Emiko Okubo-Kurihara1, Toshio Sano1,2, Takumi Higaki1, Natsumaro Kutsuna1,2 and Seiichiro
Hasezawa1,2,*
1Department
of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwanoha 5-1-5, Kashiwa,
Chiba, 277-8562 Japan
2Institute for Bioinformatics Research and Development (BIRD), Japan Science and Technology Agency (JST), Japan
Keywords: γTIP • Vauolar regeneration • Cell growth •
Aquaporin • Tobacco BY-2 cell • Miniprotoplast.
Abbreviations: BY-2, Bright Yellow 2; BY-GV, BY-2 cells
expressing GFP–AtVAM3 fusion proteins; BY-TIPG, BY-2
cells expressing NtTIP1;1–GFP fusion proteins; FM4-64, N(3-triethylammoniumpropyl)-4-[6-(4-(diethylamino)
phenyl)hexatrienyl]-pyridinium dibromide; GFP, green
fluorescent protein; MIP, major intrinsic protein; Ng,
Nicotiana glauca; Nt, Nicotiana tabacum; Pf , permeability
coefficient; Pg, Panax ginseng; PM, plasma membrane; TIP,
tonoplast intrinsic protein; vag, V-ATPase subunit G; VM,
vacuolar membrane.
Downloaded from pcp.oxfordjournals.org at University of South Carolina on August 22, 2011
Aquaporin is a water channel that increases water
permeability through membranous structures. In plants,
vacuoles are essential organelles that undergo dynamic
volume changes during cell growth. To understand
the contribution of aquaporins to plant cell growth,
we developed a transgenic tobacco BY-2 cell line
overexpressing the tonoplast intrinsic protein (TIP), γTIP.
Vacuolar membranes of isolated vacuoles from γTIPoverexpressing cells showed higher water permeation
activities than those from wild-type cells. We then
examined the role of γTIP in vacuolar regeneration of
evacuolated tobacco BY-2 protoplasts (miniprotoplasts).
Vacuolar regeneration from thin to thick tube-network
vacuoles and subsequent development of large vacuoles
was accelerated in miniprotoplasts of this cell line.
A parallel increase in the rate of cell expansion indicated
a tight relationship between vacuolar development and
cellular volume increases. Interestingly, overexpression of
tobacco γTIP also enhanced cell division. Thus, increased
vacuolar aquaporin activity may accelerate both cell
expansion and cell division by increasing water permeability
through the vacuolar membrane.
Regular Paper
Acceleration of Vacuolar Regeneration and Cell Growth
by Overexpression of an Aquaporin NtTIP1;1 in Tobacco
BY-2 Cells
The nucleotide sequence of tobacco γTIP, NtTIP1;1,
reported in this paper has been submitted in GenBank
as accession number AB371711.
Introduction
Vacuoles are multifunctional organelles that are involved in
plant growth, through the storage and lysis of substances,
and also in homeostasis of cytoplasmic pH, ions and metabolites (Marty 1999, Martinoia et al. 2007). In addition, as this
organelle occupies a large volume in plant cells, vacuolar
development leads directly to the enlargement of cells. For
example, changes in the vacuolar volume and shape of guard
cells modify guard cell shape and regulate stomatal apertures (Fricker and White 1990, Gao et al. 2005, Tanaka et al.
2007). In tobacco suspension cells placed under hyper-osmotic
conditions, the vacuolar volume was found to decrease,
based on the reduced cell volume, and a complex vacuolar
network could be observed (Kutsuna and Hasezawa 2005,
Reisen et al. 2005). In contrast, the Arabidopsis mutant,
vacuoleless1, which lacks vacuoles, shows defects in cell
elongation and in the orientation of cell division of its
embryos (Rojo et al. 2001). Therefore, vacuolar development
appears to be required for proper cell growth, including cell
elongation/expansion and cell division.
During vacuolar development, a number of vacuolar
transporters are required for vacuolar enlargement and
metabolism, for example proton pump activities, ion
transporters/channels and aquaporins that are involved
in energy production, osmotic regulation by mineral
*Corresponding author: E-mail, [email protected].
Plant Cell Physiol. 50(1): 151–160 (2009) doi:10.1093/pcp/pcn181, available online at www.pcp.oxfordjournals.org
© The Author 2008. 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. 50(1): 151–160 (2009) doi:10.1093/pcp/pcn181 © The Author 2008.
151
E. Okubo-Kurihara et al.
Results
Cloning of tobacco γTIP1;1
To investigate the role of aquaporins in vacuolar development,
we identified a tobacco cDNA homolog of the Nicotiana
glauca NgMIP3 (Smart et al. 2001) that was classified as a
γTIP and was expected to function as a vacuolar aquaporin.
As shown in Fig. 1A, the deduced amino acid sequence
encoded by the isolated tobacco cDNA was 97% identical to
that of NgMIP3 and 78% to the Arabidopsis TIP1;1 (AtTIP1;1).
Furthermore, the putative protein contained two NPA
(Asn–Pro–Ala) motifs that are well conserved among aquaporins (Fig. 1A, red boxes), and six hydrophobic segments
(p1–p6) as determined by a Kyte and Doolittle hydropathy
plot of the amino acid sequence (Fig. 1B). Therefore, we
designated the tobacco cDNA as NtTIP1;1.
Overexpression of NtTIP1;1–GFP in tobacco
BY-2 cells
To investigate the role of NtTIP1;1, as well as its cellular localization, we prepared a transgenic tobacco BY-2 cell line overexpressing NtTIP1;1 tagged with the green fluorescence
protein (NtTIP1;1–GFP) and placed under the control of the
cauliflower mosaic virus 35S promoter (the cell line being
152
designated BY-TIPG on the basis of tobacco BY-2 cells stably
expressing NtTIP1;1–GFP). The GFP fluorescence predominantly labeled the vacuolar membrane (VM) as confirmed
by vital staining of the VM with FM4-64 (Fig. 2A, Kutsuna
and Hasezawa 2002). Gene expression analysis demonstrated
that NtTIP1;1 transcript levels were 3-fold higher in the
tobacco BY-TIPG cells than in non-transformed BY-2 cells
and in tobacco BY-GV cells overexpressing GFP–AtVAM3
(Fig. 2B, Kutsuna et al. 2003). Furthermore, Western blot
analysis confirmed the stable expression of NtTIP1;1–GFP
(data not shown). AtVAM3 was localized to the VM
(Sato et al. 1997), and stable expression of AtVAM3
in tobacco BY-2 cells was found not to affect their growth
(Kutsuna et al. 2003). Expression levels of Nt-vag1, encoding
a vacuolar ATPase subunit G (Rouquié et al. 1998), were
comparable in the BY-TIPG, non-transformed BY-2 and
BY-GV cells (Fig. 2B).
Water permeation activity assay
To investigate the water transport activities of NtTIP1;1, we
monitored vacuolar swelling in prepared isolated vacuoles.
Exposure of isolated vacuoles from control BY-GV cells
to hypo-osmotic conditions, from 0.70 to 0.45 M mannitol
(Fig. 3A), caused them to swell almost immediately, with
the volume increases ceasing within 3 min, whereas volumes
of the tobacco BY-TIPG vacuoles reached their maximum
within 1 min (Fig. 3B). The osmotic water permeability coefficient (Pf) of the BY-TIPG and BY-GV VMs was calculated
to be 14.3 ± 0.9 and 5.7 ± 0.6×10–6 m s–1, respectively. These
results demonstrate that NtTIP1;1 possesses water permeation activities within the VM.
Effects of overexpression of NtTIP1;1 on cell
elongation during protoplast and miniprotoplast
culture
To assess the physiological impact of NtTIP1;1 overexpression on the transgenic tobacco BY-2 cells, we induced cell
elongation by culturing the cells in a modified FMS medium
with low auxin concentrations (Fig. 4A), as previously
reported for non-transgenic tobacco BY-2 cells (Hasezawa
and Syōno 1983). The average diameters of freshly prepared
BY-TIPG and BY-GV protoplasts were 37.4 ± 0.9 and
40.9 ± 0.8 µm, respectively, with the BY-TIPG protoplasts
being 0.91-fold smaller (Fig. 4B). After a 2 d culture under
conditions promoting cell elongation, the BY-TIPG and
BY-GV protoplasts elongated to 72.8 ± 7.9 and 63.9 ± 1.8 µm,
respectively, with the rate of increase being 1.24-fold higher
in the BY-TIPG protoplasts (Fig. 4B). These results suggest
that NtTIP1;1 expression promotes cell elongation during
protoplast culture.
To confirm the acceleration of cell elongation by NtTIP1;1
expression, we developed a more efficient and synchronized
cell elongation system using miniprotoplasts, which are
Plant Cell Physiol. 50(1): 151–160 (2009) doi:10.1093/pcp/pcn181 © The Author 2008.
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transport and water transport, respectively (Martinoia et al.
2007). Of these, the aquaporins are responsible for the transport of water that directly increases vacuolar volumes. The
vacuolar aquaporins, referred to as TIPs (tonoplast intrinsic
proteins), are classified according to their localization; those
localized in the tonoplast of storage vacuoles are referred
to as δTIPs, those in seed-type storage vacuoles as
αTIPs, and those in lytic vacuoles as γTIPs. In Arabidopsis,
10 genes have now been found to encode TIPs, with the
genes encoding γTIPs, δTIPs and αTIPs being designated in
a new nomenclature as TIP1, TIP2 and TIP3, respectively,
(Johanson et al. 2001, Maurel 2007).
In this study, to investigate vacuolar development and its
contribution to cell growth, we focused on the γTIP that was
initially demonstrated to function as a plant aquaporin
through its water transport activities in Xenopus oocytes
(Maurel et al. 1993). In Arabidopsis, reduced gene expression
of AtTIP1;1 by RNA interference produced small plants that
died when severely affected (Ma et al. 2004). In contrast,
overexpression of the cauliflower γTIP, BobTIP26-1, in
tobacco cells increased cell volume (Reisen et al. 2003). In
addition, overexpression of the Panax ginseng γTIP, PgTIP1,
in Arabidopsis increased seed size and plant growth, and also
enhanced salt and drought stress tolerance as well as cold acclimation ability (Lin et al. 2007, Peng et al. 2007). We isolated
the tobacco γTIP cDNA, NtTIP1;1, from BY-2 suspensioncultured cells and investigated its role in vacuolar development
and cell growth using miniprotoplast culture.
Vacuolar aquaporin and plant cell growth
A
NtTIP1;1
NgMIP3
AtTIP1;1
ASIAHAFGLFVAVSVGANISGGHVNPAVTFGAFVGGNITLFRGILYIIAQLLGSTVACFL 120
ASIAHAFGLFVAVSVGANISGGHVNPAVTFGAFVGGNITLFRGILYIVAQLLGSTVACFL 120
AAVAHAFGLFVAVSVGANISGGHVNPAVTFGAFIGGNITLLRGILYWIAQLLGSVVACLI 120
*::******************************:******:***** :******.***::
NtTIP1;1
NgMIP3
AtTIP1;1
LEFATGGMSTGAFALSAGVSVWNAFVFEIVMTFGLVYTVYATAIDPKKGDLGVIAPIAIG 180
LEFATGGMSTGAFALSAGVSVWNAFVFEIVMTFGLVYTVYATAVDPKKGDLGVIAPIAIG 180
LKFATGGLAVPAFGLSAGVGVLNAFVFEIVMTFGLVYTVYATAIDPKNGSLGTIAPIAIG 180
*:*****::. **.*****.* *********************:***:*.**.*******
NtTIP1;1
NgMIP3
AtTIP1;1
FIVGANILAGGAFTGASMNPAVSFGPALVSWTWTHQWVYWAGPLVGGGIAGVVYELIFIN 240
FIVGANILAGGAFTGASMNPAVSFGPALVSWTWTHQWVYWAGPLVGGGIAGVVYELVFIN 240
FIVGANILAGGAFSGASMNPAVAFGPAVVSWTWTNHWVYWAGPLVGGGIAGLIYEVFFIN 240
*************:********:****:******::***************::**:.***
NtTIP1;1
NgMIP3
AtTIP1;1
HSHEPLPSGDF 251
HSHEPLPSGDF 251
TTHEQLPTTDY 251
:** **: *:
B
4
Hydropathy
MPINQIAVGSHEELRQPGTLKAALAEFICTLIFVFAGQGSGMAFNKLSVDGTATPSGLIS 60
MPINQIAVGSHEELRQSGTLKAALAEFICTLIFVFAGQGSGMAFNKLSADGVATPAGLIS 60
MPIRNIAIGRPDEATRPDALKAALAEFISTLIFVVAGSGSGMAFNKLTENGATTPSGLVA 60
***.:**:* :* :..:*********.*****.**.*********: :*.:**:**::
0
p1
−4
0
p2
50
p3
p4
100
150
Position (a.a.)
p5
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NtTIP1;1
NgMIP3
AtTIP1;1
p6
200
250
Fig. 1 Multiple amino acid sequence alignment of NtTIP1;1, NgMIP3 and AtTIP1;1. Identical, conserved and semi-conserved amino acid residues are
denoted by asterisks (*), colons (:) and periods (.), respectively. Two NPA motifs are boxed in red. GenBank accession numbers of the corresponding proteins are as follows: NtTIP1;1 (AB371711), NgMIP3 (AF290619) and AtTIP1;1 (M84344). (B) Kyte and Doolittle hydropathy plot of NtTIP1;1. The six hydrophobic domains (p1–p6) are indicated.
evacuolated protoplasts generated by removal of the central
vacuole by centrifugation (Sonobe 1990). The BY-TIPG and
BY-GV miniprotoplasts elongated faster than protoplasts
cultured in a modified FMS medium with low auxin concentrations. By 12 h of culture, the miniprotoplasts increased
their cell volume but still maintained their spherical shape
(Fig. 5A, 0–12 h). From around 12 to 24 h of culture, the cells
elongated with a concomitant increase in their cell volume
(Fig. 5A, 24 h).
Measurement of cell sectional areas on micrographs
revealed that the sectional areas of freshly prepared BY-TIPG
miniprotoplasts were 0.8-fold the size of those from BY-GV
cells (2,248 ± 175 µm2 in BY-GV and 1,885 ± 197 µm2 in BYTIPG), although these became comparable after 24 h culture
(4,781 ± 486 and 4,763 ± 412 µm2 in BY-GV and BY-TIPG,
respectively, Fig. 5A, B). When the relative cell size increases
were plotted, the miniprotoplast sectional areas were found
to have increased by about 2.5-fold in BY-TIPG and 2.1-fold
in BY-GV after 24 h culture, suggesting a faster rate of size
increase in the BY-TIPG miniprotoplasts (Fig. 5C). Similarly,
the major axis lengths of freshly prepared miniprotoplasts
were 1.1-fold shorter in BY-TIPG than in BY-GV miniprotoplasts, although these elongated to comparable values after
24 h culture (Fig. 5D, E). However, the rates of miniprotoplast elongation, as evaluated from the ratios of the major
to minor axis lengths, were found to be comparable in the
BY-TIPG and BY-GV miniprotoplasts (Fig. 5E). These results
suggest that NtTIP1;1 overexpression also promotes cell
elongation in miniprotoplast culture.
Plant Cell Physiol. 50(1): 151–160 (2009) doi:10.1093/pcp/pcn181 © The Author 2008.
153
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Bright field
NtTIP1;1-GFP
A
0.70M
Mannitol
0.45M
Mannitol
BY-TIPG
FM4-64
B
1.20
Rel. diameter
BY-GV
A
1.15
1.10
BY-TIPG
BY-GV
1.05
1.00
0
Merged
1
2
3
4
5
Time (min)
Fig. 3 Swelling of isolated vacuoles from BY-TIPG and BY-GV cells. (A)
NtTIP1;1
Nt-vag1
12
A
4
BY-TIPG
BY-GV
BY-TIPG
BY-GV
BY-2
BY-2
0
0
Fig. 2 Vacuolar localization of NtTIP1;1 and its water transport activity.
(A) Localization of NtTIP1;1–GFP in tobacco BY-2 cells. Bright field (upper
left), NtTIP1;1–GFP (upper right), FM4-64 (lower left) and NtTIP1;1–GFP
and FM4-64 merged (lower right) images are shown. FM4-64 predominantly stains the vacuolar membrane. The scale bar represents 10 µm.
(B) Gene expression levels of NtTIP1;1 (left) and Nt-vag1 (right) in nontransformed tobacco BY-2 cells, in BY-GV cells that stably express GFP-AtVAM3 and in BY-TIPG cells that stably express NtTIP1;1-GFP. Results were
standardized to expression levels of the constitutive tobacco actin gene,
Tob103. Values are given as mean values (n = 3, ±SE).
Effects of overexpression of NtTIP1;1 on vacuolar
regeneration during miniprotoplast culture
The rapid increase in cell volume of the miniprotoplasts suggested that vacuoles eliminated by centrifugation were
regenerated and then expanded during the culture. Indeed,
observation of the VM labeled by GFP–AtVAM3 and
NtTIP1;1–GFP showed that only remnants of the VM could
be observed in the miniprotoplasts just after centrifugation
(Fig. 6A, vacuolar remnant) but that large vacuoles regenerated
after 24–36 h culture (Fig. 6A, large vacuoles). Interestingly, the
vacuoles formed tube-network-like structures during the
154
0d
1d
2d
BY-GV
4
8
BY-TIPG
8
B
90
Major axis length (µm)
Rel. transcripts
Rel. transcripts
12
80
BY-TIPG
BY-GV
70
60
50
40
30
0
1
2
(d)
Fig. 4 Accelerated cell elongation in BY-TIPG protoplasts. (A) Bright field
images of BY-GV (upper panels) and BY-TIPG (lower panels) protoplasts.
Scale bars represent 10 µm. (B) Changes in major axis lengths of BY-TIPG
(filled squares) and BY-GV (open squares) cells during protoplast culture.
Data represent mean values (n = 3, ±SE).
Plant Cell Physiol. 50(1): 151–160 (2009) doi:10.1093/pcp/pcn181 © The Author 2008.
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B
Confocal images of isolated vacuoles. Vacuoles from BY-GV (upper panels)
and BY-TIPG (lower panels) cells are shown in 0.70 M mannitol (left panels)
and after exposure to hypo-osmotic conditions from 0.70 to 0.45 M by
water application (right panels). Scale bars represent 10 µm. (B) Changes in
diameter of isolated vacuoles from BY-TIPG (filled squares) and BY-GV
(open squares) cells after the change from 0.70 to 0.45 M mannitol. Data
represent mean values (n = 3, ±SE), and 30 vacuoles were measured in each
experiment. The original values are shown in Supplementary Table S1.
Vacuolar aquaporin and plant cell growth
0h
12 h
BY-TIPG
C
6000
BY-TIPG
BY-GV
5000
Rel. cell sectional area
Cell sectional area (µm2)
B
4000
3000
2000
1000
0
6
12
18
24
BY-TIPG
BY-GV
3.0
2.5
2.0
1.5
1.0
0
Time (h)
BY-TIPG
BY-GV
Rel. major axis length
Major axis length (µm)
E
35
30
25
20
0
6
12
Time (h)
12
18
24
Time (h)
D
40
6
18
24
1.8
BY-TIPG
BY-GV
1.6
Downloaded from pcp.oxfordjournals.org at University of South Carolina on August 22, 2011
To examine the effects of NtTIP1;1 overexpression on
vacuolar regeneration, we time-sequentially compared the
frequency of appearance of these vacuolar types in the
BY-GV and BY-TIPG cells. The population of miniprotoplasts
with vacuolar remnants and thin tube-network vacuoles
after 9 h culture was approximately 60% in control BY-GV
cells, whereas in the BY-TIPG cells this population rapidly
decreased to about 20% (Fig. 6B). In addition, the thick
tube-network vacuoles appeared to develop faster in the
BY-TIPG cells than in the BY-GV cells, with these populations comprising up to almost 70 and 40% of the cells,
respectively, after 12 h culture (Fig. 6C). Furthermore, the
large vacuoles appeared earlier in the BY-TIPG cells than in
the BY-GV cells (Fig. 6D). These observations suggest that
overexpression of NtTIP1;1 accelerated the regeneration of
large vacuoles. A time-sequential examination of the endogenous NtTIP1;1 gene expression patterns during vacuolar
regeneration in the BY-GV miniprotoplasts showed twin
peaks of expression at 8 and 32 h after miniprotoplast preparation (Fig. 6E). These times correspond to the transition
points from the thin tube-network to the thick tube-network
vacuoles (Fig. 6C), and from the thick tube-network vacuoles
to large vacuoles (Fig. 6D), respectively.
24 h
BY-GV
A
Effects of NtTIP1;1 overexpression on protoplast
division
1.4
1.2
1.0
0
6
12
18
24
Time (h)
Fig. 5 Changes in BY-TIPG and BY-GV cell size and shape during miniprotoplast culture. (A) Bright field images of BY-GV (upper panels) and
BY-TIPG (lower panels) protoplasts. The scale bars represent 10 µm. (B–E)
Time-sequential morphometry of BY-TIPG (filled squares) and BY-GV
(open squares) miniprotoplasts. Changes in cell sectional area (B), relative
cell sectional area (C), length of the cell major axis (D) and the ratio of the
major and minor axis lengths (E) are shown. Data represent mean values
(n = 3, ±SE) and 100–150 cells were observed in each experiment.
regeneration of large vacuoles (data not shown; Fig. 6A, thin
and thick tube-network), and the series of vacuolar
structures formed during this process could be categorized
as follows (data not shown): as thin tube-network vacuoles
(Fig. 6A, thin tube-network), from conversion of the remnants of the vacuolar network; as thick tube-network vacuoles
(Fig. 6A, thick tube-network), from the isotropic swelling
and increase in diameter of the vacuolar network; and as
large vacuoles (Fig. 6A, large vacuole), from a subregion of
the vacuolar network.
To understand the additional role of NtTIP1;1 in cell growth,
we examined whether overexpression of NtTIP1;1 affected
cell division in protoplasts. After 2 d under low auxin concentrations, cell numbers reached about 6.0×104 cells ml–1
for the BY-GV protoplasts and 8.0×104 cells ml–1 for the
BY-TIPG protoplasts (Fig. 7A), whereas under high auxin
concentrations the divided BY-TIPG and BY-GV cell populations were comparable (data not shown). The higher dividing
activity of the BY-TIPG cells was confirmed by monitoring
expression of the cyclin B gene, NictaCycB1;2. The relative
NictaCycB1;2 expression level in BY-TIPG cells at 1 d of culture under low auxin concentrations was 3-fold higher than
that in the BY-GV cells under the same conditions (Fig. 7B),
but only about 0.5-fold the expression level of the BY-TIPG
cells under high auxin concentrations (data not shown).
We also monitored expression of the vacuolar proton pump
(H+-PPase), NtTVP9 (Lerchl et al., 1995), since a previous
report suggested that overexpression of the Arabidopsis
H+-PPase AVP1 promoted auxin transport and cell division
during organ development (Li et al. 2005). The relative
expression level of NtTVP9 in the BY-TIPG cells at 1 d of
culture under low auxin concentrations was 4-fold higher
than that in the BY-GV cells (Fig. 7C). These results indicate
that overexpression of NtTIP1;1 enhanced the expression of
NtTVP9.
Plant Cell Physiol. 50(1): 151–160 (2009) doi:10.1093/pcp/pcn181 © The Author 2008.
155
E. Okubo-Kurihara et al.
A
vacuolar
remnant
thick
tube-network
large vacuole
BY-TIPG
BY-GV
thin
tube-network
100
BY-TIPG
BY-GV
80
*
60
*
40
*
20
0
0
6
12 18
Time (h)
Rel. transcripts
E 1.5
D
thick tube-network
24
BY-TIPG
BY-GV
80
*
60
40
20
0
0
6
12 18
Time (h)
24
Cells with the respective vacuole (%)
100
C
Cells with the respective vacuole (%)
vacuolar remnant and
thin tube-network
large vacuole
100
80
BY-TIPG
BY-GV
*
60
40
*
20
0
*
0
6
12 18
Time (h)
24
NtTIP1;1
1.0
0.5
0
4
8 12 16 20 24 28 32 36
Time (h)
Fig. 6 Vacuolar regeneration in tobacco miniprotoplasts and effects of NtTIP1;1 overexpression. (A) Typical vacuolar structures in BY-GV (upper panels)
and BY-TIPG (lower panels) cells during miniprotoplast culture. The series of vacuolar structures observed could be categorized as: thin tube-networks
(conversion from remnants of the vacuolar network); thick tube-networks (isotropic swelling and increase in the diameter of the vacuolar network); and
large vacuoles (from a subregion of the vacuolar network). Scale bars represent 10 µm. (B–D) Changes in populations of miniprotoplasts with: (B) vacuolar
remnants or thin tube-network vacuoles, (C) thick tube-network vacuoles and (D) large vacuoles, in BY-TIPG (filled squares) and BY-GV (open squares)
cells. Data represent mean values (n = 3, ±SE). From 100 to 150 cells were observed in each experiment. Significance was determined by Student's t-test
with *P < 0.05. (E) Quantitative reverse transcription–PCR analysis of NtTIP1;1 gene expression during vacuolar regeneration in BY-GV miniprotoplasts.
The results were standardized on the basis of expression levels of the constitutive tobacco actin gene, Tob103. Data represent mean values (n = 3, ±SE).
Discussion
NtTIP1;1 possesses aquaporin activity
As vacuoles occupy a large volume of plant cells, vacuolar
enlargement would be expected to promote cell enlargement
156
directly. In this study, we investigated vacuolar development
and subsequent cell growth by focusing on a particular vacuolar aquaporin, NtTIP1;1. We have demonstrated that NtTIP1;1
is localized primarily on the VM (Fig. 2A) and that it has water
permeation activities on the VM, as shown by the rapid
Plant Cell Physiol. 50(1): 151–160 (2009) doi:10.1093/pcp/pcn181 © The Author 2008.
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Cells with the respective vacuole (%)
B
Vacuolar aquaporin and plant cell growth
swelling of vacuoles isolated from BY-2 cells stably expressing NtTIP1;1–GFP (BY-TIPG) (Fig. 3). The calculated osmotic
water permeability coefficient (Pf) of the VM in BY-TIPG
cells was about 2.5-fold that of BY-GV cells, and this corresponds to differences in NtTIP1;1 gene expression levels
between the cells (Fig. 2B). These results demonstrate that
NtTIP1;1–GFP possesses aquaporin activity.
10.0
Cell number (×104 cells / ml)
A
8.0
6.0
4.0
BY-TIPG
BY-GV
Acceleration of cell growth and vacuolar
regeneration by NtTIP1;1 overexpression
2.0
0.0
0
1
2
(d)
B
NictaCycB1;2
1.0
Rel. transcripts
0.8
0.6
0.4
0.2
0
0
1
2
(d)
C
NtTVP9
10.0
BY-TIPG
BY-GV
Rel. transcripts
8.0
6.0
4.0
2.0
0
0
1
2
(d)
Fig. 7 Accelerated cell division in BY-TIPG protoplasts under low auxin
conditions. (A) Cell numbers of BY-TIPG (filled squares) and BY-GV (open
squares) during protoplast culture. Data represent mean values (n = 3, ±SE).
(B) and (C) Cyclin and V-PPase gene expression levels in BY-TIPG (filled
boxes) and BY-GV (filled boxes) protoplasts. Expression of the (B) tobacco
cyclin B gene, NictaCyc1;2 , and of the (C) V-PPase gene, NtTVP9 , was
examined by reverse transcription–PCR. The results were standardized on
the basis of expression levels of the constitutive tobacco actin gene, Tob103,
and data represent mean values (n = 3, ±SE).
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Downloaded from pcp.oxfordjournals.org at University of South Carolina on August 22, 2011
BY-TIPG
BY-GV
We have shown in this study that NtTIP1;1 overexpression
promoted cell volume increases during protoplast and miniprotoplast culture (Figs. 4, 5), in agreement with previous
reports of γTIP overexpression (Reisen et al. 2003, Lin et al.
2007). In addition, however, we found that NtTIP1;1 overexpression enhanced vacuolar regeneration in miniprotoplasts
(Fig. 6A–D), and that the peak expression levels of endogenous NtTIP1;1 corresponded to the transition points from
the thin tube-network to thick tube-network vacuoles
(Fig. 6E). These results suggest that vacuolar aquaporin
activity could enhance vacuolar and cellular enlargement in
protoplasts and miniprotoplasts.
To increase vacuolar and cellular volumes, water uptake
through the plasma membrane (PM) as well as through the
VM is required. Most previous studies have discussed the
high water permeability of the VM compared with that of
the PM (Url 1971, Kiyosawa and Tazawa 1977, Maurel et al.
1997, Niemetz and Tyerman 1997), and indeed the water
permeability of the VM was found to be about 100-fold that
of the PM (Maurel et al. 1997, Gerbeau et al. 1999). However,
Murai-Hatano and Kuwagata (2007) measured the water
permeability of the PM and VM using radish protoplasts and
their isolated vacuoles, and demonstrated comparable and
high levels of osmotic water permeability in the PM and VM,
suggesting that vacuolar aquaporins may be a limiting factor
of water uptake by plant cells.
The factors generally limiting plant cell expansion are
thought to be cell wall pressure and the osmotic potential
that are generated by osmotic substances present
in the vacuoles. In our experimental system using protoplasts and miniprotoplasts, there was an absence of cell wall
pressure at least at the start of the culture. In normal cultured cells with cell walls, NtTIP1;1 overexpression had no
effect on cell size (data not shown). Therefore, cell wall pressure may be considered as the primary factor limiting cell
enlargement in BY-2 cells, whereas water permeability
through the VM may be only a secondary factor.
In addition, it is possible that overexpression of NtTIP1;1
indirectly affects the accumulation of osmotic substances
in vacuoles and also vesicular transport to vacuoles, as suggested by previous studies (Reisen et al. 2003, Ma et al. 2004,
Lin et al. 2007). In Arabidopsis, repression of AtTIP1;1 gene
157
E. Okubo-Kurihara et al.
expression by RNA interference affected global transcript
abundance, including of genes involved in carbon metabolism and cellulose synthesis (Ma et al. 2004). Indeed, as
NtTVP9 expression was up-regulated by NtTIP1;1 overexpression in this study (Fig. 7C), there is a similar possibility of
global transcript changes in the BY-TIPG cells. Future transcriptome and metabolome approaches will clarify this
possibility.
Acceleration of cell division by NtTIP1;1
overexpression
Materials and Methods
Plant materials and culture conditions
A tobacco BY-2 (Nicotiana tobacum L. cv. Bright Yellow 2)
suspension culture was diluted 95-fold with modified Linsmaier and Skoog medium at weekly intervals as described
(Nagata et al. 1992). The cell suspension was agitated on a
rotary shaker at 130 r.p.m. at 27°C in the dark. Transgenic
BY-2 cell lines stably expressing GFP-AtVAM3 (BY-GV cells,
Kutsuna and Hasezawa 2002) or NtTIP1;1-GFP (BY-TIPG
cells) were maintained similarly to the non-transformed
BY-2 cell line.
FM4-64 staining
To observe the VM in BY-TIPG cells, N-(3-triethylammoniumpropyl)-4-[6-(4-(diethylamino)phenyl)hexatrienyl]pyridinium dibromide (FM4-64; Molecular Probes,
Invitrogen, Carlsbad, CA, USA) was added to the cell suspensions to a final concentration of 32 µM. The cells were
incubated for 2 min, washed with fresh culture medium, and
then observed as described previously (Kutsuna and
Hasezawa 2002).
158
The tobacco γTIP cDNA, NtTIP1;1, was PCR amplified from
a tobacco BY-2 cDNA library using primers designed on the
basis of the N. glauca NgMIP3 sequence (accession No.
AF290619). The open reading frame region of NtTIP1;1 was
first subcloned into pENTR/D-TOPO vector (Invitrogen)
and then into the pGWB5 binary vector (Nakagawa et al.
2007) using the Gateway system (Invitrogen). The tobacco
BY-TIPG cell line was established by transformation with the
binary vector construct using the Agrobacterium tumefaciens strain, LBA4404, according to the method of Mayo
et al. (2006).
Real-time quantitative PCR was conducted in a Smart
Cycler 2 system (Cephid, Sunnyvale, CA, USA) using
SYBR Premix Taq (TAKARA BIO INC., Shiga, Japan) according to the manufacturer's specifications. Values of relative
transcripts were calculated with actin Tob103 fragments
(Moniz de Sa and Drouin 1996) amplified with primers
5′-ATTACCATTGGTGCCGAGAG-3′ and 5′-ATCCTCCGATC
CAGACACTG-3′. A 292 bp fragment of the NtTIP1;1 cDNA
was amplified with the PCR primers 5′-TTCGCAGGTCAG
GGTTCTGG-3′and 5′-CACCGAAGGTAACGGCGGGATT-3′.
A 285 bp fragment of the Nt-vag1 cDNA (Rouquié et al.
1998) was PCR amplified with primers 5′-TGCAAAGATCGA
GCACCTGAAA-3′ and 5′-CACGGGTTGAAACGAGTTC
GAG-3. A 202 bp fragment of the NictaCycB1;2 cDNA was
amplified with the PCR primers 5′-CATTGTGGATCGC
TACCTTGC-3′ and 5′-CACCGAAGGTAACGGCGGGATT-3′.
A 188 bp fragment of the NtTVP9 cDNA (Lerchl et al.
1995) was PCR amplified with primers 5′-GGTGTTCT
TGCCGGTGCTTTGG-3′ and 5′-CAGAGGGTCGCCAATG
GTGTCA-3.
Preparation of protoplasts, miniprotoplasts and
isolated vacuoles
To prepare BY-2 protoplasts, 3-day-old cell cultures were
incubated with an enzyme solution [1% cellulase Y-C (Kyowa
Chemical Products Co., Ltd., Osaka, Japan), 0.1% pectolyase
Y-23 (Kyowa Chemical Products Co.), 0.35 M mannitol,
pH 5.5] at 30°C for 60 min. Miniprotoplasts were prepared
from BY-2 protoplasts by density gradient centrifugation
with Percoll solution [30% Percoll (GE Healthcare Ltd,
Amersham, UK), 0.7 M mannitol, 20 mM MgCl2] at 15,000×g
for 30 min. Miniprotoplasts were collected from the pellet,
washed twice with 0.7 M mannitol and the vacuoles then
collected from the supernatant. Protoplasts and miniprotoplasts, in a state of elongation, were cultured at 27°C in a
modified FMS medium [1% sucrose, 4.6 mg l–1 Murashige and
Skoog Plant Salt Mixture (Wako Pure Chemical Industries
Ltd., Osaka, Japan), 0.001% thiamine-HCl, 0.1% myo-inositol,
5.4 × 10–7 mM naphthalene acetic acid, 4.4 × 10 –6 mM
Plant Cell Physiol. 50(1): 151–160 (2009) doi:10.1093/pcp/pcn181 © The Author 2008.
Downloaded from pcp.oxfordjournals.org at University of South Carolina on August 22, 2011
In the BY-TIPG protoplast cultures, cell division activity
was found to be enhanced under low auxin concentrations
(Figs. 4B, 7). In Panax ginseng, the PgTIP1 gene, encoding a
γTIP, was found to be constitutively expressed in habituated
cells that could grow in phytohormone-free medium, but
not in non-habituated cells (Lin et al., 2007). These results
are indicative of the potential involvement of vacuolar aquaporins in cell division. One possible explanation for the acceleration of cell division by vacuolar aquaporins might be that
the increased cell volumes would drive cell division. However,
the volumes of dividing cells were not significantly larger
than those of elongating cells (data not shown). Another
possibility is that up-regulation of NtTVP9 promoted auxin
transport in the BY-TIPG cells and resulted in an acceleration
of cell division, as previously reported for overexpression of
Arabidopsis H+-PPase AVP1 (Li et al., 2005). Further investigations are needed to clarify which factor(s) enhance cell
division in the BY-TIPG cells.
Tobacco transformation and gene expression
analysis
Vacuolar aquaporin and plant cell growth
benzyladenine (Hasezawa and Sy¯ono 1983)] with 0.35 M
mannitol for protoplasts and 0.5 M for miniprotoplasts.
Protoplasts, in a state of division, were cultured at 27°C in
a modified FMS medium [1% sucrose, 4.6 mg l–1 Murashige
and Skoog Plant Salt Mixture (Wako Pure Chemical Industries Ltd.), 0.001% thiamine-HCl, 0.1% myo-inositol,
5.4 × 10–6 mM naphthalene acetic acid, 4.4 × 10–6 mM benzyladenine (Hasezawa and Sy¯ono, 1983)] with 0.35 M mannitol
for protoplasts and 0.5 M for miniprotoplasts.
The osmotic water permeability (Pf) was calculated
according to Ohshima et al. (2001) from the following
equation:
Pf = (Vt − V0 )/( S ⋅ t ⋅ Vw ⋅ ∆π )
Morphometric analysis of cells
To quantify the shape of cells, regions of the cell images were
manually segmented using Adobe Photoshop. Based on
these images, the cell shape was approximated as an ellipse
and the size and major and minor axis lengths of the cell
calculated using the plugin; KBI MorphoMeasure (http://
of
hasezawa.ib.k.u-tokyo.ac.jp/zp/Kbi/ImageJKbiPlugins)
ImageJ (Abramoff et al., 2004).
The sequence of NtTIP1;1 has been deposited in GenBank
under the accession number, AB371711.
Supplementary data
Supplementary data are available at PCP online.
Funding
The Japan Society for the Promotion of Science (Scientific
Research grant No. 04392 to T.H.); the Ministry of Education,
Culture, Sports, Science and Technology, Japan Grant-in-Aid
for Scientific Research on Priority Areas (No. 19039008 to
S.H.); Institute for Bioinformatics Research and Development
(BIRD), Japan Science and Technology Agency (JST) (to S.H.)
Acknowledgments
We are grateful to Dr. T. Nakagawa (Shimane University)
for the kind gift of the pGWB5 binary vector, and Dr. M.
Maeshima (Nagoya University) for the kind gift of the antiγTIP antibody. We thank Dr. Y. Watanabe (Tokyo University)
for experimental support with Western blot analysis. We
also thank Drs. M. Murai-Hatano (National Agricultural
Research Center for the Tohoku Region) and T. Kuwagata
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