Visualization of Plastids in Pollen Grains: Involvement of
FtsZ1 in Pollen Plastid Division
Lay Yin Tang1, Noriko Nagata2, Ryo Matsushima1, Yuling Chen3,4, Yasushi Yoshioka3 and
Wataru Sakamoto1,*
1Research
Institute for Bioresources, Okayama University, Kurashiki, Okayama, 710-0046 Japan
of Chemical and Biological Sciences, Japan Women's University, Bunkyo-ku, Tokyo, 112-8681 Japan
3Division of Biological Science, Graduate School of Science, Nagoya University, Chikusa-ku, Nagoya, 464-8602 Japan
2Department
Short Communication
Visualizing organelles in living cells is a powerful method
to analyze their intrinsic mechanisms. Easy observation
of chlorophyll facilitates the study of the underlying
mechanisms in chloroplasts, but not in other plastid
types. Here, we constructed a transgenic plant enabling
visualization of plastids in pollen grains. Combination of a
plastid-targeted fluorescent protein with a pollen-specific
promoter allowed us to observe the precise number, size
and morphology of plastids in pollen grains of the wild
type and the ftsZ1 mutant, whose responsible gene plays a
central role in chloroplast division. The transgenic material
presented in this work is useful for studying the division
mechanism of pollen plastids.
Keywords: FtsZ1 • Fluorescent protein • Male gametophyte
• Plastid division • Pollen.
Abbreviations: ARC, accumulation and replication of
chloroplasts; GFP, green fluorescent protein; IF2, translation
initiation factor 2; PD, plastid-dividing; RFP, red fluorescent
protein; VC, vegetative cell.
Plastids develop into various types according to plant development and perform various cellular activities. Proplastids
are undifferentiated plastids in stem cells and are capable of
giving rise to different plastid types. Chloroplasts for photosynthesis are one plastid type in which thylakoid membranes
are organized as granal stacks in mesophyll cells. Other plastid types include, for example, amyloplasts for storage synthesis in roots, and chromoplasts for carotenoid in fruits
(Sakamoto et al. 2008). Plastids are not created de novo, but
arise from pre-existing plastids by fission (Aldrige et al. 2005).
Chloroplast division occurs by the action of the plastiddividing (PD) ring. Several components have been identified
as being associated with the site of the PD ring. These include
proteins with a GTPase domain such as the dynamin-like
ARC5 (accumulation and replication of chloroplasts 5),
ARC6 and FtsZ1 (Robertson et al. 1995, Osteryoung et al.
1998, Gao et al. 2003). In contrast to extensive studies in
chloroplasts, the division machinery in other plastid types is
poorly understood. Very little is known about the division of
plastids in reproductive organs including pollen grains.
In Arabidopsis, a tricellular mature pollen grain consists
of one vegetative and two sperm cells. The vegetative cell,
which makes up the bulk of a mature pollen grain, contains
plastids that accumulate starch (Van Aelst et al. 1993). At
the initial stage of pollen formation from microspores, plastids are poorly differentiated, with an indistinguishable inner
membrane, in contrast to the double membrane structure
of proplastids in the meristem (Robertson et al. 1995, Kuang
and Musgrave 1996). Plastid differentiation and division
occur alongside pollen maturation. In mature pollen, the
final plastid structure contains a double membrane structure with several starch grains and simple thylakoid structures (Kuang and Musgrave 1996). Pollen grains exist in the
homogenous developmental stage in anthers, whereas the
shoot meristem contains cell layers where the cells contain
plastids with various morphologies and nucleoid structures
(Mascarenhas 1989, Fujie et al. 1994). Thus, pollen can
provide a homogenous tissue appropriate for the study of
plastid division. However, a technical problem is that the
unpigmented plastids in pollen can only be visualized using
fixed samples.
To achieve direct visualization of plastids in pollen, we
attempted to express green fluorescent protein (GFP;
Haseloff et al. 1997) in plastids. A chimeric GFP gene was
constructed in which GFP was fused to the DNA fragment
4Present
address: College of Life Science, Hebei Normal University, Shijiazhuang 050016, PR China.
*Corresponding author: E-mail, [email protected]; Fax, +81-86-434-1206.
Plant Cell Physiol. 50(4): 904–908 (2009) doi:10.1093/pcp/pcp042, available online at www.pcp.oxfordjournals.org
© The Author 2009. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.
All rights reserved. For permissions, please email: [email protected]
904
Plant Cell Physiol. 50(4): 904–908 (2009) doi:10.1093/pcp/pcp042 © The Author 2009.
Visualization of pollen plastid
corresponding to a transit peptide sequence derived from
plastid translation initiation factor 2 (IF2) (Miura et al. 2007).
The LAT52 promoter from tomato was used to drive transgene expression in vegetative cells (VCs) (Twell 1990). The
resulting chimeric construct was designated as VC-ptGFP
(Fig. 1A). Transgenic Arabidopsis plants were generated and
GFP signals in mature pollen grains were examined in the T3
generation onwards by fluorescence microscopy. Under the
normal light microscope, plastids in Arabidopsis pollen are
not visible because of the absence of chlorophyll (Fig. 1B).
In the VC-ptGFP plants, plastids in mature pollen grains
showed many discrete fluorescent globules corresponding
to plastids (Fig. 1C). Within each plastid there were nonfluorescing structures. These structures were most probably
starch grains. Starch grains develop in pollen plastids from
the vacuolated microspore stage and remain in mature
pollen grains (Kuang and Musgrave 1996). In the wild-type
pollen, the plastids were present in a variety of shapes while
the size did not show large variations (Fig. 1D–G). Occasionally, dumbbell-shaped plastids were observed (Fig. 1G). The
existence of dumbbell-shaped plastids indicates that the
division is active in pollen vegetative cells.
The area of a single plastid in wild-type pollen grains was
1.90 ± 0.54 µm2 (mean ± SD; n = 233). To estimate the plastid
number, three image planes were captured from a single
pollen grain and the total number of non-overlapping GFP
signals was scored. On average, each wild-type pollen grain
contained approximately 43 ± 15 plastids (mean ± SD;
n = 16). To confirm the correct targeting of the VC-ptGFP
transgene, we introduced the VC-mtRFP transgene into
VC-ptGFP plants by conventional crossing. The VC-mtRFP
transgene allows visualization of mitochondria in pollen VCs
with red fluorescent protein (RFP) (Matsushima et al. 2008).
Double visualization of plastids and mitochondria was
achieved in the same pollen (Fig. 1H–J). No overlapping
signals of mtRFP and ptGFP were detected in these pollen
grains. This confirmed that the GFP signals were indeed
localized in plastids. During pollen germination, VC plastids
were observed throughout the pollen tube, except at the tip
region (Fig. 1K, L). This is consistent with previous observations that the pollen tip region is free of large organelles
(Rosen et al. 1964, Pierson et al. 1990).
Although the components of the plastid division machinery have been identified by previous molecular genetic studies, most of them are involved in chloroplasts rather than
other types of plastids such as proplastids and amyloplasts
(Aldridge et al. 2005). Studying plastid division in the plastid
types other than chloroplasts is difficult, because they
are not visible by chlorophyll autofluorescence. We thus
considered that VC-ptGFP allows us to study plastid division.
The plant FtsZ1 gene is a critical factor in the chloroplast
division machinery. The Arabidopsis ftsZ1 mutants showed
enlarged chloroplasts in mesophyll cells (Yoder et al. 2007).
Fig. 1 Fluorescent images of the pollen of VC-ptGFP plants.
(A) Plasmid construct of plastid-targeted GFP that is expressed under
the control of pollen-specific promoter, LAT52-P (LAT52 promoter).
‘TP’ represents plastid-targeted transit peptide. ‘Nos-T’ is a terminator
of nopaline synthese. The plasmid name is VC-ptGFP. (B) Light
microscopic image of a mature pollen grain. Bar = 20 µm. (C) GFPlabeled plastids in a VC-ptGFP plant. Bar = 20 µm. (D–G) Different
morphologies of each plastid. Bars = 1 µm. (H–J) Visualization of
mitochondria and plastids in the same pollen by crossing VC-mtRFP
and VC-ptGFP plants. (H) RFP-labeled mitochondria. (I) GFP-labeled
plastids. (J) Merged image of (H) and (I). Bar = 20 µm. (K) Fluorescent
GFP-labeled plastids in a vegetative cell during pollen germination.
Bar = 20 µm. (L) Differential interference contrast image of the same
field of (G). Bar = 20 µm.
The mutant line that has a T-DNA insertion at the FtsZ1
locus, SALK_124371, was found using the SIGnaL ‘T-DNA
Express’ database search. As the T-DNA was inserted at the
first exon, this line is likely to be a null allele (producing no
FtsZ1 protein). We checked the genotype of the FtsZ1 locus
based on PCR analysis, and the homozygous line for T-DNA
insertion was obtained (Fig. 2A). We named this line ftsZ1.
The homozygous ftsZ1 plants showed enlarged chloroplasts in
leaf mesophyll cells compared with wild-type plants (Fig. 2B),
consistent with previous work. No FtsZ1 protein was detected
in the ftsZ1 mutant when analyzed through immunoblot
using AtFtsZ1-specific antibodies (Fig. 2C) (Stokes et al.
2000). We introduced the VC-ptGFP gene into the ftsZ1
mutant by crossing, and captured pollen images (Fig. 3A, B).
Plant Cell Physiol. 50(4): 904–908 (2009) doi:10.1093/pcp/pcp042 © The Author 2009.
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Fig. 2 Identification of an ftsZ1 null mutant. (A) Structure of the FtsZ1
gene. Black and gray boxes indicate the translated and untranslated
regions, respectively. T-DNA is inserted at the first exon of the FtsZ1
gene in the SALK_124371 line. (B) Autofluorescent images of
chloroplasts in a leaf mesophyll cell of the wild type and the ftsZ1
mutant. Bars = 10 µm. (C) Immunoblot analysis of protein extracts
from the wild type (WT) and the ftsZ1 mutant. The arrow indicates
the position of the FtsZ1 protein. The top band is a non-specific
signal.
The average area size of a ftsZ1 pollen plastid was
3.11 ± 1.15 µm2 (n = 233), showing a 64% significant increase
compared with the wild type (Mann–Whitney U-test,
P < 0.05). In addition, the plastid number was reduced to
15 ± 4 in ftsZ1 pollen (n = 16). This result clearly shows that
plastid division occurs in pollen grains, and FtsZ1 is involved
in the division process.
To compare the ultrastucture of plastids between wildtype and ftsZ1 pollen, we performed electron microscopic
observation (Fig. 4A–D). Plastids in both lines had an oval
shape surrounded by double membranes, and multiple
starch grains were present in each plastid. Occasionally,
simple thylakoid structures were also observed in both lines
(Fig. 4C, D). Thus, pollen plastids of the ftsZ1 mutant showed
an enlarged size but no other significant changes.
Before this report, Arabidopsis proplastids were observed
through microscopy sections of meristematic tissues
(Robertson et al. 1995, Robertson et al. 1996). Arabidopsis
pollen plastids were observed by electron microscopy of
fixed materials (Van Aelst et al. 1993, Kuang and Musgrave
1996). The transgenic material presented here provides a
convenient alternative for rapid determination of sizes,
numbers and morphology of pollen plastids. Due to the evident structural differences between plastid types, it is likely
906
Fig. 3 Enlarged pollen plastids in the ftsZ1 mutant. (A) GFP-fluorescent
plastids of the wild type (WT) and the ftsZ1 mutant. Bars = 10 µm.
(B) Mean value of plastid areas of the wild type (WT) and the ftsZ1
mutant (Mann–Whitney U-test; *P < 0.05).
that the division machinery is also different. This is supported by the observation that proplastid division is normal
in the arc5 mutant where chloroplast division is severely
affected (Robertson et al. 1995, Robertson et al. 1996). Studying pollen plastids in other chloroplast division mutants
using VC-ptGFP will be promising for a deeper understanding of plastid division.
Materials and Methods
Arabidopsis plants were germinated and grown according to
Miura et al. (2007). The ftsZ1 T-DNA seeds were obtained
through the Arabidopsis Biological Resource Center
(SALK_124371). T-DNA mutants were identified by PCR.
The presence of the wild-type allele was determined using
primer sets FtsZ1-1u2 (5′-CTGAGATTCCTTGTACTCC
TTG-3′) and FtsZ1-1d2 (5′-CTTCGTCGACACTAAAACC
CTA-3′) that flanked the insertion site, while the combination of the primer sets FtsZ1-1u2 and LBb1 (LBb1, 5′-GCG
TGGACCGCTTGCTGCAACT-3′) was used to identify the
mutant allele.
Western blot of FtsZ1 protein was performed as described
previously (Nishihama et al. 2001). We used the FtsZ1specific antibody that was described in Stokes et al. (2000) at
1 : 3,000 dilution. The secondary antibody, that was described
in Nishihama et al. (2001), was used at 1 : 2,000 dilution.
To generate transgenic plants with plastid-localized GFP
signals in the VCs of pollen, chimeric gene constructs were
introduced into ftsZ1 backgrounds by crossing. The constructs were all derived from the LAT52-GFPN plasmid
Plant Cell Physiol. 50(4): 904–908 (2009) doi:10.1093/pcp/pcp042 © The Author 2009.
Visualization of pollen plastid
Fig. 4 Ultrastructural analysis of pollen plastids. (A) and (B) Wild-type and ftsZ1 pollen, respectively. Bars = 5 µm. (C) and (D) Highly magnified
images of wild-type and ftsZ1 pollen plastids, respectively. Bars = 0.5 µm. Starch granules were labeled with ‘S’. The simple thylakoid membrane
and surrounding double membrane structures are indicated by an arrowhead and arrow, respectively.
donated by Dr. Sheila McCormick (University of Berkeley,
USA) and Dr. Shinichi Nishikawa (Nagoya University, Japan).
LAT52-GFPN plasmid contains the GFP gene inserted
between the LAT52 promoter and the nopaline synthase terminator. In this vector, SalI and NcoI restriction sites are
located between the LAT52 promoter and the GFP gene. To
construct VC-ptGFP, a DNA fragment containing the plastidtargeted transit peptide of IF2 (Miura et al. 2007) was produced by PCR amplification using the Columbia genome
as template and the primers 5′-TTTGTCGACATGCCA
TCGATGCTTGT-3′ and 5′-TGTCCATGGCGTCAGCAATG
AAGTCAG-3′. The fragment was treated with SalI and NcoI
and inserted into the SalI and NcoI site of LAT52-GFPN. To
generate transgenic Arabidopsis plants, Columbia wild-type
plants were transformed with the aforementioned VC-ptGFP
by the in planta method (Clough and Bent 1998).
Pollen grains were released onto glass slides by gentle
tapping and supplemented with 5% (w/v) mannitol (Nacalai
Tesque, Kyoto, Japan). GFP signals were detected with a
Disk Scanning Unit (DSU-BX61, Olympus, Tokyo, Japan)
fluorescence microscope using a GFP filter set (U-MGFPHQ,
Olympus), an excitation filter (460–480 nm), a dichroic
mirror (DM485) and a barrier filter (495–540 nm). RFP signal
detection and pollen germination were performed according to Matsushima et al. (2008).
The homozygous plants with the ftsZ1 mutation were
selected based on an enlarged plastid in leaf tissues. To view
plastids in leaf, tissue samples were placed on a glass slide,
supplemented with 50% glycerol (Nacalai Tesque) and covered with a coverslip. True leaves were sampled from 4- to
6-week-old plants. Autofluorescence signals were detected
by a filter set (U-MWIG3, Olympus), an excitation filter
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(530–550 nm), a dichroic mirror (DM570) and a barrier filter
(570 nm long pass).
The anthers of flower buds were fixed for 20 h in 4% glutaraldehyde and 4% paraformaldehyde, and then post-fixed
with 2% osmium tetroxide for 20 h. The fixed samples were
dehydrated through an ethanol series and embedded in
Spurr's resin. Ultra-thin sections were double-stained with
uranyl acetate and lead citrate, and then observed using a
JEM-1200 EX transmission electron microscope (Jeol, Tokyo,
Japan).
Images were analyzed using Metamorph (Molecular
Devices, Downingtown, PA, USA) and Adobe Photoshop
(Adobe Systems, Tokyo, Japan). Statistical analysis was performed using JMP7 (SAS Institute, www.jmp.com).
Funding
The Ministry of Education, Culture, Sports, Science and
Technology Grant-in-Aid for Scientific Research (No.
16085207 to W.S. and Nos. 18870018 and 20770036 to R.M.);
the Oohara Foundation.
Acknowledgments
The authors thank Dr. Katherine W. Osteryoung (Michigan
State University, USA) for the AtFtsZ1-specific antibodies,
Dr. Sheila McCormick (University of Berkeley, USA) and
Dr. Shinichi Nishikawa (Nagoya University, Japan) for the
LAT52-GFPN plasmid and also The Arabidopsis Biological
Resource Center for the T-DNA mutant line. We would also
like to thank Nami Sakurai-Ozato, Rie Hijiya and Sayuri
Tanabashi for their technical assistance.
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(Received January 29, 2009; Accepted March 4, 2009)
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