T

he transmembrane movement of water, a relatively polar

molecule was, until recently, thought to be accomplished
by the diffusion of water molecules across the lipid bilayer
of membranes of living organisms. In retrospect, the reason for
this assumption is not evident, as artificial lipid bilayers had been
found to be much less permeable to trans-bilayer water movement
when compared with biological membranes, such as the plasma
membranes of erythrocytes and renal epithelial cells
1
. However,
because no proteinaceous water channels or transporters had
been identified, it was believed that water crossed biological
membranes without the aid of specific channels or transporters.
The net transport of water across biological membranes is
dependent on hydrostatic and osmotic gradients
2
. The hydraulic
conductivity of a membrane is proportional to the osmotic water
permeability (P
f
), which is a property of each specific membrane,
measured by imposing an osmotic or hydrostatic gradient across
the membrane. The diffusional permeability (P
d
), which is also a
specific property of each membrane, is a measure of the free dif-
fusion of water across a membrane, as measured by the exchange
of water without an imposed gradient. For a synthetic lipid
bilayer, P
f
roughly equals P
d
. However, for biological membranes,
P
f
has been found to be greater than P
d
(Refs 3,4). For example,
much higher P
f
:P
d
ratios have been established for the movement
of water across the erythrocyte membrane. The Arrhenius acti-
vation energy (i.e. the temperature dependence) of the osmotic
water permeability of erythrocytes is similar to that for free diffusion
of water and much lower than the activation energy for diffusional
water permeability of an artificial lipid bilayer. Taken together,
this suggests that transmembrane aqueous pores are responsible
for most of the water transport across the erythrocyte membrane.
Unlike diffusional water permeability, which cannot be inhibited
by chemical means, it is possible to inhibit the osmotically driven
water permeability by chemical reagents known to covalently modify
cysteine residues, suggesting that proteins are components of the
transmembrane pores. The first protein with water transport activ-
ity to be identified was CHIP28 (channel forming integral protein
of 28 kDa), a major erythrocyte plasma membrane protein
5
.
CHIP28 had been serendipitously isolated and cloned and
found to have a high sequence homology to a previously isolated
and cloned plasma membrane protein, the major intrinsic protein
(MIP) of the bovine lens fibre cell membrane
6
. When oocytes
were injected with cRNA corresponding to the CHIP28 mRNA,
and then incubated for a few days, allowing the oocyte protein
synthesis machinery to synthesize and target CHIP28 to the
oocyte plasma membrane, the transgenic oocytes swelled much faster
than control oocytes when placed in a hypotonic solution. To-
gether with the abundance of the CHIP28 protein in cells known
to be permeable to water
7
, such as erythrocytes and epithelial cells
of the kidney, this identified CHIP28 (now renamed aquaporin
1, AQP1) as the first example of a water channel protein
5
.
When oocytes are transferred to a hypotonic solution (five
times more dilute) it typically takes wild-type oocytes an hour to
swell 50% and rupture, compared with oocytes transiently
expressing a water channel protein, which rupture within a few
minutes after a comparable increase in volume. Genes encoding
proteins homologous to the MIP of the eye lens-fibre cell mem-
brane belong to the MIP family of proteins and have been found in
organisms ranging from bacteria to fungi, plants, insects and
mammals. Using the oocyte expression system, most of the MIP
homologues tested are aquaporins, that is water channels. How-
ever, two MIP family members, GlpF in E. coli and Fps1 in yeast,
transport glycerol but not water
8,9
. Furthermore, a few MIPs have
mixed specificity, for example the human AQP3 allows the pas-
sage of glycerol as well as water
10
, and NOD26 of soybean allows
the passage of glycerol and formamide in addition to water
11
.
Based on the biochemical and structural analysis of two-dimen-
sional crystals of AQP1, a 6 three-dimensional structure has
been resolved
12
. AQP1 contains six transmembrane domains with
both the C- and the N-terminal at the cytoplasmic side of the
membrane, a topology that is conserved in all MIP homologues
(Fig. 1). AQP1 appears to exist as tetramers in the native mem-
brane, although each monomer has water transport activity
13
.
Plant aquaporins
Results consistent with the presence of proteins facilitating trans-
membrane water flow in plant cells
14
, and the identification of
several major plant membrane proteins with high sequence hom-
ology to AQP1 and MIP (also known as AQP0)
1522
, led to the
identification of a water channel protein in plant membranes
23
.
This water channel, -TIP, is localized to the tonoplast (i.e. the
vacuolar membrane)
20
. So far, all aquaporins of mammalian cells
are localized to the plasma membrane, whereas plant cells contain
308
trends in plant science
reviews
August 1999, Vol. 4, No. 8 1360 - 1385/99/$ see front matter 1999 Elsevier Science. All rights reserved. PII: S1360-1385(99)01438-7
Aquaporins and water
homeostasis in plants
Per Kjellbom, Christer Larsson, Ingela Johansson, Maria Karlsson and Urban Johanson
Aquaporins are water channel proteins of vacuolar and plasma membranes. When opened
they facilitate the passive movement of water molecules down a water potential gradient. In
Arabidopsis, 30 genes have been found that code for aquaporin homologues. Some of these
genes code for highly abundant constitutively expressed proteins and some are known to be
temporally and spatially regulated during development and in response to stress. The water
transport activity of two aquaporins is regulated at the protein level by phosphorylation and
dephosphorylation. At a given time, cells express several different aquaporins, and it is probable
that vacuolar and plasma membrane aquaporins acting in concert are responsible for the
cytosolic osmoregulation that is necessary for maintaining normal metabolic processes.
Inhibition studies of aquaporins in vivo and antisense mutant studies suggest that, in addition
to cytosolic osmoregulation, aquaporins are important for the bulk flow of water in plants.
309
trends in plant science
reviews
August 1999, Vol. 4, No. 8
aquaporins in both the tonoplast and the plasma membrane
2427
. In
Arabidopsis, there are at least 30 members of the MIP family
28
(U.
Johanson et al., unpublished). According to amino acid sequence
similarities, Arabidopsis MIPs either belong to the MIPs of the
tonoplast (i.e. the tonoplast intrinsic proteins, TIPs), to the MIPs
of the plasma membrane (i.e. the plasma membrane intrinsic pro-
teins, PIPs), or to the NLMs (NOD26-like MIPs). The PIPs, TIPs
and NLMs form three distinct phylogenetic groups (Fig. 2). The
PIPs can be further divided into two subfamilies PIP1 and PIP2
(Ref. 29). All the TIPs fall into three subfamilies: TIPs, TIPs
and TIPs, except for Nt-TIPa, which forms a separate group (M.
Karlsson et al., unpublished). Apart from a few conserved amino
acid residues at specific positions characteristic of either the PIP1
or the PIP2 subfamily, the PIP1 subfamily members have
extended N-termini and the PIP2 subfamily members have
extended C-termini. Both PIP subfamilies have extended N-
termini compared with the TIPs (Ref. 25).
Twelve of the expressed MIPs identified in Arabidopsis are
TIPs, 12 are PIPs and the remaining six Arabidopsis MIP homo-
logues are NLMs (Ref. 28, U. Johanson et al., unpublished). The
six NLMs have most similarity with NOD26, an aquaporin of the
soybean root nodule peribacteroid membrane
30
, although the sub-
cellular location of the Arabidopsis members of this group (e.g.
NLM1) is not known. The plant MIP homologues that have been
tested for water transport activity have been found to be water
channels
28
, except for NOD26 (Ref. 11), Nt-TIPa (Ref. 31) and
Nt-AQP1 (Ref. 32), which, in addition to water, have been
reported to be permeable to small uncharged solutes. It is reason-
able to assume that the majority of the remaining Arabidopsis
MIPs will be aquaporins too. However, not enough is known
about what determines transport specificity for discerning channel
specificity based on the primary sequence alone. Regardless of
whether all or only the majority of the plant MIPs are aquaporins,
it is clear that a large number of aquaporins are present in plants,
some localized in the tonoplast, some in the plasma membrane
and some possibly localized in endomembranes
33,34
.
Transcriptional and post-translational regulation
of aquaporins
Promoter-GUS fusions, in situ mRNA hybridizations, northern
blots and immunological studies have shown that the expression
of aquaporins is developmentally regulated in a cell-type-specific
manner. Some aquaporins are constitutively expressed and those
quantified have been found to constitute up to 20% of total inte-
gral membrane protein
27
. Some aquaporin genes are up-regulated
by drought [clone 7a (Ref. 35), rd28 (Ref. 22)] whereas others are
down-regulated [MipA and MipC (Ref. 36)], and some are regu-
lated by plant hormones, such as abscisic acid [AthH2 also named
PIP1b (Refs 37,38)] and gibberellic acid [At-TIP (Ref. 39)].
AQP2 of the kidney collecting duct epithelial cells, NOD26 of
the peribacteroid membrane, -TIP of the bean seed protein stor-
age vacuolar membrane, and PM28A of the spinach plasma mem-
brane have all been shown to be phosphorylated
27,4042
. In the case
of -TIP and PM28A, it has been shown that phosphorylation and
dephosphorylation regulates water transport activity when the
aquaporins are transiently expressed in oocytes
26,43
. In vivo, the
degree of phosphorylation of Ser274 of PM28A, seven amino
acids from the C-terminal, is decreased upon decreased apoplastic
water potential simulating drought conditions
27
(Fig. 3). In vitro,
using isolated plasma membrane vesicles, the same serine residue
is phosphorylated by a plasma membrane-bound Ca
2
-dependent
protein kinase
27
, possibly a CDPK (calmodulin-like domain pro-
tein kinase)
44
. By using the oocyte system and site-directed muta-
genesis, Ser115 was also shown to influence the water transport
activity of PM28A (Ref. 26). Ser115 is located in the first cyto-
plasmic loop at a consensus phosphorylation site (Arg-Lys-x-Ser-
x-x-Arg/Lys or only Lys-x-Ser-x-x-Arg, where x represents any
amino acid) for mammalian protein kinase A or C. This site is
conserved in all plasma membrane aquaporins (i.e. in all members
of the PIP1 and PIP2 subfamilies). A similar phosphorylation site
is also found in most of the tonoplast aquaporins, although most
TIPs have a threonine instead of a serine at this position (M.
Karlsson et al., unpublished). In addition to PM28A, -TIP is
phosphorylated at this site (Ser99) when expressed in oocytes
43
.
Thus, the position of the phosphorylation site corresponding to
Ser115 of PM28A and Ser99 of -TIP is conserved (Fig. 3) but
the amino acid consensus sequence differs when comparing PIPs
with TIPs.
A consensus phosphorylation site (Lys-x-x-x-Ser-x-Arg)
around Ser274 is conserved in all PIP2 plasma membrane aqua-
porins. A similar consensus site (Lys-x-x-Ser-x-x-Lys) is also found
in NOD26, which is phosphorylated at Ser262 in the C-terminal
region by a peribacteroid membrane-localized CDPK (Ref. 45).
All six members of the Arabidopsis NLM subfamily have putative
Fig. 1. Basic structure of aquaporins. The model is valid for both
plasma membrane intrinsic proteins (PIPs) and tonoplast intrinsic
proteins (TIPs). The side with the N- and C-termini always faces the
cytosol whereas the other side either faces the apoplast, in the case
of PIPs, or the vacuole, in the case of TIPs. There are six transmem-
brane helices. The first cytosolic loop and the third extracytosolic
loop, each of which contain a conserved Asn-Pro-Ala (NPA) box, are
relatively hydrophobic and probably dip into the membrane from
opposite sides creating a seventh transmembrane structure. The colour-
ing of the helices reflects the internal homology, where the N-terminal
half of the protein is homologous to the C-terminal half, although the
two halves are inserted inversely in the membrane. Thus, helix 1 cor-
responds to helix 4, helix 2 to helix 5, and helix 3 to helix 6.
Conserved serine residues that are known to be phosphorylated and
dephosphorylated in vitro, in planta and in the oocyte system,
thereby regulating the water transport activity of aquaporins, are also
depicted. The serine residue (Ser) in the first cytosolic loop is present
in all PIP1 and PIP2 subfamily members. In place of this serine, the
majority of TIP subfamily members have a threonine residue. The
serine in the C-terminal domain is present in all PIP2 subfamily members.
This schematic model is based on the structure for AQP1 (Ref. 61).
5 4
COOH
6
3
Asn
Asn
Pro
Pro
Ala
Ala
NH
2
Cytosol
Apoplast
or vacuole
Ser
Ser
1
2
T
r
e
n
d
s

i
n

P
l
a
n
t

S
c
i
e
n
c
e
phosphorylation sites (Lys/x-x/Arg-x-Ser-x-x-Lys/Arg) at this
position (U. Johanson et al., unpublished). The PIP1 and TIP sub-
families lack this C-terminal phosphorylation site. The highly
conserved nature of the consensus phosphorylation sites corre-
sponding to Ser274 and 115 among classes of plant MIPs
strongly suggest that these residues are also phosphorylated and
dephosphorylated in planta, thereby regulating the water channel
activity (Fig. 4).
The aquaporin AQP2 is expressed in the principal cells of the
renal collecting duct of mammalian kidneys and the hormone
vasopressin regulates the transcellular water permeability in this
tissue. Vasopressin binding to extracellular sites of a receptor in
the basolateral plasma membrane activates adenylate cyclase. The
cAMP-dependent protein kinase A, phosphorylates AQP2 resid-
ing in periplasmic vesicles causing them to fuse with the apical
plasma membrane of the principal cells, thereby increasing the
water permeability
46
. A related regulatory mechanism might be
operating in plants because plasma membrane aquaporins of the
PIP1 subfamily have been shown by immunogold electron
microscopy to be associated with plasmalemmasomes invagi-
nations of the plasma membrane protruding into the cytosol and
into the central vacuole of Arabidopsis mesophyll cells
33
. Recent
immunological results also indicate that
PIP1 subfamily members are present in
intracellular membranes as well as in the
plasma membrane
34
. However, direct tar-
geting of periplasmic vesicles, in line with
the mechanism described for AQP2, has
not been shown in plants. Because
periplasmic vesicles are inside-out they
would not end up in the plasma membrane
fraction obtained by the aqueous polymer
two-phase technique, although the results
for PM28A, which show high constitutive
abundance in the plasma membrane, do not
exclude regulation by vesicle-targeting for
other aquaporins. The PIP1 subfamily
members
33
have a shorter C-terminal
region compared with the PIP2 subfamily
members and, therefore, lack the consensus
phosphorylation site corresponding to
Ser274 of PM28A. This points to a differ-
ent regulatory mechanism for the PIP1 sub-
family members, which might include
vesicle targeting. Vesicle targeting, as a
consequence of transcriptional induction
of aquaporins encoded by genes known
to be drought-induced, probably occurs
but this mechanism is distinct from the
phosphorylation-induced vesicle fusion
described for AQP2, although the final
result an increased number of aquaporins
in the membrane is similar.
Specific aquaporins are generally ex-
pressed in several tissues and organs of a
plant
27,47
. However, within tissues and
organs the expression is strongly correlated
with cells known to be associated with
large fluxes of water, such as root epider-
mal, exodermal and endodermal cells,
xylem parenchyma cells immediately adja-
cent to xylem vessels, phloem companion
cells and cells surrounding the phloem in
leaf blades and stems
48
(R. Kaldenhoff,
pers. commun.), guard cells
38
, and in cells at certain developmen-
tal time points associated with cell expansion or elongation
21,38,47
.
Buffering osmotic fluctuations of the cytosol
By measuring the increase in light scattering or the increase in
fluorescence quenching by stopped-flow spectrophotometry, it is
possible to follow volume reduction of tonoplast and plasma
membrane vesicles when placed in a hypertonic solution. When
membrane vesicles from a tobacco cell suspension culture were
monitored in this way, a 100-fold difference in osmotic water per-
meability between the tonoplast and the plasma membrane ves-
icles was recorded, with the tonoplast being the most permeable
membrane
49
. Wheat root tonoplast vesicles are seven times more
permeable to water
50
compared with wheat root plasma membrane
vesicles, and eight times less permeable to water compared with
tonoplast vesicles from tobacco cell suspensions. It is concluded
that water transport across the tobacco plasma membrane occurs
mainly by diffusion through the lipid bilayer. By contrast, the
osmotic water permeability of the wheat plasma membrane is
three times higher than the diffusional permeability, implicating
the involvement of water channels. Nevertheless, the osmotic
water permeability of the plasma membrane is surprisingly low, in
310
trends in plant science
reviews
August 1999, Vol. 4, No. 8
Fig. 2. Phylogenetic analysis of 27 members of the major intrinsic protein (MIP) family. The
glycerol facilitators of E. coli (Ec-GlpF) and yeast (Fps1) transport glycerol but not water, and
the mammalian aquaglyceroporin AQP3 transports glycerol as well as water. Among the plant
MIPs, NOD26 of the soybean nodule peribacteroid membrane, belonging to the NLM
(NOD26-like MIP) subfamily, together with Nt-AQP1 and Nt-TIPa, have also been reported
to be permeable to small, uncharged solutes as well as to water. The remaining MIP family
members included in the analysis are either aquaporins or their specificity is unknown. The
plant tonoplast intrinsic proteins (TIPs) comprise three subfamilies, plus Nt-TIPa, which
forms a group of its own, whereas the plant plasma membrane intrinsic proteins (PIPs) form a
divided cluster representing the PIP1 and the PIP2 subfamilies. MIPs that are able to transport
glycerol or other small uncharged solutes are indicated in grey. The sequences were aligned using
PileUp [Wisconsin Package Version 10.0, Genetics Computer Group (GCG), Madison, WI,
USA], the tree was constructed using PAUP(phylogenetic analysis using Parsimony, Version
3.1, distributed by the Illinois Natural History Survey, Champaign, IL, USA), and the numbers
next to the nodes are bootstrap values
62
from 100 replicates. The bootstrap values represent the
proportion of times that a branch appears in the tree.
AQP3
Ec-GlpF
Fps1
NLM1
NOD26
At-aTIP
Pv-aTIP
At-dTIP
So-dTIP
At-gTIP
Zm-TIP1
PIP1a
PIP1b
PIP1c
MipB
PM28B
MipA
PIP3
PM28A
PIP2a
RD28
MipC
AQP0
AQP2
AQP1
Nt-TIPa
Nt-AQP1
Aquaglyceroporin
Glycerol facilitators
PIP1
PIP2
NLMs
Mammalian aquaporins
100
65
100
56 98
99
77
100
62
86
64
72
81
95
99
77
80
73
99
72
100
100
aTIP
dTIP
gTIP
TIPs
PIPs
Trends in Plant Science
both wheat root and tobacco suspension
cells, indicating that the aquaporins are
either not expressed or are closed under the
experimental conditions used.
In spinach, plasma membrane aquapor-
ins are highly abundant with PM28A and B
together comprising as much as 20% of the
total integral plasma membrane protein
27
.
In this case, the water transport activities of
the aquaporins in the two membranes
would be required to be different in order
for the tonoplast to have a seven- to 100-
fold higher osmotic water permeability
compared with the plasma membrane.
Oocyte expression studies do not indicate
large differences in water transport rates
between TIPs, PIPs [-TIP (Ref. 23),
RD28 (Ref. 51) and PM28A (Ref. 26)],
although unknown levels of expression in
the oocyte membrane, and uncertainty as to
whether the channels are open or not, does
introduce possible errors into such a com-
parison. However, for the tonoplast to be
able to buffer osmotic fluctuations of the
cytosol
49,50
, it would be logical if the tono-
plast had a higher osmotic water permeabil-
ity than the plasma membrane. This could
either be accomplished by a constitutively
higher osmotic water permeability for the
tonoplast compared with the plasma mem-
brane
49,50
, or by dynamic post-translational
regulation of the tonoplast and plasma
membrane-located aquaporins, as exempli-
fied by the regulation of -TIP (Ref. 43)
and PM28A (Refs 26,27).
Water uptake and whole plant
water balance
There are basically three possible routes for water movement in
plant tissue: the apoplastic, the symplastic and the transcellular
route
52
. The latter is defined as the transport of water across each
cell (i.e. the transport across the plasma membrane and across the
vacuolar membrane of each cell without the involvement of plas-
modesmata). However, because it is experimentally difficult to
discriminate between symplastic and transcellular movement of
water, they are collectively referred to as the cell-to-cell move-
ment of water
52
. The roots of most angiosperms contain a layer of
exodermal (also called hypodermal) cells, which is the outermost
cell layer of the cortex just inside the epidermal cell layer, which
is the outermost layer of root cells. The exodermal cells form a
water-impermeable Casparian strip on the radial walls in the same
way as the endodermal cells (the innermost cell layer of the cor-
tex). However, the exodermal cells form a Casparian strip at a
later developmental stage (i.e. further away from the root tip
compared with the endodermal cells). Until the exodermal Cas-
parian strip is formed, the cortex is permeable to water, which can
move apoplastically until it reaches the endodermal Casparian
strip. The cells in the exo- and endodermis later develop water-
impermeable suberin lamellae entirely covering the cells, and in
addition they also form thicker cell walls. The Casparian strips of
the exo- and endodermis create apoplastic discontinuities and
necessitate the transmembrane transport of water into the exo- and
endodermal cells in order for water to reach the xylem. Within
the cortex and the stele, where cells typically are connected by
plasmodesmata, water can move symplastically, transcellularly or
apoplastically, or by a combination of these routes. For water to
enter the xylem, it must ultimately exit from the endodermis or
from the xylem parenchyma cells of the stele, and thus cross a
plasma membrane, similar to the way in which K

has been shown

to be released into the xylem sap
53
. The expression patterns for sev-
eral aquaporins, which are abundant in the outer root cell layers, the
endodermis, and the xylem parenchyma cells [PIP1b (Ref. 38),
MIPA, MIPB, MIPC (Ref. 36), ZmTIP1 (Ref. 48), and NtAQP1,
a tobacco PIP1b homologue, R. Kaldenhoff, pers. commun.], are
consistent with the anatomic constraints for water movement and
suggest where transmembrane water transport must be facilitated.
Heavy metal ions, such as Hg
2
, are known to inhibit water trans-
port activity of some mammalian aquaporins
54
, some tonoplast
aquaporins
23
and some plant plasma membrane aquaporins
29

although some plasma membrane aquaporins are mercury-

insensitive
51
. Using site-directed mutagenesis, it has been shown that
Hg
2
binds to cysteine residues in or near the aqueous pore of the
water channels, thereby inhibiting water transport
55,56
. Mercury inhi-
bition has been shown for aquaporins expressed in oocytes
5,23,29,56
,
and for aquaporins in native membrane vesicles as monitored by
stopped-flow
11,50
. Overall water transport in intact plants or plant
organs is also dramatically affected by mercury. By adding sub-mM
concentrations of HgCl
2
to the growth media of hydroponically
grown plants, the hydraulic conductivity (L
p
) in roots is reduced by
6090% compared with the controls
57,58
. This reduction in water flux
311
trends in plant science
reviews
August 1999, Vol. 4, No. 8
Fig. 3. General topology of aquaporins. This basic topology, represented here by the primary
sequence of the plasma membrane aquaporin PM28A, is valid for plasma membrane intrinsic
proteins (PIPs) and tonoplast intrinsic proteins (TIPs). The N- and C-termini are located at the
cytosolic side, whereas the three extracytosolic loops either face the apoplast (PIPs) or
the vacuole (TIPs). The two conserved Asn-Pro-Ala (NPA) boxes are depicted in black, and
the conserved locations of the consensus phosphorylation sites are represented here by Ser115
and Ser274 of PM28A. Phosphate groups are depicted in white on grey.
V
M
S K E V
E
E
A Q A H
Q
H
G
K
D
Y V
D
P
P
P
A
P
F
F
D
L
G
E
L
K
L
W
F
W
R A
A
I
A
E
A T
I
F
F
L
L
T I
Y L
T A
V
V
I
G
H
S
K
E
T
V
V
C
G
S
G F
T
V
I
H
G
G
S
I
G
A T
C
Y
V
L V
F
I
F
G
G
M
A
W A
L
L
G
I
V
G
L
F
L
A
R
K
V
L
L
R
A
L
V
Y
M
I
A
Q
C
L
G
A
I
C
G
V
G
L V
K
A
F
M
K
G
P
Y
N
F G
G
G
A
N
S
R
K
P
D
T
A
R
D
H
V
A
S
F
V
Y
T
T
F
V L
I
L
I
G
G
A
E
A
T
G
K
N
Y
G
L
A
V
P
I
L
A
P
L
F
G
P
I
F
V A
L
H
Q
V
M
P
I
T
G
T
G
I
R
S
F
G P A V
I
F
N
S
N
N
K
A
A
S
S
A
R
L
V
Y
Q
H
A
A
Y
V
A
A
G
I
F
P
G
V
W
F
I
W
Q
D
D
W
V
K
COOH
NH
2
Apoplast or vacuole
Cytosol
A
I
A
L
G
F
R
S
N
P
T
N
A
I
T A
A
P
N
P
S
S
S
S
P
P
T
r
e
n
d
s

i
n

P
l
a
n
t

S
c
i
e
n
c
e
across the roots does not affect ion fluxes into the xylem. The inhibi-
tion is largely reversible upon the addition of -mercaptoethanol, and
the inhibition, as well as the recovery phase, is 1520 min
57
. Further-
more, pressure-probe techniques have been used to measure water
permeabilities of single cells of the giant alga Chara corallina and
inhibition by HgCl
2
was found to be reversible
4
. These results sug-
gest that the main part of the water flux in roots, as well as in sin-
gle cells, is dependent and limited by aquaporin-mediated water
transport. The remaining part of the water flux that cannot be inhib-
ited by HgCl
2
can be attributed to mercury-insensitive aquaporins
51
and to passive diffusion across the lipid bilayer.
When placed in a hypotonic solution, protoplasts from leaves
of Arabidopsis that had been transformed with an antisense con-
struct for the plasma membrane aquaporin PIP1b, survived con-
siderably longer compared with wild-type protoplasts
38
. Around
50% of the antisense protoplasts from two independent lines were
still viable (i.e. they had not burst) after 2 h of incubation in a
hypotonic solution, by which time all of the control wild-type
protoplasts had burst. The antisense lines had reduced leaf and
root expression of not only the PIP1b but also the PIP1a transcript.
The osmotic water permeability of the antisense protoplasts was
reduced about three times compared with the wild-type proto-
plasts. A comparison of the phenotypes of antisense and wild-type
plants showed that the aerial parts of the plants had the same mor-
phology, independent of developmental stage, whereas the root
systems of the antisense transformants, calculated as root mass,
were five times larger compared with the wild-type plants
59
(Fig. 4).
Furthermore, the water uptake rate and the root pressure for the
antisense plants was not altered compared with the wild-type
plants, suggesting that antisense plants compensate for reduced
plasma membrane water permeability by increasing the size of the
root system.
Conclusions and outlook
Regardless of the fact that transporting water in the xylem from
the roots to the upper parts of a plant is driven by a negative
hydrostatic pressure as a result of transpiration in the photosyn-
thesizing parts of a plant, water has to reach the stelar apoplast
adjacent to the xylem vessels to be accessible for xylem transport.
Hypotheses about water homeostasis mechanisms and water
transport within tissues and organs, such as roots and leaves, have
changed considerably over time and might continue to do so. How-
ever, the evidence for the importance of aquaporins in the bulk
flow of water and for water homeostasis cannot be ignored. The
plasma membrane and tonoplast aquaporins are highly abundant
and are encoded by many genes, which are known to be tempo-
rally and spatially regulated during development and in response
to drought-stress.
A systematic study of the expression patterns of Arabidopsis
MIP homologues (30 have been identified) is required to generate
a comprehensive view of the overall expression pattern. This
should be done both at the transcript and at the protein level
because aquaporin turnover appears to be variable, such as when
comparing constitutively expressed and inducible aquaporins.
Such a study should yield information concerning which cells are
limiting for the bulk flow of water and, therefore, important for
water homeostasis. Lateral heterogeneity of the different aqua-
porins in the plasma membrane is probably important for the pas-
sage of water across cell types representing barriers between
different organ compartments, such as the endodermal cell layer
separating the cortex and the stele. Furthermore, the water
transport activity of some aquaporins is also regulated at the
protein level by phosphorylation and dephosphorylation in a
turgor-dependent manner.
312
trends in plant science
reviews
August 1999, Vol. 4, No. 8
Fig. 4. Short-term and long-term regulation of water flow. (a) A
model for cytosolic osmoregulation at the single cell level involving
regulation of water transport activity of plasma membrane aquapor-
ins by phosphorylation and dephosphorylation. When the plant cell
senses a lowered apoplastic water potential, the aquaporins become
dephosphorylated, thereby lowering the water permeability of the
plasma membrane and minimizing water loss. The aquaporins of the
vacuolar membrane remain open and allow water to flow into the
cytosol to compensate for water lost to the apoplast. Abbreviations:
PK, protein kinase; P, phosphate group. (b) Comparison of the
Arabidopsis antisense phenotype (left) with the wild type (right).
The plants were transformed with a construct targeting PIP1b
(Ref. 59). In both shoots and roots, the antisense lines had reduced
levels of PIP1b and PIP1a mRNA. The aerial parts had the same
morphology, independent of developmental stage, whereas the root
systems of antisense plants were five times larger than wild-type
plants. The rate of water uptake as well as the root xylem pressure
of the antisense plants was not altered, suggesting that the antisense
plants compensate for the reduced plasma membrane water perme-
ability in the roots by increasing the size of the root systems. This
interpretation is supported by the fact that the osmotic water perme-
ability of antisense protoplasts was three times lower than wild-type
protoplasts. Adapted from Refs 26 and 59.
PK
Low water potential High water potential
Ca
2+
Vacuole
Cytosol
H
2
O H
2
O
P
(a)
(b)
Ca
2+
H
2
O H
2
O H
2
O H
2
O
Trends in Plant Science
With regard to the possible upstream signal transduction path-
ways responsible for regulating the opening and closing of aqua-
porins it is probable that specific membrane proteins monitor the
water potential difference across the membrane (i.e. function as
osmosensors), and either directly interact and regulate aquaporins
or regulate aquaporins indirectly, such as by regulating protein
kinases and phosphatases. In yeast, two independent and unre-
lated osmosensors have been identified. Both are membrane pro-
teins that regulate a phosphorylation cascade called the HOG
(high osmolarity glycerol) pathway, via phosphorylation path-
ways. An Arabidopsis homologue of one of the yeast osmosensors
has been identified and cloned, and its function has been verified
by functional complementation of a yeast mutant
60
, thus identify-
ing a possible plant osmosensor (K. Shinozaki, pers. commun.).
Taken together, the large number of genes encoding aqua-
porins, the differential expression of aquaporin genes in time and
space, the probable vesicle targeting of some aquaporins and the
probable lateral heterogeneity within individual cells, suggest that
aquaporins are involved in a versatile and dynamic regulation of
water movement. The available results thus suggest that, in con-
cert, plasma membrane and tonoplast aquaporins are not only
buffering osmotic fluctuations of the cytosol but are also orches-
trating the bulk flow of water in plants.
Acknowledgements
We thank Ralf Kaldenhoff, Christophe Maurel and Kazuo Shinozaki
for disclosing unpublished results and Bengt Widegren for help
with sequence comparisons. Grants from SJFR, NFR, the EU-
Biotech program (BIO4-CT98-0024) and the Swedish Strategic
Network for Plant Biotechnology are gratefully acknowledged.
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Per Kjellbom*, Christer Larsson, Ingela Johansson,
Maria Karlsson and Urban Johanson are at the Dept of Plant
Biochemistry, Lund University, PO Box 117, S-221 00
Lund, Sweden
*Author for correspondence
(tel 46 46 2224195; fax 46 46 2224116;
e-mail per.kjellbom@plantbio.lu.se).
Listening to the silent genes: transgene silencing and new mechanisms
of gene regulation and pathogen control, J.M. Kooter,
M.A. Matzke and P. Meyer
Macromolecular trafficking in the phloem,
G.A. Thompson and A. Schulz
14-3-3 proteins: cellular regulators of plant metabolism,
H-J. Chung, P.C. Sehnke and R.J. Ferl
The role of the ER and the cytoskeleton in viral trafficking,
C. Reichel, P. Ms and R.N. Beachy
Brassinosteroid biosynthesis inhibitors, T. Asami and S. Yoshida
Forthcoming articles in Trends in Plant Science