Download The Plasma Membrane of Arabidopsis thaliana Contains a Mercury

Plant Physiol. (1994) 106: 1325-1333
The Plasma Membrane of Arabidopsis thaliana Contains a
Mercury-Insensitive Aquaporin That I s a Homolog of the
Tonoplast Water Channel Protein TIP’
Mark J. Daniels, T. Erik Mirkov, and Maarten J. Chrispeels*
Department of Biology, University of California, San Diego, 9500 Gilman Drive, La Jolla, California 92093-01 16
The vacuolar membranes of plant cells and the plasma
membranes of mammalian cells contain aquaporins, proteins
that form water-selective channels (see Chrispeels and
Maurel, 1994, for review). These 27-kD integral membrane
proteins belong to a family of proteins that has cognates in
mammals, yeasts, and bacteria; they are part of the larger
MIP family (see Reizer et al., 1993, for a recent review). In
plants, more than half a dozen derived amino acid sequences
and/or proteins have been recently identified. Some of them
are expressed in a tissue-specific manner, whereas others are
induced by specific physiological conditions. For example, aTIP is a seed-specific protein found in the tonoplasts of
protein storage vacuoles (Johnsonet al., 1990), tobRB7 mRNA
is found in the roots of tobacco (Nicotiana tabacum)
(Yamamoto et al., 1991; Opperman et al., 1994), NOD26 is
specific to the peribacteroid membranes of soybean (Glycine
max) nodules (Sandal and Marcker, 1988), and trg-31 (Guerrero et al., 1990; Guerrero and Crossland, 1993) and rd28
(Yamaguchi-Shinozaki et al., 1992) transcripts are induced
by desiccation of pea (Pisum sativum) and Arabidopsis thaliana, respectively. Dip is expressed in mature seeds and darkgrown seedlings of Antirrhinum majus (Culianex-Macia and
Martin, 1993) and MIP cognates tmp-A and tmp-B (Shagan
and Bar-Zvi, 1993; Shagan et al., 1993) are highly expressed
in etiolated A. thaliana plants.
Amino acid sequence comparisons of these plant proteins
and of all members of the MIP family show that they are
evolutionarily related. All proteins have six membranespanning domains and a small number of amino acids that
are absolutely conserved, including the signature sequence
NPAXT. This signature sequence is partially repeated as NPA
in the second half of the protein. Maurel et al. (1993) recently
showed that y-TIP, a protein that is present in the tonoplast
of cells in shoots, roots, and flowers of Arabidopsis, is an
aquaporin. When y t i p cRNA was injected into Xenopus laevis
oocytes and the eggs moved to a hypotonic solution 2 to 3 d
later, we observed a 5- to 10-fold increase in the osmotic
water permeability of the eggs. The presence of y-TIP in the
plasma membrane of the oocyte causes them to rapidly swell
and burst when bathed in a hypotonic solution. Similar
results have been reported for three mammalian proteins in
the MIP family, the aquaporins CHIP28 (Preston et al., 1992),
WCH-CD (Fushimi et al., 1993), and MIWC (Hasegawa et
al., 1994). The injection of cRNAs for other membrane proteins-including homologs of MIP that are not aquaporins,
such as the bacterial glycerol channel GlpF-do not have
this effect (Maurel et al., 1994). We now report that the
desiccation-induced MIP homolog rd28 of A. thaliana also
encodes an aquaporin that creates water channels in oocyte
membranes. Furthermore, we show that the RD28 protein is
a plasma membrane protein expressed throughout the plant,
except in seeds. Therefore, we refer to it as RD28-PIP (plasma
membrane intrinsic protein).
Supported by a grant from the US. Department of Agriculture
(Competitive Research Grants Organization).
* Corresponding author; fax 1-619-534-4052.
Abbreviations:MIP, major intrinsic protein; mosmol, milliosmolal;
Pr, osmotic water permeability; PIP, plasma membrane intrinsic
protein; TIP, tonoplast intrinsic protein.
Plant cells contain proteins that are members of the major
intrinsic protein (MIP) family, an ancient family of membrane
channel proteins characterized by six membrane-spanningdomains
and two asparagine-proline-alanine (NPA) amino acid motifs i n
the two halves of the protein. We recently demonstrated that y TIP, one of the MIP homologs found i n the vacuolar membrane of
plant cells, i s an aquaporin or water channel protein (C.Maurel, J.
Reizer, 1.1. Schroeder, M.J. Chrispeels [1993] EMBO J 12: 22412247). RD28, another MIP homolog in Arabidopsis thaliana, was
first identified as being encoded by a turgor-responsive transcript.
To find out if RD28 i s a water channel protein, rd28 cRNA was
injected into Xenopus laevis oocytes. Expression of RD28 caused a
10- to 15-fold increase i n the osmotic water permeability of the
oocytes, indicating that the protein creates water channels in the
plasma membrane of the oocytes and i s an aquaporin just like i t s
homolog ?-TIP. Although RD28 has several cysteine residues, i t s
activity is not inhibited by mercury, and in this respect it differs
from y-TIP and all but one of the mammalian water channels that
have been described. Introduction of a cysteine residue next to the
second conserved NPA motif creates a mercury-sensitive water
channel, suggestingthat this conserved loop i s critical to the activity
of the protein. Antibodies directed at the C terminus of RD28 were
used in combination with a two-phase partitioning method to
demonstrate that RD28 is located i n the plasma membrane. The
protein is present in leaves and roots of well-watered plants,
suggesting that i t s presence i n plants does not require a specific
desiccation regime. These results demonstrate that plant cells contain constitutively expressed aquaporins i n their plasma membranes (RD28), as well as i n their tonoplasts (?-TIP).
1325
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Daniels et al.
1326
MATERIALS AND METHODS
Plant Physiol. Vol. 106, 1994
218
rd28 cDNA
I
Growing Conditions for Arabidopsis Plants
Arabidopsis thaliana (Columbia ecotype) was grown from
seed under continuous bright illumination at 22 to 24OC after
2 d of vemalization at 4OC. Plants were grown in 4-inch pots
filled with Terra-Lite RediEarth (W.R. Grace and Co., Ajax,
Ontario, Canada) and subirrigated with 0.05% Peter's peatlite fertilizer (J.M. McConkey, Sumner, WA). Plants for
plasma membrane isolation were grown using Terra-Lite
Vermiculite (W.R. Grace and Co., Cambridge, MA) instead
of RediEarth.
219 220 221
222
223 224 225 226
221 228
... A n CCG ATC ACC GGA ACC GGT ATC AAC' CCG GCT '.'
primer T223C (sense)
P
I
T
G
T
**
G
I
N
P
A
5' - CCG ATC ACC GGA TGC GGT ATC AAC CC - 3'
C
Figure 1. Site-directed mutagenesis of rd28 cDNA to create rd28
T22317. The sequence of the sense orientation mutagenic primer is
shown. Substituted nucleotides are marked with asterisks. Amino
acids are given by their one-letter codes and are numilered from
the putative amino terminus of the protein.
sequenced to verify that the desired mutation of 1'223C had
been introduced.
Plasmid Constructions and In Vitro RNA Synthesis
DNA fragments encoding 7-TIP (Hofte et al., 1992) and
RD28-PIP (Yamaguchi-Shinozakiet al., 1992) were cloned
into the BglII site of a pSP64T-derived Bluescript vector
carrying 5' and 3' untranslated sequences of a &globin gene
of Xenopus (Preston et al., 1992). pSP64T-derived vectors
provide high mRNA stability and translation efficiency in
Xenopus oocytes (Krieg and Melton, 1984). The cloned DNA
fragments were (a) a BamHI-XbaI fragment containing the
entire 7-TIP coding sequence plus 7 bp and 1 bp at the 5'
and 3' termini, respectively, and with the XbaI end recessed
and ligated to an 8-bp BamHI linker (New England Biolabs,
Beverly, MA); and (b) a NdeI-BamHI fragment containing the
entire RD28-PIP coding sequence plus 1 bp and 217 bp at
the 5' and 3' termini, respectively. Capped RNA encoding
?.-TIP and RD28-PIP were synthesized in vitro using T3 RNA
polymerase and purified as described (Preston et al., 1992).
DNA Sequence Analysis
Aquaporin protein sequences were aligned using
NEWAT87 software within the DNASYSTEM (Smith, 1988)
package with adjustments made by eye where appropriate.
Amino acid similarities and consensus sequence were determined by the LINEUP and PRETTY programs of the Genetics
Computer Group (Devereux et al., 1984) sequence analysis
package.
Site-Directed Mutagenesis to Create a Mercury-Sensitive
RD28-PIP
The pSP64T-derived Bluescript vector containing the
RD28-PIP coding sequence was used for site-directed mutagenesis by recombinant PCR as described by Higuchi (1990).
The sequence of the mutagenic oligonucleotideprimer (sense
orientation) is given in Figure 1. The sense oligonucleotide
was used in combination with an oligonucleotide complementary to the T7 promoter in the pSP64T-derived Bluescript
vector, whereas the antisense oligonucleotide was used in
combination with an oligonucleotide complementary to the
T3 promoter in the pS64T-derived Bluescript vector. The
products of these two separate PCR reactions were gel purified and used as templates for a secondary PCR using only
the T7 and T3 primers. The resulting PCR product was
digested with Hind111 and PsfI and cloned into the corresponding sites of pBS (Stratagene). The resulting clone was
Oocyte Preparation and Injection
Fully grown oocytes (stage V and VI) were isolated from
Xenopus llievis and incubated in Barths solution (88 m NaCl,
1 m KCl, 2.4 IT~MNaHC03, 10 mM Hepes-NaOH:, 0.33 m
Ca[N03],, 0.41 m CaC12, 0.82 m MgS04, pH 7.4) as
described (Cao et al., 1992). In vitro transcripts (0.5-1 mg/
mL) or nuclease-free water were injected in a volume of
50 nL.
Osmotic Water Permeability Assay
Oocytes were transferred 3 d after cRNA injection from
Barth's solution (200 mosmol) at room temperature to the
same solution diluted to 40 mosmol with distilled water.
Changes in cell volume were followed with a microscope by
taking photographs at 30- to 90-s intervals. Oocyte diameters
were measured four times along two sets of perpendicular
axes. The volume V was estimated as the mean of two
ellipsoid volumes. The osmotic permeability coefficient
was calculated from Pr = V,[d(V/V,)/df]/[Sx V , (Osm, Osm,,,)], where initial oocyte volume V , = 9 x
cm3,
initial oocyte surface area S = 0.045 cm', and mohr volume
of water V , = 18 cm3/mol (Zhang and Verkman, 1991).
Antiserum to RD28-PIP
A peptide representing the carboxy terminus of the derived
amino acid sequence of rd28 was chemically synthesized and
coupled to BSA with glutaraldehyde using the procedures
described by Harlow and Lane (1988). The sequence of the
synthetic peptide is H2N-KSLGSFRSAANV-COOH.The coupled peptide was injected into a New Zealand White rabbit
and the peptide was boosted at 4-week intervals. After three
boosts, we obtained a highly specific serum that w , used
~ in
a 1:250 dilution for the preparation of immunoblols.
lmmunodetection
For immunoblotting, appropriate quantities of protein were
fractionated by SDS-PAGE and transferred to nitrocellulose
and the proteins were detected using the rabbit anti-RD28PIP antiserum. Goat anti-rabbit IgG coupled to horseradish
peroxidase (Bio-Rad) was used as the secondary antibody.
Extracts were incubated in denaturing buffer for 'LO min at
room temperature or for 5 min at 55OC before loading the
gel.
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Plasma Membrane Aquaporin
Chimeric Gene Constructs and Tobacco Transformation
rd28 was cloned as an EcoRV to SstI fragment between the
cauliflower mosaic virus 35s promoter and the transcriptional
terminator from the 3’ end of the nopaline synthase gene
contained in pBIl2l (Clonetech, Palo Alto, CA) after removal
of the @-glucuronidasegene as a SmaI to SstI fragment. The
pBI121-derived constructs were transformed into Agrobacterium tumefaciens strain LBA4404 as described by Hofgen and
Willmitzer (1988). Transformants were selected on medium
containing 50 pg/mL kanamycin.
Leaf discs of Nicotiana tabacum (cv Xanthi) were transformed with A. tumefaciens harboring the chimeric rd28 gene
cloned in pBI121 as described by Voelker et al. (1989).
Transformants were selected by their resistance to kanamycin, and transformed plants were regenerated from shootlets
by transfer to a root-inducing, kanamycin-containing agar
medium as described by Voelker et al. (1989). The kanamycin-resistant plants were transferred to soil and grown in the
greenhouse. Young leaves were collected for immunoblot
analysis. The highest expressors were used for further
analysis.
Protein Extraction and Preparation of Microsomes
Protein extracts were obtained by homogenizing plants in
grinding buffer (100 mM Tris [pH 7.51, 1 mM EDTA, 12%
[w/v] SUC,0.2 mM aminoethylbenzenesulfonylfluoride [Calbiochem-Novabiochem, La Jolla, CAI, 2 pg/mL aprotinin, l
pg/mL leupeptin, 1 &mL pepstatin A) and collecting the
supernatant after centrifugation at 15,OOOg for 10 min. After
centrifugation, the supernatant was filtered through a single
layer of cheesecloth.
Microsomes were prepared by centrifuging protein extracts
at 100,OOOg for 2 h and resuspending the resulting pellet in
grinding buffer.
Purification of Plasma Membranes by
Two-Phase Separation
Plasma membranes from 3-week-old A. thaliana plants or
leaves of transgenic tobacco expressing RD28-PIP were separated from other cellular membranes according to Kjellbom
and Larsson (1984) with some modifications. The U3 upper
phase containing the plasma membranes was repartitioned
an additional time with fresh lower phase to give U4. The L1
lower phase was repartitioned three additional times with
fresh upper phase to give L4 containing the intracellular
membranes. The upper phases produced by the lower phase
repartitioning were discarded. Purification of plasma membranes from tobacco transgenic for rd28 was as above, except
that the last repartitioning was omitted so as to produce a U3
phase and an L3 phase. The @-glucansynthase I1 assay was
performed as reported by Kauss and Jeblick (1987).
1327
raised above the box floor by sections of 2-cm-tall plastic
tubing. Three different regimes of desiccation were applied
by varying the amount of water present in the box. Under
the first (low-stress) treatment, plants dried to 96% of their
original weight; under the second (medium-stress) treatment,
plants dried to approximately 60% of their original weight;
and under the third (high-stress) treatment, plants dried to
approximately 10% of their original weight. During treatment, plants were left under continuous dim illumination
and collected after 24 and 48 h, at which time they were
weighed and frozen at -7OOC.
RESULTS
RD28-PIP Forms Water Channels in Xenopus Ooctyes
In vitro-transcribed cRNAs encoding ?-TIP or RD28-PIP,
each with the 5’ and 3’ untranslated regions of a P-globin
gene from X . laevis, were injected into Xenopus oocytes and
osmotic water transport was investigated 3 d after injection.
For this, oocytes were exposed to hypoosmotic conditions
(160 mosmol gradient) and initial changes in cell volume
were measured. Control oocytes that were injected with water
instead of cRNA swelled very slowly after the sudden decrease in osmotic strength (Fig. 2A) and burst only after
approximately 40 min (data not shown). This result illustrates
a limited osmotic water flux through the oocyte plasma
membrane, whose permeability can be accounted for mainly
by lipid-mediated water transport (Zhang and Verkman,
1991). In contrast, oocytes injected with rd28 cRNA or y-tip
cRNA rapidly increased their volume by up to 40% (Fig. 2A)
and ruptured in 2 to 3 min, displaying the appearance of a
new pathway for facilitated diffusion of water into the hypertonic cell. With RD28-PIP and y-TIP, the Princreased 13to 16-fold and 11- to 14-fold, respectively, over the control
value (Fig. 2B). Recent evidence indicates that the 7-TIP
protein is responsible for the formation of pores highly
permeable to water (Maurel et al., 1993), similar to the
mammalian proteins CHIP28 (Preston et al., 1992; van Hoek
and Verkman, 1992; Zeidel et al., 1992), WCH-CD (Fushimi
et al., 1993), and MIWC (Hasegawa et al., 1994). Such
proteins are referred to as aquaporins.
It has been thought that a character of water channels was
their inhibition by mercurial sulfhydryl reagents. Mercury
readily complexes with protein sulfhydryls (Webb, 1966;
Vallee and Ulmer, 1972) and it is presumed that this complex
physically blocks water from passing through the aquaporin
channel (Preston et al., 1993). Three water channels known
so far (CHIP28, WCH-CD, and y-TIP) appear to be sensitive
to mercury, but along with the recently discovered MIWC
(Hasegawa et al., 1994), aquaporin RD28-PIP is refractory to
mercury inhibition (Fig. 2B) in spite of the presence of Cys
residues at positions 73, 95, 131, and 136 of the protein.
Water-Stress Time Course
Introductionof a Cys Residue Creates a
Mercury-Sensitive Channel
Four-week-old rosette A. thaliana plants were harvested
and washed gently to remove soil from the roots. Plants were
then placed into a chamber consisting of a magenta box with
a I-mm nylon mesh insert upon which the plants were laid,
A comparison of the amino acid sequences of the four
known aquaporins and RD28-PIP is shown in Figure 3. The
NPAXT signature sequence of these MIP family proteins is
shown in boldface, as is the corresponding NPA in the second
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Daniels et al.
1328
Plant Physiol. Vol. 106, 1994
the presence of mercury ions (1 mM HgC12).-/-TIP also lacks
a Cys residue in this position; however, it is likely that a Cys
at a different position is responsible for the mercury sensitivity of this aquaporin.
A
---o- - - water
RD28-PIP Protein I s Present in All Organs But Not
in Seeds
__._.._.o..YTIP
......
A
RD28
---
------o-
---*
I . I . I . , .
O
h
N
3.0
1
2
3
4
5
time (min)
6
7
,
8
I
water
Y TIP
RD28
Figure 2. Pf of rd28- and y-tip-injected oocytes. A, Time course of
osmotic swelling of individual oocytes injected with water or in
vitro-synthesized cRNA encoding RD28 or y-TIP. Oocytes in Barth's
buffer were perfused at t = O with a 5-fold dilution of Barth's buffer
with distilled water. Measurements of oocytes injected with rd28
and y-tip cRNA stopped at time of cell rupture. B, Pf values of
individual oocytes derived from volume change measurements
made over three independent preparations of oocytes. When indicated, the assay was performed in the presence of mercury ions
(Hg, 1 m M HgCI2, 10 min preincubation). Data are expressed as the
mean SE, with the number of replicates above.
*
To determine the tissue and subcellular location 13f RD28PIP, we prepared a monospecific antiserum. The antiserum
was raised against a peptide consisting of the amino-terminal
12 amino acids of RD28-PIP coupled to BSA. This region of
the protein has no sequence identity with other MII' proteins
of A. fhaliana. Fractionation of whole organ extracts by SDSPAGE followed by immunoblotting showed that the serum
detected a single band around 27 kD in all organs examined
except seeds. Loading the lanes with equal amounts of protein, we found the strongest signal in the floral bolt (Fig. 5).
In samples of total protein, RD28-PIP appears in monomeric
form (Fig. 5 ) , whereas in microsome samples (Figs. 6, 7, and
8) RD28-PIP was observed as a dimer or as a rruxture of
monomeric and dimeric forms. Additional reducing agent in
the samples partially solved the aggregates. The highly hydrophobic nature of the protein, the presence of inaccessible
disulfide bonds between monomers, or a combination of
these two forces may cause this aggregation. From what is
known, expression patterns of plant MIPS vary among the
family members. Immunodetection methods show that a-TIP
is exclusive to seeds, whereas y-TIP appears in most other
plant tissues (Hijfte et al., 1992). The tobRB7 promoier is root
specific, driving expression in meristematic and immature
vascular cylinder tissue normally, and is induced by nematode infection in the giant cells from which the parasite feeds
(Opperman et al., 1994). The trg-31 promoter of pea caused
P-glucuronidase expression in leaves but not in roots of
dehydrated transgenic tobacco (Guerrero and Crossland,
1993).
Abundance of RD28-PIP Protein I s Not Altered by
Water Stress
half of the protein. This sequence is present in nearly all
members of the MIP family and its repetition is an indication
of the internal duplication of the gene (Reizer et al., 1993).
Both sequences are present in loops on opposite sides of the
membrane: NPAXT on the cytoplasmic side and NPA on the
extracellular or vacuolar side. By using site-directed mutagenesis, Preston et al. (1993) were able to show that Cysla9,
located just in front of the second NPA sequence, is responsible for the mercury sensitivity of CHIP28, indicating that
this is a functionally important region of the protein. Replacement of this Cys residue by Ser created a mercury-insensitive
water channel. RD28-PIP and MIWC lack a Cys residue at
the position homologous to that of CHIP28 where a Cys was
found to cause mercury sensitivity. Through site-directed
mutagenesis, an RD28 T223C mutant was made in which
the Thr residue present was changed to Cys (Fig. 1). The
resulting mutant RD28 T223C retains the original water
channel activity but is also sensitive to mercury inhibition,
showing an inhibition of Pfof approximately 80% (Fig. 4) in
Previous results have found induction of rd28 mRNA transcription beginning 10 h after A. thaliana plants w12re dehydrated to 10% of their initial fresh weight (YamaguchiShinozaki et al., 1992). rd28 is very closely relakd to the
trg-31 (clone 7A) gene from Pisum sativum (Rei2:er et al.,
1993). RNA transcript levels of trg-31 also respond to water
stress and increase shortly after dehydration (Guenero et al.,
1990). Microsomal fractions of well-watered Arabidopsis
plants contain RD28-PIP protein, and dehydration of the
plants using three different dehydration regimes did not
result in an increase in the level of RD28-PIP over the course
of 48 h (Fig. 6). Low levels of rd28 mRNA were detected in
Arabidopsis under normal growing conditions (K. Shinozaki,
personal communication) and these may sustain the apparently constitutive level of RD28-PIP abundance in wellwatered control plants. It is likely that this semi-quantitative
method to detect protein did not detect subtle changes in
RD28-PIP abundance resulting from water stress.
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Plasma Membrane Aquaporin
1
CHIP28
MIWC
WCH-CD
gTIP
RD28
consensus
70
H. ........
M. ........
M. ........
M. ........
Makdvegpdg
..........
..........
..........
.pirniaigr
fqtrdyedpp
......asEf
....vafkgv
.......wEl
p......dEa
ptpffdaeEl
kkklFwrAVv
wtqaFwkAVt
rsiaFsrAVl
trpdalkAal
tkwsLyrAVi
AEFlATtlFV
AEFlAmLiFV
AEF1ATL1FV
AEFisTLiFV
AEFvATLlFl
FisiGSalgF
LlsvGStinW
FfglGSalqw
vagsGSgmaF
YvtvltvigY
kypv....Gn
g.......Gs
........as
nklte..nGa
kiqsdtkaGg
AEF-
M71
CHIP2B
MIWC
WCH-CD
gTIP
RD28
consensus
CHIP28
MIWC
WCH-CD
gTIP
RD28
consensus
140
nqtavqdnvk
enplpvdmvl
sppsv...lq
ttpsglvaaa
vdcggvgilg
VslAFGLsIa
IslcFGLsIa
lavAFGLglg
VahAFGL..f
lawAFGgmlf
tLaqsvghlS
tMVqcfghlS
iLVqalghvs
vaVsvganIS
iLVyccaglS
GaHlNPAVTl
GgHINPAVTv
GaHINPAVTv
GgHVNPAVTf
GgHINPAVTf
gILlscqISi
amvctrkisi
acLvgchVSf
gaFiggnltl
glFlarkVSl
—————————
———FG———
————————S
G-H-NPAVT- — — — — — — — —
141
LsgiTssl.t
LylvTpps.v
LheiTpve.i
LkfaTg....
vkafqsshyv
gnslgrndLa
vgglgvttvh
rgdlavnaLh
glavpafgLs
nyggganfLa
dgvnsGqgLg
gnltaGhgLl
nnataGqavt
agvgvlnaFv
dgyntGtgLa
iEilgTLqLV
vEHiTFqLV
vElflTMqLV
fEiVmTFgLV
aEilgTFvLV
LcVLAt .Dr
FtlFAs .Ds
LcIFAs .De
YtVYAt. iDp
YtVF.s top
fRalMYiiAQ
aksvFYitAQ
IRaaFYvaAQ
IRgiLYwiAQ
iRavLYmvAQ
cvGAIvatal
cLGAIigagl
ILGAVagaal
ILGsVvaclI
cLGAtcgvgf
————Y —AQ
--G——————
rRrdlgGS..
kRtdvtGS..
rRgdnlGS..
k....nGSlg
kRnardshvp
209
..apLalGLs
..vaLalGFs
..paLsIGFs
tiapialGFi
vlapLpIGFa
-IG-
-LV
210
T
279
CHIP28
MIWC
ValghLlald YTGcgiNPAR SFGsAVI... thnFsnHWIF WVGPFIGgal AvLiYdFiLa prssdltdrv
ValgpLfaln YTGasmHPAP SFGpAVI... mgnWenHWIY WVGPilGavl AgalYeYvFc p.dvelkrrL
WCH-CD
VtlghLlgly FTGcsmSPAR SLapAW... tgkFddHWVF
gTIP
RD28
consensus
1329
Figure 3. Amino acid comparison of five aquaporins, CHIP28 (Preston and Agre, 1991),
MIWC (Hasegawa et al., 1994), WCH-CD
(Fushimi et al., 1993), 7-TIP (gTIP) (Hofte et al.,
1992), and RD28 (Yamaguchi-Shinozaki et al.,
1992). The single-letter amino acid code is
used. Gaps added for aligning sequences are
denoted by periods (.). Numbers indicate the
distance from the RD28 protein amino-terminal
end. Similar and conserved amino acids are in
uppercase letters, others are in lowercase. Con-
sensus line shows those residues conserved in
all sequences. The MIP family signature NPAXT
and NPA residues contained in extramembrane
loops (Reizer et al., 1993) are shown in boldface
type. Cys189, the mercury-sensitive Cys residue
of CHIP28, is marked with an arrowhead.
WIGPLVGaii gsLlYnYlLf psakslqerL
VganiLagga FsGasmNPAv aFGpAW. . . swtWtnHWVY WaGPLVGggi AgLiYevfF. ..inttheqL
vfmvhLatlp iTGtgiNPAR SFGaAVIfnk skpWddHWIF WVGPFIGati AaFyhqFvLr asgskslgsF
V———L——— --G——NPA- ———AV——— —————HW-- W-GP--G—— ———————— ————————
285
CHIP2B
MIWC
WCH-CD
gTIP
RD28
k. ........
keafskaaqq
a.........
pttdy.....
rsaanv....
.......... vwtsgqveey
tkgsymeved nrsqvetedl
......vlkg lepdtdweer
.......... ..........
.......... ..........
dldad.....
ilkpgvvhvi
evrrrqsvel
..........
..........
dinsrvemkp
didrgdekkg
hspqslprgs
..........
..........
k.........
kdssgevlss
ka........
..........
..........
.
v
.
.
.
consensus
RD28-PIP Is Localized to the Plasma Membrane
meability (cm/s x 102)
To determine the subcellular location of RD28-PIP and to
investigate the possibility that it may be a plasma membrane
aquaporin, we purified plasma membranes with the twophase partition method of Kjellbom and Larsson (1984). This
method is based on the observation that vesicles derived
from different membranes are separated into different polymer phases as a result of their unique surface properties (see
Sandelius and Morre, 1990, for a review). The purified membranes were assayed for the presence of the plasma membrane marker /3-glucan synthase II. Ninety-five percent of
the glucan synthase activity was found in the plasma mem-
brane fraction when compared to the intracellular membrane
fraction. This purity of plasma membranes is typical with the
two-phase partitioning method (Sandelius and Morre, 1990).
As seen in Figure 7, the plasma membrane fraction is greatly
enriched in RD28-PIP compared to the internal membranes.
The slight amount of RD28-PIP in the intracellular membranes may be attributed to newly synthesized protein that
is in transit through the secretory pathway and/or residual
plasma membrane in the intracellular fraction. The same
membrane fractions were assayed for pyrophosphatase, a
tonoplast marker (Rea and Sanders, 1987), and as an immunoblot using pyrophosphatase-specific antibodies (kindly
supplied by P.A. Rea). The results showed that the concentration of pyrophosphatase was 2 to 4 times greater in the
c
10
13
in
14
Q - Hg++
o
m + Hg++
i
l
l
-46.3
In
.l ,
=
t.
.
.
o
'
r>
osmotic wat
u
,0
- 27.6
10
9 r^|
r— — 1..;.;..;,-v/.j
water
-18.6
RD28
RD28 T223C
Figure 4. PI values of re/28- and re/28 T223C-injected oocytes derived from volume-change measurements made over four indeFigure 5. Immunodetection of RD28-PIP in tissues of bolting A.
pendent preparations of oocytes. When indicated, the assay was
thaliana. Protein extracts were fractionated by SDS-PAGE and transperformed in the presence of mercury ions (Hg, 1 mM HgCI2, 10
ferred to nitrocellulose and the blot was developed with anti-RD28
min preincubation). Data are expressed as the mean ± SE, with the
antibodies. Samples were loaded for equal protein in each lane.
number of replicates above.
Molecular
massbymarkers
are indicated on the right.
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- Published
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Copyright © 1994 American Society of Plant Biologists. All rights reserved.
Plant Physiol. Vol. 106, 1994
Daniels et al.
1330
95%
60%
10%
Oh 24h 48h 24h 48h 24h 48h
Untr»n»formed
kD
-46.3
MIC ICM PM MIC ICM PM
-27.6
74.7
kD
-18.6
46.3
kD
27.6
kD
18.6
kD
Figure 6. Immunodetection of RD28-PIP in a water-stress time
course of A. thaliana. Plants were dehydrated to and maintained at
the percentage of their original fresh weight as indicated. They
were harvested at the time after desiccation shown. Microsomes
were fractionated by SDS-PACE and transferred to nitrocellulose
and the blot was developed with anti-RD28-PIP antibodies. Samples
were loaded for equal protein in each lane. Molecular mass markers
are indicated on the right. Both the 27- and the 54-kD bands
represent authentic RD28-PIP, which aggregates under these
conditions.
intracellular membranes compared to the plasma membraneenriched fraction (data not shown).
N. tabacum transgenic for RD28 was grown and membranes
were purified from the leaves by two-phase partitioning.
RD28-PIP in transgenic tobacco accumulates in the plasma
membrane, with very little protein in evidence in the intracellular membranes. No RD28-PIP was detected in any membranes of nontransgenic tobacco (Fig. 8). The lack of any
cross-reacting species in nontransgenic tobacco immunodetection, and the poor amino acid conservation of C-terminal residues in MIP family proteins (Reizer et al., 1993),
indicate that the anti-RD28 antiserum is highly specific to
detect the presence of RD28-PIP. Tobacco cells probably also
have a plasma membrane aquaporin, but there is no crossreactivity between the tobacco and A. thaliana proteins with
this antiserum.
RD28
Transformed
1
2
3
4
5
6
Figure 8. Immunodetection of RD28-PIP expressed in transgenic
tobacco. Plasma membranes (PM), intracellular membranes (ICM),
or total microsomes (MIC) purified from nontransgenic tobacco or
tobacco transgenic for rd28 were separated by SDS-PAGE and
transferred to nitrocellulose. Blots were probed with anti-RD28-PIP
antibodies. Samples were loaded for equal protein in each lane.
DISCUSSION
In this report we show RD28-PIP to be a mercuryinsensitive aquaporin of the plasma membrane that is expressed in all organs but not in seeds. rd28 was first discovered as an mRNA induced by water stress in A. thaliana
(Yamaguchi-Shinozaki et al., 1992). The protein encoded by
this gene is a member of the MIP family of proteins whose
members are functionally related in facilitating diffusion of
small molecules across a membrane (Reizer et al., 1993).
Recently, two other cDNAs were identified in etiolated seedlings of A. thaliana (Shagan and Bar-Zvi, 1993; Shagan et al.,
1993) that are members of the MIP family and are closely
related to this gene, as well as to the turgor-responsive gene
found in pea by Guerrero et al. (1990).
RD28-PIP Is a Mercury-Resistant Aquaporin
Based on the amino acid sequence identity between RD28PIP and the aquaporin 7-TIP, we examined RD28-PIP for its
activity as a water channel. rd28 cRNA injected into X. laevis
oocytes resulted in the oocyte plasma membrane becoming
approximately 10-fold more permeable to water than can be
accounted for by intrinsic water permeability alone. Oocytes
injected with water swell very slowly after being shifted into
hypotonic conditions, bursting only after 40 min, whereas
oocytes injected with RD28-PIP cRNA swell rapidly when
introduced into a hypotonic solution, bursting within 3 to 4
-74.7
min. We assume that this increase in P, is caused by the
appearance of RD28-PIP protein in the Xenopus oocyte
-46.3
plasma membrane. This increase in Pf is not the result of a
foreign protein that disrupts membrane integrity; oocytes
-27.6
injected with glycerol facilitator GlpF cRNA or potassium
channel Xsha2 cRNA have plasma membranes with novel
Figure 7. Immunodetection of RD28-PIP in membranes of A. thalproperties but there is no increase in membrane water permeiana. Plasma membrane and intracellular membrane samples were
ability (Maurel et al., 1993, 1994).
taken from fractions U4 and L4, respectively, of a two-phase sepaIn the presence of mercury ions RD28-PIP water channel
ration purification of plasma membranes. These fractions and a
activity
was not inhibited. It was thought that a pharmacomicrosome sample were fractionated by SDS-PAGE and transferred
logic character of water channels was their sensitivity to
to nitrocellulose and the blot was developed with anti-RD28-PIP
inhibition by mercury (Zhang and Verkman, 1991; Maurel et
antibodies. Samples were loaded for equal protein in each lane.
al., 1993;byPreston
et al., 1993); however, RD28-PIP and the
Molecular mass markers are indicated
on
the
right.
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Copyright © 1994 American Society of Plant Biologists. All rights reserved.
Plasma Membrane Aquaporin
recently discovered MIWC (Hasegawa et al., 1994) lack this
property. There is as yet no evident biological significance
for mercury insensitivity. The mercury-sensitivesite of ./-TIP
is likely to be the single Cys residue (CYS"~)on the cytoplasmic side of the membrane. Our finding that the T223C
mutant is mercury sensitive indicates that the second NPA
region, in an extracellular loop of the protein, is equally
important for the correct functioning of plant aquaporins as
it is for mammalian aquaporins. Characterization of additional PIP aquaporins will be necessary to confirm this idea.
It is interesting to note in this respect that TMP-A, TMP-B,
and TRG-31, the three plant MIP sequences most closely
related to RD28-PIP, also lack a Cys residue in this position
(Guerrero and Crossland, 1993; Shagan and Bar-Zvi, 1993;
Shagan et al., 1993). However, it is not yet known whether
they are plasma membrane proteins and whether they are
aquaporins.
RD28-PIP Is a Plasma Membrane Aquaporin
./-TIP, the only plant aquaporin characterized to date
(Maurel et al., 1993), is a tonoplast protein. In mammalian
cells, aquaporins are present in the plasma membrane (see
Chrispeels and Maurel, 1994, for review). Preliminary evidence presented by Kammerloher and Schaffner (1993) indicated that the plant plasma membrane probably contains
MIP proteins. To find out where RD28 is located, we used
the two-phase separation technique of Kjellbom and Larsson
(1984) to separate plasma membranes from intemal membranes. The technique was applied to leaves of A. thaliana
and to leaves of transgenic tobacco plants that expressed
RD28 from the cauliflower mosaic virus 35s promoter. Immunoblots of the subcellular fractions showed clearly that
the protein was located in the plasma membrane fraction,
with relatively little cross-reactivity in the internal membranes. The plasma membrane fraction was identified not
only by its partitioning characteristics in the two-phase system, but also by the presence of glucan synthase 11, a known
plasma membrane marker. Because the intracellular membrane fraction reacted positively with RD28 antibodies even
after several partitioning steps, we cannot rule out the possibility that RD28 does not also occur in the tonoplast.
However, it seems very unlikely that the same membrane
protein would be targeted to these very divergent cellular
destinations. It is more likely that cells contain distinct tonoplast and plasma membrane aquaporins.
The antiserum used in these studies detects a single 27-kD
protein in the whole tissue extracts, but in membrane preparations it also detects aggregates of this protein. This conforms to similar findings with other MIP proteins: they
readily form aggregates even when treated with SDS (see
Johnson et al., 1989, for a discussion). The related protein
CHIP28 is known to form a homotetramer in membranes
(Verbavatz et al., 1993). Although we cannot exclude the
possibility that the serum reacts with more than the 27-kD
RD28-PIP protein, the serum was made to a carboxy-terminal
peptide, and the carboxy termini of the MIP proteins are
generally poorly conserved (Reizer et al., 1993). There is little
amino acid sequence conservation at the carboxy terminus
between RD28-PIP and TmpA and TmpB, two other closely
1331
related sequences present in A. thaliana (Shagan and Bar-Zvi,
1993; Shagan et al., 1993).
Our finding that RD28-PIP is a plasma membrane protein
has two important implications. First, cells contain aquaporins in both membranes that delimit the cytoplasm: ./-TIP in
the tonoplast and RD28-PIP in the plasma membrane. This
confirms the view that transcellular water movement is an
important component of water movement through living
tissues. It is likely, but this remains to be investigated, that
water transport through aquaporins can be regulated, since
it is known that water transport through the plant can be
regulated (see Steudle, 1992, for a recent review). Second,
plant cells can target two homologous proteins with considerable amino acid sequence identity to two different locations
in the cell. At this time we do not know which destination
requires information and which one requires only bulk flow
(Gomez and Chrispeels, 1993). However, domain swap experiments and site-directed mutagenesis should reveal which
domains and specific residues are responsible for correct
targeting.
Abundance of RD28-PIP in Different Organs
Our results show that RD28-PIP is expressed throughout
the plant and is present in all organs except seeds. The protein
is most abundant in floral bolts. In this respect, its distribution
matches that of the tonoplast aquaporin ./-TIP, which is also
present everywhere except in seeds (Hofte et al., 1992). We
do not know whether RD28-PIP and -/-TIP are expressed in
the same tissues or cell types. Seeds contain a-TIP, but this
protein has not yet been shown to be an aquaporin. The
mRNA for RD28-PIP was first identified as a water stressinduced sequence (Yamaguchi-Shinozaki et al., 1992), and it
is homologous to trg-31 from peas (Guerrero et al., 1990),
which is also turgor responsive. Although trg-31 is markedly
and rapidly induced in leaves when pea plants are subjected
to water stress (Guerrero et al., 1990), RD28 is only weakly
induced and only after prolonged stress (YamaguchiShinozaki et al., 1992). Our immunoblot showed no change
in the total abundance of RD28-PIP protein after 24 h of
water stress of the A. thaliana plants, indicating that any
change in protein may be restricted to cells that make up a
relatively small fraction of the total tissue mass.
What Is the Role of Aquaporins, Such as RD28-PIP
and y-TIP?
In animal cells, aquaporins are found wherever water
transport through the membrane is high and/or regulated for
physiological reasons. In plants, water flows through living
tissues because cells are losing water as a result of the
transpiration stream or because expanding cells need to increase their volume. Such water flow can go through the
apoplast or through the symplast. Measurements with pressure probes indicate that the preferred route appears to
depend on the plant organ or tissue, the plant species, and
the physiological condition of the plant (see Steudle, 1992).
Symplastic water movement is thought to be transcellular,
mezning that it passes through the plasma membrane, as
well as through the tonoplast. We speculate that the presence
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Copyright © 1994 American Society of Plant Biologists. All rights reserved.
Daniels et al.
1332
of aquaporins allows water transport through living tissues
to be regulated i n plants, a s it is in animals. Regulation could
be achieved by altering the expression level of the aquaporins
(more or less protein in each of the two membranes) or their
activity (more or less water passing through a single channel).
Plant MIP proteins are n o w being discovered in many laboratories (Fray e t al., 1994) a n d a number of these will undoubtedly be found to be aquaporins. Further research will
reveal the physiological role of this important n e w class of
proteins.
NOTE ADDED IN PROOF
While this paper was in press, Kammerloher et al. (Plant J 6: 187199, 1994) presented evidence that the plasma membrane of A.
thaliana contains aquaporins (called PIPS). The C termini of PlP2a
and PIP2b are identical to the C terminus of RD28-PIP, and the
antibodies used in our study will therefore in all likelihood crossreact with those PIP proteins. Our results therefore confirm the
subcellular location of PIP2a and PIP2b but do not rule out the
possibility that RD28-PIP is a turgor-responsive protein, as originally
suggested by the results of Yamaguchi-Shinozakiet al. (1992).
ACKNOWLEDGMENTS
The authors would like to thank Elad Gil and Audrey Ichida for
sharing their preparations of Xenopus oocytes and Julian Schroeder
for use of the equipment to carry out the oocyte swelling assays. We
thank K. Shinozaki and K. Yamaguchi-Shinozaki for providing us
with the RD28 cDNA clone and Lyn Alkan for typing the manuscript. We thank Phil Rea for providing antibodies to tonoplast
pyrophosphatase.
Received May 27, 1994; accepted August 23, 1994.
Copyright Clearance Center: 0032-0889/94/106/1325/09.
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