Download Contribution of the Plasma Membrane and Central Vacuole in the

Plant Cell Physiol. 45(7): 951–957 (2004)
JSPP © 2004
Short Communication
Contribution of the Plasma Membrane and Central Vacuole in the Formation
of Autolysosomes in Cultured Tobacco Cells
Kanako Yano 1, Sumiko Matsui 1, Tomohiro Tsuchiya 2, Masayoshi Maeshima 2, Natsumaro Kutsuna 3,
Seiichiro Hasezawa 3 and Yuji Moriyasu 1, 4
1
School of Food and Nutritional Sciences, University of Shizuoka, Shizuoka, Japan
Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, Japan
3
Department of Integrated Biosciences, Graduate School of Frontier Sciences, University of Tokyo, Kashiwa, Japan
2
;
using the endocytosis markers FM4-64 and Lucifer Yellow CH
and three resident proteins of the central vacuole (AtVam3p,
sporamin, and gamma-VM23), which were tagged with green
fluorescent protein (GFP).
Tobacco BY-2 cells were subcultured and subjected
to sucrose starvation conditions as described previously
(Moriyasu and Ohsumi 1996). The GFP(S65T)-AtVam3p/TDNA vector was kindly provided by Dr. M.H. Sato (Kyoto
University) (Uemura et al. 2002). Plasmids harboring the 35S
promoter-preprosporamin-GFP were constructed as follows.
Genomic DNA was isolated from the tubers of sweet potato
(Ipomoea batatas, cv. Kokei 14). The DNA region encoding
one of the preprosporamins, which has no introns, was amplified by PCR using the isolated DNA as a template and the
following primers: sense primer, 5′-cccctcgagatgaaagccctcacactcgcac-3′, which includes the XhoI site and 22 bases from
the 5′-end of the coding region in the coding strand of preprosporamin DNA; antisense primer, 5′-gtgatcaaacctaccgttgtgtccatggcatg-3′, includes the NcoI site and 22 bases from the 5′-end
of the coding region in the anticoding strand of preprosporamin DNA. The amplified DNA was digested with XhoI and
NcoI, and was then ligated into the SalI–NcoI site of a GFP
expression vector, 35Somega-sGFP(S65T) plasmid (Niwa et al.
1999), in order to obtain a plasmid expressing preprosporaminGFP fusion protein under the control of the cauliflower mosaic
virus 35S promoter. This plasmid was designated pKY1. It was
then cleaved with HindIII and EcoRI to obtain a DNA fragment
that included the 35S promoter-preprosporamin DNA-synthetic
GFP DNA-poly A signal of the nopaline synthase gene. The
DNA region from the HindIII to EcoRI sites of the binary
vector pSMAB701 (see Niwa et al. 1999) was replaced with
this DNA fragment. The resulting plasmid was designated
pKY101.
Plasmids harboring the 35S promoter-gamma-VM23-GFP
were constructed by amplifying the DNA region encoding
gamma-VM23 by PCR using pBluescript containing gammaVM23 cDNA (Higuchi et al. 1998) as a template and the
following primers: sense primer, 5′-tacctcgagatctcccacaactctccttatc-3′, which includes the XhoI and BglII sites and 18 bases
Autolysosomes accumulate in tobacco cells cultured
under sucrose starvation conditions in the presence of a
cysteine protease inhibitor. We characterized these plant
autolysosomes using fluorescent dyes and green fluorescent
protein (GFP). Observation using the endocytosis markers,
FM4-64 and Lucifer Yellow CH, suggested that there is a
membrane flow from the plasma membrane to autolysosomes. Using these dyes as well as GFP-AtVam3p, sporamin-GFP and gamma-VM23-GFP fusion proteins as
markers of the central vacuole, we found transport of
components of the central vacuole to autolysosomes. Thus
endocytosis and the supply from the central vacuole may
contribute to the formation of autolysosomes.
Keywords: Autolysosome — Autophagy — Central vacuole
— Endocytosis — FM4-64 — Green fluorescent protein.
Abbreviation: GFP, green fluorescent protein.
Autophagy is the process by which cellular components
are degraded in lysosomes/vacuoles through the rearrangement
and/or synthesis of cell membranes. Autophagy can be induced
in plant cells cultured under nutrient starvation conditions (as is
the case in mammalian and yeast cells), and such cultured cells
have been used to analyze the mechanisms of autophagy in
plant cells (Aubert et al. 1996, Moriyasu and Hillmer 2000 for
review). Lysosome-like structures accumulate in tobacco cells
cultured under sucrose starvation conditions and in the presence of a cysteine protease inhibitor (Moriyasu and Ohsumi
1996). Several lines of evidence suggest that these structures
correspond to the autolysosomes observed in mammalian
autophagy (Moriyasu and Ohsumi 1996). This includes the
acidic pH within the lumen and the presence of acid phosphatase and protease as well as partially degraded cytoplasmic
components such as mitochondria. However, the mechanism of
autolysosome formation has yet to be elucidated in plant cells.
In this study, we characterized these plant “autolysosomes”
4
Corresponding author: E-mail, [email protected]; Fax, +81–54–264–5099.
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Autolysosomes in tobacco cells
in the noncoding region of gamma-VM23 cDNA; antisense
primer, 5′-tgaccatgggcagtcctccgtaatca-3′, which includes the
NcoI site and sequence similar to 18 bases around the stop
codon in the anticoding strand of gamma-VM23. Amplified
DNA was digested with XhoI and NcoI, and was integrated into
the SalI–NcoI site of the 35Somega-sGFP(S65T) plasmid
(Niwa et al. 1999). This plasmid was cut with BglII and NotI,
and the fragment containing gamma-VM23 cDNA-GFP DNA
was integrated into the BglII–NotI site of the binary vector
pMAT137 (provided by Ken Matsuoka, RIKEN) in order to
obtain pMAT (gamma-VM23)-sGFP.
The GFP(S65T)-AtVam3p/T-DNA vector and the plasmids pKY101 and pMAT (gamma-VM23)-sGFP were used
for Agrobacterium-mediated transformation of tobacco cells
(Matsuoka and Nakamura 1991) in order to prepare cells
expressing GFP-AtVam3p, sporamin-GFP and gamma-VM23GFP fusion proteins, respectively.
In mammalian cells, the autophagic and endocytic pathways are known to converge at the endosomes (Liou et al.
1997). It was also suggested that a similar process occurs in
plant cells (Record and Griffing 1988, Herman and Lamb
1992), but, confirmative evidence appears to be lacking. We
thus examined whether autolysosomes in tobacco cells are
located on the endocytic pathway using a fluorescent tracer of
endocytosis, FM4-64. Tobacco cells at 4 d in culture, i.e., in the
logarithmic growth phase, were transferred to sucrose-free
Murashige and Skoog culture medium and cultured for 1 d in
the presence or absence of 10 µM of the cysteine protease
inhibitor E-64c. As previously reported (Moriyasu and Ohsumi
1996), autolysosomes accumulated in cells cultured in the presence of E-64c, but not in control cells cultured without E-64c
(data not shown). The plasma membranes of these two types of
cells were stained by incubation in culture medium containing
100 µM FM4-64 at 0°C for 30–60 min. At this temperature,
endocytosis does not occur (Vida and Emr 1995), and thus the
plasma membrane was stained with FM4-64 in both cell types
(Fig. 1A; E64, 0d and MeOH, 0d). Cells were washed to
remove the excess dye and then cultured in fresh sucrose-free
medium without the dye at 26°C for 1 d. FM4-64 fluorescence
moved to the membranes of the central vacuoles in the control
cells in which autolysosomes had not accumulated (Fig. 1A;
MeOH, 1d). In contrast, in the cells in which autolysosomes
had accumulated, the fluorescence moved from the plasma
membrane to autolysosomes as well as to the membranes of the
central vacuoles, with the majority of fluorescence located to
the autolysosomes leaving weaker fluorescence on vacuolar
membranes (Fig. 1A; E64, 1d).
In this pulse-labeling experiment, it was difficult to maintain the time resolution of observations because we were unable to completely block the process of endocytosis during the
washing step. We thus omitted the washing step and simply
increased the incubation temperature to 26°C after pre-labeling
at 0°C for 30 min. After 30 min at 26°C, numerous dotted
structures with a strong FM4-64 fluorescence appeared in the
peripheral cytoplasm in both types of cells (Fig. 1B; arrowheads). These punctated structures have been reported to be
endosomes (Ueda et al. 2001). Most of these putative endosomes emerged far away from the perinuclear region where
autolysosomes were accumulating. While these putative endosomes possessed a strong fluorescence, the autolysosomes did
not show fluorescence with comparable intensity (Fig. 1B;
compare fluorescence and Nomarski images in E64, 30 min).
Although we occasionally observed dotted structures that possessed FM4-64 fluorescence in the perinuclear region, the fluorescence images of the E-64c-treated cells were similar to those
of control cells (Fig. 1B; compare E64, 30min and MeOH,
30min). These results suggest that autolysosomes exist downstream of endosomes on the endocytic pathway from the
plasma membrane to the vacuolar membrane.
The presence of autolysosomes on the pathway of endocytosis can also be demonstrated using another fluorescent
marker, Lucifer Yellow CH, which is incorporated into the vacuoles in some plant cells not by endocytosis but through anion
transporters. In our experiment, however, pretreatment of
tobacco cells with 10 µM wortmannin for 2 h prevented the
uptake of Lucifer Yellow into the central vacuoles (data not
shown). This confirms that most of the Lucifer Yellow is taken
up into the central vacuoles by endocytosis under the present
experimental conditions.
The same types of cells as used in the FM4-64 experiment were incubated in sucrose-free culture medium containing 2 mg ml–1 Lucifer Yellow (Fig. 1C). At 2 h, faint
fluorescence was observed in the central vacuole (Fig. 1C;
MeOH, 2h). In the E-64c-treated cells, autolysosomes were
observed to have a stronger fluorescence in addition to the fluorescence in the central vacuole (Fig. 1C; E64, 2h). At 4 h, both
the central vacuoles and autolysosomes accumulated Lucifer
Yellow at higher concentrations (Fig. 1C; MeOH, 4h and E64,
4h). However, the intensity of fluorescence in autolysosomes
seemed to be saturated before that of the central vacuole. This
supports the observations obtained using FM4-64 and shows
that autolysosomes are present upstream from the central vacuole on the endocytic pathway. Autolysosomes have also been
reported to fuse with the central vacuole and pass undegraded
cytoplasmic components to the central vacuole in E-64c-treated
cells (Moriyasu and Ohsumi 1996). All these data support the
notion that plant autolysosomes exist on the endocytic pathway.
In the course of our experiments on endocytosis using
FM4-64, we noticed the possibility that there is a flow of membrane materials from the vacuole to autolysosomes. To confirm
this possibility, we stained the plasma membrane of 3-day-old
cells and then cultured these cells for 1 d under nutrient-sufficient conditions. This resulted in the preparation of cells whose
vacuolar membranes were stained with FM4-64, but whose
plasma membranes were not (Fig. 2A; 0d). When these cells
were cultured under sucrose starvation conditions in the presence of E-64c, autolysosomes that were stained with FM4-64
accumulated in the cells (Fig. 2A; E64, 1d). Concomitant with
Autolysosomes in tobacco cells
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Fig. 1 The localization of autolysosomes in cultured
tobacco cells on the endocytic pathway by the use of the
fluorescent dyes FM4-64 and Lucifer Yellow CH. (A)
Tobacco cells (4-day-old) were cultured under sucrose
starvation conditions for 1 d in the presence of 10 µM
E-64c (E64, 0d and E64, 1d) or in the presence of 1%
(v/v) methanol as a solvent control (MeOH, 0d and
MeOH, 1d). The plasma membranes of these two kinds
of cells were pulse-labeled with 100 µM FM4-64. The
cells were immediately observed (E64, 0d and MeOH,
0d), or cultured for another 1 d and then observed (E64,
1d and MeOH, 1d) using a confocal laser microscope
(MRC-1024, Bio-Rad) to obtain the image of FM4-64
fluorescence (red). Arrows indicate the accumulation of
autolysosomes. n denotes the location of the nucleus.
Bar represents 20 µm. (B) Two kinds of cells, prepared
in an identical manner as A, were incubated in sucrosefree culture medium containing 100 µM FM4-64 at 0°C
for 30 min and then for 30 min at 26°C. These cells
(E64, 30min and MeOH, 30 min) were observed using a
confocal laser microscope (LSM510, Zeiss) to obtain
FM4-64 fluorescence (red, upper) and Nomarski
(lower) images. Arrows indicate the accumulation of
autolysosomes. Arrowheads indicate putative endosomes. n denotes the location of the nucleus. Bar represents 20 µm. (C) Two kinds of cells, prepared in an
identical manner as A, were incubated in sucrose-free
culture medium containing 2 mg ml–1 Lucifer Yellow
CH. At 2 and 4 h, the cells were observed using a conventional epifluorescence microscope fitted with
Nomarski optics (OptiPhoto, Nikon). For each treatment indicated in the figure, the images of Lucifer Yellow fluorescence are shown on the left; the Nomarski
images on the right. Arrows indicate the accumulation
of autolysosomes. n denotes the location of the nucleus.
Bar represents 20 µm.
staining of the autolysosomal membranes was a drastic
decrease in FM4-64 fluorescence from vacuolar membranes
(Fig. 2A; E64, 1d). In contrast, when cells with FM4-64labelled vacuolar membranes were subjected to sucrose starvation in the absence of E-64c, movement of FM4-64 was not
observed, although a slight decrease in the intensity of fluorescence on the vacuolar membrane was apparent (Fig. 2A;
MeOH, 1d). This suggests that a net membrane flow occurs
from the central vacuole to the autolysosomes that accumulate
in the presence of E-64c.
Because the vacuolar membrane remains at the original
position even after a drastic decrease in FM4-64 fluorescence,
some membrane material must be supplied to the vacuolar
membrane compensating for the net transfer of vacuolar mem-
brane to the autolysosomes. We are not certain how such compensation occurs, but it is known that vacuoles have complicated intravacuolar membrane structures (Saito et al. 2002,
Uemura et al. 2002). We suppose that these structures function
as a membrane reservoir and compensate for the outflow of
vacuolar membranes in this case. The flow of membrane from
the central vacuole to the autolysosomes also occurs in the
absence of E-64c. Hence, we believe that the autolysosomal
membrane returns to the central vacuole during the process of
autophagy in the absence of E-64c. In the presence of E-64c,
autolysosomes cannot accomplish the degradation of enclosed
cytoplasm and so may be unable to return to the vacuolar membrane resulting in abnormal accumulation of autolysosomes.
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Autolysosomes in tobacco cells
Fig. 2 Transport of the fluorescent dyes
FM4-64 and Lucifer Yellow from the central
vacuole to autolysosomes. (A) The plasma
membrane of 3-day-old tobacco cells was
pulse-labeled with 100 µM FM4-64 for
30 min at 0°C. The cells were further cultured at 26°C for 1 d (0d). These cells were
transferred to sucrose-free culture medium
and cultured for another 1 d in the presence
of 10 µM E-64c (E64, 1d), or in the presence of 1% (v/v) methanol as a solvent control (MeOH, 1d). A confocal laser
microscope (MRC-1024, Bio-Rad) was used
to obtain the images of FM4-64 fluorescence (red). Arrows indicate the accumulation of autolysosomes. n denotes the location
of the nucleus. Bar represents 20 µm. (B)
Tobacco cells (3-day-old) were incubated in
nutrient-sufficient medium containing 2 mg
ml–1 Lucifer Yellow CH for 4 h. The dye was
then washed out of the culture medium, and
the cells were transferred to sucrose-free culture medium (0d). The cells were then cultured for another 1 d in the presence of
10 µM E-64c (E64, 1d), or in the presence of
1% (w/v) methanol as a solvent control
(MeOH, 1d). A conventional epifluorescence microscope equipped with Nomarski
optics was used to obtain fluorescence
(upper) and Nomarski (lower) images. n
denotes the location of the nucleus. Arrows
indicate the accumulation of autolysosomes.
Bar represents 20 µm.
Like the membrane marker FM4-64, the lumenal marker
Lucifer Yellow CH also moved from the central vacuole to the
autolysosomes. Tobacco cells were incubated in Lucifer Yellow for 4 h to prepare cells containing the dye in the central
vacuoles (Fig. 2B, 0d). When such cells were kept in sucrosefree culture medium in the presence of E-64c, Lucifer Yellow
accumulated in the newly formed autolysosomes (Fig. 2B; E64,
1d). The intensity of fluorescence in the autolysosomes was
higher than that in the central vacuole. This may be due to the
adsorption of Lucifer Yellow by inclusions in autolysosomes.
Lucifer Yellow fluorescence was not altered significantly when
accumulation of autolysosomes did not occur in the absence of
E-64c (Fig. 2B; MeOH, 1d).
Further evidence for the membrane flow from vacuolar
membranes to autolysosomes was obtained using transgenic
tobacco cells expressing a GFP-AtVam3p fusion protein
(Kutsuna and Hasezawa 2002). AtVam3p belongs to the syntaxin family of Arabidopsis and is localized in the vacuolar
membrane (Sato et al. 1997). This fusion protein has been
shown to be useful for observing the detailed structure of vacu-
Autolysosomes in tobacco cells
955
Fig. 3 Transport of vacuolar resident proteins
from the central vacuole to autolysosomes. (A)
Four-day-old tobacco cells expressing a fusion
protein of AtVam3 and GFP (0d) were cultured
under sucrose starvation conditions for 1 d in the
presence of 10 µM E-64c (E64, 1d) or in the presence of 1% (v/v) methanol as a solvent control
(MeOH, 1d). A confocal laser microscope
(LSM510, Zeiss) was used to obtain GFP fluorescence (green, left) and Nomarski (right) images.
Arrows indicate the accumulation of autolysosomes. n denotes the location of the nucleus. Bar
represents 20 µm. (B) Four-day-old tobacco cells
expressing a fusion protein of sporamin and GFP
(0d) were cultured under sucrose starvation conditions for 1 d in the presence of 10 µM E-64c
resulting in the accumulation of autolysosomes
(E64, 1d) or in the presence of 1% (v/v) methanol
as a solvent control (MeOH, 1d). A confocal laser
microscope (LSM510, Zeiss) was used to obtain
the images of GFP fluorescence (green). Arrows
indicate the accumulation of autolysosomes.
Arrowheads indicate the Golgi apparatus and/or
ER. n denotes the location of the nucleus. Bar represents 20 µm. (C) The plasma membrane of 3day-old tobacco cells expressing a fusion protein
of gamma-VM23 and GFP was pulse-labeled with
100 µM FM4-64 for 30 min at 0°C. The cells
were then cultured at 26°C for 1 d (0d). These
cells were transferred to sucrose-free culture
medium and cultured for another 1 d in the presence of 10 µM E-64c (E64, 1d), or in the presence of 1% (w/v) methanol as a solvent control
(MeOH, 1d). A confocal laser microscope (MRC1024, Bio-Rad) was used to obtain GFP (gammaGFP; green, left) and FM4-64 (FM4-64; red, middle) fluorescence images. The two fluorescence
images are combined (Merge; right). Arrows indicate the accumulation of autolysosomes. n denotes
the location of the nucleus. Bar represents 20 µm.
olar membranes in tobacco cells and Arabidopsis plants
(Kutsuna et al. 2003, Uemura et al. 2002). In the present
study also, the membrane of the central vacuole, including
membranes surrounding the transvacuolar strands, could be
recognized in 4-day-old cells (Fig. 3A; 0d). The number of
transvacuolar strands decreased in both E-64c-treated and
methanol-treated control cells under sucrose starvation conditions (Fig. 3A; E64, 1d and MeOH, 1d). This is consistent with
our previous finding (Moriyasu and Ohsumi 1996) and shows
that the transgenic cells respond to sucrose starvation in a
manner similar to wild-type cells. Interestingly, autolysosomes that accumulated in the cells exhibited a strong GFP
fluorescence (Fig. 3A; E64, 1d; arrows). In contrast, the morphology as determined by GFP fluorescence was hardly altered
when the cells were cultured in the absence of E-64c (Fig. 3A;
MeOH, 1d).
Using a sporamin-GFP fusion protein as a vacuolar lumenal marker, we obtained results similar to those shown using
the GFP-AtVam3p fusion protein (Fig. 3). Sporamin is a storage protein in tuberous roots of sweet potato (Maeshima et al.
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Autolysosomes in tobacco cells
1985). It is localized in the vacuolar lumen (Hattori et al.
1988). Its precursor consists of a signal peptide, N-terminal
propeptide which contains a vacuolar targeting signal, and the
mature protein. When the precursor is expressed in tobacco
cells, sporamin targets the central vacuole (Matsuoka et al.
1990). A fusion protein of a polypeptide consisting of a signal
peptide and N-terminal propeptide similar to those of sporamin and GFP has recently been shown to target the central
vacuole in Arabidopsis (Tamura et al. 2003). In 4-day-old
transgenic cells expressing the fusion protein, GFP fluorescence was mainly observed in the lumen of central vacuoles
(Fig. 3B, 0d) and also in other organelles such as ER and/or
Golgi apparatus (Fig. 3B, arrowheads in all panels). When
these transgenic cells were cultured under sucrose starvation
conditions in the presence of E-64c, strong GFP fluorescence
was observed on autolysosomes in addition to the central vacuole, ER, and Golgi apparatus (Fig. 3B; E64, 1d; arrows). These
results suggest that GFP-AtVam3p and sporamin-GFP move
from the central vacuole to autolysosomes in accordance with
the incorporation of components of the central vacuole into
autolysosomes.
To examine whether the autolysosomes accumulating the
GFP fusion proteins are the same as autolysosomes stained
with FM4-64, we used tobacco cells expressing a gammaVM23-GFP fusion protein, another marker for the vacuolar
membrane. VM23 is a major hydrophobic membrane protein in
vacuoles of radish taproots (Maeshima 1992). The gammaVM23-GFP fusion protein mainly localized to the vacuolar
membranes of 4-day-old transgenic cells (Fig. 3C; gammaVM23, 0d). The vacuolar membranes of these cells stained
with FM4-64 (Fig. 3C; FM4-64, 0d) showed fluorescence colocalized with GFP fluorescence (Fig. 3C; Merge, 0d), while
the plasma membranes and nuclear membranes showed GFP
fluorescence but not FM4-64 fluorescence. Sucrose starvation
of these cells in the presence of E-64c, resulted in accumulation
of autolysosomes with both GFP and FM4-64 fluorescence
(Fig. 3C; E64, 1d). This clearly shows that the autolysosomes
having gamma-VM23-GFP correspond to the same population
of autolysosomes stained with FM4-64.
It should be noted that the contribution of de novo synthesis and transport of the GFP fusion proteins from the ER via
the Golgi apparatus to autolysosomes cannot be excluded. The
fluorescence of sporamin-GFP in the autolysosomes was
indeed stronger than that in the central vacuole, suggesting this
possibility. However, this is not the only explanation for this
phenomenon; the intensity of GFP fluorescence may be
affected by several factors such as the turnover rate and quantum yield of sporamin-GFP in each organelle. We were unable
to design an experiment where the biosynthetic pathway was
blocked by cycloheximide because this reagent inhibits the
autophagic pathway in tobacco cells (Takatsuka et al. 2004).
All the data obtained in this study strongly suggest that the
components of the vacuolar membranes and sap moved to
autolysosomes that are newly formed under sucrose starvation
conditions. We interpret these data as showing that the central
vacuole contributes to autophagy by supplying components,
such as membranes and hydrolytic enzymes, to the autophagosomes/autolysosomes. By acquiring these hydrolytic enzymes
from the central vacuole, autophagosomes are believed to
transform to autolysosomes. In the autophagic process of mammalian cells, an autophagosome fuses with a pre-existing lysosome to become an autolysosome, in which the degradation of
the enclosed cytoplasm is completed. To date, the presence of
lysosomes has not been described in mature plant cells,
although it has been reported that the central vacuole itself
fuses with autophagosomes in autophagy of cultured sycamore
cells (Aubert et al. 1996). The present study, however, revealed
that a sort of “retrograde” transport from the central vacuole to
autophagosomes occurs in the autophagic pathway in tobacco
cells and that plant cells possess a lytic compartment, autolysosomes, which is distinct from the central vacuole.
Acknowledgments
We thank Dr. Masa H. Sato, Dr. Hiroaki Ichikawa, and Dr.
Ken Matsuoka for providing the vectors, GFP(S65T)–AtVam3,
pSMAB701, and pMAT137 respectively. We also thank Dr. Alison
Hills for critical reading of the manuscript. This work was supported in
part by ISAS Grant for Basic Biology Study oriented to utilization of
Space station, and “Ground-based Research Announcement for Space
Utilization” promoted by Japan Space Forum.
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(Received October 29, 2003; Accepted April 24, 2004)