Plant Cell Physiol. 49(1): 30–39 (2008)
doi:10.1093/pcp/pcm162, available online at www.pcp.oxfordjournals.org
ß The Author 2007. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.
All rights reserved. For permissions, please email: [email protected]
Tissue and Cell-Specific Localization of Rice Aquaporins and
Their Water Transport Activities
Junko Sakurai
1, 2,
*, Arifa Ahamed 1, Mari Murai 1, Masayoshi Maeshima
2
and Matsuo Uemura
3
1
Climate Change Research Team, National Agricultural Research Center for Tohoku Region, Morioka, 020-0198 Japan
Laboratory of Cell Dynamics, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, 464-8601 Japan
3
United Graduate School of Agricultural Sciences and Cryobiosystem Research Center, Faculty of Agriculture, Iwate University,
Morioka, 020-8550 Japan
2
environment with abundant water, they can suffer from
water stress (Ishihara and Saito 1987, Jiang et al. 1988,
Hirasawa et al. 1992). During the daytime, under high
irradiance, leaves lose a large quantity of water. Imbalance
between the high transpirational demand from shoots and a
low supply from roots, due to limiting water transport, may
be a possible reason for this phenomenon (Hirasawa et al.
1992).
Water flows through vascular and non-vascular tissues
in plants. In non-vascular tissues, water moves through
a series of cell layers. Steudle (2000) has established a
composite transport model that explains the water movement through non-vascular tissues in roots. In his model,
three parallel pathways are considered. The first is an
apoplastic pathway, which means a flow through cell walls.
The second is a symplastic pathway from cell to cell through
plasmodesmata. The third is called a transcellular pathway,
where water and solutes have to cross cell membranes,
including the plasma membrane and tonoplast. The second
(symplastic) and the third (transcellular) pathways cannot
usually be distinguished and are considered as a cell–cell
pathway.
In roots, water flows radially from the root surface to
the central cylinder. Roots have apoplastic barriers, such
as exodermis and endodermis with Casparian strips and
suberin lamellae, which restrict the movement of water and
solutes through the apoplastic pathway (Miyamoto et al.
2001, Ranathunge et al. 2003, Schreiber et al. 2005, Franke
and Schreiber 2007). Rice has extremely well developed
apoplastic barriers compared with maize. Cell walls
of endodermis and exodermis in rice roots contain 6- and
34-fold, respectively, more aliphatic suberin than in maize
(Schreiber et al. 2005). Compared with intense studies on
the apoplastic pathway in rice roots, little is understood
about the cell–cell pathway, which is the other crucial
component for radial water movement.
Water flowing through the leaves constitutes
almost 30% of the whole plant hydraulic resistance
(Sack and Holbrook 2006). In leaves, water is supplied
from xylem and finally evaporates from stomata. It is still
controversial as to whether water movement in leaf blades is
Water transport in plants is greatly dependent on the
expression and activity of water transport channels, called
aquaporins. Here, we have clarified the tissue- and cell-specific
localization of aquaporins in rice plants by immunoblotting
and immunocytochemistry using seven isoform-specific
aquaporin antibodies. We also examined water transport
activities of typical aquaporin family members using a yeast
expression system in combination with a stopped-flow spectrophotometry assay. OsPIP1 members, OsPIP2;1, OsTIP1;1
and OsTIP2;2 were expressed in both leaf blades and roots,
while OsPIP2;3, OsPIP2;5 and OsTIP2;1 were expressed
only in roots. In roots, large amounts of aquaporins
accumulated in the region adjacent to the root tip (around
1.5–4 mm from the root tip). In this region, cell-specific
localization of the various aquaporin members was observed.
OsPIP1 members and OsTIP2;2 accumulated predominantly
in the endodermis and the central cylinder, respectively.
OsTIP1;1 showed specific localization in the rhizodermis
and exodermis. OsPIP2;1, OsPIP2;3 and OsPIP2;5 accumulated in all root cells, but they showed higher levels of accumulation in endodermis than other cells. In the region at 35 mm
from the root tip, where aerenchyma develops, aquaporins
accumulated at low levels. In leaf blades, OsPIP1 members
and OsPIP2;1 were localized mainly in mesophyll cells.
OsPIP2;1, OsPIP2;3, OsPIP2;5 and OsTIP2;2 expressed in
yeast showed high water transport activities. These results
suggest that rice aquaporins with various water transport
activities may play distinct roles in facilitating water flux and
maintaining the water potential in different tissues and cells.
Keywords: Aquaporin — Cell-specific localization —
Rice — Water transport activity.
Abbreviations: NIP, nodulin 26-like intrinsic protein; PIP,
plasma membrane intrinsic protein; RT–PCR, reverse transcription–PCR; SIP, small and basic intrinsic protein; TIP, tonoplast
intrinsic protein.
Introduction
Most of the rice plants cultivated in the world are
grown in paddy fields. Even in such a fortunate
Corresponding author: E-mail, [email protected]; Fax, þ81-19-641-7794.
30
Rice aquaporin localization in tissues and cells
mainly through the apoplastic pathway or the cell–cell
pathway, because details of water movement within leaves
remain poorly understood (Sack and Holbrook 2006).
Recent observations that leaf hydraulic conductance
varies rapidly with changes in temperature (Matzner and
Comstock 2001, Sack et al. 2004) and irradiance (Sack et al.
2005, Tyree et al. 2005, Cochard et al. 2007) have
highlighted an involvement of the cell–cell pathway in this
phenomenon (Sack and Holbrook 2006).
Key factors regulating cell–cell water movement are
membrane proteins called aquaporins (Hachez et al. 2006b,
Kaldenhoff and Fischer 2006, Maurel 2007). Aquaporins
facilitate a passive water movement across biological
membranes. Addition of a potent aquaporin inhibitor,
HgCl2, reduced root water uptake by 87% in rice (M. Murai
et al. unpublished data), supporting the notion that water
movement is tightly linked to aquaporin activities. Higher
plants have 430 members of the aquaporin family: 35 in
Arabidopsis (Johanson et al. 2001); 33 in maize (Chaumont
et al. 2001); and 33 in rice (Sakurai et al. 2005). They are
divided into four subfamilies, called plasma membrane
intrinsic proteins (PIPs), tonoplast intrinsic proteins (TIPs),
nodulin 26-like intrinsic proteins (NIPs) and small and basic
intrinsic proteins (SIPs).
The 33 rice aquaporins are composed of 11 PIPs,
10 TIPs, 10 NIPs and two SIPs (Sakurai et al. 2005).
Reverse transcription–PCR (RT–PCR) results showed that,
among rice PIP and TIP genes, 12 and 10 genes were
expressed at higher levels in roots and leaf blades,
respectively (Sakurai et al. 2005). However, detailed cellspecific localization of these aquaporin members and their
water transport activities have been elusive.
To enhance our understanding of rice aquaporin
function in the cell–cell pathway, we analyzed tissue- and
cell-specific localization of major rice aquaporins, in both
roots and leaf blades, using isoform-specific antibodies.
We also measured their water transport activities. We found
that aquaporins had various water transport activities and
showed tissue- and cell-specific accumulation, suggesting
that they may play distinct roles in facilitating water
movement in different tissues and cells.
Results
Binding specificities of rice aquaporin peptide antibodies
In order to analyze expression levels and localization of
rice aquaporin members, isoform-specific aquaporin antibodies were used. In the present study, seven antibodies
(anti-OsPIP1s, anti-OsPIP2;1, anti-OsPIP2;3, anti-OsPIP2;5,
anti-RsTIP1;1, anti-OsTIP2;1 and anti-OsTIP2;2) were
used. These aquaporin members were selected for this
work because their gene expression is relatively high in
31
rice plants (Sakurai et al. 2005). To raise the antibodies,
we selected unique sequences in either the N- or
C-termini, as these tail parts show the lowest sequence
similarities to each other compared with other regions,
including the six transmembrane domains (Fig. 1).
The binding specificities of these antibodies were
examined against recombinant aquaporin proteins
expressed in yeast cells. The antibodies showed target
specificities and did not cross-react with close homologs
of the target aquaporin (Fig. 2). Anti-OsPIP1s was designed
to recognize a common sequence among three members
of the OsPIP1 subgroup (Fig. 1A) and its specificity
was demonstrated (Fig. 2A). For OsTIP1;1, we used an
anti-RsTIP1;1 antibody to the C-terminal sequence
(INQNGHEQLPTTDY) of radish RsTIP1;1 (Suga and
Maeshima 2004), which partially shares the sequence with
OsTIP1;1 (ISHTHEQLPTTDY). As expected, this antibody recognized OsTIP1;1 specifically, and OsTIP1;2 to a
lower extent (Fig. 2C).
Comparison of aquaporin protein expression in leaf blades
and roots
Accumulation of aquaporins in crude membrane
fractions was compared between leaf blades and roots by
immunoblotting using the isoform-specific antibodies
described above. OsPIP1 members, OsPIP2;1, OsTIP1;1
and OsTIP2;2 accumulated both in leaf blades and in
roots (Fig. 3). On the other hand, OsPIP2;3, OsPIP2;5 and
OsTIP2;1 accumulated only in roots (Fig. 3).
In the case of some aquaporin antibodies, two or more
bands were detected. For example, judging from their calculated molecular weights, bands at 28 and 56 kDa of OsPIP2;1
are considered to be its monomeric and dimeric forms,
respectively (Fig. 3). These immunostained bands disappeared when the corresponding authentic peptide was added
to the reaction solution, supporting the reliability of the
immunological detection. On the other hand, a faint band at
23 kDa observed in leaf blades with anti-OsPIP2;5 antibody
did not disappear in the presence of peptide antigen. This
band was considered to be a non-specific artifact.
Aquaporin localization in roots
Accumulation of each aquaporin in root tissues was
analyzed by immunocytochemistry (Fig. 4). Root crosssections were prepared from tissues approximately either 4
or 35 mm away from the root tip in order to compare
aquaporin accumulation in different regions with different
tissue structures. We also examined aquaporin accumulation using longitudinal sections. At 4 mm from the root
tip, band plasmolysis occurred in endodermis (Fig. 4I).
In this region, all aquaporin antibodies stained the tissues
well (Fig. 4B–H) compared with pre-immune control serum
(Fig. 4A). Immunostained cells varied with the antibodies.
32
Rice aquaporin localization in tissues and cells
Fig. 1 Partial amino acid sequences of rice aquaporins and peptide sequences of antigens for each rice aquaporin antibody. Four OsPIP
and two OsTIP antibodies were raised from the peptide sequences (doubly underlined) of N- (A) and C-terminal (B) sequences,
respectively. An underline in OsTIP1;1 shows a high sequence similarity to an antigen peptide sequence (INQNGHEQLPTTDY) for antiRsTIP1;1 antibody (Suga and Maeshima 2004). Transmembrane domains (TM1, 5 and 6) and a helix (HE) are boxed. Amino acid residues
that are commonly preserved in all members of the listed aquaporins are marked by asterisks.
With the anti-OsPIP1s antibody, endodermis was exclusively stained, whereas rhizodermis, exodermis and sclerenchyma were found to be less stained (Fig. 4B). With
anti-OsPIP2;1, anti-OsPIP2;3 and anti-OsPIP2;5 antibodies, most cells were stained well, but the endodermis
was relatively well stained (Fig. 4C–E). Accumulation of
OsPIP2;1 in endodermis was also confirmed in the longitudinal section (Fig. 4K). Outer parts of the roots and the
root cap were also stained well by anti-OsPIP2;1 antibody
(Fig. 4K). OsTIP1;1 showed localization in rhizodermis and
exodermis (Fig. 4F). Accumulation of OsTIP1;1 in the outer
part of the roots was also observed in the longitudinal
section (Fig. 4L). The root cap was also stained with antiTIP1;1 antibody (Fig. 4L). Accumulation of OsTIP2;1 was
less intense and was seen in endodermis and the central
cylinder (Fig. 4G). OsTIP2;2 was localized in the central
cylinder (Fig. 4H).
At 35 mm from the root tip, all aquaporins examined
here showed less accumulation (Fig. 4P–V) compared with
the region at 4 mm (Fig. 4B–H). In this region, most of
the cortex had disappeared and was transformed into
aerenchyma. Aquaporins accumulated slightly in exodermis, sclerenchyma, aerenchyma, endodermis and the
central cylinder (Fig. 4–V). Low abundance of aquaporins
in this region was also confirmed using the longitudinal
sections; OsPIP2;1 accumulated less in the region at around
30–40 mm from the root tip (Fig. 4N) compared with the
region adjacent to the root tip at around 1.5–4 mm
(Fig. 4K).
Aquaporin localization in leaf blades
Two aquaporin antibodies, anti-OsPIP1s and antiOsPIP2;1, were used to analyze the aquaporin localization
in leaf blades, because these aquaporin members were
expressed at a relatively higher level in leaf blades (Fig. 3).
Both antibodies hybridized well in mesophyll cells
(Fig. 5B, C). Xylem and phloem were barely stained by
either antibody (Fig. 5B, C). Bundle sheath and epidermis
were slightly stained by anti-OsPIP1s and anti-OsPIP2;1
antibody, respectively (Fig. 5B, C).
Water transport activities of several aquaporin members
In order to measure the water transport activity, we
used a yeast heterologous expression system in combination
with stopped-flow spectrophotometry (Suga and Maeshima
2004, Sakurai et al. 2005). We adopted this method because
we can evaluate accurately the values of water transport
Rice aquaporin localization in tissues and cells
Os
;2
PI
P1
Os
Os
PI
P1
PI
P1
Os
;3
PI
P2
;1
Recombinant aquaporins
;1
A
(kDa)
28
Anti-OsPIP1s
B
Os
PI
P1
;1
Os
PI
P2
;1
Os
PI
P2
;2
Os
PI
P2
;3
Os
PI
P2
;4
Os
PI
P2
;5
Recombinant aquaporins
Anti-OsPIP2;1
(kDa)
29
Anti-OsPIP2;3
29
Anti-OsPIP2;5
28
33
typical results of the experiments. Compared with a slow
increase in the scattered light intensity of the empty vector
myc-pKT10, a rapid increase was observed in membrane
vesicles containing OsPIP2;3, indicating that OsPIP2;3 has
high water transport activity. The relative stimulation of
water transport activity of each aquaporin member was
calculated from the fitted exponential rate constants of the
reaction curves on the basis of expressed protein amounts
(Fig. 6C). OsPIP2;1, OsPIP2;2, OsPIP2;3, OsPIP2;5 and
OsTIP2;2 showed42.5-fold higher water transport activities
than that of the vector control. On the other hand, water
transport activities of OsPIP1;2, OsTIP1;1 and OsTIP1;2
were less stimulated (Fig. 6C).
Discussion
Anti-RsTIP1;1
;2
P2
;1
P2
TI
Os
;2
P1
TI
Os
P1
;1
TI
Os
TI
Os
Os
PI
P2
;1
C
Recombinant aquaporins
(kDa)
24
Anti-OsTIP2;1
21
Anti-OsTIP2;2
20
Fig. 2 Binding specificities of rice aquaporin antibodies. Crude
membrane fractions were prepared from yeast that expressed each
rice aquaporin and then subjected to immunoblotting with the
aquaporin antibodies for OsPIP1 members (A), OsPIP2 members
(B) and OsTIP members (C); 0.2 mg protein was applied for
each lane.
activities on the basis of the amount of expressed protein in
the membrane fractions. Each aquaporin was expressed in
yeast cells. Expression levels of aquaporins were compared
by immunoblotting using anti-myc antibody, since they
were tagged with a myc epitope at the N-termini. This myc
epitope was reported to have no effect on water channel
activities (Suga and Maeshima 2004). The membrane
fractions prepared from the yeast cells expressing rice
aquaporins showed immunostained bands at close to the
calculated molecular sizes of monomers (26–29 kDa for
PIPs and 20–24 kDa for TIPs) and dimers (52–57 kDa for
PIPs and 37 kDa for OsTIP2;2) (Fig. 6A). The expression of
each aquaporin was also confirmed by immunoblotting
with the isoform-specific antibodies (data not shown).
Water transport activities of crude membrane fractions
from the yeast cells were measured with a stopped-flow light
scattering spectrophotometer. The shrinking rate of membrane vesicles in hypertonic solution was monitored as an
increase in the scattered light intensity. Fig. 6B shows the
In this study, we examined the water transport activity
and the cell-specific localization of typical members of the
rice aquaporin family to clarify their individual roles, both
in the roots and in the leaf blades. We focused on six PIPs
(OsPIP1;1, OsPIP1;2, OsPIP1;3, OsPIP2;1, OsPIP2;3 and
OsPIP2;5) and three TIPs (OsTIP1;1, OsTIP2;1 and
OsTIP2;2) in the present study, because their gene expression levels were relatively high in rice plants (Sakurai et al.
2005). Although aquaporins have high sequence similarity
within each subfamily, we proved that careful selection of
peptide sequences makes it possible to raise isoform-specific
antibodies (Fig. 2). Immunoblot analysis using these
antibodies revealed the organ-specific accumulation of
aquaporins in roots and/or leaf blades (Fig. 3). The result
was consistent with their mRNA expression patterns
reported in our previous paper (Sakurai et al. 2005).
To clarify the function of aquaporin in the roots, we
examined aquaporin accumulation in root tissues by immunocytochemistry (Fig. 4). As far as it is known, these are the
first detailed data for the isoform-specific localizations of
seven aquaporins in roots. We used cross-sections from two
different root regions; one from the region adjacent to the
root tip (around 4 mm from the root tip) and the other from
a region away from the root tip (around 35 mm from the
root tip). There are differences between the two regions in
tissue structure; at 35 mm from the root tip, most of the
cortex observed at 4 mm (Fig. 4A–H) had disappeared
and was transformed into aerenchyma (Fig. 4O–V), which
is a structure specific to the roots of rice and other
hydrophytes. Different patterns of aquaporin accumulation
were observed in these two regions.
At 4 mm from the root tip, different aquaporin members were localized in different cells (Fig 4B–H). OsPIP1
members, OsPIP2;1, OsPIP2;3, OsPIP2;5 and OsTIP2;1
accumulated at high levels in endodermis (Fig. 4B–E, G).
In endodermis, band plasmolysis occurs (Fig. 4I) due to the
tight connection between plasma membranes and cell walls
34
Rice aquaporin localization in tissues and cells
(kDa)
150
100
75
LB R
LB R
LB R
LB R
LB R
LB R
LB
R
LB
R
(kDa)
150
100
75
50
50
37
37
25
20
25
20
Peptide −
Peptide +
AntiOsPIP1s
(kDa)
150
100
75
LB R
LB R
Peptide −
Peptide +
AntiOsPIP2;1
LB R
LB R
Peptide −
Peptide +
Peptide −
AntiOsPIP2;3
LB R
LB R
(kDa)
150
100
75
50
50
37
37
25
20
25
20
Peptide −
Peptide +
AntiRsTIP1;1
Peptide −
Peptide +
AntiOsTIP2;1
Peptide −
Peptide +
AntiOsPIP2;5
Peptide +
AntiOsTIP2;2
Fig. 3 Comparison of aquaporin protein amounts in the leaf blades and the roots. Crude membrane fractions were prepared from leaf
blades (LB) and roots (R) and then subjected to immunoblotting. Each aquaporin antibody was reacted with a membrane after
pre-incubation with (peptide þ) or without the peptide antigen (peptide ). The amounts of protein applied to each lane were 0.5 mg
(for anti-OsPIP2;1 and anti-RsTIP1;1 antibody), 1.5 mg (for anti-OsPIP1s, anti-OsTIP2;1 and anti-OsTIP2;2 antibody) and 5.0 mg (for antiOsPIP2;3 and anti-OsPIP2;5 antibody), respectively. Black and white arrowheads represent monomeric and dimeric forms of each
aquaporin, respectively.
at the positions where two endodermal cells come into
contact with each other (Ma and Peterson 2001). The
connection is attributed to Casparian bands with suberin or
lignin in the cell walls of the endodermal cells. Therefore,
these aquaporin members may facilitate water flux from
the cortex to the central cylinder by partially compensating
the apoplastic barriers of Casparian bands in endodermis.
In the outer part of the root, such as rhizodermis, exodermis and sclerenchyma, OsPIP2;1, OsPIP2;3, OsPIP2;5
and OsTIP1;1 accumulated at relatively higher levels
(Fig. 4C–F). These aquaporins may promote water influx
from the root surface to the cortex, since the outer part of
the roots is another potential apoplastic barrier to water
movement (Morita and Abe 1999, Miyamoto et al. 2001).
In cortex, OsPIP2;1, OsPIP2;3 and OsPIP2;5 accumulated
(Fig. 4C–E), suggesting their roles in facilitating the
water flow through the cortex. High levels of OsTIP2;2
accumulated in the central cylinder (Fig. 4H). OsTIP2;2
may contribute greatly to the promotion of water movement in cells of the central cylinder together with other
central cylinder-localized aquaporins such as OsPIP2;3 and
OsTIP2;1 (Fig. 4D, G). The isoform-specific aquaporin
localization has also been reported for other plants, such as
maize (Hachez et al. 2006a), Mesembryanthemum crystallinum (Kirch et al. 2000) and radish (Suga et al. 2003).
The results using cross-sections from the two different
regions and longitudinal sections revealed that aquaporin
was abundant in the region adjacent to the root tip, at
around 1.5–4 mm (Fig. 4B–L), and gradually decreased
toward the basal part of the roots at around 30–40 mm
(Fig. 4N–V). One possible function of aquaporins in the
region adjacent to the root tip is considered to be their role
in cell elongation, since this region includes the cell
elongation zone. The other possible function is a contribution to radial water movement. Kawata and Lai (1968)
examined water absorption in different parts of rice roots
using micropotometers. They demonstrated that, in rice
roots, the most water-absorbing region is where mature
Casparian bands with clear band plasmolysis are observed
in endodermis. The result of their study is consistent with
Rice aquaporin localization in tissues and cells
A
B
35
C
D
G
H
CC
CO
RH
EX
SC
E
EN
F
J
I
CO
CC
EN
K
RC
EN
CC
M
L
O
CO
N
P
Q
R
T
U
V
CC
EN
AE
S
SC EX
Fig. 4 Localization of aquaporins in the primary roots. Cross-sections (7 mm) were prepared approximately 4 mm (A–I) and 35 mm (O–V)
from the root tip of 38-day-old rice plants. Longitudinal sections (7 mm) were prepared from 0–5 mm (J–L) and 30–40 mm (M, N) from the
root tip. The sections were hybridized with each aquaporin antibody and then visualized with a horseradish peroxidase-linked second
antibody and DAB. A, J, M, O, pre-immune (100-fold dilution); B, P, anti-OsPIP1s (50-fold); C, K, N, Q, anti-OsPIP2;1 (C, K, N, 300-fold,
Q, 100-fold); D, R, anti-OsPIP2;3 (50-fold); E, S, anti-OsPIP2;5 (100-fold); F, L, T, anti-RsTIP1;1 (F, L, 300-fold; T, 100-fold); G, U, antiOsTIP2;1 (G, 50-fold; U, 25-fold); H, V, anti-OsTIP2;2 (100-fold); I, a close-up view of D around the endodermis. AE, aerenchyma; CC,
central cylinder; CO, cortex; EN, endodermis; EX, exodermis; RH, rhizodermis; SC, sclerenchyma. Arrowheads in I indicate band
plasmolysis. Bars represent 100 mm (A–H, O–V), 50 mm (I), 1 mm ( J, K, L) and 500 mm (M, N).
PI
P1
PI
My
A
Os
EP
c-p
KT
10
A
;2
P2
Os
;1
PI
P2
;
2
Os
PI
P2
;
Os
3
PI
P
Os 2;5
TI
P
Os 1;1
TI
P1
;2
Os
TI
P2
;2
Rice aquaporin localization in tissues and cells
Os
36
(kDa)
75
PX
SV
BS
LV
50
MX
ME
ST
37
PH
SC
25
20
B
Scattered light intensity
B
Scattered light intensity
Anti-myc
C
1.94
1.92
1.90
1.88
1.86
1.84
Myc-pKT10
1.82
1.98
1.96
1.94
1.92
1.90
1.88
OsPIP2;3
1.86
0
2
4
6
8
10 12 14 16 18 20
our present observation that high levels of aquaporins
accumulated in the region where band plasmolysis occurred
in endodermis (Fig. 4B–L). Therefore, it is considered that
aquaporins in the region adjacent to the root tip may
greatly contribute to the cell–cell pathway of radial water
movement. This result contrasts with the recent report on
maize aquaporin localizations (Hachez et al. 2006a).
The expression of maize PIP genes was low at 0–10 mm
from the root tip, and gradually increased toward the zone
at 50–60 mm, corresponding to the mature root tissues,
where both suberized endodermis and exodermis were
7
6
5
4
3
2
1
0
My
Fig. 5 Localization of aquaporins in leaf blades. Cross-sections
(7 mm) were prepared from the middle part of the uppermost leaves
of 18-day-old rice plants. The sections were treated in the same
way as indicated in Fig. 4. A, pre-immune (100-fold dilution);
B, anti-OsPIP1s (200-fold); C, anti-OsPIP2;1 (300-fold). BS, bundle
sheath; EP, epidermis; LV, large vascular bundle; ME, mesophyll;
MX, metaxylem; PH, phloem; PX, protoxylem; SC, sclerenchymatous cell; ST, stomata; SV, small vascular bundle. Bars represent
100 mm.
8
c-p
K
Os T10
PI
P
Os 1;2
PI
P
Os 2;1
PI
P
Os 2;2
PI
P2
;3
Os
PI
P2
;5
Os
TI
P1
;1
Os
TI
P1
;2
Os
TI
P2
;2
C
Relative stimulation of
osmotic water permeability
Time (sec)
Fig. 6 Comparison of water transport activities of eight typical rice
aquaporins. Crude membrane fractions were prepared from yeasts
and then subjected to immunoblotting with anti-myc antibody (A);
0.35 mg proteins were applied to each lane. Typical changes in
scattered light intensities during the stopped-flow analysis are
shown for vector control myc-pKT10 and OsPIP2;3 (B). Each
reaction curve shows the average trace of 14 independent
determinations. The relative stimulation fold of osmotic water
permeability was calculated as the ratio of the rate constant of each
aquaporin to that of the vector control on the basis of the expressed
amount of protein (C). Values are expressed as mean SE
calculated for three independent experiments.
Rice aquaporin localization in tissues and cells
observed (Hachez et al. 2006a). The authors concluded that
the decrease in the hydraulic conductivity of maize root due
to the development of suberized apoplastic barriers may be
somewhat balanced by the presence of a high level of
aquaporin expression (Hachez et al. 2006a). Future
examination using roots at various developmental stages
under different growth conditions will help to clarify the
differences in aquaporin accumulation observed in maize
and rice.
In leaf blades, high levels of both OsPIP1 members
and OsPIP2;1 accumulated in mesophyll cells (Fig. 5).
Accumulation of PIP proteins in mesophyll cells was also
reported for Arabidopsis (Robinson et al. 1996) and
tobacco (Otto and Kaldenhoff 2000). On the other hand,
several plant PIPs were reported to accumulate in other
tissues. PIP localization in guard cells was found in
Arabidopsis (Kaldenhoff et al. 1995) and spinach
(SoPIP1;1) (Fraysse et al. 2005). Another spinach PIP
(SoPIP1;2) accumulated in phloem sieve elements (Fraysse
et al. 2005). Mesembryanthemum crystalluinum PIP was
mainly localized in vascular tissues (Yamada et al. 1995).
Two possible functions are considered for mesophyll
cell-specific rice aquaporins. One is that they may play
crucial roles in the maintenance of water potential in
mesophyll cells. Recently, Cochard et al. (2007) showed that
light-induced up-regulation of hydraulic conductance of
walnut leaves may be correlated with an increase in the
amounts of PIP aquaporin in leaves. The other possible
function is that they may contribute to CO2 diffusion in the
mesophyll cells, since some plant PIP proteins were reported
to transport CO2 (Uehlein et al. 2003, Hanba et al. 2004,
Flexas et al. 2006). Overexpression of barley aquaporin
HvPIP2;1 in rice caused an increase in internal CO2
conductance in leaf blades (Hanba et al. 2004). The
possibilities of CO2 transport by OsPIP members remains
to be examined, and may improve our understanding of
their physiological significance.
Measurement of water transport activities revealed that
rice aquaporin members had diverse activities (Fig. 6C). We
have already demonstrated that OsPIP2;4 and OsPIP2;5
(PIP2 members) have high water transport activities
compared with OsPIP1;1 and OsPIP1;2 (PIP1 members)
(Sakurai et al. 2005). Similar results were obtained in the
present study; high activities were confirmed in other PIP2
aquaporin members (OsPIP2;1, OsPIP2;2 and OsPIP2;3)
(Fig. 6C). These low and high activities of PIP1- and PIP2type aquaporins, respectively, were also found in maize
using an oocyte expression system (Chaumont et al. 2000,
Fetter et al. 2004) and in radish using a yeast system (Suga
and Maeshima 2004). OsTIP2;2 had a high water transport
activity, which agreed with the result obtained with maize
ZmTIP2;3 (Lopez et al. 2004). However, OsTIP1;1 and
OsTIP1;2 had low activity. This result is in contrast to
37
previous reports on other plant TIP1 members. Arabidopsis
AtTIP1;1 (Maurel et al. 1993) and radish RsTIP1;1 (Suga
and Maeshima 2004) have high water transport activities.
We need to clarify whether the low activities of two OsTIP1
members applies only in rice, or if the activity was inhibited
in the yeast expression system we used in the present study,
since several researchers reported regulation of TIP
activities by phosphorylation (Maurel et al. 1995) and
membrane trafficking (Vera-Estrella et al. 2004, Boursiac
et al. 2005).
We conclude that, in roots, rice aquaporins accumulate
abundantly in the region adjacent to the root tip. In this
region, aquaporins showed different cell-specific localization, indicating that they may play distinct roles in the cell–
cell water movement in different cells. In leaf blades, high
levels of PIP aquaporins accumulated in mesophyll cells.
Further, we demonstrated that OsPIP2 members and
OsTIP2;2 can act as ‘active’ water channels. Future analysis
of aquaporin mutants which either lack or overexpress
an individual aquaporin will help to uncover the precise
function of each aquaporin member for water transport in
rice plants.
Materials and Methods
Plant materials and growth conditions
Rice (Oryza sativa L. cv. Akitakomachi) seeds were germinated in the dark for 3 d at room temperature and then grown in
a growth chamber under 12 h light/12 h dark (light period;
450 mmol s1 m2) and with day/night temperatures set at
25/208C and a relative humidity of 75%. Plants were grown in
tap water for the first 5 d and then in the hydroponic solution. The
solution was changed every other day until the plants were used for
experiments. The content of the culture solution was reported
previously (Sakurai et al. 2005). For immunoblot analysis, leaf
blades (upper three leaves) and roots (apical half) were harvested
from 15-day-old plants. These tissues were immediately frozen in
liquid nitrogen and were stored in a deep freezer until required. For
immunocytochemistry, root and leaf blade samples were harvested
from 38- and 18-day-old plants, respectively.
Preparation of crude membrane fractions
The frozen tissues were ground into powder with a mortar
and pestle in liquid nitrogen. The powder was mixed with 3 vols.
of a homogenizing medium that contained 50 mM Tris-acetate
(pH 7.5), 0.25 M sorbitol, 2 mM EGTA, 10 mM 4-amidinophenylmethanesulfonyl fluoride hydrochloride (p-APMSF) (SigmaAldrich, St Louis, MO, USA), 1% (w/v) polyvinylpyrrolidone
K-30 (Nacalai Tesque, Kyoto, Japan) and 2 mM dithiothreitol
(DTT). The homogenate was centrifuged at 2,500 g for 5 min
(himac CF15RX, Hitachi, Tokyo, Japan), and the resulting
supernatant was centrifuged at 10,000 g for 15 min (Avanti HP25I, Beckman, Fullerton, CA, USA). The second supernatant was
then centrifuged at 150,000 g for 30 min (Optima XL-90,
Beckman) to obtain the pellet of crude membrane fractions.
The pellet was washed with suspension buffer that contained
50 mM Tris-acetate (pH 7.5), 2 mM EGTA, 10 mM p-APMSF and
2 mM DTT. The final pellet was suspended in the same suspension
38
Rice aquaporin localization in tissues and cells
buffer with a teflon homogenizer and was used as the crude
membrane fraction for immunoblotting.
Preparation of isoform-specific peptide antibodies
Aquaporin antibodies were prepared against peptides that
corresponded to the N- or C-terminal parts of each OsPIP and
OsTIP protein (Fig. 1). Each peptide sequence was specific to the
corresponding aquaporin family member. The peptides were
coupled to keyhole limpet hemocyanin (KLH) and the resulting
conjugates were injected into rabbits. Anti-RsTIP1;1 antibody was
raised against a peptide (INQNGHEQLPTTDY) and specifically
reacted with radish TIP1;1 (Suga and Maeshima 2004).
Immunoblotting
Protein samples were subjected to SDS–PAGE (12.5% gels)
and then transferred to a polyvinylidene fluoride (PVDF)
membrane with a semi-dry blotting apparatus for 40 min at 15 V.
The membrane was blocked by 4% (w/v) skimmed milk in Trisbuffered saline (TBS). Then the membrane was reacted with each
aquaporin or anti-myc antibody (Nacalai Tesque) (1,000-fold
dilution). After washing, the membrane was visualized with a
second antibody, horseradish peroxidase-linked protein A (2,000fold dilution) (GE Healthcare Biosciences, Waukesh, WI, USA)
and ECL detection reagents (GE Healthcare Biosciences).
Immunocytochemistry
Root tissue samples of 38-day-old plants were excised at
0–1 cm and 3–5 cm from the root tip of primary roots. Leaf blade
samples were taken from the middle part of the uppermost leaves
of 18-day-old plants. After fixation with 3.7% formaldehyde, 5.0%
acetic acid and 50% ethanol, the tissues were dehydrated through
an ethanol series. The ethanol was displaced by xylene and the
samples incubated at 608C in melted paraffin (Paraplast plus,
Sigma-Aldrich). After five changes of paraffin over a 48 h period,
the tissues were embedded in paraffin blocks, and sectioned into
7 mm slices by a Leica RM2125RT microtome (Leica Microsystems
GmbH, Nussloch, Germany). After dewaxing, the sections were
gradually hydrated by an ethanol series from 100 to 75%. Then
sections were treated with peroxidase block solution (0.3% H2O2 in
methanol) for 30 min to block the intrinsic peroxidase activity.
After washing with distilled water and phosphate-buffered saline
(PBS) they were blocked with 1% bovine serum albumin in
PBS–Tween-20 (0.2%) and then reacted with aquaporin antibodies
(25- to 300-fold dilution). The sections were visualized with a
second antibody, horseradish peroxidase-linked protein A (200fold dilution), in combination with a 0.1% solution of DAB
(3, 30 -diaminobenzidine tetrahydrochloride, Nacalai Tesque).
Expression of rice aquaporins in yeast
EcoRI–SalI fragments of rice aquaporin cDNA (OsPIP2;1,
OsPIP2;2, OsPIP2;3, OsTIP1;1, OsTIP1;2 and OsTIP2;2) were
inserted into a yeast expression vector, myc-pKT10 vector (Tanaka
et al. 1990, Suga and Maeshima 2004), and were introduced into
the yeast Saccharomyces cerevisiae strain BJ5458 as described
previously (Sakurai et al. 2005). The yeast strain is deficient in
major vacuolar proteases and functional aquaporins (Suga and
Maeshima 2004).
Determination of the osmotic water permeability of membranes
Yeast crude membrane fractions that contained each recombinant rice aquaporin were prepared as described previously
(Nakanishi et al. 2001). The membranes were finally suspended
in 50 mM mannitol, 90 mM KCl, 1 mM EDTA, 1 mM EGTA and
20 mM Tris–HCl, pH 7.2. Osmotic water permeability of the
membranes was measured by a stopped-flow spectrophotometer
(model SX18MV, Applied Photophysics Ltd, Leatherhead, UK).
The yeast membrane vesicles (0.4 mg ml1) containing rice aquaporin in the 50 mM mannitol solution were quickly mixed with an
equal volume of 500 mM mannitol solution. The shrinking rate
of membrane vesicles at 108C was monitored as an increase of
scattered light intensity. An average was calculated from 14
reactions for each membrane sample. Initial rate constants were
calculated from exponential fittings. Relative stimulation fold of
osmotic water permeability was determined by the ratio of the rate
constant of each aquaporin to that of the vector control on the
basis of the expressed amount of proteins calculated from the
results of immunoblotting using anti-myc antibody. Expression
levels of each aquaporin protein were visualized by Light-Capture
(ATTO, Tokyo, Japan) and calculated from signal intensities using
an image analyzing software CS analyzer (ATTO).
Funding
Grants-in-Aids for Scientific Research from the Ministry of Education, Sports, Culture, Science and Technology
of Japan (17780196, 18380151) to J.S. and M.M.; the
Program for Promotion of Basic Research Activities for
Innovative Biosciences (PROBRAIN); the Bio-Design
Program of the Ministry of Agriculture, Forestry, and
Fishes of Japan.
Acknowledgements
We are grateful to Dr. Masumi Okada and Dr. Kensaku
Suzuki for their valuable discussions throughout this work. We
also thank Mr. Masahiro Mizutani and Mr. Satoshi Watanabe for
their valuable advice and technical support in conducting
measurements of water transport activities. We would like to
thank Dr. Ai Oikawa, Dr. Takami Hayashi and Dr. Nobuyo
Yoshida for their helpful advice regarding the immunocytochemistry assay. We greatly appreciate Mrs. Katsuko Takasugi for
providing experimental assistance.
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(Received August 30, 2007; Accepted November 16, 2007)