Journal of Experimental Botany, Vol. 47, No. 303, pp. 1475-1490, October 1996
Journal of
Experimental
Botany
Chemical regulation of gas exchange and growth
of plants in drying soil in the field
I.C. Dodd 1 , R. Stikic 2 and W.J. Davies1-3
1
2
Division of Biological Sciences, Lancaster University, Lancaster LA14YQ, United Kingdom
Faculty of Agriculture, University of Belgrade, Nemanjina, 11081 Belgrade-Zemun, Serbia
Received 19 September 1995; Accepted 29 April 1996
Abstract
There is now substantial evidence that chemical regulation of shoot physiology occurs in draughted plants
in the field. The evidence that ABA may play a role in
such regulation is considered, and topics of relevance
to the worker interested in determining the ABA relations of plants in the field; such as the methods used
for ABA quantification, the relevance of quantifying
ABA in various plant tissues, methods of xylem sap
collection and timing of sap collection are reviewed.
A possible role of tissue sensitivity to ABA in controlling the diurnal changes in stomatal conductance and
leaf growth rate seen in the field is also considered.
Key words: ABA, drought, stomatal conductance, leaf
growth, hormonal sensitivity, xylem sap.
1. Why be concerned with chemical regulation of
shoot physiology?
Drought is one of the most common stresses experienced
by plants. The conventional view is that soil drying
induces restriction of water supply and this results in a
sequential reduction of the tissue water content, water
potential and turgor, growth and stomatal conductance
(g,). While this is undoubtedly the case, it appears that
in some cases, changes in leaf physiology are more closely
linked to changes in the soil water content than to the
leaf water status. One of the best examples of this type
of plant response is presented by Jones (1985), who found
that over a period up to 10 weeks, midday water potential
values were higher in unwatered than in irrigated apple
seedlings. The higher water potential values in draughted
plants were associated with lower g,, indicating that
stomata controlled leaf water status rather than the
converse, which is generally assumed to be the case.
This kind of stomatal reaction requires that the plants
have some mechanism for sensing the availability of the
water in the soil and regulating stomatal behaviour
accordingly. Jones (1980) and Cowan (1982) have suggested that this involves transfer of chemical information
from roots to the shoots via the xylem. Such control has
been termed non-hydraulic or chemical signalling. This
distinguishes it from hydraulic signalling, which represents
transmission of reduced soil water availability via changes
in the xylem sap tension.
Substantial evidence obtained from a large number of
different species in both growth cabinet studies and under
field conditions suggests that plants can use chemical
signals to regulate growth and development as the soil
dries (and perhaps also in response to other stresses).
This regulation can occur even under circumstances where
shoot water relations are not changed, but under most
circumstances probably occurs in conjunction with
hydraulic regulation. Chemical signals may move from
roots to shoots or from one leaf to another via the roots.
Whatever the source of the chemical regulator, it seems
important that some assessment of chemical regulation
should be included in studies of the effects of soil drying.
Studies of drought stress effects would not be conducted
'To whom correspondence should be addressed. Fax: +44 1524 843854. E-mail: w.davies©lancaster.ac.uk
Abbreviations. ABA, abscisic acid; DW, dry weight; ECD, electron capture detector; E2, elongation zone; [EZ-ABA], elongation zone ABA concentration;
ELJSA, enzyme-linked immunosorbent assay; FID, flame-ionization detector; GA, gibberellic acid; GC or GLC, gas or gas liquid chromatography, g.,
stomatal conductance; HPLC, high-performance liquid chromatography; IA, immunoaffinity-, IAA, indole acetic acid; [L-ABA]. bulk leaf ABA concentration;
LER, leaf elongation rate; V,, leaf water potential; MS, mass spectrometry; MePA and MeDPA, methyl esters of phaseic acid and dihydrophaseic add;
NAA, naphthyl acetic acid; QTL, quantitative trait loci; [root ABA], root ABA concentration; RIA, radioimmunoassay; SIM, selectedranmonitoring; TLC,
thin layer chromatography; VPD, vapour pressure deficit; [X-ABA], xylem sap ABA concentration.
© Oxford University Press 1996
1476
Dodd et al.
without assessment of plant water relations variables. It
is argued that it is equally unrealistic to consider such
studies without some estimate of chemical regulation.
Chemical signals, according to the terminology of
Jackson and Kowalewska (1983), can be negative or
positive messages. Negative messages are supplied by
turgid roots and promote stomatal opening and shoot
growth, therefore production and transport of these messages would decrease as the soil dries. A restricted supply
of cytokinins from dehydrating roots may be the best
example of this kind of message. Meinzer et al. (1991)
and FuBeder et al. (1992) have shown cytokinin involvement in root signalling of sugarcane and almond, respectively. Positive messages, whose production increases as
the soil dries, can be inhibitors such as abscisic acid
(Jones, 1980). Changes in mineral composition or pH of
the xylem sap (Gollan et al., 1992; Schurr et al, 1992)
may provide additional signals. Bradford and Hsiao
(1982) have pointed out possible roles for gibberellins,
auxins and ethylene in draughted plants.
2.
Why ABA?
Since the pioneering work of Mittelheuser and van
Steveninck (1969) and Jones and Mansfield (1970), there
has been much interest in the potent stomatal closing
action of ABA. This hormone also has an inhibitory
effect on leaf growth (van Volkenburgh and Davies, 1983)
and has recently been shown to play an important regulatory role in maintaining root growth in drying soil
(Sharp et al., 1994). Wright (1977) and subsequently
many others have demonstrated that ABA synthesis is
stimulated as cells dehydrate. The combination of all of
these observations suggests that ABA produced by dehydrating cells can move to guard cells to restrict water loss
and slow further dehydration and exert a similar effect
by limiting leaf area development and sustaining root
growth. As such, ABA acts as a 'signal' to activate a
range of turgor maintaining processes.
Originally it was thought that ABA synthesized in the
mesophyll of the leaf moved from there to the guard cells
and to the growing cells to exert an effect (Mansfield and
Davies, 1981). More recently, the importance of xylemborne ABA has been emphasized (Loveys, 1984). The
experiments of Zhang et al. (1987) and Zhang and Davies
(1989a, 1990a, b) provided evidence that increased xylem
ABA concentration ([X-ABA]) was root-sourced and
quantitatively sufficient to account for the physiological
changes (reduction of stomatal conductance and leaf
growth rate) observed in the shoots of plants draughted
under controlled conditions. Similar evidence has come
from field experiments (Wartinger et al., 1990; Tardieu
et al., 1991, 1992A). Application of ABA externally by
root-feeding plants with different concentrations of synthetic ABA (Zhang and Davies, 1990a) or stem injection
of ABA (Tardieu et al., 1993), also demonstrated relationships between stomatal conductance, leaf growth and
[X-ABA] that were closely comparable to those caused
by roots in drying soil. All these results confirmed that
ABA had all the prerequisites for a root signal molecule;
being root-sourced, able to move from the root to the
shoot and able to affect shoot physiology. Nevertheless,
in the field, it seems likely that much xylem-borne ABA
is recirculated from the shoot. In a recent simulation,
Slovik et al. (1995) have suggested that ABA sequestered
in the leaf mesophyll chloroplasts is released as a result
of a stress treatment, moves to the root and only from
there to sites of action in the leaf.
3. What evidence is necessary to support an
unequivocal case for the involvement of ABA (or
any other chemical regulator) in a physiological or
developmental response?
Correlations between ABA and shoot physiology of the
type described above are not sufficient to allow us to
ascribe cause and effect (Tardieu and Davies, 1993). Even
when the solute in the xylem has no influence on g,, an
apparent relationship can occur between g, and the solute
of interest as a result of passive dilution as transpiration
increases. If it is considered that increased ABA modifies
shoot responses, experiments which follow the time-course
of changes in physiology and ABA are crucial. Too often,
changes occur when sampling times are infrequent and it
is not possible to establish that changes in the concentration of the regulator either precede the physiological or
developmental response of interest, or that the two
variables change simultaneously.
Jackson (1987) has proposed some modifications of
the original criteria (Jacobs, 1959) for testing the physiological significance of hormone effects. Correlation and
duplication can indicate a potential regulatory role of a
particular hormone in a particular process. In this kind
of experiment the link between xylem ABA concentration
and stomatal conductance generated by soil drying can
be compared with a relationship generated by external
application, as described in Section 2. Deletion and
reinstatement are more certain criteria to test the specificity of hormone action, achieved by manipulating endogenous hormone levels. Sharp and co-workers have used
this approach to indicate a role for endogenous ABA in
maintaining root growth under low water potential (Sharp
et al., 1994), by deleting ABA through genetic (use of the
vp5 ABA-deficient mutant) and chemical (use of the
carotenoid biosynthesis inhibitor fluridone which reduced
tissue ABA concentration) means. Another deletion
experiment has employed the split-root system (Gowing
et al., 1990) to allow excision of the putative source for
extra ABA (i.e. roots in drying soil). The reinstatement
criterion is usually supplied by external ABA treatment.
Chemical regulation of gas exchange and growth of plants
1477
However, the most specific type of deletion and reinstatement experiment in assessing the role of ABA in controlling stomatal conductance and leaf growth is the use of
the immunoaffinity (IA) column to remove ABA from
xylem sap, as discussed below.
Before basing a substantial research programme around
the possibility that xylem-borne ABA may have an exclusive role as a chemical regulator, it is desirable to perform
an experiment where a portion of the xylem sap of interest
is passed through an IA column (composed of ABA
antibodies) to remove ABA. The anti-transpirant activity
of the xylem sap can then be tested in the presence
5 10
100
1000
(unprocessed sap) and absence (after passage through the
ABA concentration (nW)
IA column) of ABA by feeding the sap to detached leaves
and gravimetrically monitoring transpiration (a transpirFig. 1. Immunoaffinity column experiment (from Zhang and Davies,
ation bioassay), or incubating epidermal strips in the sap
1991). Transpiration of detached leaves is plotted against the ABA
and measuring stomatal aperture (an epidermal strip
concentration of feeding solutions, which were synthetic ABA m an
artificial xylem solution (O), or xylem sap collected from well-watered
bioassay). In a similar manner, the growth inhibitory
( • ) or unwatered (A) maize plants. Xylem sap from unwatered maize
activity of the sap can be tested using a leaf elongation
plants which was passed through the immunoaffinity column to remove
assay (Munns, 1992).
ABA is indicated as (A). Values are mean±SD of 5 leaves.
Using a transpiration bioassay system, Munns and
King (1988) and Zhang and Davies (1991) obtained
transpirant activity on particular days) makes it difficult
contradictory results. Munns and King (1988) demonto be certain.
strated that ABA added to distilled water, at a concentraTo summarize the evidence from immunoaffinity
tion comparable to that found in the xylem sap of
column experiments, in 2 out of 3 cases, xylem sap
unwatered wheat plants, failed to reduce transpiration
stripped of its ABA still had anti-transpirant activity,
rate in detached leaves by as much as xylem sap from
which considerably weakens the case for measuring ABA
unwatered plants. They concluded that 100 times more
in the field. However, the IA experiments employ nonABA than was apparently present in sap of unwatered
water-stressed tissues and thus do not address the importplants was needed to promote the same effect. They also
ant concept of tissue sensitivity (see Section 8) which may
showed significant anti-transpirant activity in xylem sap
be crucial in arguing a regulatory role for ABA. In
after removing ABA. On the basis of these results they
designing such experiments, attention should also be
proposed that xylem sap of wheat plants contained an
drawn to the method of collection of xylem sap (see
unidentified compound with anti-transpirant activity.
Section 6). Another criticism of such immunoaffinity
Zhang and Davies (1991), however, demonstrated that
column experiments is that they may collect sap from
removal of ABA, by means of an immunoaffinity column,
relatively stressed plants. It is noted that the effectiveness
eliminated the anti-transpirant activity of maize xylem
of ABA diminishes with increasing severity of stress
sap (Fig. 1). Like Munns and King (1988), Trejo (1994)
(Correia and Pereira, 1995) and thus xylem sap may
showed that ABA was unable to account for all the anticontain a compound which is important in maintaining
transpirant activity in Phaseolus vulgaris xylem sap.
long-term stomatal closure and does not regulate stomatal
Several possible explanations for the anti-transpirant
behaviour in the early stages of a drying cycle.
activity of 'ABA-stripped' sap have been proposed including the redistribution of existing ABA (especially in plants
containing high levels of ABA in the well-watered state,
4. How should ABA be quantified?
e.g. Phaseolus), high sensitivity of stomata to small
The aim of this paper is not to discuss the theoretical
changes in ABA concentration and the presence of an as
background of techniques currently in use to quantify
yet unidentified regulator of stomatal control. Recent
ABA in samples of plant material; for this purpose the
results of Munns et al. (1993) apparently confirmed the
following texts and reviews are recommended (Neill and
third supposition for wheat and barley plants, although
there is some doubt that their compound occurs in vivo, Horgan, 1987; Parry and Horgan, 1991; Walker-Simmons
and Abrams, 1991). In what follows, some considerations
since significant anti-transpirant activity only developed
with sap storage at — 20 °C. The anti-transpirant activity in the choice of a method to quantify ABA are discussed.
Methods of ABA analysis are of two main types,
of freshly collected sap is apparently explicable by ABA
physico-chemical and immunological; the chief procedconcentration, although the variability of the transpirures of which are illustrated in Fig. 2. Since field studies
ation bioassay sensitivity (up to a 20% change in anti-
1478
Dodd et al.
IMMUNOASSAY
PHYSICO-CHEMICAL
EXTRACTION
EXTRACTION
ASSESS EXTRACT QUALITY
PURIFICATION
• Dilution/Spike Test
- Gas Chromatography (GC)
- Immunohistogram
- Thin Layer Chromatography (TLC)
- Validation with a physico-
- Pre-packed Chromatograptiic
chemical technique
Columns (PPCC) eg SEP-PAK
- High Performance Uquid
Chromatography (HPLC)
[ PURIFICATION ]
IMMUNOASSAY
DERIVATIZATION (for GC-based detector)',
DETECTION / IDENTIFICATION
- Electron Capture Detector (ECD)
- Flame lonizatjon Detector (FID)
- Ultra-Viotet Detector (UV) - HPLC only
- Mass Spectrometry (MS)
Fig. 2. Procedures of ABA analysis.
of the ABA relations of plants generally require the
processing of a large number of samples of potentially
small volume, immunoassays are often preferred as it
may be possible to analyse samples, such as crude aqueous
extracts, with minimal sample purification. When extensive purification procedures are necessary for a valid ABA
analysis, physico-chemical methods may be preferred.
Potential disadvantages of physico-chemical techniques
are that they may require a large volume of sample, and
the availability of a suitable internal standard to quantify
non-predictable losses of ABA during purification.
Another major disadvantage of physico-chemical methods
is the time-consuming preparation and purification of
samples, which usually involves partitioning of the extract
against an organic solvent followed by chromatographic
separation of the extract portion containing ABA. The
chief advantages of TLC (thin layer chromatography)
over HPLC (high-performance liquid chromatography)
are its simplicity, cheapness and speed of analysis (Parry
and Horgan, 1991). However, HPLC offers greater
sample capacity and resolving power (Brenner, 1981) and
the chance to do both the preparatory sample clean up
and the analytical quantitation (by use of a UV detector:
254 nm) with the same instrument (Ciha et al., 1977). In
some samples, if ABA is present at a level similar to that
of a UV-absorbing interfering compound, HPLC will be
of limited use since the UV detection is not selective
(Parry and Horgan, 1991). Additional purification (Parry
and Horgan, 1991), or verification by another form of
analysis, will be necessary for such samples.
Gas and gas-liquid chromatography (GC and GLC)
are usually linked to detection of ABA by electron capture
(ECD), flame-ionization (FID) or mass spectrometry
(MS). Use of GC demands that the ABA first be derivatized by the addition of diazomethane, as ABA is not
volatile. The chief consideration in the above detectors is
their selectivity, and it may first be necessary to establish
identity of the ABA-containing peak with other methods
before reliable quantitation can be achieved. As with UV
detection, the FID is non-selective. Similarly, the ECD
will detect other electron-capturing compounds such as
the ABA metabolites MePA and MeDPA (Neill and
Horgan, 1987). Even using MS, the preferred technique
for 'the definitive quantification of ABA' (Parry and
Horgan, 1991), it is necessary to use information on GC
retention times to distinguish between methyl ABA and
its 2-trans isomer which have identical mass spectra (Gray
et al., 1974). The sensitivities of such detectors are given
in Table 1, which may influence the choice of method
when dealing with small samples.
The chief advantages of immunoassays are that they
allow the operator to analyse a large number of small
samples in a short time, without highly sophisticated
instrumentation, with a high degree of sensitivity and,
most importantly, often without extensive sample purification procedures. However, before advantage can be
taken of these techniques, it is first necessary to ensure
that their use provides a true measurement of ABA, free
from contamination by interfering substances, in each
new tissue type and treatment (e.g. drought) combination.
Chemical regulation of gas exchange and growth of plants
1479
Table 1. Sensitivities (upper and lower limits of the working ranges) of physico-chemical and immunological means of ABA detection.
Immunoassay types are given as solid-phase (RIA-Solid) or liquid-phase (RJA-Liq) radioimmunoassays and direct (D-ELISA) or
indirect (I-ELISA) enzyme-linked immunosorbent assays
Abbreviations of physico-chemical techniques in text.
Technique
Assay type
Lower limit (pg)
HPLC-UV
500
1000-2000
10
50
0.3
50
Upper limit (pg)
Reference
100000
10000
Ciha el al., 1977
Durley el al., 1978
Ciha et al., \911
Neill and Horgan, 1987
Brenner, 1981
Belefant and Fong, 1989
5000
2500
2000
8000
8000
250000
25000
10000
Weiler, 1979
Walton et al., 1979
Weiler, 1980
Weiler, 1980
Weiler, 1982
Daie and Wyse, 1982
Rosher et al., 1985
Leroux et al., 1985
Physico-chemical
GLC-ECD
GC-MS-SIM
Immunoassay—Polyclonal Sera
Detection of
RIA-Liq
Total ABA
Total ABA
RIA-Liq
RIA-Liq
Free ABA
RIA-Liq
Total ABA
D-ELISA
Total ABA
D-ELISA
Total ABA
RIA-Liq
Total ABA
Total ABA
I-ELISA
Immunoassay—Monoclonal Sera
Free ABA
Antibody
16-I-A4
RIA-Liq
I-ELISA
16-I-C5
15-I-C5
I-ELISA
I-ELISA
(IDETEK)
D-ELISA
D-ELISA
I-ELISA
I-ELISA
MAC 62
RIA-Liq
I-ELISA
DPBA 1
RIA-Liq
RIA-Solid
I-ELISA
100
100
50
50
25
25
50
10
5
0.05
20
5
2.5
5
0.8
5
100
500
25
8
50
This can be done by construction of a dilution curve
from the mixture of the hormone and extract (the dilution-spike test) (Jones, 1987); assessing the distribution
of immunoreactivity in a sample extract by TLC; and
comparison of data with that obtained by physicochemical methods. Ideally, all three of these procedures
should be performed, as it is possible that interference
may be masked in one technique. For example, aqueous
extracts of sunflower showed no significant interference
in a dilution-spike test, yet a significant amount of
immunoreactive contamination on a TLC plate (Palmer,
unpublished observations). TLC is usually preferred over
HPLC as the latter relies upon eluting all the extract
constituents from the stationary phase, which may allow
immunoreactive contaminants to remain in the stationary
phase (Quarrie, 1991).
Several types of monoclonal antibodies are in current
use (the earlier polyclonal antisera in Table 1 having
largely been superseded), allied to either radioimmunoassay (RIA) or enzyme-linked immunosorbent assay
(ELISA). ELISAs can be direct or indirect depending on
whether they use a second antibody to amplify the
1000
2.5
500
250
2500
800
250
250
4000
6000
1500
800
2000
Mertens et al., 1983
Harris et al., 1988
Ross et al, 1987
Walker-Simmons, 1987
Morris et al., 1988
Belefant and Fong, 1989
Belefant and Fong, 1989
Walker-Simmons et al., 1991
Quarrie et al., 1988
Walker-Simmons et al, 1991
Vernieri et al, 1989
Vernieri et al, 1989
Walker-Simmons et al, 1991
detection process (indirect), which can increase assay
sensitivity (Belefant and Fong, 1989). The most commonly used methods (RIA and ELISA) have certain
advantages and disadvantages and the choice between
them will mainly depend on available equipment or
antibodies. Quarrie (1991) indicated that the advantages
of RIA include time efficiency, since RIA is usually
quicker (3-4 h) than ELISA (which routinely needs overnight incubation). RIA can be speeded up even further
using a scintillation proximity assay (Whitford and
Croker, 1991). Other advantages of RIA are that antibody affinity for the tracer antigen is usually the same as
for the sample antigen, the tracer is not affected by other
components present in the sample extract and replicate
reproducibility is often better than it is in ELISA (Quarrie,
1991). Radioactively labelled antigens are readily available commercially. As the sample size and extract volume
can be adjusted, the same tubes can be used for both
collecting and extracting the tissue. The major disadvantage of ELISA is that the enzyme tracer can be affected
by other components of the sample extract, reducing the
efficiency of the colour reaction. However, ELISA is less
1480 Dodd etal.
expensive than RIA and does not require radioactive
tracer or much specialized laboratory equipment (WalkerSimmons and Abrams, 1991).
The sensitivities of various immunoassays are given in
Table 1 and clearly rival physico-chemical methods.
Particular mention should be made of the ability to
measure ABA in individual guard cell pairs (Harris et al.,
1988), coupling an indirect ELISA to single cell sampling
methodology.
Using published values of plant ABA concentrations
and the lowest value of the assay working range of the
three principal monoclonal antibodies, the amount of
tissue (or xylem sap) that will be required for sampling
have been calculated (Table 2). Clearly, the availability
of plant material for destructive harvest may influence
the choice of antibody. This will especially be the case
when sampling leaf epidermes or the elongation zones of
grasses or xylem sap from small plants.
Despite the arsenal of techniques available to measure
ABA accurately, the frequently observed lack of correlation between measured tissue ABA concentration and the
observed response points to the importance of suitable
techniques for ABA localization and quantitation, both
at a tissue and cellular level. Although some immunocytochemical methods for tissue ABA localization have been
published (Sossountzov et al., 1986; Bertrand et al., 1992),
they have not been widely repeatable. On a more macro-
scopic scale, correlations between ABA and physiological
response may depend on where the hormone is quantified.
5.
In which tissues should ABA be measured?
This is an important question that has not received very
much attention in the literature. The answer will depend
to a large extent on the processes that are of interest to
the experimenter. In general terms, it is obviously important to assess the ABA status as close to the site of action
as possible. Those interested in signalling will need to
assess the possible nature of the signal (concentration
versus flux—see Section 8) and then decide whether to
assess this quantity close to the putative source and/or
close to the site of action.
Roofs
Due to the lack of accessibility, few studies have attempted
to measure root ABA concentration ([root-ABA]) in the
field (but see Tardieu et al., \992a). It is well established
that isolated roots are capable of producing ABA in
response to dehydrative or osmotic stress (Milborrow
and Robinson, 1973; Walton et al., 1976) and that plants
show enhanced [root-ABA] in response to a number
of stresses (e.g. drought, nitrate deprivation, salinity).
However, detailed investigations are necessary to show
Table 2. How much tissue (LEAF or ROOT) or xylem sap (X-SAP) needs to be collected from well-watered (WW) or water-stressed
(WS) plants to allow ABA quantification with a given monoclonal antibody?
Data were calculated using the lower working range of liquid-phase radioimmunoassays (from Table 1) and from published ABA concentrations
for different tissues (see Reference). Calculations as follows. Sap required (/il) = [lower limit of assay range (pg)x lO^xylem sap ABA
concentration (nmol I" 1 ) x molecular weight of ABA (g mol~')]. Tissue required (mg) = lower limit of assay range (pg)/tissue ABA concentration
( n g g - 1 DW).
Antibody
Reference
Lower limit of assay working range using RIA-liq
16-I-A4
Mertens et al., 1983
Min: 5 pg/vial
MAC 62
Quarrie et al., 1988
Min: lOOpg/vial
DPBA 1
Vernieri et al., 1989
Min: 25 pg/vial
Species/Reference
Tissue
Concentration
Required sample
Xylem sap (/J)
Required sample
Xylem sap (^1)
Required sample
Xylem sap (/J)
Triticum aestivum
Munns and King, 1988
Zea mays
Zhang and Davies, 1990a
Phaseolus vulgaris
Trejo and Davies, 1991
Lupinus albus
Correia and Pereira, 1995
Acer pseudoplalanus
Khali! and Grace, 1993
X-SAP-WW
X-SAP-WS
X-SAP-WW
X-SAP-WS
X-SAP-WW
X-SAP-WS
X-SAP-WW
X-SAP-WS
X-SAP-WW
X-SAP-WS
1 nM
50 nM
10 nM
160 nM
90 nM
200 nM
5nM
900 nM
190 nM
413nM
Zea mays
Zhang and Davies, 1990a
Phaseolus vulgaris
Trejo and Davies, 1991
Zea mays
Zhang and Davies, 1989a
Phaseolus vulgaris
Trejo and Davies, 1991
LEAF-WW
LEAF-WS
LEAF-WW
LEAF-WS
ROOT-WW
ROOT-WS
ROOT-WW
ROOT-WS
90ngg"
160 n g g "
300 ng g"
900 ng g"
40ngg"
200 ng g"
400 n g g "
800 n g g " 1
19.0
0.38
1.90
0.12
0.210
0.095
3.78
0.02
0.100
0.046
Dry weight (mg)
0.056
0.031
0.017
0.006
0.125
0.025
0.013
0.007
379
7.5
38
2.4
4.2
1.9
75 7
0.42
2.00
0.92
Dry weight (mg)
1.11
0.63
0.33
0.11
2.5
0.5
0.25
0.13
95
1.9
9.5
0.59
1.05
0.47
18.9
0.11
0.50
0.23
Dry weight (mg)
0.28
0.16
0.083
0.028
0.625
0.125
0.063
0.031
DW
DW
DW
DW
DW
DW
DW
DW
Chemical regulation of gas exchange and growth of plants
that elevated [root-ABA] is a result of synthesis and not
redistribution of ABA as a result of perturbed leaf water
relations in older leaves. Growth cabinet studies have
shown that this is probably the case, with enhanced [rootABA] in the upper part of the soil profile preceding
decreases in leaf water relations and stomatal conductance
of droughted plants (Zhang and Davies, 1989a; Trejo
and Davies, 1991). A tight relationship between [rootABA] and soil water content can exist over a drying cycle
and for roots from different parts of the soil profile
(Zhang and Davies, 1989a). However, this may occur
only under growth cabinet conditions when VPDs and
water fluxes are low. In contrast to growth cabinet studies,
where dehydration of only part of the soil profile increased
root and xylem ABA concentrations to initiate stomatal
closure, field studies show appreciable increases in [rootABA] only when the whole soil profile was close to
depletion (Tardieu et al., 1992a). Root ABA content can
be related to soil water status when the plants are not
transpiring (Tardieu et al., 1992a), but it is not clear that
roots of transpiring plants in drying soil will necessarily
contain a lot of ABA. Roots in very dry soil may
contribute little to the transpiration stream, but presumably will rehydrate at night and then dehydrate again the
following day, thereby supplying some ABA to the shoot.
Given the evidence for ABA production in roots in
drying soil, there is some attraction in the possibility of
relating root ABA concentration to soil water status. This
is not an easy thing to attempt, particularly in the field,
and the correlation of shoot processes with ABA measured closer to the site of action (growing leaf or stomatal
complex), makes it unlikely that many workers will have
cause to measure root ABA under field conditions. In
sampling, the worker should be aware of the heterogeneity
of environments experienced by different parts of the root
system, which will be reflected in root ABA contents.
Even under similar soil conditions, different orders of
roots may show differing ABA contents. Nodal roots of
maize contain more ABA than seminal roots (Pekic and
Quarrie, personal communication). The importance of
nodal roots in regulating shoot physiology has been
shown by comparison of quantitative trait loci (QTL),
which gives a striking correlation between nodal root
number and [X-ABA] (Lebreton et al., 1995). Finally,
spatial gradients of ABA concentration have been characterized within a single root (Saab et al., 1992), with ABA
concentration maximal at the root tip. Spatial differentiation of root ABA content may be important to those
interested in the control of root growth.
Leaves
Bulk leaf ABA content ( [ L - A B A ] ) has been routinely
measured in drought-stress studies because of the accessibility and abundance of tissue. However, its physiological
1481
significance in the early stages of soil drying is questionable. Time-course studies of growth cabinet-grown plants
show that [ L - A B A ] increases only slightly and much later
than [X-ABA] (Zhang and Davies, 1989ft, 1990a). It
is common for g, in split-root maize plants to decline
by 30-40% before changes in [ L - A B A ] are detected
(Blackman and Davies, 1985; Zhang and Davies, 1990a).
In other studies, [ L - A B A ] increases only once the stomata
have closed (Trejo and Davies, 1991). An increase in [LABA] may therefore be a consequence and not a cause
of chemical signalling. In the field, a relatively loose
relationship is found between [ L - A B A ] and stomatal
conductance (Burschka et al., 1983; Tardieu et al., 19926)
in contrast to the tight relationship between [X-ABA]
and conductance (Tardieu et al., 1991, 1992A).
Leaf ABA may generate a chemical signal in the
phloem, since changes in the water relations of leaves
liberates ABA to the apoplast and this is then taken up
by the alkaline phloem (Slovik et al., 1995). Comparative
studies of leaf water relations at different points of
insertion indicate that older leaves are less able to control
their water status in some species (Zhang and Davies,
19896), perhaps as a result of reduced sensitivity to a
chemical signal (Atkinson et al., 1989). Perturbed water
relations may, therefore, allow older leaves to be a source,
rather than a sink, for ABA (Wolfe/ al., 1990).
The leaf epidermis
Part of the reason for the lack of relationship between
bulk leaf ABA concentration and stomatal responses is
the relatively crude nature of the sampling. Leaves commonly have a substantial ABA content in an unstressed
condition, with the ABA being sequestered in mesophyll
chloroplasts in the light (Heilmann et al., 1980) where it
can have no effect on stomata or on growth. Measurement
of ABA closer to the site of action on guard cells is
possible in some species where the epidermis can be
stripped from the mesophyll. Using this approach, Zhang
et al. (1987) showed that afternoon stomatal closure in
well-watered Commelina plants could be explained by
increased epidermal ABA concentration even though no
change could be detected in bulk leaf ABA concentration.
Epidermal ABA concentration was also correlated with
stomatal closure over the course of a drying cycle.
Measurement of epidermal ABA concentration has also
been shown to account for the different dose-response
relationships observed when Commelina stomata were fed
the same ABA concentration using different assay
methods (Trejo el al, 1993; Fig. 3) When isolated epidermes were incubated on 10~3 mol m~ 3 ABA, stomata
closed by 76% while incubation of leaf pieces on the same
concentration closed stomata by only 13%. The difference
in the degree of closure was accounted for by differential
ABA accumulation in the epidermis and suggests that the
1482 Dodd etal.
1400
1200 -
so
Epidermal strips^*
1000
60 -
40 Transpiration buasay
20
•
Pwcasoftoif
0 *
6
8
10
200 -
EPIDERMAL ABA (jifl / g DW)
Fig. 3. Apparent differences in stomatal responses to 2 x 10 3 mol m 3
(±)-ABA in different bioassay systems can be accounted for by
measurement of epidermal ABA content (redrawn from Trejo el ai,
1993).
mesophyll exerts a controlling influence over epidermal
ABA accumulation. Although there can be little doubt
of the value of measuring epidermal ABA content to help
explain stomatal behaviour, its application may be limited
since collection of sufficient tissue for analysis is both
destructive and laborious, and the technique is limited
only to certain species.
The grass leaf elongation zone
For studies which seek to relate changes in leaf growth
and ABA content over a drying cycle, it is important to
measure ABA content in cells that are actually growing.
Grasses are especially amenable to this analysis as the
elongation zone (EZ) is restricted to a basal portion of
each leaf. However, there are particular problems. It will
be difficult to sample ABA specifically at the site of
action, since the epidermis is usually considered to be the
tissue limiting expansion (Kutschera, 1992). There are
also spatial gradients of cell dimensions due to expansion
in the EZ, which may complicate interpretation of any
change in ABA concentration at a particular point. Saab
et ai (1992) have analysed the spatial distribution of
growth and ABA in the maize mesocotyl, and failed to
show any relationship between these two variables on a
local scale. This occurs since ABA concentration is relatively stable across the growing zone apart from enhanced
amounts close to the leaf base. The same pattern exists
for intact maize plants grown in drying soil and detached
shoots fed ABA via the sub-crown internode in a leaf
elongation assay (Palmer, Dodd and Davies, unpublished
observations; Fig. 4). This stability of ABA across the
EZ permits the sampling of a bulk elongation zone
(Fig. 4, 10—40 mm from crown), which allows sufficient
plant material for RIA analysis of small grass samples
(Table 2).
0
10
20
30
40
50
DISTANCE FROM CROWN (mm)
Fig. 4. Spatial distribution of ABA concentration in the leaf elongation
zone of well-watered ( • ) and draughted (A) maize plants and detached
maize shoots fed an artificial xylem solution (O) or 2 x 10"3 mol m~3
(±)-ABA (A) for 8 h. Samples are from the third main-stem leaf and
are bulked from 6-8 leaves (Palmer, Dodd and Davies, unpublished
observations).
The necessity of measuring [EZ-ABA] was made apparent by the observation that detached wheat shoots in a
leaf elongation assay showed a variable growth inhibition
at the same ABA concentration when the assay conditions
(temperature and VPD) were varied (Dodd and Davies,
1994). It now appears that the increased sensitivity of
leaf growth to ABA at high temperature can be explained
by the greater accumulation of ABA at high transpiration
rates (Fig. 5). However, the usefulness of [EZ-ABA] in
accounting for soil-drying induced leaf growth inhibition
has not been established since soil drying can apparently
promote growth reductions of up to 35% in the absence
of substantial ABA accumulation in the EZ (Dodd and
Davies, 1996).
Xylem sap
The lack of correlation between bulk leaf ABA concentration and stomatal responses has been a source of frustration to physiologists arguing for a role for ABA in the
control of plant water loss. Although ABA was detected
in xylem sap as early as 1968 (Lenton et ai, 1968) and
shown to increase when the roots were placed under
osmotic stress (Hoad, 1975); correlations between
[X-ABA] and stomatal conductance or leaf growth rate
were not published until the 1980s (Loveys, 1984; Zhang
and Davies, 19896, 1990a, b). It should be noted that the
xylem sap appears to be directly in contact with the leaf
apoplast and thus the apoplast surrounding the guard
cells, which would intuitively suggest a relationship
between g, and [X-ABA]. However, the role of metabol-
Chemical regulation of gas exchange and growth of plants
120
T = 7°C, VPD = 0 25 kPa
100
I
80
£
60 T = 15°C, VPD = 0 9kPa
o
UJ
= 20°C,VPD = 1 1 kPa
40
20
T = 30°C, VPD = 3 2 kPa
200
400
600
800
1000
1200
14O0
ELONGATION ZONE ABA (ng / g DW)
Fig. 5. Apparent temperature-dependence of leaf growth response to ABA
in detached barley shoots may be accounted for by measurement of bulk
elongation zone ABA. The shoots were fed 2 x 10~3 mol m~ 3 (±)-ABA
via the sub-crown internode and the bulk elongation zone of the third
main-stem leaf (a portion 1 0 - 4 0 mm from the node) excised 6 h later.
Each point consists of leaf growth measurements from 8 leaves ±SE and
5 ABA determination ±SE (redrawn from Dodd and Davies, 1966).
ism in modifying the composition of xylem sap as it
passes to the guard cells may be crucial in regulating the
[ABA] at the guard cells (Trejo et ai, 1993, 1995). Despite
this caution, [X-ABA] has been shown to correlate with
stomatal conductance in the field despite differences
caused by plant-to-plant variability, different soil conditions and changes over a drying cycle (Tardieu et ai,
\992b). Xylem ABA concentration responds sensitively
to mild soil drying although the controversy of whether
ABA is the only chemical signal regulating shoot physiology continues (see Section 3). It would therefore appear
that xylem sap ABA concentration is a valuable measurement to make; although this is not without its problems.
6.
1483
transpiration. Although this technique cannot be used in
the field, it can be extremely useful to validate other
sampling techniques for field use. Rather surprisingly, there
has been little attempt to do this. This technique has been
used in a number of laboratory studies to investigate the
influence of chemical signalling on shoot physiology
(Gollan et ai, 1986; Passioura, 1988; Schurr et ai, 1992).
Although the root pressure chamber is not compatible
with field use, it is possible to express a sample of xylem
sap from detached leaves and shoots using the Scholander
pressure bomb. However, there are concerns that application of pressure to the leaf will force symplastic water
into the xylem vessels, thereby changing the concentration
of ABA in the xylem sap. If the expressed sap shows the
same ABA concentration at various pressures (= flow
rates), there can be reasonable confidence that the fluid
existing in the vessels prior to detaching the leaf has been
obtained. This has been demonstrated for woody almond
twigs (Wartinger et ai, 1990) and large maize leaves
(Fig. 6). Most studies, however, usually apply an arbitrary overpressure (above the balancing pressure) and do
not determine the effect of various overpressures on the
ABA concentration.
Evidence of contamination of the sample by cell sap
(from crushed cells or phloem exudation) can be obtained
by assaying the sample for sugars, in species which are
known not to transport sugars in the xylem (Correia and
Pereira, 1994, 1995; Correia et ai, 1995). By plotting the
regression of ABA on sucrose concentration for a number
of samples, it is possible to obtain an estimate of ABA
concentration at zero sucrose. The value of such an
approach will be species-dependent and rely on previous
knowledge of carbohydrate transport in the plant.
How should xylem sap be collected?
Section 3 showed that xylem sap ABA concentration is
highly dependent on the flux of water through the plant.
In a non-transpiring plant, ABA concentration will be
substantial. Ideally, xylem sap should be collected at the
same flow rate as it would move in the transpiration
stream. In the absence of specialized equipment, this will
only be possible in the field when the plants guttate predawn. Although ABA can be detected in guttation fluid
and its concentration increases over a drying cycle (Dodd
and Davies, unpublished observations), it is uncertain
exactly what this fluid represents.
The development of the root pressurization chamber
(Passioura and Munns, 1984) has made it possible to
sample xylem sap at the same flow rate as occurs during
0.2
03
04
05
0.6
07
1 1
OVERPRESSURE (MPa)
Fig. 6. Effect of different sap collection overpressures on measured
xylem sap ABA concentrations of maize leaves. Each line is drawn
between 3 leaves from the same plant sampled at different overpressures
(Dodd and Davies, unpublished observations).
1484 Dodd etal.
Another caution in the use of xylem sap expressed
from leaves concerns the amount of sample taken relative
to the xylem vessel volume in the leaf. This may especially
be a problem with the large amounts of sap required for
RIA in some species (Table 2). One can have more
confidence in xylem sap samples collected from large
leaves with a substantial xylem volume.
In small-leaved plants, obtaining sufficient xylem sap
for analysis will be problematical and the worker may be
forced to resort to collection from detopped stumps. This
method has been frequently criticised (Munns, 1990;
Jackson, 1993) since it overestimates [X-ABA] due to the
low sap flow rate. Parallel use of the root pressure chamber
has shown that sampling at low flow rates from detopped
plants can over-estimate ABA concentration by 5 (Schurr
and Schulze, 1995) and 3.3 (Else et ai, 1995) times. ABA
enrichment of the first sap samples collected also occurs
via the radial pressure exerted by the tubing fitted to allow
xylem sap collection (Else et ai, 1994), which leads some
workers to discard these samples. However, sequential
sampling of xylem sap from detopped shoots of maize
plants showed no such enrichment (Zhang and Davies,
19906) thus this effect may be species specific. The use of
sucrose validation analysis with root exudates (as outlined
above) has produced ABA concentration values within
the range obtained from sap samples expressed under
pressure from leaves (Correia and Pereira, 1995). Thus,
when there is no alternative, in certain species it may be
possible to determine [X-ABA] from root exudates, but
substantial validation will be necessary.
7.
When should ABA be measured?
Measurement of ABA concentration in non-transpiring
plants before dawn may be a useful indicator of soil water
availability since [X-ABA] has been shown to correlate
with pre-dawn water potential (Wartinger et ai, 1990;
Tardieu et al., 1992a).
A number of studies have examined changes in xylem
ABA concentration over the course of the day. Although
a single relationship between g, and [X-ABA] is not
found over the course of the day, average daily ABA
concentration has been shown to set the maximum conductance achieved that day (Wartinger et al., 1990;
Correia et al., 1995). While diurnal variations in ABA
(Loveys, 1984; Wartinger et al., 1990) or cytokinins
(FuBeder et al., 1992) concentrations undoubtedly occur
on certain days, most field data indicate a lack of diurnal
variation (Loveys et al., 1987; Wartinger et ai, 1990;
FuBeder et al., 1992; Tardieu et al., 1992a; Correia et ai,
1995). Therefore alternative explanations must be sought
for the often-published diurnal changes in stomatal conductance and leaf growth rate of unwatered plants.
8. Does measurement of ABA concentration
provide useful information?
Concentration versus flux
In attempting to answer whether stomata or growing
leaves are responding to a chemical signal, one should
first know what measure of the hormone the cells are
responding to. Concentration is the number of molecules
in a given volume while flux is the concentration multiplied by the flow rate (of the transpiration stream). The
autocorrelation of these two variables means that covariance analysis may help in the interpretation of field data.
More useful information has been obtained from laboratory studies, where ABA is fed to detached leaves and
shoots. In analysing stomatal responses to a pulse of
ABA applied via the petiole to detached cherry leaves,
Gowing et ai (1993) found that concentration alone
could account for 30% of the variance, while flux
accounted for 74%. Use of a simple model to calculate
apoplastic concentration showed that this variable had
the greatest explanatory power, but information from the
model cannot be validated in the absence of data on the
rate of ABA metabolism. It seems likely that this rate
will be highly variable depending on environmental variation and probably also on the water status of the plant.
Trejo et al. (1995) were able to change ABA flux at a
given ABA concentration fed by altering the temperature
and VPD surrounding detached shoots. At a given concentration, a 3-fold variation in ABA flux had no effect
on the restriction of conductance; while the same flux
achieved by increasing the ABA concentration was able
to decrease conductance further. The same authors also
used a microscopic system to vary the ABA flux to
detached epidermes. Significantly, stomata were only capable of responding to an increase in flux of an order of
magnitude. As transpiration and VPD increase as the day
progresses, changes in ABA flux will undoubtedly occur.
It seems likely that ABA delivery will contribute to local
ABA concentration (Gowing et ai, 1993) and therefore
influence stomatal behaviour and growth. Our data (Trejo
et ai, 1995) suggest, however, that once the stomata are
effectively open then variation in flux will have little
influence on stomatal behaviour.
Jackson (1993) has stressed the importance of calculating
delivery rate of ABA with the calculation taking account
of sap flow rate, ABA concentration measured at a reasonable transpiration rate and even the size of the source and
sink for ABA. Such calculation will undoubtedly be important for estimating the nature of any signalling process and
showing whether or not the supply of a particular regulator
is actually augmented by the cultural treatment.
Same concentration, different effect
Tardieu and Davies (1992) demonstrated for field-grown
maize plants that the relationship between xylem ABA
Chemical regulation of gas exchange and growth of plants
(a)
0-3-
0
¥ , > -1-3 MPa
02-
01 A
•>-n
1
09
u
-
I o
0 3 -t
(b)
-1-6< x i',< -1-3 MPa
111
0 2 * • >
O
E.
Q
0 1-
•tit
*"•*& '
a^T —j*. •
0 0 - — ^r
.»
0 0
200
400
600
Xytem (ABA] (jimol m"3)
800
Fig. 7. Stomatal response to ABA in field-grown maize plants is
dependent on current leaf water potential (V, from Tardieu el al., 1993).
Each point is a paired measurement of g, and xylem ABA concentration
for one leaf. Different symbols represent plants subject to different soil
water availabilities and sampled at different times of day, Data have
been sorted according to whether V, was less than - 1 . 3 MPa (a)
between —1.3 and —1.6 MPa (b) or greater than —1.6 MPa (c).
concentration and g, was greatly affected by the time of
day. Their results showed that a given dose of xylem
ABA was more effective in the restriction of g, later in
the day than early in the morning (Fig. 7). These differences have been described as an increase in stomatal
sensitivity to xylem ABA at lower water potential. The
progressive decline in leaf water potential to a minimum
at midday apparently sensitized stomata to the ABA
signal. The same tendency was observed with epidermal
tissue of Commelina incubated in solutions with different
ABA and PEG solutions. The study of Saab et al. (1990)
suggested that the influence of ABA on root cell growth
can also be modified with variations in tissue water status.
These results suggest that the response of stomata or
growth to endogenous ABA is not a fixed characteristic
for a species and that responses cannot be explained by
measuring concentration only.
The concept of hormone sensitivity is well developed
in the literature (Trewavas, 1981, 1991) although the
basis of any variation is not well understood. Variation
in sensitivity can be a problem for the worker trying to
1485
demonstrate that the hormone of interest can account for
the observed physiological effect; it is important that
Jacobs' Rules (see Section 3) are tested under appropriate
environmental conditions. An examination of the literature will reveal that ABA can interact with a large number
of environmental variables to influence the effect on
stomata and leaf growth rate (Table 3). It is likely that
the number of cases where no interaction was detected is
much greater than the Table would indicate, due to the
bias of only reporting significant interactions. In assessing
the physiological relevance of such data, attention must
be drawn to the few examples existing from whole plant
studies. Even within the laboratory, detection of an
interaction can be highly dependent on the species and
assay method used. For an interaction to be considered
physiologically relevant, its existence should be demonstrated in different assay systems which mimic the whole
plant as closely as possible in terms of environmental
conditions and hormone concentrations. Despite these
cautions, it would seem undeniable that variation in
hormone sensitivity does occur; the challenge being to
consider a role for hormone sensitivity in the context of
diurnal changes in g, and growth seen in the field.
Stomata typically show morning opening with afternoon
closure (Loftfield, 1921; Correia et al., 1995), with variation in the photoenvironment usually considered to be
the driving variable. This behaviour cannot generally be
accounted for by increasing xylem ABA concentration
over the course of the day (see Section 7) and if ABA is
implicated, then a role for other factors modifying the
response to xylem ABA concentration must be considered.
One possible explanation for the afternoon closure could
be that decreases in bulk leaf water potential ( f , ) in the
afternoon are able to sensitize stomata to a given ABA
concentration (Tardieu and Davies, 1992; Fig. 7). The
generality of this interaction has been demonstrated in the
Commelina epidermal strip bioassay (Tardieu and Davies,
1992) and a transpiration bioassay where the water potential was decreased in the leaf by attaching the shoot to a
capillary (Trejo and Davies, 1994). However, no such
interaction was demonstrated for sunflower when f , and
xylem ABA were manipulated in the field and in detached
leaves (Tardieu et al., 1996). Correia et al. (1995) were
similarly unable to account for the afternoon closure of
grape stomata in terms of a Wx x ABA interaction.
Studies of leaf growth in the field have shown that
temperature can exert a very significant effect on leaf
elongation rate (LER) (Gallagher and Biscoe, 1979;
Ong, 1983). Unwatered plants show a divergence from
the expected growth-temperature relationship (Fig. 8;
Gallagher and Biscoe, 1979) which is unlikely to be
accounted for by diurnal changes in xylem ABA concentration (see Section 7). The observation that leaf growth
responses to ABA were temperature-dependent (Dodd and
Davies, 1994) appeared to provide an interpretation of
1486
Dodd et al.
Table 3. Factors which may modify the stomatal or leaf growth response to ABA
Experimental systems are as follows: ES, epidermal strip bioassay; ES(P), leaf fragments incubated on solutions, then epidermal strips removed to
allow stomatal measurement; TB, transpiration bioassay; LE, leaf elongation assay; WP, whole plant. NT indicates no interaction between ABA
and the variable.
Factor
Species
System
Reference
Temperature
Assay
Zea mays
Phaseolus vulgaris
Triticum aestivum
Phaseolus vulgaris
Zea mays
Triticum aestivum
Bellis perennis
Cardamine pratensis
Commeluia communis
Commelina communis
Commelina communis
Zea mays
Phaseolus vulgaris
Helianthus annuus
ESES(P)
TB
TB
TB
WP
LE
ES
ES
ES
ES
ES
WP
TB
WP, TB
Rodriguez and Davies, 1982
Eamus and Wilson, 1983
Ward and Lawlor, 1990
Comic and Ghashghaie, 1991
Tardieu et al., 1993
Dodd and Davies, 1994
Honour et al., 1995
Honour et al., 1995
Honour et al., 1995
Allan et al, 1994
Tardieu and Davies, 1992
Tardieu and Davies, 1992
Trejo and Davies, 1994
Tardieu et al., 1996
Vicia faba
Vicia faba
Gossypium hirsutum
Commelina communis
Commelina communis
Xanthium strumarium
Xanthium strumarium
Commelina communis
Triticum aestivum
Zea mays
Commelina communis
Triticum aestivum
many spp
maize cultivars
wheat cultivars
Commelina communis
Commelina communis
ES
WP
TB
ES
ES
TB
TB
ES
TB
ES(P)
ES
TB
TB
ES(Pj
WP.TB
ES ES(P)
ESES(P)TB
Davies, 1978
Davies, 1978
Ackerson, 1980
Wilson, 1981
Peng and Weyers, 1994
Raschke, 1975
Mansfield, 1976
Wilson, 1981
Mittelheuser and van Stevemnck, 1969
Blackman and Davies, 19846
Willmer et al., 1988
Atkinson et al., 1989
Kriedemann et al., 1972
Rodriguez and Davies, 1982
Quarrie, 1983
Blackman and Davies, 1983
Trejo et al, 1993
Helianthus annuus
WP
Schurr et al, 1992
Gossypium hirsutum
Gossypium hirsutum
TB
TB
Radin et al, 1982
Radin, 1984
Commelina communis
Commelina communis
Phaseolus vulgaris
Commelina communis
Commelina communis
Commelina communis
Triticum aestivum
Commelina communis
Hordeum vulgare
Commelina communis
Zea mays
Commelina communis
Commelina communis
Aster tripolium
Vicia faba
Commelina communis
Commelina communis
ES
ES
LE
ES
ES
ES
TB
ES
TB
ES ES(P)
ES(P)
ES
ES
ES
ES
ES
ES
Wilson et al, 1978
Snaith and Mansfield, 1982A
Van Volkenburgh and Davies, 1983
De Silva et al, 1985
Jarvis and Mansfield, 1980
Paterson et al, 1988
Mittelheuser and van Steveninck, 1969
Tucker and Mansfield, 1971
Cooper el al, 1972
Blackman and Davies, 1983
Blackman and Davies, 1983, 1984a, b
Tucker and Mansfield, 1971
Snaith and Mansfield, 1982a, b
Perera et al, 1994
Dunleavy and Ladley, 1995
Snaith and Mansfield, 1984
Tucker and Mansfield, 1971
NI
Pretreatment
Water status
NI
Water stress history
NI
CO,
NI
Leaf age
Genotype
Assay type
Xylem sap composition
Nutrient status of plants
Nitrogen
Phosphorous
Incubation solution composition
Potassium
Calcium
Sodium
PH
Cytokinin
NI
NI
IAA
NI
NAA
GA 3
NI
such diurnal phenomena. However this temperaturedependent response may simply be a function of accumulated ABA (Fig. 5), which indicated that ABA accumulation in the EZ may have a role in regulating the growth of
intact plants. Although enhanced accumulation in the EZ
did not occur in growth-cabinet plants which showed a
35% reduction in leaf growth rate (Dodd and Davies,
1996), there are no reports known which have measured
[EZ-ABA] in the field when ABA flux rates will be much
higher. Another possible explanation of the diurnal changes
Chemical regulation of gas exchange and growth of plants
Allan AC, Flicker M D , Ward JL, Beale M H , Trewavas AJ. 1994.
2-5 r
Two transduction pathways mediate rapid effects of abscisic
acid in Commelma guard cells. The Plant Cell 6, 1319-28.
Atkinson CJ, Davies WJ, Mansfield TA. 1989. Changes in stomatal
conductance in intact ageing wheat leaves in response to abscisic
acid. Journal of Experimental Botany 40, 1021-8.
Betefant H, Fong F. 1989. Abscisic acid ELISA: organic acid
interference. Plant Physiology 91, 1467-70.
2.0
I
1.5
Bertrand S, Benhamou N, Nadeau P, Dostater D , Gosselin A. 1992.
o
i
i.o
0.5
A,'
0.0
Temperature (°C)
16
Fig. 8. Leaf growth of draughted barley plants in the field as a function
of temperature (from Gallagher and Biscoe, 1979). The line AB represents
the leaf extension response of well-watered barley plants to temperature.
Each point is the leaf elongation rate over a 2 h period which commenced
at the time indicated (e.g. 17 is the measurement commencing at 17.00 h).
in leaf growth of water-stressed plants is the existence of a
water potential x ABA interaction (Dodd and Davies,
1996). As "F, declines during the day, the effect of the
interaction will be greater, with leaf growth being further
inhibited.
9.
1487
Conclusions
Substantial evidence suggests that it is necessary to consider chemical signals in the regulation of stomatal behaviour and leaf growth in the field. Most research has centred
around ABA since other major growth inhibitory or antitranspirant compounds have not been positively identified. Emphasis has also been placed on ABA because of
the relative ease with which it can be measured using
modern immunological techniques. Although xylem sap
ABA concentration often correlates well with g, and LER
of draughted plants, it has been difficult to establish
whether ABA concentrations in samples of xylem sap
provide a true measure of concentrations in the transpiration stream. When ABA concentration is insufficient to
explain the observed physiological response, consideration should be given to possible variation in tissue sensitivity to ABA. Such variation may have an important role
in controlling the diurnal variation in g, and LER
observed in draughted plants growing in the field.
Acknowledgements
ICD thanks the Commonwealth Scholarship Commission for
financial support and RS thanks the Royal Society for grant
support. We thank Mr S Palmer for access to unpublished data.
References
Ackerson RC. 1980. Stomatal response of cotton to water stress
and abscisic acid as affected by water stress history. Plant
Physiology 65, 455-9.
Immunogold localization of free abscisic acid in tomato root
cells. Canadian Journal of Botany 70, 1001-11.
Blackman PG, Davies WJ. 1983. The effects of cytokinins and
ABA on stomatal behaviour of maize and Commelina. Journal
of Experimental Botany 34, 1619-26.
Blackman PG, Davies WJ. 1984a. Modification of the CO 2
responses of maize stomata by abscisic acid and by naturallyoccurring and synthetic cytokinins. Journal of Experimental
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