The Plant Journal (1997) 12(5), 1103-1111
Two TIP-like genes encoding aquaporins are expressed in
sunflower guard cells
X. Sarda 1, D. Tousch 1,*, K. Ferrare 1, E. Legrand 2,
J.M. Dupuis 3, F. Casse-Delbart 1 and T. Lamaze 2
1Biochimie & Physiologie Mol~culaire des P/antes,
ENSA-M / INRA / CNRS URA 2133 / UM II, 2 place Via/a,
34060 Montpellier, France,
2Centre de Biologie et Physiologie V6g6tales, Universit~
Paul Sabatier, 118 route de Narbonne, 31062 Toulouse,
France, and
3Rh6ne-Poulenc Agrochimie, rue Pierre Baizet 69009,
Lyon, France
Summary
SunTIP7 and SunTIP20 are closely related sunflower cDNAs
showing a deduced amino acid sequence homologous to
proteins of the tonoplast intrinsic protein (TIP) family.
Their expression in Xenopus oocytes caused a marked
increase in osmotic water permeability (demonstrating
that they are water channels) which was sensitive to
mercury. In leaves, in situ hybridization revealed that both
SunTIP7 and SunTIP20 mRNA accumulated in the guard
cells. The possible involvement of SunTIPs in stomatal
movement was examined by comparing the time course
of transcript accumulation and leaf conductance during
the daily cycle and following a water limitation. SunTIP7
mRNA fluctuations fitted changes occurring in leaf conductance. The transcript levels were markedly and systematically increased during stomatal closure. It is suggested
that aquaporin SunTIP7 facilitates water exit associated
with a decrease in guard cell volume. In the same conditions, the transcript level of SunTIP20 remained constant
indicating that SunTIP genes are differentially regulated
within the same cell.
Introduction
Despite the relatively high water permeability of biological
membranes, they contain water-selective channels which
can facilitate and regulate the passage of water. Aquaporins
are water selective channels which belong to the Major
Intrinsic Protein (MIP) family that has representatives in all
kingdoms (reviewed in Chrispeels and Agre, 1994; Chrispeels and Maurel, 1994; Reizer et al., 1993). In plants, MIP
are divided into two major subfamilies, one corresponds to
proteins assumed to be located in the plasma membrane
(termed plasma membrane intrinsic protein, PIP, Daniels
Received5 May 1997;revised18 June 1997;accepted3 July 1997.
*For correspondence([email protected]).
et aL, 1994; Kammerloher et al., 1994) and the other to proteins assumed to be located in the tonoplast (TIP, Ludevid
etaL, 1992; Daniels etaL, 1996; Johnson, 1990).The effective
location in the membranes has only been demonstrated
for a few MIP proteins. A water transport activity has been
characterized for some members of both the PIP and TIP
subfamilies (Daniels et aL, 1996; Kammerloher et aL, 1994;
Maurel et aL, 1993; Yamada et aL, 1995). The expression of
plant MIP genes can be regulated at both transcriptional
(H6fte et al., 1992) and post-translational (Johansson et al.,
1996; Maurel et aL, 1995) levels. This expression can be
organ and tissue-specific with a pattern suggesting that
aquaporins are involved in both cell expansion and vascular
water permeability (H6fte et al., 1992; Jones and Mullet,
1995; Daniels et al., 1996). Water channels are assumed to
largely contribute to hydraulic conductivity in Chara cells
(Henzler and Steudle, 1995), Arabidopsis leaf protoplasts
(Kaldenhoff et al., 1995), the tomato root system (Maggio
and Joly, 1995), and sunflower hypocotyls (Hejnowicz and
Sievers, 1996). Indeed, expression of some aquaporin genes
was affected by osmotic stress, strengthening the hypothesis that they play a significant role in the control of water
movement (Fray et al., 1994; Guerrero et al., 1990; Yamada
et aL, 1995; Yamaguchi-Shinozaki et aL, 1992).
Stomatal movements occur by modifying the turgor pressure of the guard cells which is achieved by water fluxes
following changes in cellular solute concentration. In this
way, stomatal closure results mainly from efflux of K+ and
CI-to the apoplast, driving osmotic water loss and decrease
in guard cell turgor pressure. Guard cells are a multi-compartment system that includes a prominent vacuole where
osmotica and water are primarily accumulated. During
stomatal closure, K÷ and CI-effluxes occur through selective
and highly regulated ion channels at both the vacuolar and
plasma membranes (Schroeder, 1995; Ward and Schroeder,
1994). AthH2, a plasma membrane aquaporin gene, has
been shown to be strongly expressed within the guard cells
(Kaldenhoff etaL, 1995). However, the presence of tonoplast
aquaporins has not been demonstrated in these cells and
there is no indication that aquaporins mediate water flux
during stomatal movements.
In this report, we show that two TIP-like genes, SunTIP7
and SunTIP20, encode aquaporins expressed in guard cells
of sunflower leaves. Despite their high homology, these
genes display differential regulation. SunTIP7 transcript
accumulation was enhanced during the phase of stomatal
closure, associated with either the day-night cycle or following water limitation, while that of SunTIP20 was not modified
in the same conditions. We suggest that a finely tuned regu1103
1104
X, Sarda et al.
Figure 1. Alignmentof SunTIP7and SunTIP20sequences.
(a) Nucleotidesequences,shadowedareasindicatesequencehomologies.The initiationand stop codonsare ringed.The sequencescorrespondingto the
oligonucleotidesare underlined.
(b) Alignmentof SunTIP7(embl accessionnumberX95950),SunTIP20(EMBLaccessionnumberX95952)and Arabidopsis5TIP(EMBLaccessionnumber
U39485) deducedamino acid sequences.Shadowedareas indicatesequencehomologies,MIP family signaturesare ringed, mercury-sensitivecysteineis
shown in dark shadow.
lation of water transport via aquaporins occurs during
stomatal movements as already described for ion transport.
Results
Cloning and sequencing of sunflower TIP homologs
A partial cDNA clone was isolated from a subtractive cDNA
library constructed using root RNA from water-stressed and
control plants (Sarda et al,, unpublished data). It showed
70% homology to the Arabidopsis 5TIP sequence (Daniels
et aL, 1996) which belongs to the TIP subfamily of MIP. This
cDNA clone was used to screen 60000 plaques of a sunflower
leaf cDNA library. The screening yielded two clones, the
inserts of which were termed SunTIP7 and SunTIP20 (for
Sunflower Tonoplast Intrinsic Protein). SunTIP7 and Sun-
TIP20 cDNA are 904 and 932 bp in length respectively and
share a high (80%) nucleic acid identity (Figure la). They
show 74% and 75% sequence identity over 724 pb to Arabidopsis (STIPcDNA, respectively. Their open reading frames
were found to encode polypeptides of 248 amino acids with
calculated molecular weights of about 25 000 Da. Their
amino acid sequences contain the MIP family signature
motif (SGxHxN PAVT) and present more than 90% homology
over 239 amino acids to (STIP(Figure lb).
Water transport activity of Sun TIP7 and 20 proteins in
Xenopus oocytes
In vitro transcription and RNA capping of SunTIP20 cDNA
were performed from the initial plasmid (pBluescript ® SK
(-)). The cRNA contained its own 5' and 3' untranslated
Expression of TIP genes in sunflower guard cells
1105
Figure 3. RNA gel blot analysis of sunflower total RNA extracted from
distinct tissues and probed with 32p-labelled specific SunTIP7 and 20
antisenseRNAs.
25 S rRNAaccumulationis shown below as a control for loading. R: root;
ML: matureleaf;YL: youngleaf;SL: senescentleaf;B: bud,L: influorescence.
Figure Z. Osmoticwater permeability(Pf) of injected oocytes.
Oocyteswere injectedwith SunTIP7, SunTIP20and ArabidopsisyElPcRNAs
or water. HgCI2was added to Barth's solution to a final concentrationof
1 mM. Pf values were derived from volume change measurements of
individual oocytesfor three different injections.
region including a 55 polyA tail. Capped RNAs corresponding to the SunTIP7 or Arabidopsis yTIP cDNA were transcribed in vitro from the pBSTA and pSP64T plasmids
respectively and thus contained the untranslated 5' and 3'
regions of the ~-globin gene. Control oocytes were injected
with water. Three days after the injection, when exposed
to hypoosmotic conditions (160 mOsm kg -1 gradient),
control oocytes slowly swelled without any rupture within
the first 45 min. In contrast, oocytes injected with SunTIP7
and SunTIP20, or Arabidopsis 7TIP cRNA as a positive
control (Maurel et aL, 1993), showed a volume which
increased by up to 60% within 6-10 min (SunTIP7 and
20) and 2-5 min (TTIP), and they burst. The swelling rate
increments corresponded to a 6.4, 6.6 or 17.5 fold enhancement in osmotic water permeability ofthe oocyte membrane
for SunTIP7 (6.21 10-3 cm -1 sec), SunTIP20 (6.44 10-3
cm -1 sec) and yTIP (17.05 10-3 cm -1 sec, Figure 2), respectively. These permeabilities were lowered by 90% in the
presence of 1 mM HgCI2 which is a characteristic inhibitor
of water channel proteins (Macey, 1984).
The increase in osmotic water permeability was systematically about 60% smaller for both the SunTIPs than for
yElP. A weaker increase in permeability after expression in
Xenopus oocytes was reported for the ice plant MIP related
genes as compared with yElP (Yamada et aL, 1995).
Accumulation of Sun TIP mRNA in tissues
RNA gel blot analysis was performed on total RNA isolated
from a number of distinct tissues. The SunTIP7 and 20
transcripts were seen in all tissues but primarily in leaves
(young, mature and senescent, Figure 3).
Location of SunTIP mRNA in leaves
In situ localization of SunTIP7 and SunTIP20 transcripts
was carried out using specific oligonucleotide probes.
These short probes (30 nucleotides in length) easily penetrate the plant tissues and do not contain vector sequences
(often present in in vitro synthesized RNA probes) which
could enhance background. No specific signal was detected
in leaf sections hybridized with sense oligonucleotide
probes (Figure 4a). In contrast when sections were hybridized with anti-sense oligonucleotide probes, intense hybridization signals were observed only in guard cells on both
surfaces of the leaf for SunTIP7 and SunTIP20 (Figure 4b,
c and d). This specific expression of SunTIP genes in guard
cells was observed at various times of the day, therefore
while leaf conductance was increasing, at a stable high or
decreasing (see below).
Stomatal aperture and Sun TIP transcript accumulation
during a day-night cycle and following water stress
Northern analyses were carried out with a specific antisense RNA probe corresponding to the 3' untranslated
region of SunTIP7 and SunTIP20.
Diurnal fluctuations. In the glasshouse, daily time courses
of leaf conductance (gs) showed that the gs of well watered
plants increased steadily from 3:30 a.m. (dawn) to about
10 a.m., then it progressively diminished, displayed a
plateau around 3 p.m. and subsequently diminished again
so that stomata were fully closed after 7 p.m. (Figure 5a).
In these leaves, SunTIP7 transcripts were of low abundance
in the morning. However, the level of accumulation was
markedly enhanced between 11 a.m. and 7 p.m. The time
course in SunTIP7 transcript accumulation paralleled that
of stomatal closure (Figure 5b). This was always found
although the extent of gs variations was dependent on
climatic conditions. SunTIP20 transcript accumulation was
strongly enhanced at 5 a.m. as compared with dawn, then
1106
X. Sarda et al.
Figure4. In situ location of SunTIPtranscripts.
Transversalleaf sectionswere hybridizedwith senseoligonucleotideprobe (a) or with SunTIP7(b and c) and SunTIP20(d) antisenseoligonucleotideprobes.
Bars = 40 pm, GC : guard cells, P : pallisadeparenchymacells, S : spongy parenchymacells, × : xylem.
remained high throughout the day and started to decline
after 5 p.m. (Figure 6).
Response to water stress. Under steady environmental
conditions in a growth chamber, stomata of hydroponic
plants (control) opened rapidly at the onset of the photoperiod and then remained almost unchanged up to the dark
period (not shown). When water limitation was imposed
2 h into the photoperiod, a significant reduction in leaf
conductance started 2 h after water withdrawal, continued
over the following h and ended after 5 h reaching a value
of 40% (Figure 7a). SunTIP7 transcript amount in leaves of
control plants was comparable throughout the experiment.
In contrast, in leaves of water stressed plants, level of
SunTIP7 mRNA was transitorily increased (threefold) 2 h
after stress imposition (Figure 7b). The SunTIP20 transcript
level remained unchanged in leaves of both water stressed
and control plants at all times during the experiments (0,
2, 5, 7 and 24 h, Figure 8).
Discussion
According to their sequences, SunTIP7 and SunTIP20 must
be assigned to the TIP subfamily and appeared to be highly
homologous with Arabidopsis (~TIP, a tonoplast-located
aquaporin (Daniels etaL, 1996). Expression of SunTIP7 and
SunTIP20 in Xenopus oocytes led to a sixfold increase in
the membrane osmotic water permeability indicating that
they are also aquaporins.
Protein-modifying sulfhydryl reagents such as mercuric
chloride are characteristic inhibitors of many aquaporins
(Daniels et al., 1996; Maurel et al., 1993; Preston et al.,
1992; Yamada et al., 1995). SunTIP7 and SunTIP20 water
channel activities in Xenopus oocytes were about 90%
Expression of TIP genes in sunflower guard cells
(a)
1107
s0o
600'
400"
200"
,
3
"
i
5
•
!
7
°
|
9
•
i
11
•
=
13
•
.
15
°
|
17
•
|
19
.
P
21
Time of the day (hours)
Figure 6. SunTIP20transcript accumulation in sunflower leavesduring a
day-night cycle.
Northern analysisof total RNAextractedfrom the youngestfully-expanded
leaves during a day-night cycle and probed with 32P-labelledSunTIP20
antisenseRNA. RelativemRNA accumulationis shown below.
Figure 5. Leafconductanceand SunTIP7transcript accumulationduring a
day-night cycle.
(a) Conductance(gs) of the youngestfully-expandedleavesof sunflowers.
Data (+/- standard deviation)are the means of 4 measurementsper plant
for 30 individual plants.
(b) Northern analysis of expression of SunTIP7 in leaves. Total RNA
extracted from the youngestfully-expandedleavesof R1 sunflowerswere
probed with 32p-labelled SunTIP7 antisense RNA. Relative mRNA
accumulation is shown below.
inhibited by 1 mM HgCI 2. In a recent paper, Daniels et al.
(1996), demonstrated that cysteine residues in transmembrane domain at positions 116 and 118 play a role in the
mercury sensitivity of ~TIP and 7TIP, respectively. SunTIP7
and SunTIP20 also contain a cysteine residue at position
116 that is thus also likely to be a mercury-sensitive site
(Figure lb).
SunTIP7 and SunTIP20 transcripts were preferentially
found in leaves but also in roots though at lower levels.
This indicates that SunTIP gene expression is not tissue-
specific. This is consistent with the fact that the SunTIP
cDNAs were obtained from a root cDNA library.
The tissue expression patterns of 8TIP, and SunTIPs were
different. In situ hybridization for SunTIPs was distributed
in the guard cells whereas expression of 5TIP correlated
with developing vascular tissues (Daniels et al., 1996). This
is the first report showing TIP-like aquaporins in the guard
cells. Interestingly, SunTIP7 transcript in leaves was found
at a high level when the stomata were closing whereas
the transcript was present close to basal levels when the
stomata were either opening or in a steady, either opened
or closed, state. This was true for a relatively rapid decline
in leaf conductance resulting from a sudden water limitation and for the progressive decrease linked to the daynight cycle. In contrast, the expression of SunTIP20 was
not subject to variation during stomatal closure. These
expression patterns show that the two genes which encode
highly homologous aquaporins are differentially regulated
within the same cells.
We also performed in situ hybridization on leaf tissue
showing varying SunTIP7 mRNA levels and since expression remained exclusive to the guard cells it would appear
that the fluctuations observed on Northern blots resulted
from fluctuations in the levels of expression in the guard
cells. From the concordance between SunTIP7 transcript
abundance in the leaves and the time course of leaf
conductance, we propose that SunTIP7 is involved in
stomatal closure. Indeed, assuming that changes in transcript levels lead to similar changes in protein level, and
assuming that SunTIP7 is located in the tonoplast, this
aquaporin may play an important role by accelerating
water exit from the guard cell vacuole. Although SunTIP7
and 20 showed a striking sequence homology and were
found expressed within the same cells and exhibited similar
1108
X. Sarda et al.
(a)
120
100
o
o
80
o
60
(;}
40
o
4
6
Time since stress was applied (hours)
Figure8. Northern analysis of SunTIP20transcript accumulation in the two
youngest fully-expanded leaves of sunflower plants. Total RNA extracted
from water stressed (s) and control (c) plants after 2, 5, 7 and 24 h of
treatment were probed with 32p-labelledSunTIP20 antisense RNA. Relative
mRNA accumulation is shown below.
to w a t e r l i m i t a t i o n m a y occur even t h o u g h the w a t e r status
of the leaves is not disturbed. In our e x p e r i m e n t a l design
based on the split-root t e c h n i q u e (Neales et al., 1989),
w a t e r limited plants e x h i b i t e d no significant differences in
leaf w a t e r and o s m o t i c potentials as c o m p a r e d with control
plants o v e r a 24 h t r e a t m e n t duration (Sarda et al., in
preparation). As just discussed, r o o t - t o - s h o o t c o m m u n i c a tion appears to be the o n l y factor influencing the p h y s i o l o g y
of the aerial parts. Consequently, the effects on SunTIP7
expression could result (directly or not) f r o m root signals
as is the case for leaf conductance.
Figure 7. Leaf conductance and SunTIP7 transcript accumulation in
response to water stress.
(a) Conductance (gs) of the two youngest fully-expanded leaves of
hydroponically grown sunflower plants. Data (+/- standard deviation) are
means of four measurements per plant with 48 plants for the first point
and 6 for the last.
(b) SunTIP7 expression in sunflower leaves after water deprivation: Total
RNA extracted from stressed (s) and control (c) plants after 2, 5, 7 and 24 h
of treatment were probed with S2p-labelledSunTIP7 antisense RNA. Relative
mRNA accumulation is shown below.
w a t e r t r a n s p o r t activity in Xenopus oocytes, t h e y are likely
to be i n v o l v e d in distinct functions in planta, as suggested
by the different e x p r e s s i o n patterns of the c o r r e s p o n d i n g
genes.
Early control of s t o m a t a l c o n d u c t a n c e by roots is a well
characterized p h y s i o l o g i c a l p h e n o m e n o n that can occur in
response to soil drying. Roots are a s s u m e d to perceive the
v a r i a t i o n in w a t e r a v a i l a b i l i t y and to c o m m u n i c a t e this
i n f o r m a t i o n to the shoots t h r o u g h chemical signals inducing s t o m a t a l closure. This a d a p t a t i v e response of leaves
Experimental procedures
Plant culture
Sunflower (Helianthus annuus L, R1 RPA) seeds were germinated
for 3 days at 25°C in the dark on filter paper moistened with distilled
water. The seedlings were then transplanted into individual pots
of sand (5 I) placed in a greenhouse where they received daily
application of a complete nutrient solution• Environmental conditions were: 25/22°C and 60/80% RH (day/night, respectively) with
a minimum of 15 h photon flux density of 250 ~tmol m -2 sec-1 PAR
(enhanced by fluorescent lamps depending on solar irradiance).
Experiments were carried out on 5-week-old plants. At the time
of sampling, leaves were rapidly harvested, weighed, frozen
in liquid nitrogen and stored at -80°C until extraction. Entire
experiments were performed at least in duplicate (distinct cultures)
and representative results are shown.
Daily time course of stomatal conductance (gs)
Abaxial stomatal conductance of the four youngest fully-expanded
leaves of 20 individual plants was measured (diffusion porometer
AP4, Delta-T Devices, UK). At each time of sampling, the two
Expression o f TIP genes in sunflower guard cells
youngest fully-expanded leaves of eight individual plants were
harvested for RNA extraction.
1109
subclones generated using existing restriction sites. Sequence
comparisons with databases were performed at the US NCBIs
Genlnfo network BLAST (Altshul et eL, 1990; Gish and States,
1993) server.
Water stress application
Plant roots were carefully removed from the sand, washed in
distilled water and the plants transferred to a growth chamber for
hydroponic culture. Six plants were placed in each 8-1 container
covered with a tray that allowed the roots to be immersed in an
aerated solution in the dark. Environmental conditions were: 22/
18°C (day/night), 40% RH, 16-h photoperiod (300 Ilmol m-2 s-1
PAR, cool white fluorescent lamps).
The experiments took place 18 h after transferring the plants to
hydroponic culture (2 h into the photoperiod) in order to allow
acclimation. Stress was applied by lowering the level of nutrient
solution so that less than 10% of the entire root system remained
immersed. The bubbling of the nutrient solution was reduced in
order to avoid any wetting of the emerged roots but the air in the
pot was kept humid. The emerged roots were thus suspended in
the dark, at 20°C in the air the humidity of which was monitored
at 97-98% RH for the remainder of the experiment (24 h). This
corresponded to a water potential of around -3.5 MPa. The root
system of control plants was maintained in the aerated nutrient
solution. Physiological measurements were performed at regular
intervals on the youngest fully-expanded leaves and included:
abaxial conductance, photosynthesis (Licor 6200, US) and water
relations (disc psychrometers connected to a Wescor HR-33T). At
each sampling, six plants per treatment were harvested.
Nucleic acid isolation and cDNA library construction
Leaf and root total RNA were isolated using the procedure
described by Ausubel (1990) except that the extraction buffer
was pre-heated to 80°C and contained 0.5% (w/v) polycler ATTM.
Poly(A)+ RNA was purified from total RNA using oligo dT columns
(Bioprobe system, France) with the standard procedure (Kingston,
1987). Double stranded (ds) cDNA synthesis was performed
according to (Klickstein, 1988) using SuperScript TM II reverse
transcriptase (Gibco BRL). Ds cDNA was ligated to specific linkers
(EcoRI, Promega) and cloned in Lambda Zap II/EcoRI/CIAP arms
(Stratagene). The ligation products were packaged in vitro using
the Gigapack II Packaging Extract (Stratagene) according to the
supplier's instruction manual.
Isolation of Sun TIPs cDNA
Library was plated on XL1-Blue E. coli cells (Stratagene). Plaque
blotting was performed according to the HybondTM N instruction
manual (Amersham). Hybridization was carried out in 7% SDS
(Church and Gilbert, 1984) at 65°C, using a random 32p-labelled
cDNA as probe (T7QuickPrimeTM kit, Pharmacia). Selected phages
hybridizing preferentially to the probe were converted to
pBluescript ® SK (-) by in vivo excision using ExAssist helper
phage (M13) according to the supplier's instructions (Stratagene).
RNA blot analysis
Fifteen ilg of denatured total RNA were separated on a 1.5%
MOPS-formaldehyde agarose gel (Sambrook et aL, 1989) and
transferred by capillarity onto Hybond-N filters (Amersham) in
10 x SSC. Probes corresponding to the 3' untranslated region of
SunTIP7 and SunTIP20 were generated by T7 RNA polymerase
run-off transcription of the BsiHKA I and Nla III restriction products
of pSunTIP7 and pSunTIP20 respectively in the presence of 32p.
radiolabelled UTP. Hybridization was then performed at 62°C for
16 h in 60% formamide buffer. Hybridized membranes were
washed for 15 min in 0.1% SDS, 0.1% SSC buffer at 62°C (high
stringency). Wet filters were autoradiographed at -80°C using
Kodak X-OMAT and BioMax films. Between hybridizations with
different probes, the filters were washed in 0.2% SDS at 95°C for
15 min. Precise quantification of the RNA blotted was carried out
with a 32p-random primed BamHI fragment of HA89 25S rDNA
(H. annuus) hybridized in 50% formamide buffer at 46°C. The
autoradiographs were quantitatively analysed with Biolmager
(Appligene) and the NIH Image/68K 1.57 program. The experiments
were duplicated as follows : RNA was extracted from plants from
two separate cultures, and at least four independent hybridizations
with distinct membranes were realized for each culture. These
procedures allowed us to accurately compare SunTIP transcript
levels.
Plasmid construction and in vitro cRNA synthesis
A fragment corresponding to the open reading frame of SunTIP7
was amplified by PCR from the initial pBluescript ® clone using
the folowing primers which contain the Bglll restriction sites:
Forward primer 5'-'I-I-GCGGCCGCAGATCI I I I II I II ICCAAAATGCCT-3' and reverse primer 5'-TTGCGGCCGCAGATCTAAATAGCCTGGAATCATAC-3'. The PCR product was cloned into the Bglll
site of pBSTA derived pBS-SK (+) vector carrying the 5' and 3'
untranslated sequences of a J~-globingene from Xenopus. Clones
with inserts in the correct orientation were determined by restriction mapping and sequencing, pBSTA containing SunTIP7 insert
was linearized with Notl and used for in vitro transcription with
T7 RNA polymerase and capping RNA as described (Titus, 1991).
pBluescript ® containing SunTIP20 insert was linearized with Sail
and used for in vitro transcription as above, pSP64T plasmid
containing Arabidopsis 7TIP insert (pGTIP~2, Maurel et eL, 1993)
was kindly provided by Dr Christophe Maurel. This plasmid was
linearized with Xbal and used for in vitro transcription with T3
RNA polymerase.
Preparation of Xenopus laevis oocytes and cRNA
injection
DNA sequencing and analysis
cDNA inserts in recombinant plasmids were sequenced on both
strands on an automated DNA sequencer (Applied Biosystems)
using a Taq Dye primer Cycle Sequencing Core kit (Applied
Biosystems). The cDNA sequences were constructed by analysis
of both overlapping exonuclease III deletions (Henikoff, 1984) and
Oocytes (stage V and Vl) were prepared as described by Dascal
(1987), injected with 50 nl of in vitro transcribed mRNA (1 mg
m1-1) and incubated at 20°C for 3 days in Berth's solution (10 mM
HEPES-NaOH, pH 7.4, 88 mM NaCI, 1 mM KCI, 2.4 mM NaHCO3,
0.33 mM Ca(NO3)2, 0.41 mM CaCI2, 0.82 mM MgSO4, Cao et aL,
1992) supplemented with 50 mg 1-1 gentamycin.
1110
X. S a r d a et al.
Osmotic water permeability m e a s u r e m e n t
Water transport was assayed by transferring individual oocytes
from Barth's solution (Osmin = 200 mosmol kg-1) to the same
solution diluted to 40 mosmol kg-1 with water as described by
Maurel et al. (1993). Cell swelling was followed by video microscopy, oocyte diameters were measured at 30 to 120 sec intervals.
Cell volume and water permeabitities were calculated from cell
section areas (Preston et al., 1992).
In situ hybridization
Tisssues of the youngest-fully-expanded leaves were harvested
and immediately fixed 1 h in phosphate-buffered 0.4% glutaraldehyde and 4% paraformaldehyde solution, embedded in
Paraplast Plus (Monoject Inc., St Louis, MO) and 10 p.m sections
cut. Specific oligonucleotides of the SunTIP7 3' untranslated
region (5'-CAATCCAACACACAAGAGCACAAACATTGG-3') and 5'
untranslated region of SunTIP20 (5'-CAGAACTTAAATAATTAAAAGAAATGGAGA-3') were radiolabelled with 35S-yATP and T4 polynucleotide kinase. No cross-hybridization was observed between
SunTIPs oligonucleotides and their respective targetted sequences
in either Northern or Southern experiments (data not shown). Ten
pmot (107 cpm) of radiolabelled oligonucleotides were introduced
per ml of hybridization solution (3M TMAC, 0.1 M Na3PO4 pH 6.8,
10% (w/v) dextran sulfate, 0.6% (w/v) SDS, 0.5 M EDTA,
1 × Denhart's, 120 mM DTT, 1 mg m1-1 poly A, 0.3 mg m1-1 E. coil
tRNA). Hybridizations were carried out with 0.1 mt per slide at
50°C for 48 h. Slides were then rinsed in 2 x SSC, 0.1% SDS,
5 mM DI-I for 30 min, dehydrated with ethanol, coated with NTB2 photographic emulsion (Kodak, Rochester, NY) and exposed at
4°C for 2 weeks. After development, the slides were stained with
toluidine blue for histochemicat observations.
Acknowledgements
We thank Profressor Pont-Lezica and Dr Cannut for critical comments on the manuscript and B. Sarrant for technical assistance.
We thank Dr Maurel for providing Arabidopsis yTIP clone. This
work was financed in part by the Bio Avenir programme supported
by Rh6ne-Poulenc, the Research Ministry and the Industry Ministry.
References
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EMBL Data Library accession numbers X95950 (SunTIP7) and X95952 (SunTIP 20).