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PCEPlant, Cell and Environment0016-8025Blackwell Science Ltd 2002
252February 2002
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ABA-based chemical signalling
S. Wilkinson & W. J. Davies
10.1046/j.0016-8025.2001.00824.x
Original ArticleBEES SGML
Plant, Cell and Environment (2002) 25, 195–210
ABA-based chemical signalling: the co-ordination of
responses to stress in plants
S. WILKINSON & W. J. DAVIES
The Lancaster Environment Centre, Lancaster University, Bailrigg, Lancaster, LA1 4YQ, UK
ABSTRACT
There is now strong evidence that the plant hormone abscisic acid (ABA) plays an important role in the regulation of
stomatal behaviour and gas exchange of droughted plants.
This regulation involves both long-distance transport and
modulation of ABA concentration at the guard cells, as well
as differential responses of the guard cells to a given dose of
the hormone. We will describe how a plant can use the
ABA signalling mechanism and other chemical signals to
adjust the amount of water that it loses through its stomata
in response to changes in both the rhizospheric and the
aerial environment. The following components of the signalling process can play an important part in regulation: (a)
ABA sequestration in the root; (b) ABA synthesis versus
catabolism in the root; (c) the efficiency of ABA transfer
across the root and into the xylem; (d) the exchange of
ABA between the xylem lumen and the xylem parenchyma
in the shoot; (e) the amount of ABA in the leaf symplastic
reservoir and the efficiency of ABA sequestration and
release from this compartment as regulated by factors such
as root and leaf-sourced changes in pH; (f) cleavage of
ABA from ABA conjugates in the leaf apoplast; (g) transfer of ABA from the leaf into the phloem; (h) the sensitivity
of the guard cells to the [ABA] that finally reaches them;
and lastly (i) the possible interaction between nitrate stress
and the ABA signal.
Key-words: abscisic acid (ABA), apoplast, calcium, drought
stress, drying soil, leaf, long-distance signalling, low
temperature, nitrate, pH, PPFD, root, stomatal guard cells,
symplast, VPD, xylem.
ABA AS A STRESS RESPONSE HORMONE
It has long been known that in order to conserve the water,
nutrients and carbohydrates required for survival, plants
respond to stresses such as soil drying by reducing leaf
expansion and closing stomatal pores. In addition, root
growth rates may be maintained in order that the plant may
continue to access water. Traditional explanations for
drought-induced regulation of gas exchange and leaf
growth have emphasized the importance of the decline in
shoot water status, which commonly accompanies severe
Correspondence: Sally Wilkinson. Fax: +44 01524 843854;
E-mail: [email protected]
© 2002 Blackwell Science Ltd
soil drying (e.g. Kramer 1969). It is now accepted, however,
that many of the plant’s responses to soil drying can occur
in the absence of changes in shoot water status, via chemical
signals. In one very clever series of experiments, pressure
has been applied to roots to counteract the increasing soil
suction that occurs as soil dries. This generated shoot water
relations in droughted plants that were comparable to those
of well-watered plants. Despite this, rates of gas exchange
and leaf growth were still restricted compared to rates
shown by well-watered plants (e.g. Gollan, Schurr &
Schulze 1992).
Another apparent demonstration of root-sourced chemical signalling lies in the work of Gowing, Davies & Jones
(1990). Here, the roots of young apple trees were split
between two containers, the soil in one of which was
allowed to dry, while the other container was irrigated as
normal. This treatment restricted the rate of leaf growth,
which could be restored to control rates by removing the
roots in contact with the drying soil, i.e. by removing the
source of the chemical signal. Blackman & Davies (1985)
used the same technique to demonstrate that chemical signals sent from drying soil could close the stomata of wheat
leaves.
It is important to note, however, that several groups
have used the root pressure vessel technique to restore the
shoot water relations of a range of species growing in dry
soil, and have demonstrated that stomata re-open (e.g.
Saliendra, Sperry & Comstock 1995; Fuchs & Livingstone
1996; Comstock & Mencuccini 1998). These results indicate
that drought-induced limitations in stomatal opening in
these species are mostly hydraulic, suggesting that the relative importance of hydraulic versus chemical signalling
may differ between species. It might be surprising if this
were not the case, but we should exercise care in using the
root pressurization technique to differentiate between
chemical and hydraulic signalling mechanisms, as pressurization of plant parts is known to change the pH of different
compartments of the shoot and root (Hartung, Radin &
Hendrix 1988). We will see below the possible significance
of such a change on the modification of the signalling process, which raises the possibility that root pressurization can
minimize both hydraulic and chemical limitation of stomatal behaviour simultaneously.
It has long been apparent that the major plant growth
regulating hormone ABA (see Addicott & Carns 1983)
strongly promotes stomatal closure (Jones & Mansfield
1970), and can often inhibit shoot growth. The synthesis of
ABA is stimulated by the dehydration of plant cells
195
196 S. Wilkinson & W. J. Davies
(Wright 1977) including root cells. Leaf cells also synthesize
ABA (Cutler & Krochko 1999) and leaf dehydration
caused by severe soil water shortages massively increases
bulk leaf [ABA], which often correlates well with stomatal
closure. If we are to argue that ABA acts as a long-distance
chemical signal which can provide information on water
availability in the soil, it is important to be able to differentiate between such shoot-water deficit-induced increases
in leaf ABA, and those that arise due to ABA import from
the root. Zhang & Tardieu (1996) showed that all tissues of
maize roots, regardless of their age and position, could synthesize ABA when dried. Much research has now shown
that the amount of ABA in the xylem sap can increase substantially as a function of reduced soil water availability,
and that this increased delivery to shoots can increase ABA
concentrations in different compartments of the leaf (e.g.
Loveys 1984; Zhang & Davies 1989). In order to ascribe an
important stress detection function to the roots we need to
show that the delivery of ABA by the xylem is sufficient to
bring about the observed degree of stomatal closure seen in
response to soil drying. This is only sometimes the case, but
for reasons that will be explained below we can still argue
that ABA plays an important regulating role in the
droughted plant in response to perturbations at the root,
even when xylem ABA concentrations are not increased at
all.
Increases in the ABA concentration in the leaf in
response to perturbations around the root are now well
documented (see Davies & Zhang 1991). The xylem vessels
give up their contents (including their ABA) to the leaf
apoplast (Weyers & Hillman 1979), thereby increasing the
concentration of the hormone in this compartment. The
ABA is carried with the transpiration stream inside the leaf
around and/or through the mesophyll cells (see below) so
that it reaches the target cells (the stomatal guard cells) in
the epidermis, which contain ABA receptors with external
(and possibly internal) loci (see below) in their plasma
membranes (pm). Once bound, the hormone then induces
an internal signal transduction cascade usually involving
increases in both externally and internally sourced cytoplasmic calcium, which eventually reduces guard cell
osmotic potential via loss of K+ and Cl– to cause stomatal
closure (for reviews see McAinsh, Brownlee & Hetherington 1997; Assmann & Shimazaki 1999). Although the ABA
receptor has yet to be unambiguously identified in plants
(Assmann & Shimazaki 1999) there is much evidence in the
literature for an apoplastic locus of ABA perception, indicating that the apoplastic fraction of ABA will be the major
determinant of stomatal aperture (Hartung 1983; Hornberg
& Weiler 1984). Anderson, Ward & Schroeder (1994) demonstrated a failure of direct microinjection of ABA into the
guard cells of Commelina communis L. to cause closure
(but see Allan et al. 1994 and Schwartz et al. 1994). In addition, external ABA has been found to stimulate inward
Ca2+ conductance and to induce elements of the IP3 signalling cascade inside guard cells that leads to the release of
Ca2+ from internal stores (see MacRobbie 1997). Zhang &
Outlaw (2001a) found that stressing Vicia faba L. roots
could change the ABA concentration at the guard cell apoplast (2·3 µM) in the absence of bulk apoplastic changes in
[ABA] (185 nM), and that the apoplastic guard cell ABA
fraction correlated with changes in stomatal aperture more
effectively than the guard cell symplastic fraction. Even
more recently, ABA supplied via the xylem to non-stressed
V. faba was seen to accumulate at the guard cell apoplast
without a concomitant increase in symplastic guard cell
ABA, and this correlated with stomatal closure in the
absence of any other stress-induced signals (Zhang & Outlaw 2001b). These data show that apoplastically-facing
guard cell ABA receptors seem to be important in the
responses to very subtle ‘stress’ signals experienced by
plants. Such signalling mechanisms may not be important
when shoot water stress develops. Harris et al. (1988) found
that when severe leaf water deficit develops, most ABA
accumulated in the guard cell symplast, and it may be that
ABA receptors with an internal locus (Allan et al. 1994;
Schwartz et al. 1994) only operate when shoot water relations are also affected.
MODIFICATION OF THE XYLEM
ABA SIGNAL
Although we can accept that xylem ABA will modify leaf
functioning, it is clearly not just a simple matter of the
[ABA] in the xylem being that perceived by the target cells,
given that the ABA concentration in apoplastic microsites
around the guard cells is that to which stomata respond. In
fact, there is often great variation in the apparent sensitivity
of leaf conductance to a given concentration of ABA in the
xylem stream (e.g. Correia & Pereira 1995). The reasons for
these findings begin to become clear in the work of Trejo
and colleagues (Trejo, Davies & Ruiz 1993; Trejo, Clephan
& Davies 1995). Their data confirmed early work by Incoll
& Jewer (1987a, b) that stomata in isolated epidermis were
often sensitive to concentrations of ABA as low as 10−9 M,
much lower than that found in the xylem sap even of wellwatered plants. If the stomata were directly exposed to the
concentrations of ABA normally seen in well-watered
xylem sap (usually between 1·0 and 50 nM, e.g. Schurr, Gollan & Schulze 1992) they would be permanently closed. So
the cells of the leaf must ‘filter out’ some of the ABA arriving in the transpiration stream before it reaches the stomata
in the epidermis. By inhibiting the catabolic degradation of
ABA in the mesophyll, Trejo et al. (1993) found that the
amount of ABA reaching the epidermis was increased, and
that the concentration of ABA supplied in the xylem
exerted an increased effect on stomatal aperture. In other
words the mesophyll normally removes (by metabolism
and sequestration) much of the ABA passing through it in
the transpiration stream before it reaches the guard cells.
This would also explain why the same authors noted that
there was a linear relationship between the [ABA] in the
epidermis and the stomatal response, but a very poor relationship between bulk leaf ABA and stomatal aperture.
Daeter & Hartung (1995) demonstrated that the epidermal
© 2002 Blackwell Science Ltd, Plant, Cell and Environment, 25, 195–210
ABA-based chemical signalling 197
Figure 1. The effect of temperature on the [ABA] in the abaxial
epidermis and in the total leaf of Commelina communis L. when
detached leaves were incubated under a PPFD of
350 µmol m−2 s−1 in the presence or absence of 1 µM ABA for
3 h. Results are means of five separate experiments at each
temperature (± SE), and in each experiment the epidermis (and
the remaining tissue) from 15 leaves was bulked for ABA analysis
by radioimmunoassay. From Wilkinson et al. (2001).
symplast is an even better sink for apoplastic ABA than the
mesophyll, because it has a catabolic rate that is five-fold
greater. So once the ABA has reached the epidermis, there
is still potential for ABA to be removed from its site of
action (the guard cell apoplast). The importance of the contributions of ABA sequestration and catabolism in the symplast is made clear by the work of Kefu, Munns and King
(1991). These authors calculated that the amount of ABA
carried in the transpiration stream of cotton plants each day
is nine times in excess of the amount of ABA actually
detected in the leaves at the end of the day.
In the past it has been argued (e.g. Jackson 1993) that as
the transpirational flux increases, for example with increasing VPD or temperature, the delivery of ABA to the leaf
will increase, and that stomata will respond to the total flux
of ABA into the leaf rather than to the concentration in the
xylem stream. However, the fact that the leaf has a large
reservoir (the mesophyll cells) in which to sequester and/or
catabolize ABA entering via the xylem could explain why
several lines of evidence have failed to support this theory.
For example, Trejo et al. (1995) found that increases in VPD
and temperature (19·9–36·1 °C) increased transpirational
water loss from Phaseolus acutifolius L. leaves, but did not
affect stomatal aperture. This was despite an increased
delivery of ABA, as evidenced by greater than two-fold
increases in bulk leaf [ABA]. Presumably the leaf symplastic reservoir for ABA sequestration is large, and only when
© 2002 Blackwell Science Ltd, Plant, Cell and Environment, 25, 195–210
the reservoir is full and/or catabolic enzyme activity is saturated, will increases in flux increase the [ABA] seen at the
guard cells (see below). Some related work was carried out
by Wilkinson, Clephan & Davies (2001). This showed that
the [ABA] penetrating to the abaxial epidermis of detached
C. communis leaves supplied with a concentration as high
as 10−6 M via the transpiration stream, was kept uniformly
low as the temperature was increased from 11 to 28 °C. As
temperature, transpiration flux and ABA flux into the leaf
increased, the rest of the leaf acted as a reservoir storing the
extra ABA entering the leaf tissues and keeping it away
from the epidermis (Fig. 1). The change in ABA flux with
temperature (except between 7 and 11 °C) failed to change
the epidermal [ABA].
Movements of ABA between the xylem and the leaf
symplastic reservoir provide a powerful point at which the
ABA stress signal response can potentially be modulated.
Xylem sap/apoplastic sap pH changes are one way in which
accessibility to the mesophyll (and epidermal) reservoir can
be controlled. Evidence that xylem and apoplastic sap pH
can increase in response to soil drying has been reviewed by
Wilkinson (1999). It is important to note that sap pH
changes can occur even when the shoot water status of
plants in drying soil is maintained using the root pressure
vessel (Schurr et al. 1992) or under partial root drying
(PRD, Wilkinson & Davies, unpublished; Stoll, Loveys &
Dry 2000). Schurr et al. (1992) found that correlations
between xylem sap ABA from droughted sunflowers and
reductions in stomatal conductance were poor, but when
sap pH or sap [nitrate] or sap [calcium] were also taken into
account the correlation was improved (Gollan et al. 1992).
The authors proposed that increasing xylem sap pH
reduced the ability of the leaf symplastic compartment to
sequester ABA, so that during drought more of the ABA
that enters the leaf will ultimately reach the guard cells in
the epidermis. This proposal was based on very detailed
information available in the literature, demonstrating that
the extent of ABA uptake by all leaf cell types is reduced
when the pH outside the cell is increased (e.g. Heilmann,
Hartung & Gimmler 1980; Kaiser & Hartung 1981). Models
were generated by this group, based on measurements of
ABA uptake in response to pH in isolated cells and tissues.
These predicted that the drought-induced pH increases
detected in vivo would be enough to cause stomatal closure
in an intact leaf, by preferentially concentrating the ABA
that already exists in the leaf into the apoplastic as opposed
to the symplastic compartment (Slovik & Hartung 1992a,
b). It was not even necessary to incorporate an increase in
xylem-sourced [ABA] into the model for stomatal closure
to occur. These predictions have since been substantiated to
some extent by Wilkinson & Davies (1997) and Wilkinson
et al. (1998) in C. communis and tomato. We were able to
conclude that as well as potentially magnifying an effect of
xylem ABA on stomatal closure during drought, a droughtinduced increase in xylem sap pH alone could constitute a
root-sourced signal to the leaf, inducing stomatal closure. In
essence, well-watered xylem sap has a pH around 6·0 and
sap from droughted plants has a more alkaline pH of
198 S. Wilkinson & W. J. Davies
around 7·0 (see Wilkinson 1999). Artificial xylem sap buffered to pH 7·0 supplied to whole detached C. communis or
tomato leaves via the xylem stream reduced stomatal aperture in comparison to controls supplied with pH 6·0 buffer.
The reduction of transpirational water loss by high pH in
this system was found to require absolutely the presence of
a very low ‘well-watered concentration’ of ABA (c. 10−8 M)
in the xylem stream, a concentration that does not affect
stomatal aperture at control pH. The transpiration rate of
the ABA-deficient tomato mutant flacca could not be
reduced by increasing xylem sap pH up to 7·75 (highest
tested), but the inclusion of a ‘well-watered’ concentration
of ABA in the artificial sap buffer restored the pH response
seen in the wild-type. It was concluded that the increased
pH caused stomatal closure by allowing more of the ABA
that enters the leaf to penetrate to the stomatal guard cells
as a result of a pH-induced reduction in sequestration into
the symplastic reservoir as described above.
We now know that soil drying-induced modulation of
ABA sequestration into the leaf symplast via xylemsourced pH changes is only one example out of many
whereby the ‘basic’ ABA signal can be influenced by the
environment around the plant. Further examples are given
below.
Whole-plant modulation of the ABA signal
The root
ABA may gain entry to plant roots in the following ways: it
can be synthesized in the root (or released from conjugated
forms), it can be taken up from soil water surrounding the
root, or it can be delivered to the root from the shoots by
the phloem. Once inside the root the ABA can be taken up
by the symplast and stored or degraded, or it may be transferred from cell to cell towards the xylem vessels, or carried
apoplastically with the transpiration stream towards the
xylem. Alternatively, ABA may be lost from the root by diffusion into the soil water (rhizosphere). If root ABA is not
degraded, stored in the symplast or lost into the soil water
it is transferred into the xylem vessels for transport to the
leaf. ABA loading into the xylem can either occur directly
from the apoplast, or indirectly via specialized xylem
parenchyma cells found along the length of the root xylem.
There are several points along this root ABA uptake and
transport pathway that can be influenced by the environment such that the strength of the root-sourced ABA signal
in the xylem can be enhanced or reduced. These are
described below.
Root cells constitutively synthesize a low ‘well-watered’
amount of ABA, and as described above this rate of synthesis is massively increased by root tissue dehydration,
classically associated with soil drying. However the strength
of this ‘basic’ ABA signal sent from roots to shoots can be
modified in other less obvious ways. Firstly the soil around
the roots may be influential, not only as regards the direct
effects of its moisture or nutrient content (see last section).
Hartung et al. (1996) suggested that when rhizospheric
ABA is high (for example at low soil water contents) the
outwardly directed concentration gradient for ABA
between the root and the soil could be reduced. This makes
it less likely that root ABA will diffuse out of the plant,
potentially strengthening the xylem ABA signal further
along the transpiration stream. In addition, different soil
types are characterized by different pH values, and as
described above ABA tends to accumulate in alkaline compartments as a result of the anion trapping effect (see below
and Wilkinson 1999). Degenhardt et al. (2000) have shown
that in alkaline soil substrates considerable portions of the
ABA synthesized in the roots can be leached out into the
soil solution, although species with an exodermis (e.g. Zea
mays) were less susceptible to this type of stress. In this way
alkaline soil could weaken the root-sourced ABA signal.
More usually though, rhizospheric ABA is transported
into the root with the soil water. Water flow into and across
the root (see Steudle 2000) is a result of the tension created
by the transpiration stream (in which case water flows apoplastically), or of the decreased osmotic potential of the
xylem sap in the absence of transpiration from the leaf (in
which case water flows from cell to cell). Anything that
increases water flux across the root should promote the
transfer of ABA across this organ towards the xylem,
because the uptake and radial transport of ABA occurs
alongside that of water (Freundl, Steudle & Hartung 2000).
However, until recently this prediction could not have been
made, as it was believed that radial transport of ABA across
roots was entirely trans-cellular due to the perceived impermeability of the exo- and endodermis. This would have
meant that increased flux of apoplastic water into the xylem
upon increased transpiration would dilute the xylem
[ABA] (e.g. Else et al. 1994). However, Freundl, Steudle &
Hartung (1998, 2000) found that filtration of ABA from the
apoplastic flow of water as it crossed the endodermis built
its local concentration up high enough to cause ‘solvent
drag’ through this potential barrier into the xylem. This is
has been termed the ‘apoplastic by-pass’ for ABA.
Different species transport root water to different
extents within each pathway (symplastic or apoplastic), and
when the symplastic route is necessarily dominant (e.g. in
heavily suberized roots), root cell plasma membranes contain large concentrations of water channels or ‘aquapoprins’ which can be actively regulated. Interestingly, ABA
itself can increase the flow of water into and through the
root, otherwise known as its hydraulic conductivity (Hose,
Steudle & Hartung 2000). This is believed to result from
ABA-induced opening of these inwardly directed water
channels (Tyerman et al. 1999; Netting 2000). Soil drying
and salinity have been found to increase root hydraulic conductivity, and the induction of ABA biosynthesis in the root
by these stresses may effectively maximize the transport of
the newly synthesized ABA within the root towards the
xylem (positive feedback). As described above, water and
ABA flux across the root is also regulated by the osmotic
potential of the xylem sap. Again, ABA itself may reduce
the xylem osmotic potential and thereby increase water and
ABA flux across the root, by inducing ion loading into the
© 2002 Blackwell Science Ltd, Plant, Cell and Environment, 25, 195–210
ABA-based chemical signalling 199
xylem, e.g. Glinka (1980) – potentially another positive
feedback mechanism to maximize the root-sourced ABA
signal.
Because root cells may degrade or store the ABA that
they synthesize or take up as it flows past them, not all the
ABA that enters the root reaches the xylem vessels. Conditions that reduce the ability of root cells to degrade or
take up and store ABA will increase the amounts that penetrate to the xylem. The catabolic degradation of ABA
takes place constitutively in most plant cell types (see Cutler & Krochko 1999 for review), but there is some evidence
that soil drying reduces the rate of this process in roots.
Liang, Zhang & Wong (1997) found that the half-life of 3HABA fed to maize roots was extended from 1·15 to 02·27 h
by drying the soil around them. Thus, more of the fed ABA
could potentially penetrate to the xylem.
Soil drying also leads to changes in the pH of the various
compartments of the root. That of the apoplast increases,
and root dehydration causes the cell cytoplasm to acidify
(Daeter, Slovik & Hartung 1993). This has multiple effects
on the root ABA signal, all based on the fact that ABA
always becomes concentrated in the most alkaline compartments, and all leading to an increase in the amount of ABA
that finally reaches the xylem, by at least two or three-fold
(Slovik, Daeter & Hartung 1995). Firstly, these pH changes
inhibit ABA loss from the root to the rhizosphere. Secondly, all shoot-sourced ABA arriving at the root in the
phloem is compartmentalized in the apoplast. Both Liang et
al. (1997) and Neales & Mcleod (1991) have reported that
root dehydration gives rise to increases in phloem-sourced
ABA in the xylem sap of 25–30%, not a negligible amount.
Finally, cytoplasmic acidification causes ABA synthesized
in the cytoplasm or previously stored there to be released to
the apoplast (see below). The fact that more root ABA
becomes compartmentalized in the apoplast as a result of
drought-induced pH changes means that it is less likely to
be catabolized or simply stored, and a greater percentage
finally reaches the xylem vessels.
To summarize, a soil drying ABA signal classically associated simply with the induction of synthesis in the root can
be intensified by the simultaneous effects of dry soil to
reduce ABA catabolism, to prevent rhizosphere- and
phloem-sourced ABA from entering the symplast, to
induce more effective efflux of the synthesized ABA to the
apoplast, and potentially to increase water and therefore
ABA flux towards the xylem.
Once ABA has reached the xylem vessel its transfer into
this conduit can also be modulated by the environment.
Xylem vessels have the ability to modify the pH of solutions
passing through them (Clarkson, Williams & Hanson 1984).
Fromard et al. (1995) showed that H+-pumping ATPases are
much more concentrated in the plasma membranes of
xylem parenchyma cells than in any other plant cell type.
They showed that these pumps were involved in the control
of vascular sap pH, and that this control fluctuated with season. Xylem pH values were close to neutrality in winter
(when ATPase activity was low), whereas they were acidic
(pH 5·5) at the beginning of spring. Hartung & Radin
© 2002 Blackwell Science Ltd, Plant, Cell and Environment, 25, 195–210
(1989) reported that plant water stress could reduce the
activity of these H+-ATPases associated with the root
xylem, and suggested that this may be one of the causes of
the increased alkalinity often observed in this compartment
under stress (see below for other hypotheses). Increases in
xylem pH enhance ABA loading to the root xylem by
increasing the capacity of the xylem sap to trap anions.
The xylem stream
As described above, soil drying can increase the pH of the
xylem stream. Recently, work by Hartung’s group determined that not only will this pH change maximize the initial
amount of ABA loaded into the xylem at the root, but also
that ABA loading into the xylem lumen from stores found
to exist in the stem parenchyma occurred more effectively
(Hartung, Sauter & Hose 2002). However, this only
occurred when the initial xylem [ABA] loaded in at the root
was low, i.e. it will be an early response to soil drying. Nevertheless, the possibility exists that the ABA concentration
arriving at the leaf in the xylem stream may be greater than
that loaded in at the root, and this effect may be intensified
in drying soil.
The leaf
It is described above how ABA entering the leaf via the
xylem does not all reach the guard cells because of sequestration into the symplastic ‘reservoir’, and that soil dryinginduced changes in xylem and therefore in apoplastic sap
pH modulate the rate of this process. However, several
additional stress-induced phenomena occur in the leaf to
intensify the root-sourced ABA signal that finally reaches
the epidermis. One, or a combination of the following,
could explain why the extent of stomatal closure observed
in droughted plants is often greater than can be attributed
alone to the concentration of ABA found in the xylem (e.g.
Correia & Pereira 1995).
A full symplastic reservoir. Under severe drought, shoot
water potentials drop and ABA biosynthesis occurs in the
cells of the leaf as well as in those of the root. This means
that the leaf symplast contains a great deal more ABA than
that of a turgid leaf. This rapidly alters the ABA concentration at the guard cells not only as a result of the penetration
of this newly synthesized ABA, but also because ABA
entering the leaf at the xylem cannot enter the symplastic
reservoir and therefore ends up at the epidermis. Filling of
the symplastic reservoir therefore increases the sensitivity
of the stomata to the ABA coming from the xylem. For
example, when xylem ABA concentrations return to wellwatered values after a period of water deficit, stomatal reopening is often sluggish (e.g. Trejo et al. 1995), and this
may be because the symplast is still full of ABA. Thus, even
the low well-watered concentrations of ABA carried by the
xylem may be capable of maintaining stomata in the closed
state until normal rates of symplastic sequestration are
restored. In related work, Heckenberger, Schurr & Schulze
(1996) found that when ABA influx to sunflower leaves was
200 S. Wilkinson & W. J. Davies
large, either due to long-term feeding of low concentrations
or short-term feeding of high concentrations via the xylem,
stomatal opening after the cessation of feeding took longer
to occur. ABA flux into the leaf may therefore be an important determinant of stomatal aperture under severe but not
incipient drought stress, when the capacity of the symplastic reservoir to remove ABA from the apoplast is minimized by a reduction of the inwardly-directed pm
concentration gradient for ABA.
Loss of ABA from the symplast. The symplastic ABA
reservoir in the leaf may also lose its ability to hold on to
the ABA that it contains. The occurrence of ABA release
from root cells has already been described above. There is
plenty of evidence in the literature that leaf cells are also
capable of releasing ABA to the surrounding medium, and
Hartung and co-workers have suggested that droughtinduced increases in xylem sap pH cause leaf cells to release
ABA to the apoplast. This could cause stomatal closure in
the absence of any increase in bulk leaf ABA concentration, as has often been described (references in Wilkinson
& Davies 1997). However, Wilkinson & Davies (1997)
could find no evidence for ABA efflux from tissue isolated
from turgid leaves in response to an increase in pHext. On
the other hand, when leaves become dehydrated, the cell
cytoplasm is acidified. Hartung, Kaiser & Burschka (1983)
showed that wilting induced the efflux of 14C-ABA from the
mesophyll of preloaded Valerianella locusta leaf tissue so
that it was able to penetrate to the lower epidermis. Hartung & Radin (1989) showed that cotton leaf dehydration
reduced the activity of outwardly rectifying pm H+ATPases,
which would acidify the symplast. This mechanism was similar to, but separate from that involved in the alkalinization
of the xylem sap of plants in drying soil referred to above.
Kaiser & Hartung (1981) demonstrated that the efflux
of ABA from the cytoplasm of isolated mesophyll cells of
Papaver somniferum increased with pHext in the presence of
KNO2, which acidified the cytoplasm. However, they were
unable to detect an effect of pHext, from 5 to 8, on ABA
release in the absence of this salt. ABA is usually present in
its dissociated form (ABA–) in the normally high pH of the
cytoplasm. This form cannot cross the plasma membrane
and it is therefore trapped in the symplast. ABAH formed
upon cytoplasmic acidification is lipophilic, and can diffuse
out of the symplast such that ABA accumulates in the apoplast if this is alkaline enough.
Thus, episodes of soil (or air) drying that are severe
enough to induce reductions in shoot water potential may
increase the penetration of ABA to the guard cells in one of
three ways. Firstly, the up-regulation of ABA biosynthesis
in leaves means that bulk leaf ABA concentrations
increase, but it also means that ABA arriving at the leaf via
the xylem is more easily able to penetrate to the epidermis
as the symplastic reservoir will be full. Finally, leaf dehydration-induced cytoplasmic acidification induces more
effective release of symplastic ABA to the apoplast,
increasing its likelihood of penetrating to the guard cells in
the epidermis. These leaf-sourced hydraulically induced
chemical changes may occur either in addition to or instead
of the root-sourced increases in xylem sap pH, which
increase the penetration of ABA to the guard cells by
reducing its symplastic uptake, even in leaves that are still
fully turgid.
Evidence for chemical signals originating in leaves that
may control ABA penetration to guard cells. Recently
it has been found that the leaf may be able to affect the penetration of ABA to the guard cells in the absence of leaf
water deficits and/or root-sourced changes in xylem pH. In
other words, chemical changes originating in leaves that are
not obviously hydraulically induced may influence the
ABA signal. This was first indicated by the finding that
changes in VPD (and mild soil drying episodes) reduced
leaf growth and stomatal aperture in some species without
affecting water potential, apparently by sensitising growth
receptors and guard cells to xylem ABA (Tadieu & Davies
1992, 1993). This is an entirely different response to that
described above, in which large soil water deficits (or
increases in VPD) induce obvious hydraulic signals that
reduce leaf water status prior to stomatal closure. The
authors coined the term ‘isohydric’ to describe the resultant
stability of leaf water status that they described in the field,
in the face of fluctuating VPD and soil water content. They
proposed that mild stresses induce small hydraulic changes,
possibly undetectable in bulk shoot water measurements,
that can directly sensitize guard cells to ABA so that stomata close and maintain leaf water status. We have proposed here and elsewhere that soil drying-induced pH
changes can often be responsible for that heightened sensitivity of leaf physiological responses to xylem ABA sometimes observed, such as those described by Tardieu &
Davies (1992, 1993). It is also possible that leaf-sourced
changes in pH can occur in response to variable VPDs in
the absence of VPD-induce leaf water deficits, possibly
alongside the VPD-induced hydraulic influence proposed
by Tardieu & Davies (1992, 1993). Some evidence that this
does indeed occur is provided below; however, it is by no
means a universal phenomenon. For example, in sunflower
ABA and plant water status do not interact at all even when
leaf water deficits are clearly detectable (Tardieu, Lafarge
& Simonneau 1996) and plant water status is variable and
unregulated (anisohydric).
Recent work at Lancaster University has shown that the
aerial environment can change leaf sap pH in both Forsythia × intermedia cv Lynwood and Hydrangea macrophylla cv Bluewave. Increases in sap pH correlated with
reductions in stomatal conductance in the absence of measurable changes in leaf relative water content in F. intermedia. Aerial conditions that increase transpiration (high
VPD, PPFD and leaf surface temperature) increased the
pH of sap expressed under pressure from the upper parts of
F. intermedia and H. macrophylla shoots, and the stomata
were more closed (see Fig. 2A and B for data from H. macrophylla, see Davies, Wilkinson & Loveys 2002 for data
from F. intermedia). The same conditions that caused stomatal closure in the afternoon did not do so in the morning
© 2002 Blackwell Science Ltd, Plant, Cell and Environment, 25, 195–210
ABA-based chemical signalling 201
in F. intermedia (and vice versa in H. macrophylla), suggesting that the sensitivity of guard cells to pH (and ABA)
change over the course of the day.
In F. intermedia, high VPD/PPFD, increased pH and
reduced stomatal conductance correlated with increases in
bulk leaf (but not xylem sap) ABA, and we suggest that
ABA removal by the phloem may have been inhibited (see
below). In H. macrophylla the same set of conditions had
no effect on bulk leaf [ABA], and it can only be hypothesized that the increased pH increased the penetration of
xylem-sourced ABA to the guard cells and/or induced its
release from the symplast as a result of the types of processes described above. That the climatically induced
changes in sap pH originated from changes within the leaf
apoplast rather than from the incoming xylem sap itself
were indicated by effects of soil drying on the response.
Unusually (but see below), soil drying acidified the xylem
sap of F. intermedia plants. In plants in drying soil the correlation between apoplastic sap pH and both the changes in
aerial conditions and changes in stomatal conductance were
poor (Davies et al. 2002). This was hypothesized to result
from the combination of the sap from the two different
sources (root and leaf, see also Mühling & Lauchli 2001),
which would partially override the climate-induced pH
change. There is plenty of evidence in the literature that
such gradients in pH exist between the leaf apoplast and the
xylem, with pH often being higher in the apoplast (Hoffmann & Kosegarten 1995; Mühling & Lauchli 2000). Regulation of gas exchange will be the result of a processing by
the guard cells of environmental information received by
both the roots and the shoot. Changes in sap pH may be a
unifying influence on guard cells through which both classes
of information may act.
It is as yet unknown which aerial factor caused the
changes in pH and stomatal aperture described above. High
VPD may have reduced leaf cell pm H+-ATPase activity by
causing slight (localised) changes in water relations (see
above, Hartnung & Radin 1989). Alternatively (or additionally) at high PPFD, increased removal of CO2 from the
apoplast for photosynthesis may alkalize this compartment.
Stahlberg, Van Volkenburgh & Cleland (2001) found that
photosynthetic removal of CO2 from Coleus leaves induced
an increase in apoplastic pH that could be propagated over
a distance of 2 cm. A high PPFD- and/or temperatureinduced increase in nitrate uptake by leaf cells could have
the same effect (see below).
The multiple effects of pH (in the root, stem and leaf) on
the ABA signalling process raise an interesting possibility
that species that exhibit a very low stomatal sensitivity to
ABA may do so because the xylem/apoplastic pH has not
become more alkaline with soil drying/climatic changes.
Schurr & Schulze (1995, 1996) have shown that there are
marked differences between species in the effects of soil
drying on hormone and nutrient fluxes to shoots. In our
experience, this is also true for changes in pH (soil drying
increases sap pH in tomato, C. communis and barley;
decreases pH in F. intermedia, and has no effect on sap pH
from H. macrophylla and Cotinus coggyria cv Royal Pur-
(b)
Xylem sap pH
Stomatal conductance (mmol m–2 s–1)
(a)
PPFD (mmol m–2 s–1)
PPFD (mmol m–2 s–1)
Figure 2. Ambient PPFD (n = 6 ± SE) incident on the first fully expanded leaf of well-watered Hydrangea macrophylla cv Bluewave plants
of approx. 50 cm in height (three or four branches) growing in 3 L pots in polythene tunnels in July–September 2000, plotted against (a) the
stomatal conductance of the leaves (n = 6 ± SE) and (b) the pH of xylem sap expressed immediately afterwards from the top 10 cm of a
dominant shoot under pressure (n = 3 ± SE). Each point represents measurements taken approximately every other day (a) or every 5–
6 d (b). Second-order regressions are shown, along with 95% confidence intervals (dotted lines) and r2 curve coefficients, as calculated on
Sigmaplot version 1·02.
© 2002 Blackwell Science Ltd, Plant, Cell and Environment, 25, 195–210
202 S. Wilkinson & W. J. Davies
ple). These results suggest that the differences in signalling
responses to drought are especially significant between
woody and herbaceous species (see below).
Release of ABA from inactive transport forms within
the leaf. Another way in which the leaf may modify the
ABA signal has been described by Dietz et al. (2000). ABA
conjugates, which are themselves unable to affect stomatal
aperture or plant growth, have been detected in the xylem
sap of stressed plants (Netting, Willows & Milborrow 1992).
ABA-GE (ABA-glucose ester) seems to be the most common of these. Glycosylation of ABA is thought to be a
mechanism to allow for the export of ABA from cells independently of the prevailing transmembrane proton gradient. In addition, its release from root stelar cells and
liberation to the xylem seems to be greatest under conditions of inhibited ABA metabolism (Sauter & Hartung
2000). ABA-GE transport may therefore be important as a
root-sourced stress signal during soil flooding when ABA
synthesis becomes inhibited. ABA-GE in barley xylem sap
has also been shown to increase under salinity, whereas its
concentration in the apoplast of primary leaves was
unchanged (Dietz et al. 2000). This is because the barley
leaf apoplastic sap contained the enzyme β-glucosidase,
which rapidly cleaved ABA from the inactive ABA-GE
pool arriving at the leaf apoplast via the xylem. The data
support the hypothesis that ABA-GE is a long-distance
transport form of ABA. Anything that affects the activity of
β-glucosidase in the leaf will therefore affect the [ABA] in
the leaf apoplast, presumably thereby affecting stomatal
behaviour. There is also some evidence that ABA can be
released from its conjugated form in the root before transport in the xylem (Sauter & Hartung 2000).
Removal of ABA from leaves by the phloem. Finally,
leaves may alter the ABA signal by influencing the rate at
which ABA is removed from them via the phloem. If less
ABA exits the leaf then presumably there is an increased
likelihood that the remaining ABA will penetrate to the
guard cells. Jia & Zhang (1997) detected a large reduction of
ABA transport out of detached maize leaves in the phloem
when buffers at a ‘droughted’ pH of 7·4 as opposed to a ‘well
watered’ pH of 5·5 were injected into the xylem. Wilkinson
& Davies (1997) found that detached C. communis leaves
taking up 3 µM ABA from the external medium had a
greater bulk leaf [ABA] 3 h later when this was fed at a
‘droughted pH’ of 7 as opposed to pH 6. These data suggest
that an alkaline apoplastic pH prevents ABA movement out
of the leaf via the phloem. This could either result from the
decreased pH gradient between the apoplast and the
phloem, trapping the ABA in the leaf apoplast (if apoplastic
ABA transfer is important), or from a decrease in the
amount of ABA present in the sieve element companion
cell (if symplastic phloem transfer is predominant). Phloem
sieve tubes are living cells with an internal pH of 7·5 (Baier
& Hartung 1991), so that the phloem’s efficiency as an ABA
reservoir (and export conduit) will depend on the same
types of processes described above. Support for an effect of
apoplastic pH on loading of ABA into the phloem comes
from work by Peuke, Jeschke & Hartung (1994), who
showed that ABA flux in the phloem from the leaves of
ammonium-treated plants is extremely large. As will be
explained below, ammonium nutrition reduces the apoplastic pH of leaves.
Other work has shown that the ABA concentration actually increases in the phloem in response to stress (Wolf,
Jeschke & Hartung 1990). This presumably occurs only
under more severe and/or prolonged stress in which [ABA]
will be high in all tissues due to both root and shoot biosynthesis.
THE SENSITIVITY OF GUARD CELLS TO A
PERCEIVED CONCENTRATION OF ABA
As described above, plants control the amounts of ABA
that reach the guard cells in the leaf epidermis via processes
that originate in the root, the stem or the leaf. However,
they also control the sensitivity with which guard cells
respond to the ABA concentration that finally reaches
them. Changes in guard cell sensitivity to ABA occur over
the day/night cycles and as a plant matures, often in
response to changes in electrical and osmotic membrane
potentials generated by guard cells themselves (for reviews
see Assmann & Shimazaki 1999; Blatt 2000).
Effects of apoplastic composition on guard cell
sensitivity to ABA
Calcium
Calcium ions are known to enter guard cells and promote
stomatal closure and inhibit opening when applied to isolated epidermis, and apoplastic calcium sensitizes guard
cells to ABA (De Silva, Hetherington & Mansfield 1985).
This is because ABA-induced stomatal closure involves
increases in cytosolic-free Ca 2+ in guard cells as part of the
signal transduction chain leading to loss of turgor
(McAinsh, Brownlee & Hetherington 1990). The addition
of 8 mM calcium to the transpiration stream of detached
leaves initiates rapid reductions in stomatal conductance
(Ruiz, Atkinson & Mansfield 1993). Variations in calcium
and ABA concentration in the xylem with soil drying
(Schurr et al. 1992) may interact to limit stomatal opening.
To add further complexity, guard cell sensitivity to external
calcium can also change under some circumstances (see
below).
Potassium
Potassium is centrally involved in the up-regulation of turgor-driven stomatal opening, and the effectiveness of ABA
at closing stomata may be reduced when its application to
the medium bathing epidermal strips exceeds a certain concentration (Wilson, Ogunkanmi & Mansfield 1978; see
below). The significant reductions in xylem sap [K+]
© 2002 Blackwell Science Ltd, Plant, Cell and Environment, 25, 195–210
ABA-based chemical signalling 203
observed at high bulk soil density and in dry soil (Mulholland et al. 1999; Roberts & Snowman 2000) may have maximized a potential reduction by ABA of stomatal
conductance. It is also interesting to note that ABA in the
root inhibits potassium loading into the xylem by xylem
parenchyma cells (Roberts & Snowman 2000), indicating
that ABA in the root could potentially sensitize stomata to
ABA in the leaf by reducing K+ availability as a guard cell
osmoticum.
Protons
Protons can also directly affect stomatal aperture and/or its
sensitivity to ABA. There may be an optimum apoplastic
pH for stomatal opening related to the activity of guard cell
pm H+-ATPases and other pm ion channels that directly
control turgor. Wilkinson & Davies (1997) and Wilkinson et
al. (1998) have provided evidence that the direct effect of
pH on stomatal aperture in isolated epidermis can be very
different to that seen when buffers of differing pH are supplied to intact leaves via the xylem. Increasing the pH of the
medium on which isolated epidermal strips of C. communis
were floating actually opened stomata and reduced their
sensitivity to ABA (see also Schwartz et al. 1994). Feeding
artificial sap of pH 7 to intact detached leaves of the ABAdeficient tomato mutant flacca increased stomatal aperture
and transpirational water loss compared to when the fed
sap was buffered to pH 6, but the response to pH was
reversed when ABA was supplied in the fed sap. Several
other studies have shown that, whereas pH per se had no
effect on stomata in isolated epidermis, reduced pH did
sensitize stomata to ABA (e.g. Anderson et al. 1994), as
described above for C. communis. These authors proposed
that because guard cells take up ABA more efficiently at
more acidic pH, its contact with internally located receptors
would be increased, thereby inducing more sensitive stomatal closure. However, given the evidence that guard cell
symplastic ABA is not always necessary for stomatal closure, it may instead be the case that an acidic external pH
directly modifies the ionic balance and thereby the turgor
of the guard cell, as suggested above, to predispose it
toward stomatal closure. For example, there is already evidence that under some circumstances (but see below)
reduced external pH can directly reduce K+ influx and
increase K+ efflux at the guard cell plasma membrane
(Roelfsema & Prins 1998). The reason for the fact that in
some cases (Wilkinson & Davies 1997) but not others
(Anderson et al. 1994) acidic pH closes stomata in isolated
epidermis in the absence of ABA could be related to the
ionic composition of the bathing medium used. Thompson
et al. (1997) describe how in the presence of 75–100 mM KCl
stomatal aperture no longer varied with pHext as it did in the
presence of 50 mM KCl, whereas the sensitivity of aperture
to ABA varied with pH as usual in both media. It must be
supposed, however, that the more potent effect of pH in the
intact plant is on the distribution of ABA between the compartments of the leaf. This must normally override the
opposing direct effect of pH to modulate guard cell sensi© 2002 Blackwell Science Ltd, Plant, Cell and Environment, 25, 195–210
tivity to local ABA, otherwise we would find that high
xylem sap pH opens stomata, and this is often clearly not
the case (e.g. Patonnier, Peltier & Marigo 1999, Wilkinson
1999).
Sugars
There is some evidence in the literature that apoplastic sugars could potentially increase guard cell sensitivity to ABA.
As the water potential in soil around ash seedlings
decreased, there was a significant increase in the concentrations of malate and mannitol in the xylem and a concomitant decrease in maximum stomatal conductance
(Patonnier et al. 1999). A transpiration bioassay using
detached leaves from control plants found that mannitol
had no effect on stomata, whereas malate between 0·5 and
3 mM (the range found in xylem sap from the droughted
plants) prevented stomatal opening. Other structural
malate analogues (citrate, aspartate or shikimate) had the
same effect, whereas neutral amino acids did not. The
authors suggested that the effect of malate on stomata was
to specifically increase the activity of the anion efflux channel at the pm of the guard cells (GCAC1), as described by
Hedrich & Marten (1993) for V. faba. As ABA also induces
anion loss and therefore reduces turgor in guard cells, it
may be supposed that malate and ABA act synergistically
at GCAC1 to close stomata. Alternatively, because of the
non-specificity of the effect in ash, Patonnier et al. (1999)
suggest that the effects of the inorganic acids on stomatal
aperture were directly dependent on the negative charge of
the molecules. However, given that under some circumstances increasing the local apoplastic pH at the guard cell
induces stomatal opening and/or reduced guard cell sensitivity to ABA as described above, it is possible instead that
the negative charge of the inorganic acids exerted an indirect effect on stomata. Namely, the increased pH that they
induce (see below) may cause preferential accumulation of
ABA in the apoplast, rather than directly affecting guard
cell sensitivity to a given ABA concentration.
High CO2 in the substomatal cavity (see below) also
reduces stomatal aperture and may enhance the response
of guard cells to ABA. Hedrich & Marten (1993) have suggested that high CO2 induces photosynthetically derived
malate to be released from mesophyll cells adjacent to
guard cells, which, as described above may directly induce
loss of guard cell turgor. Mesophyll-derived apoplastic
sucrose is another CO2-sensing candidate which can reduce
stomatal aperture, by virtue of its osmotic effect to draw out
guard cell water, but this will presumably occur only in species which load sucrose into the phloem apoplastically (Lu
et al. 1997). Apoplastic malate or sucrose may signal the
plant that mesophyll-derived photosynthetic products are
high and stomata can therefore close to limit water loss.
However, Schmidt & Schroeder (1994) have suggested that
the efflux of osmotically active malate from guard cells
themselves (which also contain photosynthetic apparatus)
via anion channels may provide a positive feedback mechanism for ‘plentiful photosynthate’-induced stomatal clo-
sure. As described above, it may be supposed that ABA and
malate or sucrose therefore act synergistically to reduce
guard cell turgor, and this may be the basis of a high CO2induced sensitization of stomata to ABA. Alternatively, the
apoplastic mode of sucrose loading into the phloem could
remove H+ from the apoplast and increase its pH (see Tetlow & Farrar 1993), which in an intact leaf would cause
ABA to compartmentalize in the apoplast. However,
Zeiger & Zhu (1998) provide evidence that in the light high
CO2 detection occurs at the blue light photoreceptor in the
guard cell chloroplast. It is therefore possible that CO2 and
ABA could interact directly at the guard cell pm H+ATPase, as its down-regulation comprises part of the signal
response chain of both ABA and CO2 perception in guard
cells. Apoplastic sugars may only be involved in the stomatal response to CO2 when its concentration is high due to
high light-saturated photosynthesis, whereas the direct
effect of CO2 to close stomata may occur when its concentration is high because photosynthesis is limiting at low
light intensities (which include the blue light wavelengths).
However, because stresses such as drought increase the
concentration of sugars in the xylem as described above,
they may still exert an important influence on stomatal sensitivity to ABA.
Other hormones
Cytokinins in the xylem have been shown to decrease stomatal sensitivity to xylem ABA in cotton (Radin, Parker &
Guinn 1982), and their presence at high concentrations in
the transpiration stream can act directly on stomata to
increase their aperture (Incoll & Jewer 1987a, b). Blackman
& Davies (1985) showed that these effects became larger as
leaves aged. Fusseder et al. (1992) report data from field
studies of almond trees under desert conditions that suggest
an ameliorating influence of increasing xylem cytokinin
concentration on ABA’s effect on stomatal aperture. More
recently, Stoll et al. (2000) showed that under partial root
drying there was an increase in xylem sap and leaf ABA in
grape vines which correlated with reduced stomatal conductance. Concomitant increases in xylem sap pH and
reductions in cytokinins were observed, both of which
could have enhanced stomatal sensitivity to ABA, the latter
via direct sensitization.
Effects of the aerial microclimate around the leaf
on guard cell sensitivity to ABA
Temperature
We have shown recently that chilling temperatures applied
to epidermal strips reduce the sensitivity of C. communis
stomata to ABA in the bathing medium (Fig. 3). This is
despite the fact that the same conditions induce stomatal
closure in the intact leaves of this chill-tolerant species to
counteract chill-induced reductions in water uptake at the
root (Wilkinson et al. 2001). Given much evidence in the literature that chilling temperatures can eventually induce
Stomatal aperture (mM)
204 S. Wilkinson & W. J. Davies
[ABA] (M)
Figure 3. The effect of [ABA] on stomatal aperture in detached
abaxial epidermal strips of Commelina communis L. at two
different temperatures. Strips (approx 35–40) were pre-incubated
on 25 cm3 70 mM KCl at room temperature for 3 h, then
transferred to treatment solutions, pre-chilled where necessary, of
70 mM KCl (4·0 cm3) ± ABA for 1·5 h. All leaves received a PPFD
of 350 µmol m−2 s−1 at all times. Stomatal aperture was measured
under the microscope. Each point represents the mean of 25
apertures in each of three strips per treatment (n = 75 ± SE). From
Wilkinson et al. (2001).
ABA biosynthesis, it seems counterintuitive that stomata
become less responsive to this hormone. This finding may
explain why some research has shown that low temperatures can actually open stomata in vivo in chill-sensitive
species. Although we found that stomata remained open
when C. communis epidermis was isolated from the rest of
the leaf and floated on pre-chilled media, the closing
response to low temperatures could be restored (Wilkinson
et al. 2001) by supplying the epidermal strips with the calcium concentration estimated to exist in the leaf apoplast
(0·01–0·05 mM, De Silva, Honor & Mansfield 1996). Stomata in strips floating on the same calcium concentration
remained open at room temperature. Thus, chill-induced
stomatal closure occurs via a sensitization of guard cells to
calcium in the apoplast, even though stomatal sensitivity to
ABA is reduced (Wilkinson et al. 2001). In the context of
this review, it might be more pertinent to state that increasing temperature increases the sensitivity of guard cells to
ABA (Fig. 3), presumably by increasing ABA binding to its
receptors in the guard cell pm, and/or by increasing the
activity of the ion transporters involved in the ABA signal
response chain.
Although early responses to chilling temperatures
directly reduce guard cell sensitivity to ABA, there is evidence in the literature that a previous period of chilling
© 2002 Blackwell Science Ltd, Plant, Cell and Environment, 25, 195–210
ABA-based chemical signalling 205
stress followed by recovery can sensitize guard cells to close
in response to a second stress such as drought (Wilson 1976)
– ‘chill-hardening’. Allan et al. (1994) found that guard cells
in epidermis isolated from cold-acclimated C. communis
leaves did not require cytoplasmic increases in calcium for
ABA to cause stomatal closure. Our work (Wilkinson et al.
2001) demonstrates that chilling temperatures may replenish internal stores of calcium in guard cells. In effect, cells
are ‘primed’ by cold pre-treatment for closure in response to
a subsequent exposure to ABA, as internal calcium is centrally involved in the ABA signal transduction chain.
VPD
There is much evidence in the literature that increasing
VPD closes stomata and increases their sensitivity to ABA,
although again the underlying cellular mechanisms for
these responses remain under debate. We have proposed
some explanations for this response in the sections above
(see also Zhang & Outlaw 2001a), based on ABA redistribution. However, Assmann, Snyder & Lee (2000) found
that increasing VPD closed stomata in both ABA-deficient
and ABA-insensitive mutants of Arabidopsis thaliana,
showing that, at least in some species, it is necessary to
invoke the more traditional view that dry air has a more
direct effect on guard cell and/or epidermal cell turgor.
Like responses to soil drying, responses to VPD may differ
between species, and could be hydraulic or chemical or a
combination of both. Saliendra et al. (1995) found that in
western water birch,restoration of shoot water potential
using a root pressure vessel fully reversed stomatal closure
in response to both a soil water deficit and increased VPD.
By contrast, Fuchs & Livingstone (1996) found that stomatal responses to VPD in Douglas fir and alder seedlings
were only partially reversed by restoration of shoot water
relations.
Light
It is well established that indirect responses of stomata to
light involve photosynthetic effects on intercellular CO2
concentrations (see above). Stomata are also directly
responsive to light (for reviews see Assmann & Shimazaki
1999 and Zeiger & Zhu 1998): photosynthetically active
radiation (400–700 nm) induces guard cell photosynthesis
and opens stomata via the production of osmotically active
solutes such as malate. In addition, blue light (425–475 nm)
induces stomatal opening by transposing a signal cascade in
guard cells that includes a stimulation of H+ efflux to the
apoplast via pm H+-ATPases. This provides the driving
force for uptake of K+ and Cl– and therefore increases
guard cell turgor (depending on the prevailing membrane
potential, see Roelfsema & Prins 1998). Stomata may be
more sensitive to ABA under conditions of low light
because: (a) the intercellular [CO2] will be higher (less photosynthetic removal); (b) reduced guard cell photosynthesis
means lower guard cell [malate]; and/or (c) the blue-lightdependent H+-ATPase in the guard cell pm, which is
directly down-regulated as part of the ABA signal response
© 2002 Blackwell Science Ltd, Plant, Cell and Environment, 25, 195–210
chain, will already be less active when light is limiting. However, work by Clephan in our laboratory could only detect
evidence for a sensitization of C. communis stomata to
ABA at low light intensities when leaves were intact, and
not in isolated epidermis (personal communication). This
suggests that only indirect effects of light (e.g. on the mesophyll-determined [CO2]) affect the response of stomata to
ABA. The same series of experiments revealed that more
ABA reached the epidermis of intact leaves at lower light
intensities (despite decreased ABA fluxes into them). It
was suggested that light-induced alkalinization of the mesophyll chloroplast stroma and therefore ABA retention in
chloroplasts (Kaiser & Hartung 1981) may have inhibited
ABA penetration to the epidermis in saturating light, such
that stomata were less sensitive to the [ABA] supplied.
These responses occur over very low light intensities,
and will be very different to those suggested to result from
stressfully high PPFD and/or VPD described earlier, which
correlate with apoplastic alkalinization and stomatal
closure.
NITRATE STRESS AND ABA SIGNALS MAY
INTERACT AS SOIL DRIES
Plants growing in dry soil frequently show a sensitive
reduction in the delivery of nitrate to shoots and this signal
can regulate leaf processes independently from a variation
in plant water relations (Shaner & Boyer 1976). When
nitrate supply is limiting (regardless of soil water availability), stomata close, leaves grow more slowly, root growth is
maintained, and is often characterized by greater lateral
root proliferation. All of these are also symptoms of water
stress. It may be that the soil drying response that is often
characterized is at least in part a response to a limitation in
N supply.
One way in which limiting nitrate could reduce leaf
growth would simply be the result of a decrease in the supply of N-derived amino acids and structural proteins. However, there is much evidence that responses to Ndeprivation are governed instead by fast root–shoot (or
shoot–root) chemical signals. Krauss (1978) found marked
increases in ABA in the xylem sap of rooted sprouting
potato tubers deprived of N, and increases in shoot and leaf
ABA in N-deprived plants have since been observed in several species (see Radin et al. 1982; Clarkson & Touraine
1994). However Peuke et al. (1994) found that ABA in low
nitrate-grown Ricinus communis was only increased in the
xylem and not in the leaves. Similar data were reported by
Brewitz, Larsson & Larsson (1995) for barley. Some studies
detected no long-term effect of reduced nitrate supply on
tissue ABA concentrations at all (Chapin et al. 1988, barley
and tomato). When Peuke et al. (1994) grew R. communis
on ammonium as a substitute N source, rather than on low
N, they were able to demonstrate large increases in leaf and
xylem ABA, and also large increases in phloem ABA transport, but the stomata in the leaves did not close in response
to this increase in ABA flux around the plant.
206 S. Wilkinson & W. J. Davies
To explain these rather variable responses, it has been
suggested that rather than inducing increases in [ABA] per
se, low nitrate availability may modulate responses in the
shoot via a sensitization of stomata/leaf growth receptors to
ABA. Radin et al. (1982) have shown that both N and P
deficiency can enhance stomatal sensitivity to an ABA signal. Schurr et al. (1992) found that the concentration of
nitrate in the xylem sap of droughted sunflower plants regulated stomatal sensitivity to ABA. It is possible that ABA
and nitrate can interact to influence stomata via nitrateinduced changes in the pH relations of the leaf (Raven &
Smith 1976).
There is now much evidence available for effects of Ncontaining compounds on plant pH balance. Mengel,
Planker & Hoffmann (1994) found that young sunflower
plants growing on a sole NO3– (or bicarbonate) medium
had chlorotic pale leaves, but those on ammonium nitrate
did not. On NO3– (or bicarbonate), the pH of the leaf apoplast was increased in comparison to that of plants on
ammonium, and this reduced leaf chlorophyll content by
inhibiting iron reduction in the apoplast (this process has an
acidic pH optimum). Tagliavini et al. (1994) reported similar findings in field-grown Kiwi fruit: many of the plants displayed chlorotic pale leaves, and these were found to
contain xylem sap with a higher pH than those with healthy
green leaves. The plants were growing on calcareous soils,
which contain high levels of HCO3– and NO3–. When the
leaves were sprayed with citric acid or H2SO4, to counteract
the (presumably) nitrate-derived alkalinization, re-greening occurred. More recently, Kosegarten, Hoffmann &
Mengel (1999) found that nitrate nutrition (and nitrate plus
HCO3–) gave rise to a high apoplastic pH in immature but
not mature sunflower leaves, especially at distinct interveinal sites. This did not occur in sole NH4+ or NH4/NO3
nutrition. Mühling & Lauchli (2001) also found that nitrate
nutrition gave rise to more alkaline leaf apoplastic sap than
ammonium nutrition, in both Phaseolus vulgaris and sunflower, but not in Vicia faba or Zea mays. Mengel et al.
(1994) hypothesize that NO3– may alkalize apoplastic sap
because it is co-transported into the leaf cytoplasm with a
proton (Ullrich 1992), and this presumably occurs to a
greater extent in young leaves because these are the site of
greatest N reduction. HCO3– may simply neutralize H+
pumped into the xylem sap by ATPases. Soil-sourced NH4+
is reduced in roots, and the neutral amino acids which are
produced are transported in the xylem to the leaf instead of
NO3– (see below). Amino acids are already protonated at
apoplastic pH values, so uptake into the mesophyll will
remove fewer protons from the apoplast than NO3–/H+ cotransport, presumably giving rise to a more acidic apoplast.
These results could help to explain the findings of Peuke et
al. (1994) described above, that even though ammoniumgrown R. communis plants contain more root-sourced ABA
than nitrate-grown plants, they have open stomata and
poor water use efficiencies. Ammonium nutrition may have
given rise to a more acidic leaf apoplast than nitrate nutrition, as it did in sunflower, which as described above can
reduce the accessibility of ABA to guard cells by increasing
its symplastic sequestration and increasing the efficiency of
its transfer into the phloem and out of the leaf. Similarly,
Allen & Raven (1984) found that R. communis with NH4+
as the N source transpired more water per g dry matter synthesized than when NO3– was the N source. Raven, Griffiths
& Allen (1984) went a step further and showed that
ammonium-grown plants actually had increased stomatal
apertures.
So far, the data described above would tend to suggest
that a reduction in nitrate availability, by whatever means
(soil nutrient deficiency, soil moisture deficiency, soil compaction or soil flooding), would tend to decrease the leaf
apoplastic pH, as nitrate nutrition alkalizes this compartment. This is counterintuitive because, as discussed above,
acidic apoplastic sap pH reduces the accessibility of ABA to
stomata, and would tend to oppose stomatal closure (as in
ammonium nutrition). However, all of these stresses are
characterized by stomatal closure and increased sensitivity
of stomata to ABA. Some of them are characterized by
increases in xylem sap pH. How can we explain these discrepancies?
The answer lies in the fact that N availability influences
the species of N-containing molecule that is carried in the
xylem. Different N-containing molecules have different
abilities to influence the xylem and/or apoplastic pH. When
nitrate is plentiful, nitrate is transported in the xylem to the
leaf, where it is reduced in the mesophyll. When nitrate is
limiting, either due to low soil availability or to soil
drought/flooding/compaction, then nitrate reduction is
switched from the shoot to the root (see Lips 1997). Root
nitrate reductase activity produces hydroxyl ions, which are
then converted to COO– (often as malate). This is carried in
the xylem to the leaf. Malate and related compounds alkalize apoplastic sap to an even greater extent than nitrate
can. It is well known that organic anion salts such as malate
have higher pH values than mineral anion salts such as
nitrates (Wallace, Wood & Soufi 1976). Kirkby & Armstrong (1980) showed that under nitrate nutrition, organic
acids were present in only trace amounts in the xylem sap of
castor oil plants, which had a pH of 5·6. Under NO3– stress
the pH of the sap rose to 7·3, and considerable amounts of
organic acid anions were present. Patonnier et al. (1999)
describe drought-induced increases in xylem sap pH and in
the organic acid fraction of the xylem (that presumably
result from a drought-induced switch of nitrate reduction to
the root) that could reduce stomatal aperture in ash. Neutral amino acids (the product of ammonium nutrition that is
transported in the xylem) fed into the xylem of detached
leaves had no affect on stomatal aperture. These authors
suggested that the organic acids had a direct effect on guard
cells (see above), but it is just as likely that their effect to
increase xylem sap pH allowed ABA more access to the
guard cells by reducing symplastic sequestration.
Rather than having a direct affect on xylem and therefore leaf apoplastic pH, organic acids could increase apoplastic sap pH by virtue of their di-anionic form: upon
transfer of these to the symplast, the uptake of two protons
and therefore their removal from the apoplast will be
© 2002 Blackwell Science Ltd, Plant, Cell and Environment, 25, 195–210
ABA-based chemical signalling 207
required. Whether they directly increase xylem sap pH, or
increase apoplastic sap pH by removing protons during
symplastic sequestration, the result will be the same:
greater penetration of ABA to its site of action at the guard
cells. This could explain why much research shows
increased stomatal sensitivity to ABA in response to Ndeficiency, whereas N-deficiency does not always lead to
increases in leaf [ABA]. It could also be one explanation
for the effect of soil drying to increase xylem sap pH.
To summarize, the literature shows that xylem/apoplastic sap seems to be most alkaline under N deficiency when
nitrate reduction in the root produces inorganic anions and
loads them to the xylem. It is of intermediate alkalinity
when nitrate is carried to leaves in the xylem stream, and
most acidic when ammonium is reduced in roots and the
neutral amino acids produced are loaded to the xylem
stream.
An explanation for differences between species in the
effects of rhizospheric stresses on xylem sap pH could lie in
the inherent species response to low nitrate availability.
Most crop species exhibit very active nitrate uptake and
xylem loading, carry out nitrate reduction in the leaf and
have fast growth rates. However, some slow-growing trees
carry out N assimilation in their roots (see Lips 1997). Perhaps fast-growing crop species will exhibit xylem sap pH
changes as N deprivation switches nitrate reductase activity
to the root where, e.g. malate will be transferred to the
xylem. However, in species that reduce N in the root as a
matter of course, N deficiency or soil drying will not change
the position of N reduction, such that xylem sap concentrations of organic acids show little change as stress develops,
and xylem pH remains stable. Our finding that xylem sap
pH in woody species is unresponsive to soil drying supports
this hypothesis.
CONCLUSIONS
Early work on the chemical control of plant growth and
functioning under stress led to the seductively simple
hypothesis that ABA produced in the roots of plants undergoing rhizospheric stresses moved to the shoot to provide a
measure of resource availability. Shoot responses could
then be modified as a function of the intensity of this signal.
We show here how a range of influences interact to modify
the ‘root signal’ and much of this modification occurs via a
modulation by the environment of the pH relations of the
different compartments of the root and the leaf. This provides some understanding of how rhizospheric and aerial
influences may interact to influence leaf growth and functioning. We are now moving towards a more complete
understanding of the whole-plant signalling process and an
understanding of why the intensity of chemical signalling
and the response to these signals may vary between environments and plant species. To aid in our thinking we now
have available a range of new technology to assess pH and
local accumulation of ABA in the apoplast adjacent to the
© 2002 Blackwell Science Ltd, Plant, Cell and Environment, 25, 195–210
guard cells. This technology will allow us to test some of the
hypotheses proposed in this review.
ACKNOWLEDGMENTS
The authors would like to thank the members of MAFF
Horticulture Link project HL0132LHN/HNS 97 for
financial support towards the work with ornamental
species. We are grateful to the American Society of Plant
Biologists for permission to reprint Fig. 1 (represented as
Fig. 2 at source) from: Wilkinson, Clephan & Davies (2001).
This work is copyrighted by the American Society of Plant
Biologists.
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Received 12 June 2001; received in revised form 7 September 2001;
accepted for publication 10 September 2001
© 2002 Blackwell Science Ltd, Plant, Cell and Environment, 25, 195–210