai, Tsukuba, 305 8642 Japan
Plant Cell Physiol. 51(9): 1469–1479 (2010) doi:10.1093/pcp/pcq116, available online at www.pcp.oxfordjournals.org
© The Author 2010. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.
All rights reserved. For permissions, please email: [email protected]
Plant Cell Physiol. 51(9): 1469–1479 (2010) doi:10.1093/pcp/pcq116 © The Author 2010.
1469
M.-S. Yun and Y. Kawagoe
Errington 2009). Almost all prokaryotes have a single FtsZ gene,
whereas plants and green algae have two nuclear-encoded FtsZ
genes, FtsZ1 and FtsZ2, which are distinguished by conserved
differences in the amino acid sequences of their products
(Stokes and Osteryoung 2003). Phylogenetic analyses indicate
that the FtsZ1 and FtsZ2 families originated long before the
evolution of land plants. FtsZ1 and FtsZ2 appear capable of
polymerizing independently of one another, and interact with
ARC3 and ARC6, respectively, indicating that the two FtsZ
proteins have diverged to acquire unique binding activities for
various assembly regulators (Yang et al. 2008). Although the
Z ring formation at the division site is a critical step in chloroplast division, FtsZ null mutants are viable and fertile, suggesting that an FtsZ-independent mode of plastid partitioning may
occur in higher plants (Schmitz et al. 2009). We have revealed,
by fusing rice FtsZ2-1 to a fluorescent protein, that FtsZ2-1 is
targeted to multiple constriction sites in dividing amyloplasts
in the rice endosperm (Yun and Kawagoe 2009), suggesting
that FtsZ2-1 is a component of the Z ring formed at division
sites. Transgenic potato tubers expressing a high level of StFtsZ1
produce fewer but larger spherical amyloplasts (de Pater et al.
2006), suggesting that StFtsZ1 plays a role in amyloplast division. In a recent study, we revealed that rice ARC5, a dynaminrelated protein that forms a polymer at the cytosolic side of
the OEM, is recruited to constriction sites of dividing amyloplasts and plays a role at the late stage of amyloplast division
(Yun and Kawagoe 2009). These results suggested that the
inner envelope protein ARC6 (Vitha et al. 2003) and the outer
membrane proteins PDV1 and PDV2 (Miyagishima et al. 2006,
Okazaki et al. 2009) may also function in amyloplast division.
The Min system in bacteria contributes to medial Z ring
placement in cell division by inhibiting the Z ring assembly at
the cell poles (Lutkenhaus 2007). In E. coli, MinE drives MinC
and MinD, negative regulators of Z ring assembly, to oscillate
from pole to pole, which creates a symmetrical concentration
gradient of negative regulators such that the time-averaged
concentration of the negative regulators is highest at the cell
poles and lowest at the cell center, permitting Z ring assembly
only at the midcell (Lutkenhaus, 2008). In B. subtilis, which lacks
MinE, DivIVA and MinJ recruit MinC and MinD to division sites
and retain them at the new cell poles (Bramkamp et al. 2008).
In plastid division in Arabidopsis, Z ring placement is also controlled by MinD and MinE; however, MinC appears to have
been lost in most eukaryotic lineages. Arabidopsis mutant
analyses indicate that the division inhibitor in the Min system
is functionally replaced by ARC3, which exhibits both polar
and medial localizations in chloroplasts, and interacts with
FtsZ1, MinD and MinE (Shimada et al. 2004, Maple et al. 2007).
Overexpression of AtMinD1 or CrMinD1-YFP results in severe
inhibition of chloroplast division in Arabidopsis mesophyll
cells, generating one or two enlarged chloroplasts per cell
(Colletti et al. 2000, Kanamaru et al. 2000, Adams et al. 2008).
Although initial studies showed that AtMinD1 localizes as
one or two discrete loci in the Arabidopsis chloroplast (Maple
et al. 2002), recent studies revealed that AtMinD1 localizes at
1470
the constricting sites of chloroplasts (Fujiwara et al. 2009b,
Nakanishi et al. 2009). AtMinE1 overexpression results in division site misplacement, giving rise to multiple constrictions
along the length of plastids in Arabidopsis seedlings (Itoh et al.
2001, Maple et al. 2002). Fujiwara et al. (2008) pointed out
that the balance between the inhibitory role of AtMinD1 and
the antagonistically facilitative role of AtMinE1 in the formation and/or maintenance of FtsZ filaments determines the
symmetric binary fission of chloroplasts.
In this study, we detected green fluorescent protein (GFP)
fused to Brittle1 (BT1), an ADP-glucose transporter, between
granules in the amyloplast of rice endosperm, suggesting the
possibility that a septum-like structure, or a cross-wall, divides
granules in the rice amyloplast. Loss-of-function and overexpression analyses suggested that plastid division proteins
including FtsZ, Min and PDV2 play critical roles not only in
amyloplast division, but also in the synthesis of septum-like
structure between granules in the amyloplast.
Results
A septum-like structure divides granules in
the amyloplast
While conducting studies on amyloplast division employing
amyloplast stroma-targeted fluorescent proteins, we puzzled
over what mechanisms might generate small amyloplasts containing two or three sharp-edged granules, as shown in Fig. 1.
Because tpGFP was targeted to the amyloplast stroma, it is
probable that the stroma was present between the granules.
We obtained no evidence indicating that the stromal space
between granules was generated by degrading large granule at
the equatorial plane. Because some dividing amyloplasts looked
like spherical-celled cyanobacteria containing a division septum
at the midcell (e.g. Marbouty et al. 2009), we hypothesized that
there might be a septum-like structure, or a cross-wall, between
granules, and that granules could grow independently and
become sharp edged in the stroma section divided by the
septum. To see whether the IEM is present between granules,
we expressed rice BT1, which is an ADP-glucose transporter
and integral IEM protein (Leroch et al. 2005), fused to GFP.
BT1–GFP was detected at the outer limit envelope of the
amyloplast and between granules (Fig. 2, upper panels), suggesting that a septum-like structure containing IEM is present
between granules. Serial Z-sections of the amyloplasts confirmed that BT1–GFP was localized in the septa between
granules (Supplementary Fig. S1). We noted that the signal
intensities of BT1–GFP in the septa were not uniform (Fig. 2,
upper panels; Supplementary Fig. S1).
The sugary1 mutant of rice, a mutant of the starchdebranching enzyme isoamylase1 (ISA1), does not synthesize
typical granules, but instead synthesizes water-soluble phytoglycogen (Kawagoe et al. 2005). Because it is unclear whether
amyloplasts containing phytoglycogen also synthesize septum,
we expressed BT1–GFP in the endosperm with reduced ISA1
Plant Cell Physiol. 51(9): 1469–1479 (2010) doi:10.1093/pcp/pcq116 © The Author 2010.
Compound starch granule synthesis in rice
Fig. 1 Dividing amyloplasts in the endosperm (10 DAF) were visualized with a stroma-targeted GFP (tpGFP). Arrows point to division sites.
Arrowheads indicate the stromal space between granules. Bars = 2 µm.
BT1-GFP
tpCherry
Merged
Fig. 2 Septum-like structure between granules contained the IEM. BT1–GFP, an IEM-spanning protein, was targeted to the outer limit envelope
and septum between granules in the wild-type amyloplast (WT, 7 DAF, upper panels), whereas the fusion protein was localized only at the outer
limit envelope of the amyloplasts containing phytoglycogen (ISA1-KD, 10 DAF, lower panels). The fluorescence signals of BT1–GFP (left panels)
and stroma-targeted tpCherry (middle panels) are converted to green and magenta, respectively, and merged (right panels). Arrowheads point
to septa between granules. See also serial Z-sections of the wild-type amyloplasts in Supplementary Fig. S1. Bars = 5 µm.
Plant Cell Physiol. 51(9): 1469–1479 (2010) doi:10.1093/pcp/pcq116 © The Author 2010.
1471
M.-S. Yun and Y. Kawagoe
expression (ISA1-KD). Starch granules of large size were not
detected in the amyloplast stroma of ISA1-KD endosperm
(Fig. 2, lower panels), confirming that the debranching activity
of ISA1 is necessary for granule synthesis. Although BT1–GFP
was targeted to the outer limit envelope of the amyloplast containing phytoglycogen, the fusion protein was not detectable
in the stroma. These results suggested that granules promoted
septum synthesis in the amyloplast.
We found disk-like walls with a strong fluorescence signal
at the rim on the surface of small developing amyloplasts
(Fig. 3, arrowheads). The size of the disk was similar to that of
granules being synthesized in the amyloplasts. Such a disk was
not observed on the surface of large, developed amyloplasts.
FtsZ2-1 and FtsZ1 have roles in amyloplast
division and septum synthesis
The plastid division protein FtsZ2-1 is targeted to the constriction sites of dividing amyloplasts in the rice endosperm (Yun
and Kawagoe 2009). We thus asked the question of whether
FtsZ2-1 and FtsZ1 play roles in septum synthesis. We repressed
the expression of the respective genes by RNA interference
(RNAi) under the control of the maize Ubq (ZmUbq) promoter
(Fig. 4A). When FtsZ1 expression was repressed (FtsZ1-KD),
amyloplast division was inhibited, and pleomorphic granules
were synthesized (Fig. 4C). Consistent with the specific localization of FtsZ2-1 at the constriction sites (Yun and Kawagoe
2009), amyloplast divisions were significantly inhibited in
BT1-GFP
FtsZ2-1-repressed seed (FtsZ2-1-KD, Fig. 4D). We then analyzed
the effects of FtsZ1-KD or FtsZ2-1-KD on BT1–GFP localization
in the amyloplast (Fig. 5A). FtsZ1-KD apparently induced
septum synthesis at aberrant sites and produced pleomorphic
granules (Fig. 5B). When FtsZ2-1 expression was repressed
(FtsZ2-1-KD), we consistently found pleomorphic amyloplasts,
which appear to have been produced by incomplete divisions
(Fig. 5C). In addition, we noted that the localization of BT1–
GFP was not like a partition wall between granules, but was
detected on the granule surface (Fig. 5C, arrowheads). Serial
Z-sections revealed that the IEM containing BT1–GFP wrapped
the granules (Supplementary Fig. S2). Similar results were
obtained when the expression of PDV2, which encodes an outer
envelope-spanning protein that recruits ARC5 (Miyagishima
et al. 2006), was repressed (PDV2-KD) (Fig. 5D, E; Supplementary Fig. S2).
We then analyzed the effects of overexpressing FtsZ1
(FtsZ1-OX) or FtsZ2-1 (FtsZ2-1-OX) on amyloplast division and
granule synthesis. FtsZ1-OX and FtsZ2-1-OX both generated
pleomorphic amyloplasts, which were most probably generated
by incomplete division processes. In addition, these amyloplasts
of failed divisions contained granules of different size and shape
(Supplementary Fig. S3A, B). The phenotypes of simultaneous
overexpression of the two proteins were additive (Supplementary Fig. S3A, B). Together, these results suggest that FtsZ1
and FtsZ2-1 play significant roles not only in amyloplast
division, but also in septum synthesis in the amyloplast.
tpCherry
Merged
Fig. 3 Multiple disks on the surface of developing amyloplasts. The fluorescence signals of BT1–GFP (left panels) and stroma-targeted tpCherry
(middle panels) in the wild-type amyloplast (7 DAF) are converted to green and magenta, respectively, and merged (right panels). Arrowheads
indicate the disk structure beneath the outer limit envelope. Bars = 2 µm.
1472
Plant Cell Physiol. 51(9): 1469–1479 (2010) doi:10.1093/pcp/pcq116 © The Author 2010.
Compound starch granule synthesis in rice
A
W
T
D
D -K
-K -1
Z1 sZ2
s
Ft Ft
B
C
WT
FtsZ1-KD
D
FtsZ2-1-KD
FtsZ2-1
FtsZ1
ISA3
Fig. 4 Loss-of-function analyses of FtsZ1 and FtsZ2-1. (A) Western blot analysis of FtsZ1-repressed (FtsZ1-KD) or FtsZ2-1-repressed (FtsZ2-1-KD)
transformants induced by RNAi under the control of the ZmUbq promoter. Total proteins were extracted from 5-day-old seedlings, and
immunoblotting was conducted with the indicated antibodies. Isoamylase3 (ISA3), a starch-debranching enzyme, was used as a control protein
(Yun and Kawagoe 2009). (B–D) Fluorescence images of stroma-targeted tpGFP in wild-type (B), FtsZ1-KD (C), and FtsZ2-1-KD (D) endosperm
(10 DAF). Typical amyloplasts in the wild type were oval and granules were homogeneous in size and shape (B). Arrowheads point to amyloplasts
with a disordered septum in the FtsZ1-KD endosperm (C). Arrows indicate division sites that apparently failed in the constriction of the OEM in
FtsZ2-1-KD endosperm (D). Bars = 5 µm.
W
T
A
D
D -K
-K 2-1
1
sZ sZ
Ft Ft
tpCherry
Merged
BT1-GFP
tpCherry
Merged
B
FtsZ1-KD
FtsZ1
BT1-GFP
FtsZ2-1
ISA3
FtsZ2-1-KD
C
D
KD
E
PDV2
Actin
PDV2-KD
W
T
2-
V
PD
Fig. 5 BT1–GFP localization in FtsZ1-KD, FtsZ2-1-KD or PDV2-KD endosperm. (A) Western blot analysis of FtsZ1-repressed (FtsZ1-KD) or FtsZ2-1repressed (FtsZ2-1-KD) transformants induced by RNAi under the control of the ZmUbq promoter. Total proteins were extracted from the seed
(10 DAF), and immunoblotting was conducted with the indicated antibodies. Isoamylase3 (ISA3) was used as a control protein (Yun and Kawagoe
2009). (B and C) Fluorescence images of BT1–GFP (left panels) and stroma-targeted tpCherry (middle panels) in the endosperm (10 DAF) of
FtsZ1-KD (B) and FtsZ2-1-KD (C) are converted to green and magenta, respectively, and merged (right panels). (D) RT-PCR analysis of PDV2repressed (PDV2-KD) transformant induced by RNAi under the control of the ZmUbq promoter. Total RNA was isolated from the immature
seeds (7 DAF) of the wild type and PDV2-KD. Actin was used as a control. (E) Fluorescence images of BT1–GFP (left panel) and stroma-targeted
tpCherry (middle panel) in the endosperm (7 DAF) of PDV2-KD are converted to green and magenta, respectively, and merged (right panel). The
asterisk shows an amyloplast containing pleomorphic granules of heterogeneous size and shape. Arrows indicate putative division sites that
appear to have failed in the constriction of the OEM. Arrowheads indicate IEM containing BT1–GFP on the granule surface. Bars = 5 µm.
Plant Cell Physiol. 51(9): 1469–1479 (2010) doi:10.1093/pcp/pcq116 © The Author 2010.
1473
M.-S. Yun and Y. Kawagoe
MinD and MinE play roles in amyloplast division
and septum synthesis
We next analyzed the localizations of MinD and MinE in the
developing amyloplasts. MinD–GFP was localized as discrete
spots at some corners of peripheral granules in the developing
amyloplasts (Fig. 7A). Similarly, MinE–GFP was detected as
spots at some corners of peripheral granules and along the
circumference of the amyloplast (Fig. 7B). Serial Z-sections of
the amyloplasts confirmed that MinD–GFP and MinE–GFP
were not uniformly localized in the stroma between granules
(Supplementary Fig. S5A–F). We further analyzed the effects
of overexpression of MinD or MinE on amyloplast division
and septum synthesis. Overexpression of MinD-GFP (MinD-OX)
reduced the protein level of FtsZ1, whereas overexpression of
MinE-GFP (MinE-OX) increased the protein level of FtsZ2-1
(Supplementary Fig. S4A). The effect of MinD-OX on amyloplast morphology was insignificant, compared with those of
FtsZ1-OX and FtsZ2-1-OX (compare Supplementary Figs. S3B
and S4B). However, the granules in the MinD-OX amyloplast
were heterogeneous in size and shape (Supplementary
Fig. S4B, arrowheads), suggesting that the septa were synthesized at irregular sites. In contrast, MinE-OX promoted amyloplast division, generating clusters of small amyloplasts containing
granules of different sizes (Supplementary Fig. S4C).
We next studied the possible roles of MinD and MinE in amyloplast division and septum synthesis by generating plants with
reduced levels of MinD or MinE by RNAi under the control
of the ZmUbq promoter. Immunoblotting experiments with
antibodies against recombinant MinD and MinE proteins
failed to detect the endogenous proteins in developing seeds
(Supplementary Fig. S4A), indicating low levels of these proteins in the seed. We thus screened for plants with reduced
expression of MinD or MinE by reverse transcription–PCR
(RT–PCR) (Fig. 6A). In MinD-repressed endosperm (MinD-KD),
amyloplast division was promoted, generating clusters of small
amyloplasts (Fig. 6B). The small dividing amyloplasts contained
several small granules, suggesting that septum synthesis may
also have been induced when the MinD level was low (Fig. 6B).
In contrast, in MinE-repressed endosperm (MinE-KD), elongated amyloplasts containing septa parallel to one another
were a characteristic phenotype (Fig. 6C). These results suggest
that the Min system plays critical roles in both amyloplast
division and septum synthesis in the dividing amyloplast.
A
D
T
W
D
-K
K
Ein
D
M
in
M
MinD
MinE
Actin
B
MinD-KD
C
MinE-KD
Fig. 6 Loss-of-function experiments of MinD and MinE. (A) Semi-quantitative RT–PCR analysis. Total RNA was isolated from the immature seeds
(7 DAF) of the wild type, MinD-repressed (MinD-KD) and MinE-repressed (MinE-KD) transformants. Actin was used as a control. (B and C)
Fluorescence images of stroma-targeted tpGFP in MinD-KD (B) and MinE-KD (C) endosperm (7 DAF). Amyloplast division was enhanced in
MinD-KD (B). Arrows point to small dividing amyloplasts containing small granules. Arrowheads in (C) point to elongated amyloplasts containing
parallel septa. Bars = 5 µm.
1474
Plant Cell Physiol. 51(9): 1469–1479 (2010) doi:10.1093/pcp/pcq116 © The Author 2010.
Compound starch granule synthesis in rice
Discussion
Compound granule synthesis involves septum
synthesis in the amyloplast
We showed that BT1–GFP, a marker protein for the IEM, was
targeted not only to the outer limit envelope, but also to the
septum between granules (Fig. 2; Supplementary Fig. S1). It is
important to note that pioneering studies on rice amyloplast
development by electron microscopy (EM) have detected a
wall between granules (Hoshikawa 1968, Sato and Ehara 1974).
However, subsequent EM studies with higher resolutions have
not definitely confirmed the wall between granules (e.g. Kim
et al. 2004, Kang et al. 2006). Therefore, there has been no
generally accepted view as to whether granules are divided by
a cross-wall, or septum, in the rice amyloplast. In this study
we employed a membrane-targeted GFP as a sensitive probe
to detect a septum between granules. Because BT1 is an IEMspanning protein, it is probable that the septum between granules contains the IEM. However, it is not clear whether the
septum also contains the OEM. Further studies are needed to
elucidate the components of the septum between granules.
The fluorescence signal of BT1–GFP in the septum diminished
as the amyloplast matured, suggesting that BT1–GFP was
unstable or degraded. We also point out that BT1–GFP was not
always detectable between granules in developing amyloplasts
(Figs. 2, 3; Supplementary Fig. S1). It is thus possible that not
all segments of the septum were continuous from the outer
limit envelope, which may have limited the movement of BT1–
GFP from the outer limit envelope to the septum between
granules. Alternatively, it is possible that there may have been
septa of different compositions in the amyloplasts. If so, BT1–
GFP may have been targeted only to certain types of septum
in the amyloplasts. We noted that BT1–GFP in the FtsZ2-1-KD
and PDV2-KD endosperm was localized on the granule surface
(Fig. 5C, E; Supplementary Fig. S2). The reason for the aberrant localizations of BT1–GFP in FtsZ2-1-KD and PDV2-KD
amyloplasts is not clear; however, because both FtsZ2-1 (stromal
protein) and PDV2 (OEM-spanning protein) are components
of the division complex involved in fission of the Arabidopsis
chloroplast, it is possible that FtsZ2-1 and PDV2, together with
as yet unidentified proteins, may form complexes across the
IEM and OEM such that the two envelope membranes are
held in an undetachable fashion. If so, depleting FtsZ2-1 or
A
MinD-GFP
tpCherry
Merged
B
MinE-GFP
tpCherry
Merged
Fig. 7 Localization of MinD–GFP and MinE–GFP in the developing amyloplast (7 DAF). GFP-fused MinD (A) or MinE (B) was co-expressed with
the stroma-targeted tpCherry, and confocal images of fluorescence signals of GFP fusion proteins (left panels) and tpCherry (middle panels) are
converted to green and magenta, respectively, and merged (right panels). Both MinD–GFP and MinE–GFP were targeted to some corners of
peripheral granules in developing amyloplasts. Serial Z-sections of the amyloplasts are shown in Supplementary Fig. S5A–F. Bars = 5 µm.
Plant Cell Physiol. 51(9): 1469–1479 (2010) doi:10.1093/pcp/pcq116 © The Author 2010.
1475
M.-S. Yun and Y. Kawagoe
PDV2 from the complex may detach the IEM from the OEM,
enabling the IEM to wrap around the granule.
It is important to note that BT1–GFP was localized at multiple disks on the surface of the developing amyloplast (Fig. 3).
Because such a disk was not found on the surface of large,
developed amyloplasts, we speculate that these disks were
septa being synthesized beneath the outer limit envelope.
We further speculate that de novo granule synthesis occurs in
the stroma section divided by the septum and the outer limit
envelope. If so, the granule in the stroma may grow outwards
by concomitant outgrowth of the outer limit envelope. As
schematically illustrated in Fig. 8, we propose that successive
synthesis of septa beneath the outer limit envelope and de
novo granule synthesis in the stroma section divided by the
septa are chiefly responsible for the synthesis of characteristic
compound granules in the rice endosperm. In this model, the
size and shape of granules are primarily determined by the
shape of the stroma section divided by the septa. This mode of
synthesis of compound granules is consistent with the fact that
adjacent granules do not fuse during granule synthesis.
At least four subfamilies in the Poaceae produce compound
granules in the endosperm, and the taxonomic relationships
in the Poaceae are not an accurate predictor of granule morphology (Shapter et al. 2008). Although we revealed that the
synthesis of compound granules is regulated, in part, by plastid
division proteins, it remains unclear how other cereals such as
maize, wheat and barley prevent the synthesis of compound
granules in the amyloplast. The simplest explanation for the
occurrence of compound granules in the four subfamilies
of Poaceae is that loss of function of an as yet unidentified
inhibitor of septum synthesis occurred independently in the
respective progenitors of the four subfamilies. Further studies
are needed to investigate whether and how such an inhibitor
functions in the amyloplast of cereals that synthesize a single
granule in each amyloplast.
Roles of FtsZ and the Min system in amyloplast
division and septum synthesis
The amyloplasts and starch granules in FtsZ1-KD, FtsZ2-1-KD,
FtsZ1-OX and FtsZ2-1-OX endosperm exhibited pleomorphic
morphologies (Fig. 4; Supplementary Fig. S3). These results
strongly suggest that both FtsZ1 and FtsZ2-1 play significant
roles not only in amyloplast division, but also in septum synthesis
in the amyloplast. FtsZ of E. coli polymerizes into protofilaments
in vitro, which then associate through lateral interactions into
rings or sheets (Erickson et al. 1996). In the rice endosperm
amyloplast, protofilaments of FtsZ1 and FtsZ2-1 may similarly
associate into sheets, and may function as a scaffold that
facilitates the recruitment of enzymes involved in septum
synthesis.
Both MinD–GFP and MinE–GFP were localized as discrete
spots at some corners of peripheral granules in the developing
amyloplast (Fig. 7A, B; Supplementary Fig. S5A–F). Loss of
function of MinD produced clustered amyloplasts and small
granules in dividing amyloplasts (Fig. 6B). It is thus conceivable
that MinD functions as a negative regulator of the assembly
of the Z ring at potential division sites and potential planes
of septum synthesis. In the Arabidopsis chloroplast, MinE
functions as a division site placement factor (Maple et al.
2002, Maple and Møller 2007b, Fujiwara et al. 2008). In the
rice endosperm, MinE-OX exhibited enhancement of division,
similar to the phenotypes of MinD-KD amyloplasts (Fig. 6B;
Amyloplast division
constriction
separation
Compound granule synthesis
Fig. 8 A possible mechanism of compound granule synthesis in the rice endosperm. Amyloplast division (upper panels) and compound granule
synthesis (lower panels) are related processes that share common factors. Appropriate amounts of FtsZ1 and FtsZ2-1 are necessary for proper
division and septum synthesis. MinD inhibits Z ring formation and thus regulates both division and septum synthesis. MinE controls the
localization of MinD. Amyloplast division: when an amyloplast containing compound granules divides simultaneously at the two separate sites as
indicated, two FtsZ rings are independently assembled beneath the outer limit envelope at the potential division sites, generating a septum disk
(red bars). Granule synthesis initiates in the stroma section divided by the septum, and the new granules grow outwards with concomitant
outgrowth of the outer limit envelope. ARC5 is recruited to the constriction sites from the cytosol (Yun and Kawagoe 2009). The division process
involves constriction of the septum and invagination of the OEM, and separation of the new amyloplasts. Compound granule synthesis: a septum
disk structure is successively formed beneath the outer limit envelope. Unlike the division process (upper panels), the septum synthesis is not
accompanied by constriction of the OEM. Red bars represent new septa of disk structures being synthesized, and black bars represent old septa
that divide adjacent granules. Granule synthesis initiates in each stroma section divided by the septum, and the granules grow outwards with
concomitant outgrowth of the outer limit envelope. Low or excess amounts of FtsZ1 or FtsZ2-1 lead to the synthesis of a septum at aberrant
sites, generating pleomorphic granules of different sizes.
1476
Plant Cell Physiol. 51(9): 1469–1479 (2010) doi:10.1093/pcp/pcq116 © The Author 2010.
Compound starch granule synthesis in rice
Supplementary Fig. S4C). We noted that MinE-KD produced
an elongated amyloplast with septa parallel to one another
(Fig. 6C). It is thus conceivable that MinE influences the plane
of septum synthesis. In E. coli, successive division planes are
parallel to one another, whereas rodA mutants divide along
planes alternating at right angles to one another (Begg and
Donachie 1998). Further studies are needed to investigate
how MinE influences plane alternations in septum formation
in the amyloplast.
In summary, we showed that both amyloplast division
and granule morphologies were modulated by changing the
expression levels of plastid division proteins such as FtsZ,
Min and PDV2 (Figs. 4–6; Supplementary Figs. S3, S4). These
results strongly suggest that amyloplast division and septum
synthesis are related processes that share common factors.
It is tempting to speculate that in amyloplast division the
synthesis of septum is accompanied by constriction of the
OEM, whereas septum synthesis without constriction of
the OEM leads to the synthesis of characteristic compound
granules (Fig. 8). We noted that division and septum synthesis
were severely inhibited in the amyloplasts containing phytoglycogen (Fig. 2). These results suggest that insoluble starch
granules, but not water-soluble phytoglycogen, may promote
septum synthesis in the amyloplast.
Materials and Methods
Materials
Full-length cDNA clones of MinD (AK059897), MinE (AK070044)
and PDV2 (CI682676) were obtained from the National
Institute of Agrobiological Sciences (NIAS, Tsukuba, Japan). The
cDNA clones of Brittle1 (BT1), FtsZ2-1, FtsZ1, ISA3 and actin
have been described previously (Onda et al. 2009, Yun and
Kawagoe 2009). The fragments of MinD (N88-G306, 23.6 kDa)
and MinE (A76-Y236, 18.2 kDa) were PCR-amplified and cloned
in pET23d (Novagen). The respective polypeptides were
expressed in Rosetta2 (DE3) (Novagen) with isopropyl-β-Dthiogalactopyranoside (IPTG) induction, purified from inclusion bodies, eluted from an Ni column and injected into rats
for antibody production.
SDS (w/v), 20% (v/v) glycerol and 5% (v/v) β-mercaptoethanol
as described previously (Yun and Kawagoe 2009). Proteins were
extracted from the seedlings in 10 vols. of extraction buffer
(v/w) using a plastic homogenizer. Proteins were separated
in pre-cast 10–20% polyacrylamide gels (ATTO). The gels were
either stained with Coomassie Brilliant Blue or blotted onto
a polyvinylidene fluoride (PVDF) membrane for immunoblotting with antibodies and enhanced chemiluminescence (ECL;
GE Healthcare). Antibodies against rice recombinant proteins
FtsZ2-1, FtsZ1 and ISA3 have been described previously (Yun
and Kawagoe 2009).
RT–PCR
Total RNA was extracted from the developing seed at 7 DAF
(days after flowering) using the RNeasy Plant Mini Kit (www
.qiagen.com). First-strand cDNAs were generated by reverse
transcriptase (www.roche.com) according to the manufacturer's instructions. The resulting cDNA samples were analyzed
by PCR using the following primers: MinD (AK059897),
5′-GAACCCCAACCCTAACCCTA-3′ and 5′-CTCTTCTGCAG
GGGCAATAG-3′; MinE (AK070044), 5′-AATGGCGACGTC
GATCTC-3′ and 5′-GTGCCTCAACGATGTTCTCA-3′; PDV2
(CI682676), 5′-CGTCTGCGCTCTCCTAGC-3′ and 5′-GGCAT
GCTACCATTCCTGTT-3′ (94°C for 15 s, 62°C for 30 s, 68°C
for 30 s for 30 cycles); and Actin (AK100267), 5′-GAAGGAT
CTATATGGCAACATCGT-3′ and 5′-ATTTCCTGTGCACAAT
GGATG-3′ (94°C for 30 s, 62°C for 2 s, 74°C for 30 s for 25 cycles).
PCR products were electrophoresed on an agarose gel and
visualized by staining with ethidium bromide.
Laser scanning confocal microscopy
Sections of developing seed were prepared as described previously (Yun and Kawagoe 2009). Confocal images of fluorescence signals were examined using a TCS SP2 AOBS laser
scanning confocal microscope (Leica Microsystems).
Supplementary data
Supplementary data are available at PCP online.
Funding
Plasmid construction and rice transformation
The binary vectors for Agrobacterium-mediated rice transformation were constructed from modified plasmids derived
from the Entry and Destination vectors of the Gateway system
(www.invitrogen.com) as previously described (Yun and Kawagoe
2009). The vectors and PCR primers are listed in Supplementary Tables S1 and S2, respectively. Transformation of calli
derived from rice seeds (cv. Yukihikari) and regeneration of
plants were conducted as described (Kawagoe et al. 2005).
Protein extraction and immunoblotting
Seed proteins were extracted from a fine flour of seeds in extraction buffer consisting of 50 mM Tris–HCl (pH 6.8), 8 M urea, 4%
This work was supported by the the Bio-Oriented Technology
Research Advanced Institution [Research and Development
Program for New Bio-Industry Initiatives and a grant from
the Ministry of Agriculture, Forestry and Fisheries of Japan
(Genomics for Agricultural Innovation, IPG-0023)].
Acknowledgments
We thank Dr. Yoshiaki Nagamura (NIAS) for providing us
with cDNA clones of MinD, MinE, FtsZ1 and PDV2. We thank
Ms. Chiemi Takaboshi and Mariko Shibahara for technical
assistance.
Plant Cell Physiol. 51(9): 1469–1479 (2010) doi:10.1093/pcp/pcq116 © The Author 2010.
1477
M.-S. Yun and Y. Kawagoe
References
Adams, D.W. and Errington, J. (2009) Bacterial cell division: assembly,
maintenance and disassembly of the Z ring. Nat. Rev. Microbiol. 7:
642–653.
Adams, S., Maple, J. and M ller, S.G. (2008) Functional conservation of
the MIN plastid division homologues of Chlamydomonas reinhardtii.
Planta 227: 1199–1211.
Begg, K.J. and Donachie, W.D. (1998) Division planes alternate in
spherical cells of Escherichia coli. J. Bacteriol. 180: 2564–2567.
Bramkamp, M., Emmins, R., Weston, L., Donovan, C., Daniel, R.A. and
Errington, J. (2008) A novel component of the division-site selection
system of Bacillus subtilis and a new mode of action for the division
inhibitor MinCD. Mol. Microbiol. 70: 1556–1569.
Colletti, K.S., Tattersall, E.A., Pyke, K.A., Froelich, J.E., Stokes, K.D. and
Osteryoung, K.W. (2000) A homologue of the bacterial cell division
site-determining factor MinD mediates placement of the chloroplast
division apparatus. Curr. Biol. 10: 507–516.
de Pater, S., Caspers, M., Kottenhagen, M., Meima, H., ter Stege, R. and
de Vetten, N. (2006) Manipulation of starch granule size distribution
in potato tubers by modulation of plastid division. Plant Biotech. J. 4:
123–134.
El-Kafafi, E.S., Mukherjee, S., El-Shami, M., Putaux, J.L., Block, M.A.,
Pignot-Paintrand, I., et al. (2005) The plastid division proteins, FtsZ1
and FtsZ2, differ in their biochemical properties and sub-plastidial
localization. Biochem. J. 387: 669–676.
Erickson, H.P., Taylor, D.W., Taylor, K.A. and Bramhill, D. (1996) Bacterial
cell division protein FtsZ assembles into protofilament sheets and
minirings, structural homologs of tubulin polymers. Proc. Natl Acad.
Sci. USA 93: 519–523.
Evers, A.D. (1971) Scanning electron microscopy of wheat starch. 3.
Granule development in endosperm. Starke 23: 157–192.
Fujiwara, M.T., Hashimoto, H., Kazama, Y., Abe, T., Yoshida, S., Sato, N.,
et al. (2008) The assembly of the FtsZ ring at the mid-chloroplast
division site depends on a balance between the activities of
AtMinE1 and ARC11/AtMinD1. Plant Cell Physiol. 49: 345–361.
Fujiwara, M.T., Li, D., Kazama, Y., Abe, T., Uno, T., Yamagata, H., et al.
(2009b) Further evaluation of the localization and functionality of
hemagglutinin epitope- and fluorescent protein-tagged AtMinD1
in Arabidopsis thaliana. Biosci. Biotechnol. Biochem. 73: 1693–1697.
Fujiwara, M.T., Sekine, K., Yamamoto, Y.Y., Abe, T., Sato, N. and
Itoh, R.D. (2009a) Live imaging of chloroplast FtsZ1 filaments,
rings, spirals, and motile dot structures in the AtMinE1 mutant
and overexpressor of Arabidopsis thaliana. Plant Cell Physiol. 50:
1116–1126.
Hannah, L.C. and James, M. (2008) The complexities of starch
biosynthesis in cereal endosperms. Curr. Opin. Biotechnol. 19:
160–165.
Hoshikawa, K. (1968) Studies on the development of endosperm in
rice. 10. Electron microscopic studies on the development of starch
granules in the endosperm cells. Proc. Crop. Sci. Jpn. 37: 97–106.
Itoh, R., Fujiwara, M., Nagata, N. and Yoshida, S. (2001) A chloroplast
protein homologous to the eubacterial topological specificity
factor MinE plays a role in chloroplast division. Plant Physiol. 127:
1644–1655.
Jane, J.L., Kasemsuwan, T., Leas, S., Zobel, H. and Robyt, J.F. (1994)
Anthology of starch granule morphology by scanning electronmicroscopy. Starch-Starke 46: 121–129.
Ji, Q., Oomen, R.J.F., Vincken, J.P., Bolam, D.N., Gilbert, H.J., Suurs, L.,
et al. (2004) Reduction of starch granule size by expression of an
1478
engineered tandem starch-binding domain in potato plants. Plant
Biotechnol. J. 2: 251–260.
Kanamaru, K., Fujiwara, M., Kim, M., Nagashima, A., Nakazato, E.,
Tanaka, K., et al. (2000) Chloroplast targeting, distribution and
transcriptional fluctuation of AtMinD1, a eubacteria-type factor
critical for chloroplast division. Plant Cell Physiol. 41: 1119–1128.
Kang, H.J., Hwang, I.K., Kim, K.S. and Choi, H.C. (2006) Comparison of
the physicochemical properties and ultrastructure of Japonica and
Indica rice grains. J. Agric. Food Chem. 54: 4833–4838.
Katz, F.R., Furcsik, S.L., Tenbarge, F.L., Hauber, R.J. and Friedman, R.B.
(1993) Behavior of starches derived from varieties of maize
containing different genetic mutations. Effects of starch genotype
on granular morphology. Carbohydr. Polym. 21: 133–136.
Kawagoe, Y., Kubo, A., Satoh, H., Takaiwa, F. and Nakamura, Y. (2005)
Roles of isoamylase and ADP-glucose pyrophosphorylase in starch
granule synthesis in rice endosperm. Plant J. 42: 164–174.
Kim, K.S., Kang, H.J., Hwang, I.K., Hwang, H.G., Kim, T.Y. and Choi, H.C.
(2004) Comparative ultrastructure of Ilpumbyeo, a high-quality
Japonica rice, and its mutant, Suweon 464: scanning and transmission
electron microscopy studies. J. Agric. Food Chem. 52: 3876–3883.
Leroch, M., Kirchberger, S., Haferkamp, I., Wahl, M., Neuhaus, H.E.
and Tjaden, J. (2005) Identification and characterization of a novel
plastidic adenine nucleotide uniporter from Solanum tuberosum.
J. Biol. Chem. 280: 17992–18000.
Lutkenhaus, J. (2007) Assembly dynamics of the bacterial MinCDE
system and spatial regulation of the Z ring. Annu. Rev. Biochem. 76:
539–562.
Lutkenhaus, J. (2008) Biochemistry—tinkering with acellular division.
Science 320: 755–756.
Maple, J., Chua, N.H. and M ller, S.G. (2002) The topological specificity
factor AtMinE1 is essential for correct plastid division site placement
in Arabidopsis. Plant J. 31: 269–277.
Maple, J. and M ller, S.G. (2007a) Plastid division coordination across
a double-membraned structure. FEBS Lett. 581: 2162–2167.
Maple, J. and M ller, S.G. (2007b) Interdependency of formation and
localisation of the Min complex controls symmetric plastid division.
J. Cell Sci. 120: 3446–3456.
Maple, J., Vojta, L., Soll, J. and M ller, S.G. (2007) ARC3 is a stromal
Z-ring accessory protein essential for plastid division. EMBO Rep. 8:
293–299.
Marbouty, M., Saguez, C., Cassier-Chauvat, C. and Chauvat, F. (2009)
Characterization of the FtsZ-interacting septal proteins SepF and
Ftn6 in the spherical-celled cyanobacterium Synechocystis strain
PCC 6803. J. Bacteriol. 191: 6178–6185.
Margolin, W. (2005) FtsZ and the division of prokaryotic cells and
organelles. Nat. Rev. Mol. Cell Biol. 6: 862–871.
Matsushima, R., Maekawa, M., Fujita, N. and Sakamoto, W. (2010)
A rapid, direct observation method to isolate mutants with defects
in starch grain morphology in rice. Plant Cell Physiol. 51: 728–741.
Miyagishima, S.Y., Froehlich, J.E. and Osteryoung, K.W. (2006) PDV1
and PDV2 mediate recruitment of the dynamin-related protein
ARC5 to the plastid division site. Plant Cell 18: 2517–2530.
Nakamura, Y. (2002) Towards a better understanding of the metabolic
system for amylopectin biosynthesis in plants: rice endosperm as
a model tissue. Plant Cell Physiol. 43: 718–725.
Nakanishi, H., Suzuki, K., Kabeya, Y., Miyagishima, S. (2009) Plantspecific protein MCD1 determines the site of chloroplast division in
concert with bacteria-derived MinD. Curr. Biol. 19: 151–156.
Okazaki, K., Kabeya, Y., Suzuki, K., Mori, T., Ichikawa, T., Matsui, M.,
et al. (2009) The PLASTID DIVISION1 and 2 components of the
Plant Cell Physiol. 51(9): 1469–1479 (2010) doi:10.1093/pcp/pcq116 © The Author 2010.
Compound starch granule synthesis in rice
chloroplast division machinery determine the rate of chloroplast
division in land plant cell differentiation. Plant Cell 21: 1769–1780.
Onda, Y., Kumamaru, T. and Kawagoe, Y. (2009) ER membranelocalized oxidoreductase Ero1 is required for disulfide bond
formation in the rice endosperm. Proc. Natl Acad. Sci. USA 106:
14156–14161.
Sato, K. and Ehara, Y. (1974) Studies on the starch contained in the
tissues of rice plants. XIV. Electron microscopic observations of
plastids of various organs and tissues. Proc. Crop. Sci. Jpn. 43:
111–122.
Schmitz, A.J., Glynn, J.M., Olson, B.J.S.C., Stokes, K.D. and
Osteryoung, K.W. (2009) Arabidopsis FtsZ2-1 and FtsZ2-2 are
functionally redundant, but FtsZ-based plastid division is not
essential for chloroplast partitioning or plant growth and
development. Mol. Plant 2: 1211–1222.
Shapter, F.M., Henry, R.J. and Lee, L.S. (2008) Endosperm and starch
granule morphology in wild cereal relatives. Plant Genet. Resour. 6:
85–97.
Shimada, H., Koizumi, M., Kuroki, K., Mochizuki, M., Fujimoto, H.,
Ohta, H., et al. (2004) ARC3, a chloroplast division factor, is a chimera
of prokaryotic FtsZ and part of eukaryotic phosphatidylinositol-4phosphate 5-kinase. Plant Cell Physiol. 45: 960–967.
Stokes, K.D., McAndrew, R.S., Figueroa, R., Vitha, S. and Osteryoung, K.W.
(2000) Chloroplast division and morphology are differentially affected
by overexpression of FtsZ1 and FtsZ2 genes in Arabidopsis. Plant
Physiol. 124: 1668–1677.
Stokes, K.D. and Osteryoung, K.W. (2003) Early divergence of the FtsZ1
and FtsZ2 plastid division gene families in photosynthetic eukaryotes.
Gene 320: 97–108.
Vitha, S., Froehlich, J.E., Koksharova, O., Pyke, K.A., van Erp, H. and
Osteryoung, K.W. (2003) ARC6 is a J-domain plastid division protein
and an evolutionary descendant of the cyanobacterial cell division
protein Ftn2. Plant Cell 15: 1918–1933.
Yang, Y., Glynn, J.M., Olson, B., Schmitz, A.J. and Osteryoung, K.W.
(2008) Plastid division: across time and space. Curr. Opin. Plant Biol.
11: 577–584.
Yoder, D.W., Kadirjan-Kalbach, D., Olson, B., Miyagishima, S.Y.,
DeBlasio, S.L., Hangarter, R.P., et al. (2007) Effects of mutations in
Arabidopsis FtsZ1 on plastid division, FtsZ ring formation and
positioning, and FtsZ filament morphology in vivo. Plant Cell Physiol.
48: 775–791.
Yun, M.S. and Kawagoe, Y. (2009) Amyloplast division progresses
simultaneously at multiple sites in the endosperm of rice. Plant Cell
Physiol. 50: 1617–1626.
Plant Cell Physiol. 51(9): 1469–1479 (2010) doi:10.1093/pcp/pcq116 © The Author 2010.
1479