Journal of Experimental Botany, Vol. 53, No. 373, pp.
15031514, June 2002
Stomatal control in tomato with ABA-deficient roots:
response of grafted plants to soil drying
N. Michele Holbrook1, V.R. Shashidhar2, Richard A. James3 and Rana Munns3,4
1
Department of Organismic and Evolutionary Biology, Harvard University, Cambridge, MA 02138, USA
2
Department of Crop Physiology, University of Agricultural Sciences, Bangalore 560 065, India
3
CSIRO Division of Plant Industry, GPO Box 1600, Canberra, ACT 2601, Australia
Received 8 May 2001; Accepted 22 February 2002
Abstract
The hypothesis that ABA produced by roots in
drying soil is responsible for stomatal closure was
tested with grafted plants constructed from the
ABA-deficient tomato mutants, sitiens and flacca
and their near-isogenic wild-type parent. Three
types of experiments were conducted. In the first
type, reciprocal grafts were made between the
wild type and sitiens or flacca. Stomatal conduct-
ance accorded with the genotype of the shoot, not
the root. Stomates closed in all of the grafted plants
in response to soil drying, regardless of the root
genotype, i.e. regardless of the ability of the roots
to produce ABA. In the second type of experiment,
wild-type shoots were grafted onto a split-root
system consisting of one wild-type root grafted to
one mutant (flacca or sitiens) root. Water was
withheld from one root system, while the other was
watered well so that the shoots did not experience
any decline in water potential or loss of turgor.
Stomates closed to a similar extent when water
was withheld from the mutant roots or the wild-type
roots. In the third type of experiment, grafted plants
with wild-type shoots and either wild-type or sitiens
roots were established in pots that could be
placed inside a pressure chamber, and the pressure
increased as the soil dried so that the shoots
remained fully turgid throughout. Stomates closed
as the soil dried, regardless of whether the roots
were wild type or sitiens. These experiments demon-
strate that stomatal closure in response to soil
drying can occur in the absence of leaf water deficit,
and does not require ABA production by roots. A
chemical signal from roots leading to a change in
apoplastic ABA levels in leaves may be responsible
for the stomatal closure.
4
To whom correspondence should be addressed. Fax: q61 2 6246 5399. E-mail: rana.munns@csiro.au
Society for Experimental Biology 2002
Key words: Abscisic acid, drought, flacca, root signals,
sitiens.
Introduction
Experiments in which leaf water status was maintained
in plants as the soil dried, either by balancing pressure
techniques (Gollan et al., 1986; Schurr et al., 1992) or by
roots split between wet and dry soil (Gowing et al., 1990;
Khalil and Grace, 1993; Stoll et al., 2000), demonstrate
that stomates can close independently of the water status
of the leaves. This suggests that there is a chemical signal
produced by the roots that controls stomatal conduct-
ance. There is evidence that this signal is ABA; stomatal
closure is often associated with an increase of ABA in
xylem sap, prior to a detectable increase in leaf ABA
(reviewed in Davies and Zhang, 1991), and significant
stomatal closure can be induced in detached leaves by
feeding synthetic ABA at concentrations similar to that
occurring in the transpiration stream (Tardieu et al.,
1996; Correia and Pereira, 1994). This suggests that root-
produced ABA might regulate stomatal behaviour under
stress. However, a number of findings are inconsistent
with this hypothesis. First, there is no absolute relation-
ship between ABA and conductance even within a given
species. The relationship can vary with the time of day
(Tardieu et al., 1996), the degree of stress (Correia and
Pereira, 1995) or with the individual plant (Schurr et al.,
1992). Possible explanations of this variable relationship
include an interaction with leaf water status (Tardieu
et al., 1996), or, in the absence of changes in leaf water
status, changes in ion transport that might give rise to
changes in xylem sap pH (Gollan et al., 1992; Wilkinson
and Davies, 1997; Wilkinson et al., 1998). Second,
ABA arriving in the xylem is rapidly metabolized by
1504 Holbrook et al.
leaves; ABA fed through the xylem of detached leaves had
a half-life less than 1 h (Gowing et al., 1993), although
in intact leaves the half-life was a little longer (Jia
and Zhang, 1997). Trejo et al. showed that the meso-
phyll tissue rapidly metabolized ABA fed through the
xylem, so it is possible that the fine control of stomatal
conductance is exerted by leaf metabolic activity rather
than by the ABA arriving in the xylem (Trejo et al.,
1993). Third, the ABA appearing in the xylem may
not have been produced by the roots; it may have
been synthesized in the leaves, translocated in the
phloem to the roots, and recirculated in the xylem
(reviewed by Munns and Cramer, 1996). Fourth, experi-
ments in which normal shoots have been grafted onto
either normal roots or ABA-deficient roots have
found little or no effect of the root genotype on
stomatal behaviour (Fambrini et al., 1995; Jones et al.,
1987). Thus, the role of ABA in the xylem versus
that made in the leaf in controlling stomatal conductance
is not clear.
In order to test the hypothesis that root-produced
ABA controls stomatal conductance in drying soil,
plants with ABA-deficient roots and wild-type shoots
were constructed. If ABA produced by roots is required
for root-to-shoot signalling, then the response to
drying soil should depend upon the root genotype. The
ABA-deficient tomato mutants sitiens and flacca were
used. Previous work had shown that roots of sitiens
plants do not synthesize increased amounts of ABA
under stress (Cornish and Zeevaart, 1988; Dunlap and
Binzel, 1996), and that sitiens does not export significant
amounts of ABA in the xylem even in dry soil (R Munns
and VR Shashidhar, unpublished data). Three types of
experiments were conducted. The first type examined
patterns of water use and stomatal conductance in
reciprocal grafts of both sitiens and flacca. The second
type of experiment examined patterns of water use
when only part of the root system was dried. These
split-root experiments used plants formed from 3-way
grafts such that the shoot had a wild-type phenotype,
while the root system had both a wild-type and an
ABA-deficient part. The split-root design allowed the
effect of root genotype on patterns of water use in
response to soil drying to be examined without the
possibility of dehydration-induced ABA synthesis in
leaves. The third type of experiment examined changes
in stomatal conductance in response to soil drying of
plants whose xylem pressures were held constant using
root pressurization. Reciprocal grafts formed from
wild-type shoots and sitiens or wild-type roots were
established in pots that could fit within a Scholander-type
pressure chamber. The chambers were pressurized as
the soil dried to maintain the xylem sap at atmospheric
pressure and hence also the shoot water status at its
maximum.
Materials and methods
Plant material and growth conditions
Seeds of the ABA-deficient mutants flacca and sitiens and their
near-isogenic parental line, Rheinlands Ruhm, were obtained
from the Tomato Genetic Resources Centre, University of
California, Davis. Seeds were germinated on wet filter paper
and grown without the addition of ABA. Experiments involving
the mutant sitiens used a sandy loam (bulk density of
1.28 g cm
3; maximum water capacity of about 0.25 g cm
3)
as the rooting medium. Experiments involving the mutant flacca
were conducted using an organic-based potting medium (bulk
density of 0.54 g cm
3; maximum water capacity of about
0.4 g cm
3). The plants were grown and experimental measure-
ments carried out in growth chambers providing 30u20 8C
dayunight temperature, 60% daytime relative humidity, light
intensity of 650 mmol m
2 s
1 and a 12 h photoperiod.
Reciprocal graft experiments
The effect of the root genotype (ABA-deficient versus wild type)
on patterns of water use and stomatal conductance in response
to drying soil was investigated using reciprocal grafts. Grafts
were made by cutting the scion and stock plants diagonally,
removing the leaves, securing the union with parafilm, and
growing under a mist until new leaf growth was observed
(approximately 23 weeks). At this stage there was no indication
of reversion of the situwt (scionustock) to the shoot morphology
of the wild type, which occurs with time (Cornish and Zeevaart,
1988). In the experiments using the mutant sitiens, five
individuals of all four graft combinations, wtusit, wtuwt, situwt,
and situsit were grown in 600 cm3 pots. There was no difference
in leaf area between wtuwt, wtusit, and situwt plants (average of
378 cm2), however, situsit plants had somewhat less total leaf
area (average of 302 cm2). In the experiments using the mutant
flacca, six individuals each of wtuflc and wtuwt were grown in
5.0 l pots. There was no difference in mean leaf areauplant
between the two graft constructs (average of 2926 cm2). Both
soil volume and leaf area per plant were smaller in the sitiens
experiments than in the flacca experiments, however, the
relationship between leaf area (cm2) and soil volume (cm3) was
similar (0.55 cm
1 flacca versus 0.60 cm
1 sitiens). The plants
were watered regularly throughout their development. At the
onset of the experiment, the pots were covered with plastic to
prevent evaporation from the soil and watering ceased.
In the sitiens experiment, plants were allowed to dry the soil
at their own rate (i.e. no water was added to the pots during
the period of soil drying). The time to visible wilting varied
between graft types, but was approximately 3 d for plants with a
wild-type scion and 2 d for plants with a sitiens scion. Plants
were weighed at hourly intervals between 11.00 h and 17.00 h,
and the transpiration rate calculated. Stomatal conductance
was measured at 1 h intervals during the same period using a
porometer (AP4, Delta-T Devices, Burwell UK). The experi-
ment was terminated when the plants wilted, at which point
the plants were harvested and total leaf area and soil dry
weight determined. Soil water content was calculated assuming
a constant plant weight over the course of the experiment (3 d).
Leaves were sampled for ABA content from three individuals
of each graft type at the beginning and the end of the experiment
(method described below). Leaflets for ABA measurement were
taken from the same leaf in which stomatal conductance was
measured.
In the flacca experiment, each plant was weighed at the
beginning and end of the light period, allowing an average
 Stomatal control in tomato 1505
water loss rate for the light and dark periods to be calculated.
Differences in the rate of soil drying between plants were
minimized by adding water to the pots such that the maximum
rate of water loss did not exceed 0.05 kg d
1. At this rate,
approximately 7 d were needed before the plants began to wilt.
Measurements of water loss, stomatal conductance, and leaf
water potential were made until the plants began to wilt,
at which point the plants were harvested. Stomatal conductance
was measured on five leaves per plant between midday and
14.00 h. using a porometer (Li-1600, Li-Cor, Lincoln USA).
Measured leaves were selected from at least three different
branches per plant and only the youngest fully expanded leaves
were used. Leaf water potential was determined using thermo-
couple psychrometers (JRD Merrill Specialty Equipment Co.,
Model 8413C). Leaf discs (0.4 cm diameter) were rapidly sealed
into the psychrometer chambers in a temperature-controlled
box (30 8C), and measurements made with a Wescor HR33T
dew point microvoltmeter.

Split-root experiments
A split-root experiment was used to examine the role of root-
produced ABA on patterns of water use in response to partial
drying of the root system. Split-root plants were constructed
from a three-way graft joining together material from three
separate plants. A V-shaped cut was made at the base of a wild-
type shoot and simultaneously grafted to the root system of two
other plants, growing in separate pots, from which the shoots
had been excised (Fig. 1). The success rate for these grafts
was quite low and large numbers of plants could not be gen-
erated. Six sitiens split-root plants were generated, with wild-
type shoots and both wild-type and sitiens root systems, growing
in different pots. Two flacca split-root plants were similar to
this, i.e. had wild-type shoots and both wild-type and flacca root
systems, and in another two the root systems were the same
(either wild type or flacca). In all cases the pots were tall
cylinders (5 3 40 cm). Fig. 1. Grafted plants with split root systems. A wild-type shoot was
Time-domain reflectometry (TDR) was used to determine the grafted to the root system of a wild-type plant and a sitiens plant. The
soil water content of each pot (Fig. 1). Wave-guides, consisting rods are part of the TDR apparatus for measuring soil water content.
of three stainless steel rods, were inserted at a spacing of 2.5 cm.
Measurements using a Tektronix 1502c time-domain reflecto-
meter were taken three times each day, and the relationship
between TDR reading and volumetric soil water content deter- water was withheld from the other side of the root system.
mined empirically by growing tomato plants in pots identical to Stomatal conductance was measured at midday on the abaxial
one used for the split-root plants. surface of five young, but fully expanded leaves, per plant using
The basic experiment consisted of monitoring rates of a porometer (Li-1600). Simultaneously, leaf discs were col-
whole plant water use and leaf water status as one pot dried lected from two leaves per plant for measurements of leaf water
while the other was watered. In the sitiens experiment, the three potential using thermocouple psychrometers.
treatments (wild-type side dried, sitiens side dried, or neither
side dried) were applied sequentially to each plant. The order
in which each plant experienced these treatments was varied.
Each plant (i.e. pair of pots) was weighed three times per day, Root pressurization experiments
and TDR measurements taken for each pot. The amount of Root pressurization was used to determine whether the ability
water needed to bring the plant to its initial weight was of roots to produce ABA influenced patterns of stomatal
calculated, and the appropriate pot was watered with this closure in response to soil drying when xylem water potentials
amount. Stomatal conductance was measured between midday were maintained at a constant level. Two types of grafts were
and 14.00 h on five leaves per plant using a porometer made having wild-type shoots, but either wild-type or sitiens
(Delta-T). Leaf relative water content was determined by roots (i.e. wtuwt and wtusit), and plants were established in pots
rapidly weighing excised leaflets (three per plant), re-weighing designed to fit within a pressure chamber (9 3 19.5 cm). When
them after allowing them to hydrate fully by floating them for the plants had 78 unfolded leaves pots were placed in a
3 h on deionized water, and then drying the leaves to a constant pressure chamber and the stems were sealed according to
weight at 65 8C. methods outlined previously (Stirzaker and Passioura, 1996).
In the flacca experiment, water was withheld from one side of Plants were brought to balancing pressure by removing a
the root system of each plant for a period of 7 d. The plants leaflet from the most recent fully expanded leaf, and raising
were then freely watered for several days, following which the pressure in the chamber until the xylem sap was on the
1506 Holbrook et al.
point of bleeding, as judged from moisture appearing at the end
of the cut petiolule. No excess bleeding was observed in other
parts of the plant. The method followed that described earlier
(Stirzaker and Passioura, 1996), except that the plants were in
walk-in controlled environment chambers, and that the pressure
was monitored and adjusted manually.
Experiments were carried out on three replicate control
plants (wtuwt) and six replicate mutant grafts (wtusit). Three
different treatments were carried out sequentially on these
plants. First, the soil was allowed to dry while the plant
was maintained at balancing pressure. Stomatal conductance
was measured until the balancing pressure reached 2 MPa.
Pots were weighed at the beginning and end of the period, and
the rate of water loss calculated. Second, after allowing plants
to recover for 3 d, the soil was allowed to dry without balancing
pressure, and conductance and pot weights measured. Third,
after 9 d the soil was allowed to dry while plants were
pressurized, and leaf tissue as well as xylem sap were collected
for measurement of ABA.
Stomatal conductance was measured with a porometer
(Delta-T) as the soil dried, on each of 68 leaves, starting from
the youngest leaf whose laminae had expanded sufficiently
to measure, and finishing with the oldest that was not starting to
senesce. Conductance was measured every hour until the rate
of change increased when it was measured every 2030 min.
The soil took about 2 d to dry to the extent that conductance
was reduced by 5070%. Conductance was measured only
between the period of 10.00 h and 18.00 h, i.e. not less than
2 h into the photoperiod and 1 h before the end. However, as
conductance was maximum between 11.00 h and 17.00 h, and
decreased outside this time in well-watered plants, only data
between these times are used. When the rate of drying was such
that conductance started to fall outside these times, the
experiment was repeated.
To measure changes in ABA during the drying period, a
leaflet was detached from the most recently fully expanded leaf
at the start of the drying period, and sap was collected from its
petiolule while balancing pressure was monitored on the next
oldest leaf, for periods of 12 h, while the soil was wet,
(balancing pressures about 0.5 MPa), and at two times while it
was drying (balancing pressures about 1.0 and 1.4 MPa).
Another leaflet was sampled for ABA at the end of the drying
period.
ABA measurements in xylem sap and leaves
Xylem sap was collected from a number of different mutant
and grafted plants, growing under normal light levels in pots
designed to fit within a pressure bomb, as described above.
Plants were pressurized to bring the water potential of the
shoots to near atmospheric, so that the most recently fully
expanded leaf was on the point of bleeding. One leaflet was
then cut from this leaf, and sap was collected from its petiole,
while balancing pressure was monitored on the next oldest
leaf. Thus the sap was flowing at normal rates, through a
virtually-intact plant. This avoids the problems of collecting
sap exuding from stumps of plants with shoots removed, when
the low flow rate of the sap results in artificially high con-
centrations of solutes (Sagi et al., 1999). Leaf material was
collected as described earlier. Sap and leaflets were frozen in
liquid nitrogen and stored at
80 8C. ABA was assayed by an
indirect ELISA method (Walker-Simmons, 1987) using a mono-
clonal antibody (Idetek). All assays were made in triplicate and
possible interference with cross-reactive substances was checked
by analysing a range of dilutions. In some plants (wild type,
sitiens, and situwt), some of the sap was digested by alkaline
hydrolysis ( pH 11 for 1 h at 60 8C) (following the method of
Bano et al., 1993).
Leaf tissue was analysed for ABA by the ELISA method
after extraction with hot water (Loveys and van Dijk, 1988) and
passage through a C18 SepPak column. As a further check on
the specificity of the assay, and possible interference with cross-
reactive substances, replicate leaf samples were taken for
analysis by GCMS. Leaves were extracted in 80% methanol
with deuterated internal standard added, purified by filtration
and ether partitioning, then analysed by GCMS (Green et al.,
1997). This validated the results of the ELISA assay.
Results
ABA in xylem sap
The ABA concentration of the xylem sap from well-
watered sitiens plants was very low, and sometimes it
was undetectable (less than 0.1 nM; Table 1). Sap was
hydrolysed with alkali to see if there were conjugated
(esterified) forms of ABA present, but no significant
amount of ABA was released (data not shown). The level
of ABA increased with soil drying about 10-fold in both
wild type and sitiens, but the level in the mutant was
still low compared to the unstressed wild type (Table 1).
These data show that roots of sitiens do not export a
significant amount of ABA to the shoot.
Xylem sap was collected from reciprocal grafts of
wild-type and mutant plants, both sitiens and flacca,
grown under well-watered conditions. Self-grafted plants
(both mutant and wild type) were examined in case the
graft union interfered with the transport of ABA up or
down the stem, or produced ABA of its own. Self-grafted
sitiens (situsit) and flacca ( flcuflc) transported very little
ABA in xylem sap, and self-grafted wild-type (wtuwt) had
ABA levels similar to those found in intact wild-type
plants (compare Fig. 2 with Table 1). Reciprocal grafts
between wild type and mutant (either sitiens or flacca)
had xylem sap ABA concentrations that were several
times higher than the self-grafted mutants although much
less than self-grafted wild type (Fig. 2). The low amount
of ABA in the sap of plants with mutant shoots and
wild-type roots (situwt and flcuwt) had presumably arisen
Table 1. ABA concentrations in xylem sap collected from intact
transpiring plants of wild type and sitiens before and after the soil
was allowed to dry for 3 d
Leaf water potentials for the wild-type plants were about
0.2 MPa in
wet and
0.8 MPa in dry soil and for sitiens were
0.4 MPa in wet and

1.4 MPa in dry soil. Values are means"SE (n 4).
Xylem ABA (nM)
Well-watered Water-stressed
Wild type 38"15 242"40
sitiens 0.4"0.2 6"2

Fig. 2. ABA in xylem sap of grafted plants in well-watered conditions.
Data are the average of triplicate assays for separate plants. Bars show
standard errors (number of plants was 8, 8, 5, and 3 for the wtuwt, situsit,
flcuflc, and situwt graft types, respectively). Histograms without bars
indicate that only one plant was measured.
in the roots. However, the low amount of ABA in the
sap of plants with wild-type shoots and mutant roots
(wtusit and wtuflc) had presumably originated in the
shoots, as the roots of the situsit and flcuflc plants
exported little ABA (Fig. 2). This ABA could have been
produced by the stem or leaves below the site of collec-
tion, and transferred to the xylem either via the roots,
or by direct phloemxylem transfer in the stem above
the graft.
Reciprocal graft experiments
The stomatal behaviour of reciprocal grafts of sitiens
demonstrated that the plants responded to soil drying
according to their shoot genotype. In wet soil, there was
no statistically significant difference between the plants
with wild-type shoots (wtuwt and wtusit) in transpiration
rate (Fig. 3A) or stomatal conductance (Fig. 3B). Neither
was there any difference between the plants with mutant
shoots (situsit and situwt). However, there was the expected
difference between shoot genotype. In wet soil, plants
with sitiens shoots had almost twice the transpiration
rate and stomatal conductance of plants with wild-type
shoots, regardless of their root genotype (Fig. 3).
Stomatal closure began at about the same soil moisture
level (about 0.07 g cm
3) in all grafted plants (Fig. 3A, B).
At this stage, the plants with sitiens shoots were already
severely wilted, almost desiccated (as first noted by Neill
and Horgan, 1985). Prior to this stage, the conductance
actually increased; when the soil water content fell
below 0.15 g cm
3, the conductance increase to about
1200 mmol m
2 s
1, and remained elevated as the soil
water fell to about 0.06 g cm
3 (Fig. 3B). This occurrence
was most pronounced in the situsit plants.
Stomatal control in tomato 1507
Fig. 3. Response of sitiens reciprocal grafts to soil drying. Each point is
the mean ("SE) of five plants. (A) Transpiration (mmol m
2 s
1) in
relation to soil moisture. Plants began to wilt when the soil moisture
content fell below 0.07 g cm
3. (B) Midday stomatal conductance (mmol
m
2 s
1) in relation to soil moisture. (C) Foliar ABA concentrations
from leaf tissue collected at the beginning and end of the drying period.
The ABA concentration in the leaves of all the grafted
plants increased to different extents as the soil dried, with
the increase being greatest in the self-grafted wild-type
plants (Fig. 3C). Thus, there was little correlation
between bulk ABA in the leaves and stomatal closure.
Figure 3C also shows that although the ABA concentra-
tion in the leaves reflects most strongly the shoot geno-
type, there was some influence of the roots, as the level of
ABA in the wtuwt plants in well-watered soil at the
beginning of the experiment was greater than in the wtusit
plants, and the initial level of situwt plants was greater
than in situsit plants. As the soil dried, this pattern became
more pronounced. No water relations measurements
were made in this experiment.
The stomatal response of reciprocal grafts with flacca
produced similar results to those with sitiens. There were
no differences in transpiration rates between wtuwt and
wtuflc plants, both when the soil was well watered and as
the soil dried (Fig. 4A). Stomatal conductance was also
independent of root genotype (data not shown), and
1508 Holbrook et al.
Fig. 4. Response of flacca reciprocal grafts to soil drying. Each point is
the mean ("SE) of six plants. (A) Transpiration (mmol m
2 s
1) in
relation to soil moisture. Plants began to wilt when the soil water content
fell below 0.17 g cm
3. (B) Midday leaf water potential (MPa) in relation
to soil moisture.
declined in parallel with transpiration rates. There were
no differences in leaf water potential between the two
types of grafts (Fig. 4B), and little decrease as the soil
dried (Fig. 4B). The 4-fold decrease in transpiration as
the soil water content fell from 0.20 to 0.12 g cm
3 was
accompanied by a relatively small fall in leaf water
potential, about 0.2 MPa (Fig. 4B), indicating that the
stomatal closure was sufficient to prevent a significant
fall in leaf water status. This makes it unlikely that
stomates closed because of a leaf water deficit and
raises the possibility of regulatory signals coming from
the roots.
Split-root experiments
In the split-root experiment using the sitiens mutant,
water use per plant increased over the 6 d during which
each treatment was applied, as a result of the increase
in leaf area that occurred during the same interval. Total
plant water use increased to a lesser degree when water
was withheld from one portion of the root system,
compared with when the entire root system remained
well watered, but there was no difference if the sitiens
roots or the wild-type roots were not watered (Fig. 5A).
Stomatal conductance declined to about 80% of their
Fig. 5. Water use by sitiens split-root plants in response to water being
withheld from one side of the root system. (A) Mean change in
transpiration rate (% of initial rate) in relation to whether water was
withheld from the wild-type roots (wt dried), the sitiens roots (sit dried),
or neither (watered). Data are the mean"SE of six plants, averaged
over the course of the four experimental runs. (B) Stomatal conductance
in relation to whether water was withheld from the wild-type roots
(closed symbols) or sitiens (open symbols) roots. Data are the mean"SE
of three plants measured during the first experiment. (C) Soil moisture
content of the dried pots. Data are the mean"SE for plants over the
course of the four experimental runs (n 8).
initial values, again regardless of whether water was with-
held from the sitiens or the wild-type roots (Fig. 5B).
There was no significant difference in leaf water con-
tent when water was withheld from the wild-type or the
sitiens root system. Average leaf relative water content
("SE) for the three treatments (wild type dried, sitiens
dried, and both sides watered) were 91.6"0.6, 91.4"0.4,
and 92.7"0.1, respectively. After 4 d of drying, there
was little water uptake from the dry side (Fig. 5C), so
water was taken up almost exclusively from the wet side.
The sitiens roots were not able to extract water at the
same rate as the wild-type roots (Fig. 5C), nor to the same
final extent. For example, the amount of water used by
the wild-type root system during the first day after
cessation of watering was always about 40% greater than
that used by the sitiens root system. After 45 d, the soil


3
water content of the wild-type side reached 0.053 g cm ,
while the sitiens side reached a minimum of only
0.063 g cm
3 (Fig. 5C). Yet the sitiens root systems were
much bigger than the wild-type root systems; the ratio
of wild type to sitiens being 0.4 : 0.6 (Table 2). This meant
that the rate of water uptake per mass of root was
much greater for the wild-type than the sitiens roots. For
example, during the first day after cessation of watering,
the amount of water taken up by wild-type root systems
was about double that from the sitiens root systems.
The split-root experiment performed with flacca
gave qualitatively similar results. Partial drying of the
root system resulted in a decrease in stomatal conduct-
ance that was independent of the genotype of the roots in
the drying soil (Fig. 6). There was no difference in the
Table 2. Biomass and leaf area of split-root grafted plants, with
wild-type shoots grafted onto two root systems of wild type and
sitiens
Plants were harvested at end of the series of experimental treatments
shown in Fig. 5, in which either one or both systems were watered.
Values are means"SE (n 6).
Leaf area (cm2) 1539"55
DW of leaves (g) 7.8"0.3
DW of wild-type root system (g) 1.69"0.10
DW of sitiens root system (g) 2.47"0.05
Fig. 6. Stomatal conductance of flacca split-root plants in response to
water being withheld from one side of the root system. (A) Split-root
plants with different root systems; water was withheld from wild-type
roots (closed symbols) or flacca roots (open symbols). (B) Split-root
plants with the same root system, either wild type (closed symbols) or
flacca (open symbols). Each point represents the mean"SE of all leaves
measured on a single plant.
Stomatal control in tomato 1509
response of stomatal conductance when water was with-
held from the sitiens roots or the wild-type roots (Fig. 6A).
There were no differences in midday leaf water poten-
tial between plants (average 0.70"0.04 MPa) and no
decreases when water was withheld from one side of the
split-root system (data not shown). As with the sitiens
split-root plants, wild-type roots were better able to
extract water from the soil compared with the flacca
roots. In the plants with different roots, the minimum soil
water content in the side with the wild-type roots was
0.13 g cm
3, compared with 0.17 g cm
3 in the side with
flacca roots (Fig. 6A). However, without competition
with wild-type roots, flacca roots were able to extract
more water; in the plants with all roots of the same
genotype, the minimum soil water content resulting from
uptake by flacca roots was 0.13 g cm
3, almost as low as
that from wild-type roots (Fig. 6B).
Root pressurization experiments
Root pressurization, which was used to maintain a high
shoot water status during soil drying, did not alter
stomatal apertures when the soil was moist. When well-
watered plants were first brought to balancing pressure
(i.e. the pressure was increased such that xylem sap was
visible at the end of a recently cut petiolule) there was no
measurable change in stomatal aperture. Stomatal closure
in response to soil drying occurred in all plants, irrespect-
ive of root genotype or application of pressure to the
roots. In wtuwt plants, stomata began to close when
the soil water content dropped below 0.095 g cm
3, and
the balancing pressure was 0.81.0 MPa (Fig. 7A).
Stomatal closure of 50% occurred at 1.8 MPa, at which
point the experiment was terminated as the pressure
was approaching the pressure limit of the chamber
(2 MPa). The plant was re-watered, and some days later
was allowed to dry again, this time without the applica-
tion of balancing pressure. The relationship between
conductance and soil moisture was similar to when
it was pressurized, i.e. closure started when the soil
moisture fell to the same level (Fig. 7A). Two other
replicate plants had the same response (data not shown).
Similar patterns of stomatal closure in response to
soil drying were observed with wtusit plants. Stomatal
closure started at balancing pressures of 0.81.0 MPa
and a soil moisture content of about 0.08 g cm
3, with
closure of about 50% occurring at 1.8 MPa (Fig. 8A).
When the plant was allowed to dry without balancing
pressure, stomatal closure began when soil moisture con-
tent fell to the same extent (Fig. 8B). The same result was
found with three other replicate (wtusit) plants (data not
shown).
ABA content of xylem sap could only be determined
for plants maintained at balancing pressure as positive
xylem pressures are needed to produce exudation from
1510 Holbrook et al.

Fig. 7. Soil drying of grafted plants having wild-type roots (wtuwt). (A)
Stomatal conductance (closed circles) with balancing pressure (open
circles) to maintain leaf water status as the soil dried. (B) Stomatal
conductance of the same plant without balancing pressure. Each point
represents the mean"SE of all leaves measured on a single plant.
Fig. 8. Soil drying of grafted plants having sitiens roots (wtusit). (A)
Stomatal conductance (closed circles) with balancing pressure (open
circles) to maintain leaf water status as the soil dried. (B) Stomatal
conductance of the same plant without balancing pressure. Each point
represents the mean"SE of all leaves measured on a single plant.

transpiring plants. At the start of the drying period,

xylem sap ABA concentration in wtusit plants was a little
lower than that of wtuwt plants (Fig. 9). With soil drying,
there was a large increase in the ABA concentration of
xylem sap of the wtuwt plants, but very little increase in
the wtusit plants (Fig. 9). ABA content of leaf tissues
was also lower in the wtusit than the wtuwt plants at the
start of the drying period, being 3.20"0.24 nmol g
1 FW
for the wtuwt plants and 2.32"0.08 nmol g
1 FW for
the wtusit plants. However, the ABA concentration in
the leaves increased as the soil dried in both types of
plants to a similar extent, the increase being 0.52"0.30
and 0.60"0.06 nmol g
1 FW respectively (n 3).
Fig. 9. Xylem ABA concentrations during soil drying of grafted plants
Discussion having wild-type roots (wtuwt) or sitiens roots (wtusit). Plants were
maintained under balancing pressure as the soil dried to maintain leaf
Stomatal closure in drying soil does not require water status and thus to allow sap to be collected. Curves are quadratic
regressions. Data are for three wtuwt and six wtusit plants.
root-sourced ABA
The experiments with reciprocal grafts between ABA- from a wild-type sunflower and a wilty ABA-deficient
deficient and wild-type shoots and roots showed that mutant (Fambrini et al., 1995); they reported that the
the stomatal behaviour depended on the genotype of stomatal conductance during water stress was determined
shoot, not the root. Stomatal conductance was higher by the shoot rather than the root genotype. They are not
when the shoot genotype was either sitiens or flacca, inconsistent with those of Jones et al. with reciprocal
regardless of the root genotype, and decreased as the soil grafts of wild-type tomato and sitiens or flacca, measured
dried regardless of the root genotype. These results are in well-watered conditions (Jones et al., 1987), who
similar to those of Fambrini et al. with reciprocal grafts concluded that there was only a small and generally

insignificant effect of the root genotype after grafting
either wild-type or mutant shoots onto alternate root-
stocks. They are different from those of Borel et al. with
tobacco (Borel et al., 2001), who reported similar
stomatal responses of ABA-deficient and wild-type shoots
on a common wild-type rootstock, but in that case there
may have been ABA circulating from the wild-type shoot
to the mutant shoot, causing a reversion of the
phenotype. Phenotype reversion of ABA-deficient shoots
on wild-type roots was reported with grafts of tomato
(Cornish and Zeevaart, 1988) and of sunflower (Fambrini
et al., 1995).
Leaf ABA was not closely linked to stomatal
behaviour. Although leaf ABA was lower when the shoot
genotype was sitiens rather than wild type, and thus
correlated with stomatal conductance in unstressed
plants, it increased much more with soil drying in the
wtuwt than the wtusit plants, yet stomates of these plants
closed to the same extent (Fig. 3). As the roots of the
mutants did not export significant amounts of ABA in
the xylem, something other than root-produced ABA
was causing stomatal closure. In the experiments with
reciprocal grafts, shoot water potential was not con-
trolled, so it was possible that reduced leaf water status
influenced stomatal conductance, either directly, or by
inducing ABA accumulation. The succeeding two types
of experiments, split-root and root pressurization, were
designed to examine the effects of soil drying without
changes in shoot water status. In both types of experi-
ment, the plants had wild-type shoots and varied only in
root genotype. This avoided the morphological abnormal-
ities in leaf and stomatal architecture due to a mutant
shoot phenotype (as well as the problem of reversion of
shoot morphology of situwt grafts).
In the split-root experiments, patterns of stomatal
conductance (sitiens and flacca experiments) or transpira-
tion (sitiens experiment) were the same when the ABA-
deficient or the wild-type root system was allowed to dry,
that is, there was a small degree of stomatal closure,
independent of the root genotype. Compared to some
other split-root experiments (Jones et al., 1987; Gowing
et al., 1990; Khalil and Grace, 1993; Stoll et al., 2000), this
closure was small, but it was discernible, unlike the split-
root experiment of Saab and Sharp with maize, which
showed no closure at all, just a reduction in leaf
expansion (Saab and Sharp, 1989). In the present study,
while the soil dried around one root system, there was
increasing uptake from the watered roots until, by the
end of the experiment, all of the water transpired was
withdrawn from the watered root system. To maintain
the watered pot at its target soil water content, water
was added three times per day. Perhaps the different
degrees of stomatal closure in the various split-root
experiments reported in the literature reflect the frequency
with which the wet pot was watered.
Stomatal control in tomato 1511
It was surprising that the root system of sitiens was
larger than the wild-type root system in the split-root
grafted plants (Table 2), as roots of ABA-deficient maize
seedlings grow more slowly than those of the wild type
under water stress (Saab et al., 1990; Sharp et al., 1994).
Furthermore, addition of ABA can largely restore root
and shoot growth in flacca in unstressed conditions
(Sharp et al., 2000), and presumably would also in
stressed conditions. The greater growth of sitiens roots
in the grafted plants described here was possibly due to
signals coming from the wild-type shoots. However,
the hydraulic conductance of the sitiens root system
appeared to be impaired, the maximum rate of water
transport per unit mass of root being about half that
of wild-type roots. The inability to produce ABA was
therefore still having an effect on architecture, which is
consistent with suggestions that ABA enhances hydraulic
conductance in roots (Zhang et al., 1995). Nevertheless,
despite the greater root system, the actual rate of water
uptake from the pot with the sitiens roots was less than
from the wild type. The greater mass of the sitiens root
system did not compensate for the lower hydraulic
conductance.
In the experiments run under balancing pressure,
stomatal conductance decreased as the soil dried regard-
less of the root genotype (stomatal behaviour was the
same in wtusit and wtuwt). This showed there was a signal
coming from the roots that influenced stomatal con-
ductance independently of leaf water status and, at least
in the case of wtusit, that this signal was not ABA
produced by the roots. What is the signal?
Signals that could be produced by ABA-deficient roots
It is possible that roots of the ABA-deficient mutants
are able to export precursors of ABA (such as the
ABA-adduct identified by Netting et al., 1997) or other
precursors that would presumably accumulate in
mutants blocked, like sitiens and flacca, at the final step
of ABA synthesis (as found in aba3; Schwartz et al.,
1997). These would not have been detected by the assay
system used here for ABA. Other products of the ABA
biosynthetic pathway may accumulate in ABA-deficient
mutants. For instance, t-xanthoxin accumulates in
unstressed sitiens, and increased in water-stressed flacca
to a similar extent as ABA in the wild type (Parry et al.,
1988). Whether t-xanthoxin carries biological activity
is unknown.
Other types of hormonal stress signals are also
possible. Cytokinin concentrations are often lower in
xylem sap from droughted plants (Bano et al., 1993, 1994;
Shashidhar et al., 1996; Stoll et al., 2000), and can
influence stomatal opening (Bradford, 1983). Spraying
grapevines exposed to partial root water deficit with
cytokinin restored stomatal conductance to that of fully
1512 Holbrook et al.
irrigated controls (Stoll et al., 2000). Other compounds in
xylem sap may influence stomatal conductance, for
example, an unidentified inhibitor of transpiration was
detected in xylem sap of wheat seedlings (Munns et al.,
1993).
Changes in pH in xylem sap occur in droughted
plants, and there is some evidence for these being part
of a root signalling system. Changes in pH could be
caused by changes in ion transport induced by the lower
water status of the roots as the soil dries. A decrease in
cell volume or turgor of root cells could activate mechano-
sensitive Ca2q channels which could have profound
effects on ion transport systems such as KqHq symport
(Netting, 2000). This could affect the ion relations of
the apoplast of the root and hence the pH of xylem sap
(Gerendas and Schurr, 1999). Increases in the pH of the
xylem sap were found in sunflower (Gollan et al., 1992),
tomato (Wilkinson et al., 1998) and grapevine (Stoll et al.,
2000), although decreases were found in Ricinus (Schurr
and Schulze, 1996). If the xylem flowed unmodified to the
guard cells, the pH would affect apoplastic ABA levels
in guard cells via carrier-mediated transport, without
a change in the bulk leaf ABA (Wilkinson and Davies,
1997; Hartung et al., 1998).
Regardless of the nature of the signal coming from
roots, the fact remains that ABA levels rose in leaves as
the soil dried in both wtuwt and wtusit plants, i.e. in
plants whose roots could not produce ABA. It is possible
that an increase in xylem pH caused an increase in ABA
synthesis in leaves, or reduced its catabolism (Wilkinson
and Davies, 1997). An increase in pH could release ABA
from a precursor pool within the leaf (Netting, 2000), or
trigger release of ABA from inactive ABA-conjugates via
an extracellular b-glucosidase which can release ABA
from glucose esters in barley leaves (Dietz et al., 2000).
ABA circulation in grafted plants
Although the roots of the wtusit plants cannot have
produced ABA (situsit plants did not export any ABA in
the xylem), there was a significant amount of ABA in the
xylem sap. The source of this ABA in the xylem of the
wtusit plants must have been the leaves. The ABA could
enter the xylem either from the stem below the point of
collection and above the graft junction by direct phloem
xylem transfer, or after recirculation via the roots. ABA is
readily mobile in phloem sap, and has been found to
increase several-fold under water stress (Hoad, 1973) or
salinity (Wolf et al., 1990; Jeschke et al., 1997). Evidence
that leaf-derived ABA is exported to roots in the phloem
and recirculates in the xylem comes from experiments
with labelled ABA, showing that ABA synthesized in
leaves appeared later in roots and xylem sap (Hoad, 1975;
Loveys, 1984). In lupin, it was calculated that only 70%
of the ABA in the xylem had been synthesized by the
roots, the remaining part being recirculated from the
leaves, and that in salt-treated plants the proportion was
even lower at 55% (Wolf et al., 1990). Furthermore,
defoliation and girdling experiments have indicated that
leaves can contribute a substantial proportion of the ABA
moving in the xylem (Liang et al., 1997; Neales and
McLeod, 1991). Alternatively, the ABA in the xylem from
the wtusit plants may have not arrived via the roots. The
sap was collected from the most recently expanded leaf,
inserted into the stem some 5 cm above the graft. The
ABA may have been made within the stem above the
graft, or made by the older leaves and transferred into
the xylem within the stem internodal tissue.
The fate of the ABA in the xylem, whatever its origins,
is unclear. Calculations of the amount of ABA in the
xylem sap of the wtuwt plants that was delivered to leaves
throughout the drying period showed that this exceeded
the actual increase in the leaf by about 6-fold (values
derived from the data for the last experiment presented
above, given in the text and in Fig. 9, and assuming a
transpiration rate of 6 g g
1 FW of leaves d
1). This
indicates that there was considerable metabolism of the
incoming ABA. Earlier studies showed that the mesophyll
tissue rapidly metabolized ABA fed through the xylem,
limiting the supply to the epidermis (Trejo et al., 1993).
ABA fed through the xylem of detached leaves had a
half-life less than 1 h (Gowing et al., 1993; Zhang
et al., 1997a), although in intact leaves the half-life was
a little longer, 2.2 h in well-watered maize (Zhang et al.,
1997b) and increased under water stress to 3.6 h (Jia
and Zhang, 1997). It is possible that the control of
stomatal conductance by ABA is exerted by the metabolic
activity in the leaves, not by the amount arriving in
the xylem.
Conclusion
Overall, these experiments show that, in tomato, as in
other species, root signals operate to control stomatal
conductance in drying soil. However, these data show
that this closure was not related to xylem ABA con-
centrations, nor to the ability of roots to produce ABA,
and indicate that stomates could be responding to ABA
that is made in situ rather than transported from the roots
via the xylem. The signal from the roots that causes this
increase in ABA, or otherwise influences stomatal
conductance, remains unknown, but may be a precursor,
or related to pH changes that have been observed in
xylem sap.
Acknowledgements
We thank Andrew Poole for verification of leaf ABA by GCMS,
and Andy Netting, Ian Dodd, John Passioura, Rod King, and
Brian Loveys for helpful comments on the manuscript.

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